(Advances in Parasitology 92) Xiao-Nong Zhou, Shi-Zhu Li, Jürg Utzinger and Robert Bergquist (Eds.)-Schistosomiasis in the People's Republic of China From Control to Elimination-Academic Press (2016)

VOLUME NINETY THREE
ADVANCES IN
PARASITOLOGY
Haemonchus contortus and
Haemonchosis – Past, Present
and Future Trends
SERIES EDITOR
D. ROLLINSON J. R. STOTHARD
Life Sciences Department Department of Parasitology
The Natural History Museum, Liverpool School of Tropical
London, UK Medicine Liverpool, UK
d.rollinson@nhm.ac.uk russell.stothard@lstmed.ac.uk
EDITORIAL BOARD
T. J. C. ANDERSON R. C. OLIVEIRA
Department of Genetics, Texas Centro de Pesquisas Rene Rachou/
Biomedical Research Institute, CPqRR - A FIOCRUZ em Minas
San Antonio, TX, USA Gerais, Rene Rachou Research
Center/CPqRR - The Oswaldo Cruz
M. G. BASA NEZ
~ Foundation in the State of Minas
Professor of Neglected Tropical Gerais-Brazil, Brazil
Diseases, Department of Infectious
Disease Epidemiology, Faculty of R. E. SINDEN
Medicine (St Mary’s Campus), Immunology and Infection
Imperial College London, Section, Department of Biological
London, UK Sciences, Sir Alexander Fleming
Building, Imperial College of
S. BROOKER Science, Technology and
Wellcome Trust Research Fellow Medicine, London, UK
and Professor, London School of
Hygiene and Tropical Medicine, D. L. SMITH
Faculty of Infectious and Tropical, Johns Hopkins Malaria Research
Diseases, London, UK Institute & Department of
Epidemiology, Johns Hopkins
Bloomberg School of Public Health,
R. B. GASSER Baltimore, MD, USA
Faculty of Veterinary and
Agricultural Sciences, The R. C. A. THOMPSON
University of Melbourne, Parkville, Head, WHO Collaborating Centre
Victoria, Australia for the Molecular Epidemiology
of Parasitic Infections, Principal
N. HALL Investigator, Environmental
School of Biological Sciences, Biotechnology CRC (EBCRC), School
Biosciences Building, University of of Veterinary and Biomedical
Liverpool, Liverpool, UK Sciences, Murdoch University,
Murdoch, WA, Australia
J. KEISER
Head, Helminth Drug X.-N. ZHOU
Development Unit, Department Professor, Director, National
of Medical Parasitology and Institute of Parasitic Diseases,
Infection Biology, Swiss Tropical Chinese Center for Disease Control
and Public Health Institute, Basel, and Prevention, Shanghai, People’s
Switzerland Republic of China
VOLUME NINETY THREE
ADVANCES IN
PARASITOLOGY
Haemonchosis – Past, Present
and Future Trends
Edited by
ROBIN B. GASSER
Faculty of Veterinary and Agricultural Sciences,
The University of Melbourne, Parkville, Victoria, Australia
GEORG VON SAMSON-HIMMELSTJERNA

Institute for Parasitology and Tropical Veterinary Medicine,
Freie Universit€
at Berlin, Berlin, Germany
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CONTRIBUTORS
L.I. Alvarez
Laboratorio de Farmacología, Centro de Investigaci
on Veterinaria de Tandil (CIVETAN),
CONICET-CICPBA-UNCPBA, Campus Universitario, Tandil, Argentina
R.B. Besier
Department of Agriculture and Food Western Australia, Albany, WA, Australia
C. Britton
University of Glasgow, Glasgow, United Kingdom
I. Chan-Perez
Universidad Aut
onoma de Yucatan, Merida, Yucatan, Mexico
J.A. Cotton
Wellcome Trust Sanger Institute, Cambridge, United Kingdom
M.M. Dakheel
University of Reading, Reading, United Kingdom
R.B. Gasser
The University of Melbourne, Parkville, VIC, Australia
T.G. Geary
McGill University, Québec, Canada
J.S. Gilleard
University of Calgary, Calgary, AB, Canada
J.F. Gonzalez
Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain
A. Harder
WE Biology, Heinrich-Heine-University D€
usseldorf, D€
usseldorf, Germany
E.P. Hoberg
US National Parasite Collection and Animal Parasitic Disease Laboratory, Agricultural
Research Service, USDA, Beltsville, MD, United States
N. Holroyd
H. Hoste
INRA, UMR 1225 IHAP, Toulouse, France; Université de Toulouse, Toulouse, France
L.P. Kahn
University of New England, Armidale, NSW, Australia
xi j
xii Contributors
D.S. Kommuru
Fort Valley State University, Fort Valley, GA, United States
P.K. Korhonen
A.C. Kotze
CSIRO Agriculture, Brisbane, QLD, Australia
R. Laing
University of Glasgow, Glasgow, Scotland, United Kingdom
C.E. Lanusse
A.L. Lifschitz
N.D. Marks
A. Martinelli
E.N. Meeusen
Federation University, Churchill, VIC, Australia; Monash University, Melbourne, VIC,
Australia
I. Mueller-Harvey
A.J. Nisbet
Moredun Research Institute, Edinburgh, United Kingdom
D.M. Piedrafita
Federation University, Churchill, VIC, Australia; Monash University, Melbourne, VIC,
Australia
R.K. Prichard
McGill University, St Anne-de-Bellevue, QC, Canada
J. Quijada
INRA, UMR 1225 IHAP, Toulouse, France; Université de Toulouse, Toulouse, France
E. Redman
B. Robertsa
aPresent address: Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow,
United Kingdom
Contributors xiii
N.D. Sargison
University of Edinburgh, Roslin, Midlothian, United Kingdom
E.M. Schwarz
The University of Melbourne, Parkville, VIC, Australia; Cornell University, Ithaca, NY,
United States
T.H. Terrill
J.F.J. Torres-Acosta
Universidad Aut
onoma de Yucatan, Merida, Yucatan, Mexico
A. Tracey
W. Tuo
USDA, Agricultural Research Service, Beltsville, MD, United States
J.A. Van Wyk
University of Pretoria, Hatfield, South Africa
N.D. Young
D.S. Zarlenga
Animal Parasitic Disease Laboratory, Agricultural Research Service, USDA, Beltsville, MD,
United States
PREFACE
Nematodes are one of the most diverse groups of organisms on the planet.
Some are free-living, and many are parasitic, causing devastating diseases and
socioeconomic problems worldwide. For example, nematode infestations of
livestock animals cause substantial financial losses to farmers due to poor pro-
ductivity, failure to thrive and deaths. Haemonchus contortus (the barber’s pole
worm) and related species are very important parasites of livestock, and
belong to a large order of nematodes (Strongylida) of animals, including
humans.
Haemonchus contortus is arguably one of the most important parasites of
small ruminants due to its high pathogenicity and widespread occurrence,
particularly in tropical, subtropical and temperate climatic regions of the
world. This nematode infects hundreds of millions of ruminants, particularly
sheep and goats, and causes major production losses globally, each year. This
nematode feeds on blood from capillaries in the stomach (abomasal) mucosa,
and causes haemorrhagic gastritis, anaemia, oedema and associated compli-
cations, often leading to the death of severely affected animals. Particularly
young animals are vulnerable to clinical disease during their first grazing sea-
son, and usually protective immunity develops only in lambs of more than
six months of age. Haemonchus contortus is transmitted orally from contami-
nated pasture to the host through a complex life cycle involving three
free-living larval stages, of which the infective third larval stage is ingested
during grazing. After a histotropic phase in the host animal, the larvae
develop to fourth-stage larvae and then to adults, which both feed on blood
and cause pathogenic effects.
Over the years, there has been extensive research of this parasite and the
disease that it causes (haemonchosis), but there has been no major review of
published information. The purpose of this Thematic Issue was to review
salient aspects of Haemonchus/haemonchosis research. The topics include
fundamental areas, such has the evolution, biogeography, genetic diversity,
population genetic structure, biochemistry, pathophysiology, ecology and
epidemiology of the parasite, and the diagnosis, treatment and management
of haemonchosis as well as the interactions between nutrition and infections
with H. contortus and/or related nematodes, as well as immunity to
H. contortus.
xv j
xvi Preface
The emergence of anthelmintic resistance in H. contortus and related

nematodes necessitates an understanding of its mechanisms, at the molecular
level, and requires the development of new interventions, which is why this
Thematic Issue also covers key aspects of drug discovery, vaccine develop-
ment and the latest information on the pharmacology of anthelmintics and
improved approaches for the control of haemonchosis. The advent of
molecular and bioinformatics technologies have led to major progress,
which is why new information on the genome and transcriptome of
H. contortus has been reviewed, providing new insights into the genome
structure, organization, developmental and reproductive biology, biochem-
istry, biological pathways, anthelmintic resistance and gene functions. The
intent here was to provide a useful resource for scientists and students
working in and outside of the field of Parasitology. We hope that we
have achieved this goal.
We sincerely thank the authors for their contributions to this Issue (see
Contents), and Professors David Rollinson and Russell Stothard (Editors
of Advances in Parasitology) and Helene Kabes (Elsevier) for their support.
The Alexander von Humboldt Foundation is also gratefully acknowledged
for support (Editors RBG and GvS-H).
Robin B. Gasser and Georg von Samson-Himmelstjerna

December 2015
CHAPTER ONE
Evolution and Biogeography of

Haemonchus contortus: Linking
Faunal Dynamics in Space and
Time
E.P. Hoberg*, 1, D.S. Zarlengax
*US National Parasite Collection and Animal Parasitic Disease Laboratory, Agricultural Research Service,
USDA, Beltsville, MD, United States
x
Animal Parasitic Disease Laboratory, Agricultural Research Service, USDA, Beltsville, MD, United States
1
Corresponding author: E-mail: Eric.Hoberg@ars.usda.gov
Contents
1. Introduction 2
2. Haemonchus: History and Biodiversity 3
3. Phylogeny and Biogeography: Out of Africa 4
4. Domestication, Geographical Expansion and Invasion 7
5. Host Range for Haemonchus contortus 9
5.1 Host colonization, ecological fitting and sloppy fitness space 12
5.2 Generalists and specialists: an obsolete nomenclature 14
6. Host and Geographical Colonization in Faunal Assembly 17
7. Climate Impacts Integrating Historical Perspectives 19
8. Understanding Diversity: Some Recommendations 22
Acknowledgements 24
References 25
Abstract
History is the foundation that informs about the nuances of faunal assembly that are
essential in understanding the dynamic nature of the hosteparasite interface. All of
our knowledge begins and ends with evolution, ecology and biogeography, as these
interacting facets determine the history of biodiverse systems. These components,
relating to Haemonchus, can inform about the complex history of geographical distri-
bution, host association and the intricacies of hosteparasite associations that are
played out in physiological and behavioural processes that influence the potential
for disease and our capacity for effective control in a rapidly changing world. Origins
and evolutionary diversification among species of the genus Haemonchus and Hae-
monchus contortus occurred in a complex crucible defined by shifts in environmental
structure emerging from cycles of climate change and ecological perturbation during
the late Tertiary and through the Quaternary. A history of sequential host colonization
Advances in Parasitology, Volume 93
j
ISSN 0065-308X
http://dx.doi.org/10.1016/bs.apar.2016.02.021 2016, Published by Elsevier Ltd. 1
2 E.P. Hoberg and D.S. Zarlenga
associated with waves of dispersal bringing assemblages of ungulates from Eurasia into
Africa and processes emerging from ecosystems in collision and faunal turnover
defined the arena for radiation among 12 recognized species of Haemonchus. Among
congeners, the host range for H. contortus is exceptionally broad, including species
among artiodactyls of 40 genera representing 5 families (and within 12 tribes of Bovi-
dae). Broad host range is dramatically reflected in the degree to which translocation,
introduction and invasion with host switching, has characterized an expanding distri-
bution over time in North America, South America, southern Eurasia, Australia and
New Zealand, coincidental with agriculture, husbandry and global colonization by hu-
man populations driven particularly by European exploration after the 1500s. African
origins in xeric to mesic habitats of the African savannah suggest that historical con-
straints linked to ecological adaptations (tolerances and developmental thresholds
defined by temperature and humidity for larval stages) will be substantial determinants
in the potential outcomes for widespread geographical and host colonization which
are predicted to unfold over the coming century. Insights about deeper evolutionary
events, ecology and biogeography are critical as understanding history informs us
about the possible range of responses in complex systems under new regimes of envi-
ronmental forcing, especially, in this case, ecological perturbation linked to climate
change. A deeper history of perturbation is relevant in understanding contemporary
systems that are now strongly structured by events of invasion and colonization. The
relaxation of abiotic and biotic controls on the occurrence of H. contortus, coincidental
with inception and dissemination of anthelmintic resistance may be synergistic, serving
to exacerbate challenges to control parasites or to limit the socioeconomic impacts of
infection that can influence food security and availability. Studies of haemonchine
nematodes contribute directly to an expanding model about the nature of diversity
and the evolutionary trajectories for faunal assembly among complex hosteparasite
systems across considerable spatial and temporal scales.
1. INTRODUCTION
Biodiversity information is a cornerstone for developing a nuanced
understanding and picture of the distribution and history of complex
hosteparasite associations (eg, Brooks and Hoberg, 2000; Brooks and
McLennan, 1993, 2002; Brooks et al., 2014; Hoberg, 1997; Poulin, 1998;
Poulin and Morand, 2004). The current regime of extensive environmental
perturbation across biodiverse assemblages globally, including the recog-
nized convergence of accelerating climate change, new or altered patterns
of land use, and extensive globalization drive ecosystems in collision with
anticipated cascading effects on the distribution of animal pathogens and
emergence of diseases (eg, Brooks and Hoberg, 2013; Harvell et al., 2002;
Hoberg, 2010; Hoberg et al., 2008; van Dijk et al., 2009). History, encom-
passing phylogeny, explorations of hosteparasite coevolution, ecology and
Evolution and Biogeography of H. contortus 3
biogeography (with phylogeographical approaches) provide a foundation to

recognize or identify the drivers and responses to perturbation. Historical in-
sights also provide a pathway for anticipating and mitigating the outcomes of
accelerating change at regional to landscape scales.
2. HAEMONCHUS: HISTORY AND BIODIVERSITY

Species of the genus Haemonchus Cobb, 1898 occur in this complex
intersection of history, ecology and biogeography (Cerutti et al., 2010;
Giudici et al., 1999; Hoberg et al., 2004; Jacquiet et al., 1995; Troell
et al., 2006). Comparative morphological studies initially served to define
a framework for the recognition of nine species in the genus (Gibbons,
1979). Although some species were defined based on relatively few speci-
mens, consistent structural differences were apparent especially in attributes
of the spicule tips and dorsal ray among male nematodes. Subsequently,
evaluation of the synlophe (a system of longitudinal cuticular ridges present
in male and female nematodes; eg, Durette-Desset, 1983) served to provide
separation of species based on female specimens, provided the possibility of
linking male and female conspecifics in mixed infections and recognition of
hybrids between Haemonchus contortus and Haemonchus placei (Lichtenfels
et al., 1986, 1994, 2002). These studies also were essential in validating prior
conclusions regarding the inadequacy of variation in the morphology of
vulval flaps as a defining character in the genus and among proposed subspe-
cies and varieties of H. contortus (eg, Gibbons, 1979).
Expanding knowledge of structural characters and application of molecular
methods to establish and explore species criteria have further resolved limits
among 12 species-level taxa currently regarded as valid (Hoberg et al.,
2004). Within this assemblage, most species can be separated and distin-
guished by unequivocal structural attributes among adult male and female
nematodes (eg, Gibbons, 1979; Giudici et al., 1999; Jacquiet et al., 1995,
1997; Lichtenfels et al., 1994, 2001, 2002). Species limits based initially on
partitions derived from comparative morphology have been confirmed in
those situations where genetic diversity has been explored, and especially
for example in the differentiation of H. contortus and H. placei (chapter: The
identification of Haemonchus Species and Diagnosis of Haemonchosis by
Zarlenga et al., 2016, in this volume). Although considerable genetic diversity
has been demonstrated among global populations of H. contortus at varying
spatial scales, an indication of an unrecognized assemblage of cryptic species
has not been revealed (eg, Cerutti et al., 2010; Jacquiet et al., 1995; Morrison
and H€ oglund, 2005; Troell et al., 2006). There remains, however, a need to
expand the development of synoptic information about population genetic
diversity and possible genetic partitions at landscape to regional scales beyond
those taxa [H. contortus (Rudolphi, 1803), H. placei (Place, 1893) Haemonchus
similis Travassos, 1914 and Haemonchus longistipes Railliet and Henry, 1909]
that most often circulate among domesticated ruminants (eg, chapter: The
Identification of Haemonchus Species and Diagnosis of Haemonchosis by
Zarlenga et al., 2016, in this volume). Collectively these species remain
among the most economically significant on the global stage. Patterns of cir-
culation for these species often cross ecotones or the interface between
managed and natural ecosystems, with consequences for domesticated and
free-ranging ungulates (eg, Hoberg, 2010; Hoberg et al., 2001, 2008).
Defining the parameters responsible for faunal assembly and species di-
versity on varying temporal and spatial scales remains critical for demon-
strating the pathways and directionality for parasite transmission among
assemblages of ungulates occurring in sympatry or in temporal overlap
(Brooks et al., 2014; Cerutti et al., 2010; Haydon et al., 2002; Hoberg,
2010). Multispecies infections attributable to Haemonchus in single hosts
are not uncommon, particularly in Africa, denoting complexity in evolu-
tionary history, ecological structure and factors influencing circulation
(Budischak et al., 2015; Hoberg et al., 2004; Jacquiet et al., 1998). For
example, 8 of 12 species of Haemonchus have been reported in impala [Aepy-
ceros melampus (Lichtenstein)] from the African savannahs (Boomker, 1990).
Emphasized by these interactions is the importance of ecotones and trans-
mission among domestic and free-ranging wild ungulates for H. contortus
and other species. On a global scale, elucidating an intersection for processes
of invasion and colonization in evolutionary and ecological time addresses
contemporary challenges transcending interactions for responses to acceler-
ating climate change, potential geographical colonization, and the origins,
routes of dissemination and persistence of drug-resistance genes at the intra-
specific level in H. contortus and within species assemblages of Haemonchus
(Chaudhry et al., 2015).
3. PHYLOGENY AND BIOGEOGRAPHY: OUT OF AFRICA

Haemonchines (species of Haemonchus, Mecistocirrus Railliet and
Henry, 1912 and Ashworthius Le Roux, 1930) had origins among Eurasian
and African ungulates during the Miocene (Durette-Desset et al., 1999),

although radiation among species of Haemonchus was subsequently limited
to Africa. Diversification among species of Haemonchus demonstrates a
geographically restricted history in sub-Saharan Africa, highlighted by the
absence of endemic faunas in the Western Hemisphere (Nearctic and
Neotropical regions) and the Palearctic encompassing Eurasia and the Indian
Subcontinent. Substantial climatological controls on species radiation and
geographical distribution are apparent (chapter: The Pathophysiology, Ecol-
ogy and Epidemiology of Haemonchus contortus Infection in Small Ruminants
by Besier et al., 2016, in this volume; Hoberg et al., 2002, 2004; O’Connor
et al., 2006).
Phylogeny, biogeography and host distribution are consistent with
African origins for species of Haemonchus, initial radiation associated with
colonization among grazing and browsing antelopes (in the absence of
cospeciation), and a downstream history of sequential host switching to ar-
tiodactyls among the Caprinae, Bovinae, Giraffidae and Camelidae (Fig. 1).
Radiation occurred against a backdrop of climatological variation, shifting
structure for habitats, pulses of ecological transition in sub-Saharan environ-
ments and independent episodes of biotic expansion/isolation (Hoberg
et al., 2004). Faunal turnover, circumscribed in time, influenced recurrent
zones of contact and defined opportunities for chronological and sequential
geographical and host colonization. Episodes of colonization represent dif-
ferential times for arrival from Eurasia and establishment of respective ungu-
late groups, extending from the Middle Miocene (14e15 MYBP) through
the Pliocene (3e2.5 MYBP) and Quaternary (after 2.6 MYBP) (Hernandez
Fernandez and Vrba, 2005; Vrba, 1985, 1995; Vrba and Schaller, 2000). The
history for H. contortus is complex, and although recognized globally as a
dominant nematode pathogen of domestic sheep and goats (tribe Caprini),
the origin of this species is linked to an assemblage of antelopes in Africa dur-
ing the late Tertiary.
The complexity of radiation for species of Haemonchus among ungulates
demonstrates interacting and episodic mechanisms in evolution and biogeog-
raphy that drive development and assembly of Macroevolutionary Mosaic Faunas
(eg, Araujo et al., 2015; Hoberg, 2005, 2010; Hoberg and Brooks, 2008;
Hoberg et al., 2008, 2012). At a minimal level of simplicity, mosaics in
ecological time represent admixtures of endemic (indigenous) and introduced
species (often invasive exotic taxa) or populations resulting from anthropo-
genic introduction and establishment. Mosaic structure is also manifested as
a macroevolutionary process involving parasite assemblages on continental,
H. krugeri Camelidae
Suidae
H. lawrenci Tayassuidae
Tragulidae
H. dinniki
Antilocapridae
H. horaki
Giraffidae
RU
H. contortus
Cervidae
H. placei
Moschidae
Bovini
H. bedfordi Bovinae
Boselaphini
Tragelaphini
H. similis Cephalophinae
Peleinae
Reduncinae
BOVIDAE
H. longistipes Aepycerotinae
Antilopini
H. okapiae Neotragini
Alcelaphinae
H. vegliai Hippotraginae
Antilopinae
Pantholopinae
Caprini
Ovibovini
Caprinae
H. mitchelli Rupicaprini
Figure 1 Phylogenetic perspective for host-group distribution and coevolutionary his-

tory for species of the genus Haemonchus among ungulates. Initial diversification
among all Haemonchus species was associated with antelopes among Cephalophinae,
Peleinae, Reduncinae and Antilopinae; secondarily radiation and faunal assembly was
driven by sequential host colonization among ruminants (¼RU) and other artiodactyles
including Camelidae (Hoberg et al., 2004). Relationships are shown for species assem-
blages linked to putative ‘core’ hosts based on empirical data for prevalence and abun-
dance; incidental associations representing postulated contemporary host-switching
events since European colonization are not shown (Hoberg et al., 2004). Phylogeny
for species of Haemonchus is from Hoberg et al. (2004). Ungulate and ruminant phylog-
eny is derived and modified from currently available sources (Hassanin and Douzery,
2003; Hernandez Fernandez and Vrba, 2005; Vrba and Schaller, 2000). Host taxonomy
among ungulates is consistent with Grubb (2005).
regional and landscape scales, resulting from episodic dispersal and geograph-
ical colonization in deeper evolutionary time, encompassing populations,
species and faunas (eg, Hoberg and Brooks, 2008, 2010, 2013; Hoberg
et al., 2012). The dynamics of episodic environmental perturbation, recurrent
invasion, geographical colonization, isolation and faunal radiation are
described in the Taxon Pulse which provides a macroevolutionary perspective
for evolution of complex systems (Araujo et al., 2015; Erwin, 1985; Halas
et al., 2005; Hoberg and Brooks, 2008, 2010). Among species of Haemonchus,
African origins and radiation in xeric to mesic habitats of the African savannah
suggest that historical constraints linked to ecological adaptations (tolerances
and developmental thresholds defined by temperature and humidity) will
be substantial determinants in the potential outcomes for widespread

geographical and host colonization which are predicted to unfold over the
coming century (chapter: The Pathophysiology, Ecology and Epidemiology
of Haemonchus contortus Infection in Small Ruminants by Besier et al., 2016, in
this volume). As a consequence, insights about deeper evolutionary events,
ecology and biogeography are critical as understanding history informs us
about the possible range of responses in complex systems under new regimes
of environmental forcing, particularly, in this case, ecological perturbation
linked to climate change (eg, Hoberg et al., 2008).
4. DOMESTICATION, GEOGRAPHICAL EXPANSION

AND INVASION
Considering H. contortus, H. placei and H. similis, the broad assemblage
of hosts has resulted from initial diversification in Africa and subsequent
events of colonization in ecological time. Introduction, establishment and
dissemination in new ecological situations were coincidental with jump
and long-range dispersal as mechanisms for breakdown in ecological isola-
tion (Capinha et al., 2015; Hoberg, 2010; Hoberg and Brooks, 2013;
Hoberg et al., 2004; Wilson et al., 2009). Thus, cosmopolitan distribution
is a consequence of recurrent anthropogenic invasion, leading to the devel-
opment of complex mosaic faunas and populations. As a generality for Hae-
monchus, these assemblages have not involved admixtures of endemic and
introduced species (relative to source and recipient regions), but may involve
genetic structuring and partitions in local populations among conspecifics
(Cerutti et al., 2010; Giudici et al., 1999; Hoberg, 2010; Hoberg et al.,
2004, 2012; Thompson, 1994, 2005; Troell et al., 2006).
Diversification among species of Haemonchus was not associated with the
process of domestication for sheep, goats or cattle, and these economically
dominant ungulates were absent from sub-Saharan Africa during the history
of radiation for these nematodes (eg, Caramelli, 2006; Chessa et al., 2009;
Hanotte et al., 2002). The development of currently recognized breeds or
lineages of domestic sheep has a complex history initially focused in south-
western Asia about 11,000 years before present (KYBP); sheep and goats
expanded with agriculture into Africa by at least 8 KYBP. Considering cat-
tle, initial domestication occurred in isolated centres of southwestern Asia
and the Indian subcontinent reflecting the origins, respectively, of taurine
and zebu lineages about 10 KYBP (Caramelli, 2006; Loftus et al., 1994).
Taurine cattle were established in Africa from sources in southwestern
Asia and possibly via exchange with Europe, whereas zebu (along with
camels, Camelus dromaderius Linnaeus) appear associated with Arabian expan-
sion and possibly development of early sea routes and trade (Caramelli,
2006). Near 10 to 6 KYBP, expansion of pastoralists and Neolithic agricul-
tural systems led to a widening distribution for isolated domesticated breeds
extending from Scandinavia in the north to the region of North Africa, sug-
gesting the potential for early patterns of exchange and dissemination of
H. contortus, H. placei, H. similis and H. longistipes among free-ranging and
domestic ungulates (eg, Balter, 2014; Chessa et al., 2009).
A signature for human-mediated invasion for H. contortus, H. placei and
H. similis is well established, reflecting the history of early trade routes
following ungulate domestication, later European colonization and explora-
tion after the 1500s, and accelerating globalization over the past two cen-
turies (Brooks and Hoberg, 2013; Giudici et al., 1999; Hoberg, 2010;
Morrison and H€ oglund, 2005; Rosenthal, 2009; Troell et al., 2006;
Zarlenga et al., 2014). Patterns of genetic diversity at intercontinental scales,
and possibly extending to local landscapes, are consistent with recurring ep-
isodes of geographical invasion often involving limited founding populations
and varying levels of gene flow (eg, Hunt et al., 2008; Jacquiet et al., 1995;
Troell et al., 2006). It has been suggested that, once established in a new
continental arena, intercontinental gene flow has been minimal for H. con-
tortus (and perhaps other nematodes in domestic ungulates). Reflected is a
history of anthropogenic introductions that influence distribution for para-
sites, dependent on hosts for dispersal relative to otherwise impermeable
geographical barriers (eg, Leignel and Humbert, 2001; Poulin, 1998; Troell
et al., 2006). In contrast, at landscape scales, where populations have been
explored in regions of sympatry for domestic sheep, free-ranging caprines
and cervids, evidence of extensive cross-transmission has been revealed,
and raises substantial questions and implications about the nature of parasite
circulation in zones of contact (Cerutti et al., 2010). Contemporary (and
near-time) introductions at global, regional and landscape scales for species
of Haemonchus are largely dependent on human-facilitated movement of do-
mestic caprines and cattle, or in some situations free-ranging artiodactyls, as a
function of vagility and permissive environments (Troell et al., 2006). The
dynamics of transmission following establishment, however, may often
involve host colonization and circulation in novel (and endemic) ungulates
associated with particular regional ecosystems (eg, host colonization and cir-
culation among cervids). For example, H. contortus is now a dominant nem-
atode established in species of Odocoileus Rafinesque and particularly in
white-tailed deer, O. virginianus (Zimmermann), across the southern lati-

tudes of North America, where it is a significant pathogen (Hoberg et al.,
2001; Prestwood and Pursglove, 1981).
Defining specific parameters for the development of single and multiple
species of Haemonchus and other nematodes in an array of disparate host taxa,
for example, relative to fecundity, longevity and fitness for parasites and de-
mographics and density for hosts, are essential in establishing the role of
different ungulates as sources or sinks for population persistence on local
to regional scales (eg, Fenton et al., 2015; Holt et al., 2003; Jacquiet et al.,
1998). Among species of Haemonchus, including H. contortus, in multi-host
assemblages, it is apparent that differential contributions to population
persistence and circulation are often attributable to a limited spectrum of
host species across a larger array of ungulates in sympatry (eg, Boomker,
1990; Jacquiet et al., 1998). In a historical perspective, susceptibility and
competence for hosts as well as capacity and opportunity for parasites are
essential components in establishing lineage persistence and evolutionary
trajectories that are associated with downstream patterns of diversification
(Hoberg and Brooks, 2008, 2013; Hoberg et al., 2004). In this arena, climate
and abiotic controls determining the availability of infective larval nema-
todes, and the potential for infections, interface with multispecies host
assemblages as well as aspects of parasite ontogeny and selection, to deter-
mine the limits for diversity and distribution (eg, Jacquiet et al., 1998).
Thus, the potential for population bottlenecks for parasites across space
and time emerges from interactions with host vagility, competence and de-
mographics in an arena defined by environmental permissiveness, the latter
which may shift incrementally in the long term, or be strongly influenced by
extreme and ephemeral events associated with accelerating climate warming
(Hoberg and Brooks, 2015; Hoberg et al., 2008; van Dijk et al., 2008).
5. HOST RANGE FOR HAEMONCHUS CONTORTUS

Understanding the limits of host range for H. contortus prior to the 1990s
was confounded by our abilities for accurate identification and prevailing tax-
onomy (Gibbons, 1979). This understanding also may have been conflated
with respect to reports that were undocumented by voucher specimens and
which now cannot be validated (Hoberg et al., 2009). Although clear
morphological and molecular attributes for female and male conspecifics
have been developed and have been available for the past 25 years, these
have not always been applied in the process of identification (chapter: The
Identification of Haemonchus Species and Diagnosis of Haemonchosis by
Zarlenga et al., 2016, in this volume). In reports published prior to the advent
of reliable morphological or molecular-based identification, those that ‘docu-
ment’ H. contortus in various host species need to be carefully considered.
Many appear to be correct based on ecological context; however, other re-
cords may be in error, representing known taxa such as H. placei, nominal
taxa reduced as synonyms, or cryptic diversity that had not been previously
distinguished from H. contortus. For example, the ‘long-spicule’ form of H.
contortus reported from South Africa (Boomker et al., 1983) was later shown
to be a distinct species, H. horaki Lichtenfels, Pilitt, Gibbons and Boomker,
2001, with an apparently restricted host range in grey rhebuck, Pelea capreolus
(Forster) (Lichtenfels et al., 2001). Similarly, H. okapiae van den Berghe, 1937
in African giraffids was resurrected from synonymy with H. contortus based on
structural attributes (Lichtenfels et al., 2002). These latter taxonomic revisions
would not have been possible in the absence of type specimens and vouchers
that were historically archived in museum repositories. Such also highlights
the critical importance of integrated methods in systematics that incorporate
comparative morphology and specific sequence data derived from archival
specimen collections (vouchers with authoritative identification) as the foun-
dation to define species limits and the distribution of global diversity (eg,
Hoberg et al., 1999, 2001). Caveats aside, and correcting for these modifica-
tions in taxonomy, the host range for H. contortus is recognizably broad,
including species among artiodactyls of 40 genera across 5 families (and within
12 tribes of Bovidae) (summarized in Hoberg et al., 2004) (Fig. 2).
An expansive host range for H. contortus is observed in endemic regions of
Africa, encompassing ungulate species among 23 host genera, including
domestic sheep, goats and cattle. The broad host range is further dramatically
reflected in the degree to which translocation, introduction and invasion with
host switching, among 20 additional host genera, in North America, South
America, southern Eurasia, Australia and New Zealand has characterized an
expanding distribution over time, coincidental with agriculture, husbandry
and global colonization by human populations (Fig. 2) (Hoberg, 2010;
Hoberg and Brooks, 2013; Hoberg et al., 2004, 2008; Wilson et al., 2009;
Zarlenga et al., 2014). In comparison, other species of Haemonchus are char-
acterized by considerably less variation in host associations (Gibbons, 1979;
Hoberg et al., 2004), with 5 of 12 species having three or fewer recognized
hosts in Africa (eg, H. dinniki Sachs, Gibbons and Lweno, 1973, H. horaki,
H. kruegeri Ortlepp, 1964, H. lawrenci Sandground, 1933, and H. okapiae).
H. krugeri Camelidae
Suidae
H. lawrenci Tayassuidae
Tragulidae
H. dinniki
Antilocapridae
H. horaki
Giraffidae
RU
H. contortus
Cervidae
H. placei
Moschidae
Bovini
H. bedfordi Bovinae
Boselaphini
Tragelaphini
H. similis Cephalophinae
Peleinae
Reduncinae BOVIDAE
H. longistipes Aepycerotinae
Antilopini
H. okapiae Neotragini
Alcelaphinae
H. vegliai Hippotraginae Antilopinae
Pantholopinae
Caprini
Ovibovini
Caprinae
H. mitchelli Rupicaprini
Figure 2 Phylogenetic perspective of host-group distribution for H. contortus among

ungulates. Associations for H. contortus encompass a considerable array of ungulate
families, subfamilies, tribes, genera and species denoting a complex history of natural
expansion and anthropogenic events of global translocation, introduction and estab-
lishment with geographical and host colonization. Translocations of domestic caprines
with global introduction, for example, were the drivers of host colonization among Cer-
vidae, Antilocapridae and free-ranging Caprinae in the Western Hemisphere and Cam-
elidae and Cervidae across Eurasia and South America. Dissemination out of Africa and
globally reflects events tracking early routes of cultural interchange and later European
colonization, exploration and trade. Phylogeny for species of Haemonchus is from
Hoberg et al. (2004). Ungulate and ruminant phylogeny is derived and modified
from currently available sources (Gatesy and Arctander, 2000; Hassanin and Douzery,
2003; Hernandez Fernandez and Vrba, 2005; Vrba and Schaller, 2000). Host taxonomy
among ungulates is consistent with Grubb (2005).
Further, H. longistipes occurs in six species of ungulates, including camelids,

and less often in cattle, sheep, goats and antelopes. Haemonchus mitchelli Le
Roux, 1929 occurs in six species of bovids, especially antelopes, and H. vegliai
Le Roux, 1929 occurs in nine hosts, particularly antelopes, tragelaphines and
cephalophines. Only H. bedfordi Le Roux, 1929 occurs among a diverse
assemblage of 19 bovids or giraffids; however, host ranges of all congeners
do not approach that seen for H. contortus (see Hoberg et al., 2004). Haemon-
chus contortus is one of three haemonchines, including H. placei (seven host spe-
cies, primarily among Bovinae) and H. similis (nine host species primarily
among Bovinae), which have been widely translocated, introduced and
established globally, coinciding with the expansion of trade routes and move-
ment of domestic stock since the 1500s. The distribution of H. longsitipes,
although influenced by anthropogenic translocation out of Africa, remains
relatively limited to Eurasia and India.
In this arena, H. contortus might be considered as a generalist parasite,
whereas congeners exhibiting varying degrees of apparent restriction to a
more limited spectrum of host species or host groups would be regarded
as specialists among the ungulates (eg, Walker and Morgan, 2014). In this
conventional definition, generalists contrast with specialists relative to the
apparent number of hosts in which parasites may successfully develop. Un-
derstanding the spectrum of hosts involved in persistence of H. contortus is
essential, particularly in defining the competence of free-ranging artiodactyls
to maintain viable populations in the absence of sheep and cattle, and thus to
serve as significant reservoirs for infection of domestic stock. Among nem-
atodes of ungulates, including H. contortus, the structure of host assemblages
and dynamics for transmission are essential drivers for persistence and the po-
tential for emergence when suitable conditions are conducive relative to a
basic reproductive number of R0,tot > 1 across the community (Dobson,
2004; Fenton and Pedersen, 2005; Fenton et al., 2015; Haydon et al.,
2002). Although the basic reproductive number does represent the potential
for establishment and persistence, relying on this measure is nondimensional
and substantially changes the focus to outcomes, in contrast to process.
Designations as generalist or specialist parasites based on convention, or an
R0,tot > 1, serve to diminish the adequacy of explanations reflecting the
dynamic complexity of temporal, spatial, evolutionary and ecological pro-
cesses, and mechanisms that determine host range in deep and shallow
time (Agosta et al., 2010; Araujo et al., 2015; Brooks and McLennan,
2002; Hoberg and Brooks, 2008; Jacquiet et al., 1995, 1998).
5.1 Host colonization, ecological fitting and sloppy

fitness space
Colonization requires a convergence of opportunity and compatibility, or
capacity, on the part of parasites to successfully infect, establish and be
maintained in a novel host species or host group (see Combes, 2001). In
a simplistic sense, opportunity is established through ecological perturba-
tion and the disruption or breakdown of physical, biological or historical
barriers (on a range of temporal and spatial scales) that previously limited
exposure to infection or were the determinants for ecological isolation of
populations, species, faunas and biotas in space and time (eg, Araujo
et al., 2015; Elton, 1958; Hoberg, 2010; Hoberg and Brooks, 2008). For
example, intercontinental and regional barriers have historically limited
dissemination and establishment for H. contortus. Breakdown in ecological
isolation has emerged secondarily from anthropogenic events of transloca-
tion and introduction with domestic sheep and potentially other ungulates
for conservation and game ranching that have established opportunity in
new regional settings beyond Africa.
Opportunity converging with capacity in the context of Ecological Fitting
defines events of colonization through the interaction of potential and real-
ized host range, determined by a capability to utilize phylogenetically
conserved resources by parasites (Brooks and McLennan, 2002; Janzen,
1985). Ecological fitting may be manifested by host colonization through
resource tracking where similar attributes are presented by ancestral and novel
hosts (Agosta and Klemens, 2008; Agosta et al., 2010). For example, sequen-
tial host-group acquisition and radiation demonstrated for species of Haemon-
chus among ungulates in Africa from the Miocene into the Quaternary appears
consistent with this pathway. Alternatively, ecological fitting in ‘sloppy fitness
space’ facilitates colonization through the exploitation of novel host-based
resources that are beyond or outside of the range of conditions in which
the species evolved, but may be characterized by a range in positive fitness
encompassing suboptimal to optimal associations (Agosta and Klemens,
2008; Agosta et al., 2010; Araujo et al., 2015). H. contortus may occur in
this variable or sloppy fitness space as reflected in the considerable array of
ungulate hosts in which the parasite species may persist and which have
been acquired through colonization in distant ecological settings following
a history of translocation and introduction. Highlighted is the variation in
competence across a broad spectrum of potential artiodactyl hosts and in
host groups, which have been documented for H. contortus and other species
of Haemonchus (eg, Boomker, 1990). Also apparent are the interrelationships
for phenotypic plasticity, correlated trait evolution and phylogenetic conser-
vatism that contribute to potential host-switching abilities of parasites, irre-
spective of the degree of specialization or specificity (Agosta and Klemens,
2008; Agosta et al., 2010; Araujo et al., 2015). Ecological fitting in broad
sloppy fitness space facilitates translocation (geographical colonization and in-
vasion), introduction and host switching, and has been an essential character-
istic of faunal assembly on evolutionary and ecological time-scales (Agosta and
Klemens, 2008; Agosta et al., 2010; Hoberg and Brooks, 2008, 2010, 2013).
The contemporary host range for species of Haemonchus contrasts the
widespread versus restricted or narrow distributions for infections among
ungulates (Figs 1 and 2). An apparently extensive fitness space for H. contor-
tus, coinciding with opportunity and capacity to infect a broad spectrum of
endemic and introduced ungulates (with divergent trajectories on all conti-
nents, except Antarctica) has facilitated anthropogenic dissemination out of
Africa. Congeners, including those that have been translocated, such as
H. similis and H. placei, however, appear to be characterized by a smaller
fitness space associated with a reduced assemblage of hosts; among African
endemics, limited host range appears to be typical. Thus, a pertinent ques-
tion is whether this assemblage of species has not had opportunity through
breakdown in ecological isolation to utilize a broader spectrum of hosts, or if
they are actually limited relative to the host groups in which they occur.
Considered from a parallel perspective, how broad or narrow is the fitness
space in which species other than H. contortus exist? A discussion of fitness
space and ecological fitting appropriately changes the focus from explicit
determination of generalists or specialists to an increasingly integrated
view of ecology and evolution in the dynamics of host association and faunal
assembly (eg, Brooks and McLennan, 2002).
5.2 Generalists and specialists: an obsolete nomenclature

Brooks and McLennan (2002) proposed that ecological fitting, in conjunc-
tion with the stochastic nature of opportunity would eliminate host range
as a reliable indicator of whether a parasite is a specialist or generalist. Par-
asites are ecological specialists, irrespective of host range, as demonstrated
by specific microhabitat preferences, conservative life cycles and transmis-
sion dynamics. Ecological fitting provides the mechanism that accounts for
extensive host range and host switching, even in situations of specialization,
and resolves these contrasting or conflicting relationships that are at the
core of the Parasitological Paradox (Agosta et al., 2010; Araujo et al., 2015;
Brooks and McLennan, 2002). Further, a property of parasites is consider-
able conservation in the degree of phylogenetic relatedness among hosts,
although a clear relationship for host range and ecological specialization
is equivocal. According to convention in these circumstances, parasites
with a single or narrowly defined spectrum of hosts are considered as spe-
cialists, whereas those with multiple hosts are regarded as generalists (eg,
Walker and Morgan, 2014) e an observation that is nondimensional in
the context of evolutionary time. Consequently, applying restrictive
nomenclature, such as generalist or specialist, is obsolete, and does not
adequately reflect the evolutionary and ecological dynamics involved in
the origins of faunal structure among complex assemblages of parasites in
multi-host associations, including species of Haemonchus (Brooks and

McLennan, 2002).
Not all hosts are equivalent or optimal, and thus may represent different
contributions to the maintenance and persistence of parasites among sympat-
ric and multispecies assemblages, as exemplified among species of Haemon-
chus (eg, Achi et al., 2003; Fenton et al., 2015; Jacquiet et al., 1998).
Domestic sheep and goats, however, are the source of H. contortus globally
through introduction, establishment and host colonization (eg, in cervids,
particularly Odocoileus in North America and also camelids in South Amer-
ica). Persistence and maintenance often in suboptimal hosts (irrespective of
introduced versus endemic populations) are indicated by patterns of preva-
lence and abundance (Boomker, 1990; Hoberg et al., 2004; Jacquiet et al.,
1998). Critically, these relationships determine the potential circulation of
H. contortus in wild free-ranging ungulate hosts and the degree of ‘threat’
to domestic stock in ecotones involving overlap in managed and wild sys-
tems. As a function of ecological context, deer or pronghorn [Antilocapra
americana (Ord)] can represent a source for colonization of domestic stock
in southwestern North America; for example, putative circulation of H. con-
tortus in cattle in the absence of sheep (E.P. Hoberg, P.A. Pilitt and D.S. Zar-
lenga, unpublished field data). Concurrently, expanding degrees of
environmental perturbation that alter the field of ecological isolation and
thus constitute emergent opportunity would be anticipated to drive bouts
or events of switching among species of Haemonchus and ungulate host as-
semblages globally where conditions are suitable for transmission (chapter:
The Pathophysiology, Ecology and Epidemiology of Haemonchus contortus
Infection in Small Ruminants by Besier et al., 2016, in this volume).
Observations of contemporary host associations are most often viewed
through a lens established by a slice of ecological time, rather than as a
comprehensive picture across the expanse of evolutionary history. Such a
perspective arises in discussions of specificity and host range, and has conse-
quences for our understanding of the temporal definition and processes that
determine host associations. An application of prevailing and convenient la-
bels of generalist or specialist (based on the number of recognized hosts) to
particular parasites reflects a limited temporal view or a window in time (eg,
Walker and Morgan, 2014). Essentially, these designations depict a static
snapshot of otherwise long-term and dynamic processes, extending across
evolutionary into ecological time, and a misconception about the nature
of hosteparasite relationships (Araujo et al., 2015; Brooks and McLennan,
2002; Hoberg and Brooks, 2008, 2015).
Alternating trends for generalization and specialization emerge in the

context of the Oscillation Hypothesis (Janz and Nylin, 2008), which has
only been applied over during the past decade and less to systems of parasites
and vertebrate hosts (eg, Hoberg and Brooks, 2008). Oscillation interacts
with ecological fitting and constitutes the continuum for capacity that deter-
mines the limits for host exploitation. A temporally restricted snapshot,
consequently, will reveal variation in the capacity to utilize hosts as fitness
space changes over time (an intrinsic capacity of parasites) and interacts
with local ecological structure. Such variation in observed associations is
reflected in the existence of ‘faux generalists’ and ‘faux specialists’, where
relationships are influenced by ecological context, further emphasizing
that we cannot rely on host range even of the snapshot (Brooks and
McLennan, 2002). Concurrently, oscillation tells us that specialists can pro-
duce generalists through alternating trends in relative specialization.
Oscillation embodies the dynamic nature of microevolutionary aspects of
coevolution represented by co-accommodation (or coadaptation) (Brooks,
1979) that influences the degree of specialization (or specificity) demon-
strated by parasites through reciprocal adaptation in associated lineages at
any point in time. Trends in specialization/generalization interact with
changing opportunities that are influenced by spatial/ecological dynamics,
or the temporal and geographical arena for relative/apparent ecological
isolation (Araujo et al., 2015; Hoberg and Brooks, 2008). Thus, opportunity
and capacity determine host range at any point in time (constituting the
limited temporal snapshot). Dynamics across evolutionary time, however,
controls outcomes downstream, irrespective of the apparent picture or
perspective within a particular temporal window, and are influenced at local
scales by Geographic Coevolutionary Mosaics (Thompson, 2005) that determine
the complexity of evolutionary interactions linking hosts and parasites
through co-accommodation and cospeciation (Brooks, 1979). A focus on
limited or nondimensional concepts in isolation, such as specificity, host
range or even population parameters and fitness, provides an incomplete
view of interactions and dynamics involved in multi-host associations and
masks the considerable complexity resulting in faunal structure (eg, Fenton
et al., 2015; Walker and Morgan, 2014). Each component alone is insuffi-
cient in providing broad explanatory power about diversification and faunal
assembly, and is analogous to descriptions of the world that focus on a
limited spectrum of mechanisms (eg, Hoberg et al., 2015).
Static snapshots or pictures of diversity in a contemporary arena do not
accommodate historical processes; that is, the dynamic nature of change,
perturbation and episodic events that have structured faunal assemblages.

Furthermore, this static picture results in the conceptual problem of gener-
alists and specialists and host distribution within a temporally narrow
context. It neither accounts for past change nor does it accommodate future
dynamic change (how ecological fitting, sloppy fitness space and oscillation
play out over time), but provides an inappropriate basis for interpretation of
host associations that emerge from spatially and temporally discrete inven-
tories. What we observe, or think we observe, is determined by the lens
or perspective of spatial and temporal scale.
Current and widely held concepts of specificity or narrow host range
(these terms are not synonymous) imply stasis and a static association or
end point in hosteparasite relationships. Specificity and stability/stasis are
linked in the wider paradigm of cospeciation that does not adequately repre-
sent or account for the origins of complexity through ecological, biogeo-
graphical and evolutionary dynamics (eg, Hoberg and Brooks, 2008,
2010, 2013). Specificity in this realm becomes an observation about static
phenomena, with implications that host switching and dispersal are rare.
A paradigm view over the past century is apparent in the context of cospe-
ciation, where diversification was most often linked to modification by
descent in co-associated lineages occurring in a biosphere in relative stability
governed by gradual change (reviewed in Brooks, 1979; Brooks and
McLennan, 1993, 2002; Klassen, 1992). As a corollary, these assumptions
conceptually established the parasitolgical paradox about the apparent
enigma of the pervasive nature of host switching in associations dominated
by host-specific parasites (see Agosta et al., 2010). This view of the biosphere
is countered by considerable empirical observations and the nature of
episodic perturbation, dispersal and host switching as factors central to diver-
sification and assembly (eg, Araujo et al., 2015; Hoberg and Brooks, 2008,
2015). Recognizing the importance of complexity in the biosphere has
considerable implications for anticipating and managing/mitigating re-
sponses related to invasion and emergence of disease among intricate assem-
blages of hosts and parasites, including species of Haemonchus in ungulates,
across environments under increasing change.
6. HOST AND GEOGRAPHICAL COLONIZATION IN

FAUNAL ASSEMBLY
The history of radiation among species of Haemonchus and the devel-
opment of expansive host associations for H. contortus are broadly consistent
with processes defined in the Stockholm Paradigm, which constitutes a synthe-

sis and formal integration of macro- and microevolutionary dynamics, ecol-
ogy and biogeography involved in diversification and faunal assembly
(Araujo et al., 2015; Galbreath and Hoberg, 2015; Hoberg and Brooks,
2015). A synoptic approach or view of host range and specificity, and the
central significance of geographical and host colonization emerges from
this perspective, being one that is fundamental in understanding invasion
and emergent disease (eg, Agosta et al., 2010; Brooks and Hoberg, 2013;
Brooks and McLennan, 2002; Hoberg and Brooks, 2008, 2015).
Considered for diversification among species of Haemonchus, four primary
and interrelated drivers, as interacting components of the Stockholm Paradigm,
are involved as outlined in the previous sections: (1) opportunity and drivers
of sequential (or episodic) geographical colonization and subsequent isolation
in Africa connecting events, initiated during the Miocene and extending to
the Quaternary, that are consistent with the Taxon Pulse that defines the
ecological context and faunal outcomes of environmental perturbation/
stability; (2) a capacity for host switching is established by Ecological Fitting
and the potential for exploitation of phylogenetically conserved resources,
and is seen in shifts to arrays of novel ungulate host groups arriving in Africa
from Eurasia; (3) alternating trends for broadening (generalization) and nar-
rowing (specialization) of host range in evolutionary time, associated with
the potential for switching, occurs as a function of Oscillation; and (4) spec-
ificity may emerge downstream as a narrowing of host range during periods
of relative stability and arises through co-accommodation (reciprocal co-
adaptation in associated lineages) as specified in development of Geographic
Coevolutionary Mosaics. Host colonization and a stepping-stone dynamic dur-
ing diversification for species of Haemonchus are also evident among a consid-
erable assemblage of ungulates in evolutionary time (eg, Araujo et al., 2015).
A deep history of sequential host colonization associated with waves of
biotic expansion, bringing assemblages of ungulates from Eurasia into Africa,
processes emerging from ecosystems in collision and faunal turnover defined
the arena for radiation of Haemonchus (Hernandez Fernandez and Vrba,
2005; Vrba, 1995). Secondarily, episodic waves of dispersal associated
with human activities of agriculture, exploration and globalization, linking
Africa, Europe, Eurasia, the Americas, Australia and New Zealand, only
over the past 500 years demonstrate the importance of anthropogenic forces
as determinants of distribution and invasion (eg, Capinha et al., 2015). This
history is relevant in contemporary systems that are increasingly structured
by events of invasion and colonization, which reveals the significance of
perturbation and ecological fitting as drivers of faunal assembly across all

temporal and spatial scales. It is evident that these invasion processes are,
to a large degree, equivalent, and that history informs about the potential
range of responses that may be anticipated in contemporary systems across
managed and natural habitats (Hoberg, 2010). Ecological fitting with respect
to H. contortus accounts for what must be considered an extraordinary range
of contemporary hosts. As such, this system, for species of Haemonchus,
strongly validates the process and mechanisms outlined for faunal assembly
by Hoberg and Brooks (2008, 2010, 2013), and also instructs about the
emerging generality for the role of expansion and geographical colonization
relating to the development and structure of chronological and spatial mo-
saics (Hoberg et al., 2012).
7. CLIMATE IMPACTS INTEGRATING HISTORICAL

PERSPECTIVES
The origin of the assemblage of Haemonchus species provides historical
context for environmental/ecological regimes and selective arenas for evo-
lution and radiation in Africa over the late Tertiary and through the Quater-
nary. It is apparent that H. contortus initially emerged in association with
antelopes in relatively xeric to mesic savannah habitats of Africa, empha-
sizing the importance of selection and adaptations for persistence in subtrop-
ical environments (Hoberg et al., 2004). Conversely, radiation under
tropical regimes would pose historical constraints for development and
expansion into Temperate/Boreal and Sub-Arctic regions, serving to
explain the absence of endemic species of Haemonchus in the Western Hemi-
sphere. Faunal continuity at high latitudes was strongly influenced by
climate and cold-based filter bridges such as that across Beringia, linking
the Nearctic and Eurasia, that limited the potential for dispersal during
glacialeinterglacial stages of the late Pliocene and Pleistocene (Hoberg
et al., 2012). Host switching among and dissemination within now domestic
caprines, bovids and camelids occurred secondarily. Sequential introductions
out of Africa, and among the continents where considerable animal hus-
bandry has expanded, now serve to define the global distribution for these
nematodes (Fig. 2).
Broad geographical patterns of occurrence suggest that the constraints
posed by temperature (resilience, tolerances, metabolic upper and lower
thresholds for development of third-stage infective larvae) and moisture
are critical to geographical persistence, and as determinants of distribution
and emergence (chapter: The Pathophysiology, Ecology and Epidemiology

of Haemonchus contortus Infection in Small Ruminants by Besier et al., 2016,
in this volume; O’Connor et al., 2006; Troell et al., 2005; van Dijk et al.,
2008, 2009). Limitations created by variation in moisture, humidity and
pulses of precipitation (seasonally, and at finer temporal and spatial scales)
could be decisive in establishing permissive conditions conducive for intro-
duction/invasion, establishment and population amplification on the pe-
ripheries of current core distributions (eg, in Eurasia, North America and
South America). Precipitation rather than elevated temperature may be a
primary constraint on the distribution of H. contortus and other gastrointes-
tinal nematodes in circulation among domestic ungulates, at least in some
regions (Beck et al., 2015; Wang et al., 2014). Scenarios and models for sub-
stantial alteration in patterns of temperature and precipitation encompassing
incremental and extreme events emerging from accelerated climate warm-
ing suggest complex responses (expansion/retraction, local extinction)
with respect to geographical range occupied by Haemonchus nematodes
(eg, chapter: The Pathophysiology, Ecology and Epidemiology of Haemon-
chus contortus Infection in Small Ruminants by Besier et al., 2016, in this
volume; Hoberg et al., 2008; IPCC, 2013, 2014; van Dijk et al., 2008,
2009). In Europe, climate-driven increases in infection pressure are pre-
dicted for H. contortus, shifting from the south to north in response to envi-
ronmental change related to increasing temperature and decreasing moisture
over this century (Rose et al., 2015). An expanded window for transmission
in northern Europe by 2e3 months is also predicted, which is consistent
with general expectations for altered patterns and extension of seasonal dy-
namics for development and transmission of ungulate nematodes in the
Temperate and Boreal zones (eg, Hoberg et al., 2001, 2008).
Aside from direct environmental forcing, Waller et al. (2004) demon-
strated that the establishment and persistence of H. contortus in sheep at
high latitudes of Sweden, above the Arctic Circle near 66 N, were depen-
dent on apparent selection, resulting in a prolonged period of arrested devel-
opment that may be of 7 months duration. Interactions across biotic and
abiotic mechanisms result in populations of H. contortus sequestered as early
fourth-stage larvae in overwintering ewes. Behavioural patterns of parasites
led to a shift towards a single parasitic generation per year associated with
peri-parturient emergence, subsequent pasture contamination and infection
of lambs in the spring cycle. Epidemiology is consistent with absence of
winter survival for eggs or larval stages at Swedish latitudes, although genetic
signatures for selection and adaptations related to new life history pathways
could not be recognized (Troell et al., 2005). In these environments, char-

acterized by extreme cold temperature, persistence is currently associated
with populations that undergo long-term inhibition that carries each para-
sitic generation through extended periods of adverse ambient temperature.
Changing temperature regimes, however, can alter the potential for survival
of larval stages of H. contortus across northern environments as a consequence
of incremental warming (chapter: The Pathophysiology, Ecology and
Epidemiology of Haemonchus contortus Infection in Small Ruminants by Bes-
ier et al., 2016, in this volume; Hoberg et al., 2008; O’Connor et al., 2006).
Coincidental with environmental shifts driven by warming, seasonally
defined bimodal peaks for transmission, characteristic of core distributions
in temperate environments, may be reestablished and influence expansion
and population amplification at increasingly high latitudes (Rose et al.,
2015). Shifting epidemiological trajectories for H. contortus are expected
and further demonstrate the considerable phenotypic plasticity and capacity
for selection leading to persistence in the dual adverse environments repre-
sented by hosts and the external environment (chapter: The Pathophysi-
ology, Ecology and Epidemiology of Haemonchus contortus Infection in
Small Ruminants by Besier et al., 2016, in this volume; Crofton et al., 1965).
Persistence of H. contortus in xeric environments and under historically
elevated temperatures characteristic of Africa represents a contrast to condi-
tions in the Temperate and Boreal zones (eg, Jacquiet et al., 1998). Seasonal
effects such as strongly defined wet and dry periods and variation in the dis-
tribution and degree of sympatry for assemblages of domestic ungulates
through the annual cycle (sheep, goats, zebu cattle and dromedary camels)
result in selection pressures that may determine circulation of different spe-
cies of Haemonchus nematodes. Persistence appears linked to extended time
frames (8e9 months) for arrested development spanning the duration of a
6-month dry season (H. placei and H. longstipes) or is associated with
increased longevity or perhaps delayed senescence of adult parasites (H. con-
tortus). Either trajectory provides a capacity for survival and circulation in
otherwise harsh environmental conditions, and parallels observations from
the Northern Hemisphere that may involve extension of seasonal hypobio-
sis, when conditions would directly limit the longevity of developing and
infective larval stages.
Apparently rapid selection within small effective populations and at fine
geographical scales leading to measurable genetic and phenotypic diver-
gence demonstrates the potential for development of considerable popula-
tion heterogeneity across landscapes (Hunt et al., 2008). Recognition of
such population mosaics has implications for patterns of potential emergence

of disease conditions and should be considered in decisions about manage-
ment and husbandry at local scales. These dynamics are consistent with local
effects and the mosaic occurrence of disease in space and time that may result
from selection and adaptation on landscape scales in convergence with
changing environmental conditions for temperature and moisture (eg,
Hoberg and Brooks, 2008; Hunt et al., 2008; Thompson, 2005).
Regimes of perturbation driving origins of new ecotones, sympatry
among domestic and free-ranging wild ungulates, and dissolution of mech-
anisms for ecological isolation in combination with expansion of permissive
environments can be associated with amplification of populations, emer-
gence and disease (Brooks and Hoberg, 2007; Hoberg and Brooks, 2015;
Hoberg et al., 2008; Mas Coma et al., 2008). Relaxation of abiotic and bi-
otic controls on the occurrence of H. contortus, coincidental with inception
and dissemination of anthelmintic resistance may be synergistic, serving to
exacerbate challenges to control expansion of parasite populations or to limit
the socioeconomic impacts of infection that can influence the security and
availability of food (eg, Hoberg et al., 2008; Rose et al., 2015).
8. UNDERSTANDING DIVERSITY: SOME

RECOMMENDATIONS
Although considerable advances have been achieved in recognizing
the global extent of Haemonchus diversity and distribution, a definitive un-
derstanding of biogeography and host association remains complicated by
several interacting factors: (1) a considerable morphological homogeneity
has led to often superficial or incorrect identification when unequivocal
structural or molecular criteria are not applied and where assumptions about
host association drive concepts for elucidation of species diversity; (2) an
occurrence of unrecognized cryptic diversity and incompletely defined
limits for morphological variation conflate species identities that can only
be revealed through integrated morphological/molecular approaches; (3)
an uneven sampling across host taxa and geography may lead to biased or
incomplete assumptions about diversity and distribution, demonstrating a
justification for continued survey and inventory, especially in poorly known
areas of the Neotropical region, Eurasia and North America; (4) an absence
of broad-based landscape level assessments of genetic diversity, population
structure and gene flow hinders the recognition of relationships or linkages
for local and regional faunas; and (5) an ambiguity about transmission
pathways results from patchy information about the genetic structure of

H. contortus and other species in multi-host assemblages in circulation among
domestic and free-ranging ungulates. Such ambiguity is heightened in zones
of sympatry or across ecotones, and among wild artiodactyls in isolation from
managed systems. Additional conflation over the identity of H. contortus and
related species of Haemonchus has also been introduced by a culture in para-
sitology and disease ecology that has not developed and applied a uniform
strategy for archival deposition of voucher specimens in museum repositories
(eg, Brooks et al., 2014; Hoberg et al., 2009). Absence of an unequivocal
picture of diversity confounds the identification of routes and pathways
for the dissemination of drug resistance, and in establishing robust models
for species responses to environmental perturbation and accelerating climate
change.
Proactive assessments of diversity are necessary and a proposal for broad-
based capacities to assess and understand diversity of complex hosteparasite
systems was outlined in the Documentation-Assessment-Monitoring-Action
(DAMA) protocols (reviewed in Brooks and Hoberg, 2000; Brooks et al.,
2014; Hoberg et al., 2015). DAMA is a proposal which codifies articulation
of a proactive and collaborative capacity for biodiversity informatics, linking
field collections, archived specimens, morphology and sequence data in
museum resources, to understand, anticipate and respond to the outcomes
of accelerating environmental change and globalization. Envisioned is an
expansive platform to develop and provide essential information addressing
ecology, evolution and epidemiology for hosts and parasites linked across
temporal and spatial scales, which codifies an ongoing discussion of the
nature of diversity and biodiversity information that extends into the
1990s (eg, Hoberg, 1997). Relevant to Haemonchus and more generally
across hosteparasite systems, the past decades have demonstrated the nature
of critical information emanating from biodiversity inventories that estab-
lishes the evolutionary/ecological context necessary to recognize and docu-
ment (baselines) the cascading influence of climate change and emerging
disease (Brooks et al., 2014). Inventories at regional scales provide the mech-
anism to identify new or continuing pathways for anthropogenic invasion
and climate-driven modifications, and to monitor host and geographical as-
sociations through shifting spatial and ecological boundaries and expanding
(or contracting) distributions. Informatics emerging from inventory pro-
cesses is an essential key that links evolutionary and ecological history.
The development of timely and effective responses that mitigate emergent
parasitic infections will directly depend on integrating knowledge across
the past, present and the future of systems in dynamic change (chapter: The
Pathophysiology, Ecology and Epidemiology of Haemonchus contortus Infec-
tion in Small Ruminants by Besier et al., 2016, in this volume; Brooks et al.,
2014; Hoberg and Brooks, 2013).
Understanding diversity remains important. Translocation, establish-
ment and invasion of otherwise exotic parasites continue in a regime of
globalization (Brooks and Hoberg, 2013; Hoberg, 2010; Hulme, 2014).
Habitat perturbation, transitions, and shifting distributions due to acceler-
ating climate warming are analogous (or equivalent) to historical episodes
of climate fluctuation and environmental disruption in Africa during the
Miocene, Pliocene and Quaternary, which had influential contributions
to the distribution and radiation among species of Haemonchus in ungulates
(Hoberg and Brooks, 2010, 2013; Hoberg et al., 2004). Species of Haemon-
chus radiated in savannah environments of sub-Saharan Africa under rela-
tively xeric conditions and elevated temperatures. Controls on current
distributions, for example in South America and North America, may
reflect this evolutionary and ecological trajectory with thresholds for devel-
opment, tolerances and resilience as conservative constraints linked to
particular regimes of temperature and moisture. Consequently, climate,
manifested in long-term incremental change and short-term extreme
events for temperature and precipitation (IPCC, 2013, 2014), must be
accounted for in anticipating responses in complex hosteparasite systems
that can influence patterns of persistence, emergence and disease across a
broad spectrum of ungulate hosts (eg, Hoberg et al., 2008; Mas Coma
et al., 2008; van Dijk et al., 2009). All of our knowledge starts with
evolution, ecology and biogeography, as these interacting facets determine
the history of biodiverse systems. These components, relating to Haemon-
chus, can inform about the nuanced history of geographical distribution,
host association and the intricacies of the hosteparasite interface that are
played out in physiological and behavioural processes that influence the
potential for disease and our capacity for effective control in a rapidly
changing world.
ACKNOWLEDGEMENTS
Thanks are extended to D.R. Brooks for long-term collaborations extending over 30 years,
and for the continuing insights and discussion about evolution, biogeography and the nature
of hosteparasite associations in a world undergoing accelerating change. Further, we are
grateful for revealing discussions within the Stockholm Group, S.B.L. Araujo, M.P. Braga,
D.R. Brooks, S. Agosta, F. von Hathental and W.A. Boeger, in explorations of evolutionary
and ecological patterns and processes of host and geographical colonization and faunal
assembly in complex systems across the biosphere. Concepts explored in our paper reflect dis-
cussions held at the workshop: “Changing species associations in a changing world: a Marcus
Wallenberg Symposium” (MWS 2015.0009) with funding to S€ oren Nylin; organized and
hosted by S€oren Nylin and Niklas Janz at the Tovetorp Field Station near Stockholm, Swe-
den, 11e13 March 2016.
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CHAPTER TWO
Genetic Diversity and Population

Structure of Haemonchus
contortus
J.S. Gilleard1, E. Redman
1
Corresponding author: E-mail: jsgillea@ucalgary.ca
Contents
1. Introduction 32
2. Background Information on Reproduction and Genetics 33
3. Genetic Diversity and Population Structure of Haemonchus contortus in the Field 34
3.1 Many factors influence genetic diversity and population structure of 34
Haemonchus contortus
3.2 Extremely high levels of genetic diversity are seen within Haemonchus 35
contortus populations
3.3 Large population size is a major determinant of the high genetic diversity 37
within Haemonchus contortus populations
3.4 Haemonchus contortus has substantial global population structure 40
3.5 Haemonchus contortus has a low but discernable regional population structure 41
within countries
3.6 Current evidence regarding genetic differentiation between Haemonchus 45
contortus populations from different host species
3.7 Effect of anthelmintic selection on the overall genetic diversity of Haemonchus 46
contortus populations in the field
4. Consequences of Haemonchus contortus Population Structure for the Emergence 47
and Spread of Anthelmintic Resistance in the Field
4.1 Consequence of high genetic diversity 47
4.2 Consequence of low regional population structure within a country 48
4.3 Consequence of substantial global population structure 49
4.4 Consequence of low population structure between hosts 50
5. Genetic and Phenotypic Variation in Laboratory Strains 50
5.1 Genetic variation within and between laboratory strains 52
5.2 Phenotypic variation within and between laboratory strains 54
5.2.1 Variation in gene expression and function 55
5.2.2 Variation in morphological traits 57
5.2.3 Variation in life history traits and pathogenicity 59

© 2016 Elsevier Ltd.
j
ISSN 0065-308X
http://dx.doi.org/10.1016/bs.apar.2016.02.009 All rights reserved. 31
32 J.S. Gilleard and E. Redman
6. Concluding Remarks 61
Acknowledgements 62
References 62
Abstract
Haemonchus contortus is one of the most successful and problematic livestock parasites
worldwide. From its apparent evolutionary origins in sub-Saharan Africa, it is now found
in small ruminants in almost all regions of the globe, and can infect a range of different
domestic and wildlife artiodactyl hosts. It has a remarkably high propensity to develop
resistance to anthelmintic drugs, making control increasingly difficult. The success of
this parasite is, at least in part, due to its extremely high levels of genetic diversity
that, in turn, provide a high adaptive capacity. Understanding this genetic diversity is
important for many areas of research including anthelmintic resistance, epidemiology,
control, drug/vaccine development and molecular diagnostics. In this article, we review
the current knowledge of H. contortus genetic diversity and population structure for
both field isolates and laboratory strains. We highlight the practical relevance of this
knowledge with a particular emphasis on anthelmintic resistance research.
1. INTRODUCTION
Current knowledge indicates that Haemonchus contortus evolved in wild
ungulates in sub-Saharan Africa before being translocated around the globe
by anthropogenic livestock movement (Hoberg et al., 2004). Over this time,
it has adapted to a wide range of different host species and climatic zones,
and is now essentially ubiquitous in grazing small ruminants worldwide.
This parasite has a remarkably high propensity to develop anthelmintic
drug resistance, even within a few years of drug use (Gilleard, 2013;
Prichard, 2001). This adaptive capacity is largely due to the very high level
of genetic variation in parasite populations upon which selection can act
(Gilleard and Beech, 2007; Prichard, 2001). Understanding this genetic vari-
ation, and how it is partitioned within and among populations, is central to
understanding how parasite populations respond to selective pressures, such
as drug treatments, host genetics, immune responses, climate change and
other environmental factors (Gilleard and Beech, 2007). It is also important
for interpreting apparent associations of particular genetic markers with a
drug resistance phenotype and for applying genome-wide approaches to
identify novel drug resistance loci (Gilleard, 2013; Gilleard and Beech,
2007). In this article, we review the current understanding of genetic
variation and population structure of H. contortus. We first consider field
populations and then laboratory strains.
Genetic Diversity and Population Structure of H. contortus 33
2. BACKGROUND INFORMATION ON REPRODUCTION

AND GENETICS
Haemonchus contortus is sexually dioecious and undergoes obligate
sexual reproduction. The karyotype of H. contortus has been defined for a
number of different isolates, and comprises of five pairs of autosomes and
one sex chromosome pair (Le Jambre and Royal, 1980; Redman et al.,
2008a). The sex chromosomes have been identified by sperm karyotyping
and confirmed by genotyping single female broods using sex-linked micro-
satellite markers (Le Jambre and Royal, 1980; Redman et al., 2008a). The
male sex karyotype is XO, and the female sex karyotype is XX. As in the
case for Caenorhabditis elegans, all five autosomes and the X-chromosome
are of a similar size, whilst in the closely related species Haemonchus placei,
the X-chromosomes are considerably larger than the autosomes (Bremner,
1954; Le Jambre, 1979). Inheritance studies using both autosomal and
X-linked microsatellite markers have demonstrated that mating is polyan-
drous with each female mating with multiple males (Redman et al.,
2008a). Genotypes derived from up to four different parental males have
been identified in broods from single females derived from experimental in-
fections (Redman et al., 2008a). A similar level of polyandry has also been
shown for Teladorsagia circumcincta, suggesting that it may be a common
feature of the trichostrongyloid group (V. Grillo and J.S. Gilleard, unpub-
lished data). The relative costs and benefits of polyandry are an ongoing
subject of debate for a variety of organisms (Jennions and Petrie, 2000;
Tregenza and Wedell, 2000). In the case of H. contortus, the potential costs
of polyandry include the expenditure of energy in finding and copulating
with multiple mates, together with the associated disruption of mucosal
attachment and feeding. However, a major benefit might be greater genetic
variation associated with an increased opportunity for recombination be-
tween different parental haplotypes at each generation. Polyandry is also
expected to reduce the impact of population bottlenecks on genetic diver-
sity within populations, and so reduce genetic drift between populations.
The extent to which polyandry occurs in natural H. contortus infection has
not been investigated and may vary with infection intensity.
It is possible to undertake genetic crosses of H. contortus by experimental
transplantation of male and female adult worms from different strains into
the sheep abomasum (Le Jambre et al., 1979; Redman et al., 2015; Sangster
et al., 1998). This approach has allowed the inheritance of anthelmintic resis-
tance to be studied and now offers the potential for forward genetic mapping
of anthelmintic resistance loci utilizing the rapidly improving H. contortus

genomic resources and sequencing technologies. This aspect will not be dis-
cussed here as it has been recently reviewed elsewhere (Gilleard, 2013).
3. GENETIC DIVERSITY AND POPULATION STRUCTURE

OF HAEMONCHUS CONTORTUS IN THE FIELD
3.1 Many factors influence genetic diversity and
population structure of Haemonchus contortus
Population genetic structure essentially describes the total genetic
diversity and its distribution within and among a set of populations. It is
shaped by many factors, including life history, population size, geographical
or environmental barriers, gene flow, selection and population crashes or
bottlenecks (Charlesworth, 2009; Slatkin, 1987; Wright, 1931). These fac-
tors are more complex for parasites than for free-living organisms, since the
interactions between parasite and host have additional impacts. The popu-
lation dynamics of parasites is intimately associated with that of their hosts,
since changes in host numbers and/or geographic range can drive associated
changes in parasite populations (Blouin et al., 1995; Donnelly et al., 2001;
Morrison and Hoglund, 2005). This aspect is particularly relevant to live-
stock parasites, since their hosts are commonly subject to major changes in
their number and distribution due to human activity. For example, parasites
of human-associated hosts, including H. contortus, show evidence of recent
population expansions in their mitochondrial (mt)DNA sequences more
often than do hosts not directly subject to human intervention (Mes,
2003; Morrison and Hoglund, 2005).
Haemonchus contortus has both parasitic and free-living stages of its life cy-
cle, and these stages are subject to very different environmental influences
(Gilleard, 2013). The free-living stages can be exposed to dramatic fluctua-
tions in temperature and humidity that will affect population size and will
differ depending on geographical location and season. In contrast, the host
provides a much more stable environment, allowing a proportion of the
parasite population to avoid adverse external environmental conditions,
and this has been an essential element in the successful expansion of H. con-
tortus around the world. The parasite originates from sub-Saharan Africa and
is consequently best adapted to warm and humid conditions (Gilleard, 2013;
Hoberg et al., 2004). Hence, its ability to survive inside the host during pe-
riods when the external environment is inhospitable has allowed it to estab-
lish, and even thrive, following its introduction into much colder and more
arid regions (chapter: The Pathophysiology, Ecology and Epidemiology of

Haemonchus contortus Infection in Small Ruminants by Besier et al., 2016).
However, the host environment is not completely benign from the parasite’s
perspective. The parasitic stages are subject to host immune responses, and
drug treatments and these responses will also influence population size
and apply strong selective pressures to the parasite. Hence, a large number
of factors, both inside and outside of the host, affect parasite populations
and apply selection pressure.
Another major factor that contributes to the population genetic structure of
parasites is their dispersal by host movement that can potentially lead to high
rates of gene flow, even across large distances. This factor disrupts the pattern
of isolation by distance that is often seen for free-living organisms as a result
of their dispersal capacity being less than their geographical distribution
(Koop et al., 2014). In the case of H. contortus, anthropogenic movement
of its livestock hosts can be extensive, long range and complex. In summary,
there are many variables that shape the population genetic structure of H. con-
tortus and these will differ between regions, seasons and production systems.
3.2 Extremely high levels of genetic diversity are seen within

Haemonchus contortus populations
The high genetic diversity of nematodes in the superfamily Trichostrongy-
loidea was first suggested by a restriction fragment polymorphism analysis
of mtDNA of Ostertagia ostertagi in US cattle (Dame et al., 1993; Tarrant
et al., 1992). This was followed by a second, more detailed, study of
sequence diversity in the nicotinamide adenine dinucleotide dehydroge-
nase subunit 4 (nad4) gene in five species of trichostrongyloid nematodes
from four or five different locations in the United States; H. contortus and
T. circumcincta from sheep, O. ostertagi and H. placei from cattle, and Maza-
mastrongylus odocoilei from the white-tailed deer (Blouin et al., 1995).
Extremely high levels of within-population genetic diversity were found
in all of these species (Blouin et al., 1995). In the case of H. contortus, the
within-population diversity was 0.026 substitutions per site of the nad4
sequence, which was much higher than that observed for other taxa (Lynch
and Crease, 1990). Numerous other studies have subsequently confirmed
these findings using a variety of different approaches or nuclear DNA
markers, including amplified fragment length polymorphism (AFLP) anal-
ysis, transposons, single nucleotide polymorphisms (SNPs), indels and
microsatellites. (Hoekstra et al., 1997, 2000a,b; Otsen et al., 2000a,b;
Redman et al., 2008b; Silvestre et al., 2009; Troell et al., 2006a). For
example, a study of laboratory strains of H. contortus reported the diversity

of AFLP patterns between individual H. contortus worms to be similar to the
level of variation found between closely related mammalian species, such as
cattle and bison (Otsen et al., 2001). Similar levels of within-population di-
versity of AFLP markers have also been reported for field populations
(Troell et al., 2006a). Microsatellite markers also show high levels of ge-
netic diversity with a high proportion being polymorphic and having
imperfect repeat structures (Hoekstra et al., 1997; Otsen et al., 2000b;
Redman et al., 2008b). The high genetic diversity within H. contortus pop-
ulations is also reflected by the frequent presence of null alleles for micro-
satellite markers (Hunt et al., 2008; Otsen et al., 2000b; Redman et al.,
2008b, 2015; Silvestre et al., 2009). This observation has also been made
for other trichostrongyloid nematodes, including T. circumcincta and Tri-
chostrongylus tenuis (Grillo et al., 2007, 2006; Johnson et al., 2006). For
several loci, these null alleles have been directly shown to be due to
sequence polymorphisms within the flanking primer sites (Redman
et al., 2015). In spite of extensive screening, even the best available H. con-
tortus microsatellite markers contain null alleles in at least some populations,
resulting in heterozygote deficiencies in population genetic data (Otsen,
2000b; Redman et al., 2015). Although methods are available to detect
and partially compensate for null alleles in population genetic data, the
presence of null alleles still results in some limitations in the analyses that
can be performed and the interpretation of data produced (Chapuis and
Estoup, 2007). Nevertheless, microsatellite markers have proved to be use-
ful tools for studying the genetic diversity and population structure of H.
contortus, and this work is reviewed in further detail below (Chaudhry
et al., 2015a; Hunt et al., 2008; Redman et al., 2015; Silvestre et al., 2009).
As yet, there are no published studies of genetic diversity of H. contortus in
natural field populations using genome-wide data. However, whole
genome sequencing of laboratory strains provides some insight into the
overall levels of genome-wide variation present in this parasite. Both of
the H. contortus genome sequencing consortia have found a very high level
of sequence polymorphism in the raw sequence reads used to produce the
consensus reference genome sequences (Laing et al., 2013; Schwarz et al.,
2013). Indeed, these high levels of sequence polymorphism have been a ma-
jor challenge for genome assembly (chapter: Haemonchus contortus: Genome
Structure, Organization and Comparative Genomics by Laing et al., 2016;
chapter: Understanding Haemonchus contortus Better Through Genomics
and Ranscriptomics by Gasser et al., 2016 e in this volume). To provide
some indication of the level of sequence polymorphism between laboratory

strains, genome-wide short (100 bp) Illumina sequence reads generated from
20 to 30 adult worms from each of a number of strains were aligned to the
338,499,134 bp MHco3(ISE) reference genome assembly (14.11.2014
version). The number of SNPs identified across the genome assembly, after
filtering for read depth and quality with the vcf_annotate script (http://
vcftools.sourceforge.net), was 921,246 (1/367 bp) for Hco3(ISE),
1,650,368 (1/205 bp) for Hco4(WRS), 1,671,886 (1/202 bp) for Hco10
(CAVR), 1,194,992 (1/283 bp) for MHco18(UGA2004) and 1,373,491
(1/246 bp) for MHco16 (A. Martinelli, J. Cotton and J. Gilleard, unpub-
lished data). This information illustrates the high density of SNPs across
the genome, relative to MHco3(ISE) reference genome assembly for labo-
ratory strains derived from field populations from different countries. In
addition, there is likely to also be a large number of indels across the ge-
nomes as indicated by studies of specific genes (Otsen et al., 2000a; Rufener
et al., 2009).
3.3 Large population size is a major determinant of the high

genetic diversity within Haemonchus contortus
populations
Genetic diversity in a population is a function of mutation rate (m) and effec-
tive population size (Ne) and, in an idealized diploid population, the pair-
wise nucleotide diversity is equal to 4 mNe (Charlesworth, 2009; Wright,
1931). Consequently, high levels of genetic diversity within H. contortus
populations could be due to high mutation rates and/or due to large effec-
tive population sizes. An accurate determination of mutation rates is difficult
for parasitic species, and we do not have meaningful values for H. contortus.
However, mutation rates have been directly measured for the free-living
nematode C. elegans, and were originally estimated to be 2.1 108 and
1.6 107 mutations per site per generation for the nuclear and mitochon-
drial genomes, respectively (Denver et al., 2004, 2000). Whilst these esti-
mates are significantly higher than those of many other organisms, more
recent estimates suggest the nuclear genome mutation rate in C. elegans is
actually lower. Genome-wide analysis, and conversion of the data to a
per-germ-line cell division mutation rate, yielded an estimate of
3.2 1010 mutations per site per-cell division. This value is very similar
to the per-cell division mutation rate estimate for Saccharomyces cerevisiae
(3.3 1010) and only approximately threefold higher than those for
Drosophila melanogaster (1.5 1010) and humans (1.0 1010) (Denver
et al., 2009). Consequently although it is possible that the mutation rate in

H. contortus is higher than that of C. elegans, the current evidence suggests
that it is unlikely that mutation rate alone accounts for the high genetic di-
versity of trichostrongyloid nematode populations. Instead, population size
is likely to be a key factor.
The effective population size (Ne) is the size of an idealized, sexually repro-
ducing population that would provide the same outcome of a random sampling
of alleles as that observed in the real population under study (Charlesworth,
2009; Wright, 1931). A number of different outcomes can be used to calcu-
late Ne for a population including levels of heterozygosity, genetic drift and
inbreeding. Ne is generally much smaller than the actual number of individ-
uals in a population e referred to as the census population size (N) e due to
factors including nonrandom mating, breeding sex ratios, overlapping
generations and nonuniform spatial dispersion (Charlesworth, 2009). Howev-
er, Ne is a useful concept, since it, rather than the census population size,
determines how a population is likely to respond to selection. Values of Ne
will generally be large for H. contortus populations by virtue of the high levels
within-population genetic diversity that are observed and this has a number of
important consequences. For example, genetic drift is likely to be low in
populations with high Ne and this, along with migration, helps explain
why genetic differentiation is generally low among different H. contortus pop-
ulations within a region (Charlesworth, 2009). In addition, positive selection
has more impact when effective population sizes are large, which helps
explain why anthelmintic resistance alleles commonly arise in H. contortus
populations (Charlesworth, 2009; Gilleard and Beech, 2007).
One important question is whether the remarkably high levels of genetic
diversity, and consequently large Ne, observed within each H. contortus
population is dependant on migration of genotypes between populations.
Blouin et al. (1995) was the first to note that, although sequence diversity
in nad4 mtDNA within the four populations of H. contortus examined in
the United States was extremely high (0.026 substitutions per site), the
diversity among populations was very low (0.0004 substitutions per site)
(Blouin et al., 1995). Hence, more than 96% of the genetic diversity was
within, and not among, separate H. contortus populations. This lack of pop-
ulation structure was suggested to be a consequence of gene flow between
populations as a result of anthropogenic animal movement. It was further
suggested that this gene flow might result in a single ‘meta-population’
across the United States with a huge effective population size. However,
in the same study, even higher levels of genetic diversity were seen within
populations of M. odocoilei, in spite of there being substantial partitioning of

this variation among populations, suggesting lower levels of gene flow
(Blouin et al., 1995). Hence, it seems unlikely that gene flow was solely
responsible for the high diversity observed in the trichostonglyoid nematode
populations examined in that study. Furthermore, two subsequent studies,
using microsatellite markers, have shown that sheep and goat farms, in
France and Pakistan, which have been closed to animal movement for
more than 30 years have similarly high levels of diversity as farms open to
animal movement (Chaudhry et al., 2015b; Redman et al., 2015; Silvestre
et al., 2009) and (U. Chaudhry, E. Redman, K. Ashraf, M. Shabbir, M.
Rashid, S. Ashraf and J. Gilleard, unpublished data). It is also noteworthy
that laboratory isolates passaged for many years typically retain very high
levels of genetic diversity, in spite of being effectively closed to gene flow
in this wildlife parasites (Hunt et al., 2008; Otsen et al., 2001; Redman
et al., 2008b). Hence, a high level of contemporary gene flow between pop-
ulations does not seem necessary to maintain high levels of genetic diversity
within H. contortus populations.
One other factor that could potentially increase observed levels of genetic
diversity within H. contortus populations is admixture (ie, when individuals
derived from previously allopatric and genetically differentiated populations
are mixed in a single population) (Dlugosch et al., 2015). The large amount
of long-distance livestock movement that has historically occurred in many
parts of the world might be expected to result in such admixture being
commonly seen. However, there are no obvious discontinuities in phyloge-
netic relationships of nad4 haplotypes reported for H. contortus populations and
little evidence of admixture from the various studies that have been
performed in different countries using microsatellite markers (Chaudhry
et al., 2015b; Redman et al., 2015; Silvestre et al., 2009). However, one
caveat to this evidence is that the presence of null alleles for microsatellites
used in these studies makes definitive testing of admixture difficult.
Nevertheless there is little evidence to suggest that admixture of diverse
populations is a major feature of most H. contortus populations.
The balance of evidence overall suggests that the high genetic diversity
observed within H. contortus populations is largely due to their large census
population sizes. This information is consistent with our knowledge of the
life history of the parasite. A single small ruminant host can contain
thousands, or tens of thousands, of adult female H. contortus worms, each
of which can produce up to 4000 eggs per day (Fleming, 1988). Conse-
quently, a single pasture grazed by a flock of several hundred sheep will
be seeded with billions of new progeny every few days. Although only some
of these progeny are then ingested by a host and contribute to the next gen-
eration, the census population size of H. contortus is generally very large, even
at a single location. If census population size is the most important determi-
nant of the high genetic diversity of H. controtus populations, one would pre-
dict that parasite species with lower infection intensities (lower numbers of
adult worms per host) would have lower levels of genetic diversity. There
are few studies to date that have directly addressed this question, but there
is some evidence that mtDNA diversity in sexually reproducing nematode
species with direct life cycles is positively correlated with mean infection in-
tensities (Criscione et al., 2005). For example, the ascaridoid nematodes
Ascaris suum and Ascaris lumbricoides, which have much lower infection inten-
sities than trichostrongyloid nematodes, also have much lower levels of
mtDNA diversity. In addition, an estimate of the effective population size
(Ne) of A. lumbricoides in a village in Nepal, using microsatellite data, was
just w1300 compared with estimates of several million on a single farm
for trichostrongyloid nematodes such as O. ostertagi (Blouin et al., 1992;
Criscione, 2013).
3.4 Haemonchus contortus has substantial global population

structure
Haemonchus contortus has substantial population structure on a global scale.
Troell et al. (2006a,b) examined AFLP profiles and nad4 mtDNA sequences
from 8 to 10 worms from each of 19 isolates distributed across 14 different
countries (Troell et al., 2006a). Of a total of the 150 individual worms
analysed, there were no identical AFLP profiles and 94 different nad4
haplotypes, reflecting the high overall genetic diversity. For the AFLP
data, genetic differentiation between continental areas was significant at
P < 0.001 for all pairwise comparisons. For the nad4 sequence data,
38.0% of the genetic variation was among individuals within populations,
27.1% among populations within continents and 34.8% among continents.
When the AFLP data were used to construct phylogenetic trees, almost all
individuals from the same isolate clustered together and, in most cases,
isolates from the same continent were also clustered. The mitochondrial
nad4 marker showed less phylogenetic resolution overall, but broadly sup-
ported the phylogenetic relationships determined using AFLP data. In sum-
mary, this study not only found very high levels of overall genetic diversity,
but also demonstrated significant genetic differentiation between H. contortus
populations from different countries, suggesting strong barriers to gene flow
at this scale. A few findings did not fit the expected pattern based on
geographical location. Most notably, the Greek isolate clustered with the
Australian rather than the other European isolates, suggesting a possible
introduction of H. contortus to Greece from Australia. However, a limitation
of the study was that only a single isolate was examined from each country,
and so further work is needed to test this hypothesis.
We have genotyped H. contortus populations from the UK, southern In-
dia and Pakistan with panels of microsatellite markers in separate population
genetic studies (Chaudhry et al., 2015b; Redman et al., 2015) (U.
Chaudhry, E. Redman, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf and
J. Gilleard, unpublished findings). Although different microsatellite marker
panels were used in each of these studies, five markers were common to
all three panels. To assess the genetic differentiation of H. contortus popula-
tions between countries, we have analysed the data from these five markers
for six H. contortus populations from each country. The populations clearly
cluster by country on principal coordinate analysis (PCoA), even using as
few as five microsatellite markers (Fig. 1A). The populations from southern
India and Pakistan appear more closely related to each other than they are to
the UK population, as expected based on their geographical relationships.
Eight microsatellite markers were shared among the panels used in the
Pakistan and southern India studies, and the populations clustered clearly
by country when these eight markers were applied to all populations from
the two studies (Chaudhry et al., 2015b; Redman et al., 2015) (Fig. 1B).
This latter point illustrates that the detection of genetic differentiation in-
creases in sensitivity as a greater number of discriminatory markers are
used. Hence, the future application of genome-wide approaches is expected
to reveal finer scale population structure that can be detected using the mi-
crosatellite marker panels employed to date.
The characterization of H. contortus laboratory strains is also suggestive of
significant population structure among countries since there is substantial ge-
netic differentiation between laboratory strains derived from different coun-
tries (Redman et al., 2008b). Consequently, laboratory strains may not be
representative of field populations if originally isolated from a different
geographical region. This aspect is discussed in more detail in Section 5.1.
3.5 Haemonchus contortus has a low but discernable

regional population structure within countries
Although, on a regional level, most genetic variation in H. contortus is
within and not between populations, some population structure is still
Figure 1 Principal coordinate analysis based on Fst values calculated from microsatellite
genotype data from UK, southern India and Pakistan Haemonchus contortus populations
using Arlequin 3.11. Panel (A) Six loci e Hcms36, Hcms25, Hcms33, Hcms3086,
Hcms53265, Hcms8a20 e were used to genotype 25e30 worms from six populations
from three different countries, UK, southern India and Pakistan. Each data point repre-
sents a different population with the country of origin coded by its colour. Panel (B) Eight
loci e Hcms36, Hcms25, Hcms33, Hcms3086, Hcms53265, Hcms22193, Hcms2561 and
Hc8a20 e were used to genotype 25e30 worms from 13 H. contortus populations
from southern India and 11 H. contortus populations from Pakistan. Each data point rep-
resents a different population with the country of origin coded by its colour and the
colour of the text label indicating the host species of origin.
evident. Although the study of Troell et al. (2006a,b) only examined a sin-
gle isolate from most countries, four different isolates were examined from
Sweden. There was low but significant genetic structure among these pop-
ulations, with an overall Fst of 0.13 based on the AFLP data and an Nst of
0.16 based on the mtDNA nad4 data. In addition, in the minimum-

evolution tree constructed using AFLP data, all eight worms from each
Swedish isolate clustered together but separately from those of the other
Swedish isolates (Troell et al., 2006a). A study in southern and central
France also revealed detectable population structure at the regional scale.
Pairwise Fst values, based on a panel of seven microsatellite markers, ranged
from 0.045 to 0.183, with 11 of 15 being significantly different from zero
(p ¼ 0.08 with Bonferroni correction) (Silvestre et al., 2009). Although
this is a clear example of genetic differentiation between H. contortus
populations within a region of a country, the herds of goats studied had
been closed to animal movement for more than 30 years.
Consequently the lack of gene flow may mean that genetic drift of these
H. contortus populations may be higher than is typical for most farms that
are open to animal movement. However, in a separate study of seven H.
contortus populations on UK sheep farms, genetic differentiation could
also be detected on the regional scale using a panel of 10 microsatellite
markers (Redman et al., 2015). In this case, pairwise Fst values ranged
from 0.0198 to 0.0757, with 10 of 21 being significantly different from
0 (p ¼ 0.01). In contrast to the French study, these sheep flocks were
not closed, and so even with the considerable movement of sheep that
occurs in the UK, low but detectable population substructure of H. contor-
tus occurs at a regional level.
One hypothesis to potentially explain regional population structure of
H. contortus is suggested by comparison with the closely related trichostron-
gyloid nematode T. circumcincta. In both the French and UK studies
described above, T. circumcincta was also examined on the same farms. In
contrast to H. contortus, this nematode species showed no significant genetic
differentiation between farms in either of the studies. In the French study,
pairwise Fst values between T. circumcincta populations ranged from 0.001
to 0.057, with none being significantly different from 0 (p ¼ 0.08). In the
UK study, pairwise Fst values between T. circumcincta populations ranged
from 0.0269 to 0.0340, with only 2 of 21 being significantly different
from zero (p ¼ 0.08). In this latter study, H. contortus and T. circumcincta
were collected from the same individual hosts, suggesting that their contrast-
ing population structures must be directly related to differences in the life
histories of the two parasites. Haemonchus contortus is primarily adapted to
warmer climates (being originally native to sub-Saharan Africa), and so in
temperate and colder regions, very few infective larvae survive on pastures
over the winter (Falzon et al., 2014; Sargison et al., 2007; Thomas and
Waller, 1979; Waller et al., 2004). Instead, the parasite primarily overwinters
inside the host, which is likely to represent a population bottleneck, partic-
ularly if hosts are treated with anthelmintic drugs when larval counts on
pasture are low. In contrast, T. circumcincta is native to temperate regions,
and so a larger numbers of infective larvae usually survive on pastures over
the winter, making population bottlenecks less likely. The relative preva-
lence and infection intensities of these two parasite species in UK sheep
are consistent with this model. In a survey of 118 UK sheep farms, T. circum-
cincta was found to be present in all flocks and, in most cases, at high
frequencies (Burgess et al., 2012; Redman et al., 2015). In contrast,
H. contortus was only detected in w50% of flocks and was present at a
very low frequency (<5%) in most cases.
If the population structure of H. contortus observed in temperate regions
(UK, France and Sweden) is predominantly due to population bottlenecks
caused by the death of larvae on winter pastures, one would predict less pop-
ulation structure in countries with year-round warm humid climates. Pre-
liminary evidence suggests that this might be the case. Haemonchus
contortus populations show little population structure in southern India;
our recent study reported pairwise Fst values, based on microsatellite data,
to be very low, ranging from 0.0244 to 0.0351, with only 5 of 66 pairwise
comparisons being significantly different from 0 (p ¼ 0.01) (Chaudhry et al.,
2015b). We also see a similar lack of genetic structure for H. contortus
populations in Pakistan using a similar panel of microsatellite markers
(U. Chaudhry, E. Redman, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf
and J. Gilleard, unpublished data). In that case, pairwise Fst values were
not significantly different from zero, even among three government farms
closed to animal movement for more than 30 years
In summary, most of the genetic variation is within, and not among,
H. contortus populations from an individual country. However, a low level
of regional genetic differentiation is sometimes discernable, even with rela-
tively small microsatellite marker panels. It is hypothesized that population
genetic structure is more marked in temperate than in tropical regions due
to an increased population bottleneck occurring in regions with colder
climates. However, this suggestion is based on relatively few studies, and
more work comparing H. contortus, and other trichostrongyloid nematodes,
in different climatic zones is needed to test this hypothesis. The use of larger
sets of genetic markers, such as genome-wide SNPs, should also provide
more discriminatory power for such studies.
3.6 Current evidence regarding genetic differentiation

between Haemonchus contortus populations from
different host species
Although its ‘preferred’ hosts are sheep and goats, H. contortus has been
reported to infect a range of wild and domestic ungulate species (Hoberg
et al., 2004). In the few reports published to date, there is little evidence
to suggest the presence of cryptic species or of discernable genetic differen-
tiation between H. contortus populations in different host species. For
example, there was no clustering by host species of nad4 mtDNA sequences
derived from 78 H. contortus specimens from alpine chamois (Rupicapra r.
rupicapra), roe deer (Capreolus capreolus), alpine ibex (Capra ibex ibex), domes-
tic goat (Capra hircus) and sheep (Ovis aries) (Cerutti et al., 2010). To date,
most comparisons have been between populations of H. contortus from sheep
and goats. Troell et al. (2003) investigated the species identity of Haemonchus
specimens isolated from a sheep and goat from Sweden and a sheep from
Kenya using pyrosequencing of the ITS-1, ITS-2 and the 5.8S rRNA
regions of the rDNA cistron (Troell et al., 2003). The worms from the
Swedish sheep and goat were more closely related to each other than to
the worms from the Kenyan sheep. Similarly, in the study of 19 H. contortus
isolates from 14 different countries, the only 2 isolates from goats (from
Cambodia and Guadaloupe) clustered by geographical region and not by
host species for both the AFLP and mtDNA sequence data (Troell et al.,
2006a). This finding is further supported by our study of H. contortus in
southern India in which six populations were from sheep and six from goats
(Chaudhry et al., 2015b). No differences were found between H. contortus
populations in the two host species based on a panel of eight polymorphic
microsatellite markers. Indeed, the Fst values were lower for many of the
pairwise comparisons between the H. contortus populations from sheep
and goats than between those from the same host species (Chaudhry
et al., 2015b). The same result was found for our recent study of H. contortus
in Pakistan, with no Fst values for pairwise comparisons between the two
host species being significantly different from zero (U. Chaudhry, E. Red-
man, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf and J. Gilleard, unpub-
lished data). This lack of clustering by host species in both of these studies
is illustrated by PCoA (Fig. 1B).
There is only one study that has suggested genetic differences can occur
between H. contortus from sheep and goats. This is a phylogenetic analysis of
H. contortus mtDNA nad4 sequences from sheep and goats in Malaysia and
Yemen (Gharamah et al., 2012). In this study, marked clustering of

sequences between the two countries revealed significant geographical pop-
ulation substructuring. Although all of the nad4 sequences from H. contortus
from Malaysia and most from Yemen did not show any evidence of clus-
tering by host species, there was one separate clade of nad4 sequences that
was derived exclusively from H. contortus from goats from Yemen (Ghara-
mah et al., 2012). This information suggested the possibility of a subpopu-
lation of genetically distinct parasites present in Yemen goats that was not
present in sheep in the same region. The authors speculated that this might
be evidence of goat-specific cryptic species of H. contortus in Yemen, anal-
ogous to that which has been described for Teladorsagia cricumcincta in France
(Grillo et al., 2007). However, these authors also noted that there is signif-
icant importation of small ruminants into Yemen from Africa, and so the re-
sults could also be explained by admixture of allopatric parasites with the
indigenous H. contortus populations. A subsequent, detailed morphometric
analysis of the same populations, based on eight morphological characters,
revealed significant grouping based on country but not by host species in
either country (Gharamah et al., 2014). In summary, most of the evidence
suggests that H. contortus is generally freely shared between sheep and goats,
with little or no host species barrier or population substructuring.
3.7 Effect of anthelmintic selection on the overall genetic

diversity of Haemonchus contortus populations in the field
In general, field populations of parasitic nematodes that are resistant to
anthelmintic drugs appear have a similar level of overall genetic diversity
as susceptible populations. For example, H. contortus populations on two
farms with a low frequency of benzimidazole resistance mutations showed
no significant difference in allelic richness or expected heterozygosity
compared with five farms with much higher frequencies of resistance
mutations assessed using a panel of 10 microsatellite markers (Redman
et al., 2015). Similarly 2 H. contortus populations from southern India,
from which benzimidazole resistance mutations were absent, showed no sig-
nificant difference in allelic richness or expected heterozygosity for a panel of
8 microsatellite markers compared with 10 populations in which benzimid-
azole resistance mutations were present (in some cases at high frequency)
(Chaudhry et al., 2015b). In these two examples, there was significant ani-
mal movement between farms that might have reduced the impact of
anthelmintic selection on overall genetic diversity. However, we have
recently compared H. contortus populations on three government (small
ruminant) farms in Pakistan with a history of intense benzimidazole drug se-

lection pressure and that have been closed to animal movement for more
20 years with those from four pastoral locations with little or no drug treat-
ment. Although the frequency of benzimidazole resistance mutations in H.
contortus was very high on the government farms and very low for the pas-
toral locations, consistent with their respective drug treatment histories,
there was again no significant difference in allelic richness, expected hetero-
zygosity or inbreeding coefficient for a panel of eight microsatellite markers
(U. Chaudhry, E. Redman, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf and
J. Gilleard, unpublished data). In contrast to these studies, we are not aware
of any evidence to date showing an overall loss of genetic diversity of H. con-
tortus populations in response to drug selection in the field.
4. CONSEQUENCES OF HAEMONCHUS CONTORTUS

POPULATION STRUCTURE FOR THE EMERGENCE AND
SPREAD OF ANTHELMINTIC RESISTANCE IN THE FIELD
4.1 Consequence of high genetic diversity
The high level of genetic diversity within H. contortus populations un-
derlies the remarkable adaptive capacity of this parasite. Assuming a muta-
tion rate of w2.1 mutations per genome per generation (based on data
for C. elegans), mutations for each position in the >300 Mb genome of
H. contortus will occur many times within the billions of progeny seeded
on to a typical pasture grazed by small ruminants every few days. This pro-
cess results in both a large amount of standing genetic variation and a con-
stant supply of new mutations on which selection can act. It also provides the
parasite with an extraordinary capacity to respond not only to drug selection,
but also to other changes, such as climate, geographical location and host
species.
The consequence of this high genetic diversity of H. contortus for anthel-
mintic resistance is best illustrated by studies of the population genetics of
benzimidazole resistance. There are three mutations that occur in the H. con-
tortus isotype-1 b-tubulin gene: F200Y (TAC), E198A (GCA) and F167Y
(TAC) (Ghisi et al., 2007; Prichard, 2001; Silvestre and Cabaret, 2002).
The F200Y (TAC) mutation is commonest and present in most geographic
locations studied to date, the F167Y (TAC) is less common but can be at
high frequency in some regions and the E198A (GCA) is the rarest based
on current studies (chapter: Anthelmintic Resistance in Haemonchus contortus:
History, Mechanisms and Diagnosis by Kotze and Prichard, 2016 e in this
volume). For the common F200Y (TAC) mutation, several studies

(Chaudhry et al., 2015b; Redman et al., 2015; Silvestre and Humbert,
2002; Silvestre et al., 2009) have reported a high level of haplotype diversity
for resistance alleles within H. contortus populations. Phylogenetic network
analysis of these haplotypes suggests that the F200Y (TAC) mutation has
originated multiple independent times within a region and is derived from
both recurrent mutation and from the standing genetic variation (Chaudhry
et al., 2015b; Redman et al., 2015; Silvestre et al., 2009). The same results
have been found even for H. contortus populations that have been closed to
animal movement for many years and, hence, in the absence of contempo-
rary gene flow (Silvestre et al., 2009). This hypothesis, and the evidence for
it, is discussed in more detail in Redman et al. (2015). This repeated appear-
ance of an anthelmintic drug resistance mutation is a direct consequence of
the high genetic diversity of H. contortus. This information provides a persua-
sive argument that the emergence of anthelmintic resistance is inevitable
when intensive drug selection is applied to a parasite that has such high levels
of genetic diversity.
4.2 Consequence of low regional population structure

within a country
As discussed in Section 3.5, the low levels of population structure of H. con-
tortus within a region, at least in part, reflects high levels gene flow between
populations. This observation is consistent with the high levels of anthropo-
genic host movement for sheep for most farms studied. If gene flow is high,
then even rare resistance mutations have the potential to spread widely in
regions under the influence of drug selection. This observation has been
made recently in a study (Chaudhry et al., 2015b) showing that the
E198A (GCA) mutation is relatively widespread in southern India, in spite
of being rare or absent from most countries studied to date (Barrere et al.,
2013; Prichard, 2001; Redman et al., 2015; Silvestre and Humbert,
2002). Phylogenetic analysis of the resistant and susceptible isotype-1 b-
tubulin haplotypes showed that this E198A (GCA) mutation was repre-
sented by a single haplotype in the region, despite high levels of susceptible
haplotype diversity. This finding strongly suggests that this mutation has
arisen once and has subsequently spread throughout populations of
H. contortus in this region of India (Chaudhry et al., 2015b). The spread of
a relatively rare mutation, such as E198A (GCA), can be clearly demon-
strated by phylogenetic analysis of resistant and susceptible haplotypes.
However, it is more difficult to demonstrate for a common mutation
with multiple origins, such as the F200Y (TAC), because of the diversity of
resistance haplotypes. Further, if resistance is well established, the lack of sus-
ceptible haplotypes makes the interpretation of the phylogenetic analysis
difficult. Nevertheless the population genetic data, overall, is also consistent
with the spread of the F200Y (TAC) between farms in the UK (Redman
et al., 2015). This conclusion emphasizes the role of animal movement in
spreading anthelmintic resistance, and the need for stringent biosecurity
and quarantine dosing procedures in minimizing the spread of resistance be-
tween farms.
4.3 Consequence of substantial global population structure

The high level of population genetic structure of H. contortus among
different countries has a number of consequences for anthelmintic resistance.
It suggests that there is more limited gene flow between parasite populations
in different countries. Consequently, the spread of resistance mutations be-
tween countries is likely to be much less than that which occurs at the
regional level. In addition, the genetic background on which selection
acts is different among countries, such that one might expect different mu-
tations to be important in different regions. In the case of benzimidazole
resistance, we know this to be true; although the F200Y (TAC) mutation
appears to occur in all countries examined to date, the rarer E198A
(GCA) and F167Y (TAC) mutations differ markedly among regions. For
example, in the UK, the F167Y (TAC) mutation is almost as frequent as
F200Y (TAC), but has not yet been detected in southern India (Redman
et al., 2015). Conversely, the E198A (GCA) mutation is widespread in
southern India, but the F167Y (TAC) has not yet been found (Chaudhry
et al., 2015b). This information has important implications for the use of
molecular diagnostic tools and the surveillance of resistance. It also empha-
sizes the importance of biosecurity measures for imported livestock, such as
anthelmintic dosing in quarantine to avoid the importation of new resistance
mutations to a particular geographical region.
The global population structure of H. contortus also has a number of con-
sequences for anthelmintic resistance research. One cannot assume that a
mutation implicated as an important cause of resistance in one region is
necessarily important in another region or is of general global importance.
Specific regional studies will always be needed. In addition, care must be
taken when comparing the diversity or association of candidate gene haplo-
types between resistant and susceptible parasite isolates from different
geographical regions. It is likely that differences will be found between
such populations, even at neutral genetic loci, and, therefore, it is critical to

take into account the genetic background of parasite populations in such
studies. This will also be important for future genome-wide population
genomic and association studies.
4.4 Consequence of low population structure between hosts

The low population structure found between H. contortus populations in
different host species in the same region suggests that the parasite is freely
shared with little or no host species barrier. Hence, it is likely that the
same resistance mutations will be found in the different host species within
the same geographical region. There have been few studies directly testing
this aspect, but it is supported by the observation that the same benzimid-
azole resistance mutations were found in H. contortus from sheep and goats,
both southern India and Pakistan (Chaudhry et al., 2015b) (U. Chaudhry,
E. Redman, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf and J. Gilleard,
unpublished data).
5. GENETIC AND PHENOTYPIC VARIATION IN

LABORATORY STRAINS
Understanding and monitoring the genetic and phenotypic variation
between laboratory strains is an important and neglected aspect of
H. contortus research. The substantial levels of genetic diversity present
within H. contortus field populations will be inevitably reflected in laboratory
strains, since they are derived from field populations. The mode of obligate
sexual reproduction of this parasite means that clonal lines cannot be estab-
lished. Instead, laboratory strains are typically maintained as populations of
large numbers of interbreeding parasites, which are serially passaged through
experimentally infected hosts. Consequently, there is often considerable ge-
netic and phenotypic variation both within and among laboratory strains,
and there is potential for this variability to change over time.
There is no generally accepted definition of what constitutes an ‘isolate’
or a ‘strain’ for a sexually reproducing organism, such as H. contortus. In this
chapter, we use the term ‘isolate’ for a population of parasites recovered
directly from the field and the term ‘strain’ for an isolate that has been sub-
sequently serially passaged by experimental infection, and then studied and
archived in the laboratory. In the case of H. contortus, isolates are generally
recovered from an infected animal from the field by harvesting infective
third-stage larvae (L3s) from faecal cultures. Such field isolates are sometimes
contaminated with other species. Although these can be removed by trans-

plantation of morphologically identified adult worms into the abomasum of
a recipient sheep, they are often ignored if only present in trace amounts.
Harvested L3s can be stored for several months in water or exsheathed
L3s can be cryopreserved in liquid nitrogen where they remain viable and
infective for many years (Van Wyk et al., 1977). Laboratory strains are usu-
ally passaged every few months by oral or rumenal infection of sheep or
goats, typically using between 2000 and 5000 L3s (Wood et al., 1995).
Faeces from such animals are then cultured to obtain the next generation
of infective L3s. There are a number of aspects of these processes that
may have important impacts on experimental work.
First, strain contamination can occur in a variety of ways, including
donor animals that are not parasite-free, by contamination of feed or
bedding with infective L3s or by human error during strain handling or
archiving. If contamination occurs with a different nematode species, it
should be readily detectable. However, if contamination occurs with a
different H. contortus population, then there is a significant risk that it will
go undetected, and could lead to erroneous experimental results. Second,
there is an ongoing risk of population bottlenecks due to variability in infec-
tive dose or rates of establishment in the host animal. For example, larvae
that have been stored incorrectly, or for too long a period, can lose infec-
tivity, leading to experimental infections with low parasite numbers. This
reduction in population size could result in a loss of overall genetic diversity
or to genetic drift of the population. Third, the number of passages of a strain
is not always recorded and captured in the parasite strain nomenclature and,
hence, experiments performed on the same strain over time may not be
equivalent. Fourth, strains are often exchanged between laboratories
without any monitoring of genetic integrity, and so contamination events
or errors may only be detected if clear differences exist in phenotype or if
specific molecular markers are used. Finally, there is no standardized nomen-
clature system as there is for model organisms, such as C. elegans and
D. melanogaster (Attrill et al., 2015; Harris et al., 2004). As a result, strains,
such as the ‘ISE’ or ‘McMaster’, which are commonly used in experimental
studies around the world, may differ genetically and phenotypically among
laboratories. We use a nomenclature system for the reference strains that are
maintained at the Moredun Research Institute, Scotland, to help minimize
some of these problems (Gilleard, 2013). For example, the version of the
CAVR strain that is passaged at this institute is named MHco10(CAVR),
to distinguish it from other versions of this isolate passaged in other
laboratories (Redman et al., 2008b). There have been relatively few publi-
cations specifically addressing genetic and phenotypic variation of H. contor-
tus laboratory strains. However, some information is available in a variety of
papers that are reviewed here.
5.1 Genetic variation within and between laboratory strains

In spite of some of the limitations discussed in Section 5, H. contortus is still
one of the best-characterized parasitic nematode species, in terms of genetic
variation within and between laboratory strains. As for natural field popula-
tions, there is a substantial amount of genetic variation within H. contortus
laboratory strains. The earliest work examining genetic diversity of
H. contortus laboratory strains focused on sequence polymorphisms in candi-
date anthelmintic resistance genes (Beech et al., 1994; Blackhall et al.,
1998a,b; Kwa et al., 1993; Prichard, 2001; Sangster et al., 1999). Typically,
these studies compared the frequency of particular haplotypes of candidate
genes between resistant and susceptible strains or between populations of
the same strain before and after drug selection. In all cases, high levels of hap-
lotypic diversity have been reported both within and between strains. A
number of these studies have reported increased frequencies of particular
haplotypes in resistant relative to susceptible strains or following drug selec-
tion (chapter: Anthelmintic Resistance in Haemonchus contortus: History,
Mechanisms and Diagnosis by Kotze and Prichard, 2016 e in this volume).
However, to interpret such studies, it is important to consider differences
and changes that occur throughout the genome as well as at the locus or
loci under investigation. A variety of marker systems have been developed
for H. contortus that can be used for this purpose, including random amplified
polymorphic DNA, restriction fragment length polymorphism, AFLP,
transposon-associated markers, SNPs, indels and microsatellites (Hoekstra
et al., 1997, 2000a,b; Hunt et al., 2008; Otsen et al., 2000a, 2001; Redman
et al., 2008a; Roos et al., 1998).
Panels of well-characterized microsatellites are available to assess,
compare and monitor the genetic diversity within and among laboratory
isolates (Hoekstra et al., 1997; Otsen et al., 2000b; Redman et al., 2008b,
2015). For instance, Redman et al. (2008a,b) used a panel of eight microsat-
ellite markers to characterize five laboratory isolates that had been passaged
by serial experimental infection for many years, following their original field
isolation from different countries: MHco1(MOSI) and MHoc3(ISE) of
unknown field origin (possibly UK); MHoc4(WRS) from South Africa;
MHco10(CAVR) from Australia and HcSwe(VAST) from Sweden.
All pairwise Fst values were very high (0.1385e0.333), except for MHco1(-
MOSI) and MHoc3(ISE) (0.1008), which was lower. This observation is
consistent with our understanding of global population structure of the para-
site in the field (Troell et al., 2006a), as discussed in Section 3.4. MHo-
c3(ISE) is derived from the MHco1(MOSI) strain, and so the closer
relationship of these two strains is consistent with their known history
(Roos et al., 2004). Amplification of the five microsatellite markers from
pools of worms generates repeatable genetic ‘fingerprints’ for individual
strains, and provides a convenient and rapid system with which to monitor
strain integrity during passage and exchange between laboratories (Redman
et al., 2008b). Hunt et al. (2008) used a number of different microsatellite
markers to characterize six commonly used laboratory strains (called
McMaster1931, Wallangra2003, Gold Coast2004, Arding2005 and Canna-
wigara2005), originally isolated from the field in south eastern Australia.
Depending on the markers used, pairwise Fst values varied from
0.00007 to 0.04532 (Hunt et al., 2008). Although these values are lower
than those reported by Redman et al. (2008a,b), many pairwise comparisons
were statistically significant. This information demonstrates that there can be
significant genetic differentiation between laboratory strains, even when iso-
lated from different regions of the same country (Hunt et al., 2008). These
results suggest that a laboratory strain is likely to be more representative of
field populations located in the same region from it was originally isolated.
This hypothesis has not been rigorously tested, but is supported by compar-
ison of a Swedish laboratory isolate with global field populations (Troell
et al., 2006a). Also our recent results show that field populations of
H. contortus, isolated from the south-east United States, are genetically closer
to the UGA2004 laboratory strain than to the MHoc3(ISE), MHoc4(WRS)
and MHco10(CAVR) laboratory strains (M. Miller, E. Redman, R. Kaplan
and J. Gilleard, unpublished data).
Although there are no published studies specifically comparing
laboratory strains and field populations, the overall evidence suggests that
laboratory strains are generally as genetically diverse as field populations. Mi-
crosatellite markers typically show similar levels of allelic richness, expected
heterozygosity and inbreeding coefficients in studies of passaged H. contortus
laboratory strains as they do for field populations (Hunt et al., 2008; Redman
et al., 2008b, 2015; Silvestre et al., 2009). As discussed in Section 4.6, from
the limited data available, it appears that anthelmintic selection does not lead
to an overall reduction of genetic diversity in H. contortus populations in the
field. Similarly from the limited analyses conducted to date, drug selection
does not seem to substantially reduce the overall genetic diversity of labora-
tory strains. To date the most direct analysis to address this question used
AFLP analysis of individual worms, to monitor changes in genetic diversity
within and between strains during consecutive stages of selection for
increased benzimidazole or levamisole resistance (Otsen et al., 2001). In
the case of benzimidazole selection, eggs from a susceptible laboratory strain
were incubated at a drug concentration (ED80) such that w20% of the eggs
survived and were used to infect a donor sheep following culture to L3. Five
rounds of such in vitro selection resulted in a significantly increased ED50,
but no reduction in the overall genetic diversity was detected by AFLP anal-
ysis. In the same study, six rounds of levamisole selection were applied to a
susceptible laboratory strain by in vivo drug treatments of experimentally
infected animals. Although a small reduction in overall diversity was
detected by AFLP analysis after the first round of selection, there was no
further loss of diversity detected even by the sixth generation (Otsen
et al., 2001). In addition, as discussed in Section 3.2, genome-wide SNP
analysis has revealed that several anthelmintic laboratory strains, namely
Hco4(WRS), Hco10(CAVR), MHco18(UGA2004) and MHco16, retain
very high levels of sequence polymorphism across the genome.
Although microsatellite markers have been useful for the genetic charac-
terization of H. contortus strains, more extensive genome-wide marker ana-
lyses using various methods, such as SNParrays, restriction site-associated
DNA markers and whole genome sequencing, should provide much greater
resolution in the future (Davey et al., 2011; Salgotra et al., 2014). Recent
progress in the assembly of the H. contortus reference genome (Laing et al.,
2013; Schwarz et al., 2013), together with the rapidly diminishing costs of
next-generation sequencing, is now making such approaches increasingly
feasible.
5.2 Phenotypic variation within and between laboratory

strains
Although much of the genetic variation within and between H. contortus lab-
oratory strains consists of sequence polymorphisms in noncoding regions,
there is also a substantial number of nonsynonymous mutations in coding
regions. For example, in an analysis of 927 gene models, in a 11.2-Mb region
of the H. contortus draft genome sequence, nonsynonymous SNPs resulted in
2104 and 1666 amino acid substitution mutations in the MHco10(CAVR)
and MHco4(WRS) strains compared with the MHco3(ISE) reference
genome, respectively (A. Martinelli, A. Rezansoff, J. Cotton and J. Gilleard,
unpublished data). Hence, there is clear potential for phenotypic variation

between laboratory strains. Here, we review the information currently avail-
able on phenotypic variation among laboratory strains.
5.2.1 Variation in gene expression and function

Most of the work investigating differences in gene expression and function
between H. contortus laboratory isolates has been aimed at understanding the
mechanisms of anthelmintic resistance (chapter: Anthelmintic Resistance in
Haemonchus contortus: History, Mechanisms and Diagnosis by Kotze and
Prichard, 2016 e in this volume). Anthelmintic resistance research has
provided a number of examples of genes that are differentially expressed
or transcribed between different H. contortus isolates and strains. For
example, differences in gene expression between levamisole resistant and
susceptible H. contortus populations has been described for the nicotinic
acetylcholine receptors, Hco-unc-29.3 and Hco-unc-63 and acr-8 as well as
ancillary proteins Hco-unc-74, -unc-50, -ric-3.1 and -ric-3 (Sarai et al., 2013;
Williamson et al., 2011). Similarly, expression differences have been
described between ivermectin resistant and susceptible populations for dyf-
7 e a gene that encodes a protein involved in amphid sensory neuron devel-
opment e and several members of the P-glycoprotein efflux pump family
(Urdaneta-Marquez et al., 2014; Williamson et al., 2011; Xu et al., 1998).
This topic is not discussed in detail here, as it is reviewed by Kotze and
Prichard (2016) (chapter: Anthelmintic Resistance in Haemonchus contortus:
History, Mechanisms and Diagnosis e in this volume) as well as in other
articles (Gilleard, 2006; Gilleard and Beech, 2007; Kotze et al., 2014;
Prichard, 2001).
Other than anthelmintic resistance research, there has been relatively
little work investigating differences in gene expression between H. contortus
laboratory strains. One study compared the soluble proteome of adult female
worms of the MHco3(ISE) and MHco10(CAVR) strains (Hart et al., 2012).
The data from three replicate two-dimensional (2-D) gels for each strain
identified 23 protein spots appearing to differ in abundance between the
two strains. Four of these had a greater than twofold difference and were sta-
tistically significant; a cysteine protease, a glutathione-S-transferase, an actin
and a heat shock protein 60. This paper also reported some differences in the
antigens detected by immune sera taken from experimentally infected sheep
(Hart et al., 2012). One caveat to these experiments is that the data appear to
be derived from a single aqueous worm homogenate extract for each strain
examined on 2-D gels (run three times) rather than from three independent
bio-replicates for each strain. We have compared the transcriptomes of three

independent bio-replicates for each the MHco3(ISE), MHco4(WRS) and
the MHco10(CAVR) laboratory strains using DESeq2 analysis of RNAseq
data (Love et al., 2014). A total of 1239, 1803 and 718 transcripts were
greater than twofold differentially expressed (significance: p ¼ 0.01) be-
tween MHco3(ISE)/MHco4(WRS), MHco3(ISE)/MHco10(CAVR) and
MHco4(WRS/MHco10(CAVR), respectively (A. Martinelli, A. Rezansoff,
J. Cotton and J. Gilleard, unpublished data).
Other than anthelmintic resistance candidates, the molecules most thor-
oughly investigated for strain differences in expression and function are the
secreted and intestinal microvilli proteases. This is primarily because of the
interest in these molecules as vaccine candidates and concerns about poten-
tial antigenic variation among different geographical regions. Initial evidence
of geographical variation came from differences in the protease profiles of
excretoryesecretory (ES) products between strains derived from the United
States and the UK (Karanu et al., 1993). Subsequent analysis of the
ES proteases from one US and two Kenyan strains revealed differences in
the mobility of the major enzyme species detected on substrate gels and in
the effect of inhibitors (Karanu et al., 1997). The cysteine protease inhibitors
E64 and iodoacetic acid abolished substrate gel protease activity of ES
products from the US strain but had little effect on either of the Kenyan
strains. Conversely, protease activity from the Kenya strains, but not the
US strain, was completely inhibited by the metallo- and serine protease
inhibitors ethylenediaminetetraacetic acid, 1,10-phenanthroline and phe-
nylmethylsulfonyl fluoride (PMSF). Redmond and Wyndham (2005) char-
acterized the protease activity profiles of integral membrane protein extracts
isolated from the gut of three different H. contortus strains (MOSI, ISE and
WRS) (Redmond and Windham, 2005). At pH 5, there were three clear
zones of proteolysis on gelatin substrate gels in all three strains, with only mi-
nor differences in mobility. However, at pH 7 and pH 9, although there was
a high level of activity at >200 kDa in MOSI and WRS, it was completely
absent from ISE. Hemoglobinase activity was detected in the MOSI and
WRS strains but not in the ISE strain, and fibrinogen b-degradation was
observed at much higher levels for the MOSI than for the WRS and ISE
strains. Overall the results suggested a more limited enzymatic profile for
the ISE strain, that the authors speculated might be related to its inbred
nature (Redmond and Windham, 2005).
The genetic basis for differences in protease activity is presently unknown.
The proteases and amino-peptidase gene families are much larger in
H. contortus than in most other organisms including other nematode species

(Jasmer et al., 2004; Laing et al., 2013). For example, the cathepsin D aspartic
protease and cathepsin B cysteine protease gene families are predicted to
consist of 83 genes and 63 genes, respectively (Laing et al., 2013). These
numbers appear to relate to a recent evolutionary expansion, since the gene
families predominantly consist of large monophyletic clades with many of
the genes organized in large tandem arrays in the genome (Laing et al.,
2013). Differences in these gene families, either in copy number, sequence
polymorphism or expression have not yet been examined in detail. Indeed,
analyses are a difficult proposition for such large and complex gene families
encoded in a draft reference genome. Some differences in expression are
suggested by the observation that only 60% of the 194 cathepsin B-like
ESTs (50 clusters) sequenced from a UK isolate were present in a data set
of 686 ESTs (123 clusters) from a US isolate (Jasmer et al., 2004). However,
our RNAseq comparisons of these strains reveal just 5, 2 and 1 of 74
annotated cathepsin B genes that are greater than fourfold differentially
expressed (significance: p ¼ 0.01) between MHco3(ISE)/MHco10(CAVR),
MHco3(ISE)/MHco4(WRS), and MHco4(WRS)/MHco10(CAVR),
respectively (A. Martinelli, A. Rezansoff, J. Cotton and J. Gilleard, unpub-
lished data). In summary, we still have a poor understanding of differences
in gene expression and transcription between H. contortus laboratory strains,
but recent progress in the H. contortus reference genome assembly should
make systematic analyses more feasible.
5.2.2 Variation in morphological traits

The simple body plan of nematodes limits the amount of visible morpholog-
ical variation apparent within and among species. However, detailed
morphological and morphometric analyses reveal that significant variation
occurs. For example, 25 distinct morphological characters were used to
study the phylogenetic relationships between 12 different species of Haemon-
chus (Hoberg et al., 2004). Although there is within-species variation for
some of these traits, care must be taken when interpreting some of the earlier
studies due to the lack of a definitive specific identification. For example,
Gibbons (1979) considered H. contortus and H. placei to be the same species,
and a number of studies before that time classified both these species as
H. contortus (Gibbons, 1979). Nevertheless subsequent studies have shown
significant variation for a number of morphological characters within and
among H. contortus isolates. The traits most commonly examined are the
series of ridges on the anterior region of the cuticle called the synlophe,
the male bursa and the female vulva. Although these traits are generally used
to distinguish between different Haemonchus species, there is significant
within-species variation. For example, the number of ridges comprising
the synlophe varies between 22 and 30 among H. contortus individuals,
and there is significant variance in the morphometrics of the spicules among
individual H. contortus worms (Jacquiet et al., 1997; Lichtenfels et al., 1994).
One clear example of variance of morphometric traits in different geograph-
ical isolates of H. contortus comes from a study in Yemen and Malaysia (Ghar-
amah et al., 2014). In that case, the majority of 200 male H. contortus worms
taken from sheep and goats were separated into two distinctive groups by
PCoV analysis using morphometric data of body length, length of cervical
papillae and spicule length. Most worms clustered by country of origin,
with only a slight overlap between countries. This differentiation was sup-
ported by molecular analysis, where mtDNA sequences also clustered by
country (Gharamah et al., 2012). Similarly we have found a number of sta-
tistically significant morphometric differences between the MHco3(ISE),
MHco4(WRS) and MHco10(CAVR) strains, including oesophagus length
and spicule length in males as well as the extent of the synlophe cuticular
ridges in females (E. Hoberg, E. Redman and J. Gilleard, unpublished data).
The clearest example of a morphological trait that varies between isolates
is vulval morphology. At least 14 morphological types have been described
for the H. contortus female vulval, which have been grouped into three major
types; smooth, knobbed and linguiform, the proportions of which differ
among different isolates (Das and Whitlock, 1960; Hunt et al., 2008; Le
Jambre, 1977). Although some of the minor variations are suggested to be
environmentally determined e since they vary with parasite population
density e there is evidence that the major morphotypes are genetically
determined (Le Jambre, 1977; Le Jambre and Ractliffe, 1976). Experiments
with US isolates have shown that several generations of selection for
offspring of one vulva type increases the frequency of that phenotype in
the population. In addition, test crosses between female worms of one vulva
type with male worms derived from an isolate with a different predominant
vulva type have suggested a genetic basis and an order of dominance of lin-
guiform over knobbed over smooth morphotypes (Le Jambre, 1977).
Similar genetic crosses using Bulgarian isolates also supported a genetic basis,
but suggested a different dominance hierarchy, with the linguiform type be-
ing recessive to both knobbed and smooth morphotypes (Daskalov, 1975).
These apparent geographical differences in the respective dominance of
these traits were suggested to be due to differences in the genetic
backgrounds between different regions (Le Jambre, 1977). In conclusion,

there are clear differences in morphology and morphometrics between H.
contortus isolates and strains derived from different geographical regions,
consistent with the pronounced global population genetic structure.
5.2.3 Variation in life history traits and pathogenicity

There have been relatively few detailed studies investigating differences in
life history traits or pathogenicity of H. contortus isolates. Aumont et al.
(2003) investigated whether there was evidence of H. contortus being better
adapted to host breeds derived from the same geographical region. These
authors compared H. contortus populations from two different geographical
regions, one isolate from France and another derived by pooling five
different isolates from Guadeloupe. Groups of 10 lambs of two different
sheep breeds were infected with each parasite isolate: the ‘Martinik’ Black
Belly breed, derived from the Barbados (BB) and the INRA 401 breed
from France (Aumont et al., 2003). In both breeds, the resultant faecal
egg counts (FECs) were significantly higher in lambs infected with the
H. contortus population from Guadaloupe than those infected with the
French parasite population (p ¼ 0.008). The establishment rate was same
for both H. contortus populations in INRA 401 lambs (w50%); however,
in the more resistant BB lambs, it was higher for the ‘sympatric’ Guadaloupe
population (15.2%) than for the ‘allopatric’ French population (7.4%). There
was no significant difference in haematocrit or eosinophil count between the
two H. contortus populations in either breed, indicating that the two strains
had a similar pathogenicity. In another study, Troell et al. (2006b)
investigated potential differences between isolates adapted to temperate
and tropical climates. Groups of six sheep were infected with either a Swed-
ish or a Kenyan isolate using larvae freshly developed from eggs or following
storage at 5 C for 9 months. For the fresh larvae only, there were significant
differences in both the prepatent period (p ¼ 0.025) and the proportion of
larvae undergoing hypobiosis; 70% of the Swedish larvae underwent
inhibition compared with 36% for Kenyan isolate (p ¼ 0.0104). This result
is consistent with the high propensity of H. contortus to arrest development
inside the host in Sweden and the suggestion that this arrest may be the
parasite’s ‘genetic default’ in that region as an adaptation to survive cold
winters (Waller et al., 2004). No other traits, including worm length,
establishment rate, sex ratio or any of the haematological parameters reflec-
tive of pathogenicity, were significantly different between the two isolates
(Troell et al., 2006b).
A number of studies have suggested differences in pathogenicity occur

between different H. contortus isolates. For example, Poeschel and Todd
(1972a,b) undertook a series of experimental infections using 18 different
H. contortus isolates obtained from different regions of the United States.
These authors reported that three isolates had statistically significant reduced
pathogenicity, and two isolates had increased pathogenicity with respect to a
control isolate (based on a reduction in blood haemoglobin concentrations,
corrected for adult worm number). Although these experiments were repli-
cated several times for the main isolates of interest, the host group size was
small (three animals per group), and minimal detail of statistical analyses was
reported. More recently, Hunt et al. (2008) compared five laboratory strains
(McMaster1931, Wallangra2003, Gold Coast2004, Arding2005 and Canna-
wigara2005) that were originally derived from south eastern Australia and
showed significant genetic differentiation based on microsatellite markers
(Hunt et al., 2008). An experiment, in which 10 sheep were infected
with each strain, suggested a number of differences in life history traits
and pathogenicity. There were significant differences, at least between
some of the isolates, in the establishment rate, the rate of increase in FEC
and in worm fecundity (FECs divided by the number of adult worms)
(p < 0.001). In addition, significant differences in erythrocyte and neutro-
phil counts as well as wool growth between isolates were reported. ANOVA
analysis suggested that these differences were only partially due to the inten-
sity of infection, suggesting differences in pathogenicity among the isolates.
Angullo-Cubilan et al. (2010) compared experimental infection of groups of
six Spanish Manchego breed lambs with a ‘sympatric’ Spanish H. contortus
isolate, Aran 99, with infections with two ‘allopatric’ non-Spanish isolates
MRI (Moredun Research Institute, Edinburgh UK) and MSD (Merck
Sharp and Dohme) (Angulo-Cubillan et al., 2010). The prepatent period
of the Aran 99 isolate was significantly longer (mean 28.1 days) than those
of the MSD and MRI isolates (means of 21.3 and 21.7 days, respectively)
(p < 0.05). Although there were no differences in the intensity of infection
between the isolates, the MRI infected group had significantly lower packed
cell volume values than those infected with the other two isolates, again sug-
gesting differences in pathogenicity.
We have compared the genetically divergent MHco3(ISE),
MHco4(WRS) and MHco10(CAVR) laboratory strains for differences in
basic life history traits in an experiment, in which groups of 15 sheep were
infected with each isolate (Redman et al., 2012). Only a small difference in
the prepatent period was detected between the MHco3(ISE) and the other
two strains (significantly more animals positive for eggs at 18 days after infec-
tion). Day 18 was the only day on which there was a statistically significant
difference in FEC between the groups up to day 36 after infection
(D. Bartley, N. Sargison, E. Redman and J. Gilleard, unpublished data).
We have also recently investigated whether there are competitive differences
between these strains during coinfection. We coinfected sheep with 4000 L3s
of each of two different strains, to test for differences in overall fitness or
fecundity by genotyping F1 progeny with microsatellite markers to determine
their parental strain identity. Two sheep were coinfected with strains
MHco3(ISE) and MHco4(WRS) and two sheep with strains MHco3(ISE)
and MHco10(CAVR). In both cases, MHco3 (ISE) homozygous progeny
were significantly overrepresented compared with progeny homozygous or
heterozygous for the second strain, suggesting a competitive advantage to
the MHco3(ISE) strain during experimental coinfection (N. Sargison, E.
Redman, D. Bartley and J. Gilleard, unpublished data).
In summary, a number of studies have suggested phenotypic differences
in life history traits (including establishment rate, prepatent period and worm
fecundity) between different field isolates and laboratory strains. Several
studies (Angulo-Cubillan et al., 2010; Hunt et al., 2008) have also suggested
that observed differences in the extent of anaemia induced by different
strains was not completely accounted for by differences in infection inten-
sity. However, other studies (Aumont et al., 2003; Newton et al., 1995)
have found no difference in pathogenicity between isolates. Hence, only
a very limited number of studies to date have suggested phenotypic differ-
ences in life history traits between isolates and strains of H. contortus.
6. CONCLUDING REMARKS
Genetic diversity and population structure are poorly understood for
most parasitic nematode species. However, a substantial amount of research
has been undertaken of H. contortus, and this parasite serves as a useful model
for the trichostrongyloid nematode group. Studies have consistently shown
that H. contortus field isolates have remarkably high levels of genetic diversity,
which is predominantly due to extremely large population sizes. There is also
usually substantial anthropogenic gene flow among populations within a
geographical region. Although most of the genetic diversity occurs within
populations, there is low, but discernable population structure within a
region and substantial genetic differentiation among populations from different
countries. This population genetic structure underlies the propensity of the

parasite to develop resistance to anthelmintic drugs and predisposes to the
repeated emergence and potentially rapid spread of drug resistance mutations.
There is also substantial genetic diversity within and among H. contortus
laboratory strains. It is important that this diversity is considered during
experimental studies, particularly when investigating apparent associations
between candidate genes and drug resistance or other phenotypes. There
has been much less research on phenotypic variation between field isolates
and laboratory strains, other than for drug resistance and certain morpholog-
ical traits. There is some suggestion in the literature of potential variation in
life history traits and pathogenicity, but these aspects are still poorly defined
and more research is needed.
Most of the information on genetic diversity and population structure of
H. contortus to date is based on the study of specific genes or the application
of panels of relatively low-coverage, neutral genetic markers. Whilst such
studies have been very informative, these marker systems are of limited
resolution. However, in recent years, there have been substantial improve-
ments in H. contortus genomic resources, together with major advances in
sequencing technologies. Consequently larger scale genome-wide
approaches, using much larger panels of genetic markers, are becoming
both practically and economically feasible. This context should not only
enable a more detailed view of genetic variation and population structure
of the parasite, but also allow the application of more powerful population
genomic approaches to identify drug resistance loci and to study the emer-
gence and spread of drug resistance.
ACKNOWLEDGEMENTS
We are grateful to Charles Criscione, James Cotton, Axel Martinelli, Andrew Kotze, Ray
Kaplan, Melissa Miller and Andrew Rezansoff for discussion and for sharing unpublished
data and information. We are also grateful to Robin Gasser for his valuable comments.
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CHAPTER THREE
The Biochemistry of Haemonchus

contortus and Other Parasitic
Nematodes
A. Hardera
WE Biology, Heinrich-Heine-University D€
usseldorf, D€
usseldorf, Germany
E-mail: achim_harder@hotmail.de
Contents
1. Introduction 70
2. Ecosystems of Haemonchus contortus Life Cycle Stages 72
3. Gene Expression in Parasitic Life Cycle Stages 73
4. Energy Metabolism in Nematodes 74
4.1 Energy metabolism in larval nematodes 74
4.2 Energy metabolism in adult nematodes 75
4.3 Anthelmintic drugs targeting energy and/or carbohydrate metabolism 77
5. Amino Acid Metabolism 78
5.1 Polyamines, nitrogen excretion in parasites 78
6. Nucleic Acid Metabolism 79
6.1 Purine metabolism 79
7. Lipid Metabolsim 79
8. Structure and Biochemical Composition of the Cuticle 80
9. Tubulin as a Major Structural Component and Drug Target 81
10. Nervous System in Nematodes 83
10.1 Nicotinic AChRs in Haemonchus contortus 83
10.2 Inhibitory neurotransmitters in nematodes 85
10.2.1 g-Aminobutyric acid-A receptors 85
10.2.2 Glutamate-gated chloride channels and macrocyclic lactones 85
10.2.3 Calcium-activated voltage-gated potassium channel SLO-1 87
11. Biochemistry of Drug Resistance 87
11.1 Specific resistance mechanisms 88
11.1.1 Benzimidazole resistance 88
11.1.2 Levamisole resistance 89
11.2 Nonspecific resistance mechanisms e drug metabolism and efflux 89
12. Conclusions 90
Acknowledgement 91
References 91
a
Dedication: Prof. Dr. Hildegard Debuch (1919e1993), former Professor of Physiological Chemistry
at the University of Cologne.
j
ISSN 0065-308X
70 A. Harder
Abstract
Different life cycle stages of Haemonchus contortus adapt to different ecosystems. This
adaptation is accompanied by alterations in gene transcription and expression associ-
ated with the energy, amino acid, nitrogen, lipid and/or nucleic acid metabolism of the
respective stages. For example, the aerobic metabolism of larvae depends on an effi-
cient citric acid cycle, whereas the anaerobic metabolism of adults requires glycolysis,
resulting in the production of volatile fatty acids, such as acetic acid and propionic acid.
There are only few anthelmintics targeting nematode energy metabolism. In addition,
H. contortus has reduced pathways for amino acid metabolism, polyamine metabolism
and nitrogen excretion pathways. Moreover, nucleic acid metabolism comprising pu-
rine and pyrimidine salvage pathways as well as lipid metabolism are reduced. In addi-
tion, nematodes possess a particular composition of their cuticle. Energy production of
adult worms is mainly linked to egg production and complex regulation of the neuro-
muscular system in both females and males. In this context, microtubules consisting of
a- and b-tubulin heterodimers play a crucial role in the presynaptic vesicle transport.
Due to the significant distinction of its quarternary structure in nematodes in compar-
ison to other organisms, b-tubulin was identified as a major target for benzimidazoles
used for anthelmintic treatment. Concerning the function of the neuromuscular sys-
tem, acetylcholine, a ligand of the nicotinic acetylcholine receptor (nAChR), is the major
excitatory neurotransmitter in H. contortus. In contrast, glutamate-gated chloride chan-
nels, calcium- and voltage-dependent potassium channels as well as g-aminobutyric
acid (GABA)A and its receptors act as inhibitory neurotransmitters and thus opponents
to nAChR. For example, the calcium- and voltage-dependent potassium channel SLO-1
is an important target of emodepside, which is involved in the sensitive regulation of
activatory and inhibitory receptors of the nervous system. Most of the modern anthel-
mintics target these different neuromuscular receptors. The mechanisms of resistance
to anthelmintics, either specific or non-specific, are associated with changes in the
molecular targets of the drugs, changes in metabolism of the drug (inactivation,
removal or prevention of its activation) and/or increased efflux systems. The biochem-
ical and molecular analyses of key developmental, metabolic and structural process of
H. contortus still require substantial efforts. The nAChR, glutamate-gated chloride chan-
nel and calcium- and voltage-dependent potassium channel SLO-1 have long been
known as being essential for nematode survival. Therefore, future research should be
intensified to fully resolve the three-dimensional structures of these receptors, as has
already been started for glutamate-gated chloride channel. With this knowledge, it
should be possible to design new anthelmintics, which possess improved binding
capacities to corresponding receptors.
1. INTRODUCTION
Haemonchus contortus establishes and lives in different ecosystems. The
development, migration and establishment of this parasite are accompanied
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 71
by adaptions to diverse macro- and micro-environments. These adaptions

are characterized by differential gene transcription and expression patterns
that have significant influences on the energy, amino acid, nitrogen, lipid
and nucleic acid metabolism of the respective stages. As an example, the
aerobic metabolism of larvae depends on an efficient tricarboxylic acid
(TCA) cycle, whereas the anaerobic metabolism of adults is reliant predom-
inantly on glycolysis (Kapur and Sood, 1987). As a common energy-saving
feature for parasites, H. contortus has considerably reduced pathways for
amino acid, nucleic acid as well as lipid metabolism, thus being reliant on
source materials from their hosts (K€ohler, 2006).
The energy acquired via anaerobic carbohydrate degradation in adult
worms is mainly used for egg production in female worms and the complex
regulation of the neuromuscular system in both females and males (K€ ohler,
2006). A well-functioning neuromuscular system relies on the controlled
and accurate presynaptic release of neurotransmitters via vesicles and their
interaction with postsynaptic receptors (Dalliere et al., 2015).
Microtubules play an important role in axonal vesicle transport. They
consist of a- and b-tubulin dimers. Due to the significant distinction of its
quarternary structure in nematodes, in comparison with other organisms,
b-tubulin was identified as a major target for anthelmintic therapy (Harder,
2002). In addition, the chemical signal represented by the major excitatory
neurotransmitter acetylcholine is translated into an electrical signal via its
postsynaptic receptors, the nAChRs. Glutamate-gated chloride channels,
calcium- and voltage-dependent potassium channels as well as g-aminobu-
tyric acid (GABA)A and its receptors act as opponents via their inhibitory
effects on the neuromuscular signal transmission (Dalliere et al., 2015). In
this context, the SLO-1 receptor, a voltage-gated and calcium-dependent
potassium channel, a target of emodepside, is involved in the regulation
of activatory and inhibitory receptors of the nervous system (Dalliere
et al., 2015). The composition of this receptor is highly complex and specific
to nematodes, such as H. contortus, and its specificity makes it an attractive
drug target for a number of anthelmintics.
As there are serious resistance problems in parasitic nematode popula-
tions against many modern anthelmintics, it is of great importance to
know and understand the biochemical mechanisms of resistance to these
anthelmintics to prevent resistance. The resistance mechanisms are multifac-
torial and relate to a number of alterations, including (1) changes in the
molecular target of a drug, resulting in a loss of interaction with the drug
72 A. Harder
target; (2) changes in metabolism that inactivate or remove a drug, or pre-

vent its activation; (3) changes in the distribution of a drug, preventing it
from reaching its target or increasing its efflux; (4) amplification of target
genes to circumvent drug action; and (5) compensation of the molecular
target via an expression of closely related proteins which are not sensitive
to the drug. The purpose of the present chapter is to review salient infor-
mation on the biochemistry of H. contortus and other parasitic nematodes
as a foundation for future anthelmintic discovery and drug resistance
research.
2. ECOSYSTEMS OF HAEMONCHUS CONTORTUS LIFE

CYCLE STAGES
The ecosystems in which H. contortus reside vary and change
considerably during the parasite’s life cycle. Free-living stages, including
eggs, first-stage larvae (L1), second-stage larvae (L2), third-stage larvae
(L3) and fourth-stage larvae (L4), are confronted with very different physi-
cochemical, environmental conditions, such as pO2, pCO2, pH, osmotic
pressure, redox potential and temperature (K€ ohler, 2006).
Adult nematodes usually have access to abundant water and food
resources in the mammalian host, but O2 is present in very variable
amounts, ranging from almost anoxic conditions (eg, mammalian gut e
distal ileum, colon, bile duct and rumen contents) to conditions with
enriched oxygen (duodenal fluid), which are comparable with those in
venous blood. On the other hand, the CO2 tension is high in the gut, which
greatly influences metabolic pathways in adult parasites, and also the hatch-
ing of nematode larvae (K€ ohler, 2006). In addition, high pH values (of up to
9) may be present in the rumen, through which larval stages of H. contortus
have to pass on their way to the abomasum and/or intestine. In the
abomasum, there are considerable variations in pH (between 1 and 6),
and adult H. contortus resides under these harsh conditions. Moreover, early
(free-living) life cycle stages of nematodes are often faced with major tem-
perature changes in the environment. Eggs and larvae of cattle and sheep
trichostrongyloids spend the winter outside of their host animals, whereas
adult stages live inside their warm-blooded hosts. A particular feature of
H. contortus is that the L4 and adult stages feed on blood from the abomasal
mucosa and, thus, have access to abundant food and oxygen sources
(K€ohler, 2006).
3. GENE EXPRESSION IN PARASITIC LIFE CYCLE

STAGES
Distinct life cycle stages of H. contortus are adapted to their environ-
ments, which is reflected in differences in gene transcription among stages
(Laing et al., 2013; Schwarz et al., 2013; see also chapter: Haemonchus contor-
tus: Genome Structure, Organization and Comparative Genomics and
Understanding Haemonchus contortus Better Through Genomics by Laing et
al., 2016; chapter: Ranscriptomics” by Gasser et al., 2016 e this issue).
Therefore, significant variation in transcription patterns for the genes occur
among eggs, L1, L2, L3, L4 larval stages and adult nematodes (Laing et al.,
2013). Overall, variations in transcriptional patterns represent genes in L3
larvae and genes in adult males (Laing et al., 2013; Schwarz et al., 2013).
Haemonchus eggs do not take up any external food and, therefore, rely on
endogenous energy stores. These have already been obtained from the pre-
ceding parasitic stages in their host. In the eggs, the transcription of genes
associated with oxidoreductase activity, apoptosis, body morphogenesis
and development, larval and embryo development, DNA replication and/
or chromosome organization are upregulated (cf Laing et al., 2013; Schwarz
et al., 2013). During the transition from egg to the L1 stage, other genes
associated with muscle development and motor activity are upregulated
(Laing et al., 2013; Schwarz et al., 2013); these gene alterations go in parallel
with the high motility of the actively feeding larval stage compared with
embryonated eggs (Laing et al., 2013).
During the transition from L2 to the L3 stage, a decrease in the transcrip-
tion of genes associated with the myosin complex, motor activity and
various metabolic processes can be observed (Laing et al., 2013; Schwarz
et al., 2013). In addition, genes associated with oxygen transport and haeme
binding and also oxidoreductase enzymes are upregulated (Laing et al., 2013;
Schwarz et al., 2013). This upregulation might be explained by an increased
need to detoxify endogenously accumulated metabolic compounds associ-
ated with higher cytochrome P450 (CYP450) activity in the H. contortus
L3 than in L1 or adult stages (Laing et al., 2013). CYP450 genes are also
upregulated in response to reduced food intake, and a significant increase
in gluconeogenesis from L1 to L3 takes place, together with an upregulation
of acetyl-CoA metabolic processes (Laing et al., 2013; Schwarz et al., 2013).
In addition, genes associated with the binding of cobalamin (vitamin B12)
are also upregulated. Cobalamin accumulates and is stored in the infective
74 A. Harder
L3 (Laing et al., 2013), and may be required for rapid larval development
after ingestion by the host.
The L4 is the first blood-feeding stage of H. contortus. The transition from
L3 to the L4 stage is accompanied by a significant upregulation of many
genes associated with motor activity, the myosin complex and locomotion
as well as various metabolic processes (Laing et al., 2013; Schwarz et al.,
2013). Genes for oxygen binding proteins, lipid and sugar metabolism,
possibly associated with active feeding, are also upregulated. Moreover,
there are changes in the expression of genes linked to response to oxidative
stress, reflecting the reactivation of the parasite from its dormant stage (Laing
et al., 2013; Schwarz et al., 2013). An increase in the expression of genes
associated with collagen and cuticle development and body morphogenesis
can also be observed and are (likely) linked to parasite growth (Laing et al.,
2013; Schwarz et al., 2013). The transition from the L4 to the adult stages is
also accompanied by multiple changes that are, however, different between
females and males. During the transition to the female stage, various genes
are upregulated and relate to gender-specific development and embryogen-
esis, as adult females contain oocytes and eggs at various developmental
stages (Laing et al., 2013; Schwarz et al., 2013).
Male L4 and adults of H. contortus are characterized by low transcription of
genes linked to body morphogenesis, moulting, collagen and cuticle devel-
opment, oxidoreductase activity, haeme-binding and response to oxidative
stress (Laing et al., 2013; Schwarz et al., 2013). By contrast, there is an in-
crease in transcription of a number of major sperm protein genes (Laing
et al., 2013). In the intestine of the adult stage of H. contortus, the major organ
of digestion and detoxification, there is a high level of transcription of genes
with protein kinase, cysteine-type peptidase and cysteine-type peptidase in-
hibitor activities as well as those encoding proteins involved in sugar and
cobalamin binding, the transport of cations, anions and oligopeptides or asso-
ciated with oxidoreductase activity, which accords with the transcriptional
profile for detoxification genes (Laing et al., 2013).
4. ENERGY METABOLISM IN NEMATODES

4.1 Energy metabolism in larval nematodes
The transition through the egg, L1, L3 and L4 stages of H. contortus is
accompanied by considerable alterations in transcription profiles linked to
various enzymes. From L1 to L3, the genes of most enzymes are
downregulated, including those involved in carbohydrate, lipid and energy

metabolism, but many of them are upregulated in the transition from L3 to
L4 (Laing et al., 2013). This finding can be explained by the fact that L3
development is arrested, analogous to the dauer larva of Caenorhabditis elegans
(see Crook, 2014; Laing et al., 2013). Interestingly, the L3 stage synthesizes
glycogen from lipids during ageing (Kapur and Sood, 1987) and, in general,
in larval stages, substrate degradation and energy production are usually
dependent on O2, and larvae are able to degrade carbohydrates via the
TCA-cycle to CO2 and H2O (Kapur and Sood, 1987; K€ ohler, 2006).
4.2 Energy metabolism in adult nematodes

As for many organisms, carbohydrates are the main energy source for
H. contortus (see Kapur and Sood, 1987). Haemonchus contortus feeds on
blood-containing carbohydrates (Harder and Wunderlich, 1991), but in
times of nutrient shortage, glycogen stores are degraded. The adult stage
of H. contortus is capable of synthesizing carbohydrates from acetate, fatty
acid, CO2 and glucose (Kapur and Sood, 1987). Acetate is the most, and
fatty acid is the least efficient precursor (Kapur and Sood, 1987). It is possible
that propionate, a main end product of glucose degradation in H. contortus,
is, by means of CO2 fixation, converted to succinate that is glycogenic and a
precursor to glycogenesis pathway.
As a haematophagous gastric nematode, H. contortus has ready access to
O2 sources, although the gut environment is hypoxic. The blood of mam-
mals is characterized by high physiological stability; as it is rich in O2, it has a
relatively constant pH, and contains abundant glucose, amino acids, vitamins
and other nutrients, which can easily be absorbed across the nematode
cuticle (K€ ohler, 2006).
Adults of H. contortus are not able to degrade their carbohydrates
completely to CO2 and H2O via the TCA-cycle (Harder and Wunderlich,
1991). They use the glycolytic pathway up to the step of phosphoenolpyr-
uvate (PEP). At a high CO2 tension (>600 mm Hg) in the intestine of H.
contortus, CO2 is directly bound to PEP by producing 1 mol ITP (inosine
triphosphate) (Harder and Wunderlich, 1991; K€ ohler, 2006). Oxaloacetate
synthesized via this reaction is then transferred to malate. Thereafter, ma-
late is transported to mitochondria via a shuttle mechanism, and plays a key
role in the further energy production (Harder and Wunderlich, 1991). The
so-called malate dismutation, intensively studied in Ascaris suum (see
Harder and Wunderlich, 1991), is a generally accepted metabolic reaction
in H. contortus and all gastrointestinal nematodes (Harder and Wunderlich,
76 A. Harder
1991). In this reaction step, malate is metabolized further via pyruvate and
acetyl-CoA to acetate, or via fumarate and succinate to propionate. Both
pathways lead to the production of acetate and propionate and are tightly
connected with each other: per 1 mol of acetate, 2 mol of propionate are
produced. The two NADþs, which are reduced during the acetate forma-
tion, become completely reconstituted during the formation of propionate
in the transition from fumarate to succinate, catalysed by NADHefumarate
reductase. Fumarate is the terminal electron acceptor in the following elec-
tron flow: NADH / flavoprotein 1 / rhodoquinone / cytochrome
b558 / flavoprotein 2 / fumarate (Harder and Wunderlich, 1991).
Thus, adult H. contortus is able to degrade glucose to acetate and propi-
onate under anaerobic conditions. Via substrate chain phosphorylation,
approximately 5 mol of adenosine-tri-phosphate (ATP) can be produced
per mole of glucose, in total. In addition, ATP can be produced via branched
respiratory chains. In some nematode species, even threefold-branched elec-
tron transport chains are present (Harder and Wunderlich, 1991).
Again, NADHefumarateereductase, together with a rhodoquinone/
cytochrome b558 complex, plays a central role. Two distinct respiratory
chains are connected through this complex. One respiratory chain corre-
sponds to the mammalian respiratory chain and contains cytochrome a/a3
(Kita et al., 1997). The other respiratory chain contains cytochrome o.
H2O2 is produced as the end product of this step, rather than H2O. How-
ever, it is unknown how H2O2 becomes detoxified and what function it has.
Of note is that this alternative respiratory chain is 100 times more active in
A. suum than in mammals (Kita et al., 1997). The co-existence of different
respiratory chains represents a useful means for H. contortus and other gut-
dwelling nematodes to adapt relatively quickly to changing O2 tensions in
the environment. Parasitic nematodes possess characteristically large fractions
of B-type cytochromes and small fractions of A-type cytochromes (Bryant
and Behm, 1989). Although present in all life cycle stages, A-type cyto-
chromes are subordinated in anaerobic metabolism. The information on
A-type cytochromes allows possibilities of further adaptations to the aerobic
conditions in some life cycle stages.
Haemonchus contortus appears to be particularly sensitive to inhibitors
of fumarate reductase (Barrett, 1981). By contrast, the intestinal (non-
haematophagous) nematodes, Trichostrongylus colubriformis and Cooperia curti-
cei, can much more readily tolerate the inhibition of this enzyme than can
H. contortus, since the former two species possess several alternative oxidases,
which are presumably absent from the latter species (Barrett, 1981). This
information might explain why no broad-spectrum anthelmintic acts against

haematophagous and non-haematophagous gastrointestinal nematodes
simultaneously, with fumarate reductase as a target.
Each nematode species excretes its specific pattern of end products.
Thereby, the pyruvate kinase/PEP carboxykinase ratio determines whether
lactate is formed preferentially or different volatile fatty acids are synthesized.
Correspondingly, this ratio is high in species that produce lactate (eg, Nippos-
trongylus brasiliensis, Dictyocaulus viviparus and Dirofilaria immitis) and low ratio
in those that produce volatile fatty acids (eg, Ascaris lumbricoides and, very
likely, H. contortus) (Barrett, 1981). Haemonchus contortus produces acetate,
propionate, propanol, traces of lactate, succinate and ethanol (Barrett,
1981; Kapur and Sood, 1987).
As indicated above, rhodoquinone is the electron carrier instead of ubi-
quinone, which functions in the mammalian respiratory chain. Rhodoqui-
none can be found only in a few other organisms: in free-living nematodes
(eg, C. elegans), in Rhodobacter sphaeroides (purpura bacteria), Euglena gracilis
and fungi (Barrett, 1981). The main difference between rhodoquinone
and ubiquinone is in their redox potential, which is 63 mV for rhodoqui-
none and þ113 mV for ubiquinone. The fumarate/succinate system has a
redox potential of þ33 mV, which is in between both electron carriers.
As this reaction system is a general feature of nematodes (Barrett, 1981), it
can be assumed that it should also function in H. contortus. This means
that different fluxes of electrons and hydrogen operate in the metabolism
of nematodes and mammals.
4.3 Anthelmintic drugs targeting energy and/or

carbohydrate metabolism
Carbohydrate and/or energy metabolism do not appear to be an important
target of drugs against nematodes (Harder, 2002); there are presently only
two narrow-spectrum anthelmintics e disophenol and closantel e which
are occasionally used to treat H. contortuseinfected animals (Harder,
2002). These drugs are uncouplers of oxidative phosphorylation and are
taken up by the nematodes via blood. The respiratory chain of nematodes
could be a target for new inhibitory compounds, such as quinazoline or
atpenin (Sakai et al., 2012). Recently, crystallization of mitochondrial rho-
doquinolefumarate reductase from A. suum was successful (Osanai et al.,
2009), and the fungicide flutolanil was shown to act as a specific inhibitor.
Moreover, fluopyram (fungicide Luna) exhibited activities against H. contor-
tus in sheep (A. Harder, personal observations).
78 A. Harder
5. AMINO ACID METABOLISM

The transition of L4 to adult male H. contortus is accompanied by an
increased amino acid metabolism (Laing et al., 2013). Nematodes, like all
other organisms, use amino acids for protein synthesis, as precursors for spe-
cific biosynthetic pathways and, also, but in a very limited manner, for the
production of ATP. Essential amino acids are absorbed from host diet and/
or hydrolysed by proteinases or peptidases before they are further degraded
in the intestinal lumen of nematodes (K€ ohler, 2006). Amino acid meta-
bolism of parasitic nematodes resembles that of free-living nematodes.
Nematodes are unable to synthesize porphyrins from glycine and
succinyl-CoA, both TCA-cycle intermediates, or purines from glycine
and aspartate (K€ ohler, 2006). Although neurotransmitters and neurohor-
mones are widely distributed among different helminths, almost nothing
is known about their biosynthesis from amino acids. Histamine, serotonine
and catecholamines are primarily absorbed from the host animal. Haemonchus
contortus contains, at least, the physiologically active amines, adrenalin and
noradrenalin (Barrett, 1981). In nematodes, including H. contortus, there is
a variety of amino acids that serve as donor compounds for transamination
reactions.
5.1 Polyamines, nitrogen excretion in parasites

In general, there is only limited information on H. contortus regarding
polyamines and nitrogen excretion. Predominant polyamines of helminths
are spermidine and spermine. Nematodes, such as H. contortus, possess
only a limited capacity for the biosynthesis of polyamines from ornithine
and are thus dependent on a polyamine supply from the host (Barrett,
1981; K€ ohler, 2006). However, absorbed polyamines can be transferred
into other polyamines by direct oxidation of acetylated intermediates,
such as from spermine into spermidine or spermidine into putrescine
(K€ohler, 2006).
In nematodes, a fraction of amino nitrogen is excreted in the form of
distinct amino acids (K€ohler, 2006). Another means of excretion is via
ammonia by transaminations and deamination processes. An oxidative
deamination occurs according to the reaction L-amino acid þ H2O þ
O2 / 2-ketoacid þ NH3 þ H2O2 (K€ ohler, 2006). The excretion of
ammonia, which is toxic to cells, is important in the main excretory/
secretory pathways (Barrett, 1981; K€ohler, 2006). Most likely, nematodes
do not contain a functional urea cycle (Barrett, 1981; K€

ohler, 2006). In
nematodes, the largest proportion of urea comes from purine degradation,
and both larval and adult nematodes excrete amines, such as alkylamines
or ethanolamine (Barrett, 1981; K€ohler, 2006). This excretion occurs by
yet unkown enzymatic mechanisms.
6. NUCLEIC ACID METABOLISM

Parasitic nematodes, like other eukaryotic parasites, are characterized
by substantial cellular multiplication rates associated with high nucleic acid
synthesis. One adult female of H. contortus can produce up to 10,000 eggs
per day (Veglia, 1916). In comparison, A. lumbricoides can produce
2 105 eggs per day (Wehner and Gehring, 1995a,b).
6.1 Purine metabolism

Nematodes including H. contortus are not able to synthesize purines de novo
and, therefore, are dependent on the supply of suitable purine precursors
from the host (K€ ohler, 2006). Purine bases are converted to the different
purine nucleotides, either by phosphoribosyltranserases (PRTases) or by
reactions involving nucleoside phosphorylases and nucleoside kinases. The
pattern of salvage pathways for purines can vary considerably, depending
on nematode species and developmental stage (Barrett, 1981; K€ ohler,
2006). Nematodes are able to synthesize pyrimidine nucleotides de novo,
but can also produce these compounds via salvage pathways. There are
substantial differences among nematode species in synthesis capacities
(K€ohler, 2006).
7. LIPID METABOLSIM
During the transition from L4 to adult male H. contortus, there is a
decreased lipid metabolism coupled to an increase in amino acid metabolism
(Laing et al., 2013). In nematode eggs, long-chain fatty acids from triacylgly-
cerols are used for the resynthesis of carbohydrates via a functional glyoxylate
cycle (Barrett, 1981; K€ ohler, 2006). The presence of this pathway in the
developing eggs of some helminths is unique, and is not seen in other ani-
mals studied to date (K€ ohler, 2006).
The lipid metabolism of most adult nematodes is limited (K€ ohler, 2006);
the worms are usually not able to synthesize long-chain fatty acids and
80 A. Harder
sterols. Therefore, they exclusively rely on the absorption of these com-

pounds from the host diet. For most nematodes studied, the use of lipids
as an energy source in adult stages is either very limited or absent (K€ ohler,
2006). A plausible explanation is that nematodes usually lack effective termi-
nal oxidases (K€ohler, 2006). As the TCA-cycle and cytochrome oxidases are
lacking, NADH, which is produced in large amounts during fatty acid
degradation, cannot be reoxidized at sufficient quantities. In nematodes,
the oxidative capacity is restricted to some larval parasitic and most free-
living stages (K€ohler, 2006).
Fatty acids absorbed by nematodes from exogenous sources are rapidly
incorporated into triglycerides and phospholipids (K€ ohler, 2006). These
steps seem to be very similar to those that occur in other animals. Most en-
doparasites are able to synthesize phospholipids and sphingolipids de novo, if
they have access to the corresponding fatty acids and sugars. The activation
of fatty acids to acetyl-CoA thioesters occurs via acyl-CoA-synthetases,
which are relatively widely distributed in the nematodes studied to date
(K€ohler, 2006). The further steps of synthesis and conversions of complex
lipids are similar to those in higher animals. Moreover, nematodes are not
able to synthesize sterols, such as cholesterol, de novo. However, they are
able to produce farnesol and ecdysteroids and juvenile hormones (K€ ohler,
2006). In most nematodes, a mevalonic acid pathway is active and is used
for dolichol biosynthesis, which is important for protein glycosylations,
quinone isoprene side-chain synthesis and the synthesis of geranylgeranyl
pyrophosphates as substrates for isoprenylations of proteins (K€ ohler, 2006).
8. STRUCTURE AND BIOCHEMICAL COMPOSITION OF

THE CUTICLE
The cuticle of parasitic nematodes forms the exoskeleton and consists
mainly of cross-linked collagens (Page and Johnstone, 2007); its overlaying
surface coat represents the primary interface between the pathogen and the
host’s immune system (Page et al., 1992). However, nematodes, including
H. contortus, also absorb nutrients as well as relatively large amounts of
some anthelmintic drugs (eg, levamisole and macrocyclic lactones) through
the cuticle, whereas other anthelmintics are absorbed via the alimentary tract
(eg, benzimidazoles, morantel and pyrantel) (Mehlhorn, 2008a,b). The
cuticle of nematodes is excreted by the epidermis and covers the mouth parts
and pharynx as well as distal parts of intestine, vagina and excretory pores,
and is usually renewed four times during growth and development (Bird
and Zuckerman, 1989). The cuticle has several layers, consisting of an inner
fibrillar layer, followed by a matrix and the outer cortex, which is covered by
a 20-mm-thick epicuticle as well as an additional lipid layer in some nema-
tode species (Mehlhorn, 2008a,b). Numerous structures of the cuticle (eg,
lips, pores, grooves, leaf crowns or thorns as well as lateral or sublateral
caudal or cervical alae or a copulatory bursa) can be present (Mehlhorn,
2008a,b).
Glycolipids are present predominantly in the outer layer of membranes,
where the sugar moieties participate in the structure of the glycocalyx (Bird
and Zuckerman, 1989). The glycocalyx contains multiple-branched
oligosaccharide chains of glycolipids and glycoproteins, which gives the gly-
cocalyx major biochemical complexity (Mehlhorn, 2008a,b). The robust
cuticle renders nematodes relatively resistant against host immune attack
(Maizels, 2013). For example, larvae of Toxocara canis produce a biophysical
barrier between their surface and host immune effector cells (Page et al.,
1992). They produce a glycocalyx that surrounds the worm. This layer,
sometimes called ‘fuzzy coat’ (Maizels, 2013) consists of various mucins
with different chain lengths; it is produced by oesophageal and secretory
glands (Mehlhorn, 2008a,b) and binds eosinophils (Maizels, 2013). Such a
similar situation may also occur in H. contortus. However, the immune cells
do not reach the nematode’s surface, as the worms are continuously
excreting new glycocalyx and slouging the coat (Mehlhorn, 2008a,b). In
addition, absorptive surfaces of nematodes contain various enzymes, such
as Naþ-/Kþ-ATPases, Caþþ-ATPases and Naþ-/Hþ-exchange proteins,
for the transport of organic ions. These are membrane-bound proteins
that facilitate the active transport of ions and maintain a balanced ratio of
ion concentrations inside and outside of cells and, hence, the osmotic pres-
sure in the worm (Mehlhorn, 2008a,b).
9. TUBULIN AS A MAJOR STRUCTURAL COMPONENT

AND DRUG TARGET
Microtubular functions are important for numerous cellular processes,
such as cell division, axoplasmic transport, cell movement and cell-to-cell
communication. The cytoskeleton is intimately involved in the growth of
axons, and microtubuli are involved in axonal transport of compounds
(Wehner and Gehring, 1995a,b). Almost all biosynthetical activities of the
neuron can be found in the cell soma, which contains a highly developed
endoplasmic reticulum. Via the Golgi apparatus, located at the origin of
82 A. Harder
the axon near the cell nucleus, the synthesized products are introduced into
an axoplasmatic flux. There are two components of filamentous proteins
differentiated according to velocity and mechanism. In a slow mass flux
(1e5 mm d1), the filamentous proteins and cytosolic enzymes move
from the soma to the synaptic region. Enzymes required for the synthesis
of transmitters or neurotransmitters as well as membrane proteins follow a
rapid transport mechanism (200e400 mm d1). In this case, single parti-
cles move along the microtubules (Wehner and Gehring, 1995a,b).
Microtubuli are polymers of tubulin. Tubulin itself is a dimer, consisting
of a- and b-tubulin subunits. In mammals, a microtubule usually consists of
13 protofilaments. By contrast, intestinal or nerve cells of T. colubriformis and
N. brasiliensis each contain 11 and 12 protofilaments, respectively, whereas
corresponding cells of Ascaridia galli, Heligmosomoides polygyrus and larvae
of H. contortus contain microtubuli with 11 protofilaments (Gull et al.,
1986). Microtubuli of specialized nerve cells of T. colubriformis and H. contor-
tus contain 14 and 15 protofilaments, respectively (Gull et al., 1986). In
nematodes, microtubuli have been shown to be involved in a variety of
physiological functions, such as egg laying, egg hatching, larval develop-
ment, substrate transport, enzyme activity and enzyme secretion (Rew
and Fetterer, 1986), but detailed studies are warranted to provide better in-
sights into the structures and functions of microtubules of different species of
nematodes.
Anthelmintic benzimidazoles play a major role in veterinary as well as in
human medicine for the treatment of nematodiases. A variety of benzimid-
azoles, benzimidazole carbamates and prebenzimidazoles, entered the drug
market between the early 1960s and late 1980s. They exert their inhibitory
activity by interacting with b-tubulin of the tubulin dimer (Roos, 1997).
The tubulinebenzimidazole complex unfolds the carboxy terminal region
of b-tubulin, and the abnormally unfolded loop of b-tubulin prevents
further addition of a- and b-tubulin subunits and, consequently, microtu-
bule polymerization (Roos, 1997). However, the exact binding site/s of
benzimdazoles on b-tubulin is/are still unknown. Benzimidazole resistance
is associated with a phenylalanine-to-tyrosine substitution at amino acid po-
sition 200 of H. contortus b-tubulin isotype-I. In addition, studies of other
parasitic nematodes have shown that other mutations (ie, amino acid
positions 166, 167 and 198) in this region of b-tubulin may have an influ-
ence on the interaction of benzimidazoles with b-tubulin (von Samson-
Himmelstjerna et al., 2007). However, a problem is that the residue 200
and the other reported residues responsible for benzimidazole binding are
‘hidden’ within the protein. Therefore, there must be an additional

mechanism by which these residues may become accessible to the drugs
(Robinson et al., 2004).
10. NERVOUS SYSTEM IN NEMATODES

Of all nematodes, the nervous system of C. elegans is the best under-
stood. It contains 302 neurons with 118 neurone classes (Joyner, 2010).
Approximately 5000 chemical synapses and 600 electrical synapses (gap
junctions) are functional (Bargmann, 2006; Thomas and Lockerly, 1999).
More than one-third of the neuronal cells in C. elegans release acetylcholine
(ACh), the major excitatory neurotransmitter, causing contraction of the
body wall muscle by opening sodium-gated ACh receptors (Holden-Dye
et al., 2013; Joyner, 2010). Important ACh-mediated behaviours are loco-
motion, pharyngeal pumping, egg laying and developmental timing (Joyner,
2010). The main inhibitory neurotransmitters in C. elegans are g-butyric acid
(GABA) and glutamate with the corresponding GABA- and glutamate-
gated chloride channels. A further inhibitory receptor is SLO-1, discovered
during intensive research activities of the mode of action of emodepside
(Kr€ucken et al., 2012; Walker et al., 1996). In H. contortus and other nem-
atodes, the stimulatory action of the nicotinic AChRs are counterbalanced
by the inhibitory action of GABA- and glutamate-gated chloride channels
as well as the calcium-activated voltage-gated potassium channel SLO-1
(Amliwala et al., 2004). Any disturbance of one of these receptors by anthel-
mintics leads to an impairment of one or more physiological activities of the
nematodes and death of the respective parasites.
10.1 Nicotinic AChRs in Haemonchus contortus

The nAChRs of H. contortus and other parasitic nematodes are targets of
anthelmintics such as levamisole, pyrantel, morantel, oxantel, monepantel
and tribendimidine (Holden-Dye et al., 2013). From experiments using
A. suum, three pharmacologically distinct nAChRs types can be
distinguished. The L-type is activated by levamisole and pyrantel; N-type
is activated by nicotine, oxantel and methyridine and the B-type is activated
by the old anthelmintics bephenium and thenium (Martin et al., 2012).
These subtypes were delineated using the antagonists paraherquamide
and 2-deoxy-paraherquamide, the latter being a constituent of derquantel,
together with abamactin (Puttachary et al., 2013). The nAChR subtypes
84 A. Harder
reveal differences in single ion channel properties (mean conductance and

opening times) as well as different responses to anthelmintic agonists and
antagonists (Holden-Dye et al., 2013; Martin et al., 2012; Qian et al.,
2006).
Genes orthologous to those in C. elegans, such as unc-38, unc-63, unc-29
and lev-1, have been identified in H. contortus (see Beech and Neveu, 2015;
Holden-Dye et al., 2013). There are two main nAChR clades representing
H. contortus. Specific subunit genes of nAChR in the unc-38 clade are Hco-
acr-12, Hco-acr-8, Hco-acr-6, Hco-unc-38 and Hco-unc-63. The unc-29 clade
represented in H. contortus comprises the subunits Hco-lev-1, Hco-unc-29.1,
Hco-unc-29.2, Hco-unc-29.3, Hco-unc-29.4, Hco-acr-2 and Hco-acr-3 (Beech
and Neveu, 2015). The recombinant expression of the subunits encoded
by genes Hco-acr-8, Hco-unc-29.1, Hco-unc-38 and Hco-unc-63a, together
with Hco-ric-3.1, Hco-unc-50 and Hco-74, proteins involved in L-nAChR
function in C. elegans, was shown to result in functional receptors, being sen-
sitive to ACh and levamisole. These receptors were named Hco-L-nAChr1
(Holden-Dye et al., 2013). When Hco-ACR-8 was removed from the com-
bination of subunits, this receptor was named Hco-L-AChR2. This receptor
was less sensitive to ACh and levamisole. It is not known whether either of
these receptors is expressed in vivo. Hco-RIC-3.1, Hco-UNC-50 and Hco-
UNC-74, ancillary proteins involved in L-nAChR function, were essential
for the expression of both receptors Hco-L-nAChr1 and Hco-L-nAChr2
(Holden-Dye et al., 2013).
Interestingly, the pharmacology of both receptors varies for different
anthelmintics. Hco-L-nAChr2 is more sensitive to pyrantel, but insensitive
to bephenium which selectively activates Hco-L-nAChr1 (Holden-Dye
et al., 2013), suggesting that the activation by bephenium involves ACR-
8. Pyrantel was more active on Hco-L-nAChr2 than ACh, while levamisole
was approximately equally active with nicotine, but more than 100 times less
active compared with Hco-L-nAChr1 (Holden-Dye et al., 2013).
Recently, two receptors for the amino-acetonitrile derivative monepan-
tel (MPTL) have been proposed for H. contortus (see Baur et al., 2015;
Holden-Dye et al., 2013), one containing MPTL-1 and one containing
DEG-3 and DES-2. The latter is activated by choline, which is potentiated
by monepantel. The corresponding homologue for MPTL-1 in C. elegans is
ACR-23. Moreover, it could be shown that all 11 levamisole-resistant
C. elegans mutants assessed were also resistant to tribendimidine, a new
L-nAChR agonist anthelmintic, indicating the same mode of action for
this receptor (Hu et al., 2009).
Since there are many nAChR subunits in nematodes that are not current
anthelmintic targets, some of these subunits might represent future drug tar-
gets. While C. elegans provides a useful model for the study of nematode
nAChRs, there are differences in the subunit composition of these receptors
between C. elegans and parasitic nematodes, including H. contortus. As an
example, while LEV-1, LEV-8 and UNC-63 are required for electrophys-
iological functions in C. elegans, both ACR-8 and UNC-63 as well as
UNC-38 and UNC-29 are required in H. contortus (see Holden-Dye
et al., 2013). Interestingly, while LEV-1 is absent from Trichinella spiralis,
A. suum, Brugia malayi, it is present in H. contortus, Teladorsagia circumcincta
and T. colubriformis. However, LEV-8 is absent from all of these nematodes,
while both LEV-1 and LEV-8 are present in C. elegans (see Holden-Dye
et al., 2013). On the other hand, ACR-26 is present in a number of parasitic
nematodes, but absent from C. elegans.
There is evidence that a number of ancillary proteins are important for
nAChR function, and these may also provide useful targets for new anthel-
mintics. The pharyngeal nAChR subunit, EAT-2, is a possible target for
new anthelmintics, since the disruption of feeding results in morbidity and
mortality in C. elegans (see Holden-Dye et al., 2013). Another protein
that is linked to L-nAChR function in C. elegans is UNC-68, which is a rya-
nodine receptor and target of the anthranilic diamide insecticides, and might
have anthelmintic potential (Holden-Dye et al., 2013).
10.2 Inhibitory neurotransmitters in nematodes

10.2.1 g-Aminobutyric acid-A receptors
Piperazine causes flaccid, reversible paralysis of body wall muscle in A. suum,
acting as a weak GABA-mimetic. Electrophysiology has shown that piper-
azine is a partial agonist with low efficacy acting on GABA-gated chloride
channels (Joyner, 2010). Flaccid paralysis leads to the expulsion of the
worm from the host gut. The GABA receptor seems to be of minor impor-
tance in the nerve muscle transduction in nematodes, since piperazine is the
only anthelmintic drug that exerts its effect via this target (Harder, 2002). By
contrast, this target is of great importance in arthropods (eg, Beugnet and
Franc, 2012).
10.2.2 Glutamate-gated chloride channels and macrocyclic lactones

Glutamate is the most important excitatory neurotransmitter in mammals.
By contrast, in invertebrates, glutamate acts as an inhibitory neurotrans-
mitter. The glutamate-gated chloride channels (GluCls) are linked to the
anthelmintic activity of ivermectin (IVM) (Joyner, 2010; Kr€ ucken et al.,
86 A. Harder
2012). GluCls are evolutionary related to GABA(A)-receptors and are target

sites for the avermectin/milbemycin macrocyclic lactone anthelmintics
(Portillo et al., 2003). Macrocyclic lactones (MLs) cause paralysis of the
somatic and pharyngeal muscles in nematodes. Four GluCl subunits,
HcGluCla, HcGluClb, HcGluCla3A and HcGluCla3B, have been identi-
fied in H. contortus (see Portillo et al., 2003). All of these subunits are
expressed in the motor nervous system, particularly motor neuron commis-
sures (Portillo et al., 2003). HcGluCla and HcGluClb are expressed on the
same commissures and they may be inhibitory motor neurons, and the
HcGluClb subunits are also detected in lateral and sublateral nerve cords
(Portillo et al., 2003). The expression of HcGluCla3A and HcGluCla3B
subunits, products of an alternatively spliced gene, is seen in different neu-
rons (Portillo et al., 2003) Thus, GluCls are widely distributed in the H. con-
tortus nervous system, suggesting critical roles in controlling locomotion,
pharyngeal function, feeding, egg laying and possibly sensory processing
in parasitic nematodes (Portillo et al., 2003).
MLs (including IVM, avermectin, abamectin, eprinomectin, doramec-
tin, moxidectin, milbemycin oxime and selamectin) activate the anionic
channels and, typically, inhibit neuronal transmission and muscle contrac-
tion (Joyner, 2010). There are two H. contortus subunit genes, glc-5 and
glc-6, which encode glutamate-sensitive channels that are absent from C. ele-
gans. Both of these subunits are targets for MLs, and changes in their
sequence or expression have been associated with drug resistance in parasites
of veterinary importance. Most of the other anionic channel subunits in
H. contortus have direct orthologues in C. elegans (see Joyner, 2010). A family
of five genes encodes GluCl channel subunits in the latter species.
Specifically, the glc-1 gene encodes GLuCla1; avr-15 encodes alterna-
tively spliced GLuCla2A and GLuCla2B; avr-14 encodes alternatively
spliced GLuCla3A and B; glc-3 encodes GluCla4; and glc-2 encodes GluClb
(Joyner, 2010). When GluCla and GluClb are expressed together, GluCla
is found to respond to IVM but not to glutamine, whereas GluClb has the
opposite pharmacological effect (Joyner, 2010). Receptors containing both
subunits respond to glutamate and are positively allosterically modulated by
IVM. Genes encoding GluCla are involved in the regulation of locomotion
patterns in C. elegans, particularly reversal behaviour, suggesting that the sub-
units GluCla1-3 may form a heteroligomeric receptor. Recently, the three-
dimensional structure of a GluCl was solved, the first for any eukaryotic
ligand-gated anion channel, revealing an ML-binding site between the
channel domains of adjacent subunits (Hibbs and Gouaux, 2011;
Wolstenholme, 2012). This information highlights some unique features of

the GluCls and contributes to knowledge of the entire cys-loop ligand-gated
ion channel superfamily.
10.2.3 Calcium-activated voltage-gated potassium channel SLO-1

SLO-1 has an important role in the regulation of neuronal and muscle cell
excitability in vertebrates and invertebrates (Kr€
ucken et al., 2012). SLO-1
regulates the neuronal networks that control behaviour, including locomo-
tion (Kr€ucken et al., 2012). It is suggested that the cyclooctadepsipeptide
anthelmintic, emodepside, facilitates the opening of SLO-1 in the course
of the pleiotropic actions of this anthelmintic (Kr€ ucken et al., 2012;
Holden-Dye et al., 2012). An increase in activity would tend to inhibit
neuronal and muscle activity via a membrane hyperpolarization and provide
an explanation for the inhibition of pharyngeal muscle, body wall muscles
and muscles linked to egg-laying that results from exposure to emodepside.
Emodepside has also been shown to interact directly with GABAA-R
(Chen et al., 1996) and latrophilin-1 (LAT-1) (Saeger et al., 2001). Howev-
er, C. elegans strains lacking these putative emodepside receptors show only
modest decreases in their sensitivity to emodepside relative to the worms
that lack SLO-1 (Kr€ ucken et al., 2012). This information suggests that
SLO-1 is a major determinant of the paralysing activity of emodepside, a
statement supported by the fact that ectopic expression of SLO-1 in pharynx
muscles in an SLO-1-deficient worm is sufficient to confer emodespside sus-
ceptibility to this organ (Holden-Dye et al., 2012; Kr€ ucken et al., 2012).
SLO-1 is found on presynaptic nerves innervating body wall and pharynx
and also in postsynaptic body wall muscles, but not in pharyngeal muscles
(Kr€ucken et al., 2012). Recently, it could be shown that emodepside binds
directly to the SLO-1 receptor (Kulke et al., 2014). In another study, the
proposed direct interaction of emodepside with C. elegans SLO-1 was
confirmed (Crisford et al., 2015).
11. BIOCHEMISTRY OF DRUG RESISTANCE

In principle, nematodes can employ a range of different strategies to
achieve a state of reduced susceptibility to a particular anthelmintic drug.
These strategies include the modification of a drug target (eg, binding
site), increased target site numbers (eg, neuronal receptors), increased drug
efflux (eg, through transmembrane pumps), increased metabolization (eg,
through CYP450) and/or sequestration of the drug (James et al., 2009).
88 A. Harder
11.1 Specific resistance mechanisms

11.1.1 Benzimidazole resistance
Resistance-associated changes in the drug target would generally be consid-
ered as specific mechanisms of resistance, since only the respective drug class
will be affected. An example of this is benzimidazole resistance. It was shown
by investigations who compared the drug target of susceptible and resistant
nematodes (eg, Robinson et al., 2004; Roos, 1997) that b-tubulin is the true
target of benzimidazoles. A target-oriented approach to analyse drug resis-
tance detected specific changes in the b-tubulin gene sequence that corre-
lated with resistance. Furthermore, benzimidazole resistance could be
conferred by changing the b-tubulin sequence at one position (codon
200; Kwa et al., 1994a,b). Susceptible worms exhibited phenylalanine at
this site, compared with tyrosine in resistant worms. This change of the
amino acid sequence is the result of a single nucleotide polymorphism
(SNP) from TTC200 to TAC200. The benzimidazole binding affinity of
b-tubulin encoding tyrosine at position 200 is considerably lower than those
expressing phenylalanine (Kwa et al., 1994a,b). However, the situation has
become more complicated in that additional mutations (eg, at codon posi-
tion 167 and 198) were reported to be associated with resistance in the same
nematode species (Ghisi et al., 2007; Silvestre and Cabaret, 2002). More-
over, it has become apparent that the relative importance of the benzimid-
azole-resistance phenotype for the different resistance-associated b-tubulin
SNPs differs between nematodes. For example, it was reported that in benz-
imidazole-resistant small strongyle populations of equines, codon 200 SNP
is not present in a large proportion of resistant individuals (Drogem€ uller
et al., 2004; Hogkinson et al., 2008).
There are structural prerequisites for benzimidazoles for optimal tubulin-
binding activity. The imidazole ring system is essential for activity (Prichard,
2001). Protonation and/or deprotonation are important for the transport of
drugs across membranes, and the carbamate moiety is essential for the inter-
action with b-tubulin (Prichard, 2001). Aliphatic side chains are essential for
a more efficient microsomal oxidation compared with aromatic ring systems.
Moreover, sulphur, instead of oxygen, as a bridge between side chain and
the benzole core, improves the pharmacokinetic properties of the drug
(Prichard, 2001). The benzole core of the benzimidazole interacts with
phenylalanine at position 200 of b-tubulin at the C-terminus and at phenyl-
alanine 167 of the N-terminus. The imidazole and the carbamate moiety are
bound to cysteine 201 at the C-terminus, and serine 166 at the N-terminus
of b-tubulin (Prichard, 2001). Both phenylalanines at positions 200 and 167

are therefore of critical importance for the binding of benzimidazoles to
b-tubulin. A replacement by tyrosine will significantly lower the affinity
of the drug to this target and, thus, may explain resistance against this
drug at the molecular level.
11.1.2 Levamisole resistance

In H. contortus, levamisole resistance has been associated with truncated forms
of UNC-63 (Martin et al., 2012). When a truncated form of Hco-UNC-63,
named Hco-UNC-63B, was co-expressed with Hco-UNC-63, the expression
of L-nAChRs was inhibited, inducing levamisole resistance. It is possible that
this situation might occur under natural conditions to produce levamisole-
resistant H. contortus phenotypes. A loss of UNC-63, however, is predicted
to lead to a loss of sensitivity to pyrantel also (Martin et al., 2012). In addition,
with a loss of or truncation of ACR-8 (Hco-ACR-8B), the L-nAChR is ex-
pected to be less sensitive to levamisole, but still sensitive to pyrantel (Martin
et al., 2012). Thus, it is suggested that a loss of ACR-8 subunits may lead to a
selective loss of sensitivity to levamisole, but not to pyrantel.
11.2 Nonspecific resistance mechanisms e drug metabolism

and efflux
Parasitic nematodes possess a large variety of inducible metabolizing enzymes
and transporters to protect themselves against toxins. There are three main
detoxification reactions in nematodes, namely the modification, conjugation
and excretion of toxic compounds. CYP450s and the short-chain dehydro-
genases/reductases are involved in modification; the UDP-glucuronosyl
transferases (UGTs) and the glutathione S-transferases (GSTs) are involved
in conjugation; and, the ATP-binding cassette (ABC) transporters in excre-
tion. It is assumed that these detoxification systems are also involved in the
detoxification of anthelmintics or resistance of anthelmintics (eg, Laing
et al., 2013). In H. contortus, a large number of modification and conjugation
gene products have been predicted from the genome, including 42 CYPs,
44 short-chain dehydrogenase/reductases, 34 UGTs and 28 GSTs (Godoy
et al., 2015; Laing et al., 2013; Schwarz et al., 2013).
The process best investigated to date involves the transmembrane trans-
porter P-glycoprotein (Pgp), which is expressed at higher rates in ML-
resistant than in ML-susceptible populations of parasitic nematodes (Areskog
et al., 2013; Janssen et al., 2013). Pgp was found to be expressed, for
example, in the intestine or egg shell of nematodes, and is considered to
90 A. Harder
effectively reduce toxic drug concentrations within the parasite (de Graef
et al., 2013; Janssen et al., 2013, 2015). Pgp transporters are of particular in-
terest, as they have been implicated in resistance of H. contortus to IVM and
other anthelmintics (Lespine et al., 2012). Thus, the family of ABC trans-
porters is involved in the efflux of a large number of drugs including
IVM, an ML endectocide widely used in antiparasitic therapy in humans
and livestock animals (Janssen et al., 2015). In total, 10 Pgp genes have
been predicted in the draft genome of H. contortus (see Godoy et al.,
2015), and knowledge of the complement will now allow a more systematic
analysis of the role of P-glycoproteins in resistances to IVM and other an-
thelmintics. Of particular relevance are genes pgp-1, pgp-2, pgp-9, pgp-16
and pgp-17, whereby pgp-1, pgp-2 and pgp-9 have been reported in IVM-
resistant versus susceptible isolates of H. contortus (see Janssen et al., 2015)
and in pgp-9 for resistant T. circumcincta (see Laing et al., 2013). Studies of
H. contortus have indicated that repeated treatment with MLs, such as
IVM, have led to the selection of specific Pgp alleles. Many anthelmintic
drugs are known to be substrates for Ppgs and thus amenable to removal
via upregulated expression of this efflux pump (Kerboeuf et al., 2003).
12. CONCLUSIONS
Understanding the biochemistry of nematodes is central to gaining in-
sights into catabolic and anabolic pathways of these worms. Moreover, it
helps to better understand nematodeehost interactions in the habitats where
nematodes reside in the host. In addition, this research field supports the
finding of new target sites and thus anthelmintic screening. Unfortunately,
there is a paucity of information on biochemical processes in parasitic nem-
atodes in general, and also specifically in H. contortus. There are major
knowledge gaps, particularly concerning drugereceptor interactions.
With advances in molecular biology, anthelmintic target research has been
intensified. Through the use of innovative research tools new drug targets can
be identified and characterized during the development of new drugs. Such
advances have assisted in the characterization of the mode of action of benz-
imidazoles, levamisole, IVM and other MLs, monepantel and emodepside. In
the future, by identifying the three-dimensional structures of the nAChR,
glutamate-gated chloride channel and calcium- and voltage-dependent potas-
sium channel SLO-1, which are essential for nematode survival, it should be
possible to design new anthelmintics. These compounds should have
improved binding capacities to the corresponding receptors and
resistance-disrupting properties against the common anthelmintic drugs.

Moreover, the analysis of biochemical processes (including neuroreceptor
functions, metabolism and efflux) supports the understanding of anthelmintic
resistance. This is a particularly important issue, since the knowledge of the
mechanism of resistance of a nematode against an anthelmintic drug might
help to extend the respective drug’s activity and longevity. This goal might
also be achieved by using suitable drug combinations according to their spe-
cific interactive anthelmintic capacities. Furthermore, elucidating of parasitic
nematode-specific Pgp transporter substrate specificities may even help to
restore the former anthelmintic activities. Therefore, the combinatory use
of both research fields e biochemistry and molecular biology e will continue
to have a major impact in the field of anthelmintic drug research.
ACKNOWLEDGEMENT
I would like to thank Prof. Dr Robin Gasser and Prof. Dr Georg von Samson-Himmelstjerna
for their great support during writing this manuscript.
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40232e40238.
CHAPTER FOUR
The Pathophysiology, Ecology

and Epidemiology of
Haemonchus contortus Infection
in Small Ruminants
R.B. Besier*, 1, L.P. Kahnx, N.D. Sargison{, J.A. Van Wykjj
*Department of Agriculture and Food Western Australia, Albany, WA, Australia
x
{
jj
1
Corresponding author: E-mail: R.B.Besier@murdoch.edu.au
Contents
1. Introduction 96
2. Occurrence and Importance 97
2.1 Geographical distribution 97
2.1.1 Tropical and subtropical climates 98
2.1.2 Warm temperate regions 98
2.1.3 Cool temperate regions 99
2.1.4 Arid regions 99
2.2 Economic significance 100
3. Pathogenesis and Disease 101
3.1 Pathophysiology and pathogenesis 101
3.2 Clinical signs of disease 103
4. Ecology 106
4.1 Controlled environment studies 107
4.1.1 Moisture requirements for egg development and survival 107
4.1.2 Moisture requirements for the survival of infective larvae 110
4.1.3 Temperature requirements for the development of eggs to infective larvae 110
4.1.4 Temperature requirements for the survival of infective larvae 111
4.1.5 Intraspecific differences in critical requirements 112
4.1.6 Other environmental factors 113
4.2 Ecological investigations in the field 113
4.2.1 Tropical and subtropical climates 114
4.2.2 Warm, temperate and Mediterranean climates 116
4.2.3 Cool, temperate climates 117
4.2.4 Arid regions 118
4.2.5 Effect of microclimatic factors on larval development 119
4.2.6 Lateral and vertical migration of infective larvae 120

j
ISSN 0065-308X
96 R.B. Besier et al.
5. Epidemiology 122
5.1 Tropical and subtropical regions 122
5.2 Warm, temperate climates 127
5.3 Cool temperate climates 128
5.4 Arid climates 129
6. Prediction of the Occurrence of H. contortus 130
6.1 Predictive models 130
6.2 Potential effects of climate change 131
7. Conclusions 132
References 133
Abstract
The parasitic nematode Haemonchus contortus occurs commonly in small ruminants,
and it is an especially significant threat to the health and production of sheep and goats
in tropical and warm temperate zones. The main signs of disease (haemonchosis) relate
to its blood-feeding activity, leading to anaemia, weakness and frequently to deaths,
unless treatment is provided. Due to the high biotic potential, large burdens of
H. contortus may develop rapidly when environmental conditions favour the free-living
stages, and deaths may occur with little prior warning. More chronic forms of haemon-
chosis, resulting in reduced animal production and eventually deaths, occur with
smaller persistent infections, especially in situations of prolonged, poor nutrition. The
global distribution of the main haemonchosis-endemic zones is consistent with the
critical requirements of the egg and larval stages of H. contortus for moisture and
moderate to relatively warm temperatures, but the seasonal propensity for hypobiosis
(inhibition of the fourth-stage larvae within the host) largely explains the common,
though sporadic, outbreaks of haemonchosis in arid and colder environments. The
wide climatic distribution may also reflect the adaptation of local isolates to less favour-
able ecological conditions, while an apparent increase in the prevalence of outbreaks in
environments not previously considered endemic for haemonchosis e especially cold,
temperate zones e may be attributable to climatic changes. Although the risk of
haemonchosis varies considerably on a local level, even where H. contortus is endemic,
the extensive range of ecological investigations provides a sound basis for predictions
of the relative geographical and seasonal risk in relation to climatic conditions.
1. INTRODUCTION
Haemonchus contortus is a highly pathogenic helminth, primarily of
small ruminants, with a global distribution. Due to its blood-feeding behav-
iour and the potential for the rapid development of large burdens, it is a
frequent cause of mortalities in sheep, goats and occasionally other
Haemonchus contortus Infection in Small Ruminants 97
ruminants, and is the most important parasite of livestock in warm climatic

regions, and arguably on a global basis.
Although considered primarily a parasite of tropical and summer rainfall
zones, the ecological adaptability of H. contortus afforded by its high level of
genetic polymorphism and high biotic potential has seen it become increas-
ingly important over a wide range of climatic zones. The possible further
increase in its geographical range, especially due to climate change, could
lead to an increased prevalence of haemonchosis in presently low-risk areas.
In conjunction with an increasing severity of anthelmintic resistance, this
would further add to the costs of livestock production, and the necessity
to develop new and sustainable preventative strategies. This chapter reviews
the effects of H. contortus on host animals, and the ecological factors that
determine the occurrence and impact of H. contortus, as the basis for under-
standing its changing distribution and seasonality, and for developing control
strategies.
2. OCCURRENCE AND IMPORTANCE

2.1 Geographical distribution
The requirement of warm and moist environmental conditions for
the free-living stages of H. contortus governs the parasite’s geographical
and seasonal distributions. The prevalence of H. contortus and disease in
grazing animals is therefore particularly high in the tropical climatic zones
of both hemispheres, between latitudes 23.5 N and S (O’Connor et al.,
2006). However, H. contortus has proven to be remarkably adaptable
over a wide range of environments (Waller and Chandrawathani, 2005),
due to its high biotic potential which allows it to take advantage of
short periods which are favourable for the development of its free-living
stages, and the survival ability of the relatively robust infective third-stage
larvae (summarized in Table 1), and specific adaptive mechanisms, such as
hypobiosis of the fourth-stage larvae. Genetic differences in environmental
tolerance arising as a consequence of a high level of polymorphism may
confer a selective advantage to particular strains in the face of climate
change. Hence, H. contortus occurs in almost all regions where small rumi-
nants are raised, with the potential for outbreaks of haemonchosis, regard-
less of the climatic zone.
Due to its clinical and economic significance H. contortus is probably the
most studied of ruminant helminths, and the many ecological and
epidemiological studies constitute a vast literature that defines its ecological

adaptability across many different environments. In this chapter, the distri-
bution of H. contortus is considered largely in relation to the availability of
moisture (rainfall) and the typical temperature range in different types of
environments, namely tropical, subtropical, temperate (warm and cool)
and arid regions (summarized in Table 2). Although information is not avail-
able from some regions, taken as a whole, the studies detailed in the
following sections provide an indication of the prevalence of H. contortus
and the severity of haemonchosis in a range of environments.
2.1.1 Tropical and subtropical climates

Due to continually high temperatures, most locations within these climatic
zones consistently support the larval development of H. contortus, and the
presence of this nematode relates almost entirely to rainfall. In the wet tro-
pics and equatorial zones, infective larvae are present on pasture essentially
throughout the year, and haemonchosis is a significant constraint to the
raising of small ruminants (eg, Barger et al., 1994; Chandrawathani, 2004;
Dorny et al., 1995). These regions include tropical Africa, South-East
Asia, tropical Pacific Island countries, Central America and countries in
the northern parts of South America and the Caribbean.
In subtropical and similar environments, seasonal variations in rainfall
largely determine whether H. contortus is a continual or, alternatively, a
routine seasonal threat, as generally high temperatures maintain the potential
for rapid population development. However, there is extensive variation in
the risk for haemonchosis throughout this zone, depending on the relative
length of dry seasons, and, in some cases, on the effects of altitude in moder-
ating temperatures (eg, Githigia et al., 2001; Shillhorn Van Veen, 1978). In
markedly seasonal climates with long and hot dry seasons, during which there
is negligible external survival of infective larvae, hypobiosis of the fourth-stage
larvae (Gibbs, 1986; Michel, 1974) allows H. contortus to survive until more
favourable conditions resume. Regions included are to the north and south
of the true tropics in Africa, Asia and the Americas, including some southern
regions of the USA, central and southern India and the north of Australia.
2.1.2 Warm temperate regions

Haemonchus contortus is a significant seasonal threat in the warmer temperate
climatic zones, as temperatures are sufficiently high to permit development
for several months of the year, and winters not sufficiently severe for a pro-
longed, restrictive effect on infective larvae. The major restrictions are
seasonally dry conditions or droughts, although winter temperatures typi-

cally limit egg development for part of the year, particularly when combined
with altitude. The severity is greatest in summer rainfall regions (Swan,
1970; Veglia, 1915), and whether larval development is constant or sporadic
throughout summer depends mostly on the pattern of rainfall. In predom-
inately winter rainfall areas in this zone, haemonchosis usually occurs more
sporadically but is still a seasonally endemic threat, also depending on the dis-
tribution of summer rainfall. Where significant small ruminant populations
occur, affected regions extend from the tropics to around 35 N and S,
including in southern Africa, much of eastern Australia, parts of southern
USA, mid-regions of South America, southern Asia and Mediterranean
climatic zones in both hemispheres.
2.1.3 Cool temperate regions

Outside the major endemic zones, longer periods of cold conditions restrict
the annual availability of H. contortus, although seasonally dry conditions are
usually less important, as the infective larvae can survive for relatively long pe-
riods. Haemonchosis may be a significant annual, although short lived, threat
where summer temperatures are sufficiently high and sustained, including lat-
itudes >35 S in the southern hemisphere, and >45 N in Europe and north-
ern America and Asia. The risk diminishes as latitude increases, with only
sporadic outbreaks where specific circumstances favour H. contortus develop-
ment. In colder zones, H. contortus is generally of minor and brief significance,
and its annual survival usually associated with hypobiosis (Gibbs, 1986). How-
ever, there are concerns regarding an increasing importance of H. contortus in
regions in which development is constrained to short periods in summer, such
as in northern Europe, Scandinavia and Canada (Waller and Chandrawathani,
2005; Rinaldi et al., 2015). The climate change trends are likely to extend the
range of H. contortus and other parasites (Van Dijk et al., 2010) in all environ-
ments where cold or dry conditions might limit its present significance.
2.1.4 Arid regions

Due to the requirement for moisture for development of the free-living
stages, haemonchosis is of relatively lesser importance in arid zones, but out-
breaks occur in hotter climates where brief seasonal rainfall permits rapid larval
development. This may occur routinely due to the maturation of hypobiotic
larvae after annual dry seasons, such as in southern and sub-Saharan Africa
(Shillhorn Van Veen and Ogunsusi, 1978; Vercruysse, 1985; Viljoen, 1969)
and the Middle East (Altaif and Issa, 1983), or rarely following unusually
protracted periods of rainfall, such as in the dry inland of northern Australia

(De Chaneet and Mayberry, 1978). In many situations, H. contortus infection
probably persists only because anthelmintic treatment is rarely considered
justified, although it may be a threat on irrigated pastures in these zones (Altaif
and Yakoob, 1987; Pullan and Megadmi, 1983). There are few instances
where arid conditions occur in colder climates, but the dual pressures of
extremely dry conditions and low temperatures would severely restrict the
expansion of H. contortus populations (Viljoen, 1969).
2.2 Economic significance

Haemonchosis is recognized as the most economically important parasitic
nematode in its main endemic zones (McLeod, 2004; Perry et al., 2002),
chiefly due to the common occurrence and potential for heavy mortality
rates in small ruminants. Animal losses vary greatly between regions, years
and seasons, depending on environmental conditions and the effectiveness
of control measures, including the impact of anthelmintic resistance. The
immediate economic impact is greatest when animals are managed under
intensive commercial conditions in endemic areas. However, the losses
experienced in traditional livestock systems when small numbers of animals
are run under extensive conditions are proportionately greater at particular
times, and often exacerbated by periods of poor nutrition and the limited
availability and affordability of anthelmintics, as well as anthelmintic resis-
tance (Vatta and Lindberg, 2006).
In a study in an H. contortus endemic area in New South Wales, Australia,
annual mortalities in Merino ewes of >10% were largely attributed to hae-
monchosis on farms with relatively unplanned control practices (Kelly et al.,
2010), although there was a large between-year variation. The mean annual
cost of AUD 11.00/head was associated chiefly (80%) with ewe deaths but
included the control measures (anthelmintics and diagnostic tests) required
to treat and prevent haemonchosis. The impact of chronic H. contortus infec-
tion is difficult to assess, as it is most significant in extensive grazing situations
where routine monitoring is rarely conducted, but Qama et al. (2012) attrib-
uted substantial loss to the reduced value of animal production, and Fabiyi
(1987) reported substantial losses due to mixed helminth infections in a
number of African countries, mostly related to H. contortus. In many cases,
mortalities eventually occur after some months of infection and in associa-
tion with poor nutritional conditions (Allonby and Urquhart, 1975).
Anthelmintic resistance is well established in all major zones endemic for
H. contortus, often precluding the use of entire anthelmintic groups, thus
exacerbating the costs and complexity of control (chapter: Diagnosis,

Treatment and Management of Haemonchus contortus in Small Ruminants
by Besier et al., 2016; chapter: Anthelmintic Resistance in Haemonchus con-
tortus: History, Mechanisms and Diagnosis by Kotze and Prichard, 2016).
3. PATHOGENESIS AND DISEASE

3.1 Pathophysiology and pathogenesis
Haemonchus contortus is by far the most pathogenic of the common
nematodes of small ruminants, due to its blood-feeding activity and its ca-
pacity for rapid population increases during periods and under conditions
favouring the development of the free-living stages. The pathophysiology
of haemonchosis and associated clinical signs are chiefly linked to the
anaemia that develops as a consequence of the blood-feeding activity of
the parasite (Dunn, 1978; Levine, 1980; Urquhart et al., 1996). Blood loss
commences with the development of the fourth-stage larvae (M€ onnig,
1950; Veglia, 1915), with anaemia being first detectable 10e12 days after
infection (Dargie and Allonby, 1975; Hunter and McKenzie, 1982). Indi-
vidual adult worms are estimated to remove 30e50 mL of blood per day
(Clarke et al., 1962; Dargie and Allonby, 1975), and a daily blood loss of
30 mL has been reported in sheep 11 days after infection with 10,000 infec-
tive larvae of H. contortus (see Albers and Le Jambre, 1983). The severity of
disease in the host is closely related to the number of H. contortus larvae that
establish, as there is a strong correlation between blood loss and the number
of adult worms (Le Jambre, 1995). The outcome of H. contortus infection
therefore depends largely on the rate of intake of infective larvae, the ability
of the host to reject them and the capacity to replace lost blood.
Depending on the intensity of infection and the host response, haemon-
chosis has been categorized into a continuum of three general syndromes:
hyperacute, acute and chronic (Dunn, 1978; Urquhart et al., 1996). In
the relatively rare hyperacute form, massive blood loss from infection
with as many as 30,000 H. contortus causes a haemorrhagic gastritis, in
addition to terminal anaemia (Dunn, 1978). Deaths occur suddenly with
no premonitory signs of disease, but with signs of severe anaemia in many
of the survivors. The diagnosis is obvious at necropsy due to very large
numbers of worms of different developmental stages, and numerous obvious
haemorrhages on the mucosal surface.
In acute haemonchosis, significant anaemia develops over a relatively

longer period, but deaths may occur within 4e6 weeks of infection,
depending on the rate of larval intake. H. contortus burdens of 2000e
20,000 worms per sheep may be present (Urquhart et al., 1996), with faecal
worm egg counts (FWECs) of 50,000 eggs per gram (Dunn, 1978). Dargie
and Allonby (1975) defined three stages in the progression of anaemia during
acute haemonchosis, with an initial decrease in packed cell volume (PCV)
over about 6 weeks following infection, and an apparent recovery due to
compensatory erythropoiesis in animals that survived. However, over the
following weeks a dramatic and terminal reduction in PCV can occur due
to exhaustion of the capacity to replace blood cells, due in part to depletion
of the iron reserves. At necropsy, the carcass is pale with marked ascites and
submandibular oedema, reflecting the hypoproteinaemia which also results
from the blood-feeding activity of H. contortus. The blood may be watery
and fail to clot, and the abomasal mucosa is often oedematous with
blood-flecked mucous and obvious signs of parasite attachment. The histo-
pathological changes associated with acute haemonchosis include traumatic
damage to the mucosal surface and evidence of a cellular immunological
response (Hunter and McKenzie, 1982; Silverman and Paterson, 1960).
Infections with smaller but persistent H. contortus burdens have been
characterized as ‘chronic haemonchosis’ (Allonby and Urquhart, 1975;
Dunn, 1978), which may pass unnoticed or become obvious only when
larval intake and, hence, worm burdens increase, or when poor nutritional
conditions reduce the capacity of the host to tolerate the pathogenic effects.
The syndrome was first characterized on the basis of observations made in
Kenya (Allonby and Urquhart, 1975) and in pastoral grazing environments
in Australia, where rainfall is relatively low and variable between seasons,
and small burdens of worms persist (Cobon and O’Sullivan, 1992; De
Chaneet and Mayberry, 1978; Roberts and Swan, 1981). Chronic haemon-
chosis is most common in environments which are marginal for the
development of the free-living stages, or during less favourable periods in
seasonally endemic zones, and is usually accompanied by infections with
other helminths. The chronic form of haemonchosis may also occur where
overt outbreaks are common but partially effective control measures prevent
the emergence of acute haemonchosis.
Nutritional status has a major role in the tolerance of H. contortus infec-
tion, and overt haemonchosis can be precipitated by a reduction in feed
quality. A sharp differential in the tolerance (or resilience) to H. contortus
has been demonstrated in pen-kept sheep on extremely low-protein ratios
compared with those in groups receiving feed supplements (Abbott et al.,

1986a; Nnadi et al., 2009; Wallace et al., 1996), even though there was
no significant change in worm numbers (Abbott et al., 1986b; Wallace
et al., 1996). Loss of milk production might also be associated with small
but chronic burdens of H. contortus in sheep (Thomas and Ali, 1983) and
goats (Nnadi et al., 2009), and partial agalactia in ewes may add to the less
obvious effects of H. contortus infections by reducing lamb growth. The pro-
duction effects of chronic H. contortus infection in sheep on low planes of
nutrition relate to a negative nitrogen balance (Abbott et al., 1985a;
Rowe et al., 1988), and, to a degree, to inappetance (Abbott et al.,
1985a; Holmes, 1987), as commonly occurs for infections with many tri-
chostrongyle species (Fox, 1997). As expected, the benefits of protein sup-
plementation to enhance the resistance and resilience of sheep against H.
contortus infection is greatest in breeds or individual sheep that are more sus-
ceptible to helminthosis (cf. Abbott et al., 1985b; Kahn et al., 2003; Steel,
2003). In addition, the potential for differences in pathogenicity among
H. contortus strains has been implied by results from a number of studies,
including those of Hunt et al. (2008), who reported both genomic and phys-
iological differences between Australian strains, and also Angulo-Cubillan
et al. (2010) who found differences between Spanish and Scottish strains.
3.2 Clinical signs of disease

The clinical signs of H. contortus infection depend upon the number of
haematophagous adult and larvae present in the abomasum, and the varia-
tion in susceptibility among individual animals and, to an extent, on their
nutritional status. The visible evidence presented to livestock owners and
veterinarians varies considerably both over time and within a flock, and a
classic outline of the progression of haemonchosis from inapparent infection
to the commencement of mortalities was originally described by Clunies-
Ross and Gordon (1936; cited in Georgi, 1974). Detailed descriptions of
the clinical signs (and associated pathophysiology) are available in numerous
veterinary and parasitology texts (eg, Bowman, 2014; Dunn, 1978; Levine,
1980; Taylor et al., 2007; Urquhart et al., 1996).
The principal clinical signs in individual host animals relate almost entirely
to the degree of anaemia associated with the size and duration of H. contortus
infection, but this expression is mediated by a number of factors. No age or
class of animal is specifically associated with haemonchosis, although the dis-
ease is probably most common in lambs that have not acquired natural, pro-
tective immunity against helminths. However, it is also seen in lactating ewes
and does (presumably under the influence of the peri-parturient relaxation of

resistance; O’Sullivan and Donald, 1973), and occasionally in helmintholog-
ically naive adult animals which have been newly introduced into an H. con-
tortuseendemic zone. In addition to the very marked natural variation in
immunological responses among individuals within a flock or herd, which
can be exploited for the breeding of naturally resistant (Preston and Allonby,
1979; Woolaston and Baker, 1996) or resilient animals (Bissett and Morris,
1996), there is a large variation among breeds in their resistance to haemon-
chosis (Mugambi et al., 1997; Preston and Allonby, 1979), with a significant
advantage to locally adapted breeds (chapter: Diagnosis, Treatment and
Management of Haemonchus contortus in Small Ruminants by Besier et al.,
2016). As indicated above, the nutritional state of affected animals can have
a significant influence on the expression of haemonchosis (McArthur et al.,
2013), as would concurrent infection with other parasites or other diseases.
In hyperacute cases, sudden deaths occur without prior signs to alert an
owner, but the signs of anaemia typical of acute haemonchosis will be
evident in most individuals that survive. These signs include pallor of the
mucous membranes, most readily seen in the conjunctivae. The close rela-
tionship between colour of the ocular membranes and the degree of anaemia
is the basis of the FAMACHA (FAffa MAlan CHArt; Malan et al., 2001) sys-
tem for assessing the risk for haemonchosis in a group of animals, expressed
as a score of 1e5, ranging from a red-pink (normal) colour to an extreme
white in terminal situations (Van Wyk and Bath, 2002). Affected animals
become progressively weaker with increasing blood loss, and may be less in-
clined to move, or spend more time lying down than usual. On driving,
some will collapse and may die, particularly if repeatedly forced to move
(ironically, often for anthelmintic treatment). At this stage, treatment is often
undertaken, but if the disease progresses, the hypoproteinaemia due to blood
loss may lead to general ventral oedema in a proportion of animals. Subman-
dibular oedema (‘bottle jaw’) is also typically seen, although this sign is not
pathognomonic for haemonchosis, and deaths may occur before it develops.
Diarrhoea is not a feature of haemonchosis, and the faeces are typically firm,
scant and may be dark (due to melaena), although haemonchosis may occur
concurrently with infections with other nematodes that do cause diarrhoea
(Eysker and Ogunsusi, 1980). No pain is evident, but a ‘break in the wool’ of
sheep occasionally occurs, with shedding of strands of wool or even the
entire fleece in recovered or chronically affected animals.
Acutely developing outbreaks of haemonchosis are not immediately
associated with observable animal production losses, but, if allowed to
progress, substantial effects on live-weight gain and (in sheep) wool growth
can occur. Pen studies in New South Wales showed a mean reduction of
38% in animal growth rates after 9 weeks of H. contortus infection in lambs,
which led to clinical haemonchosis, although wool growth loss was not
evident for some weeks, with a mean reduction of only 7% (Albers et al.,
1989). Similarly, in observations on haemonchosis in grazing sheep, also
in New South Wales, no animal production effects were apparent in affected
sheep at the time that mortalities occurred, but with continued infection,
both live-weight gains and wool growth were significantly reduced (Cohen
et al., 1972). However, observations on the animal production implications
for the FAMACHA system indicated that, despite high FWECs of >10,000
eggs per gram in individual sheep, there was little associated reduction in
live-weight, by comparison with sheep drenched at monthly intervals
(Van Wyk, 2008). This information suggests that, if treatment can be pro-
vided when imminent haemonchosis is detected, a significant production
penalty is not inevitable.
In more chronic forms of haemonchosis, signs may be similar to malnu-
trition, seen as weight loss or poor weight gains and general ill-thrift, and a
degree of anaemia in some individuals. Depending on the nutritional status,
minor infections would need to continue for a considerable period before a
significant animal production impact is evident. Barger and Cox (1984)
observed only a small reduction (3%) in the live-weights of yearling sheep
on good pasture over a 12-week period of low-level H. contortus challenge,
and no significant effect on wool production. However, in poorly nourished
animals, chronic H. contortus infection is often associated with a loss in animal
production. In a pen experiment in Indonesia, a daily reduction in live-
weight growth (w30 g per day) was recorded in both sheep and goats
with small burdens of H. contortus (Beriajaya and Copeman, 2006). Similarly,
in pen experiments in the Philippines, Howlader et al. (1997) reported a
reduction of 25% in the growth rates of goats with subclinical haemonchosis.
Losses associated with chronic, subclinical H. contortus infection in grazing
Merino sheep were also reported from an arid environment in inland
Queensland, Australia, with a significant reduction in weight gains and
wool growth in all ages, and a reduction in both the milk yield of ewes
and lamb survival (Cobon and O’Sullivan, 1992). Similarly, continual infec-
tion with moderate worm burdens, predominately H. contortus, led to
reduced live-weight gains in grazing goats in Kenya (Githigia et al.,
2001), with mortalities during seasonal periods of poor nutrition, mostly
in animals with the poorest body condition. As noted previously, it is likely
that unsuspected chronic haemonchosis occurs relatively common in situa-

tions where small burdens are maintained for some months due to limited
larval intake or where partially effective treatment prevents its overt expres-
sion, and that the expression of clinical signs is largely nutritionally mediated.
4. ECOLOGY
The geographical and seasonal distributions of parasitic nematodes
with a free-living component of the life cycle is determined by the effects
of the external environment on the development of eggs through the first-
to third-larval stages, and by the survival of the infective larvae on herbage
(Crofton, 1963; Levine, 1980). For each species, a critical minimum require-
ment for moisture within the faecal pellet and on the herbage determines the
viability of the egg and various larval stages, and development between these
stages occurs at an increasing rate as temperature increases from a minimum
value over a defined range. In general, trichostrongyle eggs either develop to
infective larvae relatively rapidly (within one or more days) or die before
reaching this stage (Crofton, 1963; Levine, 1980; Stromberg, 1997; Veglia,
1915). In contrast, the infective larvae are considerably resilient, and can sur-
vive on pasture for periods of some months, provided that temperatures are
not extreme and moisture is sufficient (O’Connor et al., 2006).
Due to its importance, H. contortus is probably the best studied nematode
of ruminants in relation to ecological factors that determine the viability of
the egg and larval stages. In comparison with other trichostrongyles, such as
Teladorsagia circumcincta and Trichostrongylus colubriformis, the free-living stages
of H. contortus have a more stringent requirement for moisture, a lower toler-
ance of low temperatures, and a greater requirement for and tolerance for
warm temperatures (O’Connor et al., 2006). Investigations to establish crit-
ical values for the development and survival of free-living stages include
in vitro laboratory studies, in which eggs isolated from host faeces or larvae
at various stages can be exposed to controlled environmental conditions, and
field plot studies to indicate the integrated effects of ecological factors,
mostly climatic measurements. Further studies, utilizing grazing animals to
sample pasture contaminated at defined times provide a basis for explaining
the epidemiology of infections in different locations. In all cases, compari-
sons among studies must be interpreted with caution, due to differences
in observation intervals and other procedural variations, and particularly in
relation to older studies, the sensitivity of technology used to measure
environmental variables, as well as for estimations of egg and larval numbers.

The possibility of between-strain variation cannot be discounted as an expla-
nation of varying results, because evidence from direct comparisons suggests
that adaptive responses occur in relation to environmental variation
(Crofton and Whitlock, 1965; Le Jambre and Whitlock, 1976).
4.1 Controlled environment studies

In general, in vivo investigations with the free-living stages of nematodes
indicate the critical range of values for key factors, principally moisture
and temperature, and thus the absolute environmental boundaries for the
occurrence of a particular species. While observations within individual
studies extend only over a predetermined set of values and usually for a single
variable, taken together, numerous studies provide a comprehensive indica-
tion of the specific ecological requirements. A summary of the critical values
for environment variables is provided in Table 1.
4.1.1 Moisture requirements for egg development and survival

Early investigations established the critical requirement for moisture for
development beyond the egg stage, and that embryonated H. contortus
eggs are considerably more resistant to desiccation than eggs that have not
commenced development (Berberian and Mizelle, 1957; Shorb, 1944b;
Veglia, 1915). Most H. contortus eggs die if allowed to desiccate, but the sur-
vival period increases as the relative humidity or faecal moisture content
(FMC) increases (Berberian and Mizelle, 1957; Rose, 1963; Waller and
Donald, 1970), and once eggs have embryonated they hatch rapidly when
exposed to moisture (Silverman and Campbell, 1959; Waller and Donald,
1970). Desiccation tolerance appears to be determined largely by the perme-
ability of the exterior egg membrane to water, as Waller and Donald (1970)
found structural differences between the membranes of T. colubriformis and
H. contortus that related to the greater survival of T. colubriformis eggs at
low humidity values.
The interactions between moisture and temperature for H. contortus egg
development are evident from experiments in which temperature and rela-
tive humidity were cycled; over the relatively high temperature range of
20e35 C, no eggs developed at low humidity levels (70e85%), but most
eggs produced infective larvae at 100% relative humidity (Hsu and Levine,
1977). However, the period of time for which eggs were exposed to low
humidity was critical, as substantial development occurred provided that
low humidity (70%) was maintained for only 12 h before an increase to
Table 1 Effects of environmental factors on the free-living stages of Haemonchus contortus under controlled conditions (Section 4.1)
108
Aspect investigated Environmental factor Optimal conditions (key references) Limiting conditions (key references)
Development and Moisture Relative humidity 100% at 20e35 C Relative humidity <85% in faecal
survival of eggs (in faecal pellets) (Hsu and Levine, pellets at 20e35 C (Hsu and Levine,
(unembryonated) 1977) 1977; Misrah and Ruprah, 1973a;
‘Moist faeces’ (Rose, 1963) Shorb, 1944a,b)
‘Dry air, unshaded’ (Veglia, 1915)
Development and Moisture ‘Shaded faecal pellets’ in dry air Relative humidity <88% at 20 C
survival of eggs (development and survival) (Veglia, (Waller and Donald, 1970)
(embryonated) 1915)
Dry faecal pellets at room temperature
(survival) (Silverman and Campbell,
1959)
Survival of eggs Low temperature 0e4 C (<10 days) (Shorb, 1944a,b; <0 C (Jasmer et al., 1986; Rose, 1964;
(unembryonated) Silverman and Campbell, 1959; Todd et al., 1976b)
Smith-Bujis and Borgesteede, 1986)
Survival of eggs Low temperature 0e4 C (2 months) (Silverman and <0 C (Rose, 1964; Todd et al., 1976b)
(embryonated) Campbell, 1959; Todd et al., 1976b;
Veglia, 1915)
Development of eggs to Temperature (no Rapid development, high proportion Slow development, low proportion of
infective larvae moisture restriction) hatch: w15 C: 4e12 days (Misrah hatch: <8 C, no development
and Ruprah, 1973a; Rose, 1964; (Crofton and Whitlock, 1965;
Veglia, 1915) 22e25 C: 3e7 days Silverman and Campbell, 1959;
(Berberian and Mizelle, 1957; Hsu Veglia, 1915)
and Levine, 1977; Rose, 1964; 10 C: w2e4 weeks (Veglia, 1915)
R.B. Besier et al.

Silverman and Campbell, 1959; 40 C: little/no development
Veglia, 1915) 35e37 C: 3 days (Berberian and Mizelle, 1957; Jehan
(Jehan and Gupta, 1974; Silverman and Gupta, 1974; Veglia, 1915)
and Campbell, 1959; Veglia, 1915)
Haemonchus contortus Infection in Small Ruminants
Survival of infective Moisture (desiccated; Relative humidity 60e90%: w20 C: 8 Relative humidity <50%: >w20 C:
larvae <100% relative e36 weeks (Rose, 1963; M€ onnig, few days to 3 weeks (Ellenby, 1968;
humidity) 1930; Todd et al., 1970; Todd et al., Rose, 1963; Todd et al., 1970)
1976a) w30 C: 1e8 weeks (Todd
et al., 1970; 1976a)
Survival of infective Temperature (no Optimum (>20 weeks, relatively high Minimum (<20 weeks, low
larvae moisture restriction) proportion): 5e10 C: >12 months proportion): <0 C: few days (Jasmer
(Boag and Thomas, 1985; Misra, et al., 1987; Rose, 1963; Todd et al.,
1978; Rose, 1963; Todd et al., 1976b)
1976b) 35e40 C: 1e9 weeks (Jehan and
15e20 C: 32e56 weeks (Boag and Gupta, 1974; Misra, 1978; Sood and
Thomas, 1985; Todd et al., 1976b) Kapur, 1975)
25e30 C: 17e36 weeks (Boag and >40 C: few days (Jehan and Gupta,
Thomas, 1985; Jehan and Gupta, 1974; Misra, 1978; Sood and Kapur,
1974; Rose, 1963; Todd et al., 1975; Todd et al., 1976b; Veglia,
1976b) 1915)
109
100%. Incubator and small plot studies (Khadijah et al., 2013a,b; O’Connor
et al., 2007a,b, 2008; discussed in the following section) confirm the critical
role of moisture for the development of H. contortus eggs to infective larvae
during the short period of time after they are deposited onto pasture.
4.1.2 Moisture requirements for the survival of infective larvae

The remarkable survival capacity of infective larvae of trichostrongyle nem-
atodes on pasture during dry conditions is a key explanatory feature of the
epidemiology of infections in livestock. It has long been noted that the L3
(infective) larvae are relatively resistant to desiccation, and can survive stor-
age in a dry state on glass slides at room temperature (22e24 C) for
2 months (M€ onnig, 1930; Rose, 1963), provided that the relative humid-
ity is sufficiently high. With a low relative humidity but similar temperature
conditions, the survival is lower. Ellenby (1968) found desiccated infective
larvae of H. contortus to survive in vitro for a maximum of 3 weeks at 47%
relative humidity and a temperature of 18 C. The percentage surviving
declines as the relative humidity decreases (Rose, 1963; Todd et al.,
1970), although survival is closely related to temperature: at 60% relative hu-
midity, survival decreased from 64 days at 20 C, to 8 days at 35 C (Todd
et al., 1976a).
The environmental resistance of the infective larva has long been associ-
ated with the larval sheath, which is retained after the second moult. Ellenby
(1968) showed that exsheathed larvae survived for only a few hours
compared with up to 3 weeks when the sheath was retained. Survival is
also enhanced in faecal pellets, with larvae found to be viable in faeces after
256 days at 20 C and a relative humidity of 60%, compared with only
64 days for desiccated larvae on glass under the same conditions (Todd
et al., 1976a). As well as ensuring the presence of at least some moisture,
the faecal mass may moderate the rate of desiccation of larvae. The require-
ments for moisture to enable the migration of infective larvae from the faecal
mass were reviewed (Van Dijk and Morgan, 2011; see Section 4.2.6).
4.1.3 Temperature requirements for the development of eggs to

infective larvae
Provided that moisture conditions are adequate, eggs of H. contortus survive
and develop between about 10 C and <40 C, with maximum hatching
rates (and mortality) at high temperatures, but greatest survival at low tem-
peratures. Under conditions of extreme cold, freshly passed (unembryo-
nated) eggs in faecal pellets are not viable, surviving for only 24 h at 0 C
(Jasmer et al., 1986; Shorb, 1944a,b; Veglia, 1915), and for few days at 4e
5 C (Shorb, 1944a,b; Smith-Bujis and Borgesteede, 1986). The longer sur-
vival of embryonated eggs at low temperatures (2 months at 1.1 to 2.2 C;
Silverman and Campbell, 1959) confirm the environmental resistance of this
stage, although this is likely to be of marginal, practical significance, except
where diurnal temperature fluctuations permit sporadic egg development to
infective larvae. H. contortus eggs are significantly less tolerant of cold tem-
peratures than are the eggs of other major trichostrongyles of ruminants
(McKenna, 1998).
The role of temperature in determining H. contortus development rates is
clearly seen in the effects on the time required for egg hatching. At the
minimum hatching temperatures (8e10 C) reported for eggs incubated in
water, first-stage larvae were observed between 5 and 18 days (Berberian
and Mizelle, 1957; Crofton and Whitlock, 1965; Jehan and Gupta, 1974;
Veglia, 1915), but none developed at 7.2 C (Silverman and Campbell,
1959). In contrast, hatching was rapid at high temperatures: 14e16 h at
37 C (Berberian and Mizelle, 1957; Jehan and Gupta, 1974; Veglia,
1915). This temperature is close to the upper limit for hatching, because
at 40 C, little or no egg development occurs (Misra and Ruprah,
1973a; Todd et al., 1976b; Veglia, 1915).
A similar response to temperatures applies to the development of infec-
tive larvae of H. contortus, with an increasingly short period required as
temperatures increase. Silverman and Campbell (1959) reported that infec-
tive larvae appeared after 11 days at 11 C, 5 days at 21.7 C, and 3 days at
37 C. No larvae develop at extreme temperatures (w40 C) (Berberian
and Mizelle, 1957; Jehan and Gupta, 1974; Misra and Ruprah, 1973a;
Silverman and Campbell, 1959; Veglia, 1915). The consensus from these
reports indicates that optimal development with minimal mortality occurs
over the physiological range optimal for most ruminant nematodes (20e
30 C), provided that moisture is not limiting.
4.1.4 Temperature requirements for the survival of infective larvae

Although infective larvae of H. contortus can survive extremes of moisture
conditions, from desiccation to full hydration, this is largely mediated by
temperature, with limitations closely following the requirements for egg
development. Larvae stored in water at <0 survived for only a few days
(Jasmer et al., 1987; Rose, 1963; Todd et al., 1976b), but for some months
at temperatures close to freezing point, although in very small numbers
(Boag and Thomas, 1985; Misra, 1978; Rose, 1963; Veglia, 1915). Survival
in faecal cultures was longer when larvae were desiccated before storage
(Todd et al., 1976b). The survival of larvae at extremes of high temperature
(in water) was considerably shorter: only 16e33 days at 40 C (Jehan and
Gupta, 1974; Sood and Kaur, 1975).
Provided that moisture is abundant, the range of temperature for larval
survival largely corresponds with that for larval development. Periods
recorded for survival in water at 20 C varied from 140 to 256 days in several
studies (Boag and Thomas, 1985; Jehan and Gupta, 1974; Todd et al.,
1976b), and at 30 C from 91 to 118 days (Boag and Thomas, 1985; Sood
and Kaur, 1975). Although direct extrapolation to field conditions is
tenuous, these findings seem to be consistent with the wide occurrence of
H. contortus in tropical, subtropical and summer rainfall climatic zones.
4.1.5 Intraspecific differences in critical requirements

Reports of differences in egg hatching times between isolates from various
geographical regions when compared under standardiszed conditions sug-
gest the existence of environmentally mediated survival differences. Crofton
and Whitlock (1965) reported a minimum egg hatching temperature of 9 C
for an isolate from the UK, compared with a surprisingly high 15 C for H.
contortus from New York, USA, and maximum hatching temperatures vary-
ing from 36 to 41 C. Similarly, Le Jambre and Whitlock (1976) reported a
variation from 38 to 40 C for New York and Ohio isolates, respectively,
and also related morphological characteristics to temperature preferences.
A more specific link to environment was seen in Australia, where minimum
hatching temperatures varied from >10 C for H. contortus isolates from
warm-climate regions (northern Western Australia and Queensland) to
8 C for those from colder southern locations in Tasmania and southern
Western Australia (Besier, 1992). High-temperature responses also appear
consistent with environmental differences. Although comparisons between
studies must be interpreted with caution, considerable variation in egg-
hatching intervals have been reported, from 3 days for a UK isolate
(Silverman and Campbell, 1959) to 15e16 h for isolates from the USA
and India (Berberian and Mizelle, 1957; Jehan and Gupta, 1974). Similar
variation has been reported for the development of eggs to infective larvae
at high temperatures: 132 h for a US isolate at 37 C (Berberian and Mizelle,
1957) compared with 80 h in an Indian study at the same temperature (Jehan
and Gupta, 1974).
Although further evidence is needed to confirm intraspecific variation in
environmental tolerance, current information would be consistent with an
adaptation strategy that allows H. contortus to extend its geographical range,

particularly to colder environments, and thus explain the ubiquitous distri-
bution of a species of essentially tropical origin. Phenotypic variation within
H. contortus is also reflected in differences in pathogenicity among isolates
(Hunt et al., 2008; Angulo-Cubillan et al., 2010), suggested to relate to
genomic variability (Hunt et al., 2008), and in the capacity for hypobiosis
from distinct climatic regions (Troell et al., 2006). The report of genes pu-
tatively related to desiccation tolerance (Yang et al., 2015) is consistent with
the proposition of a genetic adaptation to environmental differences.
4.1.6 Other environmental factors

In comparison with the critical role of temperature and moisture, evidence
for effects of other ecological factors is limited. A requirement for adequate
oxygenation of faecal cultures under laboratory conditions has been demon-
strated (Shorb, 1944b; Veglia, 1915), but is unlikely to be a significant lim-
itation in the external environment. Different light intensities have been
postulated as a significant factor affecting the viability of larval stages, with
a deleterious effect of ultraviolet (UV) light demonstrated for preinfective
larvae (Conder, 1978; Gupta et al., 1982) and on the infective larval stage
(Krecek et al., 1992; Van Dijk et al., 2009). This is consistent with observa-
tions by Rees (1950), who related the greatest larval recoveries to periods of
low light intensity (although this was also the period of greatest pasture
moisture), and although the effect of UV irradiation would vary diurnally
and depend on the degree of shade within a pasture sward, this may have
an under-recognized effect on the availability of infective larvae to grazing
animals.
4.2 Ecological investigations in the field

In contrast to laboratory-based studies which examine the effects of various
variables under controlled conditions, field plot data integrate the effects of
ambient weather and other environmental variables on the developmental
process. For the usual experimental format, faeces containing undeveloped
nematode eggs are deposited onto small pasture plots sequentially over a
particular period, and then recovered at intervals and assayed to estimate
the numbers of various developmental stages present. The results can indi-
cate the proportion of eggs that yield larvae in relation to the number of
eggs deposited, and the period of survival of infective larvae in relation to
weather conditions and (if measured) the microclimatic conditions at ground
level. Inferences from these studies must be qualified according to the rigour
of the study design, relating mainly to the frequency and duration of depo-
sitions, the efficacy of recovery techniques, and the use of replicates to mini-
mize statistical variation among plots. Nevertheless, a large number of studies
over a wide range of environments provides a basis for explanations
regarding the observed epidemiology of infections and allows predictions
from computer simulation modelling.
Differences in ecological results related to the nature of the measure-
ments taken add some complexity to the interpretation of the data (Levine
et al., 1974). Studies in the laboratory suggest values for environment
parameters directly associated with egg and larval development; in field
plot studies, the majority of recordings are from standard meteorological
monitoring stations situated above ground level. During mid-2010s, data
loggers have been utilized to measure a wider range of variables, with
simultaneous recordings at different points, including within the pasture
microclimate and soil. An additional challenge relates to the measurement
of moisture-related variables; although, in practice, the amount and timing
of rainfall are key parameters, they do not always correlate directly with the
moisture available to the free-living stages at ground level. Factors such as
cloud cover, wind effects and evaporation rate influence the availability of
moisture, as does the nature of soil and composition of herbage. In part,
differences in the results among studies are likely to reflect the effects of fac-
tors not measured in a particular study, and comparisons require careful
consideration.
It is of interest that most studies conducted in tropical regions involved
goats, whereas those in subtropical and temperate regions were largely
with sheep.
4.2.1 Tropical and subtropical climates

Where temperatures are either continually or seasonally high, plot studies
confirm rainfall as the principal limitation on H. contortus development,
and in the true tropics, year-round rainfall permits continual H. contortus
development. For instance, in the wet zone of Fiji, larvae developed in every
month, although they survived for only 5e9 weeks under environmental
temperatures of 25e30 C (Banks et al., 1990). Similarly, in the Caribbean,
a study in Guadeloupe (Berbigier et al., 1990) found high desiccation rates to
limit larval viability under hot conditions (mean temperatures of 29 C),
but with a major difference in larval recoveries and survival periods in rela-
tion to the microclimate (short or tall grass plots) and to the time of the day
when the herbage was collected.
In more seasonal tropical locations, with distinct wet and dry seasons,
larval development is more highly seasonal. For example, in a study in the
Kenyan Highlands (Dinnik and Dinnik, 1958), development occurred
only during warm and wet periods and after rainfall of 8 mm over a 10-
day period. Larval survival was short, despite rainfall (14e65 days at maxima
of 25e29 C), but increased as temperatures decreased, with the annual
onset of dry conditions (Dinnik and Dinnik, 1961). At Hissar, northern In-
dia, infective larvae were found to survive on plots for 2 months during
rainy seasons when daily maximum temperatures were 25e44 C (Misra
and Ruprah, 1972), but even at extreme temperatures (45 C), infective
larvae developed, provided that rainfall occurred at the time of deposition
(Misra and Ruprah, 1973b). A small proportion of eggs yielded larvae during
the dry and cool season (mean minima of 3e8 C).
At different sites in Nigeria, a marked difference was similarly related
to season, with rapid larval development during the rainy season at tem-
peratures of 25e30 C and survival for 2 months in studies in Ibadan
(Okon and Enyenihi, 1977) and Vom (Onyali et al., 1990). During the
dry season, no larvae developed at either of these sites, and larvae from pre-
vious depositions died rapidly as mean temperatures rose to >25 C. This
seasonal pattern was also evident in the recovery of H. contortus larvae
from pasture plots studies in a subtropical region of Pakistan (Islamabad)
(Chaudry et al., 2008). Temperatures were conducive to substantial
development for most of the year (maxima: 22.9e37.0 C, minima:
15.0e24.0 C), but recoveries declined during short periods of cool or
dry conditions. Almost all infective larvae had died within 3 months of
faecal deposition, and in half of this time if deposited prior to or during
the dry season. The effects of dry conditions also seen in studies near Addis
Ababa, Ethiopia, where despite lower mean temperatures associated with
altitude, development was confined to the warm and wet summer season,
with little development during the dry seasons over several months
(Tembely, 1998).
The close relationship between rainfall and the migration of infective
larvae from faecal pellets was shown in plot experiments in San Paulo State,
Brazil (Santos et al., 2012), with the recovery of infective larvae at all depo-
sition times, but the greatest recoveries in the hot and wet summer months
(maxima: >40 C). Migration rates also varied with rainfall; infective larvae
were found on herbage within 24 h of experimental deposition of H. contor-
tus eggs on days when rainfall occurred, but during dry periods, larvae
remained in the faecal masses. The very small proportion of H. contortus
eggs recovered as infective larvae (typically 1e2%) raises a query as to

whether mortality is accelerated under repeated cycles of desiccation and
rehydration, in comparison to the potential for extended survival in relation
to desiccation under colder conditions (anhydrobiosis) (Lettini and
Sukhdeo, 2006).
4.2.2 Warm, temperate and Mediterranean climates

Outside of the subtropical zones, H. contortus development on pasture is
restricted when dry and hot seasons coincide, and sometimes by cooler
winter conditions. This pattern is clearly evident in a classic account of
the life history of H. contortus at the Onderstepoort Laboratory in Pretoria,
South Africa, in a summer rainfall environment. Veglia (1915) noted a
marked difference in development rates in relation to the coincidence of
temperature and moisture on field plots, with little or no development un-
der dry conditions, regardless of temperature, and significant development
only under warm and wet conditions. In the same location, M€ onnig
(1930) showed that larvae survived for 9 months through winter, but for
only a few weeks in summer. Similarly, in the uniform rainfall climate of
Sydney, Australia, Donald (1968) found little or no H. contortus egg devel-
opment on field plots during dry winter periods, although infective larvae
that had developed prior to winter could also survive for some months.
A strongly seasonal pattern was also evident in later studies involving
microclimatic effects on the development of infective larvae of H. contortus
on irrigated kikuyu grass pastures at Pretoria, with strong correlations be-
tween larval recovery and the temperature and relative humidity at ground
level (and also the wind speed and light intensity) (Krecek et al., 1992).
Infective larvae of H. contortus and H. placei were found to survive under a
grass sward for up to 10 and 16 months, respectively (Krecek et al., 1991).
This strongly seasonal pattern is also evident in Mediterranean (winter
rainfall) climatic regions, where the major restrictions on H. contortus are ex-
tremes of heat and dryness during summer, and cool winter conditions are
only a transient limitation. In plot studies in south-west Western Australia,
no development occurred during summer on dry plots with mean
maximum temperatures of 27e30 C (Besier and Dunsmore, 1993a), and
larvae that developed during spring died after a few weeks as temperatures
rose (Besier and Dunsmore, 1993b). The development of eggs ceased for
a few weeks during winter (mean minima of 6e8 C), but infective larvae
survived for some months, including through the winter period. A key
finding was the association of H. contortus larval development with the
presence of actively growing pasture, presumably as an index of the availabil-

ity of moisture at the microclimatic level; almost all depositions on to a dense
green pasture sward yielded infective larvae, compared with only 50% of
those on dry pasture plots, and not in large numbers (Besier and Dunsmore,
1993a). In Mexico, under similar temperature conditions, but in a summer
rainfall climate, Fermindez-Ruvalcaba et al. (1994) found greatest larval
development in autumn, when both rainfall and temperature were begin-
ning to decline, indicating the lethal effect of high temperatures, even
when moisture was available, as occurs in true tropical zones.
A number of investigations in northern New South Wales, Australia,
confirm the critical importance of moisture at the time H. contortus eggs
are deposited onto pasture during periods of high temperatures (O’Connor
et al., 2007a,b; 2008). During the summer period, with a range in daily air
temperatures from w10 to 36 C, simulated rainfall on to pasture plots led to
the development of infective larvae when this occurred over the first few
days following deposition, but not with a single rainfall event, regardless
of amount (O’Connor et al., 2007a). In controlled incubator studies at
similar temperatures, but under conditions of low evaporation (2 mm/
day), larval development increased with rainfall amount, and correlated
with the cumulative precipitation/evaporation (P/E) ratio (O’Connor
et al., 2007b). A further study (O’Connor et al., 2008) confirmed the lower
percentage of infective larvae developing under high evaporation rates,
regardless of the amount or distribution of simulated rainfall. From these
results, it is suggested that moisture indices other than rainfall might correlate
most closely with developmental success of H. contortus, with a P/E ratio of
1.0 required for the first 4 days following egg deposition on to pasture.
Similarly, in Texas, USA, Amaradasa et al. (2010) found a close correlation
between rainfall and larval recoveries from herbage, particularly when it
rained on the day of sampling, or on days immediately before.
4.2.3 Cool, temperate climates

A different pattern is seen in higher latitudes, where H. contortus is closer to
the edge of its geographical range, and the temperature becomes relatively
more important. Development is largely confined to the warmer summer
months, provided that the rainfall is adequate, although infective larvae of
H. contortus that develop during the colder months may remain viable for
considerable periods. In the temperate climate of New Zealand (North
Island), the results of plot studies appear to be consistent with the relatively
minor significance here of haemonchosis, with poor or zero recoveries of
infective larvae after faecal egg depositions for periods of 5e6 months, when
the mean air temperature is close to or 10 C (Waghorn et al., 2011). The
mean daily temperature was recognized as the most significant variable for
the development of H. contortus, although rainfall during the first 14 days af-
ter faecal deposition was also required (Reynecke et al., 2011).
In the more extreme climate of Beltsville, Maryland, USA, plot studies
(Dinaburg, 1944a) found eggs of H. contortus to produce infective larvae
only when the mean maximum temperature was >18.3 C (65 F), later
referred to as the ‘Dinaburg Line’ (Kates, 1950) to indicate the lower limit
of temperature for development. Larvae survived for 3 months during spring
(maximum temperatures: 17e23 C), but for only 2 weeks in summer
(maximum temperature: >30 C; Dinaburg, 1944b). Very few infective
larvae survived over winter in this environment (mean maximum tempera-
tures occasionally <0 C). In the cold winter climate of Illinois, USA (mean
monthly temperatures: <5 C), eggs developed to infective larvae only for
6 months of the year, but development proceeded when mean temperatures
ranged between 10 C and 25 C, provided that the monthly rainfall was
>50 mm (Levine, 1980). Similarly, development of H. contortus eggs failed
in winter in southern England (Weybridge), with greatest larval recoveries in
summer when mean temperatures varied from 8 C to 13 C (minima) to
14e25 C (maxima) (Rose, 1963). However, some infective larvae that
developed in summer were recorded as surviving for 40 weeks (Rose,
1963; Gibson and Everett, 1976). The critical importance of moisture was
later confirmed; H. contortus larvae developed only on plots that were either
consistently watered during summer, or where rainfall occurred for some
time after deposition (Rose, 1964). An effect of microclimate was apparent
in the greater larval development on tall (20 cm) herbage compared with on
shorter (5 cm) pasture.
4.2.4 Arid regions

Plot studies in regions with prolonged or continual hot and dry conditions
confirm the presence of H. contortus only during brief periods of seasonal
rainfall, or on irrigated pastures. A study in Utah, USA (Bullick and
Anderson, 1978) showed considerably greater recoveries of infective larvae
of H. contortus from irrigated than dry plots, with few larvae on the latter able
to migrate from faecal pellets on to dry herbage. In central Iraq, a pattern
similar to that of Western Australia (Besier and Dunsmore, 1993a) was
shown, with no larvae produced during summer, with maximum tempera-
tures of >40 C, but some development after autumn rains, although the
survival periods of infective larvae decreased as temperatures increased (Altaif

and Yacoob, 1987).
4.2.5 Effect of microclimatic factors on larval development

Although difficult to measure objectively, the microenvironment within the
herbage sward has a direct effect on strongylid nematode eggs and larvae;
data on this aspect, if quantifiable, would provide useful predictions of
developmental success in a particular circumstance. Thus, laboratory
investigations are likely to better define relationships between H. contortus
development and environmental factors than standard weather station mea-
surements. Factors, such as pasture height and density, will affect the local
microclimate. In addition to the presence of shade from trees and shrubs,
such environment-modifying factors are likely to explain differences in
the development of infective larval on a particular pasture or field.
Differences in temperature between the biome occupied by the free-
living stages and the air can be marked. Two studies (Bailey et al., 2009;
O’Connor et al., 2007a) reported consistently lower maximum air temper-
atures of 15 C in a weather station compared with those at ground level
during spring and summer, although, interestingly, there was virtually no
difference in minimum temperature values between the points of measure-
ment. Temperatures on irrigated pastures are also likely to be lower. For
instance, in Utah, USA, Bullick and Anderson (1978) recorded air temper-
atures under vegetation of 26 C on irrigated plots compared with 35 C on
dry pasture. Similarly, daily maximum temperatures under long high
(15 cm) pastures can be up to 5e10 C lower than under short (3 cm) pas-
tures (Sakwa et al., 2003). Within faecal pellets, temperatures during hot sea-
sons are typically considerably higher than in the air (up to 60 C when
weather station air temperatures are 35 C; RB Besier, unpublished data).
The most consistent relationships between microclimate and nematode
development are likely to involve the faecal pellet and the adjacent soil.
An early report (Veglia, 1915) in South Africa indicated that moisture within
4 days after faecal deposition was important for successful development to
infective larvae, a finding that was confirmed in a detailed investigation in
northern New South Wales (O’Connor et al., 2008). FMC during the
4 days after faecal deposition on to pasture was closely correlated with larval
development, which was negligible when FMC declined to <10%. From an
earlier study in Nouzilly, France, Rossanigo and Gruner (1995) suggested a
minimum FMC requirement of 39% for the development of infective larvae
of H. contortus, although a subsequent Australian study (Khadijah et al.,
2013b) found development at a considerably lower FMC, possibly due to

diurnal temperature fluctuations. A threshold effect of FMC, mediated by
(simulated) rainfall and relative humidity, was also demonstrated for larval
migration from faecal matter in laboratory studies in the UK (Wang et al.,
2014), where migration occurred at an FMC of 70%, but was negligible
at 10%. These authors concluded that rates of faecal desiccation and rehydra-
tion on pasture could explain temporal patterns of larval availability, and that
sheep faeces may act as a larval reservoir in dry conditions, with peaks of
infection following rainfall.
Investigations have also highlighted the role of soil moisture, presumably
through transfer to faecal pellets. H. contortus larval development has been
shown to be as great on plots contaminated on the day prior to simulated
rainfall as it was on plots watered on the 2 days immediately beforehand
(Khadijah et al., 2013a), and correlated closely with FMC. The interaction
between rainfall and soil moisture was also evident, as increasing the daily
simulated rainfall on dry soil from 12 to 24 mm led to a significant increase
in both soil moisture and FMC, but had little effect when the soil was
already moist (Khadijah et al., 2103b). This explains earlier field observations
from the East Cape region of South Africa, which indicated that the avail-
ability of H. contortus infective larvae varied over a property according to the
wetness of the ground (McCulloch et al., 1984); it is perhaps surprising that
this correlation had not been empirically established earlier.
4.2.6 Lateral and vertical migration of infective larvae

The migration of nematode infective larvae both laterally from faecal masses
and vertically onto the herbage is the essential final step in the ecology of the
free-living stages (reviewed by Van Dijk and Morgan, 2011). Larval move-
ment is subject to specific behavioural characteristics, and with directional
movement at random provided energy reserves are adequate, and moisture
and temperature determinants permit (Crofton, 1954).
Provided microclimatic factors are in the optimal range for larval survival,
the availability of moisture largely determines the rate and distance of migra-
tion. Moisture requirements were indicated in investigations in Bristol, UK,
using faecal masses isolated on sieves (Wang et al., 2014). At temperatures of
25e27 C, a single simulated heavy rainfall event (20 mm) within the first
few days of faecal deposition was not sufficient to permit migration, but
significant larval emergence occurred when light ‘rain’ (2 mm) over several
days coincided with high relative humidity (98%), although this was reduced
at lower levels of humidity (30e50%). It appears that, within moisture
constraints, infective larvae migrate from the faeces shortly after their devel-
opment, with maximum recovery rates reported to be within w24 h
(Crofton, 1948; Silangwa and Todd, 1964; Van Dijk and Morgan, 2011).
From various accounts and their own laboratory work, Van Dijk and
Morgan (2011) estimated that, at a constant air temperature of 20 C and
adequate moisture conditions, the numbers of larvae would remain negli-
gible after 3e4 weeks, although diurnal temperature fluctuations would
alter this time period in different seasons. A similar rapid depletion of the
faecal reservoir (2e4 weeks) during warm months was reported earlier
from pasture plot investigations in southern England (Rose, 1963), but small
numbers continued to migrate for >4 months from depositions under colder
winter conditions. A desiccated faecal mass can function as a reservoir for
infective larvae of trichostrongyles (Amaradasa et al., 2010; Stromberg,
1997), with a mass release of larvae after rainfall. It is also apparent that infec-
tive trichostrongyle larvae can remain within the soil or in the vegetation
mat at the soil surface, also providing a potential larval reservoir (Amaradasa
et al., 2010; Krecek et al., 1991; Rose and Small, 1985; Stromberg, 1997;
Van Dijk and Morgan, 2011). The rapid release of significant numbers of
infective larvae onto pasture from a desiccated faecal mass following periods
of rainfall after dry conditions would explain the sudden outbreaks of hae-
monchosis a few weeks later. However, where dung masses disintegrate after
heavy rainfall, there is little opportunity for a reservoir function.
In a study of the lateral migration of H. contortus on grass plots (unspec-
ified species) in Illinois, USA, Skinner and Todd (1980) found >90% of
infective larvae within 10 cm of the faecal mass and few beyond 20 cm,
and that migration essentially ceased during ‘hot, dry weather’. The require-
ment for moisture is at least as pertinent for the vertical migration of infective
larvae, as the microclimate becomes less favourable (drier, and a more vari-
able temperature) with the increasing height of pasture herbage. Most
studies of vertical migration of trichostrongyle larvae (not necessarily H. con-
tortus) found w90% of larvae within 5 cm of the ground in grass swards, and
very few above 20 cm (Crofton, 1948; Rees, 1950; Silangwa and Todd,
1964), although this varied with factors affecting the microclimate, such as
the herbage height (Amaradasa et al., 2010; Berbigier et al., 1990; Rose,
1964); the latter authors also considered the physical capacity of the foliage
to retain moisture to affect migration. Although Krecek et al. (1991) found
no difference in larval recovery in relation to the height (above or below 5e
7 cm) of kikuyu grass pasture, their study was conducted on irrigated plots,
where moisture was not limiting. There has long been a contention over
whether free water (rainfall, mist or dew) is essential for larval migration, but
from their own experimentation and a review of other findings, Van Dijk
and Morgan (2011) concluded that a high relative humidity is sufficient.
The diurnal pattern of larval recovery observed by Rees (1950) and Aumont
and Gruner (1989) was greatest in the early morning, presumably reflecting
considerable moisture availability, as dews, or high relative humidity at this
time, before the temperature increase during the day.
5. EPIDEMIOLOGY
Sequential observations of worm burdens in grazing animals indicate
relationships between nematode development and environmental factors,
and provide an epidemiological context. These investigations include: (1)
structured studies using ‘tracer’ animals grazing small pasture areas contam-
inated with worm eggs at specific times; (2) worm counts from flocks or
herds grazing continually contaminated pastures and (3) abattoir surveys.
Total worm counts from grazing animals also indicate the presence of hypo-
biotic larvae and their relative importance as a survival mechanism during
adverse environmental conditions. Studies based only on FWECs are not
included here, as the results are not necessarily indicative of the actual
worm burden. In the majority of studies of grazing animals or from abattoir
surveys a number of nematode species were recorded, and while interspecies
competitive effects may affect the worm numbers recovered, the comments
here relate only to H. contortus. Not all studies aimed to define the epidemi-
ology of H. contortus over the course of an entire year, and the frequency and
rigour of observations varies greatly. However, overall, they provide a good
understanding of seasonal effects on the annual pattern of worm burdens for
a range of environments, as the basis for locally applicable control pro-
grammes (Barger, 1999). A summary of the major ecological influences
and epidemiological features of H. contortus infections for each climatic
zone is provided in Table 2.
5.1 Tropical and subtropical regions

When temperatures are continually adequate for H. contortus development,
studies of worm counts in sheep have confirmed their close relationship with
rainfall patterns. In the wet tropics where rainfall occurs throughout the
year, studies in Malaysia using ‘tracer’ goats found no seasonal cessation of
H. contortus development, or a relationship with rainfall (Ikeme et al.,
Table 2 Ecological features and epidemiology of Haemonchus contortus infections in small ruminants in different climatic zones
Ecological features (References:

Climatic zonea Section 4.2) Epidemiology (References: Section 5)
Tropical, subtropical zones (tropical Temperatures are sufficiently high to Haemonchosis is a continual threat in the
Africa and the Americas, southern and permit the development of infective wet tropics, as larval populations
South-East Asia, tropical Pacific islands, larvae year-round, but these typically develop rapidly, and animals can remain
Central America, southern states of the survive on the herbage for only a continually infected. Where annual dry
USA, the Caribbean, the north of relatively short period (weeks). The seasons occur, larval availability is
Australia) availability of moisture is the key seasonal and confined to the wetter
determinant of larval occurrence, with months, with the highest burdens
little development and short survival associated with higher rainfall. Survival
periods during dry seasons. In high- as hypobiotic fourth-stage larvae occurs
altitude regions in these zones, larval routinely in seasonally dry
development and the period of survival environments, but is of minor
increases during cooler winter periods, importance or has not been reported
provided sufficient moisture is present. where infective larvae are present for
prolonged periods of the year.
Warm temperate and summer rainfall The coincidence of warm and wet Haemonchosis is a major health threat
zones (from the tropics to w35 N and conditions during summer favours the during the warmer months, although
S, including regions in southern Africa, development of infective larvae in the risk is either constant or sporadic,
higher-rainfall eastern Australia, parts of summer and adjacent months, although dependant on rainfall. Where winter
southern USA, mid-regions of South development may cease during temperatures are mild, the availability
America, southern and eastern Asia) prolonged dry periods. Development of infective larvae to livestock is less
during other seasons is dependent on sharply seasonal than in subtropical
both rainfall and temperature, and is zones. In regions with a relatively cold
limited or may cease under cold winter winter, the pattern of infection is more
conditions, such as occurs in high- seasonal. The occurrence of hypobiosis
altitude regions. appears to be variable and is mainly
associated with the avoidance of cold
123
winter periods.
(Continued)
124
Table 2 Ecological features and epidemiology of Haemonchus contortus infections in small ruminants in different climatic zonesdcont'd
Ecological features (References:
Climatic zonea Section 4.2) Epidemiology (References: Section 5)
Mediterranean climatic zones (the The hot, dry summer conditions prevent Annual infection patterns in livestock are
Mediterranean region, south-west the development or survival of infective typically bi-phasic, with the largest H.
Cape of South Africa, south-west of larvae during, and in much of this zone, contortus burdens developing from late
Western Australia, some regions in winter conditions are too cold for eggs autumn to early winter, and then from
south-east Australia) to hatch. Larval populations are late spring to early summer. The risk of
therefore largest in autumn and spring, haemonchosis varies considerably
although where winters are relatively between years and between locations
mild there is some survival of infective within this zone, depending mostly on
larvae over winter. the rainfall. Hypobiosis is reported as
either absent or variably important, and
appears to be related to the length and
severity of summer conditions.
Cool and cold temperate zones (above Low temperatures are usually a greater Haemonchosis is typically an occasional or
latitudes 40e45 N and S, including restriction on the development of rare occurrence, and restricted to the
northern Europe, Britain, Scandinavia, infective larvae than the availability of warmer months, due to the short
northern USA and Canada, south-east moisture. Development usually ceases periods of larval development and
Australia, New Zealand) completely during winter in this zone, hence availability for ingestion.
and in higher latitudes there is little or Hypobiosis is usually the major factor in
no survival of larvae through winter. overwinter survival, with the arrested
Larvae that develop when temperatures development of the majority of
R.B. Besier et al.

are adequate typically survive for some infective larvae ingested in autumn in
months until the onset of very cold frigid zones. However, under
conditions, although development may favourable weather conditions, rapid
be sporadic during dry periods in development during short periods of
summer. high summer temperatures can lead to
haemonchosis.
Arid zones (desert and very low rainfall Lack of moisture is a critical limitation, Haemonchus contortus is typically absent or
regions of southern and sub-Saharan restricting larval development to short present in livestock in very low
Africa, the Middle East, inland periods of sufficient rainfall, often numbers, and haemonchosis is rare.
Australia) varying considerably between years. However, unusually heavy and
When infective larvae develop, these prolonged rainfall may lead to short
usually survive on pasture for short periods of larval availability that
periods only. Larvae can develop on occasionally result in significant
irrigated pastures, but in many regions burdens of H. contortus. Hypobiosis
within this zone, constantly high occurs routinely in some locations but is
temperatures limit their survival. usually of limited consequence given
the hostile external environment.
Haemonchosis is a potentially greater
risk on irrigated pastures, but high
temperatures often limit the persistence
of large larval populations.
a
Regions listed are indicative of type environments based on published reports, and are not inclusive of all locations in a particular zone where H. contortus may exist.
125
1987; Dorney et al., 1995; Cheah and Rajamanickam, 1997; Chandrawa-

thani, 2004).
In tropical and subtropical zones with both wet and dry seasons, and
nonlimiting temperatures (mean monthly maxima typically >30 C), the
annual pattern and, to an extent, the amount of rainfall is closely related
to H. contortus abundance. Studies in a range of environments in tropical
Africa have indicated the sharply seasonal pattern of heavy burdens during
rainy seasons, in some cases with two peaks each year, and abrupt declines
after the sudden onset of the dry season. These investigations include
some from Cameroon (Ndamukong and Ngone, 1996), Ethiopia (Sissay
et al., 2007), Ghana (Agyei, 1997; Blackie, 2014), Kenya (Nginyi et al.,
2001), Nigeria (Anene et al., 1994; Bolajoko and Morgan, 2012; Chiejina
et al., 1988; Fakae, 1990; Nwosu et al., 2007; Shillhorn van Veen and
Ogunsusi, 1978), Senegal (Vercruysse, 1983), Tanzania (McCulloch and
Kasimbala, 1968) and the Gambia (Fritsche et al., 1993). The numbers of
adult H. contortus recovered at slaughter were considerably higher where
annual rainfall was high (>1000 mm; Ogunsusi, 1979; Fakae, 1990)
compared with low rainfall regions (300 mm; Fabiyi, 1973; Vercruysse,
1983). In Pernambuco state, Brazil, Charles (1989) found grazing goats to
be continually infected with H. contortus throughout a dry season of several
months, but new infections from pastures were largely confined to the rainy
season. Where altitude moderates conditions in this zone, however, the
seasonal pattern of H. contortus infection also appears to reflect lower winter
temperatures (mean monthly minima typically <10 C), with higher worm
recoveries than in summer, for example, in Kenya (Allonby and Urqhart,
1975) and Zimbabwe (Grant, 1981; Pandey et al., 1994).
Most published reports (Eysker and Ogunsusi, 1980; Fakae, 1990;
Fritsche et al., 1993; Gatongi et al., 1998; Sissay et al., 2007; Vercruysse,
1985) indicate that H. contortus survives throughout the dry season as
hypobiotic fourth-stage larvae, although hypobiosis appears to be absent
or less important if the dry season is short (Agyei et al., 1991; Chiejina
et al., 1988; Okon and Enyenihi, 1977; Pandey, 1990). As would be
expected, there is the potential for a continual H. contortus transmission
risk under constantly high-temperature conditions in monsoonal climates,
but with seasonal fluctuations in burdens according to rainfall. In Haryana,
northern India (mean maxima of 20e40 C), a slaughter study (Gupta et al.,
1987) found that H. contortus burdens related to local rainfall at different
locations, although the practice of irrigation maintained H. contortus
throughout seasonally dry periods, without evidence of hypobiosis.
5.2 Warm, temperate climates

Grazing studies confirm the potential for rapid population increases in
warmer temperate zones during periods when moisture is not limiting.
Under these conditions, haemonchosis is a major seasonal risk. In a study
conducted in Louisiana, USA, where rainfall occurs throughout the year
and temperatures are high for most of the year, H. contortus was recovered
from tracer sheep in all months of the year (Miller et al., 1998). A decrease
in recovery during winter was associated with cooler conditions (mean
minima of <10 C for 4 months); the highly variable pattern of recovery
of hypobiotic larvae suggested that under these climatic conditions arrested
development in the host animal was not necessary to ensure the survival of
H. contortus. However, a marked seasonal effect of cold winter conditions
was reported from other locations where the severity of haemonchosis is a
significant limitation on sheep production. In the summer rainfall region of
northern New South Wales, Australia, H. contortus development occurred
when mean temperature maxima exceeded 18 C (Southcott et al., 1976),
and heavy burdens were recovered from grazing lambs during summer
when rainfall coincided with warm temperatures (mean maxima of
25 C). However, few or no H. contortus larvae were acquired from plots
contaminated during winter, and most of the worms recovered from the
sheep during this period were as hypobiotic larvae. The poor development
in winter at this location was confirmed in later tracer sheep studies, with
negligible H. contortus worm recoveries in months when the mean minima
were consistently <5 C (Bailey et al., 2009). In a similar environment in
inland southern Queensland, Swan (1970) considered H. contortus to
require monthly minimum temperatures of >10 C, but hot summer
conditions allowed a rapid population ‘recovery’ from poor winter
development.
An even more seasonal pattern was evident in the Mediterranean climate
of southern Western Australia, with negligible larval infection from winter
egg deposition and a total cessation of intake over the hot dry summer
period (De Chaneet and Mayberry, 1978). However, the development of
lethal H. contortus burdens in tracer lambs in the warm spring months, and
a limited peak in autumn, attest to the ability of H. contortus to capitalize
on short periods when conditions are favourable for rapid development.
In this region, there is presently no evidence of hypobiosis in H. contortus
(RB Besier, unpublished findings), consistent with its absence in sheep
and goats in an abattoir study in the Nile Delta, also a Mediterranean climate
(E1-Azazy, 1990). However, in the Ebro Valley in Spain, under broadly

similar climatic conditions, Uriarte and Valderrabano (1989) recovered a
high proportion of hypobiotic larvae of H. contortus in both spring and
autumn from tracer lambs.
In South Africa, H. contortus has been shown to survive virtually
throughout the year in sheep in temperate and higher rainfall regions,
including the South-West Cape (Muller, 1968) and the Eastern Province
(Rossitter, 1964), with highest counts in summer. In the winter rainfall
region of Stellenbosch, development was poor during the winter months,
but H. contortus remained present throughout the year in sheep grazing
perennially green pastures (Reinecke et al., 1987). In drier climates, cold
winters further limit the period for effective H. contortus development,
with haemonchosis essentially confined to summer in the South-East
Cape (Barrow, 1964), particularly on kikuyu grass and irrigated pastures.
Expansions of H. contortus populations were recorded following periods
of summer rainfall in the dry Eastern Cape region (McCulloch and
Kasimbala, 1968).
5.3 Cool temperate climates

In cooler zones, where summer temperatures are warm rather than hot,
variably cold winter conditions are a critical limitation on H. contortus
development. Hypobiosis is usually a routine survival mechanism,
although apart from regions where extremely cold winter conditions
prevail, its importance in comparison to survival as the free-living stages
on pasture is not clear. In the North Island of New Zealand, development
appears to be restricted by cold winter periods (mean air temperatures of
<10 C), despite abundant moisture. In studies by Brunsdon (1970) and
Vlassoff (1973), H. contortus was recovered throughout summer, but its
availability was limited by sporadic dry conditions, and recoveries of adult
worms in lambs peaked in autumn. Hypobiosis has been demonstrated to
occur variably, but is not the sole mechanism for survival during winter
(Brunsdon, 1973; McKenna, 1974).
In colder climates, however, where mean minimum winter tempera-
tures are <0 C for some months, development is usually confined to
summer and autumn. Hypobiosis is an essential requirement for overwin-
tering in these regions, with 100% of ingested infective larvae entering an
arrested state in extreme climates (Waller et al., 2004). In the Northern
Hemisphere, numerous studies have confirmed the negligible develop-
ment of eggs and limited survival of infective larvae of H. contortus over
winter, in Canada (Alayew and Gibbs, 1973; Falzon et al., 2014; Mederos
et al., 2010), England (Connan, 1971; Gibson and Everett, 1976; Thomas
and Waller, 1979), France (Kerboeuf, 1985), Norway (Domke et al., 2013;
Helle, 1971), Sweden (Troell et al., 2005; Waller and Chandrawathani,
2005), and northern zones of the USA (Grosz et al., 2013; cf. Levine,
1980). Haemonchosis outbreaks, with considerable animal mortalities,
can occur occasionally following the development of hypobiotic larvae
to adult worms in spring (Sargison et al., 2007), but in the more extreme
climatic regions, the almost total dependence of H. contortus on hypobiosis
for survival over the winter period has raised speculation regarding the
feasibility of eradication of H. contortus (Domke et al., 2013; Waller
et al., 2006).
5.4 Arid climates

Despite the adverse conditions for larval development, grazing studies
confirm that despite temperatures as high as 40e45 C, H. contortus is
commonly present in very low rainfall zones in tropical and warm
temperate climates, although rarely in large numbers. In trials at three
different localities in the Karoo of South Africa, with maximum tempera-
tures of up to 40 C, the numbers of H. contortus recovered by Viljoen
(1969) from tracer lambs decreased as rainfall declined, with negligible bur-
dens in the severely arid Klerefontein region. Similarly, recoveries of H.
contortus were generally minimal in the Kalahari (Biggs and Anthonissen,
1982); although relatively heavy burdens were found in sheep following
only 25 mm of rainfall within a month, it was considered that the distribu-
tion of rainfall was more important than the total. Haemonchus contortus was
not found in sheep on dry pastures in Libya (Pullan and Megadmi, 1983) or
Iraq (Altaif and Issa, 1983), but small numbers were recovered on irrigated
pastures, despite maximum temperatures of 40 C (Altaif and Yakoob,
1987). Very small H. contortus burdens were found in extensively grazed
sheep and goats in the extreme climate of Saudi Arabia (summer maxima
of 45 C), and a degree of hypobiosis was evident in the autumn and sum-
mer months (El Azazy, 1995).
Presumably H. contortus populations are maintained through brief periods
of larval development when the rainfall is adequate. Local eradication of
H. contortus may be feasible in environments marginal for H. contortus
(Le Jambre, 2006), provided that treated flocks could be kept separate and
alternate hosts are not present, although in many cases the effort may not
be warranted.
6. PREDICTION OF THE OCCURRENCE OF

H. CONTORTUS
6.1 Predictive models
Attempts to define regions where H. contortus is likely to present a
threat to the livestock industries led Gordon (1948) to develop the concept
of ‘bioclimatographs’, which indicate the monthly suitability for the devel-
opment of infective larvae of H. contortus in a particular location, on the basis
of long-term climatic averages in relation to limiting temperature and rainfall
requirements. The initial models used the mean monthly maximum temper-
ature of 18 C, as proposed by Dinaburg (1944a,b), and a monthly rainfall of
51 mm, and were later modified by Swan (1970) to include a lower mean
monthly minimum of 10 C in environments with major diurnal tempera-
ture fluctuations. These criteria have proved generally suitable for indicating
the haemonchosis risk in different localities in Australia (Donald et al., 1978;
Southcott et al., 1976), although outbreaks not consistent with predictions
have occurred in some regions (De Chaneet and Mayberry, 1978). Levine
(1963) also considered bioclimatographs to provide a general indication of
the seasonal and geographical distribution of H. contortus, but indicated
that they are not sufficiently sensitive for predictions regarding specific loca-
tions, years or pasture conditions.
Since 1980s computer simulation models to indicate nematode popula-
tion changes for various locations have been developed based, initially based
mostly on the response of the free-living stages to weather inputs (eg, Barger
et al., 1972), and later including more complex factors such as host immuno-
logical effects (Barnes et al., 1995; Smith and Grenfell, 1994; Thomas, 1982;
Leathwick et al., 1992). Simulation models have evolved further to explore
strategies to avoid the development of anthelmintic resistance, including in
H. contortus (Dobson et al., 2011; Learmount et al., 2006; Van Wyk and
Reynecke, 2011). These models mostly utilize standard weather station ob-
servations, because their prime purpose is not to indicate the relative suitability
for egg and larval development within specific localities. However, the poten-
tial for using microclimatic factors to increase between-site sensitivity has been
recognized (Leathwick, 2013), including a combination of ecological, cli-
matic and topographical inputs (Van Wyk and Reynecke, 2011). Greater
sensitivity would be of significant value for predictions of the epidemiology
of H. contortus in regions where few studies have been conducted to date,
and of changes over time to its importance in different climatic zones.
6.2 Potential effects of climate change

Given the key role of climatic factors in determining the present distribution
of helminths with a free-living phase, permanent changes to climate will
affect the global occurrence and significance of parasitic helminths. For H.
contortus, the length of periods favourable for development and transmission
are likely to increase with trends towards either warmer conditions in cool
temperate zones, or for greater or less seasonal rainfall in presently drier re-
gions. An increased risk of haemonchosis is particularly great due to the high
biotic potential, which allows H. contortus to take advantage of even rela-
tively small changes to external conditions (Van Dijk et al., 2010).
Indications of climate-mediated changes are becoming apparent. Unusu-
ally high H. contortus burdens in lambs in 2003 were reported from the
Netherlands in association with relatively warm and dry conditions, and
an apparent change to the usual pattern of hypobiosis (Eysker et al.,
2005). An increase in outbreaks of haemonchosis has been reported from
Scotland, where H. contortus exists but has rarely led to clinical disease
(Kenyon et al., 2009; Sargison et al., 2007). In the UK, a survey of case re-
ports of haemonchosis indicates an increasing prevalence of H. contortus
infection over the past 30 years, and the fact that the increase has been
chiefly in northern regions, and during autumn, suggests that the parasite
has especially benefited in regions where thermal energy was most limiting
(Van Dijk et al., 2008). It was postulated that a greater proportion than usual
of the ingested larvae that enter hypobiosis were likely to develop to adult
worms, and, significantly, that there is no evidence that summer conditions
were becoming more hostile for transmission.
Mitigating the potential effects of climate changes will firstly require an
estimation of likely changes to the geographical range where significant
burdens of pathogenic helminths may develop, and the extent and season-
ality of an increased risk of disease. Where a significantly altered risk exists,
changes to present animal management practices may be necessary to avert
increased losses caused by parasites (Morgan and Van, Dijk, 2012).
Computer simulation models, suitably adapted to the local ecology of H.
contortus, will be important tools for understanding the epidemiology of
infection under changing climatic conditions, and for developing recom-
mendations for sustainable control in different environments. As with
some other parasites critically dependent on conditions in the external envi-
ronment (Polley et al., 2010), changes to the geographical distribution of H.
contortus may prove to be a useful indicator of long-term climatic changes.
7. CONCLUSIONS
Of all common nematodes of small ruminants, H. contortus has the
greatest capacity for serious pathogenic effects on a large scale and over a
wide climatic range. The expression of haemonchosis varies with the envi-
ronment and nature of the animal enterprise, from the rapid development of
heavy H. contortus burdens and potentially extensive mortalities within a
short time period, to more chronic forms, where smaller burdens are toler-
ated for long periods but become fatal when the nutritional status declines.
Whether the costs due to haemonchosis are due chiefly to heavy mortalities
and treatment costs, or to reduced animal production and occasional mor-
talities, H. contortus is generally considered the most economically significant
parasite of sheep and goats on a global scale.
The extraordinary ability of H. contortus to survive over a wide range of
climatic zones reflects unique biological characteristics that counter the sus-
ceptibility of the free-living stages to adverse environmental conditions.
Although essentially adapted to tropical or warm-temperature climates,
with a particular requirement for moisture for development from the egg
to the infective larval stage, the high biotic potential of H. contortus allows
the parasite to take advantage of transient periods when adequate moisture
coincides with sufficiently warm temperatures, in order to maximize the
probability of infection in grazing animals. Although the infective larvae
are relatively resilient, in general, their period of survival varies inversely
with the potential for population expansion. The larvae typically survive
for a relatively short period (weeks) in tropical and subtropical zones;
however, due to the continually high temperatures, a large proportion of
H. contortus eggs shed into the environment can develop rapidly. In contrast,
in cooler environments, the scale of egg development is limited or ceases
completely for prolonged periods, but the infective larvae survive for
considerable periods (months). In more hostile environments (extremes of
cold or aridity), H. contortus depends chiefly on hypobiosis (arrested develop-
ment) of the fourth-stage larvae, with the egg-laying adult worms present
mostly during the relatively brief periods when larval development is
possible. There is also evidence of intraspecific variation, with ecological
adaptations permitting H. contortus egg development outside the generally
recognized ideal climatic range; further investigations are necessary to
confirm that such variation occurs, including the existence of a genomic
basis to differences among isolates.
Observations during mid-2010s suggest an extension to the geographical

range where significant H. contortus populations develop routinely, particu-
larly in colder, temperate climates in the Northern Hemisphere, where out-
breaks of haemonchosis have been attributed to temperature increases that
may reflect long-term climate changes. In this respect, the prevalence and
extent of significant H. contortus burdens might provide a useful general indi-
cator of environmental changes, as the numerous epidemiological investiga-
tions and surveys provide an extensive record of its historical climatic range.
The availability of detailed ecological data also provides a basis for predictions
of the immediate geographical and seasonal risk of haemonchosis, as well as of
longer-term changes to the endemic range.
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CHAPTER FIVE
The Identification of
Haemonchus Species and
Diagnosis of Haemonchosis
D.S. Zarlenga1, E.P. Hoberg, W. Tuo
USDA, Agricultural Research Service, Beltsville, MD, United States
1
Corresponding author: E-mail: dante.zarlenga@ars.usda.gov
Contents
1. Introduction 146
2. Morphological Approaches for Identifying Haemonchus contortus 147
2.1 Morphology; the gold standard 147
2.1.1 Identification of adult worms 148
2.1.2 Identification of infective third-stage larvae 150
2.1.3 Identification of parasitic fourth-stage larvae 152
2.1.4 Identification of eggs 152
3. Molecular Methods for Identifying Haemonchus 155
3.1 Haemonchus contortus, Haemonchus placei or both? 155
3.2 Traditional PCR 157
3.3 Real-time PCR 159
3.4 The next generation 162
3.4.1 Loop-mediated isothermal amplification 162
3.4.2 Metagenomics and pyrosequencing 163
4. Immunological Methods for Diagnosing Haemonchosis 164
4.1 Antibody assays for the diagnosis of haemonchosis; ELISA and 165
Western blotting
4.2 Antibody assays as research tools to study haemonchosis 166
4.2.1 Eosinophils and eosinophil peroxidase assays 167
4.2.2 Mast cell and mastocytosis assays 168
4.2.3 T cell proliferation assay 168
4.2.4 Cytokine and host alarmin assays 169
4.2.5 ELISPOT and the identification of antibody-secreting cells 170
5. Final Thoughts 170
References 171
j
ISSN 0065-308X
http://dx.doi.org/10.1016/bs.apar.2016.02.023 2016, Published by Elsevier Ltd. 145
146 D.S. Zarlenga et al.
Abstract
Diagnosis is often equated with identification or detection when discussing parasitic
diseases. Unfortunately, these are not necessarily mutually exclusive activities; diseases
and infections are generally diagnosed and organisms are identified. Diagnosis is
commonly predicated upon some clinical signs; in an effort to determine the causative
agent, identification of genera and species is subsequently performed. Both identifica-
tion and diagnosis play critical roles in managing an infection, and involve the interplay
of direct and indirect methods of detection, particularly in light of the complex and
expanding problem of drug-resistance in parasites. Accurate and authoritative identifi-
cation that is cost- and time-effective, based on structural and molecular attributes of
specimens, provides a foundation for defining parasite diversity and changing patterns
of geographical distribution, host association and emergence of disease. Most tech-
niques developed thus far have been grounded in assumptions based on strict host
associations between Haemonchus contortus and small ruminants, that is, sheep and
goats, and between Haemonchus placei and bovids. Current research and increasing
empirical evidence of natural infections in the field demonstrates that this assumption
misrepresents the host associations for these species of Haemonchus. Furthermore, the
capacity of H. contortus to utilize a considerably broad spectrum of ungulate hosts is
reflected in our understanding of the role of anthropogenic forcing, the ‘breakdown’
of ecological isolation, global introduction and host switching as determinants of dis-
tribution. Nuanced insights about distribution, host association and epidemiology have
emerged over the past 30 years, coincidently with the development of increasingly
robust means for parasite identification. In this review and for the sake of argument,
we would like to delineate the diagnosis of haemonchosis from the identification of
the specific pathogen. As a foundation for exploring host and parasite biology, we
will examine the evolution of methods for distinguishing H. contortus from other com-
mon gastrointestinal nematodes of agriculturally significant and free-ranging wild ru-
minants using morphological, molecular and/or immunological methods for studies
at the species and genus levels.
1. INTRODUCTION
The differentiation of Haemonchus contortus from Haemonchus placei has
been deemed by many as inconsequential because of morphological,
biochemical and biological similarities between the organisms, as well as
similarities in the way they affect host physiology. Over time, two camps
have emerged; those wishing to define H. contortus and H. placei as distinct
species and those considering them as morphs, races or isolates of a single,
widespread species. Since comparative morphological criteria were recog-
nized, supporting their classification as distinct species (Jacquiet et al.,
1995, 1997; Lichtenfels et al., 1986, 1988, 1994), studies of the
Identification of Haemonchus Species and Diagnosis of Haemonchosis 147
epidemiology and population genetics of these organisms have become

dependent on increasingly rapid and cost-effective protocols for accurate
identification. Although there is a dearth of methods currently available
that allow accurate differentiation of H. contortus from H. placei, those that
are available are not routinely applied. Consequently, there is ongoing
confusion about the relative importance of characters, such as variation in
vulval morphology, which are often explored in the absence of clear criteria
for specific identification. Furthermore, there is persistence in the literature
of host-based identifications for species of Haemonchus which are not verified
relative to specimens, morphology or molecular data. It is important to be
mindful that the ability to readily delineate these species should be the stan-
dard, where complete and accurate identification serves, for example, as the
foundation for field-based epidemiological studies. In the absence of defin-
itive identification, the value of such studies becomes equivocal.
The importance of our capacity to identify Haemonchus species to a de-
gree parallels that for other recognized helminth pathogens. In the early
1970s and for a long time thereafter, when classification of the genus Trich-
inella was in flux, it was sufficient to present data on the epidemiology of
Trichinella spp. without having genetically characterized the isolates. Conse-
quently, today, most prior work on the circulation and biology of Trichinella
is of diminished value because the context linked to differentiation among
12 currently recognized species and genotypes was not available. Whereas
such an example may not have a demonstrable impact on the diagnosis of
haemonchosis, it can conflate and hinder a refined understanding of the
epidemiology and population genetics of these organisms.
Reviews since 2008 have holistically examined methods for diagnosing
and identifying nematode parasites infecting livestock (Gasser et al., 2008;
Preston et al., 2014; Roeber et al., 2013a,b). We have taken a more guided
examination of these studies in the hope of teasing out efforts focussing on
the genus Haemonchus and in particular H. contortus.
2. MORPHOLOGICAL APPROACHES FOR IDENTIFYING

HAEMONCHUS CONTORTUS
2.1 Morphology; the gold standard
The genus Haemonchus Cobb, 1893, was established for the large
stomach worms occurring globally in sheep, cattle and other free-ranging
artiodactyl ungulates. Recognition of these nematode parasites has a deep
history, extending over the past 200 years, consistent with their economic
and veterinary significance. Of the 12 currently recognized species, H. con-
tortus (Rudolphi, 1803) was described based on abomasal parasites in sheep,
although it has been considered to include morphologically variable nema-
todes with an otherwise exceptional range of ruminant hosts (Gibbons,
1979; Hoberg et al., 2004; chapter: Evolution and Biogeography of
Haemonchus contortus: Linking Faunal Dynamics in Space and Time by
Hoberg and Zarlenga (2016), in this volume). Species of Haemonchus are,
for the most part, well differentiated morphologically, and delineation of
adults is possible based on typical structural and meristic characteristics of
male and female worms (Gibbons, 1979). Until the 1980s, however, it
was not possible to provide reliable identification of H. contortus and H. placei
(Place, 1893) in domesticated ruminants. Conventional wisdom in the vet-
erinary literature often separated these species based erroneously on assump-
tions about host association, with the former regarded as parasites of sheep,
and the latter seen as distributed only in cattle (Giudici et al., 1999; Jacquiet
et al., 1997; Lichtenfels et al., 1994). Further creating potential confusion
were proposals for extensive partitioning of subspecies within H. contortus
based largely on the structure of the vulva and associated vulval fans and
knobs evident in female nematodes from different hosts and geographical
localities (discussed in Gibbons, 1979).
The need for efficient methods for rapid identification and separation of
H. contortus and H. placei had been evident, extending into the 1950s when
early observations were being assembled about the status of these proposed
species (summarized in Lichtenfels et al., 1986). Paramount was the appre-
ciation that an effective means of control and a clear understanding of epide-
miological patterns, host associations and geographical distribution would
emerge from an unequivocal definition of diversity for these nematodes.
In mixed natural infections, morphological differentiation of H. contortus,
H. placei and species hybrids is now based on multiple structural attributes,
including the configuration of the spicules and bursa in males and the syn-
lophe in males and females (system of cuticular ridges visible on the surface
of most trichostrongyloid nematodes (eg, Durette-Desset, 1983)).
2.1.1 Identification of adult worms

Initial development of reliable means for the morphological identification of
species and primarily limited to parasites circulating in domesticated hosts
emerged through studies of the synlophe. Criteria for identification included
the pattern of cuticular ridges, their numbers, and the extent or distribution
on the body of male and female nematodes as revealed in cross-section or
in examination of whole mounted specimens (Durette-Desset, 1983;

Lichtenfels and Pilitt, 2000; Lichtenfels et al., 1986, 1994, 2002). For
example, transverse sections at the level of the esophagealeintestinal junc-
tion reveal the presence of 30 ridges in H. contortus and 34 in H. placei
(Lichtenfels et al., 2002). Specific patterns of distribution for ridges in the
subventral and sublateral fields in the esophageal region of the body of adult
nematodes are diagnostic, and provide a capacity for robust identification of
individual males and females (Hoberg et al., 2002; Lichtenfels et al., 1994,
2001, 2002). Concurrently, morphometric protocols linked to discriminant
analysis for spicules provided an alternative means for rapid identification;
however, such protocols are limited to males of H. contortus and other species
in domesticated ruminants (Jacquiet et al., 1997). Hybrids of H. contortus and
H. placei occurring in sheep, cattle or other ungulate hosts in sympatry can
also be unequivocally identified based on the intermediate range of attri-
butes observed in adult nematodes (Lichtenfels et al., 1986, 1994).
The recurring necessity to provide authoritative and accurate identifica-
tion for species of Haemonchus that circulate among domestic and free-
ranging ungulates is emphasized by the strongly developed mosaic structure
of ruminant parasite faunas (Hoberg, 2010; Hoberg et al., 2008). Translo-
cation, introduction and successful establishment have been dominant pro-
cesses since the 1500s, associated with widespread invasion and expansion of
nematode faunas globally (Zarlenga et al., 2014). Dissemination and gene
flow associated with recurrent introductions of parasites with small and large
domestic ruminants likely extend to exchanges and trade near the time of
domestication over 10,000 years ago, but may have been maximized during
the time frame for extensive European colonization (Hoberg, 2010; Rosen-
thal, 2009). Such a history of invasion may account for founder events and
considerable population structure now partitioned globally, and contrasts
with current or contemporary intercontinental geographical barriers to
dispersal that are evident (Giudici et al., 1999; Troell et al., 2006). Although
population genetic structure is apparent on continental scales, this structure
does not coincide with identifiable morphological variation (eg, vulval
morphology) that had been the primary basis for subspecies designations
(Gibbons, 1979). Consequently, integrated morphological and molecular
evidence is consistent with H. contortus as a single, highly variable and wide-
spread species with a considerable capacity to infect a broad range of ungu-
late hosts (Hoberg et al., 2004).
Accurate species identification and an understanding of circulation and
epidemiology for nematode faunas at the intersection of managed and native
or wild ecosystems remains a priority. Identification is particularly important
in habitats under accelerating environmental change linked to climate and

other factors of anthropogenic forcing (Cerutti et al., 2010; Hoberg,
2010; Hoberg et al., 2008). Patterns of geographical invasion and host
switching between domestic and free-ranging ruminants in this arena of
perturbation are expected to influence persistence, dissemination and ge-
netic exchange among drug-resistant populations in zones of contact or
sympatry. Comparative morphological approaches provided an initial
pathway for clear identification of H. contortus and continue to constitute
relatively efficient means to explore species and faunal diversity that are at
the foundations of managing and mitigating impacts associated with parasites
and parasitism. Studies have shown that ante-mortem, morphological exam-
ination of third-stage larvae (L3) coincides well with PCR-derived data for
differentiating H. contortus and H. placei (Santos et al., 2014a). Further, a
combined parasitological and molecular barcoding assay used by Budischak
et al. (2015) to examine cultured L3s from wild hosts (due to limitations
imposed by postmortem analyses) accurately estimated both total and spe-
cies-specific worm abundance, and exhibited similar rates of parasite species
discovery as derived from postmortem analyses. Worm prevalence and com-
munity compositions were similar to those derived from lethal sampling, and
all morphological analyses were corroborated by the molecular data. Conse-
quently, morphology provides the foundation upon which other direct
methods of parasite identification are predicated; namely molecular and
biochemical-based technologies. In the following sections we review and
explore some of the techniques that have emerged.
2.1.2 Identification of infective third-stage larvae

Identification of free-living, infective stages (L3) of gastrointestinal nema-
todes of ruminants remains an important aspect of epidemiological studies
and in defining the dynamics of transmission. It is becoming increasingly
critical to understand the persistence and expansion of parasitic populations
in rapidly changing environments. Assessment of gastrointestinal parasite di-
versity has often relied on culturing larvae from eggs recovered from faeces
of naturally infected hosts (MAFF, 1986). Further, determination of pasture
contaminants and, thus, the potential for transmission across and within
seasonally defined windows has been related to egg counts and the identifi-
cation of L3s collected from rangelands populated with domestic stock.
Development of methods and criteria for identification of L3s for gastro-
intestinal nematodes in ruminants has an extensive history and, over time,
has resulted in standardized protocols based on comparative morphological
approaches (eg, Dikmans and Andrews, 1933; MAFF, 1986; van Wyk and
Mayhew, 2013). An understanding of the range of diagnostic characters
that could provide differentiation among members of the Trichostrongy-
lina and other strongylate nematodes in ruminants emerged initially from
the studies of life cycles, life history and development of free-living and
parasitic stages (Ransom, 1906; Veglia, 1915). Increasingly detailed descrip-
tions of L3s focussed on overall length, number of intestinal cells, structure
of the cuticular sheath, sheath length, tail length and cephalic morphology
including attributes of the buccal capsule have allowed the separation of tri-
chostrongylines such as Haemonchus, Cooperia, Ostertagia, Trichostrongylus,
nematodirines including Nematodirus and Nematodirella, and other strongyles
including Chabertia and Oesophagostomum (Dikmans and Andrews, 1933;
Goodey, 1922; Veglia, 1926). These studies confirmed that among genera,
it was usually possible to distinguish most nematodes circulating in rumi-
nants based on relatively constant and consistent attributes (Veglia, 1926).
Although often unequivocal identification could be achieved among four
genera typically observed in sheep (Cooperia, Haemonchus, Ostertagia e
now Teladorsagia e and Trichostrongylus), overlap in the tail and tail-sheath
lengths obviated the use of additional attributes (Dikmans and Andrews,
1933). Further, reliable characters for the definitive identification to species
have remained elusive (Dikmans and Andrews, 1933; M€ onnig, 1931).
Methods and criteria applied to parasite diversity among domestic ruminants
also could not be generally translated to free-ranging wild hosts or to a
broader understanding of parasite circulation in zones of contact or sym-
patry at the interface of managed and natural systems (Budischak et al.,
2015).
Criteria currently applied to identification of L3s have not been modi-
fied substantially over the past 80 years and primarily relate to a series of
definitive papers addressing either single species or species assemblages of
nematodes in domestic ungulates (eg, Borgsteede and Hendricks, 1974;
Dikmans and Andrews, 1933; Hansen and Shivnani, 1956; Keith, 1953).
A standardized protocol for identification has been codified in the veteri-
nary literature, as exemplified by diagnostic keys that reflect nematode
faunal diversity among domestic ruminants on a regional and global stage
(MAFF, 1986; van Wyk and Mayhew, 2013). Although comparative
morphological approaches will remain a central approach in diagnostics,
the capacity for accurate species-level identification can be directly linked
to a range of available and developing molecular-based pathways (eg,
Budishak et al., 2015).
2.1.3 Identification of parasitic fourth-stage larvae

Recognition that parasitic infection by an assemblage of gastrointestinal nem-
atodes in ruminants often involved immature or larval stages suggested the
importance of being able to identify genera and species that were involved.
Fourth-stage larvae (L4s) could be present in the abomasum or small intestine
during typical development, or could reflect the occurrence of inhibition
(Michel, 1963, 1974), thus representing distinct epidemiological processes
in transmission that have different consequences for infection and disease.
Although considerable attention had been focussed on the identification of
free-living larvae, only sporadic studies, often limited to development
observed in single species, characterize available information for parasitic
L3s and L4s (eg, Douvres, 1957a,b). Parasitic L3s and L4s of trichostrongylines
(eg, Cooperia, Haemonchus, Ostertagia and Trichostrongylus) can be differentiated
morphologically by primary attributes of the buccal capsule, tail and place-
ment of the excretory pore, and identification is limited to separation of
genera. Furthermore, prior to the separation of H. contortus and H. placei as
distinct species, most infections in sheep or cattle were attributed to the
former species, and definitive identification was not possible. Arrested devel-
opment of H. contortus occurs in the early L4, and structurally these may not
differ substantially, except in length and the degree of development of the
genital primordium in males and females, relative to conspecific nematodes
observed under typical ontogeny (Blitz and Gibbs, 1971; Veglia, 1915).
2.1.4 Identification of eggs

There exist other less conventional assays for the diagnosis of nematode
infections and, in particular, those of the genus Haemonchus. In addition to
the well-established FAffa MAlan CHArt (FAMACHA) (Bath et al.,
1996), which today has been relegated to assessing the level of H. contortus
infections in small ruminants, because of resultant anaemia, there have
been other efforts to correlate morphometric dimensions and appearances
of eggs in faeces to genus-level identification. Given improvements in
computer technology and digital imaging in the last decade, morphometrics
bears mentioning in the context of this chapter because state-of-the-art,
technological advances have not yet been fully exploited in the direct exam-
ination of faecal eggs. Further, tests have been developed to differentiate
eggs based on lectin binding, wherein Haemonchus specifically binds peanut
agglutinin. Advancements in the last 5e10 years may provide the impetus
for more common use. However, the central theme in these assays remains
genus- rather than species-level identification.
Cunliffe and Crofton (1953) were among the first to systematically

characterize and attempt to standardize egg measurements as a method to
identify parasites, though they did not pioneer this approach (Shorb,
1939; Tetley, 1941). After examining eggs derived from dissected female
worms, their data compared well with prior art, and they devised a series
of equations to define each parasite group based on the norms of length
and width measurements. However, given the ranges in size, they
concluded that, based only on these measurements, mixed populations
would be difficult to classify, even though the method was more rapid
and less variable than larval culture. In 1982, Christie and Jackson used
egg measurements, coupled to information on the stage of embryonic devel-
opment, to identify with high accuracy, Ostertagia and Trichostrongylus spe-
cies; however, they made similar conclusions regarding other species, and
suggested that many of the sheep parasites, including Haemonchus, would
require larval culture followed by the examination of L3 morphology,
depending on the composition of the infection.
Georgi and McCulloch (1989) utilized an electronic digitizer and multi-
variate analysis to combine data from length, width, area and perimeter as
well as areas and arc lengths of egg polar regions. Stepwise discriminant anal-
ysis allowed them to correctly identify H. contortus and Trichostrongylus colu-
briformis 85% of the time. Unlike prior data, eggs prepared from fresh faeces
were no different from those immediately fixed in formalin, although eggs
derived from the uterus were morphometrically distinct from those obtained
from faecal material (Tetley, 1941). Kerboeuf et al. (1996) was among the
first to adopt flow cytometry to analyse Haemonchus eggs. The investigators
used native egg fluorescence and scattering pulses to generate histograms
similar to cell histograms. Although they did not investigate the ability of
the technique to distinguish between parasite genera, they did show a strong
correlation between the level of native green fluorescence and resistance to
anthelmintics (benzimidazoles). Consequently, this test may be adaptable to
assessing the level of resistance within a flock or herd of animals.
About the same time that Kerboeuf et al. (1996) was testing flow cytom-
etry, digital imaging was employed to examine morphometric parameters
(Sommer, 1996) and egg texture (Sommer, 1998), as a means to differentiate
common bovine nematodes. Species from five common genera (Ostertagia,
Cooperia, Haemonchus, Trichostrongylus and Oesophagostomum) were examined.
Using linear discrimination analysis, the test generated a correct classification
86% of the time when 19 of 25 measured features were evaluated. Relegat-
ing this analysis to the five most important features slightly reduced the
accuracy to 82%. In contrast to the test developed by Georgi and McCulloch

(1989), this analysis did not require outlining the egg prior to taking mea-
surements. Although the test was not evaluated for quantifying eggs in
mixed populations, it, nonetheless, suggested its utility for this purpose, pro-
vided sufficient sampling was done. This same group examined egg texture
including grey colour levels throughout the egg using digital imaging
(Sommer, 1998). Of the 25 different texture parameters that were used,
10 had significant discriminatory power and collectively identified Ostertagia
ostertagi, Cooperia oncophora and Ostertagia radiatum 91% of the time. When
these data were combined with egg size and shape, correct identification
increased to 93%. At the time that this technology was developed, methods
were not available to accurately quantify mixed egg populations by PCR, to
validate or refute the morphometric and digital imaging approach for exam-
ining mixed populations. However, revisiting the assay with state-of-the-art
cameras and digital imaging software concomitant with real-time PCR
might provide better insight into the efficacy of the technology for herd/
flock-level analysis.
Nwachukwu et al. (1987) showed that egg shells from the nematode
Onchocerca gutturosa were able to bind to peanut agglutinin (PNA) and sug-
gested that nematode eggshells were capable of eliciting host-protective re-
sponses. It was not until 1996, however, that Palmer and McCombe (1996)
demonstrated that PNA was able to specifically bind to Haemonchus eggs and
that lectin binding corresponded well with data from larval cultures. Binding
was monitored using fluorescently labelled PNA, and the data provided a
good estimation of the number of H. contortus eggs in mixed populations.
Colditz et al. (2002) expanded on earlier work by extending the breadth
of species examined and incorporating flow cytometry into the analysis,
which enabled quantification of lectin staining. They showed that staining
was not altered due to the developmental stage of the egg and that the prev-
alence of Haemonchus in mixed field infections compared well with that
obtained from larval culture. In order to garner broader use, the method
was further modified by Jurasek et al. (2010) for expediency, cost and the
need for less training. In particular, the time required to purify eggs (multiple
sieving and overnight flotation in saturated salt) was a major deterrent to the
adaptation of this technique. These authors also showed that formalin
fixation could be used to preserve the eggs, but that staining intensity was
substantially diminished by 5 weeks following treatment.
Hillrichs et al. (2012) took the technology one step further and screened
19 different lectins spanning wide-ranging sugar specificities in the hope of
identifying a bank of lectins that specifically differentiated four life-cycle

stages of H. contortus and T. circumcincta, including eggs, adult worms, and
sheathed and exsheathed L3. As previously mentioned , PNA was indeed
the preferred lectin for specifically interacting with Haemonchus eggs. Lectins
that interacted with the other stages were less specific and depended on the
age of the worm. Unfortunately, differential rather than specific interactions
were routinely observed.
3. MOLECULAR METHODS FOR IDENTIFYING

HAEMONCHUS
3.1 Haemonchus contortus, Haemonchus placei or both?
Over the years, numerous ‘first-generation’ molecular and biochem-
ical methods have been developed for identifying Haemonchus species and for
examining drug-resistant genotypes. Restriction enzyme digestion followed
by agarose gel electrophoresis (Beh et al., 1989), Southern blotting (Roos
et al., 1990; Zarlenga et al., 1994), repetitive DNA hybridization probes
in conjunction with Southern blots or dot blots (Christensen et al.,
1994a,b), and isoenzyme banding profiles (Bentounsi and Cabaret, 1999;
Echevarria et al., 1992; Knox and Jones, 1992) were among the most pop-
ular examples of first-generation technologies. Sensitivity and specificity,
however, were key issues that prompted the transition to PCR-based assays
for developing more advanced tests. Also, DNA sequencing for differenti-
ating closely related species has been available for many years; however
this technology only gained popularity and momentum once PCR took
hold and problems associated with PCR inhibitors in biological samples
were addressed.
As noted earlier, the biggest misconception in the identification of Hae-
monchus species is the belief that H. contortus is a sheep parasite and H. placei is
a cattle parasite. Hence, most techniques to identify Haemonchus species
have targeted genus- rather than species-level differentiation with this lim-
itation in mind. While years ago this assumption may have held true and
may even today be appropriate in regions where cattle production is either
nonexistent or very limited, today anthropogenic forcing has globalized the
dissemination of these and many other parasite species (Hoberg, 2010;
Zarlenga et al., 2014). As an example, H. placei was found in western
Australian cattle; a geographical region believed not to be conducive to
this species because of its predilection for more tropical and subtropical
climates (Jabbar et al., 2014). Consequently, blanket assumptions of exclu-

sivity regarding hostepathogen associations can no longer be considered
unilateral, as it relates to H. contortus and H. placei. do Amarante (2011)
made special note of this issue, providing as support for differentiating
the species the need to establish proper and sustainable control strategies,
especially in light of drug-resistant parasites and the inability of H. placeie
infected animals to cross-protect against challenge infection with H. contor-
tus (see Santos et al., 2014b). It is also necessary to consider circulation
among domestic stock and free-ranging ungulates, and the growing under-
standing that populations that involve multiple species of Haemonchus can be
maintained in a broad array of wild cervids, camelids and bovids (including
caprines) that occur in sympatry in particular regions of the world (eg,
Cerutti et al., 2010; Hoberg et al., 2001, 2008).
Chaudhry et al. (2014) noted the presence of drug-resistant alleles in cat-
tle-derived H. placei obtained from mid-western and eastern southern
United States. Six of nine populations contained the characteristic P200Y
(TAC) isotype-1 polymorphism indicative of b-tubulin benzimidazole resis-
tance, albeit at low frequencies. This group also identified the presence of
naturally derived hybrids in isolates of Haemonchus obtained from Pakistan
and southern India, where numerous worms were heterozygous for fixed,
species-specific single nucleotide polymorphisms (SNP) within the internal
transcribed spacer 2 (ITS-2) of nuclear ribosomal DNA (rDNA) (Chaudhry
et al., 2015). Among these worms, one hybrid contained the H. contortus iso-
type-1 b-tubulin benzimidazole resistance allele, suggesting not only that
hybridization had occurred, but also that introgression of drug resistance
loci can transpire between the two species. This finding could only have
ensued from mixed infections. In this same study, these authors noted that
cattle in southern India were only infected with H. contortus; H. placei was
not to be found.
Other reports of mixed or dual infections have been emerging world-
wide. H. contortus and H. placei have been reported as highly sympatric
species in North Africa in both large and small ruminants based on morpho-
metric parameters and PCR (Akkari et al., 2013). Results showed that
>50% of all small ruminants tested had multiple infections, with numbers
being slightly less in cattle. There are similar reports from West Africa
(Achi et al., 2003) for small and large ruminants. In a herd in the United
States, sequence analysis of faecal eggs prior to anthelmintic treatment
revealed an H. placei infection; however, following drug treatment and a sec-
ond round of egg DNA isolation and sequencing, H. placei infection was
expelled, but a low level of drug-resistant H. contortus, not originally

detected by sequencing, was noticeably present in the herd (unpublished
data). Although one early report identified a DNA hybridization probe
(Christensen et al., 1994b) and several additional reports of species-level
PCR-based assays for differentiating H. placei from H contortus are discussed
in the following sections, it has become increasingly important to develop
methods for the differentiation of these species, and depend less on genus-
level identification.
3.2 Traditional PCR

Clearly, the biggest hurdles to generating quality PCR data have been in
obtaining amplifiable DNA or RNA devoid of inhibitors and if possible,
producing genetic material of sufficient length to allow adequate sensitivity
during amplification. Efforts to perform egg-based PCR directly from
faeces, to reduce processing time has met with sporadic success, owing to
sensitivity issues and PCR inhibitors (Demeler et al., 2013; Roeber et al.,
2012a). There are a plethora of commercial and noncommercial methods
now available for isolating nucleic acids for PCR; however regardless of
the method chosen, the presence of enzymatic inhibitors in biological sam-
ples must be addressed before or during PCR.
Over the years, numerous genes have been targeted for identifying para-
site-specific PCR primers. Roos and Grant (1993) were among the earliest
to develop a H. contortusespecific PCR test. In this assay, the investigators
synthesized primers that amplified a region of the isotype-I b-tubulin
gene spanning an intron that exhibited size variation between H. contortus
and T. colubriformis. The primers chosen did not bind to other common
sheep parasites; however, the sensitivity of the assay was low. At the time,
it was not clear whether the low sensitivity was related to PCR contami-
nants, the size of the amplicons (1300e1500 bp) or to a suboptimal copy
number of the gene. Consequently, the focus switched to using mitochon-
drial gene sequences (Blouin, 2002) and genomic spacer sequences associ-
ated with the rRNA gene repeat (for review, see Chilton, 2004) as
amplifiable targets for PCR. The compelling arguments for these choices
have been that both are highly abundant in all life-cycle stages and suffi-
ciently variable among species of gastrointestinal nematodes to attain
adequate sensitivity and specificity when designing an assay. Stevenson
et al. (1995) were among the first to assess the second internal transcribed
spacer (ITS-2) sequence for the differentiation of H. contortus from H. placei.
Several SNPs were identified among the individuals chosen, which resulted
in unique restriction enzyme digestion patterns for each species after PCR
amplification. One or more of these SNPs were later used to identify hybrid
organisms in Pakistan (Chaudhry et al., 2015) by sequencing. Though not
directly related to the delineation of species, other studies have used ampli-
fied ITS-2 sequences and denaturing gradient gel electrophoresis (DGGE)
to examine sequence heterogeneity among populations of H. contortus (see
Gasser et al., 1998).
In 1994, SNPs in the external transcribed spacers (ETS) were observed
between these same species (Zarlenga et al., 1994), as well as distinct differ-
ences in the rRNA gene repeats emanating from the external nontranscribed
spacer (NTS) that permitted the differentiation of H. contortus from H. placei.
The SNPs were generated from cloned sequences and were not validated on
larger numbers of field samples. However, Santos et al. (2014a) used and
validated the existence of multiple rRNA gene repeats within H. contortus
(cf. Zarlenga et al., 1994) by comparing PCR fragmentation patterns to
morphometric data on individual Haemonchus worms. Their results showed
that the morphology of L3s could be used as the primary method to identify
and differentiate the two species. A multiplex PCR developed by Zarlenga
et al. (2001) not only differentiated five major genera of gastrointestinal
nematodes routinely found in cattle and sheep, but provided data wherein
the chosen primers which amplified portions of the ETS were capable of
differentiating H. contortus from H. placei. Such a test would work well on
individual worms of Haemonchus; however, given the overlap in sizes be-
tween the two species, it would be problematic in the event of a mixed
infection or if performed on populations of eggs. The doublet generated
in this assay was produced only from H. contortus DNA and was the result
of either multiple-sized fragments or heteroduplex formation from sequence
variation among the repetitive units of the rRNA gene within H. contortus
(see Zarlenga et al., 1994). Given that H. contortus was shown to have mul-
tiple and distinct repeats, the former explanation is likely correct. This pro-
posal is further supported in studies showing substantially less genetic
variability among populations of H. placei than among populations of
H. contortus (see Brasil et al., 2012; Hussain et al., 2014; Jacquiet et al.,
1995). Chilton (2004) reviewed the benefits of targeting ribosomal DNA
markers for delineating bursate nematodes.
Blouin et al. (1997) showed that numerous fixed differences existed
among the mitochondrial ND4 gene sequences from H. contortus and
H. placei to allow for sequence-based or PCR-based differentiation between
the two species. These haplotypic differences were used to examine genetic
variation among worms parasitizing sheep and goats in China, where nearly
all 152 individual worms exhibited distinct haplotypes (Yin et al., 2013).
Random amplified polymorphic DNA assays were also tested (Humbert
and Cabaret, 1995; Jacquiet et al., 1995; Rabouam et al., 1999) where suf-
ficient genetic variation was observed between the two species (Jacquiet
et al., 1995). However, given the variability in the assay, the dependency
on pristine DNA and amplification conditions and inconsistencies in PCR
amplification using small, nonspecific primers, this technology was aban-
doned relatively soon after its inception.
Many assays have been developed with genus-specific rather than spe-
cies-specific detection in mind. As such, linking H. contortus and H. placei
in assay development has been a common and pervasive theme. Gasser
et al. (1994) developed a restriction fragment length polymorphism
(RFLP) linked PCR assay based on ITS-2 sequences to delineate six com-
mon trichostrongyles of ruminants, including H. contortus. Heise et al. (1999)
sequenced the ITS-2 from eight species of gastrointestinal nematodes and
later, Schnieder et al. (1999) developed a PCR assay for differentiating
five major genera of gastrointestinal nematodes infecting cattle and sheep,
among them, the genus Haemonchus; however, species-level identification
was not assessed. Bisset et al. (2014) developed a multiplex PCR capable
of differentiating 10 strongylid species that commonly infect small rumi-
nants. They combined both species-specific primers and genus-specific
primers to generate gel-banding profiles unique for each of the organisms.
The inclusion of genus-specific primers obviates the need for PCR-positive
controls (Zarlenga et al., 1999).
3.3 Real-time PCR

With the advent of real-time PCR, some new methodologies emerged for
the identification of Haemonchus spp. Real-time PCR had its inception in
the desire to quantify gene transcription; however, over time, many workers
adapted it as a means to supplant conventional PCR for identification. Some
approaches use fluorescence via resonance energy transfer between fluoro-
phore and quencher molecules bound to a DNA-probe for added specificity
(Harmon et al., 2007; Learmount et al., 2009; McNally et al., 2013; von
Samson-Himmelstjerna et al., 2002; Siedek et al., 2006). Though PCR
probes greatly enhance specificity, generating DNA probes that are dual-
labelled for proper energy transfer and fluorescence can be cost prohibitive.
Consequently, other techniques have emerged, wherein nonsequence-spe-
cific fluorescent dyes are used that intercalate and/or bind double-stranded
DNA and fluoresce either by resonance energy transfer interactions with the
helix, or by stabilization of the fluorophore when bound to DNA (Dragan
et al., 2012). Though substantially easier and less costly, many of the avail-
able fluorophores, including the most commonly used, SYBR I green, are
inhibitory to PCR to varying degrees, and can alter the melting temperature
of the DNA in a concentration-dependent manner (Gudnason et al., 2007).
This influence on melting temperature can affect studies involving melting-
curve analysis for identification and for quantification. For this reason, real-
time techniques have emerged using fluorescent dyes other than SYBR I
green (Bott et al., 2009; Roeber et al., 2011).
As the technological advances have moved towards real-time PCR, ef-
forts have begun to focus more on application rather than mere assay devel-
opment. Siedek et al. (2006) showed good correlation between probe-based
real-time PCR data and coproculture, though the study focussed only on
cultured larvae. Since this time, efforts have turned to ante-mortem
PCR-based identification of faecal eggs, rather than culturing to L3 fol-
lowed by morphological examination; a technique that has been extensively
reviewed (Preston et al., 2014; Roeber et al., 2013a,b). In conjunction with
performing molecular tests on faecal eggs, numerous reports have been pub-
lished, in which egg isolation was not preceded by purification, and DNA
isolation was performed directly on whole faeces. For instance, Sweeny
et al. (2011) used the Power Soil DNA Isolation Kit (Mol-Bio, West Carls-
bad, CA, United States) and were able to successfully perform nematode-
specific PCR on DNA isolated directly from ovine faeces. The data coin-
cided well with egg flotation assays where the epg > 50; however, the limits
of egg detection were never determined and cultures were not performed
on the faecal eggs in an attempt to confirm the PCR data. The same group
(Sweeny et al., 2012) modified the procedure in the hope of applying real-
time PCR to quantify (qPCR) larval burdens on pasture. Little correlation
was observed between qPCR Ct values and log-transformed pasture larval
counts, possibly due to a mixture of L3s and eggs on pasture. The qPCR
data was, nonetheless, encouraging. Later, McNally et al. (2013) developed
a method to extract DNA from sheep faeces that involves dehydration in
ethanol, bead-beating to disrupt faecal samples, and magnetic beadebased
DNA extraction, followed by genus-level multiplex qPCR to quantify
eggs from Haemonchus, Trichostrongylus and Teladorsagia. The assay showed
a sensitivity of 10 eggs per gram (epg) using this approach. Given that this
test also was somewhat labour intensive and exhibited a sensitivity that
was substantially less than that achievable when using purified eggs, general
laboratory practices have thus far conceded that some level of egg purifica-
tion and/or DNA dilution to reduce inhibitors is in order, to maximize
PCR sensitivity rather than isolating DNA from unfractionated, environ-
mental samples (Demeler et al., 2013; Roeber et al., 2012b).
Efforts have been made to quantify faecal eggs in mixed species infec-
tions. While it is well accepted that egg output rarely coincides with adult
worm burdens (except for Haemonchus), and bias is often generated in faecal
cultures, making it difficult to apportion egg counts to worm species
(Dobson et al., 1992), quantification nonetheless can provide information
on pasture seeding densities. This is particularly important when considering
the potential for drug-resistant worms in the flock and when establishing
pasture management programmes to reduce worm burdens within the
host. Conventional approaches to quantification, that is, larval culture, fol-
lowed by morphological identification of L3, require a person skilled in the
morphological identification of L3, and presume that the different worm
species develop at the same rate and efficiency under artificial growing con-
ditions. Some researchers would argue that molecular amplification of faecal
eggs can succumb to differences in DNA content (egg stage development)
and variations in target gene copy numbers among the species. Anecdotal
evidence indicates that the former is not an issue, and the latter point can
be addressed by properly controlling assay conditions and parameters.
Also, Harmon et al. (2007) showed that the time following egg embryona-
tion, and higher concentrations of competing DNA derived from similar
nematodes could affect egg quantification; however, changes in DNA con-
tent demonstrably affecting quantification occur only within the first 6e7 h
following embryonation.
von Samson-Himmelstjerna et al. (2002) was among the first to develop
real-time PCR for quantification of gastrointestinal nematodes of sheep.
Genus-specific probes and primers to regions within the ITS-2 were
designed to encompass common nematodes of small ruminants. The assays
exhibited good specificity and sensitivity over a large dynamic range using
DNA derived from cultured L1 and L3 parasites. The test had the advantage
of partial multiplexing due to the different labels that were chosen among
subsets of nematodes. Bott et al. (2009) developed real-time PCR method-
ology coupled to melting curve analysis to delineate seven distinct strongylids
of sheep using a single conserved reverse primer with species- and genus-
specific forward primers. In order to quantify, on a relative basis, the numbers
of eggs derived from any given species, standard curves were generated and
used in the final analysis. The technology was later applied to naturally
acquired infections in sheep for a sample size of 470 animals (Roeber et al.,
2011). The method exhibited near 98% sensitivity and 100% specificity, well
supporting the overall goal to migrate from larval cultures and morphological
examination of L3 to molecular-based analyses. The approach also demon-
strated better efficiency in assessing drug-susceptibility/resistance in strong-
ylid nematodes of sheep relative to more conventional approaches, such as
the faecal egg count reduction test (FECRT) (Roeber et al., 2012b). Iden-
tification was further advanced by automation via the development of a ro-
botic, high-throughput, multiplex tandem PCR to delineate key nematodes
infecting sheep and goats, including H. contortus. This assay again developed
primer sets targeting ITS-2. Results in field trials showed high levels of sensi-
tivity and specificity, and correlated well with the more laborious larval cul-
ture techniques.
Droplet digital PCR (ddPCR) is a methodology that provides absolute
quantification of PCR products without the need for generating standard
curves that plague many of the real-time technologies (Hindson et al.,
2011). In ddPCR, a fluorescent probeebased PCR reaction is segregated
into 1-nL reverse-micelles (water-in-oil), where zero or more copies of
the target DNA are randomly partitioned into nanoparticles along with all
other reagents needed for amplification. Following PCR, the absolute fluo-
rescence of each droplet is measured, and defined as negative or positive
based on fluorescence intensity, which accounts for droplets containing
multiple copies. The absolute number of target nucleic acid molecules is
then calculated directly from the ratio of positive droplets to total droplets
analysed. Some research has been advanced, demonstrating the applicability
of ddPCR for quantifying protozoan parasites. Yang et al. (2014) developed
such a method for identifying and quantifying Cryptosporidium in environ-
mental samples, and Wilson et al. (2015) have shown that ddPCR is more
sensitive and accurate than microscopy for quantifying Babesia spp. in blood
samples. To date, however, no studies have been generated using droplet
digital PCR (ddPCR) to quantify nematode eggs.
3.4 The next generation

3.4.1 Loop-mediated isothermal amplification
Several methodologies are on the horizon for DNA-based identification of
gastrointestinal nematodes and, in particular, those belonging to the genus
Haemonchus. In addition to ddPCR, which might find application in quan-
tifying nematode eggs in a mixed population, another technology that has
been around for 15 years, but has only recently gained traction for the
identification of gastrointestinal nematodes is loop-mediated isothermal

amplification (LAMP) (Notomi et al., 2000). Typically, this technology uti-
lizes Bst DNA polymerase rather than a thermostable enzyme, which makes
it far less subject to inhibition by contaminants. Other benefits include high
sensitivity, no requirement for expensive equipment (isothermal
reaction) and visualization that can be performed by whole sample fluores-
cence or turbidity without gel electrophoretic analysis. These benefits bode
well for adaptation to ‘in-the-field’ assays. Also, the use of four to six distinct
primers per reaction increases specificity, making it less prone to false-posi-
tive reactions from irrelevant DNA; nonetheless, the primer selection usually
requires a proprietary Primer Explorer software package. There are caveats,
however, where one has less freedom to choose primer locations and prod-
uct lengths, and where difficulties arise when differentiating closely related
organisms and multiplexing for the detection of other nematodes. Further-
more, a high false-positive rate often results inadvertently from the high
sensitivity of the assay, and displaying results via agarose gel electrophoresis
is critical when examining small amounts of target PCR product amidst an
excess of irrelevant genomic DNA. Still, Melville et al. (2014) developed a
highly specific LAMP assay for the identification of Haemonchus spp. based
on fluorescence using DNA from purified eggs. The sensitivity of this assay
was 10 times greater than conventional PCR, and the assay enabled a calcu-
lated detection level of 2 epg. In addition, the sensitivity of the assay rivals
nested PCR, and the ability to perform this test in 30 min makes it partic-
ularly appealing.
3.4.2 Metagenomics and pyrosequencing

Metagenomics is a technical innovation involving holistic genetic analysis of
an assemblage of microorganisms recovered directly from environmental
samples and obviates the need for prior culturing and/or purification. Analysis
is usually performed by high-throughput, next generation, shotgun se-
quencing methodologies. Though diagnostic metagenomics (Pallen, 2014)
has been generally relegated to discerning genomically less-complicated
organisms, such and bacteria, viruses and fungi, it is finding its way into pop-
ulation genetic studies (Mobegi et al., 2014; Wu et al., 2011) and other
organisms such as plant-parasitic (Porazinska et al., 2014) and marine nema-
todes (Carugati et al., 2015). One study showed the utility of pyrosequencing
to examine nematode biodiversity in sylvatic rats (Tanaka et al., 2014) using
18S rDNAebased metagenomics and an Illumina MiSeq sequencer.
Currently, as a test to identify gastrointestinal nematodes in faecal matter,
this approach is somewhat excessive, given the cost of supplies, equipment

and algorithms needed for data analysis. However, in the future, continuing
technological advancements in whole genome sequencing and data analyses
are likely to replace many other forms of molecular genotyping. One
example is in the adaptation of nanopore technology to high-throughput
sequencing (Maitra et al., 2012) which has the potential to substantially
reduce the time and cost of sample preparation. Furthermore, as costs
continue to moderate and as science migrates towards the philosophy of
‘One Health’, we can expect that diagnostic tests based upon metagenomics
will be developed to encompass multitudes of pathogens, among which
gastrointestinal nematodes could be included.
4. IMMUNOLOGICAL METHODS FOR DIAGNOSING

HAEMONCHOSIS
Serological methods for diagnosis can be highly informative for deter-
mining Haemonchus-specific exposure or infection. Review articles on the
immunological methods used for diagnosing haemonchosis via the specific
detection of IgG antibodies have been published (eg, Preston et al., 2014;
Roeber et al., 2013b). In general, these assays have not received wide appli-
cation because they can exhibit problems with antigen specificity and rele-
vancy where antibody levels can remain long after the infection has cleared.
In addition, the host tends to exhibit clinical signs of infection long before
Haemonchus-specific antibody titres increase to reproducibly detectable
levels. Moreover, serum antibody levels to infection can vary substantially
among outbred animals. The application of bead-based technologies for
immunodiagnosing nematode infections has shown promise for distinguish-
ing cattle infected with C. oncophora, Dictyocaulus viviparus and Fasciola
hepatica (Karanikola et al., 2015); however, like most immune-based assays
including those for haemonchosis, this test is genus rather than species spe-
cific. Consequently, haematological-based methods, such as blood packed
cell volume, eye-lid colouration (FAMACHA), and faecal egg counts
(FECs) have been used as generic indicators of nematode infection. In com-
bination with FAMACHA, which is a subjective assessment of host anaemia
resulting from blood-feeding nematodes, such as Haemonchus, the other
techniques are easy to use, practical and in some cases amenable to field
applications.
Beyond the simple diagnosis of infection, immunological methods are
important tools in research for estimating levels of exposure, population
immunity, correlating natural resistance to the level of immune response, and

in identifying animals that respond poorly to H. contortus infection. Evalua-
tion of population immunity and identification of poor- or nonresponders in
the flock can be useful for managing infections and for strategic deworming.
Herein, we cover immune-based advances that focus on indirect, sero-
logical detection of haemonchosis, and discuss less conventional immuno-
logical assays that deviate from the detection of parasite-derived diagnostic
markers. As such, the assays described here examine specific IgG and non-
IgG antibodies against Haemonchus, as well as host responses to infection,
such as eosinophilia and eosinophil peroxidase, mast cell and mastocytosis,
T cell proliferation, and changes in cytokine profiles as markers of infection.
4.1 Antibody assays for the diagnosis of haemonchosis;

ELISA and Western blotting
Antigen-specific, anti-Haemonchus antibodies can be detected and quantified
using ELISA or Western blot. These techniques involve target antigens
(whole parasite extract, secreted, purified native or recombinant proteins)
being immobilized on a solid support, followed by incubation with host
body fluids (eg, serum, mucus and saliva) containing antigen-specific anti-
bodies. Detection is followed by incubation with a labelled isotype-specific
secondary antibody, followed by an appropriate substrate. ELISA testing is
generally prone to nonspecific interactions; consequently, specificity must
often be confirmed by Western blotting. All members of sheep immuno-
globulins (Ig), including IgG, IgA, IgE and IgM, can be measured by ELISA
using isotype-specific antibodies.
Serum or mucous IgG and IgA are by far the most studied Ig classes in
sheep infected with H. contortus (see Miller, 1996). Early on, serum or mu-
cous IgG and IgA were measured by radio-immunoassay (RIA) (Duncan
et al., 1978; Smith, 1977); however, this test was rapidly replaced by ELISA,
which does not require the use of radioactive materials, and exhibits higher
throughput and sensitivity. As noted earlier, sensitivity and specificity can
pose problems where the titre of specific IgG in Haemonchus-infected sheep
is generally low (Cuquerella et al., 1995; Smith, 1977; Duncan et al., 1978),
and clinical signs normally appear before the antibody titres reach detectable
levels. Since the 1990s, more reagents have become available, so that Hae-
monchus antigenespecific IgG1 and IgG2, IgA and IgM can now be moni-
tored. Delineating subclasses has become important because IgG1 appears as
the predominant antibody species elicited by Haemonchus infection (Schallig
et al., 1995; Schallig, 2000).
Antigen-specific antibodies in body fluids can also be evaluated by

Western blot, which first separates parasite antigens by SDS-PAGE before
transferring them to a membrane and screening them with diluted host
antibodies. Though more labour intensive and not conducive to high
throughput, it has the distinct advantage of determining whether or not anti-
body binding is specific or cross-reactive in nature. This assay can also identify
the presence of isotype-specific antibodies that unambiguously recognize
known antigen(s) or particular protein bands with known molecular masses
if total antigens are used. As with ELISA, this assay has not received unilateral
use as a diagnostic test for haemonchosis; however, it has become an invalu-
able tool for discovery research on Haemonchus (García-Coiradas et al., 2009;
Hart et al., 2012; Raleigh and Meeusen, 1996; Rathore et al., 2006; Schallig
et al., 1995, 1997; Wang et al., 2014a,b; Yan et al., 2010).
4.2 Antibody assays as research tools to study

haemonchosis
Assays to detect Haemonchus-specific antibodies in body fluids (serum, tears,
saliva and faecal fluids from live animals) include ELISA and Western blot-
ting. These assays have been well described (Preston et al., 2014; Roeber
et al., 2013b), and few significant technological advances have been noted
in the literature. These assays generally target serum IgG and can be conve-
niently accomplished with ELISA; however, the detection of IgE and IgA in
the circulation, which is important in relation to understanding disease, and
are considered to be more important than IgG when assessing levels of host
protection, can be challenging due to limited availability of costly reagents.
Antigen-specific IgE and IgA from infected animals can be reliably assayed in
local mucosal tissues following biopsy or postmortem. Thus, these assays
have great utility for laboratory research, but less for the diagnosis of hae-
monchosis in live animals.
The reliable detection of low levels of IgE and IgA in blood is usually
achieved using capture/sandwich ELISA with high sensitivity, accuracy
and reproducibility. A broadly cross-reactive IgA sandwich ELISA, which
also detects ovine IgA, is commercially available (http://www.antibodies-
online.com), though it seems not to have received wide use in ovine
studies. The availability of commercially available antibodies against sheep
IgE has permitted the measurement of this Ig subtype by ELISA (Kooyman
et al., 1997; Redmond and Knox, 2004; Shaw et al., 1996). A sheep IgE
capture ELISA was developed using an antisheep IgE monoclonal antibody,
2F1, generated from a chimeric IgE protein (Bendixsen et al., 2004). This
assay was used to detect total IgE in colostrum and intestinal homogenates,
but not in serum. Of particular note, antigen-specific IgE appeared higher
in resistant than in susceptible sheep infected with T. colubriformis (see
Bendixsen et al., 2004). A similar trend in elevated IgE was seen in Gulf
Coast Native (Native) sheep known to be more naturally resistant to Hae-
monchus infection than Suffolk lambs (Shakya et al., 2011). Also, systemic
H. contortusespecific IgE was evident in sheep exposed to infection on
pasture, as determined using this same assay, and protection in H. contortus
antigen-vaccinated lambs correlated better with levels of IgE than with
IgG1 (Kooyman et al., 2000; LeJambre et al., 2008). Inasmuch as IgE facil-
itates basophil activation and IL-4/IL-13 release, which in turn are essential
for host protection against helminth infections in the mouse models
(Schwartz et al., 2014), this information suggests the need for better and
more sensitive assays to consistently measure IgE in body fluids and tissue
homogenates of ruminants infected with H. contortus. If key H. contortus an-
tigenespecific IgA and IgE can be more accurately and consistently
detected in ovine blood or other body fluids, these antibodies may be useful
in determining population mucosal immunity as well as in selecting animals
with natural resistance to H. contortus infection, as mediated by high levels of
IgA and IgE.
4.2.1 Eosinophils and eosinophil peroxidase assays

Eosinophilia is well documented in H. contortuseinfected animals as well as
in animals infected with other nematodes, and has been correlated with
protection (Fawzi et al., 2014; Huang et al., 2015; Preston et al., 2014;
Reinhardt et al., 2011). Traditionally, eosinophilia has been determined
by counting this cell population in whole blood. However, obtaining eosin-
ophil counts in blood and tissues from infected animals at postmortem can be
difficult, and data from current enumeration assays tend to be highly variable
and inconsistent. Recent advances to assess eosinophilia rely on monitoring
levels of eosinophil-specific peroxidase (EPX) in serum, tissue homogenates
or other body fluids using a sensitive sandwich ELISA. Eosinophil peroxi-
dase is specific to primary and secondary granules of mammalian eosinophils.
This sandwich ELISA utilizes a matched pair of monoclonal antibodies spe-
cific for EPX (Ochkur et al., 2012) and reflects not only eosinophil activa-
tion such as degranulation, but may also correlate with the magnitude of the
activation (eg, number of activated eosinophils). Although a good indicator
of nematode parasite-induced eosinophilia, evidence is lacking to link EPX
directly to host protection (Cadman et al., 2014; Ramalingam et al., 2005).
Given that this assay can detect ruminant EXP, it should also be useful for
assessing individual and population immunity and for selecting eosinophil-
mediated resistant breeds.
4.2.2 Mast cell and mastocytosis assays

Like eosinophilia, mastocytosis is also correlated with protection in H. contortus
and other gastrointestinal nematode infections (Hepworth et al., 2012;
Schallig, 2000; Schallig et al., 1997; Shakya et al., 2011). In the mouse model,
mast cell accumulation and, in particular, mast cell degranulation at early stages
of infection by gastrointestinal nematode parasites are critical to priming a pro-
tective Th2 response (Hepworth et al., 2012). Tryptic peptidases (tryptases),
which belong to the serine-class peptidases, are among the most abundant
proteins in mast cell secretory granules and they are released externally during
exocytosis. Thus, detection of local or systemic mast cellespecific markers,
such as tryptase (Miller and Pemberton, 2002; Pemberton et al., 2000;
Schwartz, 2006), can be useful for assessing mast cell activation/degranulation
and for determining parasite susceptibility of the host. Currently, an assay for
the specific detection of mast cell tryptase in sheep is not available; however, a
bovine ELISA tryptase appears to have broad cross-reactivity with tryptases of
other host species, including sheep and goats.
Another marker for infection is mast cell proteinase-1 (Miller and
Pemberton, 2002; Pemberton et al., 2000), which is also a serine proteinase
with dual chymase/tryptase activity. It is expressed in gastrointestinal mast
cells and transported to the surface mucosa during nematode infections.
With respect to fibrinogen cleavage and fibroblast stimulation, the sheep
mast cell proteinase (SMCP) exhibits functional similarities to mast cell tryp-
tase (Pemberton et al., 1997). ELISA targeting SMCP has been developed
(Huntley et al., 1987). The presence of SMCP has been linked to protection
in sheep infected with H. contortus, where SMCP is elevated in immune
gastric mucosa compared with that in normal tissues (Huntley et al.,
1987). Although, SMCP is abundant in homogenates of abomasal tissue of
parasite-immune sheep, it remains low to undetectable in serum and lymph,
thus limiting its application to live animals. Furthermore, sheep serum and
lymph contain inhibitors that can interfere with the SMCPeantibody inter-
actions. Consequently, the SMCP-ELISA works best with homogenates
from abomasal tissue.
4.2.3 T cell proliferation assay

T cells are one of the major components of peripheral blood mononuclear
cells (PBMC) and key players in both innate and adaptive immunity. T cells
proliferate upon activation in a recall response, which is required for protec-

tion (Haig et al., 1989; Jasmer et al., 2007; Pe~ na et al., 2006). Antigen-
specific T cell assays can be useful in determining T cell responses to
H. contortus infection, and can be used to study parasite molecules capable
of modulating host immunity (Torgerson and Lloyd, 1993). Research on
isoforms of recombinant galectin (Hco-gal-m and ef) from H. contortus,
have shown that parasite-derived galectins suppress immunity and therefore
promote the infection process by binding the surface of PBMC, including T
cells (Wang et al., 2014a,b) and, in particular, to the transmembrane protein
63A (Yuan et al., 2015). Consequently, T cell assays can be quite informa-
tive. However, the process of identification and characterization is quite
tedious, involving 3H-thymidine, homologous host cells as antigen-
presenting cells (APCs) and irradiated APCs, if T cell lines or clones are
used (Tuo et al., 1999). This type of assay is useful only for research purposes,
particularly in assessing vaccine efficacy and candidate vaccine discovery.
4.2.4 Cytokine and host alarmin assays

Cytokines are well known for their involvement in the expulsion of gastro-
intestinal nematodes. In preparation for the protective Th2 immunity
against such nematodes, the gastrointestinal epithelial cells respond to the
infection by releasing innate cytokines, such as IL-1, IL-25, IL-33, and
thymic stromal lymphopoietin (TSLP). They also release tissue/cell
injury-associated alarmins or danger-associated molecular pattern (DAMP)
molecules, such as uric acid, ATP, high mobility group box 1
(HMGB1) and S100 proteins (reviewed by Hammad and Lambrecht,
2015). Thus, characterizing the immune state of the animal can have a
demonstrable impact on delineating pathways involved in the infection pro-
cess. For example, one might expect to see ATP levels increase during Hae-
monchus infection, due to cell damage; however, levels of adenosine and
ADP were reported to be substantially reduced (Gressler et al., 2014).
This finding aligns with the importance of adenosine and ADP in control-
ling platelet activation and allowing Haemonchus to feed on blood. In addi-
tion, extracellular HMGB1 was shown to have a dual function, where it can
regulate inflammation and cellular repair, as a passively released molecule
from damaged cells or as a secreted molecule from activated immune cells
(Vande Walle et al., 2011).
Quantifying selected groups of cytokines, such as IL-4, IL-13,TNF-a,
IFN-g, either directly, or by reverse-transcription PCR (RT-PCR) has
aided in evaluating breeds of small ruminants that are resistant to H. contortus
infection (Alba-Hurtado and Mu~ noz-Guzman, 2013; Miller and Horohov,
2006; Zaros et al., 2014). Such assays can be critical for assessing vaccine and
drug efficacies. Unfortunately, the lack of commercially available immuno-
logical assays for sheep and goats has made direct investigations of many
cytokines difficult. Consequently, today RT-PCR has become the method
of choice.
4.2.5 ELISPOT and the identification of antibody-secreting cells

Immunohistochemistry can be used as a research tool to identify the spatial
localization of isotype-specific antibody-containing cells in situ. This proce-
dure involves tissue fixation, embedding, sectioning, rehydration and prob-
ing with antiisotype antibodies labelled with a reporter enzyme, followed by
detection using a substrate. For instance, Gill et al. (1992, 1993) detected
IgA-, IgG1-, IgG2- and IgM-containing cells in the abomasum of H. contor-
tuseinfected sheep, where the most abundant cell types were test positive for
IgA, IgG1 and IgM. The disadvantages of this method are that it can only
assess relative numbers of isotype-specific antibody-containing cells, and
that the antibodies detected are not necessarily antigen specific. There is
no report of the use of the enzyme-linked immunospot assay (ELISPOT)
for the detection of H. contortusespecific antibodies in small ruminants,
although the test was successfully applied to study T. colubriformisespecific
antibodies (Emery et al., 1999). This assay might be used for assessing the
frequencies of isotype-specific secreting cells in a mixed cell population in
H. contortuseinfected small ruminants.
5. FINAL THOUGHTS
When one holistically examines the identification of Haemonchus spp.
and the diagnosis of Haemonchus infection or haemonchosis, certain issues
become apparent. First, for the most part, delineation among Haemonchus
in domestic livestock has been relegated to host associations rather than
direct methods of identification. Other than for morphological identifica-
tion of adult worms and direct DNA sequencing of specific gene targets,
PCR tests to define species based on worm populations are lacking. This
aspect becomes problematic when performing epidemiological studies and
equally important, when accessioning gene sequences to worldwide data-
bases. Personal experience has instructed us that the sources of gene
sequence data derived from earlier database submissions can at times be
faulty due to nematode misidentification. In most instances, such genetic
data were not accompanied by the submission of morphologically identified
voucher specimens for archival storage in museum repositories. We suggest

that genetic data should be concurrently derived from specimens that have
an authoritative identification and which, because of archival deposition, can
be available to confirm or secondarily assess the validity of field-based obser-
vations. Voucher specimens are particularly critical in areas of sympatry for
assemblages of domestic and free-ranging ungulates, where there is a consid-
erable expectation for cross-transmission of parasites between or among
animal species. In recent years, most of the sequence databases have been
updated using highly inbred worm populations or laboratory strains of para-
site species; however, new data from epidemiological studies can no longer
rely on host associations for definitive identification/diagnosis, given anthro-
pogenic forcing and the broad host associations of members of this genus
with wild ruminants.
Second, it has become clear that antibody-based assays and other im-
mune-related tests have not been widely used for diagnosis, although they
have played insurmountable roles in understanding haemonchosis and the
host immune response against Haemonchus. Physiological parameters have
taken precedence, because of their ease of use in the field, lack of need
for expensive equipment and laboratory consumables/supplies, and because
the host exhibits symptoms of disease long before the serological tests
become functionally beneficial to use.
Finally, rapid changes and progress in molecular and proteomic technol-
ogies will continue to advance this field of research, and at a remarkable
pace. However, we need to be mindful that morphology has and will
continue to be the benchmark for defining taxa in the foreseeable future,
given the significant genetic diversity within and among populations that
defines most nematodes of the gastrointestinal tract of ruminants.
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CHAPTER SIX
Diagnosis, Treatment and

Management of Haemonchus
contortus in Small Ruminants
R.B. Besier*, 1, L.P. Kahnx, N.D. Sargison{, J.A. Van Wykjj
*Department of Agriculture and Food Western Australia, Albany, WA, Australia
x
{
jj
1
Corresponding author: E-mail: R.B.Besier@murdoch.edu.au
Contents
1. Introduction 183
2. Diagnosis and Disease Monitoring 183
2.1 Clinical signs 184
2.2 The FAMACHA system for anaemia assessment 186
2.3 Postmortem examination 187
2.4 Laboratory diagnosis 187
2.4.1 Faecal worm egg counts 187
2.4.2 Laboratory identification of eggs or larvae in faecal samples 190
2.4.3 Molecular techniques 191
2.5 Haematology 192
3. Anthelmintic Treatment 193
3.1 Anthelmintic groups 194
3.1.1 Benzimidazoles 194
3.1.2 Imidazothiazoles/tetrahydropyrimidines 195
3.1.3 Organophosphates 195
3.1.4 Macrocyclic lactones 195
3.1.5 Salicylanilides and substituted phenols 196
3.1.6 Amino-acetonitrile derivatives 196
3.1.7 Spiroindoles 197
3.1.8 Combination anthelmintics 197
3.2 Anthelmintic-resistance management 198
3.2.1 Minimizing resistance-selection treatment practices 198
3.2.2 Refugia strategies to maintain anthelmintic-susceptible populations 199
3.2.3 Anthelmintic choice 201
3.2.4 Prevention of the introduction of resistant nematodes 204
4. Nonchemical Control 205
4.1 Grazing management 205
4.2 Nutritional management 206

j
ISSN 0065-308X
4.3 Genetic selection against Haemonchus contortus 207

4.4 Biological control 208
4.5 Alternative anthelmintic compounds 210
4.6 Vaccines 211
5. Preventative Programmes 213
5.1 Haemonchosis risk assessment 213
5.2 Epidemiologically based preventative programmes 214
5.2.1 Wet tropical zones 214
5.2.2 Subtropical zones 218
5.2.3 Summer rainfall temperate zones 218
5.2.4 Mediterranean climatic zones 219
5.2.5 Cold temperate zones 219
5.2.6 Arid zones 220
5.3 Nonchemical strategies 220
5.4 Monitoring of Haemonchus contortus burdens 221
5.5 Anthelmintic choice and resistance management 221
6. Conclusions 222
References 224
Abstract
Haemonchus contortus is a highly pathogenic, blood-feeding nematode of small rumi-
nants, and a significant cause of mortalities worldwide. Haemonchosis is a particularly
significant threat in tropical, subtropical and warm temperate regions, where warm
and moist conditions favour the free-living stages, but periodic outbreaks occur more
widely during periods of transient environmental favourability. The clinical diagnosis
of haemonchosis is based mostly on the detection of anaemia in association with a
characteristic epidemiological picture, and confirmed at postmortem by the finding
of large numbers of H. contortus in the abomasum. The detection of impending hae-
monchosis relies chiefly on periodic monitoring for anaemia, including through the
‘FAMACHA’ conjunctival-colour index, or through faecal worm egg counts and other
laboratory procedures. A range of anthelmintics for use against H. contortus is available,
but in most endemic situations anthelmintic resistance significantly limits the available
treatment options. Effective preventative programmes vary depending on environ-
ments and enterprise types, and according to the scale of the haemonchosis risk and
the local epidemiology of infections, but should aim to prevent disease outbreaks while
maintaining anthelmintic efficacy. Appropriate strategies include animal management
programmes to avoid excessive H. contortus challenge, genetic and nutritional
approaches to enhance resistance and resilience to infection, and the monitoring of
H. contortus infection on an individual animal or flock basis. Specific strategies to
manage anthelmintic resistance centre on the appropriate use of effective anthelmin-
tics, and refugia-based treatment schedules. Alternative approaches, such as biological
control, may also prove useful, and vaccination against H. contortus appears to have sig-
nificant potential in control programmes.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 183
1. INTRODUCTION
The effective prevention of haemonchosis is essential for the sustain-
able management of sheep and goats in regions where Haemonchus contortus is
endemic, due especially to the threat of animal mortalities. Although the
seasonal epidemiology of H. contortus infection in relation to weather pat-
terns is well-established in the majority of climatic zones (O’Connor
et al., 2006; chapter: The pathophysiology, ecology and epidemiology of
Haemonchus contortus infections in small ruminants by Besier et al., 2016),
the high biotic potential of H. contortus can lead to rapid population in-
creases, and haemonchosis outbreaks frequently occur with little warning.
Outside of the major risk zones, however, the ability of H. contortus to
exploit short periods of favourable climatic conditions, and opportunities
related to climate change in new environments, suggest that outbreaks of
haemonchosis are increasingly likely in nonendemic regions.
Fortunately, despite the potential severity of haemonchosis outbreaks,
the presence of developing H. contortus burdens in small ruminants can be
diagnosed relatively easily both clinically and by laboratory procedures,
and easily confirmed by necropsy where deaths occur. This provides the ba-
sis for effective surveillance, treatment and preventative programmes, where
the appropriate economic and labour resources are available. Unfortunately,
in many situations, control measures are based either on intervention when
outbreaks occur or on subjectively timed, ad hoc treatments, rather than on
planned strategic or integrated programmes. Further, widespread anthel-
mintic resistance limits the effectiveness of both treatment and prevention,
particularly given that control centres largely on the use of anthelmintics.
The successful avoidance of haemonchosis relies on the early recognition
of risk situations, the periodic monitoring of H. contortus burdens, and pre-
ventative programmes which include grazing management and nonchemical
measures, in addition to anthelmintic treatments.
2. DIAGNOSIS AND DISEASE MONITORING

An accurate diagnosis is essential when haemonchosis is suspected,
given the potential for significant and ongoing animal mortalities without
appropriate and timely treatment. It is also important to exclude haemon-
chosis when it is suspected not to be involved, as some of the clinical signs
are not specific; in particular, sudden deaths of livestock in endemic zones
may be erroneously attributed to H. contortus, whereas the actual primary

cause is commonly related to other conditions, including trematodes or vec-
tor-transmitted protozoal infections. Under these circumstances, some treat-
ments may be inappropriate to the actual condition, risking further losses
until an accurate diagnosis is made.
The signs and laboratory procedures used to detect clinical disease are
also used to monitor for subclinical H. contortus infection or impending hae-
monchosis, and to indicate whether the rate of pasture contamination with
H. contortus eggs is likely to lead to overt disease in the near future. Periodic
monitoring of signs of anaemia in host animals, faecal worm egg counts
(FWECs), and opportune postmortem examinations are an important part
of integrated parasite management (IPM) programmes which aim to avoid
both serious parasitism and excessive chemical treatments. The genetic selec-
tion of animals with a greater natural resistance to nematodes, a key compo-
nent of IPM strategies, also relies on these diagnostic indicators. New
laboratory tools based on molecular technologies will further improve
both the diagnosis and management of H. contortus infections (chapter:
The identification of Haemonchus species and diagnosis of haemonchosis
by Zarlenga et al., 2016).
In all situations, diagnostic protocols must take account of the epidemi-
ological factors that influence the likelihood of a particular disease. In most
locations, the seasonal occurrence of haemonchosis and the potential effects
of specific weather events are well known, as are the classes of animal most at
risk at particular times of the year. Animal management factors, including
nutritional status, movements between pastures and the anthelmintic treat-
ment history are also pertinent to the development of disease. In general,
integrating clinical, laboratory and epidemiological information will rapidly
confirm or otherwise a diagnosis of haemonchosis, without the necessity for
prolonged or unnecessarily expensive investigations. Where a diagnosis re-
mains equivocal, it can usually be confirmed or otherwise by observing
the response to treatment with an effective anthelmintic.
2.1 Clinical signs

The signs most characteristic of H. contortus infection relate almost entirely to
the blood-feeding activities of adult and late larval stages (Bowman, 2014;
Dunn, 1978; Levine, 1980; Taylor et al., 2007; Urquhart et al., 1996),
and include deaths, anaemia, reduced exercise tolerance and subcutaneous
oedema. In cases of overwhelming infection (‘hyperacute haemonchosis’),
animals are found dead, with signs of severe anaemia in many of the
survivors. In most cases, the epidemiological picture is characteristic, in terms

of weather conditions that are especially conducive to the development of
infective larvae and the susceptibility of the animals involved; this form of
haemonchosis commonly involves juveniles (lambs or kids), and lactating
ewes or does that are subject to the peri-parturient relaxation of resistance
to nematode infection.
Usually, haemonchosis occurs in an acute form, with varying rates of
onset and mortality, depending mostly on the rate of intake of infective
larvae of H. contortus. The most specific indication is anaemia, seen as pallor
of the mucous membranes, especiallyeasily seen in the conjunctivae, and
varying from the normal, red-pink colour to extreme white in terminal sit-
uations. Affected animals become progressively weaker with increasing
blood loss, and are less inclined to move and may spend more time lying
down than usual. On driving, some will collapse and may die, particularly
if repeatedly forced to move. Without treatment, the hypoproteinaemia
due to blood loss may lead to a general, ventral oedema in a proportion
of animals. This is especially evident as submandibular oedema (‘bottle
jaw’), although this is not pathognomonic for haemonchosis, as it also occurs
on a flock or herd scale in chronic fasciolosis outbreaks and with extreme
cachexia, and deaths may occur before oedema develops. The faeces are
often firm and scant, and may be dark due to an occult blood content. Diar-
rhoea is not usual, although haemonchosis can occur concurrently with in-
fections with other nematodes that cause this clinical sign. Pain is not visually
evident, but a break in the wool of sheep occasionally occurs, with the shed-
ding of strands of wool or even the entire fleece.
If left untreated, deaths typically continue and the rate of losses increases
over several days. However, a marked variation in susceptibility to haemon-
chosis among individuals is usual, reflecting both host resistance to H. contortus
establishment and/or tolerance of its effects (presumably largely the capacity
to replace lost blood, or an initial better blood status). Hence, it is not usual or
inevitable that all individuals in a group affected by haemonchosis show the
same extent of clinical signs or die if untreated (Roberts and Swan, 1982a).
The proportion of animals within a flock or herd affected by different de-
grees of disease is largely genetically determined, and significantly mediated
by their nutritional condition (chapter: The pathophysiology, ecology and
epidemiology of Haemonchus contortus infections in small ruminants by Besier
et al., 2016).
The chronic form of haemonchosis is related to smaller but sustained
burdens of H. contortus, seen as weight loss or poor weight gain, general
illthrift and a degree of anaemia in some individuals (Dunn, 1978). This syn-
drome was first described from extensive grazing situations in Africa, where
animals are often seasonally malnourished in highly seasonal environments
(Allonby and Urquhart, 1975), and from pastoral situations in Australia
(De Chaneet and Mayberry, 1978), especially if anthelmintic treatments
are not routinely given. In such situations, haemonchosis becomes overt
when blood loss cannot be sustained as the nutritional state declines, or
when weather events incite an increased intake of infective larvae of
H. contortus. It is also likely that a chronic form of haemonchosis occurs un-
der intensive grazing conditions in less seasonal and higher rainfall zones,
where occasional partially effective treatments fail to completely remove
H. contortus burdens, but adequate nutrition ensures sufficient resilience to
infection. In such situations, it is likely that H. contortus is one of the several
nematode species contributing to suboptimal animal production.
2.2 The FAMACHA system for anaemia assessment

The visible signs of anaemia have been exploited as a simple and rapid diag-
nostic indicator through the development of the FAMACHA system in
South Africa, which involves the assessment of the colour of the conjunctival
membranes (Van Wyk and Bath, 2002). For this, animals are restrained, and
the eyes are examined and scored against a standardized set of five colours
ranging from red-pink (normal) to white (terminal anaemia) (FAMACHA
refers to ‘FAfa MAlan CHArt’; Malan et al., 2001).
The FAMACHA system provides an assessment of relative anaemia (of
any cause), and was specifically developed for the identification of animals
requiring treatment on an individual basis, to reduce the selection pressure
for anthelmintic resistance imposed by the usual (and frequent) treatment
of all animals in the group. However, the classifications can also be used
as the basis for whole-flock or herd anthelmintic treatments, when a propor-
tion of animals fall below a threshold value. This approach requires frequent
assessments to ensure the early detection and treatment of animals with
subclinical haemonchosis, with a recommended schedule of inspections at
7e10 day intervals, once anaemia is detected in a small monitor group
(Van Wyk and Bath, 2002). A significant labour input is required, which
is most feasible when animal numbers are small or sufficient labour is avail-
able, and the system has achieved wide adoption for both sheep and goats in
situations where these conditions can be met. In addition to a dramatic
reduction in the proportion of anthelmintic treatments given, ‘repeat
offender’ animals can be identified and culled, as there is a high heritability
of FAMACHA score for both sheep (Riley and Van Wyk, 2009) and goats
(Mathieu et al., 2007).
2.3 Postmortem examination

Outbreaks of haemonchosis are often first recognized when mortalities
occur, and postmortem examinations enable rapid confirmation. H. contortus
burdens are readily visible on the abomasal surface as 2 cm-long worms with
a ‘barber’s pole’ appearance where the red gut (from host-derived haemo-
globin) spirals around the white ovaries of female worms. The large burdens
(many thousands) of worms considered typical of acute haemonchosis leaves
little doubt about the diagnosis. The mucosa is oedematous and appears
covered with worms, with petechiae and often frank blood-seepage evident.
Depending on the number of worms and the stage of infection, there are
varying degrees of pallor of the carcass and of ascites, and the blood may
be watery and fail to congeal. In more chronic forms of haemonchosis,
the carcass may appear cachectic, with only a few hundred worms, and a
diagnosis requires greater support in relation to the epidemiological circum-
stances and complementary laboratory assessments.
The number of H. contortus present is rarely quantified by a total worm
count, as the presence of many worms in association with the clinical signs
and epidemiological picture are diagnostic. However, the worm numbers
quoted from original observations of different stages of haemonchosis
(Dargie and Allonby, 1975) suggest that, in the hyperacute form, massive
numbers, >30,000 H. contortus may be present; 2000e20,000 worms in
the acute form, and 100e1000 in chronic haemonchosis. As noted previ-
ously, the considerable variation in effects on individuals within a flock re-
lates especially to genetic differences in host susceptibility to infection (and
hence intensity of infection, or burden sizes), and the capacity to replace lost
blood, as well to the time that an infection is present, and in less acute forms,
the nutritional status of host animals.
2.4 Laboratory diagnosis

2.4.1 Faecal worm egg counts
FWECs can be helpful to support the diagnosis of haemonchosis when no
necropsy is conducted, or the epidemiological picture or clinical signs are
atypical. Usually, FWECs are used as a monitoring tool to indicate the rela-
tive threat of disease or the extent of pasture contamination with H. contortus
eggs. For nematode species generally, the FWEC is not a precise or sensitive
measure of infection, due to the variable relationship between the number
of worms in the gastrointestinal tract of the host and the number of eggs in
the faeces, and the typically large variation in worm burdens among different
animals in a group (Barger, 1985; Whitlock et al., 1972). In addition, the
FWEC does not account for the presence of immature (nonegg laying,
but potentially blood-feeding) worms, and is further influenced by the den-
sity of faeces due to variations in the water content (Le Jambre et al., 2007).
Nonetheless, FWEC has advantages of low cost and simplicity of technique,
and is an effective diagnostic indicator, provided that sufficient animals from
a group are sampled, the laboratory procedures are appropriate (including for
the identification of eggs) and allowances are made for other variables.
Fortunately, the diagnostic value of FWEC is greater for H. contortus than
for other trichostrongyles, because there is a relatively strong relationship be-
tween the biomass and the worm egg output (Coadwell and Ward, 1982),
and between the total H. contortus count and FWEC in sheep (Roberts and
Swan, 1981) and goats (Rinaldi et al., 2009). Le Jambre (1995) also found a
high correlation between biomass and the number of adult H. contortus, and
also with the degree of blood loss and FWEC. Of major practical impor-
tance, FWEC is most reliable when haemonchosis is imminent or present,
due to extremely high counts. In comparison with most ruminant nema-
todes, H. contortus is a prolific egg-producer (Gordon, 1948), and the high
FWECs typically seen in acute haemonchosis usually allow this disease to
be distinguished from other helminthoses.
For H. contortus, FWECs associated with significant but not immediately
dangerous burdens typically number in the thousands of eggs per gram (epg).
Levine (1980, p. 204) quotes sources that suggest 3000 epg to indicate ‘light’
infections for individual adult sheep, compared with 30,000 epg for ‘severe’
infections, and Taylor et al. (2007, p. 160) indicate that ‘moderate’ burdens
may relate to FWECs of between 2000 and 20,000 epg. These figures are
ten-times higher than FWECs relating to Trichostrongylus and Teladorsagia
burdens of similar significance, which rarely reach such high values, and
are then typically accompanied by the primary sign of diarrhoea. High
FWECs in the absence of the latter sign are hence a strong indication of
the presence of H. contortus. The interpretation of nematode FWECs is
usually based on the mean for a flock or herd, particularly if processed by
a composite (bulked) laboratory method; this value is obviously far lower
than the highest extreme counts, but the latter must be taken into consider-
ation. There is no point in withholding treatment on the basis of a moderate
mean FWEC if some individuals are likely to be at significant risk, and
where (as usual) it is not possible to individually identify them.
FWECs are less definitive when intended to indicate the presence of

H. contortus before significant burdens develop, or the degree of pasture
contamination with H. contortus eggs, particularly if no differentiation of
counts to genus or species is available. In such situations, considerably lower
FWECs become significant, and to prevent excessive pasture contamination,
treatment may be recommended at mean values of as low as 1000 epg
(www.wormboss.com.au). Sufficient animals from the flock or herd must
be sampled for confidence that the mean result reflects the group situation.
Although trichostrongylid egg counts within groups of grazing animals usu-
ally follow an aggregated (skewed) pattern, typically as the statistically nega-
tive binomial distribution (Barger, 1985; Torgerson et al., 2005), field studies
indicate that FWECs for H. contortus are moderately repeatable between in-
dividuals, indicating that the same animals tend to have a consistently high or
low ranking of counts within the group (Barger and Dash, 1987; Doligalska
et al., 1997; Van Wyk and Riley, 2009).
A key question is the number of samples necessary to account for the
typical within-flock variation in FWECs. Other sources of variation also
affect the mean count result, including variability inherent to the technique
and operator proficiency, each of which follow different statistical distribu-
tions (Van Burgel et al., 2014), and usually results in wide confidence inter-
vals around the ‘true’ mean count. The variation expected for egg
distribution within faecal suspensions (as a Poisson distribution) can be pre-
dicted, and inappropriate variation reduced by increasing the sample num-
ber, and to a degree by more sensitive detection methods (Torgerson et al.,
2012). The minimum sample size of 10 from a flock, which has become
established (Brundson, 1970; Nicholls and Obendorf, 1994), is considered
reasonable for the majority of situations, and is supported by modelling of
sample sizes for use in a composite technique (Morgan et al., 2005). How-
ever, this statement is made with the important qualification that more sam-
ples may be required if there is significant aggregation (overdispersion)
within the group. Obviously, the degree of within-flock variation cannot
be easily estimated from a small sample, but it is particularly common for
H. contortus, where individual animals with high burdens may have massively
higher FWECs than the mean of the group; to this extent, a sample number
larger than 10 is generally warranted when a ‘sensitive’ indication of
H. contortus burden or an estimate of H. contortus egg output is required.
The most commonly used techniques in the laboratory remain variations
on the McMaster procedure (Bowman, 2014; Urquhart et al., 1996;
Vadlejch et al., 2011; Whitlock, 1948), where helminth eggs are counted
by faecal salt flotation. The within-laboratory sensitivity can be increased by

counting more chambers or larger amounts of faeces, although considerably
more sensitive techniques, such as FLOTAC (Rinaldi et al., 2011) and an
earlier cuvette method (Christie and Jackson, 1982), have been developed.
If the cost of FWEC techniques is a limitation to wide adoption for routine
nematode monitoring, this can be reduced by the use of a composite
(bulked) counting technique. This entails counting a smaller number of
chambers, but there is no loss of precision of the mean count in comparison
with that from individual counts (Baldock et al., 1990; Morgan et al., 2005;
Nicholls and Obendorf, 1994), allowing the sampling and testing of a larger
number of animals at a reduced cost. Regardless of the technique used, its
apparent simplicity should not be overestimated, as evaluations have shown
considerable potential for operator-error when conducted in a laboratory
setting (Van Burgel et al., 2014) and by less-trained operators (McCoy
et al., 2005).
In summary, FWECs are an essential part of the diagnostic toolkit, as the
samples are simple to obtain and relatively inexpensive to process. Very high
FWECs in some individuals can provide rapid confirmation of cases of acute
haemonchosis, and may provide early indication of impending outbreaks,
without resorting to species identification. Less extreme FWEC results
may indicate the potential for excessive pasture contamination to lead to dis-
ease in the future. When results are not clearly indicative of H. contortus,
larger sample numbers or more precise laboratory methods may be used
to increase sensitivity. Further testing is frequently necessary to indicate
the genus or species present before a diagnostic interpretation is possible.
2.4.2 Laboratory identification of eggs or larvae in faecal samples

Confirmation of the identity of nematode eggs is a routine component of a
laboratory diagnosis, and a number of techniques exist for the species or genus
identification of the egg or larval stages present in faecal samples. The iden-
tification of H. contortus is especially relevant in situations where the size of
the estimated burden will determine the necessity or otherwise for treat-
ment, and because narrow spectrum anthelmintics are available specifically
for blood-feeding helminths. Since the 1990s, research has focussed on
the development of rapid and precise molecular tests for the quantitative in-
dicators of individual species present in faecal samples (Zarlenga et al., 2016).
As the eggs of most ruminant trichostrongyles cannot be reliably differ-
entiated based on size or morphology alone (Christie and Jackson, 1982),
their identification has depended on the use of conventional laboratory
culture of eggs in faecal samples to the infective larval stage, and the identi-
fication of the larvae on the basis of the dimensions and morphology of
various structures (e.g., Bowman, 2014; Urquhart et al., 1996). However,
despite the relative simplicity of the approach, it suffers from numerous dis-
advantages (reviewed by Roeber and Kahn, 2014). These include the
requirement for culturing of faeces for about one week; the skill required
to differentiate among some genera on the basis of larval-sheath tail length;
and that some genera cannot be reliably distinguished according to the long-
used standard identification key (Dikmans and Andrews, 1933). Various re-
visions of the identification criteria have increased the accuracy, and at least
one detailed guide to increase the speed and accuracy of differentiation has
been published (Van Wyk and Mayhew, 2013; van Wyk et al., 2004). As a
further source of potential error, different nematode species vary in their
response to faecal culture conditions, which may affect the size of infective
larvae (Rossanigo and Gruner, 1996) and the proportion of different species
from cultures (Dobson et al., 1992; McKenna, 1998).
For the specific and rapid identification of H. contortus eggs in a mixed-
genus faecal sample, many of these limitations are overcome by the lectin-
binding assay. For this procedure, nematode eggs are isolated from a faecal
suspension, and incubated briefly with a fluorescent dye conjugated to a lec-
tin (peanut agglutinin) which has a specific affinity for H. contortus eggs
(Palmer and McCombe, 1996). Eggs with a fluorescent margin are counted
and the proportion of H. contortus eggs estimated. Compared with larval cul-
ture, the lectin-based assay has a high degree of specificity for Haemonchus
species (Jurasek et al., 2010; Palmer and McCombe, 1996), requires no spe-
cial skill for identification and can be completed within 2e3 h, provided that
a fluorescent microscope is available. Lectins specific for other ovine nema-
todes have been found (Colditz et al., 2002), potentially extending the tech-
nique to other parasitic nematodes, although lectin binding appears of
limited value for the identification of infective larvae or adult worms
(Hillrichs et al., 2012). However, in most instances the prime requirement
of species identification is to indicate the presence and proportion of
H. contortus eggs following the quantification of strongylid eggs in a faecal
sample.
2.4.3 Molecular techniques

Advances in molecular technologies are now reaching practicality, with the
validation of PCR techniques for species identification, and the developing
commercialization of this and other new approaches for use in diagnostic
laboratories (chapter: The identification of Haemonchus species and diagnosis

of haemonchosis by Zarlenga et al., 2016). The basic premise is that species
can be individually identified and differentiated using genetic markers in the
nuclear ribosomal DNA (first and second internal transcribed spacers, ITS-1
and ITS-2) for a range of strongylid nematodes (eg, Gasser, 2006; Gasser
et al., 1993, 2008; Roeber et al., 2013; Wimmer et al., 2004), because of
a low level of intraspecific variation and a significantly higher variation
among species (Gasser, 2006). These markers have been exploited to detect
and identify a number of species simultaneously through various PCR tech-
niques, based on the amplification of DNA from nematode eggs in faeces
(Bott et al., 2009; Demeler et al., 2103; Learmount et al., 2009; Roeber
et al., 2012), directly from faecal DNA (McNally et al., 2013), or from in-
dividual nematode larvae (Bissett et al., 2014). The potential to quantify
worm burdens through real-time PCR has also been demonstrated, with
the prospect of replacing both the FWEC and species identification proce-
dures, providing present challenges can be met (Bott et al., 2009; Harmon
et al., 2007; H€ oglund et al., 2013; Learmount et al., 2009; McNally et al.,
2013; Melville et al., 2014).
The introduction of high-throughput molecular methods has the poten-
tial to revolutionize the application of nematode diagnostics, and automated
PCR systems are now becoming available for ruminant parasitology labora-
tories (Roeber and Kahn, 2014; Roeber et al., 2012, 2015). Their routine
use to complement traditional methods will increase as cost-efficiencies in-
crease, given the advantages of more rapid sample processing and an increased
accuracy of species identification. The prospect of quantitative nematode
detection, and possibly the molecular detection of anthelmintic resistance
and ‘point-of-care’ diagnostic systems, such as loop-mediated isothermal
amplification (LAMP) assays (Melville et al., 2014), would provide major,
additional benefits. Advances in the understanding of the genomic structure
of H. contortus will aid the development of new diagnostic tools and treatment
technologies (chapter: Functional genomics tools for Haemonchus contortus and
lessons from other helminths by Britton et al., 2016; chapter: Understanding
Haemonchus contortus better through genomics and transcriptomics by Gasser
et al., 2016; chapter: Haemonchus contortus: genome structure, organization
and comparative genomics by Laing et al., 2016).
2.5 Haematology
Although anaemia is the key pathogenic process leading to haemonchosis,
and blood loss is closely linked to the H. contortus burden (Le Jambre,
1995), it is rarely practical or efficient to utilize haematology to confirm a

diagnosis or to indicate impending disease. Critical values associated with
terminal haemonchosis are evident from field and pen observations: a fall
in haematocrit (packed cell volume) to <15% for an individual animal is
generally fatal, unless immediate treatment is given (Albers et al., 1989;
Dargie and Allonby, 1975), and despite treatment, recovery is unlikely at
values of <10e12%. Using the perhaps more consistent index of haemoglo-
bin concentration, Roberts and Swan (1982b) considered values of
<8.5 g/100 mL to be indicative of heavy H. contortus burdens.
The relationship between excreted blood in the faeces from infected
hosts and burdens of haematophagic parasites offers the potential for
pen-side tests. A simple test for the detection of faecal occult blood in
H. contortuseinfected sheep, based on a scale of colour changes on a
commercially available dipstick in relation to the concentration of blood,
has shown some promise (Colditz and Le Jambre, 2008). In practice, how-
ever, the test has proven insufficiently precise to detect low and moderate
worm burdens, and as for any simple haematological test, it is not specific
regarding the cause of faecal blood content. Nevertheless, with improved
technologies, it may prove feasible to develop this concept as a relatively
simple laboratory or field assay, without the complications of host effects
on FWEC.
3. ANTHELMINTIC TREATMENT
The necessity for effective anthelmintics for the treatment and preven-
tion of haemonchosis is hard to overestimate, given the potential for animal
mortalities if left unchecked. Although anthelmintics should always be used
in conjunction with nonchemical strategies as part of an IPM approach, the
potential for rapid increases in H. contortus populations requires effective
treatment at appropriate times. The choice of which anthelmintic and
when they should be used is a question of balance between the necessity
for treatment or prevention, the cost in terms of economics and the labour
effort required, and the potential for the development of anthelmintic
resistance.
Fortunately, there are several anthelmintic groups (ie, classes with distinct
mechanisms of effect on target helminths) available for blood-feeding para-
sites. Without considering older compounds no longer widely used, at least
six single-active anthelmintic groups are produced for use against H. contortus,
and a number of others marketed as combinations, although the range
available at any one time varies among countries. Less fortunately, there is no
guarantee that all chemicals will be uniformly effective in any one region,
due to the widespread occurrence of anthelmintic resistance. The require-
ment for frequent treatment, perceived or real, has resulted in heavy expo-
sure of H. contortus to anthelmintics, and in some situations, few effective
options remain. As there can be wide variation in the severity of resistance
among geographical regions and properties within a region, an awareness
of the likely effectiveness of the different groups is necessary for an optimal
anthelmintic choice. A summary of the global anthelmintic-resistance situa-
tion is provided in Kaplan and Vidyashankar (2012).
3.1 Anthelmintic groups

Specific reference to anthelmintics and resistance is made here only for pres-
ently available compounds and as they apply to treatment and preventative
programmes. Numerous texts and journal publications provide details of the
mode of action, pharmacokinetics and efficacy spectrum of the major an-
thelmintics (eg, Martin, 1997; McKellar and Jackson, 2004), and Chapter
“Anthelmintic resistance in Haemonchus contortus: history, mechanisms and
diagnosis” by Kotze and Prichard (2016) provides a review of the cellular
mechanisms of anthelmintic resistance.
3.1.1 Benzimidazoles
The first modern broad-spectrum anthelmintic, thiabendazole, was released
for commercial use in the early 1960s, and shown to be safe, easy to admin-
ister and highly effective (>95%) against a wide range of major ruminant
parasites (including nematodes, some trematodes and arthropods) (Gordon,
1961), and against the immature parasitic stages of some species. Other benz-
imidazoles followed, some of which are no longer in general use (parbenda-
zole, cambendazole and oxibendazole), with the current range (albendazole,
fenbendazole, oxfendazole, mebendazole) available from the late 1970s
(McKellar and Jackson, 2004). The pro-benzimidazoles, thiophanate and
netobomin, are also available in some countries. Members of this group
act on nematodes at the cellular level, mainly by inhibiting the polymeriza-
tion of microtubules, eventually causing cell death (Lacey, 1988; Martin,
1997).
Due to the time of their availability and frequent use, resistance in nem-
atodes to the benzimidazoles has been widespread globally for many years;
used alone, the group is rarely still effective against the dominant strongylid
species in a particular region (Kaplan and Vidyashankar, 2012). In most areas
endemic for H. contortus, resistance is especially severe, and the benzimid-

azoles retain a significant role only when used in combination with other
drugs.
3.1.2 Imidazothiazoles/tetrahydropyrimidines
The two families within this group share a common mode of action, as nico-
tinic agonists, against acetylcholine receptors (Martin, 1997; Robertson and
Martin, 1993). This group represented the second modern broad-spectrum
anthelmintics to be introduced (in the late 1960s), with a wide range of
activity against helminths. Levamisole is the most widely used of the group
in small ruminants, although morantel is still available in some countries for
use in sheep. Although resistance is very common in many nematode
genera, field testing results indicate that H. contortus has remained generally
susceptible to levamisole for a longer period than to the other major drugs
(eg, Playford et al., 2014). However, this is no longer the case in the main
endemic regions, and resistance must be expected to increase, although le-
vamisole typically remains effective against H. contortus in regions where it is
of lesser importance.
3.1.3 Organophosphates
An older anthelmintic group, the oral organophosphate anthelmintics, con-
tinues to be used in countries where it is still available (Campbell et al.,
1978), and includes naphthalophos, triclorfon and (as a combination prod-
uct) pyraclyfos. As with all organophosphates, they act by inhibiting acetyl-
cholinesterase activity (Martin, 1997), and are hence potentially toxic to
mammals as well as to target parasites, and caution is necessary for their
administration and handling. H. contortus is comparatively more sensitive
to these compounds than to most other ruminant nematodes (Fiel et al.,
2011), and very few cases of resistance have been reported; however, it is
less active against several other important nematodes, and especially the
larval stages of some species. Although this group is not universally available,
it can have a useful, narrow-spectrum role, particularly when resistance to
other groups is common.
3.1.4 Macrocyclic lactones

The release of ivermectin in the early 1980s introduced a new era of effec-
tiveness against most species and all stages of nematodes (but not cestodes
or trematodes) and also against some ectoparasites (Campbell et al., 1983).
Although different actives within the macrocyclic lactone (ML) group
share a major mode of action, the disruption of nervous transmission by

potentiating glutamate-gated chloride channels (Martin and Pennington,
1988; Martin, 1997), pharmacologic differences exist among avermectins,
milbemycin and moxidectin, with implications for the relative potency
and mechanisms of resistance selection (Lloberas et al., 2013; Prichard
et al., 2012). In field efficacy tests using recommended dose rates, moxidec-
tin has been shown to be more effective than other MLs once resistance to
this group appears, including against T. circumcincta (see Leathwick et al.,
2000) and H. contortus, with abamectin more effective than ivermectin
(Lloberas et al., 2013; Playford et al., 2014; Wooster et al., 2008). Resis-
tance to ivermectin is widespread in H. contortus populations in endemic
zones, and is increasing in prevalence to moxidectin (Kaplan and Vidya-
shankar, 2012; Prichard et al., 2012). The persistent effect of moxidectin
(as both the oral and long-acting injectable formulations) against H. contor-
tus offers potential control benefits, but is also reduced or eliminated when
ML resistance develops. Other MLs, such as doramectin, mostly used in
cattle, are also available in some countries, primarily for use as endectocides.
3.1.5 Salicylanilides and substituted phenols

This group comprises a number of compounds which act by inhibiting
energy metabolism (uncoupling of oxidative phosphorylation; Martin,
1997), with those active against nematodes including closantel, rafoxanide,
and (by injection) disophenol and nitroxynil (other chemicals in this group
are more useful for cestodes and trematodes). As narrow-spectrum anthel-
mintics with activity specifically against blood-feeding helminths, they are
of particular importance for the control of H. contortus, especially as some,
such as closantel and disophenol, have a prolonged activity of some weeks
after administration (Hall et al., 1981). However, the latter effect may also
have predisposed this group to resistance in H. contortus (Rolfe, 1990;
Van Wyk, 2001), which is first seen as a reduction in the persistent effect.
Resistance to closantel is now common in endemic regions where it was
intensively used (eg, Playford et al., 2014), but is generally uncommon in
lower-risk situations where the frequent use of a long-acting anthelmintic
specifically for H. contortus control has not been warranted.
3.1.6 Amino-acetonitrile derivatives

The sole member of this group, monepantel, was introduced in the late
2000s, as the first new anthelmintic type for some 30 years (Kaminsky
et al., 2008). It has a unique mode of action against nicotinic acetylcholine
receptors, and a wide spectrum of activity, similar to that of the MLs

(Hosking et al., 2010). Some instances of resistance to monepantel have
been reported, including to H. contortus (see Mederos et al., 2014; Van
den Brom et al., 2015).
3.1.7 Spiroindoles
The introduction to the anthelmintic armoury is the first member of the spi-
roindole group, derquantel (2-desoxoparaherquamide) (Little et al., 2011),
described as nicotinic cholinergic antagonists. It is produced for commercial
use only in combination with abamectin, as derqantel is itself not fully effec-
tive against all nematodes, especially the larval stages of T. circumcincta. The
combination compound has been shown in numerous trials to have high
efficacy against a range of sheep nematodes of varying resistance status (Little
et al., 2010), although a lower efficacy has been reported if abamectin is
particularly ineffective (Sager et al., 2012).
3.1.8 Combination anthelmintics

In addition to single-active anthelmintics, a wide range of combination an-
thelmintics are produced, although combination products are not accepted
in all countries. In addition to the newly introduced derquanteleabamectin
combination, various mixtures of benzimidazoles, levamisole, MLs, closantel
and organophosphates have been available. The prime purpose is to ensure
efficacy against helminths resistant to one or more of the components of a
combination, but the additional benefit of reducing the rate of selection
for anthelmintic resistance, as recognized some time ago (Anderson et al.,
1988; Barnes et al., 1995), is now the basis for recommendations for the
routine use of combinations (Bartram et al., 2012; Leathwick et al., 2009;
Le Jambre et al., 2010).
As expected, resistance to anthelmintic combinations is far less common
than to individual components, but instances of resistance occur even to
combinations of three or more actives (Mariadass et al., 2006; Playford
et al., 2014), and H. contortus populations separately resistant to several
different groups have been detected (Cezar et al., 2010; Chandrawathani
et al., 2004a; Van Wyk et al., 1997a). Plainly, the control of multiple-
resistant worms will be difficult should they become a significant part of
the total population, and procedures to prevent this from occurring are an
essential part of sustainable management programmes.
3.2 Anthelmintic-resistance management

Following the recognition of the major causal factors for anthelmintic resis-
tance (Prichard et al., 1980; Waller, 1986), strategies to minimize the further
development and impact of such resistance have been incorporated into rec-
ommended parasite management strategies. The wide recognition of the key
role of the refugia concept in explaining the development of anthelmintic
resistance has provided a general basis for sustainable control programmes
in different environments and different ruminant hosts (Leathwick and
Besier, 2014). The theoretical basis of the development of resistance and
the underlying cellular mechanisms is the subject of a more detailed review
in Chapter “Anthelmintic resistance in Haemonchus contortus: history, mech-
anisms and diagnosis” by Kotze and Prichard (2016), and the discussion here
is confined to principles for resistance management at the field level.
3.2.1 Minimizing resistance-selection treatment practices

3.2.1.1 Underdosing with anthelmintics
The potential for resistance to develop as a consequence of inadequate
anthelmintic doses has long been recognized (Prichard et al., 1980; Smith
et al., 1999). This issue appears to have been surprisingly common due
both to a lack of awareness of the importance of ensuring appropriate doses
and an underestimation of animal weights (Besier and Hopkins, 1988).
However, although underdosing may have been a factor in the development
of resistance to the older anthelmintic groups, the advice to ensure appro-
priate dosing appears to have largely been adopted in intensive, commercial
grazing situations, at least, such that underdosing should no longer play a
significant role. An exception may be where substandard products are
marketed and low doses are unwittingly administered (Van Wyk et al.,
1997b; Waller et al., 1996), and local awareness of the risk is the only
defence.
An interesting situation exists where anthelmintics have not been trialled
in animal species that require treatment, but where the market is not
perceived to justify commercial registration. It appears that, in comparison
to sheep, higher doses are required to reach or maintain the necessary blood
levels of some anthelmintic groups, and this could explain reports of
apparent suboptimal efficacy in goats (Edwards et al., 2007; Jackson et al.,
2012), South American camelids (Gillespie et al., 2010; Gonzalez-Canga
et al., 2012; Jabbar et al., 2013) and deer (MacIntosh et al., 1985; Mylrea
et al., 1991). For both the effective treatment of these species and prevention
of the development of resistance that may spread more widely, it is
unfortunate that appropriate dose rates have not been established and widely
promoted for less-commercial livestock species.
3.2.1.2 Excessive treatment frequency

Also long recognized as a major causal factor for anthelmintic resistance is a
high frequency of treatment (Prichard et al., 1980), which is a particular risk
when haemonchosis is a major risk. In environments or seasons especially
favourable for the development of the infective larvae on the pasture, treat-
ments at short intervals e often regardless of the actual immediate threat e
have been the basis of control programmes in many instances. The inevitable
occurrence of high levels of resistance to a wide range of anthelmintics un-
derlines the lack of sustainability of control regimes based largely on chem-
ical treatments.
While anthelmintic treatments will always be required, rational ap-
proaches to minimizing their frequency utilize the IPM principles of manip-
ulating the exposure of susceptible host populations to the significant intake
of infective larvae, including by using pasture management; avoiding routine
treatments by monitoring flocks and herds or individuals within them; and
incorporating nonchemical control approaches such as nutrition and ge-
netics. In particular, the different requirement for the treatment of classes
of stock at various stages of susceptibility should be recognized: young lambs
or kids require more anthelmintic support than adults until an effective ac-
quired immunity to nematodes has developed, and specific management
may be required for lactating females during peri-parturient relaxation of
resistance to helminth infection (Houdijk, 2008). Individual-animal treat-
ment strategies (see Section 3.2.2) further reduce the intensity of treatment,
despite the presence of potentially significant H. contortus challenge.
3.2.2 Refugia strategies to maintain anthelmintic-susceptible

populations
The refugia concept has been identified as a fundamental principle in resis-
tance management, to ensure that the selection advantage to resistant worms
immediately following an anthelmintic treatment does not result in a perma-
nent increase in their proportion in the total population. Strategies devel-
oped for a particular environment aim to ensure the survival of sufficient
worms in refugia from anthelmintics to dilute any resistant worms surviving
anthelmintic treatment, generally through the establishment of infective
larvae from a non(or less)-resistant source (Besier, 2008; Jackson and Waller,
2008; Kenyon and Jackson, 2012; Leathwick et al., 2009; Van Wyk, 2001).
Although a high frequency of treatment is a major causal factor, in many sit-

uations, it is not the sole or most important factor in the development of
resistance. The selective effect of any treatment regime depends largely on
the scale of dilution of resistant nematodes with newly acquired infective
larvae, and a relatively small number of treatments in a ‘low refugia’ situation
(such as a move after treatment on to helminth-free pastures) may provide
significant selection pressure (Leathwick and Besier, 2014).
Given the potentially adverse consequences of excessive parasitism,
where larger worm populations than otherwise may survive through a
refugia-based strategy, a balance is needed between the effectiveness of resis-
tance management and the efficiency of worm control. This potential con-
flict is of particular concern in intensively managed enterprises that require
highly effective worm control, and especially so in the major H. contortus
zones where there is a low margin for error in preventing excessive popula-
tion increases. The planning of refugia-based strategies requires judgements
regarding several factors: the disease risk posed by resident nematode bur-
dens; the initial resistance status; the likely intake of infective larvae
(numbers and resistance status); and the resistance selection potential of
anthelmintic treatments and animal management.
Refugia management regimes typically involve either objectively based
schedules for whole-flock/herd treatment (‘targeted treatment’) or the intro-
duction of selective treatment strategies (‘targeted selective treatment’ e
TST), by which some animals are left untreated when treatments are given
(Besier, 2012; Kenyon et al., 2009). A targeted treatment approach requires
decisions on the need for treatment for each flock or herd, rather than as
routine treatments to all groups at the same time; this ensures that some
worm populations of relatively lower resistance status (either in animals
or as infective larvae on the pasture) are present on a particular farm or
grazed area. When H. contortus is the predominant species, targeted treat-
ment can be based on visual assessments for anaemia (FAMACHA) on a
representative sample of animals, or by similarly representative FWECs,
with whole-group treatment once threshold levels are indicated. These
programmes are most effective when combined with preplanned pasture
movements based on local epidemiological patterns for H. contortus, to
minimize the need for anthelmintic treatments in comparison to contin-
uous grazing strategies (eg, Bailey et al., 2009). This approach also reduces
the requirement for continual monitoring of animals, which can involve
significant time and cost, and is generally prohibitive in intensively managed
situations. However, for all targeted treatment approaches, it is important
that treated flocks eventually graze the pasture occupied by those not treated
at the same time, to allow the uptake of infective larvae of a less-resistant
background.
‘TST strategies provide refugia for nonselected populations within a
flock or herd through individual-animal assessments, on the basis of various
indicators appropriate to the parasites involved (eg, Besier, 2012; Kenyon
and Jackson, 2012). For H. contortus, the most effective and efficient indica-
tors assess the degree of anaemia, exemplified in the FAMACHA system
(Van Wyk and Bath, 2002). As noted in Section 2.2, the procedure entails
the periodic, individual examination of the conjunctival membranes, with
colour categories providing an indication of anaemia. FAMACHA has
been demonstrated to allow a major reduction in the need for treatment
of individual sheep (Malan et al., 2001) and goats (Burke et al., 2007;
Sotomaor et al., 2012), provided that assessments are made by trained oper-
ators (Maia et al., 2014) at an appropriate frequency (Reynecke et al.,
2011a), and are used in situations where haemonchosis is the dominant nem-
atode species (Moors and Gauly, 2009). Due to the requirement for frequent
inspection of all animals during the main periods of haemonchosis threat,
FAMACHA is most applicable where labour costs are low, flocks or herds
are relatively small, and/or animal value is high. This approach has gained
acceptance in a wide range of tropical, subtropical and summer rainfall re-
gions, including in high input-cost enterprises where anthelmintic resistance
has reached extreme levels, and has a particular role in resource-poor com-
munities where the cost of whole-group treatments and the use of other
diagnostic aids is not feasible (Vatta et al., 2001). TST strategies requiring
intensive inputs are less practicable where labour is expensive and flocks
are large, and, therefore, in intensive commercial situations, a targeted treat-
ment programme based on flock or herd decisions rather than for individual
animals is usually more applicable. Although TST based on the periodic
assessment of changes in live-weight (Greer et al., 2009) or body condition
score (Besier et al., 2010; Cornelius et al., 2014) has been demonstrated to be
feasible where Teladorsagia and Trichostrongylus are the main nematode
genera, this is not appropriate for H. contortus.
3.2.3 Anthelmintic choice

Although high anthelmintic efficacy is essential for effective treatment if
haemonchosis has occurred or is imminent, it is also important to minimize
the survival of resistant H. contortus even when worm burdens and the disease
risk are low. The effectiveness of a refugia strategy is dependent on the
dilution necessary to ensure that resistant worms remain in the minority, and
the larger the number of resistant survivors of treatment, the less efficient is
the dilution effect (Leathwick et al., 2009).
There is no doubt that many livestock owners routinely use partially
effective anthelmintics, which may remove sufficient nematodes to achieve
a clinical result, but exacerbate the resistance situation by allowing resistant
worms to survive. The failure to achieve an adequate effect is of particular
consequence in regard to H. contortus, due to both the risk of animal mor-
talities and the diminishing range of anthelmintic options in many haemon-
chosis-endemic regions.
3.2.3.1 Detection of resistance

In the majority of cases, livestock owners are not aware of the anthelmintic
resistance situation on their properties. Although a variety of in vitro resis-
tance detection tests have been developed (chapter: Anthelmintic Resistance
in Haemonchus contortus: History, Mechanisms and Diagnosis by Kotze and
Prichard, 2016), at present none are available commercially as multi-
anthelmintic, multispecies systems. The faecal egg count reduction test
remains the only currently available method of simultaneously assessing
the efficacy of a range of anthelmintics against a range of species, but the
time and effort required appears to be a barrier to its wide adoption. Conse-
quently, as well as an absence of information about individual farms, in
almost all situations, there is a paucity of data for regional predictions of
likely anthelmintic efficacy patterns. Although the use of the faecal egg
count reduction test (FECRT) should continue at present to be advocated
as a routine management component, the development of practicable labo-
ratory tests, ideally with a molecular basis, is a major research priority for
livestock parasite management.
3.2.3.2 Narrow or broad-spectrum?

It is fortunate that as the most pathogenic of the common livestock nema-
todes, narrow-spectrum options with significant effects only against haema-
tophagous species are available (eg, salacylanilides and organophosphates).
The chief advantage in their use (in addition to the persistent effect of
some) is to reduce the exposure of other species to the broad-spectrum
groups, and some effort and cost is often justified to confirm that H. contortus
is the preponderant species at a particular time, and that a narrow-spectrum
anthelmintic is therefore appropriate. As for the broad-spectrum com-
pounds, resistance to some narrow-spectrum anthelmintics is common in
some regions, such as those where the salacylanilides have been used
extensively; in these situations, it is essential to assess the resistance status.

Nevertheless, the relatively wide range of treatment options for H. contortus
reduces the pressure for resistance to develop against broad-spectrum anthel-
mintics in both this and other species.
3.2.3.3 Long-acting anthelmintics

Formulations providing long-term ‘protection’ against nematodes through
persistent activity against ingested infective larvae have a particular attraction
for the control of H. contortus, given its pathogenicity and propensity for
rapid population increases. Products include those with persistent activity
for some weeks as a property of the normal formulation (the salacylanilides,
closantel and disophenol, and the ML, moxidectin), and some that are spe-
cifically formulated as long-acting injections (MLs) or slow-release capsule
formulations (avermectins and BZs, including as combinations), with activ-
ity for w3 months.
However, the ‘protective’ benefits (and economics) must be weighed
against the relatively greater potential for resistance development in compar-
ison to short-acting formulations. The chief resistance risk relates to the pro-
longed periods in which worms of susceptible genotypes are excluded in
comparison to resistant ones, due to the effect against infective larvae
(‘tail’ selection), as well as to the survival of resistant adult worms (‘head’ se-
lection) (Dobson et al., 1996; Le Jambre et al., 1999). Field studies in New
Zealand confirm the more rapid selection for resistance in a number of spe-
cies, including H. contortus, from a slow-release formulation in comparison
to multiple short-acting treatments (Leathwick et al., 2006). The first mani-
festation of resistance to persistent products is a reduction in the protective
period (Rolfe, 1990; Van Wyk et al., 1982), although this is usually not sus-
pected until reduced efficacy against adult worms is evident in an anthel-
mintic-resistance test, or more seriously, as a cause of clinical disease.
While long-acting formulations can play a beneficial role in high hae-
monchosis-risk situations, especially when used in a planned epidemiologi-
cally based programme (Dash, 1986), active steps must be taken to manage
the resistance-selection potential. These include ensuring that the adequate
dilution of resistant worms by refugia strategies, and if necessary, the removal
of resistant surviving worms with anthelmintics of alternative groups when
the persistent activity ceases. Without careful management, the continued
use of long-acting products against populations already resistant to the
anthelmintic groups involved must be expected to accelerate resistance
development (Barnes et al., 2001), and, hence, increasingly compromise
the aims of prolonged control. A high treatment efficacy (in relation to
the resistance status) is an obvious requirement for the choice of a particular,

long-acting product.
3.2.3.4 Combination anthelmintics

The beneficial effect of combining anthelmintics has long been utilized to
extend the useful lives of individual components once resistance limits their
efficacies. A range of products containing between two and four active in-
gredients are available in some countries, although the registration of prefor-
mulated combinations is not always permitted (Geary et al., 2012). The
superior efficacy of combinations is readily seen in a comparison of anthel-
mintic-resistance figures for combination products and their individual com-
ponents (McKenna, 2010; Playford et al., 2014). Of equivalent importance
is their role in delaying the rate of resistance development (Bartram et al.,
2012; Dobson et al., 2011; Geary et al., 2012; Leathwick and Besier,
2014). To maximize this effect, it may be preferable that newly introduced
anthelmintics are used in combination, as the combination advantage in
delaying resistance has been shown to decrease as resistance to the individual
actives increases (Leathwick et al., 2012).
As noted previously, a major qualification to the routine use of combi-
nation anthelmintics is the potential to exacerbate resistance to all compo-
nents, hence denying the individual use of several groups. This is a
significant risk, and combinations should always be used in the context of
refugia and effective IPM strategies, rather than considered simply as a simple
addition to the range of treatment options.
3.2.4 Prevention of the introduction of resistant nematodes

Routine measures to prevent the introduction of new forms of resistance
onto a farm through the transfer of animals appear intuitively logical.
Even in regions where resistance is highly prevalent, there is a wide variation
among farms in the severity and range of groups involved, reflecting inter-
actions between treatment practices and environmental and animal manage-
ment factors. The efficacy of anthelmintic groups that remain effective can
therefore be reduced by the inadvertent introduction of a population with a
greater degree of resistance. The presence of ivermectin resistance has been
linked to the failure to give ‘quarantine’ treatments to animals when trans-
ferred (Suter et al., 2004) and to the introduction of relatively larger numbers
of untreated stock (Hughes et al., 2007; Lawrence et al., 2006) in surveys in
Australia and New Zealand, respectively.
Given that the efficacy of anthelmintics against the nematode population

harboured by introduced animals is generally unknown, present recommen-
dations are that they should be treated with a combination of several anthel-
mintics, then placed on pastures likely to carry substantial populations of
infective larvae, to dilute any survivors of this treatment. Surprisingly, surveys
in a number of countries suggest that the adoption of a quarantine treatment
strategy is relatively poor (Lawrence et al., 2007; Morgan et al., 2012; Suter
et al., 2004). As one of the more simple elements of anthelmintic-resistance
management programmes, this strategy clearly merits more emphasis by
advisers to livestock owners.
4. NONCHEMICAL CONTROL
While a reliance on anthelmintics as the basis of helminth control is
not sustainable, the reluctance by many livestock owners in H. contortuse
endemic zones to reduce the high frequency of treatment is understandable,
unless alternatives are confirmed to be effective. A number of IPM ap-
proaches have been shown to successfully reduce the need for anthelmintic
use, by either reducing the exposure to H. contortus challenge or by
increasing the resistance or resilience of the host. While some individual
IPM elements, such as grazing management, are often used on an opportu-
nistic basis and therefore have a limited effect, objectively planned pro-
grammes incorporating a number of IPM components, in association with
appropriate monitoring of H. contortus burdens, offer the prospect of a sus-
tained reduction in both anthelmintic use and the risk of haemonchosis
outbreaks.
4.1 Grazing management

Grazing schedules based on local epidemiological information aim to mini-
mize both the intake of H. contortus infective larvae by susceptible animals
and the excessive contamination of pasture with H. contortus eggs, and hence
reduce the haemonchosis risk in the immediate and long terms. Ecological
and epidemiological data for a wide range of H. contortuseendemic zones
indicate the periods for which grazing animals must be excluded from
pasture to minimize the intake of infective larvae (chapter: The pathophys-
iology, ecology and epidemiology of Haemonchus contortus infections in small
ruminants by Besier et al., 2016). Grazing studies confirm the effectiveness of
larval-avoidance strategies, although, as expected, the time required varies
considerably between environments, from a few weeks in the wet tropics
(Fiji e Banks et al., 1990; Martinique e Mahieu and Aumont, 2009) to

some months in a summer rainfall region temperate environment (northern
New South Wales e Bailey et al., 2009; Southcott and Barger, 1975). How-
ever, the failure of many animal owners to utilize pasture management does
not necessarily relate to a lack of awareness of its potential for H. contortus
control. A major limitation is the practicality of spelling pastures for the
necessary length of time, as animal movements are largely determined by
nutritional availability. In intensive grazing situations, especially, the avail-
able pasture must be utilized for agronomic and economic reasons, and
pasture regrowth typically occurs more rapidly than the die-off of infective
larvae, although high-input, short-interval rotational systems may be feasible
(Colvin et al., 2008). A particularly effective solution is the alternation of
sheep or goats with cattle (Southcott and Barger, 1975), which have limited
susceptibility to H. contortus, although young cattle may acquire minor bur-
dens of H. contortus. Within a rotational grazing strategy, short periods of
grazing by cattle may allow some utilization of pastures, while H. contortus
larval populations diminish, and thus increase the feasibility of a pasture-
spelling strategy for sheep or goats. If grazing management opportunities
are limited, the most rational approach may be to consider the relative sus-
ceptibility of various animal classes to H. contortus, and plan to ensure that
they receive priority allocation to prepared low-risk pastures (Morley and
Donald, 1980).
4.2 Nutritional management

The ability of animals in good nutritional condition to resist infection with
nematodes and to withstand their effects has long been recognized (Coop
and Holmes, 1996), with positive responses to nutritional supplementation
demonstrated for both resistance and resilience to H. contortus (see Steel,
2003). Tolerance of H. contortus infection is significantly reduced in sheep
maintained on low-protein rations compared with animals receiving supple-
ments (Abbott et al., 1986; Nnadi et al., 2009; Wallace et al., 1996), even
though improved nutrition may not necessarily result in lower H. contortus
burdens (Abbott et al., 1986; Wallace et al., 1996). In situations of chronic
haemonchosis, overt disease may be precipitated by a reduction in nutri-
tional quality, and a loss of milk production may occur in sheep with chronic
H. contortus burdens (Nnadi et al., 2009; Thomas and Ali, 1983).
As would be expected, the benefits of supplementation in enhancing the
resistance and resilience of sheep against H. contortus infection have been
shown to be greatest in the breeds or individual animals most susceptible
to helminthosis (Abbott et al., 1985), and during periods of suboptimal

nutrition or general weight loss (Kahn et al., 2003). The relationship shown
between a higher body condition score (or fat score) and an enhanced pro-
tective immunity to H. contortus (see Macarthur et al., 2013) suggests that this
relatively simple index can be used to indicate nutritional adequacy for
worm control purposes. The necessity for effective nutritional management
to ensure adequate helminth control is a cornerstone of all worm control
recommendations, particularly when planned on the basis of nematode
epidemiology (Houdijk et al., 2012), and when combined with other con-
trol strategies (Torres-Acosta et al., 2012).
4.3 Genetic selection against Haemonchus contortus

The potential for the genetic selection of animals with a superior resistance
(Wollaston and Baker, 1996) or resilience (tolerance) (Bisset and Morris,
1996) to nematode infections, and, hence, reduced requirement for anthel-
mintic control, has been recognized for many years. Genetic strategies for H.
contortus have largely centred on resistance, as a reduction in worm burdens
decreases both the haemonchosis risk and pasture worm egg contamination.
The relatively high heritability offers significant potential for genetic selec-
tion strategies (Nieuwoudt et al., 2012; Riley and Van Wyk, 2009, 2011;
Woolaston and Baker, 1996), and although some investigations with Me-
rino sheep indicate that selection for low H. contortus FWECs may result
in marginally lower animal production (Kelly et al., 2013; Wollaston and
Baker, 1996), it has also been demonstrated that sheep that survive heavy
H. contortus challenge have lower FWECs and higher haematocrits and
body weights (Kelly et al., 2013). Taken together, it appears that selection
based on either FWEC or body weight when under significant H. contortus
challenge will identify sheep that are both resistant and resilient, and hence
most suited to haemonchosis-endemic situations, although possibly with a
minor compromise in wool production (Kelly et al., 2013).
The greater natural resistance to H. contortus of hair-breed sheep compared
with European breeds was well described from observations on Red Masai
sheep in Kenya (Preston and Allonby, 1979), and has since been confirmed
for both sheep and goats in numerous reports from a wide range of environ-
ments (eg, Amarante et al., 1999; Aumont et al., 2003; Bowdridge et al.,
2013; Burke and Miller, 2002; Chiejina et al., 2010; Courtney et al., 1985;
Gamble and Zajac, 1992; Gruner et al., 1986; Shakya et al., 2011). Presum-
ably breed variation reflects the diverse evolutionary environments, and is
consistent with demonstrated differences in immunological responses

(Amarante et al., 2005; Bowdridge et al., 2013; Shakya et al., 2011).
A key factor influencing the uptake of genetic strategies by livestock
owners is the practicality and accuracy of selection markers for worm resis-
tance. At present, selection is generally based on an FWEC index, and sig-
nificant genetic progress towards increased flock resistance has been
achieved where this has been pursued over some years. The high correlation
demonstrated between FAMACHA values for anaemia assessment and hae-
matocrit (Riley and Van Wyk, 2009, 2011) indicates this is also a practical
selection procedure, particularly when applied under significant H. contortus
challenge. Under these circumstances, a moderate correlation of FAMA-
CHA scores with resilience traits (body weight and weight gain) was seen,
and Kelly et al. (2013) also found a modest correlation of haematocrit
with resilience traits. The potential for more precise and easily utilized
genetic markers has been the subject of much investigation (Krawczyk
and Slota, 2009), including quantitative trait loci (QTLs) (De la Chevrotiere
et al., 2012; Marshall et al., 2013), algorithms based on haematological pa-
rameters (Andronicos et al., 2014), immunological indicators (Amarante
et al., 2005; Shaw et al., 2012) and molecular markers (Castillo et al.,
2011; Kathiravan et al., 2014; McRae et al., 2014). Direct genomic assess-
ment is likely to prove challenging, given the multiplicity of processes
contributing to the immunological recognition and response mechanisms,
but at least one genomic test that explains a proportion of the resistance
variation between individuals is available commercially (‘Wormstar’, Zoetis
Genetics), and further molecular technology research may provide tools to
realize the potential for genomic selection strategies.
There is no evidence for adaptation by nematodes to selection-conferred
resistance in sheep (Kemper et al., 2009; Woolston et al., 1992), and genetic
approaches are therefore considered a key element of sustainable control
strategies. Realistically, objective selection for resistance to H contortus is
likely to be most applicable in large commercial flocks, particularly in
sire-breeding enterprises, but FAMACHA assessment have also been advo-
cated for smaller and less-intensive situations (Riley and Van Wyk, 2009).
4.4 Biological control

The potential for biological control technologies to supplement the use of
anthelmintics has led to a considerable volume of research over some de-
cades, especially into the possible roles for nematophagous fungi and bioac-
tive pasture plants. The effect of naturally occurring fungi which inhabit the
soil and pasture, and form hyphae which trap and destroy nematode larvae,
has been exploited by dosing sheep with fungal spores, so that these pass into
the faeces, where they develop and predate infective larvae (Waller and
Larsen, 1993). A number of fungal species have activity against the larvae
of ruminant nematode parasites, with investigations chiefly involving
Duddingtonia flagrans, although the search for additional candidate species
continues (Kelly et al., 2009). It is envisaged that by continuous feeding
of the predacious fungi to grazing animals (not only ruminants) in feed sup-
plements over periods of weeks or months, an epidemiological effect will be
achieved due to the reduction in their larval intake. Some promising, though
variable, results have been shown in small-scale grazing studies in different
environment with sheep (eg, Chandrawathani et al., 2004b; Fontenot
et al., 2003; Waller et al., 2001) and goats (Maingi et al., 2006), including
against H. contortus, but it appears that this approach is yet to be translated
into routine control programmes for ruminants.
A large number of pasture plant species are known to contain bioactive
chemicals, especially the condensed tannin phenolic compounds, which are
associated with reduced nematode burdens and improved animal production
performance (reviewed by Hoste et al., 2006; chapter: Interactions Between
Nutrition and Infections with Haemonchus contortus and Related Gastro-
intestinal Nematodes in Small Ruminants by Hoste et al. (2016)). These
compounds, especially the condensed tannins, bind to proteins and prevent
their degradation in the rumen, and can hence have a positive nutritive
value, although in excessive concentrations or when protein intake is low,
they can also have detrimental nutritional effects (reviewed by Waghorn,
2008). There is some contention regarding the mode of anthelmintic action
of condensed tannins: whether this is a direct pharmaceutical-like effect of
various polyphenolic compounds on nematode at various life-cycle stages
(Hoste et al., 2012) or an indirect effect through an improved host immune
response due to the protein-binding properties of tannins (Athanasiadou
et al., 2005; Hoste et al., 2006; Waghorn, 2008). Generally, positive but var-
iable effects against worm infections have been demonstrated in pen feeding
and grazing studies for a number of candidate species (eg, Athanasiadou
et al., 2005; Heckendorn et al., 2006; Min et al., 2004; Moreno et al.,
2012; Niezen et al., 1998; Paolini et al., 2003), including Lotus pedunculatus
(lotus), Hedysarium coronarium (sulla), Onobrychis viciifolia (sainfoin), Cichorium
intybus (chicory) and Lolium perenne/Trifolium repens (grass/clover); useful re-
sults have also been reported for Sericea lespedeza, whether grazed or fed as
hay or pellets (Burke et al., 2012, 2014; Shaik et al., 2006). However, major
challenges remain due to the considerable variability in both animal produc-

tion and anthelmintic effects, which have been attributed to varying con-
centrations of active compounds, plant growth stages and nutritional
values, as well as the poor palatability and antinutritive effect associated
with tannins, and, for some species, significant agronomic constraints. How-
ever, the evident potential clearly warrants further investigations, especially
when the worm control and nutritional benefits are combined.
4.5 Alternative anthelmintic compounds

Observations of the use of traditional plant-based remedies against parasitic
disease have underpinned a considerable body of research into alternative
anthelmintics, initially for economic reasons, and, as a response to increasing
anthelmintic resistance. In many cases, the putative beneficial effects of eth-
noveterinary preparations, as extracts or whole plant material, are anecdotal
and are not supported when objectively investigated, but a number does
appear to have some potential (eg, Athanasiadou et al., 2007; Githiori
et al., 2006). Some widely used traditional remedies have been shown to
be inactive against H. contortus (eg, garlic and papaya; Burke et al., 2009a),
while for others the results are positive (eg, extracts of Artemisia; Irum
et al., 2015), or conflicting (eg, Azadirachta indica; Chagas et al., 2008;
Chandrawathani et al., 2006; Costa et al., 2006). In some instances, positive
effects from in vitro laboratory investigations of plant extracts have not trans-
lated to useful activity in animals. Natural compounds found to have activity
would require the development of practical deployment systems, particu-
larly regarding the frequency of administration (most are less effective
than anthelmintics), and format (as plant material or an extract).
The known effect of the element copper against nematodes, used in
various forms as an anthelmintic until the development of modern synthetic
compounds, has been the subject of numerous investigations as an alterna-
tive treatment when used as a copper oxide wire particle bolus product
(COWP). Encouraging anthelmintic effects with COWPs have been
demonstrated, especially against H. contortus (eg, Bang et al., 1990; Burke
and Miller, 2006; Knox, 2002; Spickett et al., 2012; Vatta et al., 2009),
although clarification is needed as to whether this effect is largely against
adult worm burdens or whether there is also a persistent effect against infec-
tive larvae (Galindo-Barboza et al., 2011; Vatta et al., 2012). However, as it
appears that there is no significant toxicity risk when COWPs are used at the
recommended dose (Burke and Miller, 2006; Vatta et al., 2012), there could
be a role for this form of therapy to augment conventional anthelmintics,
particularly when used in conjunction with other forms of nonanthelmintic

control (Burke et al., 2005, 2012; De Montellano et al., 2007).
The search for alternatives to synthetic anthelmintics raises the query:
how effective must they be to justify development as widely applicable con-
trol methods? In contrast to anthelmintic-based control, no single biological
control approach is generally expected to provide total efficacy, and they
will be best used in conjunction with other natural approaches or existing
strategies (Terrill et al., 2012; Torres-Acosta and Hoste, 2008). However,
within these limits, the individual methodologies require objective evalua-
tion, including across different environments and animal management sys-
tems, before acceptance for wide recommendation (Ketzis et al., 2006).
4.6 Vaccines
The prospect of vaccination against helminth parasites as an alternative to the
reliance on anthelmintics has underpinned a great deal of research over many
years, but until 2014 the only vaccine available for ruminant nematodes has
been for bovine lungworm. An effective vaccine would have a particular
role for the control of H. contortus, as continuous protection against the
development of damaging burdens would minimize the risk of animal mor-
talities, and mitigate the severity of anthelmintic resistance. Due to the
epidemiological effect of limiting or preventing the establishment of infec-
tive larvae, the efficacy criteria for vaccines differ from those of short-acting
anthelmintics; simulation modelling by Barnes et al. (1995) has suggested
that for Trichostrongylus colubriformis, a vaccine would be highly effective pro-
vided that it was 80% effective in 80% of animals.
The potential to produce an effective vaccine against H. contortus has
been evident for many years, using ‘hidden antigens’ extracted from
worm intestinal membranes (reviewed by chapter: Immunity to Haemonchus
contortus and vaccine development by Nisbet et al., 2016). Significant reduc-
tions in worm burden and worm egg counts of vaccinated sheep have been
demonstrated with this approach in numerous pen experiments in the
UK (eg, Munn et al., 1993; Smith, 1993). However, although the immuno-
logical basis has since been extensively investigated and protective antigens
characterized (Knox, 2013), it has not proved possible to reproduce the pro-
tective effects in sheep when individual proteins were produced in recom-
binant expression systems (reviewed by Cachat et al., 2010; Knox, 2013; and
Newton and Meeusen, 2003; chapter: Immunity to Haemonchus contortus and
vaccine development by Nisbet et al., 2016). Useful reductions in H. contor-
tus egg counts have been shown using prototype recombinant vaccine in
lambs (Fawzi et al., 2015) and goats (Yan et al., 2013), but the prospects for
their development to a commercial stage are not clear.
While vaccine production by recombinant technology has been unsuc-
cessful, trials in sheep have confirmed the efficacy in the field of a vaccine
produced at the Moredun Research Institute in Edinburgh from the ‘native
antigens’ H11 and H-gal-GP, extracted from adult H. contortus. Vaccination
with a combination of antigens in a trial in South Africa showed worm egg
count reductions of >80%, with simultaneous reductions in anaemia and
sheep deaths (Smith et al., 2001), and a field trial in New South Wales of
a vaccine against H. contortus based on these antigens showed comparable
results, despite clinical haemonchosis in untreated control group lambs (Le
Jambre et al., 2008). Both trials confirmed that repeated vaccination at inter-
vals of some weeks was necessary to maintain season-long protection, and
also that, as with pen trials, plasma antibody levels followed parasitological
and haematological indices relatively closely.
These investigations have led to the development by the Moredun
Research Institute of ‘Barbervax’, a native antigen-based vaccine against
Haemonchus species. The vaccine is produced in Albany, Western Australia,
in collaboration with the Department of Agriculture and Food Western
Australia (Besier and Smith, 2014). Following extensive testing, the vaccine
was released for commercial use in New South Wales in late 2014 (www.
barbervax.com.au). Initial field results appear promising (Besier et al.,
2015), and a trial in Brazil of a vaccine of a similar formulation indicated
significant protection against H. contortus in lambs when given at 3-
week intervals, although it was less effective in ewes under especially severe
challenge (Bassetto et al., 2014). Nevertheless, whether or not further devel-
opment includes production by recombinant technology, it appears that the
feasibility of vaccination against H. contortus on a commercial basis has now
been demonstrated.
In summary, the potential for vaccination to provide effective protection
and hence to significantly reduce the requirement for anthelmintics is now
evident. From the Australian experience, it appears that in regions where the
haemonchosis risk is particularly severe, many sheep owners understand the
need to minimize the development of anthelmintic resistance, and would be
prepared to follow a vaccination schedule requiring multiple doses. Exten-
sion of vaccination to other sheep classes would be expected to provide an
increased epidemiological benefit through the farm-wide reduction of
pasture contamination with H. contortus eggs, and consequent reduction in
larval challenge. Although commercial vaccine production by recombinant
technology may facilitate its availability, and a less-intensive vaccination

schedule would reduce the effort required, it appears that this approach is
now feasible as an effective alternative or adjunct to anthelmintic-based pre-
ventative programmes.
5. PREVENTATIVE PROGRAMMES
Planned preventative programmes are integral to the efficient control
of all significant parasites, and vary widely between environments in relation
to the scale and seasonality of the haemonchosis risk. Optimal helminth con-
trol programmes employ sufficient effort and resources to maintain animal
health and prevent production loss, but avoid the excessive anthelmintic
exposure that leads to anthelmintic resistance. The most sustainable and
effective programmes integrate animal management, anthelmintic treatment
and nonchemical strategies, and are most efficiently structured on the basis of
several basic elements.
5.1 Haemonchosis risk assessment

The degree of effort appropriate for H. contortus monitoring and treatment in
different situations varies among regions, and relates mostly to whether there
is a seasonally significant risk or a sporadic occurrence when local conditions
are favourable. In general, the potential risk can be gauged by an awareness
of the annual pattern of availability of infective larvae to grazing livestock in
a particular location (reviewed by O’Connor et al., 2006; chapter: The path-
ophysiology, ecology and epidemiology of Haemonchus contortus infections in
small ruminants by Besier et al., 2016), although in many environments the
seasonal favourability varies considerably between and within years. Long-
term implications for the H. contortus threat are also relevant where there
are indications that permanent climate change may occur.
Although livestock owners are usually cognizant of the general threat
level in their region, there is often considerable variation among farms
and flocks within a district due to differences in animal management and
husbandry routines, the use of nonchemical strategies (particularly genetic
and nutritional), and policies for anthelmintic use. Animals of different
species, breed, age and class vary in their susceptibility and resilience to infec-
tion. Establishing the scale and annual pattern of risk for particular environ-
ments and individual flocks is the basis of developing appropriate responses
(Reynecke et al., 2011b); to this extent, ‘one size does not fit all’.
5.2 Epidemiologically based preventative programmes

‘The epidemiology of helminth infections integrates the biology of the parasite with that
of the host as an expression of parasite abundance in relation to environmental effects,
as the basis for planning preventative measures’ (Barger, 1997). Programmes based
on the local epidemiology indicate the optimal (or minimal) requirement for
anthelmintic treatments and their timing, as well as opportunities to utilize
animal management and other approaches (Sargison, 2012). In contrast, in
regions endemic for haemonchosis, ad hoc treatment policies whereby treat-
ments are only given when clinical disease occurs or heavy worm burdens
are detected obviously risk animal mortalities. Alternatively, suppressive re-
gimes based on regular and frequent anthelmintic treatment regimens incite
and exacerbate anthelmintic resistance (Table 1).
Annual treatment and management programmes for H. contortus have
several aims:
• The removal of H. contortus burdens before they reach pathogenic levels.
• The avoidance of the excessive intake of infective larvae from pastures.
• Prevention of significant pasture contamination with H. contortus eggs.
• The management of specific risks, such as increased H. contortus burdens
due to the peri-parturient relaxation of resistance in lactating females, the
unique susceptibility to infection of young animals, and the potential for
hypobiotic worms to contribute to excessive worm populations.
Except in regions of climatic extremes, such as the wet tropics and arid or
frigid temperate zones, there is usually a clearly defined seasonal pattern to
H. contortus population development, and to the animal husbandry routines
that can be exploited to provide effective control without the excessive use
of anthelmintics. Typically, this involves the identification of periods during
the year when either helminth burdens should be monitored, or alterna-
tively, routine preventative action taken (anthelmintic treatments or pasture
movements) on the basis of objective observations and past experience.
5.2.1 Wet tropical zones

Haemonchosis is a continual threat in these zones due to the high temper-
atures and year-round rainfall (Dorny et al., 1995; Ikeme et al., 1987;
Waller, 1997), although the relatively short period of survival of infective
larvae provides the basis for rotational pasture strategies. Control strategies
appropriate for different enterprise types vary: in traditional small-holder sit-
uations, where there is typically little use of modern anthelmintics, ‘cut and
carry’ systems have been advocated. In larger commercial flocks, where the
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants
Table 1 The relative risk of occurrence of haemonchosis and management strategies appropriate in different climatic zones
(see Section 5.2)
Climatic zone Haemonchosis risk Management strategies
Wet tropical Hot and moist climatic conditions favour the development Continual monitoring of H. contortus burdens (FWEC) or
regions of H. contortus infective larvae throughout most of the their effects (anaemia, such as through FAMACHA) is
year, with only a transient reduction in larval intake essential. Management tactics to prevent the
during occasional periods of drier conditions. Small overwhelming intake of larva include short-term pasture
ruminants at pasture must be considered at continual risk rotations, or where feasible, ‘cut and carry’ feeding
of haemonchosis, but the short period of survival of systems to avoid the grazing of pastures occupied by
infective larvae under these conditions offers H. contortuseinfected ruminants. Where feasible,
opportunities for rotational gazing systems. treatments should be confined to individually identified
animals at risk, as the frequent use of anthelmintics has
led to widespread resistance in H. contortus where mass
treatments have been given routinely.
Subtropical The distinctly seasonal climate confines the H. contortus risk H. contortus is the major helminth parasite of small
regions to annual wet seasons, but during these periods hot ruminants in this zone and monitoring for infections is
conditions favour the rapid development of infective essential, although the intensity required varies
larvae, and haemonchosis is a major threat. seasonally according to the annual pattern of risk. The
Opportunities for control strategies are provided by the FAMACHA system is especially appropriate in the
seasonal nature of the high-risk periods, although their small-holder situations that predominate in this zone,
duration and timing varies, and hypobiosis of the fourth- and together with animal management strategies based
stage larvae also occurs variably within this zone. The on the seasonal variation in infective larvae availability,
likelihood of overt haemonchosis also varies seasonally this provides a sound basis for rational anthelmintic use.
with the quality of the nutrition available to grazing
animals, and is lowest in locally adapted (H. contortus-
resistant) breeds.
215
(Continued)
Table 1 The relative risk of occurrence of haemonchosis and management strategies appropriate in different climatic zones
(see Section 5.2)dcont'd
216
Climatic zone Haemonchosis risk Management strategies
Summer rainfall The development of H. contortus larvae is highly seasonal, Haemonchosis is usually the dominant parasitic risk to
temperate but haemonchosis is typically a significant threat for sheep and goats in this zone. In large intensively grazed
climates some months each year, from early summer onwards. flocks, pasture management strategies are commonly
However, the favourability for infective H. contortus used to minimize the intake of infective H. contortus
larvae typically varies considerably during this period in larvae, especially where cold winters extend the period
relation to rainfall, and winter conditions are often too when few larvae are present. These strategies require
cold for the development of larvae. In some locations, monitoring of H. contortus burdens, typically with
larvae may fail to survive through winter (especially FWECs, particularly throughout the summer risk
where temperatures are moderated by high altitudes), period. In smaller flocks or where labour resources
but in some regions hypobiosis allows the over-winter permit, monitoring by FAMACHA is also appropriate,
survival of H. contortus populations. commencing when seasonal conditions favour
H. contortus larval development. Anthelmintic resistance
is an especially severe problem in large commercial
flocks in this zone, and tactics should aim to limit
treatments to individual animals or particular flocks.
Genetic selection for nematode resistance is also an
effective strategy in intensively managed flocks.
Mediterranean The development of H. contortus infective larvae is typically The importance of H. contortus in this zone varies from
climates limited to short periods of the year, chiefly during the negligible to moderate, and its management is usually
autumn and spring months when sufficiently warm secondary to that required for other nematodes (chiefly
temperatures and rainfall coincide. However, the Teladorsagia and Trichostrongylus). In areas where
likelihood of haemonchosis outbreaks greatly from a haemonchosis occurs commonly, the times of year and
R.B. Besier et al.

seasonally endemic risk to a sporadic occurrence with classes of livestock at most risk are usually well
outbreaks mostly in years with atypical weather established, providing the basis for appropriate
conditions. In regions with particularly dry summers, preemptive treatments or management strategies.
H. contortus is only occasionally detected and disease is Where outbreaks occur only occasionally, FWEC
rare or absent. monitoring usually provides an effective indication of an
impending risk.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants
Cool temperate Cold winter conditions restrict the development of Specific measures to control H. contortus are rarely
climates infective larvae of H. contortus to relatively short summer necessary, and management programs directed at other
periods, and in general haemonchosis is of only nematodes are usually adequate. Where summer
occasional concern in this zone. However, H. contortus outbreaks occur, treatments at the end of winter (within
populations commonly survive through winter as a sustainable anthelmintic-use strategy) will reduce the
hypobiotic larvae, and the emergence of large numbers haemonchosis risk, and monitoring of FWECs (with
in spring leads to regular annual outbreaks of routine species identification) will indicate whether
haemonchosis in regions where summer temperatures H. contortus is increasing in significance. In the
are sufficiently high. Since the 2010s, increasing reports environments most hostile for H. contortus (frigid and
of haemonchosis in locations where it has previously arctic zones), local eradication may be feasible, although
been rare have led to speculation that this reflects both the economic justification and the potential for the
climatic changes that could increase the extent of the required anthelmintic treatments to lead to resistance
endemic zone. (including to nontarget species) would need to be
considered.
Arid regions The critical requirement for moisture severely limits Routine control measures are rarely required, but an
H. contortus development, although minor populations awareness of conditions favourable for larval
may remain endemic due to hypobiosis if seasonal development is appropriate in regions where there is the
rainfall occurs, or because anthelmintics are rarely potential for occasional haemonchosis outbreaks.
required (for any nematode species). Occasionally, cases Eradication may be technically feasible but would rarely
of haemonchosis occur following unusually prolonged be justified. Where H. contortus exists on irrigated
conditions that are favourable for larval development. In pasture, periodic monitoring will indicate the
common with all climates with periods of hot or warm development of significant populations.
weather, irrigated pastures carry a potential risk of
haemonchosis unless managed to prevent this.
217
heavy reliance on anthelmintics has produced severe resistance (Cezar et al.,

2010; Chandrawathani et al., 2004a), nonanthelmintic control will include
pasture rotations (Barger et al., 1994; Mathieu and Aumont, 2009). The
FAMACHA system for indicating impending disease in both flocks and in-
dividuals has particular potential in such high-risk situations (Mahieu et al.,
2007).
5.2.2 Subtropical zones

The haemonchosis risk is generally more sharply seasonal than in the true
tropics due to annual dry periods, although both the length of dry seasons
and the total rainfall, and the importance of hypobiosis, vary greatly
throughout the zone (see review by Bolajoko and Morgan, 2012). Rota-
tional grazing is a key preventative strategy to minimize animal losses or
excessive anthelmintic treatment, though appropriate regimes vary widely
between locations and seasons. As in all high-risk environments, FAMA-
CHA has a particular role for indicating H. contortus risk, especially as
although the effectiveness of frequent anthelmintic use has been demon-
strated (Fabiyi, 1987), the dominant management system involves small
flocks kept in traditional village situations.
5.2.3 Summer rainfall temperate zones

H. contortus development is also seasonal in these climatic zones, and depend-
ing on the rainfall pattern, is often the dominant livestock health risk for pe-
riods of several months each year. This is a zone where large flocks of
intensively managed sheep are grazed, and the previously general practice
of frequent anthelmintic treatment has led to especially severe anthelmintic
resistance (Dash, 1986; Van Wyk et al., 1997b). In common with other
high-risk situations, effective H. contortus preventative strategies include
pasture management to avoid excessive infective larvae intake, typically by
rotational grazing strategies whereby sheep follow cattle (Bailey et al.,
2009; Southcott and Barger, 1975) or pasture rotations at seasonally variable
intervals (Colvin et al., 2008). Where winter periods are sufficiently cold, the
periods for which H. contortus fails to develop on pasture can extend the ben-
efits of rotational grazing (Bailey et al., 2009). The use of FWEC monitoring
(Section 2.4.1) has a particular role in supporting grazing strategies, and has
become a routine management tool by sheep owners in some situations,
especially in Australia (www.wormboss.co.au).
In South Africa, where the particular threat of haemonchosis led to the
development of the FAMACHA system, the availability of adequate labour
resources has led to its application in large flocks and also by small holders in
communal grazing situations (Vatta et al., 2001). In the less seasonal southern
USA, where sufficiently warm temperatures and adequate rainfall prevail
year round, the haemonchosis risk requires continual management, and con-
trol programmes advocated for sheep and goats include rotational strategies
(Burke et al., 2009b), FAMACHA assessments (Burke et al., 2007) and a va-
riety of nonchemical approaches (Terrill et al., 2012).
5.2.4 Mediterranean climatic zones

In highly seasonal climates where rainfall is limited during the warmer
months, there are large variations in the risk among locations and among
years, but management regimes effective against haemonchosis are generally
less intensive than in summer rainfall zones. In Mediterranean climates, the
hot and dry summer conditions and relatively cool winters typically confine
H. contortus development to short periods of the year (Besier and Dunsmore,
1993), reducing the severity and duration of the threat. Preemptive
treatments prior to high-risk periods, identified through local experience
and FWEC monitoring, and confined to specific animal classes at risk, can
prevent the development of significant populations with little anthel-
mintic-resistance risk. In many cases, programmes aimed mostly at other
nematodes also control H. contortus, and specific anthelmintic treatment is
only occasionally required.
5.2.5 Cold temperate zones

In higher latitudes, H. contortus is of relatively lesser importance, due to
shorter and cooler summers and more severe winters, and hypobiosis has
been demonstrated as the major over-winter survival mechanism, in studies
such as in Canada (Gibbs, 1986; Mederos et al., 2010), Sweden (Waller
et al., 2004) and South Dakota (Grosz et al., 2013). Measures directed
primarily at more cold-adapted species are generally also effective against
H. contortus, although its potential for rapid population increases following
turn-out can lead to clinical haemonchosis. The vulnerability of the
dependence by H. contortus on hypobiosis has suggested the prospect of
the eradication by treating livestock while housed during winter (Waller
et al., 2006), although the low degree of external refugia in this situation
has significant potential for the development of anthelmintic resistance
(Grosz et al., 2013). The outbreaks of haemonchosis in atypical environ-
ments raise the query regarding changes in climatic conditions (Eysker
et al., 2005; Sargison et al., 2007), and the need to reevaluate H. contortus
control (Morgan and van Dijk, 2012).
5.2.6 Arid zones

The presence of H. contortus in arid and semidesert areas is testimony to its
survival capacity (mostly through hypobiosis) and potential for rapid
population increases. Rare haemonchosis outbreaks are chiefly due to the
occasional coincidence of favourable conditions, and it is possible that in
some situations H. contortus could be effectively eradicated on a local basis.
Eradication could be especially useful where there was a potential for
haemonchosis in irrigated pasture situations, although the justification of
any such attempt would require evaluation in terms of both the technical
feasibility and the potential to develop severe anthelmintic resistance (Le
Jambre, 2006).
5.3 Nonchemical strategies

IPM strategies are integral to sustainable preventative programmes in the
major H. contortuseendemic zones, as experience indicates that control based
chiefly on anthelmintic treatments will almost always be inadequate or
unsustainable. As noted above, the available nonchemical approaches are
not as immediately effective as anthelmintics in removing helminth burdens,
but the ‘basket of best options’ approach (Krecek and Waller, 2006) has an
additive effect, and in combination allows a significant reduction in anthel-
mintic use (Barger, 1997; Jackson and Miller, 2006; Torres-Acosta and
Hoste, 2008; Waller, 2006).
In general, the requirement for effort, planning and resources is greatest
where the haemonchosis risk is especially great, especially in intensive pro-
duction operations where maximal animal health is essential. Grazing man-
agement and pasture rotations to minimize H. contortus larval intake, along
with structured monitoring schedules, are an essential element of sustainable
programmes in tropical and summer rainfall temperate zone.
The major IPM elements, breeding for superior resistance and/or resil-
ience to haemonchosis and ensuring adequate nutrition, have been demon-
strated to have particular application for the management of the risk and
effects of H. contortus. Worm-resistant animals excrete fewer nematode
eggs, hence providing the epidemiological benefit of reduced exposure to
infective larvae, which is permanent within genetically selected flocks. The
very significant between-breed differences in H. contortus tolerance are
routinely, if not always consciously, utilized in many zones where
haemonchosis is a limiting factor on animal enterprises, although under inten-

sive commercial conditions there may be a limited role for breeds not selected
for high production efficiency. Optimal nutrition is a general recommenda-
tion to maximize animal production, and undernutrition is of greatest signif-
icance as a factor in haemonchosis outbreaks in small-holder situations.
A number of alternative nonchemical approaches have potential, and are
advocated as a suite of strategies (Terrill et al., 2012; Torres-Acosta et al.,
2012), although at present their roles in commercial situations are yet to
be realized. However, the demonstration that vaccination against H. contor-
tus is a feasible option (see Section 4.6) may provide an additional option to
reducing dependence on anthelmintic control.
5.4 Monitoring of Haemonchus contortus burdens

The legendary capacity for rapid increases in H. contortus populations
requires a stringent monitoring schedule during high-risk periods, whether
by FWEC or FAMACHA, and an objective indication of H. contortus
burdens allows a reduction in the frequency of treatment. This is most
efficiently included as part of a planned management programme, as the im-
mediate risk, and hence value of monitoring activities, varies in relation to
the time of anthelmintic treatments or pasture changes. In general, in all
except tropical environments, monitoring specifically for H. contortus needs
to be conducted only during high-risk periods: for example, whole-flock
FAMACHA inspection need not commence until anaemia is evident
from checks of a small subsample, although as with all monitoring pro-
grammes, the FAMACHA data must be interpreted in relation to prevailing
epidemiological factors (Van Wyk and Reynecke, 2011). Where H. contortus
is only occasionally significant, monitoring may most efficiently be limited
to periods of unusual weather conditions, and in many cases its presence
will only be evident after tests for species identification. In endemic situa-
tions, however, the costs and effort of monitoring are easily recouped
through the avoidance of animal losses by the timely recognition of risk,
and the maintenance of anthelmintic efficacy by minimizing exposure.
5.5 Anthelmintic choice and resistance management

As previously noted (Section 3.2), a number of factors influence the optimal
choice of anthelmintic, and on many intensively grazed properties, a number
of different anthelmintic groups and formulations may be used in any one
year. While H. contortus is the chief worm control target, the use of nar-
row-spectrum products at appropriate times is an obvious approach to
combat the development of resistance against other anthelmintic groups,

with benefits for the control of both H. contortus and other species. Where
suitable refugia tactics are feasible to manage the anthelmintic-resistance
risk, long-acting anthelmintics may be appropriate for particularly suscepti-
ble flocks.
In many situations, the anthelmintic options are limited due to the poor
efficacy of most available groups or particular formulations. Unfortunately,
in the majority of livestock situations, there is little information regarding
the efficacy of various options, and the continued use of failing products
will reduce their effectiveness. A move to combination anthelmintics,
usually to ensure adequate efficacy, will have the additional benefit of delay-
ing the onset of resistance, provided they are used within a refugia context.
The recommendation to conduct anthelmintic-resistance tests is especially
pertinent where H. contortus is a major threat.
As detailed previously , providing adequate refugia for worms of low-
resistance status is arguably the most important element of resistance-
management strategies. These strategies are integral to sustainable control
programmes, and in conjunction with monitoring of H. contortus burdens,
will not entail a reduction in animal production. Specific resistance manage-
ment tactics, including periodic testing for anthelmintic resistance and the
use of quarantine treatments for introduced animals, have long been central
elements of sustainable control recommendations.
The implementation of strategies to rationalize the use of anthelmintics
has had variable success on a worldwide scale. A balance is required between
the appropriate and excessive use of sustainable programmes, given the
potential for either inadequate parasite control or the development of resis-
tance (Bath, 2006). Programmes to achieve this are often slow to gain adop-
tion, especially those requiring a commitment of time and cost, or where the
underlying concepts appear complex (Besier, 2012; Van Wyk et al., 2006).
Objectively planned communication strategies are essential for the adoption
of effective and sustainable control programmes (Kahn and Woodgate,
2012; Woodgate and Love, 2012), requiring the close cooperation of the
scientific and advisory sectors.
6. CONCLUSIONS
Recognition of the risk of haemonchosis and the need for effective
prevention is essential to avoid serious mortalities in sheep and goats in
H. contortuseendemic zones. Haemonchosis also poses a periodic seasonal

risk in environments that are more marginal for H. contortus, and significant
losses may occur without appropriate monitoring and treatment pro-
grammes. However, despite the propensity of H. contortus for rapid popula-
tion increases under even transiently favourable conditions, a range of
diagnosis and treatment options assist in its management. Outbreaks of acute
haemonchosis are relatively easily diagnosed, as large numbers of H. contortus
are characteristic, and smaller but also lethal burdens seen in animals with
chronic haemonchosis are generally associated with typical epidemiological
and nutritional conditions. The clinical sign of anaemia is a simple indication
of the pathogenic effects in individual animals, and the FAMACHA
conjunctival colour index of anaemia provides a practical monitoring system
which can be applied at regular intervals. Where individual animal inspec-
tions are not practicable, the relatively close correlation between H. contortus
burdens and FWECs provides an effective flock or herd monitoring tool,
augmented, where necessary, by a number of techniques for the specific
identification of H. contortus in mixed-nematode populations.
Although a wide range of anthelmintic groups is potentially available for
use against H. contortus, in practice, widespread anthelmintic resistance limits
their effectiveness. Strategies to minimize the development of resistance
must be an integral component of H. contortus control programmes, and
these include measures to optimize the frequency of anthelmintic treat-
ments, and the application of refugia policies to ensure the retention of
worms of relatively lower-resistance status. Although a number of
nonchemical parasite control approaches have been considered, those
most feasible and effective include pasture management to manage
H. contortus larval intake, the provision of adequate nutrition, and the genetic
selection of host animals for superior worm resistance and resilience. The
development of a vaccine against H. contortus provides an additional control
possibility, but further work is needed before other biological control pos-
sibilities become a reality. The development of more practical and cost-
effective tests for anthelmintic resistance, nematode burdens and host
worm resistance would significantly assist with H. contortus control, and
are important research objectives.
The preventative programmes appropriate for different environments
vary according to the scale of the haemonchosis risk and the local epidemi-
ology of H. contortus infections and haemonchosis outbreaks. In the major
endemic zones, control has relied heavily on anthelmintics, with consequent
widespread and severe resistance. Sustainable approaches require the
effective detection of developing H. contortus burdens and the avoidance of

excessive larval intake through pasture changes, with the application of
refugia-based strategies on either an individual animal or a flock basis. In
environments where haemonchosis occurs more sporadically, monitoring
is particularly important to allow preemptive treatments during potential
risk periods, including where hypobiosis leads to seasonal outbreaks. In all
situations, appropriate anthelmintic choice and the use of IPM principles
are fundamental to the effective management of H. contortus, although their
perceived complexity requires a significant communication effort to achieve
wide implementation.
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CHAPTER SEVEN
Interactions Between Nutrition

and Infections With Haemonchus
contortus and Related
Gastrointestinal Nematodes in
Small Ruminants
H. Hoste*, x, 1, J.F.J. Torres-Acosta{, J. Quijada*, x, I. Chan-Perez{,
M.M. Dakheeljj, D.S. Kommuru#, I. Mueller-Harveyjj, T.H. Terrill#
*INRA, UMR 1225 IHAP, Toulouse, France
x
Université de Toulouse, Toulouse, France
{
Universidad Aut onoma de Yucatan, Merida, Yucatan, Mexico
jj
#
1
Corresponding author: E-mail: h.hoste@envt.fr
Contents
1. Introduction 241
2. Quantitative Aspects 247
2.1 Pathophysiological and nutritional consequences of H. contortus infections 248
2.2 A conceptual framework to understand and manipulate host nutrition as an 251
aid to control H. contortus infection
2.2.1 Importance of nutrition 251
2.2.2 Targeted dietary supplementation for different nutritional components 252
2.3 Supplementation with nitrogen resources to improve host resistance and 253
resilience against H. contortus
2.3.1 Effects of supplementation with different sources of dietary nitrogen in controlled 254
pen studies
2.3.2 Evaluating the role of supplementary feeding in grazing animals 261
2.4 Supplementation with dietary energy 263
2.5 Supplementation with mineral micro-nutrients and trace elements 266
2.6 A scheme to improve the control of H. contortus infection on the farm, 267
depending on the nutritional status
2.6.1 Animals on a poor nutritional plane 268
2.6.2 Animals on a good nutritional plane 269
2.6.3 Animals on an excellent nutritional plane 269

j
ISSN 0065-308X
240 H. Hoste et al.
3. Qualitative Aspects 269

3.1 Chemistry of tannins and related polyphenols 271
3.2 Methodological issues 271
3.2.1 Analytical tools for tannins 271
3.2.2 Studying the anthelmintic properties of tannins and related polyphenols 272
3.3 Impact on the biology of different, key stages of gastrointestinal nematodes 272
3.4 Impact on host resilience 293
3.5 Variability in effects of plant secondary metabolites, depending on parasitic 293
nematodes
3.5.1 Variations arising from nematode species and life cycle stages 294
3.5.2 Variations in plant tannin composition (quantity and quality) in relation to 295
activities against H. contortus
3.6 Modes of action of tannin-containing plants against H. contortus: direct versus 298
indirect effects
3.6.1 The direct (¼pharmacological-like) hypothesis 298
3.6.2 The indirect (¼immune-based) hypothesis 299
3.6.3 Emerging information on structureeactivity relationships, and possible 300
mechanisms of action
3.7 Toward the on-farm use of condensed tannin-containing nutraceuticals 309
as anthelmintic feeds
3.7.1 Temperate legume forages: sericea lespedeza and sainfoin 309
3.7.2 Dissecting the complexity of tropical legumes as nutraceuticals against 320
H. contortus and other gastrointestinal nematodes
3.7.3 Exploring the value of agroindustrial by-products 324
3.7.4 Possible combinations of resources with anthelmintic effects 326
4. Conclusions 327
Acknowledgements 328
References 328
Abstract
Interactions between host nutrition and feeding behaviour are central to understand-
ing the pathophysiological consequences of infections of the digestive tract with para-
sitic nematodes. The manipulation of host nutrition provides useful options to control
gastrointestinal nematodes as a component of an integrated strategy. Focussed mainly
on the Haemonchus contortus infection model in small ruminants, this chapter (1) illus-
trates the relationship between quantitative (macro- and micro-nutrients) and qualita-
tive (plant secondary metabolites) aspects of host nutrition and nematode infection,
and (2) shows how basic studies aimed at addressing some generic questions can
help to provide solutions, despite the considerable diversity of epidemiological situa-
tions and breeding systems.
Interactions Between Nutrition and Haemonchus contortus & Related Nematodes 241
List of Abbreviations
AH Anthelmintic
BG Bermuda grass
COWP Copper oxide wire particles
CTs Condensed tannins
EPG Eggs per gram
FEC Faecal egg count
GINs Gastrointestinal nematodes
L3 Infective third-stage larvae
ME Metabolizable energy
MP Metabolizable protein
NPN Nonprotein nitrogen
PC Procyanidins
PCV Packed cell volume
PD Prodelphinidins
PEG Polyethylene glycol
PPRI Periparturient relaxation of immunity
PSMs Plant secondary metabolites
PVPP Polyvinyl polypyrrolidone
SAR Structureeactivity relationship
SEM Scanning electron microscopy
SL Sericea lespedeza
TEM Transmission electron microscopy
UMB Urea molasses blocks
VFI Voluntary feed intake
1. INTRODUCTION
In any grazing system, from steppes or temperate grasslands to tropical
forests, wild and domestic ruminants coexist with both the forages and
browses, which are a source of both nutrients and plant secondary metab-
olites (PSMs; see Box 1), as well as with the infective larvae of parasitic
gastrointestinal nematodes (GINs) associated with grazing (See Fig. 1).
Therefore, since ruminants consume forages and browses representing
different plant communities, GINs should also be considered as normal in-
habitants of grazing ruminants. In addition, ruminant hosts are able to live,
reproduce and be productive with a moderate number of GINs in their
digestive tract.
The long-standing, close association between ruminants, plants and
GINs has shaped several features of these different organisms. In other
242 H. Hoste et al.
Box 1 A box of definitions

· Feed: Food or diet offered to livestock animals in order to cover primarily their nutri-
tional requirements (ie, macro- and micro-nutrients such as energy, protein/amino
acids, fatty acids, vitamins, minerals and so on) for survival, reproduction, and produc-
tion. However, a feed can also include ‘nonnutritional’ components (eg, PSMs).
· Feedstuff: Material that can be used by livestock animals to obtain nutrients. These
materials can originate from plants (eg, sorghum grain) or animal by-products (eg, fish-
meal). They can be used as ingredients to combine with other materials to form a feed
designed to meet the nutritional requirements of animals. Each feedstuff is character-
ized by a certain quantity of macro- and micro-nutrients and may also contain PSMs.
· Nutrients: Dietary components that meet the nutritional requirements of the animal
and include macro- and micro-nutrients.
· Nutraceuticals: Based on the definition by Andlauer and Furst (2002) for functional
foods, a nutraceutical in veterinary science can be defined as a livestock feedstuff,
which combines nutritional value with beneficial effects on animal health. This two-
pronged action is considered to stem from the presence of various PSMs or bioactive
compounds (Hoste et al., 2015).
· PSMs: Plants synthesize primary substances (eg, cell walls, proteins, lipids, DNA) for
their basic functioning. They also produce secondary metabolites to adapt to environ-
mental conditions (plant defence to diseases and aggressors, UV screens, adaptation
to physical or chemical stress). These compounds may also have numerous other roles
in animal and human health and nutrition and are the focus of much research.
· Self-medication: Self-medicative behaviours have been observed when plants that
contain natural AHs are selectively consumed by ruminants. This can be classified
into two types of feeding behaviour: prophylactic and therapeutic behaviours that
can aid in controlling intestinal parasites or can provide relief from gastrointestinal dis-
orders (Villalba and Provenza, 2007; Villalba et al., 2014).
· Resistance: This refers to the ability of an infected host to regulate nematode popu-
lations through immune responses that involve complex mechanisms (Balic et al.,
2000). The acquisition of host resistance is progressive and depends on nematode fac-
tors (eg, GIN species, frequency of contact with the GINs) and host factors (eg, host
species and age, genetic, individual factors, and host nutrition) (Hoste et al., 2010;
Van Houtert and Sykes, 1996). Four effects of host resistance on the biological traits
at different stages of the GIN life cycle have been described: (1) decrease in the estab-
lishment of infective third-stage larvae (L3), (2) reduced growth and development of
L3, L4 and S5, when established in the host; (3) reduced fertility of adult (female)
worm populations, and (4) expulsion of established worm populations (Balic et al.,
2000).
· Resilience. This refers to the ability of an infected host to maintain normal (physiolog-
ical) functions and health as measured by production and pathophysiological param-
eters when infected with GIN (derived from Albers et al., 1987). If we consider that GIN
infection, that is, H. contortus infection, can cause additional nutrient requirements,
then supplemented animals improve their resilience by using the additional macro-
and micro-nutrients from supplementary feeding to reduce the pathophysiological ef-
fects of infection, and, if available, some extra nutrients may also enhance growth rate
or milk production.
words, some features of plants, ruminants and their GIN parasites have
coevolved. For example, parasites are able to endure the conditions of
the gastrointestinal tract of ruminants, enabling GINs to reproduce
successfully and to generate offspring that will eventually invade other
generations of ruminants and allow the survival of the parasite species.
Ruminants, in turn, have evolved defences, such as physical barriers and
immunological mechanisms (resistance) and physiological responses
(resilience) (see Box 1), which enable them to regulate GINs and maintain
them at sustainable levels, conserving both the hosts and their parasite
populations. Under natural conditions, it is evident that GINs are part of
a negative feedback system, which is a natural process that helps in
regulating the population of ruminants to limit the use of forages in a
paddock. This ecological perspective of the interactions between GINs
and ruminants is crucial to understanding nutritioneparasite interactions
in the case of domestic ruminants.
Worldwide, most small ruminant production systems are extensive and
outdoors. They rely on the use of pastures and/or browses, and, hence,
are derived from the natural conditions encountered by wild ruminants.
The usage of plant resources seeks to ensure the nutrition of sheep and goats
in a wide variety of breeding systems. However, these resources also
Figure 1 Relationship between host nutrition and pathological/pathophysiological

changes caused by GIN infections, including Haemonchus contortus.
244 H. Hoste et al.
represent potential sources of GIN infections because of the presence of

infective third-stage larvae (L3). As a result, the infection of small ruminants
with GINs, including Haemonchus contortus, represents one of the main con-
straints for small ruminant production, health and welfare. Infections with
GINs are a common outcome of grazing and/or browsing systems in a
wide range of ecological settings, irrespective of the variability of local plant
resources.
The complexity of the interactions among GIN infections, plant re-
sources and host nutrition for sheep and goats relates directly to this ecolog-
ical context, and is reflected in three paradoxes.
Paradox 1: The grazed and browsed plants from fields exploited by ru-
minants are both feed resources, in terms of nutritional value, which can
help to meet the main requirements of the host. However, these plants
can also be the source of L3 of GINs. Once the infective larvae are estab-
lished in the digestive tract, a GIN infection disturbs the digestive physiology
of ruminants and, depending on the severity of the infection, nematodes
may severely impair the utilization of nutritional resources harvested from
the field.
Paradox 2: The feed exploited by domestic ruminants can provide the
nutrients, which may represent a possible solution to alleviate the negative
effects of GIN populations on the physiology of the host. This is the result
of the macro- and/or micro-nutrients obtained from the diet, which may
meet the extra nutritional requirements caused by parasitism (see Section 2;
cf. Coop and Kyriazakis, 1999, 2001).
Paradox 3: The feed harvested by domestic ruminants can also be the
source of bioactive PSMs, which can have direct anthelmintic (AH) proper-
ties. Thus, the constant consumption of feed resources containing bioactive
PSMs, above a sufficient threshold of concentration and for a sufficient time,
may affect/regulate the biology of different developmental stages in the life
cycle of GINs (see Section 3; cf. Hoste et al., 2012, 2015).
Studies of feeding behaviour and the related voluntary feed intake (VFI)
of ruminants are ongoing and reflect the complexity of interactions between
nutrition and GIN infections in ruminants. It has been established that the
feeding behaviour of grazing ruminants might govern which plant species
are eaten, how much of each plant, and which plant component is
consumed (Gonzalez-Pech et al., 2014). The latter implies that such feeding
behaviour dictates both the source of GIN infections, and also the potential
route for adaptation to the quality and quantity of PSMs in a particular
feedstuff. However, the feeding behaviour of ruminants can also be modi-

fied by high-intensity GIN infections and by high PSM content, which
complicates the scenario. A large GIN infection may cause a reduction of
VFI in ruminants, which is one of the main pathophysiological effects of
GIN infections (Coop and Holmes, 1996; Fox, 1993; Simpson, 2000).
Moreover, a high PSM content may have negative effects on the VFI in an-
imals. From the nutritionist’s viewpoint, PSMs that reduce VFI are consid-
ered antinutritional compounds. The reduction in VFI caused by GIN
infections and the antinutritional PSMs may be considered, in essence, an
important part of the natural mechanisms aiming to reduce the impact of ru-
minants on plants. The ultimate goal of nature could be to deter herbivory,
hence reducing the impact of ruminants on plants.
Evidence is now accumulating to indicate that, when infected with GINs,
ruminants can modify either the amount or the type of the feed they eat; this
may ameliorate the negative impact of parasitic infections, and has been
described as self-medication (Hutchings et al., 2003; Provenza et al., 2003;
Villalba and Provenza, 2007; Villalba et al., 2014). Hence, the study of inter-
actions between nutrition and GIN infections in domestic ruminants offers a
model to explore the complexity of interactions between some GIN parasites
and their hosts in the context of ‘sustainable interactions’ (Combes, 1995) as
well as different feedback mechanisms at the planteanimaleparasite inter-
face. These different concepts are illustrated in Fig. 1 and provide the basis
for the present chapter.
Haemonchus contortus represents a useful model to study nutritioneparasite
interactions because of its worldwide distribution, its specificities in regards
to pathogenic mechanisms, its haematophagous mode of feeding, and the se-
vere pathological consequences when animals are infected with large bur-
dens of the parasite. In addition, Haemonchus contortus represents one of the
three main nematode genera (besides Teladorsagia and Trichostrongylus spp.)
most commonly used for studies of the interactions between GIN infections
and host nutrition. Besides the peculiarities of its biology (including feeding
mode and high reproduction rate); its epidemiological features; and its path-
ophysiological effects on the host, H. contortus populations worldwide have
developed resistance to commercial AH drugs (Jackson et al., 2012). Thus,
alternative control measures are being sought to develop sustainable and in-
tegrated GIN management systems (Torres-Acosta and Hoste, 2008).
Studies of the interactions between host nutrition and H. contortus pop-
ulations are warranted because of the importance of the pathophysiological
246 H. Hoste et al.
effects caused by this particular worm. Therefore, the Haemonchus infection

model in small ruminants offers numerous scientific opportunities, as it ex-
acerbates the consequences of infection. These opportunities stem from:
1. The wide prevalence and geographical distribution of H. contortus species.
These characteristics have enabled a large number of studies focussed on
the nutritioneparasite interactions using this parasite model in sheep
and goats. Additionally, a large amount of information is available on
other aspects of hostenematode interactions (see other chapters in this
Thematic Issue).
2. A sound understanding of the pathogenic mechanisms associated with
the presence of a unique anatomical feature that allows H. contortus to
exploit the blood supply and to interfere with the blood coagulation
mechanisms in the host, in order to secure its own nutrition (Rhoads
and Fetterer, 1996a,b).
3. The anaemic syndrome that results from the presence of H. contortus is
distinct from the digestive syndromes (disturbances of digestive and
absorptive processes), such as diarrhoea, which occurs in the presence
of most other abomasal GIN species (ie, Ostertagia/Teladorsagia or
T. axei) and different genera of GIN (eg, Trichostrongylus, Cooperia, and
Nematodirus) in the small intestine. These biological aspects are related
to a better knowledge of the pathogenic mechanisms and pathophysio-
logical consequences of H. contortus infection compared with other GINs.
4. Highly significant correlations have been repeatedly established between
adult Haemonchus numbers and/or worm mass (weight) as well as
anaemia, which is the main pathogenic effect observed in the host animal.
Anaemia can be measured by packed cell volume (PCV) and haemoglo-
bin (Hb) levels (Le Jambre, 1995), and can also be estimated by clinical
signs (eg, colour of the mucosae). This latter approach led to the devel-
opment of some qualitative (although nonspecific) tools for the inference
of Haemonchus infection in sheep and goats (the FAMACHA method;
Van Wyk and Bath, 2002). Laboratory and qualitative methods provide
more reliable data on the dynamics of H. contortus infections and the effect
of nutritional improvement on host resilience (see Box 1) against such
abomasal infections compared with other GINs. However, the use of
this type of tool to identify the anaemic syndrome can be misleading
on farms with low nutritional planes. It is difficult to distinguish between
anaemia originating from malnutrition and that caused by H. contortus
and/or other GIN infections (Torres-Acosta et al., 2006). The latter
aspect illustrates the complexity of clinically differentiating haemonchosis
from malnutrition, both of which are common in many production

systems.
5. Some of the most highly prevalent parasitic nematodes of the human diges-
tive tract (namely Ancylostoma spp. and Necator americanus) are also blood-
feeders that share several biological traits with Haemonchus (cf. Hotez et al.,
2006; Williamson et al., 2003). Therefore, any data acquired on the inter-
actions between host nutrition and the infection with a haematophagous
nematode of veterinary importance might also aid in developing methods
of control against some medically important GINs of humans.
Information on the interaction between host nutrition and GIN infec-
tions has been reviewed by various authors in the past (including Coop and
Holmes, 1996; Coop and Kyriazakis, 1999, 2001; Fox, 1993; Holmes,
1993; Hoste et al., 2005; Knox and Steel, 1996; Knox et al., 2006 MacRae,
1993; Parkins and Holmes, 1989; Poppi et al., 1990; Torres-Acosta et al.,
2012; Van Houtert and Sykes, 1996). This chapter focuses on data and find-
ings for H. contortus and related congeneric species (including Haemonchus
placei, Haemonchus similis and Haemonchus longistipes), to illustrate two pri-
mary aspects, namely (1) the quantitative manipulation of nutrition to
improve the host response and (2) the effect of PSMs on worm populations
(see Fig. 1). A third aspect on the interactions between nutrition (and
feeding behaviour) and GIN infections is only evoked because reviews
have been published recently on the concept of self-medication (Villalba
and Provenza, 2007; Villalba et al., 2014). Most data will refer to H. contor-
tus of sheep and goats, because the vast majority of published studies refer to
these two small ruminant species. Reference to H. placei of cattle is made
when applicable. As far as possible, this chapter attempts to compare results
obtained for Haemonchus spp. with those for other GIN genera, and relates
to (1) their main anatomical location in the abomasum (¼Haemonchus spp.
and Teladorsagia/Ostertagia spp.) or in the small intestine (Trichostrongylus
colubriformis, Trichostrongylus vitrinus and Nematodirus spp.), and (2) the
mode of nutrition: haematophagous for Haemonchus spp. versus non-
haematophagous genera for Teladorsagia/Ostertagia/Trichostrongylus/Cooperia
and Nematodirus spp.
2. QUANTITATIVE ASPECTS
The rationale that underpins the quantitative manipulation of host
nutrition is directly based on knowledge of the various main
248 H. Hoste et al.
Figure 2 A summary of the main pathological and pathophysiological consequences

of Haemonchus contortus infection in sheep and goats.
pathophysiological processes that result from the presence of parasitic nem-

atodes in different digestive organs (see Fig. 2). Such knowledge provides the
potential for the development of complementary alternatives for improved
GIN control.
2.1 Pathophysiological and nutritional consequences of

H. contortus infections
For Haemonchus spp. (like other GIN species in small ruminants), parasitism
of the digestive tract can be described as a nutritional disease, because of the
increased nutritional demands, in much the same way as lactation or preg-
nancy might be viewed (Poppi et al., 1990). Overall, the presence of
GINs has been linked to three main effects on host digestive and general
physiology. These pathophysiological consequences, illustrated in Fig. 2,
explain the production losses, as well as the subclinical and clinical signs,
which are usually associated with GIN infection (Coop and Holmes,
1996; Holmes, 1993; Hoste et al., 1997), providing a descriptive framework
for interpreting the pathological and pathophysiological changes described
in different studies of H. contortus. The four main pathophysiological impacts
are:
1. Reduction of feed intake (VFI). When compared with infection with the
other main parasitic genera (Teladorsagia and/or Trichostrongylus species),
VFI reduction in sheep or goats with subclinical H. contortus infection
appears as milder and more transient (Bambou et al., 2009, 2011, 2013;
Dakkak, 1984; Wilson et al., 1969).
2. Decreased food digestibility due to the reduction of enzymatic functions
and changes in HCl secretion that affect digestive processes in the
abomasum (step 1).
3. Disruption of the digestive processes decreases the absorptive processes in
the small intestine (step 2).
4. Nutrients absorbed via the digestive tract can be diverted from tissues,
(including bones, muscles, udder and/or wool follicles) to maintain
blood and tissue homoeostasis, in order to compensate and replace losses
caused by GINs and to ensure host survival.
Studies of H. contortus infection demonstrated the specificity of the path-
ological changes in the host in relation to some biological traits of Haemonchus
species, relating primarily to their blood-feeding activity, and also to their
high reproductive index (number of eggs produced per female worm per
day) when compared with other GIN genera affecting small ruminants.
Early studies examined the key sequential pathological features caused by
Haemonchus in ruminants, mainly sheep (Hunter and MacKenzie, 1982;
Salman and Duncan, 1984, 1985) and, to a lesser extent, in goats (Al Quaizy
et al., 1986; Al Zubaidy et al., 1987). The major lesions in the abomasum
have been described (Simpson, 2000), and include:
• A hypertrophy of the abomasal mucosa associated with an infiltration of
inflammatory cells (mast cells, eosinophils, globule leucocytes, and
lymphoid aggregation), particularly in the case of reinfected or immu-
nized sheep (Salman and Duncan, 1985);
• A general hyperplasia of mucus-producing cells, combined with an
erosion of the surface epithelial mucous cells;
• A decrease in the number of functional parietal cells (HCl-producing)
and replacement by nondifferentiated, abnormal cells;
• For the chief cells, which produce the pro-enzyme pepsinogen, some
studies indicate the maintenance of cell numbers, but they are less differ-
entiated and functional.
The pathophysiological and pathological changes generated in the
abomasal mucosae by the presence of Haemonchus spp. (Scott et al., 1999;
Simpson, 2000) can be summarized as follows:
• Structural changes relating predominantly to the HCl-producing parietal
cells can impact pH values. An increase in abomasal pH from 2.6 to 3.6,
with transient peaks of up to 6.0, depending on the course of infection
has been described (Nichols et al., 1987; Simpson, 2000).
250 H. Hoste et al.
• These pH changes have direct effects on the abomasal digestion step,

which liberates nutrients for further digestion along the gut (small intes-
tine). The pH increase reduces the conversion of pepsinogen (produced
by the gastric chief cells) to active pepsin.
• In addition, increased abomasal pH will also favour possible invasion
(colonization) of the digestive tract with bacteria that are normally inac-
tivated by low abomasal pH in noninfected hosts.
• Detrimental effects on crude protein digestion lead to higher protein los-
ses, which have been recorded in H. contortuseinfected animals.
One of the primary traits of H. contortus infection is anaemia due to
blood-feeding activity. Several studies have evaluated blood losses as a
consequence of H. contortus infection in the host animal (Clark et al.,
1962; Dargie and Allonby, 1975; Le Jambre, 1995). In a subclinical infec-
tion, red blood cell losses range from w10 to 20 mL per day, which equates
to w50e100 mL of blood (Dargie and Allonby, 1975). These values corre-
spond to an estimated 15e50 mL of blood per worm per day (Le Jambre,
1995). These detrimental losses are explained by the fact that adult Haemon-
chus can suck blood for several minutes at any one site from the abomasal
mucosa, and can then move to a different position to feed (Le Jambre,
1995; Nichols, 1988), allowing blood to leak into the abomasum due to
the presence of anticoagulant compounds/enzymes excreted/secreted
from H. contortus (see Rhoads and Fetterer, 1996a,b). Blood losses are caused
by fourth-stage larvae and adult worms.
Highly significant correlations have been found between worm numbers
and blood loss (Le Jambre, 1995), making the PCV a valuable indirect way
of estimating the number of adult H. contortus present in a host animal.
Moreover, significant correlations were also found between blood loss
and either the parasite mass or the total number of parasite eggs produced,
indicating that the blood serves to ensure worm growth and reproduction
(Le Jambre, 1995). In addition, by using isotopic markers and related meth-
odologies (Abbott et al., 1985a,b, 1986a,b; Dargie and Allonby, 1975;
Gennari et al., 1991), three phases in the pathophysiological process have
been identified, depending on iron reserves available in the host animal:
Phase 1 corresponds to the establishment of infection and is characterized
by a regular decrease in PCV. Phase 2 corresponds to a plateau in PCV
values, because of a balance between continuous blood losses in the
abomasum and a stimulated erythropoiesis (compensation to ensure blood
homoeostasis). Finally, Phase 3 is characterized by a marked reduction in
PCV and serum iron concentration (uncompensated anaemia). These phases
suggest that iron depletion is the first limiting nutritional factor, explaining
the severity of effects linked to H. contortus infection. Increased protein losses
in the abomasum also occur. However, several studies have suggested the
possibility of reabsorption of proteins in the small intestine in the presence
of a sole or dominant H. contortus infection as a compensatory process
(Hoste, 2001; Rowe et al., 1982).
2.2 A conceptual framework to understand and manipulate

host nutrition as an aid to control H. contortus infection
2.2.1 Importance of nutrition
It is usually very generally held that nutritional factors exercise a vital influence on the
degree of infestation of sheep with Haemonchus contortus. Here again, however, resis-
tance to infestation is commonly confused with resistance to the effects of infestation.
This statement by Clunies-Ross (1932) underscored that host nutrition
has been identified as a key factor in controlling H. contortus infection
very early in the history of modern parasitology and indicated the potential
impacts on host resistance and resilience. On the one hand, dietary supple-
mentation (increased supply of feed) can provide the nutrients that
contribute to tissue maintenance and/or blood homoeostasis and host pro-
duction, despite the presence of worms (¼resilience; see Box 1). On the
other hand, it can provide the nutrients needed to improve the host response
by meeting the increasing demands of raising an immune response against
the worms (¼resistance; see Box 1), in order to regulate the worm popula-
tions in immune-competent animals. Achieving blood and tissue homoeo-
stases are first priorities, as they are essential for the survival of the host. Then,
if available, additional nutrients can be used to achieve increased production,
as suggested by Coop and Kyriazakis (1999, 2001). The latter aspect also
forms part of the improvement of resilience.
The beneficial effects of nutritional manipulation of GIN control depend
largely on the overall nutritional plane of the animals (Fig. 3). One of the
most obvious statements concerning the interactions between host nutrition
and GIN infections, particularly in the case of Haemonchus infection, is that
any combination of malnutrition and digestive parasitism will lead to un-
compensated pathophysiological disturbances, and, subsequently, to severe
clinical signs (including a high mortality rate). Under tropical conditions,
when Haemonchus is the dominant genus, this aspect is particularly evident
on farms with alternating wet (abundance of nutrition/high parasitic risks)
and dry (low nutritional value of the grazed resources/lower parasitic risk
of GIN infections) seasons. As stated by Amarante (2014), Farmers blame
252 H. Hoste et al.
Figure 3 A schematic representation of a theoretical framework to illustrate: (1) the

additional nutritional requirements and potential deficit in coverages associated with
GIN infections (increased cell turnover to maintain blood and tissue homoeostasis
and development of an immune response) and (2) the rationale for an increased gen-
eral and/or specific diet supplementation. Derived from Coop, R.L., Kyriazakis, I., 1999.
Nutrition-parasite interaction. Vet. Parasitol. 84, 187e204 and Coop, R.L., Kyriazakis, I.K.,
2001. Influence of host nutrition on the development and consequences of nematode
parasitism in ruminants. Trends Parasitol. 17, 325e330.
the worms or the lack of highly efficient chemical anthelmintic drugs (because of anthel-
mintic resistance). However the primary source of the problem is malnutrition! In this
situation, the scarcity of resources, as defined by Coop and Kyriazakis (1999,
2001), is linked to a general lack of both macro-nutrients (protein/energy)
and micro-nutrients. Under such circumstances, any improvement in nutri-
tion will be of benefit to the host in counteracting Haemonchus (and other
GIN) infections and related effects.
2.2.2 Targeted dietary supplementation for different nutritional

components
Coop and Kyriazakis (1999, 2001) introduced three main concepts to describe
the overall framework of interactions between host nutrition and GIN in-
fections (see Fig. 3). These authors proposed the following hypotheses:
1. Usually, there is one main dietary component (described as ‘limiting di-
etary factor’ or ‘scarce resource’) whose metabolism is seriously affected
by the presence of GINs. Therefore, targeted corrections of this limiting
nutritional factor should have major beneficial effects on the host
response (both resilience and resistance) to GIN infections. Nitrogen
and, to a lesser extent, energy are usually these limiting dietary resources.
2. This framework suggests that the allocation of the overall (or specific)
nutritional diet resources is needed to meet the pathophysiological
requirements of the parasitized host, and will depend on different priorities

relating to the host physiological status. The priorities are also related to
the general ecological context of the interactions between host and
GIN infections (as explained in the Introduction). These priorities for
the host populations are: (1) Survival (maintenance of body protein ¼
blood and tissue homoeostasis); (2) Transmission of genes to offspring:
reproductive efforts (including lactation) and preservation of offspring sur-
vival ¼ acquisition of immunity against GINs in young animals; (3) The
expression of host immunity, promoting growth and reproduction in the
host; (4) Storage of reserves in the host (by preservation of fat).
3. The higher the priority given to a physiological function, the less likely is
that it would be affected by host nutrition. Therefore, the framework
suggests that improved nutrition will initially lead to an increase in
host resilience (Coop and Kyriazakis, 1999), and then to an improved
host resistance.
4. Whatever the GIN species, this theoretical framework is supported by
evidence from a large number of studies of meat- and wool-producing
lambs and sheep (reviewed by Knox, 2003; Knox et al., 2006; Van
Houtert and Sykes, 1996). There is considerably less data and informa-
tion for kids and goats (Hoste et al., 2005), in particular dairy goats, as
well as for cattle.
2.3 Supplementation with nitrogen resources to improve

host resistance and resilience against H. contortus
Most results from pathophysiological studies have emphasized the fact that
protein metabolism is much more affected by GINs than other dietary com-
ponents, such as energy (Bown et al., 1991). Most studies of nutritione
parasite interactions were undertaken in the UK, Australia, and New
Zealand, mainly with European breeds of sheep used for meat and wool
production. Very few initial studies were performed on goats, cattle, or dairy
ruminants. Logically, under farming conditions in developed countries, that
is, with animals receiving a high nutritional plane when grazing on high-
quality paddocks, the nitrogenous component is usually the main limiting
factor in the ruminant diet. Therefore, early studies focussed mainly on
the effects of protein supply on the outcome of infection.
Most investigations of protein supplementation have been based on:
• Pen studies (controlled infections): Such studies are conducted under
controlled experimental infection conditions. Different infection ap-
proaches were considered, including single challenge or trickle infections
254 H. Hoste et al.
with monospecific (H. contortus) or mixed GIN infections (H. contortus þ

another, usually intestinal nematode). Pen studies allowed analyses and
detailed descriptions of the interactions between host nutrition and
GIN infections. In addition, they have provided hypotheses and argu-
ments to support the framework (Fig. 3; Coop and Kyriazakis, 1999).
• Field studies (natural infections): Animals are exposed to natural in-
fections, while browsing or grazing in the field. The interpretation of re-
sults of field studies are challenging to relate directly to H. contortus
infection. However, under tropical conditions, H. contortus is usually
one of the predominant GINs, and often occurs with T. colubriformis
and/or Oesophagostomum columbianum. The relative prevalence of each
species depends on season (wet versus dry) and management conditions
(eg, grazing system and AH treatments used).
Although dietary protein supplementation has been shown to increase
resilience and resistance to parasitism in sheep under temperate conditions,
Van-Houtert and Sykes (1996) stressed that many studies did not rigorously
assess the role of protein status of the host. These authors reported that an
increased supply of dietary crude proteins was not necessarily translated
into a better supply of metabolizable protein (MP) for the animal. The latter
could be caused by differences in the rate of degradation of dietary crude
protein in the rumen and in the conversion efficacy of dietary crude protein
into MPs. Furthermore, MP supply is not a fixed characteristic of diet, as it
varies with feed intake (AFRC, 1993), the ruminal fermentations, the feed
passage from rumen to the small intestine, and the rate of rumen escape pro-
teins. Therefore, recent studies have emphasized the need to consider both
the protein and energy components, in particular under tropical conditions
(see Section 2.4).
2.3.1 Effects of supplementation with different sources of dietary

nitrogen in controlled pen studies
The main GIN used in controlled feeding pen studies has been H. contortus.
This model was used to investigate the potential for improving the two com-
ponents of the host response (resilience and resistance) through supplemen-
tation with different sources of rumen-escape proteins (Abbott et al., 1985a,
1986a, 1988; Israf et al., 1998; Kates et al., 1962). Several studies have shown
that supplementation with dietary proteins increases the humoral and cellular
responses to infection with other GIN species, including T. colubriformis (see
Kambara et al., 1993; Van Houtert et al., 1995), Nematodirus battus (see Israf
et al., 1996), and Teladorsagia circumcincta (see Coop et al., 1995).
So far, the evidence from pen studies of tropical breeds of sheep and goats
infected with H. contortus have not shown clear effects of supplementary pro-
tein feeding on host resistance, when measured by the reduction of faecal
egg counts (FECs), worm burdens, and female worm length and fertility.
In the case of meat-producing goats, studies by Blackburn et al. (1991,
1992) did not show any reduction of H. contortus egg excretion or worm
burdens with an increased plane of nutrition. On the other hand, Nnadi
et al. (2007) showed a reduction in FECs in H. contortuseinfected West
African Dwarf goats supplemented with palm kernel cake, and Nnadi
et al. (2009) showed a longer prepatent period of this nematode in supple-
mented West African Dwarf goats. Another study (Phengvichith and Ledin,
2007) also showed reduced FECs in H. contortuseinfected Laos goats
following an improvement of their plane of nutrition using the plants Glir-
icidia and Cassava. Pen studies with sheep failed to reveal a clear reduction in
FECs in infected Menz and Horro sheep supplemented with cotton-seed
cake (Haile et al., 2002).
Although the effect of protein supplementation on resistance to GINs is
not clear, there is solid and consistent evidence showing that supplementa-
tion with rumen-escape protein significantly reduces the pathophysiological
effects of haemonchosis and, hence, improves host resilience.
Early studies with H. contortus (Abbott et al., 1985a,b; 1986a,b, 1988;
Kates et al., 1962; Preston and Allonby, 1979) showed that lambs given a
diet with high crude protein showed less severe pathophysiological effects
(anaemia evaluated by PCV) and clinical signs (weight loss, reduced feed
intake and mortality) than animals on a low-protein diet, despite similar
levels of gastroenteric blood loss. Further pen studies with sheep confirmed
this improved resilience against Haemonchus infection (Bricarello et al., 2005;
Haile et al., 2002, 2004; Khan et al., 2012). More detailed studies of H. con-
tortuseinfected sheep (Datta et al., 1998, 1999; Wallace et al., 1995, 1996,
1999) estimated the supply of ME and MP, and found similar results to those
quoted previously, which had used ‘empirical’ differences in protein supply,
as defined by Bown et al. (1991). One exception is the work by Rocha et al.
(2011), who did not show any improvement in resilience of periparturient
ewes (Santa Ines and Ile de France) when protein supplementation was
given.
Similarly, pen studies involving H. contortus infection in meat-producing
goats confirmed that a high plane of nutrition resulted in improved resilience
(Blackburn et al., 1991; Nnadi et al., 2007, 2009; Phengvichith and Ledin,
2007). For example, the studies by Blackburn et al. (1991, 1992) showed
256 H. Hoste et al.
that young goats infected with H. contortus, when offered a high-protein

diet, had better live-weight gains than those offered a low-protein diet,
despite a lack of effect on resistance.
Nowadays, on one hand, the use of meat and fish meals, which have
been widely used in previous studies, are seriously challenged in developed
countries, because of societal issues (eg, human health issues linked to prion
risk). On the other hand, the price of some feedstuffs used previously as
rumen-escape proteins is becoming prohibitive for developing countries.
This situation has led to the search for local sources of nonprotein nitrogen
(NPN) that can optimize the production of microbial proteins in the rumen.
Hence, urea and/or locally available by-products containing inexpensive
rumen-degradable proteins have been investigated.
An initial pen trial showed that the addition of urea to a basal diet
reduced the pathophysiological effects of H. contortus and improved pro-
ductivity in susceptible sheep (Wallace et al., 1994, 1998). Similar results
were then obtained in lambs trickle-infected with H. contortus and T. colu-
briformis (see Knox and Steel, 1996). However, the latter experiment used
urea-treated oaten chaff that might have suffered delignification, thus
increasing the digestibility of cellulose of the chaff and overcoming
N deficiency in the rumen (Van Soest, 1994). Studies of goats have shown
that increasing the level of urea in urea-molasses blocks (UMBs) or giving
urea supplements alone gave no production benefits or reduction in para-
site burdens or FECs. Only when urea was accompanied by cotton-seed
meal was there a beneficial effect of supplementation (Haile et al., 2002;
Knox and Steel, 1996; Singh et al., 1995). The lack of response to NPN
suggests that the rumen degradable protein supply from the basal diet
was not limiting and that the supplemented NPN was wasted via urine
(Van Soest, 1994). As previously mentioned, the sole use of NPN might
not have been sufficient to promote optimal ruminal function. The latter
function can be achieved by adding a source of rumen degradable
carbohydrates that favour the utilization of NPN sources to improve mi-
crobial efficiency (Van Soest, 1994). The lack of synthesis of specific amino
acids might have also affected animal responses (Van-Houtert and Sykes,
1996).
Based on the hypotheses that requirements for host immune response or
some production parameters can depend on specific amino acids (eg,
sulphur-containing amino acids for wool production) (MacRae, 1993)
and on their impacts on amino acid digestion in the infected small intestine,
a range of studies have evaluated diet supplementation with specific amino
acids, which were protected from ruminal degradation, on host resilience

and resistance.
Most of the pathophysiological studies of amino acids have concerned
leucine and methionine and have been performed in rodent models
(cf. Sakkas et al., 2012b). Moreover, H. contortus was rarely the GIN model
in pen studies with ruminants. Yu et al. (2000) examined the effects of T.
colubriformis infection on the metabolism of leucine in the small intestine.
Coop et al. (1997) showed that the supplementation of lambs with protected
methionine improved the rate of live-weight gain and wool growth, and
reduced the adverse effects of T. colubriformis infection when compared to
unsupplemented pair-fed controls. Increased amino acids in the diet have
also been associated with lower Te. circumcincta burdens when compared
with nonsupplemented lambs (Richardson, 2000). However, contradictory
evidence exists and indicates that protein or methionine supplementation
possibly failed to influence the rate of resistance development to Te. circum-
cincta infection (Coop et al., 1997). The lack of significant results was attrib-
uted to a high-protein level in both experimental diets. On the other hand,
the addition of protected methionine to the diet did not improve the resil-
ience or resistance of dairy goats to T. colubriformis, either as a single infection
(Chartier et al., 1998) or in a mixed infection with H. contortus (Bouquet
et al., 1997).
These studies, conducted under confined conditions, allowed the iden-
tification of several host factors that influence the interactions between host
and nutrition and, consequently, the efficiency of manipulating the host
nutritional status, in terms of resistance and resilience. These factors should
be considered when interpreting the results from supplementation studies
aimed at favouring both aspects of host response to GIN infections.
2.3.1.1 Age of animals

Effects on host resistance (and related immunological mechanisms) and resil-
ience are strongly influenced by the age of animals (Abbott and Holmes,
1990; Kambara et al., 1993). In particular, the initial establishment of nem-
atodes in young, nonimmune animals did not seem to be affected by dietary
proteins (Coop et al., 1995; Van-Houtert and Sykes, 1996). Therefore,
Coop and Kyriazakis (1999) suggested that host nutrition does not affect
the rate of the acquisition of immunity to nematode parasites. However, it is
evident that host protein and energy nutrition can affect the expression of im-
munity in wool- and meat-producing sheep at the later stages of growth in
young and adult animals.
258 H. Hoste et al.
2.3.1.2 Reproduction
In ruminant livestock, host requirements are at the highest level during
pregnancy and lactation. As far as GIN infections are concerned, the
time around parturition is usually described as the time for the ‘peripartur-
ient relaxation of immunity’ (PPRI), which is linked to significant increases
in FECs in both ewes (eg, Donaldson et al., 1998; Leyva et al., 1982) and
does (eg, Chartier et al., 1998; Rahman and Collins, 1991, 1992) compared
with nonpregnant female animals. This PPRI has been attributed to a
‘breakdown’ in host immune response under such physiological circum-
stances (Barger, 1993; Leyva et al., 1982). Apart from the hypothesis that
connects several reproductive hormones (eg, prolactin, progesterone/
oestrogen) and the host immune system, there is a second hypothesis
that proposes that the PPRI is related to the nutritional stress around the
time of parturition (Barger, 1993; Coop and Kyriazakis, 1999). Based on
the second hypothesis, it is predicted that supplementation would assist
in alleviating the PPRI (ie, restoring a degree of immunity) and the
consequences of infection (resilience) around parturition.
PPRI has been confirmed in ewes or does experimentally infected with
H. contortus (see Macarthur et al., 2013a,b; O’Sullivan and Donald, 1973;
Valderrabano et al., 2005), and in natural infections under tropical condi-
tions (Costa, 1983; Gonzalez-Gardu~ no et al., 2014; Rocha et al., 2004).
Because of the importance of PPRI in the epidemiology of GIN (Barger,
1993; Donaldson et al., 1998), nutritional manipulation around parturition
(including protein supplementation) appears to be a sustainable method of
managing infections in offsprings with less reliance on synthetic chemical
AHs (Houdijk et al., 2012). It is worth noting also that the majority of
studies of nutrition e GIN infection interactions in reproductive ewes or
does, whether under experimental or natural infection conditions, involved
either species of Teladorsagia (eg, Houdijk et al., 2003, 2005) or Trichostron-
gylus (eg, Etter et al., 1999, 2000; Kahn, 2003; Kahn et al., 2003) or both
parasites (Donaldson et al., 1998), rather than H. contortus.
In addition, some rodent models of GIN infections (eg, Nippostrongylus
brasiliensis in rats) have also been used to elucidate underlying immunological
mechanisms (Jones et al., 2011; Sakkas et al., 2011, 2012a,b). However,
regarding the interactions between the host nutritional status and GIN infec-
tions, it is important to note that interpretations of results from rodent
(monogastric) models may not apply directly to (polygastric) ruminants.
Most of the early studies focussed on the possible effect of improved host
nutrition by supplementation with MP (Etter et al., 1999; Houdijk et al.,
2003, 2005; Kahn, 2003; Kahn et al., 2003, Walden-Brown and Kahn,
2002). Thereafter, numerous results, mostly from GIN models other than
H. contortus, have stressed the complexity of the interactions between diges-
tive parasitism and host nutrition, because of the following:
1. Overall, in ruminants, despite the ‘scarce resource theory’ and associated
framework (see Section 2.2), it is difficult to separate protein and energy
metabolism, because, in ruminal processes, strong interactions occur be-
tween host protein and energy metabolism (see Section 2.4).
2. As far as the previous point is concerned, it has been shown that the
nutritional status and fat reserves of ewes prior to parturition are key factors,
which can influence the PPRI. Using the H. contortus model of infection,
Valderrabano et al. (2005) showed that an overall higher nutritional
plane of supplementation during the early ante partum period contributed
to modulating fat reserves in ewes and positively influenced host immune
response during parturition, as measured by FEC, worm numbers, and
female length and fertility. A series of studies in Australia investigated
the interactions between protein supplementation and fat score in
ewes during pregnancy, with a focus on host resistance and resilience
(Macarthur et al., 2013a,b). The conclusions were that fat and protein re-
serves of reproductive ewes during pregnancy led to better host resistance
and reduced the PPRI (estimated by FEC), particularly before lambing.
In contrast, ante partum protein supplementation did not influence host
resistance. In terms of resilience, both energy status (evaluated by fat
score) and protein supplementation have positive effects on twin-bearing
ewes and on lamb survival. Moreover, the fat score also contributed to a
higher level and better quality (measured by fat and protein content) of
milk. These results for H. contortus infection confirmed earlier data from
studies with other GIN species (Donaldson et al., 1998; Valderrabano
and Uriarte, 2003).
3. Some results that compared xylose-treated soya bean versus faba bean in
Teladorsagia-infected ewes also suggested that protein quality is an impor-
tant factor to consider (Sakkas et al., 2012a). However, it was not estab-
lished whether this was due to the amount of specific amino acids or due
to the presence of bioactive PSMs.
2.3.1.3 The level of animal production

This represents another important host factor to be considered when
exploring interactions between nutrition and host resistance and resilience
to GIN (including H. contortus) infections. GIN infections in lactating
260 H. Hoste et al.
ruminants, particularly in selected dairy breeds of small ruminants, allow the

study of (1) the negative effects of worms in the digestive tract on host nutri-
tional status and on milk production and (2) the benefits of manipulating
host diets. The interest in dairy ewes or goat models is based on the availabil-
ity of sensitive methods that can estimate effects on host resilience when
compared with fibre- and meat-producing sheep or goats. This aspect relates
to the possibility of being able to collect individual milk samples at different
times during lactation and to explore factors of variation affecting the bal-
ance between host requirements and nutritional status within a herd. The
regular collection of quantitative (milk yield) and qualitative (protein and
fat composition) data, in parallel with parasitological information, enables
an effective evaluation of the GIN effects on host resilience and resistance
parameters, and can assist in testing the theoretical framework described in
Section 2.2 (Fig. 3).
Several studies have explored the resistance and resilience of dairy goats,
depending on their level of milk production under different conditions of
GIN infections. The main results have shown that:
• Within a group of dairy goats with mixed experimental infections with
H. contortus and T. colubriformis, the does with the highest level of milk
production were less resistant (had significantly higher FECs) and less
resilient (significantly larger reductions in milk yield, changes in milk
quality, including fat content) to GIN infections. Moreover, differences
were particularly prominent in the first months after kidding (ie, the peak
of lactation), which represents a period of high nutritional stress (Chartier
and Hoste, 1997; Hoste and Chartier, 1993, 1998).
• Thereafter, it was found that a monthly scheme of AH treatment in a
dairy goat herd, under natural infection conditions, led to significantly
improved production responses, in terms of milk yield by goats with
high production potential, compared with the low-producing does
(Chartier and Hoste, 1994).
Dairy goats provide a useful model for measuring the response to protein
supplementation for improving resilience and resistance in GIN-infected ru-
minants (Chartier et al., 2000a). Under conditions of natural infections in a
dairy goat herd, an improvement of both resistance (lower FECs) and resil-
ience (higher milk yield and fat contents) was expressed when does received
a diet that covered 125% of the protein requirements versus 106% in
controls.
Overall, these results confirmed that there was an inverse relationship be-
tween the importance of milk yield of does (related either to individual
production or to stage of lactation) and the response (resilience and resistance

status) to GIN infections, most of which included H. contortus. However, it is
difficult to dissect the nutritional versus the genetic components in such
studies. The same comment applies when considering the effects of protein
and other nutritional supplementations on resilience and resistance of genet-
ically susceptible and resistant breeds.
2.3.1.4 Genetic factors

The general framework suggests that highly productive sheep or goat breeds
will be particularly susceptible to a combination of Haemonchus infection and
poor nutritional conditions. Conversely, highly productive breeds should
benefit most from any nutritional improvement. Challenges with H. contor-
tus were used in early studies to compare the effects of protein supplemen-
tation on resilience and/or resistance of two sheep breeds that differed in
their susceptibility to GIN infections. In those studies, responses to
improved planes of nutrition were reported for Scottish Blackface lambs
(the genetically ‘resistant’ breed) when compared with susceptible Finn
Dorset (Abbott et al., 1985a,b) or Hampshire lambs (Wallace et al., 1995,
1996). In both studies, high-protein nutritional planes were linked to
improved resilience in both breeds. An improvement in resistance (measured
by significant decreases in FECs) was only found in the ‘susceptible’ breeds.
Controversial results were obtained in a series of studies conducted in
Brazil (Amarante, 2014; Bricarello et al., 2005; Rocha et al., 2004, 2011),
which examined the interactions between nutrition and genotype in local
(Santa Ines) and imported (Ile de France) sheep breeds. On one hand, it
was shown that protein supplementation (mainly from soya bean meal)
resulted in significantly lower worm burdens (ie, improved resistance) in
Santa Ines lambs, but not in the Ile de France lambs (more susceptible and
more productive breed). On the other hand, there was no effect of protein
supplementation on fecal egg output of either breed (Rocha et al., 2004,
2011). Indeed, supplementary feeding might not be the best tool to regulate
the worm population per se.
2.3.2 Evaluating the role of supplementary feeding in grazing

animals
The study of the effect of supplementary feeding on resilience and resistance
against GINs is more complex under field conditions than under controlled
pen conditions. However, field studies could be more relevant in practical
terms, as they represent the real situation. However, supplementing grazing
262 H. Hoste et al.
animals may reduce their pasture intake, and, consequently, their nematode
larval intake. This substitution could confound the interpretation of the
effects of supplementation on resilience or resistance against GIN (Van-
Houtert and Sykes, 1996). However, a certain level of substitution between
pasture and supplement can be considered a positive benefit of supplemen-
tary feeding (Retama-Flores et al., 2012).
A first series of studies that assessed the link between dietary supplemen-
tation and the degree of nematode control on production responses in
grazing lambs were performed nearly 20 years ago (Shaw et al., 1995;
Van-Houtert et al., 1995a, 1996). In those studies, supplementation
(including energy sources) during parasite infection enhanced the resilience
of grazing sheep to GINs. Improved resilience achieved with oatmeal as a
low-protein supplement (Van-Houtert et al., 1996) constitutes evidence
that energy supplements might have a positive effect on the total supply
of metabolizable energy and/or proteins to the host. This finding is impor-
tant because energy supplements are normally cheaper than those based on
protein meals (Van-Houtert et al., 1996). More recently, field studies have
been performed in tropical conditions, involving goats and sheep. In all cases
it was possible to use supplementary feeding to improve resilience against
mixed GIN infections, measured as improved bodyweight gain and dimin-
ished pathophysiological effects from infection, as illustrated for Criollo kids
fed with a combined sorghum/soya bean supplement during the wet season
(Gutierrez-Segura et al., 2003; Martínez-Ortíz de Montellano et al., 2007;
Torres-Acosta et al., 2004) and the dry season (Torres-Acosta et al.,
2006). Only one study evaluated the role of UMBs (Waruiru et al.,
2004), showing that supplemented small East African goats improved body-
weight gain compared with nonsupplemented animals. The same positive
results on resilience were reported in the wet and dry seasons for Santa
Ines sheep given supplementary soya bean meal (Louvandini et al., 2006),
and Menz and Menz x Awassi lambs fed with a concentrated feed (Tibbo
et al., 2008). Meanwhile, it was shown that young Fiji ewes benefited
from UMBs, achieving higher weight at mating and positive effects on birth
rate and postnatal survival, compared with unsupplemented ewes (Knox,
2003; Knox and Steel, 1996).
It is evident that supplementary feeding, in general, is a good manage-
ment option that may benefit naturally infected sheep and goats. However,
it is important to define when and for how long supplementation is required
for grazing animals. Although Datta et al. (1998) reported that short periods
of enhanced postweaning nutrition might have long-term and perhaps
life-long effects on production in sheep infected with H. contortus, other

studies of growing goats in Mexico showed that the long-term effects of
supplementation might be too difficult to maintain beyond the period of
supplementation (Torres-Acosta, 2006). For instance, Aguilar-Caballero
et al. (2002) showed that growing kids receiving supplementary feeding
(100 g/day of sorghum:soya bean meal 70:30) during the dry season showed
a marked improvement on production and health, but this positive effect
was not maintained if the supplement was suppressed during the wet season,
when forage was abundant. Similarly, growing goats are far more resilient
when they receive supplementary feed in the wet season, but this positive
effect is not carried over to the following dry season if the supplement is
not maintained. Thus, it seems that growing animals cannot accumulate nu-
trients, which might be useful in relation to nutritional restrictions, even
during the rainy season.
So far, there is only one published field study (Faye et al., 2003) that eval-
uated the role of supplementary feeding (cotton-seed þ rice bran) on adult
goats, showing improved milk production and less pathophysiological effects
compared with nonsupplemented goats. Of all the field studies referred to in
this section, only a few showed signs of improved resistance against GINs. In
goats, Gutierrez-Segura et al. (2003) showed a reduction in the excretion of
GIN eggs and reduction of fecundity and female length, and Waruiru et al.
(2004) reported a reduction in FECs and worm burdens. In sheep, only
Louvandini et al. (2006) reported a reduction in FECs and worm burdens
in supplemented animals compared with nonsupplemented controls. In
conclusion, the few field trials of sheep and goats showed that resilience
against GIN infections can be increased by supplementation with less
degradable (rumen-escape) protein, as was expected from pen studies.
The use of UMBs has also produced encouraging results, but they were
related to higher VFI. However, the results of supplementary feeding on
resistance are less clear. In addition, it is important to indicate that the effects
of supplementation on the pathophysiology of infection in grazing calves
have been poorly evaluated, and findings have not been conclusive (Magaya
et al., 2000).
2.4 Supplementation with dietary energy

It would be inappropriate to study protein metabolism without considering
energy metabolism. Protein and energy interact because dietary proteins are
a source of dietary energy; energy is needed for rumen function, protein
turnover, and deposition, because deposited protein represents part of the
264 H. Hoste et al.
body’s energy stores. In sheep, when protein supply is adequate, body fat can
be used as a source of energy to support protein supplementation when
exogenous energy is lacking. Under these circumstances, changes in exog-
enous energy supply have little or no effect on protein metabolism, provided
the endogenous store of energy (body fat) is adequate. Thus, the use of di-
etary protein to improve resilience and resistance against GINs may be more
effective in sheep than in goats, because sheep are better able to obtain en-
ergy for extra N retention by mobilizing their more abundant fat reserves.
Meanwhile, leaner carcasses of goats might suggest that insufficient exoge-
nous energy supply might affect their protein metabolism more than that
of sheep. For instance, in a pen study with Criollo kids trickle-infected
with H. contortus and fed isoenergetic diets differing in the level of MP using
soya bean meal, information was obtained on the pathophysiological effects
of infection during a 5-month trial, together with information on diet di-
gestibilities and N balance (Torres-Acosta, 1999). The results showed that
kids had minor pathophysiological effects, irrespective of the nutritional
plane, compared with noninfected groups, suggesting that the level of infec-
tion applied was mild. However, the salient finding of this study was that,
although kids on the high-protein diet tended to retain more protein than
kids on the low-protein diet, the former did not achieve a higher growth
rate. Moreover, a large proportion of the extra N given to kids was ulti-
mately transformed into urea, and this resulted in greater elimination of N
via urine from soya beanesupplemented kids.
Under field conditions in the tropical forest of Yucatan, Mexico, several
studies have demonstrated that browsing sheep and goats eat a large quantity
of N-rich legumes (Ortega-Reyes et al., 1985). Recent studies showed that
sheep and goats eat up to >100% of their MP requirements (Gonzalez-Pech
et al., 2015). Even though some authors concluded that dietary energy was
of no use in improving resilience or resistance against GINs, it was evident
that, under these conditions, the best option was to supplement the animals
with a source or rumen fermentable energy (RFE). The approach was to
optimize the use of available protein-rich fodder from tropical forest vege-
tation to produce more microbial proteins and more volatile fatty acids to
improve animal performance (resilience) and possibly increase resistance to
GIN infections. The first attempt was performed by Gutierrez-Segura
et al. (2003), who compared nonsupplemented kids versus kids supple-
mented either with maize (108 g/day) or a combination of maize:soya
bean (70:30%; 107 g/day) during the rainy season. In this study, both sup-
plementation strategies significantly improved resilience compared with
nonsupplemented control kids. Another field study compared two sources

of RFE e maize (107 g/day) versus sugar cane molasses (150 g/day) e in
growing kids during the rainy season, showing that both sources of energy,
which represented the same amount of RFE, enhanced resilience to GIN
infection(s) compared with nonsupplemented control animals (Landa-Can-
signo et al., unpublished data). Subsequent studies determined the optimal
quantity of RFE to supplement either goats or sheep, by providing the sup-
plement as a proportion of the body weight of animals rather than giving a
set amount of supplement daily, as in previous studies (Landa-Cansigno
et al., unpublished data). The proposal was to further optimize rumen func-
tion by providing the quantity of energy required to achieve optimum an-
imal productivity, and then evaluate the effect of this strategy on a natural
parasitic infection.
A first study (Garate-Gallardo et al., 2015) evaluated four naturally
infected groups: infected not supplemented (I-NS), infected þ maize
supplement at 108 g/d (I-S108), infected þ maize supplement at 1% of
body weight (I-S1%), infected þ maize supplement at 1.5% body weight
(I-S1.5%), or infected þ supplemented (maize supplement 1.5% body
weight) þ moxidectin (0.2 mg/kg body weight subcutaneously every
28 days) (T-S1.5%). Kids browsed daily (7 h) in a tropical forest for
112 days during the rainy season and were weighed weekly to adjust supple-
mentary feeding. Haematocrit (PCV), Hb, and FECs were determined fort-
nightly. On day 112, five kids per group were slaughtered to determine
worm burdens. Kids from the I-S1.5% group showed similar body-weight
change, PCV, and Hb, compared with kids without GINs (T-S1.5%), while
showing lower FECs and T. colubriformis worm burden compared with the
I-NS group (P < 0.05). Thus, among the supplement levels tested, maize
supplementation at 1.5% body weight of kids was the best strategy to
improve their resilience and resistance against natural GIN under tropical
rain forest conditions. In the sheep field study, the treatment groups included
the I-NS (infected, not supplemented), I-S (infected, supplemented with
maize at 1.5% body weight), T-NS (treated with moxidectin 0.2 mg/kg
body weight every 28 days, and not supplemented) and T-S (treated with
moxidectin and supplemented with maize at 1.5% body weight). Again,
maize supplementation helped to improve resilience of hair sheep lambs
against GIN infections. The I-S and T-NS groups showed similar live-
weight gain, Hb, and PCV (P > 0.05), and both were higher than the
I-NS group (P < 0.05). Supplemented groups (T-S and I-S) showed higher
total Dry Matter Intake (DMI) (fodder þ maize; P < 0.05), and, hence,
266 H. Hoste et al.
higher intakes of metabolizable energy and protein than nonsupplemented

groups (T-NS and I-NS). In both studies, it was possible to establish the
cost of natural mixed parasitism due to the simultaneous presence of animal
groups with and without infection, and also with and without supplements.
Thus, under similar conditions of mixed natural GIN infections and supple-
mentation levels in the tropical forest during the rainy season, Retama-Flores
et al. (2012) estimated the average metabolic cost of GIN infections to be
43.5 g body weight/day in Pelibuey lambs. This value is similar to the differ-
ences recorded in kids by Garate Gallardo et al. (2015), with 41 g body
weight/day.
2.5 Supplementation with mineral micro-nutrients and trace

elements
There is evidence to suggest that also mineral micro-nutrients and trace el-
ements are involved in the interactions between host nutrition and GIN
infections:
1. Mineral metabolism can be seriously affected in the presence of worms
(Knox et al., 2006; Mc Clure, 2008). Sykes and Greer (2003) suggested
that few studies have shown that phosphorus, calcium, copper, and mag-
nesium metabolism are disturbed by the presence of GINs, and that
further studies are necessary to assess the role of this aspect. In haemon-
chosis, iron metabolism can be strongly modified, with increased Hb and
blood turnover in the host. Studies of lambs showed that small intestinal
infections with T. vitrinus had an effect on phosphorus absorption/meta-
bolism, leading to clinical signs (Coop and Field, 1983).
2. Some of these micro-nutrients (copper, molybdenum, cobalt, zinc, sele-
nium, and vitamin E) are important for proper immune function (Bundy
and Golden, 1987; Mc Clure, 2003, 2008; Suttle and Jones, 1989).
3. Some micro-nutrients (eg, copper) are important for nematode biology,
because they act as coenzymes for various key processes in worm meta-
bolism (eg, digestive proteinases of the worm) (Suttle et al., 1992).
However, to date, very few studies and results obtained have led to the
manipulation of mineral and trace elements to improve H. contortus control,
and more generally, GIN infections. This is explained by (1) the complexity
of physiological interactions with mineral nutrition (eg, interactions be-
tween copper and molybdenum metabolism) in the host; (2) the lack of basic
information and understanding of the interactions between mineral and
parasitic nematodes in the infected host and (3) the role of external factors,
such as soil conditions (ie, whether or not there is a deficiency in a given
trace element in feed resources). Such environmental conditions also govern

the decision whether to add trace elements to the diet, because of their po-
tential toxic effect on the hosts (ie, copper toxicity in sheep and, to a lesser
extent, in goats).
One main exception is the manipulation of copper through the use of
Copper Oxide Wire Particles (COWPs). This particular material has a long
lasting effect, as COWPs dissolve gradually in the abomasum of treated an-
imals, releasing copper over the course of several weeks. COWPs were
initially commercialized to correct copper deficiency in ruminant produc-
tion systems. Some earlier results showed that the use of COWPs could
contribute to lower GIN populations in sheep (Bang et al., 1990) and goats
(Chartier et al., 2000b). Several further studies in both small ruminant
species have also demonstrated a primary effect against abomasal species,
particularly H. contortus (see Burke et al., 2007; Soli et al., 2010). The
role of COWPs is first to prevent the establishment of incoming third-stage
infective larvae, and, second, to cure established infections, as suggested by
Knox (2003). A recent efficacy trial showed that COWPs have a significant
effect for up to 35 days against H. contortus in sheep (Galindo-Barboza et al.,
2011).
Due to the high prevalence of H. contortus in tropical and subtropical
areas, various studies focussed on examining the use of COWPs under a
wide range of conditions (Burke et al., 2004, 2006, 2007; Martínez-
Ortiz-de-Montellano et al., 2007). Overall, the data have shown that
COWPs can be a valuable alternative to AH drugs for control of H. contor-
tus, either alone, or in combination with other nutritional options
(Martínez-Ortiz-de-Montellano et al., 2007). For instance, Burke et al.
(2005) reported the complementarity of using COWPs and Duddingtonia
flagrans (nematode-trapping fungus) for H. contortus control in lambs. The
question of potential cumulative hepatic toxicity of copper has been
addressed in sheep (Burke et al., 2007) and goats, suggesting a lower risk
in the latter species (Galindo-Barboza et al., 2011; Martínez-Ortiz-de-
Montellano et al., 2007).
2.6 A scheme to improve the control of H. contortus

infection on the farm, depending on the nutritional
status
If nutrient manipulation is to be used for the control of H. contortus (and
other GINs), the following nutritional principles should be considered un-
der farm conditions (Fig. 4):
268 H. Hoste et al.
Figure 4 The outcome of supplementary feeding for the control of Haemonchus contortus
and other gastrointestinal nematodes is dependent on the level of animal nutrition. At
the lowest nutritional plane, which is the most common situation in many farming sys-
tems, there are more chances to improve health and productivity, while improvements
will be less evident in animals with a good level of nutrition and there might be very
limited improvement in those few animals, which have an excellent level of nutrition.
‘Happy factor’ correspond to those animals in good physical condition that may avoid
the use of chemical drugs to control GIN infection (Kenyon and Jackson, 2012).
2.6.1 Animals on a poor nutritional plane

It is evident that animals with poor nutritional status, such as the majority of
animals under resource-poor farming conditions, may benefit greatly from
supplementary feeding. The most important target would be to provide
nutrients that are essential to improve and optimize ruminal function.
Providing the appropriate supplement is the key. On farms where there is
insufficient feed, animals should receive a source of forage with at least 8%
crude proteins (ie, grass hay or crop stubbles). If the latter contains <8% crude
protein, it will be necessary to include a source of NPN (Leng, 1991), such as
poultry litter. But, as mentioned previously, the improvement in host resil-
ience with NPN might not be achieved, unless it is coupled simultaneously
to a source of rumen degradable energy (ie, sugar cane molasses). Similarly,
for malnourished animals browsing legume-rich foliage, as is the case in
the tropical forests of Mexico (Gonzalez-Pech et al., 2015), animals may
need a source of rumen degradable energy to increase the utilization of
digestible protein from the foliage harvested. Thus, it is evident that for most
farmers, the decision to improve the nutritional status of their animals is com-
plex. That is why computer decision support software, such as ‘Grazfeed’
(Freer et al., 1997), has been created. However, such software packages are
often not applicable under many conditions, as they require basic nutritional
information that may not be available for many ecosystems, which differ from
temperate grasslands. To avoid the complexity of nutritional management
decisions, many farmers are forced to buy expensive feeds composed of con-
ventional feedstuffs, such as maize, sorghum or soya bean. These feeds already
include the main macro-nutrients (protein, energy, fibre) necessary to supple-
ment ruminants, but are costly and also compete with the needs of humans.
2.6.2 Animals on a good nutritional plane

In the case of farming systems, where animals graze excellent quality grasses,
and where the amount of grass and grazing time is sufficient to maintain a
good level of nutrition, animals should have optimal ruminal function.
Thus, any further improvement in nutrition can only be achieved with a
source of dietary protein in the form of rumen-escape protein (eg, fish
meal and soya bean meal), which is expensive; any attempt to use such
feed for ruminants should be based on a clear cost-benefit calculation.
2.6.3 Animals on an excellent nutritional plane

For animals with an excellent level of nutrition, dietary supplementation will
not further improve the nutritional status. Thus, the only viable intervention
would be to reduce the cost of the diet without reducing its quality, and this
would be more a matter for nutritionists and economists. Under such con-
ditions, there might not be a direct improvement in the control of GINs
with supplementary feeding, except during the periparturient period or
lactation. Supplementary feeding will be coupled to very good farming con-
ditions, where GIN burdens would be mild or very low.
3. QUALITATIVE ASPECTS
Over the last 20 years, an increasing number of studies have been
focussed on exploring and validating a second aspect of the interactions be-
tween host nutrition and GIN infections, that is whether the composition or
quality of the feeds/plants eaten by ruminants can affect worm biology.
Overall, the results suggest that some natural products, also called
PSMs (see Box 1), which are either already present in or can be added to
270 H. Hoste et al.
ruminant diets, could have AH effects on nematodes (Hoste et al., 2006,

2012; Min et al., 2003; Min and Hart, 2003). The presence of these second-
ary metabolites is thought to explain the antiparasitic activities of herbal rem-
edies and/or nutraceuticals (Andlauer and F€ urst, 2002).
Due to their mode of application and objective, that is, short-term use
for treating the animals, herbal remedies (phytotherapeutic drugs) are some-
what related to pharmacological, synthetic AH drugs. In contrast, as indi-
cated by the name, the ‘nutraceutical concept’, which is based on a
combination of ‘nutrition’ and ‘pharmaceutical’, is closely linked to host
nutrition. In veterinary helminthology, a nutraceutical can be defined as a
livestock feed, which provides both nutritional value and a health benefit
to animals, and is used to prevent and control diseases. Differences between
nutraceuticals, phytotherapeutic drugs, and synthetic drugs have been thor-
oughly discussed in a review by Hoste et al. (2015). Because this chapter
covers the interaction between host nutrition and Haemonchus infection,
Section 3 focuses on the interactions between nutraceuticals, the biology
of H. contortus, and the consequences of haemonchosis in the host. We do
not consider here any data on the AH effects of phytotherapeutic drugs.
Bioactive plants or plant-based resources containing condensed tannins
(CTs) and other polyphenols are currently receiving considerable attention
and, hence, represent interesting nutraceutical models for: (1) studying the in
vivo AH effects in infected sheep and goats, (2) examining the potential
mechanisms of action of a group of PSMs against GINs, and (3) exploring
potential on farm applications, which will depend on regional epidemiolog-
ical and environmental conditions, as far as different tannin-containing re-
sources and different GINs are concerned. Chicory (Cichorium intybus) is
another interesting example of a plant that has been explored as a nutraceu-
tical. In this case, sesquiterpene lactones, not tannins, are thought to be the
main bioactive PSMs (Foster et al., 2011a,b; Molan et al., 2003b). However,
under the climatic and agronomic conditions favourable to growing chicory,
Teladorsagia, Ostertagia or Trichostrongylus spp. are the dominant GINs.
Therefore, the potential AH effects of chicory against H. contortus have rarely
been investigated (eg, Athanasiadou et al., 2007; Marley et al., 2006;
Tzamaloukas et al., 2006). In a study of lambs that compared two tannin-
containing legumes with chicory, the total reductions of H. contortus FECs
were 63% for sainfoin and Lotus corniculatus and 89% for chicory. However,
a tendency towards lower Haemonchus worm numbers was noticed with
sainfoin (35%) and birds foot trefoil (e49%), but not for the chicory
diet (Heckendorn et al., 2007).
3.1 Chemistry of tannins and related polyphenols

The plant kingdom contains an amazing array of secondary metabolites on
which herbal medicine has been founded for centuries. The plant metabolites
(ie, PSMs) range from being highly bioactive to being potentially highly toxic
and can contribute to nutraceutical effects of dietary feeds (Andlauer and
F€urst, 2002). Here, we focus on one group of important nutraceuticals,
that is, tannins (Fig. 6) and related polyphenolic compounds (Fig. 7). These
compounds are also well known for their antioxidant properties (Barbehenn
et al., 2006). The best-known tannins from terrestrial plants are condensed
tannins, gallotannins and ellagitannins and consist of monomeric building
blocks such as flavan-3-ols, gallic acid and ellagic acid. Several reviews exist
on the analysis of tannins (Huemmer and Schreier, 2008; Mueller-Harvey,
2001; Salminen and Karonen, 2011; Schofield et al., 2001), and efforts
continue to improve their detection and analysis (Engstr€ om et al., 2014;
Gea et al., 2011; Grabber et al., 2013; Zeller et al., 2015a), and to study
the relationships between their chemical structures and biological effects.
3.2 Methodological issues

3.2.1 Analytical tools for tannins
The complexity of plant polyphenol chemistry demands information on the
advantages and disadvantages of the analytical tools being used to assess their
biological, AH effects. Much research has been undertaken using nonspe-
cific colorimetric assays, which can neither distinguish between compounds
nor different compound classes (Mueller-Harvey, 2001; Salminen and
Karonen, 2011; Schofield et al., 2001). Currently, there are only a few rela-
tively simple methods that provide some useful information on tannins. The
HClebutanol assay is specific for CTs (Grabber et al., 2013), whereas
polyethylene glycol (PEG) and polyvinyl polypyrrolidone (PVPP) are
‘tannin-neutralizing’ compounds. The latter two methods are widely used
for in vitro and in vivo studies (Brunet et al., 2008a; Silanikove et al.,
2001). However, PEG or PVPP do not distinguish between condensed or
hydrolysable tannins, and they are not always perfect at ‘neutralizing’ tan-
nins, as they can also bind other polyphenols (Azando et al., 2011; Barrau
et al., 2005). Tannin analysis is now benefitting from rapid advances in
instrumentation, that is, ultra-performance liquid chromatographyetandem
mass spectrometry (UPLC/MS/MS), nuclear magnetic resonance, and other
techniques (Engstr€om et al., 2014; Mancilla-Leyton et al., 2014; Zeller et al.,
2015a).
272 H. Hoste et al.
3.2.2 Studying the anthelmintic properties of tannins and related

polyphenols
The different approaches that can be used to evaluate and interpret the AH
effects of bioactive compounds and tannin-containing nutraceuticals have ad-
vantages and disadvantages, and have been reviewed by Hoste et al. (2015). In
essence, a four-step approach is considered suitable for (1) the initial identi-
fication of likely bioactive plants; (2) subsequent in vitro screening; (3)
confirmation under controlled in vivo conditions, and (4) validation under
farm and epidemiological conditions to develop practical applications.
Haemonchus contortus has been widely used as a GIN model in these
studies, because of (1) the apparent expansion of prevalence of this GIN un-
der various epidemiological conditions; (2) the basic information that is now
available on the genetics and genomics of H. contortus, which includes iso-
lates that are resistant to synthetic AHs, and (3) the severity of the patholog-
ical/pathophysiological consequences in individual sheep and goats and
related economic costs at the whole-herd level; mortalities due to haemon-
chosis are particularly serious on small farms in the tropics.
3.3 Impact on the biology of different, key stages of

gastrointestinal nematodes
Most results have been obtained from a whole range of in vitro assays
(Jackson and Hoste, 2010) (Table 1) that were applied to different develop-
mental stages of H. contortus (Table 2). They have been followed up with in
vivo studies of H. contortuseinfected small ruminants under either confined
(Table 3) or natural conditions (Table 4). Here, we refer to in vivo studies,
where the results relate to specific effects on H. contortus, either because it
was the sole nematode or because adapted methodologies have been applied
to identify its presence and/or its specific consequences. This chapter de-
scribes findings for sheep or goats from temperate or tropical regions.
Studies of infected animals fed with tannin-containing plants have shown
that three main stages of H. contortus are targets:
1. Infective third-stage larvae (L3). In vivo results have demonstrated lower L3
establishment rates in hosts that consume selected bioactive resources.
2. Adult worms. Most studies of small ruminants that examined the effects of
CT-containing plants on adult nematode populations found that there
was a significantly lower overall FEC in animals infected with H. contor-
tus. This observation/finding relates either to a lower worm number
and/or lower fertility of female H. contortus.
3. Eggs. There are numerous in vitro studies that used egg hatch and/or
larval development assays with H. contortus. Overall, these studies suggest
Table 1 In vitro methods used to evaluate the anthelmintic activity of CT-containing resources
Interactions Between Nutrition and Haemonchus contortus & Related Nematodes

Parasite
Equipment required stage Measured anthelmintic effect References
EHA (Egg Hatch Assay) Optical microscope, dry oven Eggs Egg hatching disrupted Chan-Pérez et al. (2016),
Hernandez-Villegas et al. (2011),
Houzangbé-Adote et al.
(2005a,b), Molan (2014),
Moreno-Gonzalo et al.
(2013a,b), Paolini et al. (2004),
Vargas-Maga~ na et al. (2014a)
LDIA (Larval Development Optical microscope, dry oven Eggs e L3 Development from eggs to L3 Athanasiadou et al. (2001), Molan
Inhibition Assay) delayed/blocked (2014)
LFIA (Larval Feeding Inhibition Fluorescence microscope, dry L1 Feeding of L1 disrupted/blocked Desrues et al. (2016), Novobilský
Assay) oven et al.(2011, 2013)
LMIA (Larval Migration Optical microscope, dry oven L3 Decreased motility, paralysis Alonso-Díaz et al. (2008a,b, 2011),
Inhibition Assay) Calder on-Quintal et al. (2010),
Hernandez-Ordu~ no et al. (2008),
Hernandez-Villegas et al. (2011),
Hoste et al. (2009), Houzangbé-
Adote et al. (2005a,b), Molan
(2014), Molan et al. (2004a,b),
Moreno-Gonzalo et al.
(2013a,b), Paolini et al. (2004)
LEIA (Larval Exsheathment Optical microscope, dry oven L3 Exsheathment of L3 delayed/ Alonso-Díaz et al. (2008a,b, 2011),
Inhibition Assay) blocked Bahuaud et al. (2006),
Novobilský et al. (2011), Quijada
(2015), Quijada et al. (2015)
AMIA (Adult Motility Stereo microscope, dry oven Adult Motility (viability) of worms Hoste et al. (2009), Houzangbé-
Inhibition Assay) worms affected Adote et al. (2005a,b), Moreno-
Gonzalo et al. (2013a,b), Paolini
et al. (2004)
273
274
Table 2 Comparison of in vitro anthelmintic effects of tannin-containing resources against H. contortus and other GIN species
Botanical H. Te. T. Ostertagia Cooperia
Plant family Host CT sample contortus circumcincta colubriformis ostertagi spp. EC50 References Main findings
Temperate legumes
Lespedeza cuneata Leguminosae Goat Fraction x x Yes Quijada et al. AH effect against both species,
(2015) H. contortus was more
susceptible (lower EC50)
Lotus corniculatus Leguminosae Cattle Extract x x Yes Novobilský AH effect against both species
et al. (2011)
Lotus pedunculatus Leguminosae Cattle Extract x x Yes Novobilský AH effect against both species
et al. (2011)
Onobrychis Leguminosae Goat Fraction x x Yes Quijada et al. AH effect against both species,
viciifolia (2015) H. contortus was more
Cattle Extract x x Yes Novobilský AH effect against both species
et al. (2011)
Cattle Extract x x No Novobilský AH effect against both species,
and et al. (2013) variations between extracts
Fraction or fractions tested
Goat Extract x x x No Paolini et al. AH effect on T. colubriformis
(2004) and H. contortus L3, and on
abomasal adult worms
Cattle Fraction x x No Desrues et al. AH effect against both species,
(2016) O. ostertagi was more
susceptible
Trifolium repens Leguminosae Cattle Fraction x x No Desrues et al. AH effect against both species,
H. Hoste et al.
susceptible
Goat Fraction x x Yes Quijada et al. AH effect against both species,
Sarothamnus Leguminosae Goat Extract x x No Bahuaud et al. No AH effect for both species
scoparius (2006)
x x x Hoste et al. AH effects vary according to
(2009) the species and stage, thus
were significant for H.
contortus and T. colubriformis
adult worms but not for
Te. circumcincta L3
Tropical legumes
Acacia pennatula Leguminosae Goat Extract x x No Alonso-Díaz AH effects were similar for
et al. both parasites
(2008a,b)
Leucaena leuco- Leguminosae Goat Extract x x No Alonso-Díaz AH effects were similar for
cephala et al. both parasites
(2008a,b)
Lysiloma Leguminosae Goat Extract x x No Alonso-Díaz AH effects were similar for
latisiliquum et al. both parasites
(2008a,b)
Piscidia piscipula Leguminosae Goat Extract x x No Alonso-Díaz AH effects were similar for
et al. both parasites
(2008a,b)
Shrubs/woody plants
Betula spp. Betulaceae Cattle Fraction x x No Desrues et al. AH effect against both species,
susceptible
(Continued)
275
Table 2 Comparison of in vitro anthelmintic effects of tannin-containing resources against H. contortus and other GIN speciesdcont'd
276
Calluna vulgaris Ericaceae Goat Extract x x x Yes Moreno- Showed the higher EC50 of
Gonzalo heather’s extracts. The AH
et al. effects vary according to the
(2013a,b) parasite stage, for EHA only
T. colubriformis was affected
and it showed the lower
EC50 for LEIA also
Camellia sinensis Theaceae Sheep Fraction x x No Molan et al. AH effect against both species,
(2004a,b) Te. circumcincta was more
susceptible
Castanea sativa Fagaceae Goat Extract x x x No Bahuaud et al. Similar AH effect against H.
(2006) contortus and T. colubriformis
(L3)
Hoste et al. High AH effect vary according
(2009) to the specie and stage.
For adults of Te. circumcincta
was NS
Corylus avellana Corylaceae Cattle Fraction x x Desrues et al. AH effect against both species,
susceptible
Goat Extract x x x No Hoste et al. AH effect against the three
(2009) species (both L3, adults),
only the L3 effects on
T. colubriformis was non
significant
Extract x x x No Paolini et al. AH effect on abomasal species
(2004) (L3 and adult) and intestinal
H. Hoste et al.
adult worms, but non on L3
Fraction x x Yes Quijada et al. AH effect against both species,
Erica cinerea Ericaceae Goat Extract x x x Yes Moreno- Extract’s AH effects vary
Gonzalo according to the parasite
et al. stage, for EHA only T.
(2013a,b) colubriformis. For LEIA, the
lower EC50 were for
abomasal species
Erica erigenea Ericaceae Goat Extract x x x No Bahuaud et al. H. contortus was more
(2006) susceptible
Hoste et al. Te. circumcincta (L3 and adult)
(2009) was the less susceptible
Erica umbellata Ericaceae Goat Extract x x x Yes Moreno- Extract’s AH effects vary
Gonzalo according to the parasite
et al. stage, for EHA only T.
(2013a,b) colubriformis was affected and
showed the lower EC50 for
LEIA
Fraxinus excelsior Oleaceae Goat Extract x x x No Hoste et al. Similar AH effect on abomasal
(2009) and intestinal species (L3
only)
Junglans regia Junglandecea Cattle Fraction x x No Desrues et al. AH effect against both species,
susceptible
Newbouldia laevis Bignoneaceae Sheep Extract x x No Azando et al. Similar AH effect on both
(2011) species (L3)
Houzangbé- AH effect on L3 on both
Adote et al. species but only on T.
(2005a,b) colubriformis adult worms
(Continued)
277
Table 2 Comparison of in vitro anthelmintic effects of tannin-containing resources against H. contortus and other GIN speciesdcont'd
278
Pinus radiata Pinaceae Sheep Fraction x x Yes Molan (2014) AH effect on both species. Te.
circumcincta either egg or
larvae were more
Pinus sylvestris Pinaceae Cattle Fraction x x No Desrues et al. AH effect against both species,
susceptible
Goat Extract x x No Bahuaud et al. Similar AH effect against H.
(2006) contortus and T. colubriformis
(L3)
Extract x x x No Hoste et al. T. colubriformis was the most
(2009) susceptible (both L3 and
adult). For the two
abomasal species, AH effect
was significant on H.
contortus adult worms only
Fraction x x Yes Quijada et al. AH effect against both species,
Quercus robur Fagaceae Goat Extract x x x No Hoste et al. AH effect was NS on T.
(2009) colubriformis L3
Ribus nigrum Grossulariacea Cattle Fraction x x No Desrues et al. AH effect against both species,
susceptible
H. Hoste et al.
Ribus rubrum Grossulariacea Goat Fraction x x Yes Quijada et al. AH effect against both species,
Rubus fructicosus Rosaceae Goat Extract x x x No Hoste et al. AH effect was NS just for H.
(2009) contortus L3
Salix spp. Salicaceae Cattle Fraction x x No Desrues et al. AH effect against both species,
susceptible
Schinopsis spp. Anacardiaceae Sheep Extract x x T. vitrinus Yes Athanasiadou Similar AH effect on the 3
et al. (2001) species (EC50 were
nonsignificantly different)
Theobroma cacao Malvaceae Goat Fraction x x Yes Quijada et al. AH effect against both species,
Tilia spp. Tiliaceae Cattle Fraction x x No Desrues et al. AH effect against both species,
susceptible
Vitelaria paradoxa Sapotaceae Goat Fraction x x Yes Quijada et al. AH effect against both species,
Zanthoxylum Rutaceae Sheep Extract x x No Azando et al. Similar AH effect on both
zanthoxyloides (2011) species
Houzangbé- AH effect observed on both
Adote et al. species, H. contortus (adult)
(2005a,b) appeared less susceptible
NS, nonsignificant.
279
Table 3 Comparison of in vivo anthelmintic efficacy of tannin-containing resources against H. contortus and other GIN species under experimental conditions
of infections
Nematode parasite Main findings
Plant/ Botanical H. Te. T.
resource family Host contortus circumcincta colubriformis Other species FEC Larval culture Adult worms other References
Temperate legumes
Ceratonia Leguminosae S x x Reduced NP Lower T. Lower fecundity Manolaraki
siliqua colubriformis for T. et al.
total burden. colubriformis. (2010)
No effect on No effect on
H. contortus H. contortus
burden
S x x NS NP NS Lower fecundity Arroyo-Lopez
for H. contortus, et al.
it was NP for (2014)
T. colubriformis
Hedysarum Leguminosae D x T. axei, Cooperia Trend to be Trend to be lower Lower abomasal Fecundity did not Hoskin et al.
coronarium sp., Oesophag- lower parasite differ between (2000)
ostomum sp. burden. non species
effect one
either small or
large intestine
species
Lespedeza Leguminosae G x x x Reduced Lower percentage Lower abomasal NP Shaik et al.
cuneata of total L3 (H. contortus, (2006)
recovered Te.
(especially H. circumcincta)
contortus) and intestinal
worms (T.
colubriformis)
counts
Lotus Leguminosae S x x Reduced NP Lower Te. No inhibited L4’s Niezen et al.
pedunculatus circumcincta were observed. (1998)
burden, Fecundity ¼ no
especially effect on Te.
females. T. circumcincta
colubriformis females was
was not observed
affected increased for
T. colubriformis
females
Lotus Leguminosae D x T. axei, Cooperia Trend to be Trend to be lower Lower abomasal Fecundity did not Hoskin et al.
corniculatus sp., Oesophag- lower parasite differ between (2000)
ostomum sp. burden. No species
effect on
neither small
or large
intestine
species
S x Cooperia curticei Reduced NP Lower (but NS) NP Heckendorn
H. contortus et al.
total worm (2007)
and female
burden.
No effect on C.
curticei
Onobrychis Leguminosae S x x Reduced NP Lower H. Lower fecundity Arroyo-Lopez
viciifolia contortus total for H. contortus, et al.
burden. NS it was NP for (2014)
for T. T. colubriformis
colubriformis
S x x Lower but NS NP NS NP Girard et al.
(2013)
(Continued)
of infectionsdcont'd
S x Cooperia curticei Reduced NP Lower H. Lower fecundity Heckendorn
contortus for C. curticei. et al.
burden; C. No effect on (2006)
curticei counts H. contortus
were reduced female
but NS fecundity
S x Cooperia curticei Reduced NP Lower (but NS) NP Heckendorn
H. contortus et al.
total and (2007)
female
burden. None
effect on C.
curticei
S x x Reduced NP Lower T. Lower fecundity Manolaraki
colubriformis for H. contortus et al.
burden and T. (2010)
colubriformis
S x x x Nematodirus Reduced NP Lower Te. NP Werne et al.
circumcincta (2013)
and
Nematodirus
burden
S x x x Reduced Increasing H. NP NP Werne et al.
contortus L3 (2013)
proportion
Vicia faba Leguminosae S x x x NS Increasing H. NP NP Werne et al.
contortus L3 (2013)
proportion
Tropical legumes
Caesalpinia Leguminosae S x x NS NP NS No differences in H€ ordegen
crista fecundity for et al.
both species (2003)
Lysiloma Leguminosae G x x NP NP Lower total Lower Brunet et al.
latisiliquum worm burden establishment (2008a,b)
for both for both species
species
Woody plants
Corylus Betulaceae G x x Reduced Lower H. contortus NS Lower fecundity Desrues et al.
avellana L3 vs T. for H. contortus (2012)
colubriformis
S x x Trends for NP NS NP Girard et al.
lower FEC (2013)
(p < 0.09)
after
hazelnut
skin
addition to
sainfoin
pellets
Pistacia Anacardiaceae G x x C. ovina reduced NP NP NP Landau et al.
lentiscus (p < 0.001 (2010)
e0.0001)
S x x reduced NP NS Lower fecundity Manolaraki
for H. contortus et al.
and T. (2010)
colubriformis
Schinopsis spp. Anacardiaceae S x x x Nematodirus Reduced only NP Lower T. Lower fecundity Athanasiadou
battus in sheep colubriformis of T. et al.
infected and colubriformis and (2001)
with T. Nematodirus Nemat-
colubriformis burden. Had odirus females
or Nemato- not effect on
dirus abomasal
species burden
(Continued)
of infectionsdcont'd
G x x Reduced NP NS Lower fecundity Paolini et al.
for T. (2003a)
colubriformis and
a trend for Te.
circumcincta
G x x NP NP Lower T. The fecundity was Paolini et al.
colubriformis no affected for (2003a)
burden; trend both parasites
to lower Te.
circumcincta
worm count
(p < 0.08).
Vernonia Asteraceae S x x NS NP NS No differences in H€ordegen
anthel- fecundity for et al.
mintica both species (2003)
Quercus Fagaceae S x x Reduced NP Lower T. Lower fecundity Manolaraki
coccifera colubriformis for T. et al.
burden. No colubriformis, (2010)
effect on H. trend for H.
contortus contortus
burden (p < 0.06)
Melia Meliaceae S x x NS NP NS No differences in H€ordegen
azedarach fecundity for et al.
both species (2003)
Azadirachta Meliaceae S x x NS NP NS No differences in H€ordegen
indica fecundity for et al.
both species (2003)
Fumaria Papaveraceae S x x Reduced NP Lower H. No differences in H€ordegen
parviflora contortus and fecundity for et al.
T. colubriformis both species (2003)
burden
Embelia ribes Primulaceae S x x NS NP NS No differences in H€ordegen
fecundity for et al.
both species (2003)
Salix Salicaceae S x x x Oesophago- Reduced Decreased in both Aboma- Lower fecundity Mupeyo et al.
stomum, abomasal sum: lower H. in abomasum (2011)
Cooperia, species L3. NS contortus (male Te. circumcincta,
Trichuris, for Trichostr- and female) and small
Nemat- ongylus spp., C. and Te. intestine
odirus, C. curticei or N. circumcincta Trichostrongylus
ovina spathiger (female). Small spp. NS for the
intestine: other species
lower C.
curticei burden.
Trichostr-
ongylus and
Nemato-
dirus were not
affected
Shrubs
Ananas Bromeliaceae S x x NS NP NS No differences in H€
ordegen
comosus fecundity for et al.
both species (2003)
Erica Ericaceae G x x Reduced NP NS Reduction in Moreno-
umbellata larvae Gonzalo
establishment et al. (2012,
for both species 2014)
Erica cirenea Ericaceae G x x (a trend for T. Moreno-
colubriformis Gonzalo
p < 0.09). et al. (2012,
Lower fecundity 2014)
Calluna Ericaceae G x x and length for Moreno-
vulgaris Te. circumcincta. Gonzalo
No effect on female et al. (2012,
fertility for T. 2014)
colubriformis
G, goats; S, sheep; D, deer; NP, not performed; NS, nonsignificant.

Table 4 Comparison of in vivo anthelmintic efficacy of tannin-containing resources against H. contortus and other GIN species under conditions of natural
infections
Temperate legumes
Hedysarum Leguminosae S x x x Trichostrongylus Reduced NP Lowest abomasal and Fecundity or Niezen et al.
coronarium spp., intestinal parasite length were (1998)
Nematodirus burden. Tricho- not affected
spp., Cooperia strongylus spp. were
spp. the most affected
G x x T. vitrinus, NP NP No differences NP Pomroy and
Trichuris ovis between Te. Adlington
circumcincta, Tricho- (2006)
strongylus spp., or
Trichuris burdens
Lespedeza Leguminosae G x x x Reduced NP Lower both abomasal Lower abomasal Gujja et al. (2013)
cuneata species burden. NS L4’s in the
effects against 95% L.
T.colubriformis cuneata diet
G x x x Reduced NP Lower for both NP Mechineni et al.
abomasal species (2014)
burden (trend),
significantly for H.
contortus males
(p < 0.05). Trends
for lower burdens
(mainly male
worms) of the two
abomasal species
and T. colubriformis
G x x x Nematodirus Reduced NP Lower Te. NP Min et al. (2005)
circumcincta, H.
contortus and T.
colubriformis
burdens, but
Nematodirus was
not significant
G x x x T. axei Reduced Decreasing H. Overall reduced, NS NP Moore et al. (2008)
contortus burden. Lowest
proportion abomasal (Te.
circumcincta, H.
contortus and T.
axei) than T.
colubriformis worm
counts
G x x x Reduced Reduced L3 Abomasum: lower H. Female: male Terrill et al. (2007)
recovered, contortus burden, ratio was not
especially H. Te. circumcincta was affected
contortus not affected.
Intestine: T.
colubriformis trend
for a lower burden
(p < 0.10)
G x x x Reduced Slight reduction Lower abomasal NP Terrill et al. (2009)
on L3 burden, due to
recovered H. contortus
reduction.
Te. circumcincta and
T. colubriformis
were not affected
Lotus Leguminosae S ‘Abomasum and small intestinal spp.’ reduced NP Lower burdens of Reduced Marley et al.
corniculatus both abomasal and (2003)
intestinal species
(Continued)
infectionsdcont'd
S x x x Trichostrongylus NS NP Abomasum: Fecundity or Niezen et al.
spp., Trichostrongylus spp. length were (1998)
Nematodirus showed the higher not affected,
spp., Cooperia burden. Intestine: except for
spp. Nematodirus trend abomasal
to have higher Trichostron-
worm counts. No gylus spp. that
effect on either were longer
Trichostrongylus or
Cooperia
S x x x Trishostrongylus NS Reduced T. Lower abomasal (H. NP Ramirez-Restrepo
spp., colubriformis contortus, Te. et al. (2005)
Nematodirus, L3 circumcincta) and
Cooperia, T. intestinal
ovis, Chabertia (Nematodirus,
ovina, Cooperia) worm
Oesophago- burden, but higher
stomum, numbers of T.
Trichuris spp. colubriformis, C.
ovina,
Oesophagostomum
and T. ovis.
Onobrychis Leguminosae G x x x Reduced NP Reduced T. Lower fecundity Paolini et al.
viciifolia colubriformis burden for all (2005b)
(50%) but NS abomasal and
regard the two intestinal
other nematodes species
Lotus pedun- Legumi- S x x NS NP Abomasum: Trichostr- Fecundity or Niezen et al.
culatus nosae ongylus spp. length were (1998)
showed the higher not affected,
burden. Intestine: except for
Nematodirus trend abomasal
to have higher Trichostr-
worm counts. No ongylus spp.
effect on either that were
Trichostrongylus longer
or Cooperia
Trifolium Leguminosae S x Trichostrongylus Reduced NP Abomasum: lower Te. NP Marley et al.
pratense spp., Cooperia, circumcincta total and (2005)
Nematodirus male burden.
Intestine: trend to
have higher Tricho-
strongylus burden
(reinfection ¼
lambs treated with
AH)
Trifolium Leguminosae S x Trichostrongylus Reduced NP Lower Te. circumcincta NP Marley et al.
repens spp., Cooperia, total and male (2005)
Nematodirus burden. For
intestinal species
total adult count
were not affected,
but fewer male
were found
Tropical legumes
Acacia Leguminosae S x x Oesophagostomum, Reduced NP Lower Te. colubriformis NP Cenci et al. (2007)
mearnsii Cooperia spp., and Cooperia spp.
Strongyloides total worms count.
papillosus, T. Not differences
globulosa were found to the
other nematode
species
(Continued)
infectionsdcont'd
G x x Oesophagostomum, Reduced NP No differences related NP Costa-J
unior et al.
Trichuris to species, total or (2014)
sex worms burden
S,G x x Oesophagostomum, NS NS Sheep: lower H. NP Max (2010)
Cooperia, contortus burden.
Strongyloides, Small intestine:
Bunostomum significant increase
of worms count,
especially T.
colubriformis
S x x Reduced NP Lower H. contortus NP Minho et al. (2007)
burden. No
differences for T.
colubriformis
Woody plants
Schinopsis Anacardiaceae S x Trichostrongylus NS NP NS differences for NP Dawson et al.
spp. vitrinus abomasal or (2011)
(QUE- intestinal species
BRA-
CHO)
Trianthema Aizoaceae S x x Oesophagostumum Reduced Lower L3 for NP NP Hussain et al.
portula- spp., T. ovis the abomasal, (2011)
castrum small and
large
intestine
species
Salix Salicaceae S x x x Oesophagostomum, NS NP Abomasum: lower Te. Female: male Diaz Lira et al.
Cooperia, circumcincta burden. ratio did not (2008)
Trichuris, Small intestine: differ for
Nematodirus, lower burden abomasum
C. ovina (Nematodirus and or small
T. colubriformis). intestine
Large intestine: species
increased
proportion of male
for C. ovina and T.
ovis
S x x x Cooperia, C. ovina, NS Reduced L3 NP NP Ramírez-
Oesopha- proportion Restrepo
gostomum for abomasal et al. (2010)
species, but
the opposite
for small
(Trichostr-
ongylus spp.,
Cooperia spp.)
and large
intestine (C.
ovina,
Oesopha-
gostomum)
Rutaceae S x x NS NP NS
(Continued)
infectionsdcont'd
Zanthoxylum Trichostr- NS for H. contortus. Peneluc et al.
rhoifolium ongylus Reduced intestinal (2009)
colubrifromis, species, especially
Oesophago- Oesophago-
stomum stomum.
Shrubs
Erica spp. Ericaceae G x x C. ovina Reduced (just in T. colubriformis NP NP Osoro et al. (2007)
two months/ L3 was the
5 months most
studied) frequent. C.
ovina was not
observed in
heather
group
Calluna Ericaceae G x x C. ovina Idem Idem NP NP Osoro et al. (2007)
vulgaris
Musa Musaceae S x x Oesophago- Reduced Lower L3 for NP NP Hussain et al.
paradisiaca stumum, the abomasal, (2011)
Trichuris small and
large
intestine
species
G, goats; S, sheep; NP, Not performed; NS, nonsignificant

impaired egg hatching and/or development in the presence of CT. Some

in vivo results showed impaired development of H. contortus egg to the
L3 stage (eg, Shaik et al., 2006). However, other data were less consis-
tent, with discrepancies in significance between in vitro and in vivo
results.
3.4 Impact on host resilience

Besides the effects on H. contortus biology, multiple studies have repeatedly
demonstrated that tannin-containing nutraceuticals can have positive effects
on host resilience in sheep and goats. These investigations were conducted
in the context of controlled experimental infections or in situations where
Haemonchus was the dominant species. These beneficial effects included:
(1) better production parameters, in particular bodyweight gain in lambs,
(2) less severe signs of anaemia in goats and sheep, and (3) lower mortality
rates when challenged with parasites. Some studies also recorded the number
of AH treatments as an index of resilience required to control haemonchosis.
All references that describe PSM effects on the worm life cycle or resilience
are listed in Tables 3e4 (cf. Section 3.7).
It is difficult to separate the effects of tannins on worm biology (eg,
reduction in worm populations or in female worm fecundity) from the
beneficial effects on host pathophysiology. Moreover, apart from tannin
content, the protein content and rumen-escape protein effects, which occur
in the presence of some tannin types (Waghorn, 2008), can also be impor-
tant factors. Hence, the mechanisms for improved resilience are still unclear
and worthy of future exploration.
3.5 Variability in effects of plant secondary metabolites,

depending on parasitic nematodes
In comparison to synthetic AH molecules, tanniniferous nutraceuticals do
not result in a 99% elimination of GINs from hosts. These nutraceuticals
exert their effects mainly through combined impacts on GIN biology by
interfering with the three key stages of the life cycle (eggs, L3, and/or adult
worms). The net result is to slow down the dynamics of GIN infection and
to maintain infection at levels that are compatible with small ruminant pro-
duction and farm economy. Consumption of CT-containing plants leads to
lower environmental contamination (Min et al., 2005) or host infection
(Brunet et al., 2007; Oliveira et al., 2013); but it does not usually eliminate
worm populations from hosts. In addition, nutraceuticals and the PSM
products, unlike synthetic AH drugs, are not used in pure and standardized
294 H. Hoste et al.
P
L Season Environment Predation
A Cultivar Active concentration(s)

N Technological
Quality of the compounds
processes
T
S
EFFICACY on the NEMATODE SPECIES and/or STAGES
H Acve(s) bioavailability
O
Flow rate Physicochemistry Degradaon
S
T
Diet ingeson (VFI)
Figure 5 A summary of the main factors which might explain the variability in the ef-
ficacy of nutraceuticals against Haemonchus contortus based on the example of tannin-
containing resources.
forms. Hence, several factors can impact on their activity (Fig. 5), and these
are key issues to be considered for future farm applications.
3.5.1 Variations arising from nematode species and life cycle stages
Results from a wide range of studies conducted on CT-containing plants
suggest that PSM effects on nematode biology appear to depend on the
parasite species. This aspect was first illustrated in studies of quebracho
tannins. When established adult Haemonchus populations in sheep were
exposed to CTs, lower worm fecundity and worm numbers were found
for the intestinal species (N. battus and T. colubriformis), but there were no
changes in the species that resided in the abomasum (Te. circumcincta and
H. contortus) (Athanasiadou et al., 2001). However, in goats, the effects on
adult worms were restricted to lower female fecundity in both T. colubrifor-
mis and H. contortus, but not for Te. circumcincta (see Paolini et al., 2003a,b).
On the other hand, the establishment of infective larvae was reduced by
nearly 70% for Te. circumcincta and T. colubriformis, but only by 35% for
H. contortus (see Paolini et al., 2003a, 2005a). Since these early discoveries,
results of in vivo studies, under controlled or natural infections, have raised
various questions. Tables 3 and 4 summarize the results from different
studies, which included H. contortus, in order to examine whether this species
is particularly susceptible to tannins.
It would be reasonable to assume that intrinsic GIN species factors (eg,

differences in protein composition of sheath, cuticle, or digestive tract)
may explain differences in AH susceptibilities to tannins. If this was the
case, a comparison of results from in vitro assays (Table 2) against different
stages and nematode species should indicate which species is/are most
affected by tannins, and these results should also be consistent. However,
there is no clear trend (Table 2). Alternatively, the differences in AH effects
obtained from in vivo studies might also result from differences in bioavail-
ability, as the composition of free and bound tannins varies along the diges-
tive tract. It is hypothesized that free tannins may have a more potent AH
activity, although this has not yet been tested. The formation of CTe
protein complexes depends on tannin and protein structures, but also on
the compositions of tannin solutions. Of particular relevance is the pH of
a solution, as it affects the stability of the tannineprotein complexes. It is,
therefore, hypothesized that the different ruminal, abomasal, and intestinal
pH values may modulate tannin bioavailability. This hypothesis needs to
be tested. Unfortunately, there are few studies examining the tannin ‘phar-
macology’ along the gut (Terrill et al., 1994). These aspects need to be
further addressed. In addition, AH effects of tannins and polyphenols can
be difficult to explain because their concentration and composition adds
another dimension of complexity.
3.5.2 Variations in plant tannin composition (quantity and quality)

in relation to activities against H. contortus
Pioneering research in New Zealand (Min et al., 2003) first discovered that
the prodelphinidin tannins in Lotus pedunculatus had higher AH activities
than the procyanidin tannins in L. corniculatus (Fig. 6). However, subse-
quent progress was slow due to the complexity of plant tannins and limi-
tations of analytical techniques. Nonspecific assays, that is, colorimetric
or antioxidant assays for polyphenols, failed to explain the AH or other bio-
logical activities of tannins (Katiki et al., 2013; Salminen and Karonen,
2011).
Several factors influence the composition of bioactive compounds in
plants, with plant species, but also varieties or accessions being the most
important determinants of tannins and polyphenols (Falchero et al., 2011;
Kotze et al., 2009; Lowther et al., 1987; Mueller-Harvey, 2006; Stringano
et al., 2012). Knowing the source of such variation will be extremely useful,
because it will enable plant breeders to develop new varieties with optimized
296 H. Hoste et al.
Flavan-3-ol Monomers Condensed Tannin Examples

Monomeric units of procyanidins: A procyanidin trimer
OH OH
OH OH
HO O HO O
OH
OH OH
OH
OH OH
OH
OH HO O
Catechin
OH
HO O OH
OH
OH
OH HO O
OH
OH
Epicatechin OH
Monomeric units of prodelphinidins: A prodelphinidin trimer
OH OH
OH OH
HO O
HO O OH
OH
OH
OH
OH OH
OH
OH OH HO O
Gallocatechin OH OH
OH
OH
HO O OH
OH OH
HO O
OH OH
OH
OH
Epigallocatechin OH
A galloylated condensed tannin trimer

OH
OH
Example of a galloylated flavanol:
HO O
OH
OH
OH OH
HO O
OH
OH
OH
OH HO O
OH
OH
OH
OH
Epigallocatechin gallate (EGCg) HO O

OH
OH
Figure 6 Flavan-3-ols and tannins with anthelmintic effects against Haemonchus

contortus.
· Monomeric flavonoids: flavan-3-ols, other flavonoids (quercetin, luteolin, naringenin
and so on Fig. 7) (widespread in plants)
· Condensed tannins (forage legumes, browse and so on)
· Galloylated derivatives of CT (carob (Ceratonia siliqua), Acacia nilotica)
· Tannic acid (not shown above; for structures see Mueller-Harvey 2006)
· Taratannins (Pistacia lentiscus; not shown above. For structures see Dahmoune et al.,
2014; Rodriguez-Perez et al., 2013; Romani et al., 2002)
Other Flavonoids
OH OH
OH OH
HO O HO O
OH
OH O OH O
Luteolin (= a flavone) Quercetin (= a flavonol)

OH
OH OH
HO O HO O
OH
OH O OH O
Naringenin (= a flavanone) Taxifolin (= a dihydroflavonol)
Figure 7 Chemical structures of other anthelmintic flavonoids.
tannin traits in the future. In many plants, the composition also varies
between different plant parts, for example, sainfoin stems have more procya-
nidins and sainfoin leaves have more prodelphinidins (Malisch et al., 2015;
Theodoridou et al., 2010). Given the large phenotypic variation that typi-
cally exists within germplasm collections, it will, therefore, be possible to
enhance desirable tannin types (Falchero et al., 2011; Hayot Carbonero
et al., 2011), when the most important traits relating to AH activity are
known.
Additional factors are growth stage and environment (Azuhnwi et al.,
2013a; Grabber et al., 2014; Kotze et al., 2009; Theodoridou et al.,
2011). Fortunately, there is also some evidence that tannins and polyphenols
are affected by genotypeeenvironment interactions and that some
accessions have particularly ‘robust’ compositions, which are less subject to
environmental influences (Azuhnwi et al., 2013b; Kotze et al., 2009). Future
research will need to focus on identifying plant varieties that can deliver
consistent AH properties (L€ uscher et al., 2014).
The processing of harvested plants via drying (hay), pelleting, or ensiling
can also impact polyphenol composition. Pelleting and ensiling lead to a
higher proportion of bound tannins that are less easily extracted using
solvents (Lorenz and Uden, 2011; Minnée et al., 2002; Terrill et al.,
2007). While both processes do not affect the AH properties of Lespedeza
cuneata, others appear to significantly improve AH effects following the
ensiling of sainfoin (Manolaraki, 2011; Ojeda-Robertos et al., 2010). These
differences also require verification in future studies.
Leaves and stems of sericea lespedeza (SL) were analysed for CT concen-
tration and composition, with higher CT levels found in leaves than stems
298 H. Hoste et al.
(16.0 and 3.3 g/100 g dry weight, respectively). The CT of both leaves and
stems were almost pure prodelphinidins (98% and 94%, respectively), while
the CT polymer size was much larger in SL leaves than stems (mean degree
of polymerization: 42 and 18, respectively). It has been suggested that the
excellent AH effects of SL may stem from its unusual CT composition;
the higher proportion of prodelphinidins than procyanidins and high
molecular weights have both been linked to better antiparasitic bioactivity
(Novobilský et al., 2013; Quijada et al., 2015).
3.6 Modes of action of tannin-containing plants against

H. contortus: direct versus indirect effects
The number of studies that explore how tannins and other polyphenols
(flavonoids; Fig. 7) act against the different key parasitic GIN stages is expand-
ing. It is expected that these studies will guide the use of CT-containing
nutraceuticals under farm conditions in the future. Two general hypotheses
have been proposed to explain the AH effects of tannins and other polyphe-
nols against parasitic GINs. The main results to support these two nonexclu-
sive hypotheses are described later.
3.6.1 The direct (¼pharmacological-like) hypothesis

The bulk of the in vitro results for H. contortus tends to support the first hy-
pothesis (see Table 2), because host factors make no contribution. To a lesser
extent, short-term in vivo studies (Athanasiadou et al., 2001; Brunet et al.,
2008b) have also provided some evidence for the direct effects of PSMs
against H. contortus. In addition, some data are now emerging that tannin
structures can impact on in vitro and in vivo results. This information comes
from scanning (SEM) or transmission electron microscopy (TEM) studies,
which studied different stages of H. contortus (adult worm and/or L3 stages).
For example, structural and ultrastructural changes were induced in adult
female H. contortus after in vitro and/or in vivo contact with CT-containing
legumes, such as tzalam (Lysiloma latisiliquum), sainfoin (Onobrychis viciifolia),
or quebracho (Schinopsis spp.) extracts; none of these changes were detected
in control worms. The most evident changes detected in treated worms by
SEM were on/in the cuticle and the buccal capsule, and provided striking
evidence of shrivelled larvae/worms and of blocked orifices that probably
disabled the worm from feeding, and decreased fecundity (Hoste et al.,
2012; Martinez-Ortiz de Montellano et al., 2013; Williams et al., 2014b).
Similarly, in goats fed with SL (L. cuneata), all worms had deep cuticular
ridges and a shrunken, dishevelled appearance (Gujja et al., 2013; Kommuru
et al., 2015). Using methods of transmission electron microscopy, the main

lesions and alterations detected were in the hypodermis, and lesions were
also observed in intestinal and muscular cells of ensheathed and exsheathed
infective L3 (Brunet et al., 2011) and adult worms (Martinez-Ortiz de
Montellano et al., 2015). Taken together, the results of these different in
vivo and in vitro studies provided evidence of direct interactions between
various CT-containing resources and L3 or adult H. contortus. It is
hypothesized that the phenotypic changes in worms following exposure
to CT-containing plants relate to reduced motility, nutritional uptake,
and reproduction (Hoste et al., 2012). It is likely that the strong propensity
of tannins towards proline-rich proteins accounts for these observations.
Tannins possess numerous polyphenolic groups that confer ‘stickiness’ as
they facilitate a plethora of hydrogen bonds, and their benzene rings give
rise to hydrophobic interactions. Together, these characteristics lead to
strong interactions in proteinetannin complexes (Dobreva et al., 2011;
Kilmister et al., 2015). Further testing of the ‘direct’ hypothesis will
require a better understanding of the pharmacology of CTs and polyphe-
nols along the gut, in particular on the interactions of tannins and proteins
in the different digestive organs (Rochfort et al., 2008). Work in this direc-
tion has commenced in sheep and cattle (Desrues et al., 2015; Quijada,
2015).
3.6.2 The indirect (¼immune-based) hypothesis

CTs can also have an indirect effect on nematode biology by improving the
host’s immune response due to their ability to bind with proteins of the
digestive content. The successive processes are as follows: (1) formation of
tannineprotein complexes in the rumen, which decrease ruminal protein
degradation; (2) then, an increased flow of rumen-escape proteins into the
abomasum and increased absorption of amino acids and peptides in the small
intestine; and (3) consequently, a better nutrition of the host. Based on the
concepts described in Section 2 of this chapter, this succession of events can
explain the improved host immune response against GINs, including
H. contortus (Hoste et al., 2012). Recently, Tedeschi et al. (2014) reviewed
in detail the currently available information of the impact of CTs for rumi-
nant production.
Moreover, in vitro studies have shown that tannins have immune-
modulating effects. CTs can prime both human and ruminant gd T-cells
in vitro (Holderness et al., 2007; Tibe et al., 2012). Interestingly, tannic
acid, which is a mixture of gallotannins, has been shown to modulate the
300 H. Hoste et al.
immune responses of sheep, which were stimulated by H. contortus antigen,

by inhibiting Th1 cytokines and by increasing Th2 cytokine expression in
vitro in white blood cells (Zhong et al., 2014).
• In vivo study of circulating immune potent cells have confirmed these
results in vitro. An increased number of gd T-cells in sheep fed with
CT-containing Salix was also reported (Ramírez-Restrepo et al.,
2010). According to Singh et al. (2015), the humoral and cell-mediated
immune responses are higher in animals receiving feed containing CTs (a
mixture of Eugenia jambolana/Psidium guajava), and lower H. contortus
FECs were noticed in goats.
• In the case of H. contortus infection, only a few in vivo studies have been
completed by pathological examinations that have tested this ‘indirect’
hypothesis. These experiments compared the numbers of different
effector cells (eosinophils, mast cells, globule leucocytes and goblet cells)
against GINs (Balic et al., 2000) in the mucosae of the digestive tract of
sheep (Martínez-Ortíz-De-Montellano et al., 2010; Rios de Alvarez
et al., 2010) or goats (Paolini et al., 2003a,b) which had been given a
CT resource or control diet. The most convincing evidence of an
enhanced local/mucosal immune response showed that when lambs
grazed on sulla or chicory, they had a reduced development of worms
and significantly higher numbers of mast cells and globule leucocytes
in the abomasal mucosa. However, Te. circumcincta was the main parasitic
species involved (Tzamaloukas et al., 2006). In other studies involving
H. contortus infection, results were less convincing. These include studies
of sheep fed the CT-containing L. latisiliquum browse (Martinez Ortiz de
Montellano et al., 2010) or goats receiving quebracho (Paolini et al.,
2003c) or SL diets (Joshi et al., 2011). Usually, insignificant differences
in the number of effector cells in the digestive mucosae of small rumi-
nants were measured in response to CT-containing diets. From these
studies, the authors concluded that SL and other CT-containing legumes
had little effect on the local host immune responses, and that the
observed differences were mainly due to the direct action of CT on
H. contortus.
3.6.3 Emerging information on structureeactivity relationships, and

possible mechanisms of action
Most forage legumes possess procyanidin (PC)eprodelphinidin (PD) mix-
tures (Fig. 6), which are extremely difficult to separate, and this precludes
structureeactivity relationship (SAR) studies (Stringano et al., 2012).
Furthermore, we have noticed certain trends in the biosynthesis of tannins:

many plants tend to produce smaller PCs and larger PDs, and high-tannin
contents are often associated with higher percentages of PD tannins e
although there are some exceptions. Therefore, these associations can
confound SAR studies. However, a few plants are biosynthetic specialists,
and by isolating their tannins, it is possible to investigate features that
contribute most to AH activity. Several studies have now explored AH dif-
ferences between small versus large tannins, different PC/PD, and cis/trans-
flavanol ratios, between A- and B-type interflavanol linkages and effects of
galloylated tannins (Fig. 6; Mueller-Harvey, 2006; Williams et al., 2015).
Thus far, the conclusions are that higher AH activity against many
different GINs, including H. contortus, is found in CTs that have:
• A high proportion of prodelphinidins rather than procyanidins
(Desrues et al., 2016; Novobiliský et al., 2013; Quijada et al., 2015;
Williams et al., 2014a);
• Larger rather than smaller tannins (Desrues et al., 2016; Novobiliský
et al., 2013; Quijada et al., 2015).
• Stereochemical differences arising from cis-versus trans-flavanol units in
tannins do not appear to be important, although it must be pointed
out that only a few plants have tannins, which are composed largely of
trans-flavanols in, for example, the leaves from black currant (Ribes
nigrum) and a few Salix species. This statement limits the range of tannins
that has been tested so far.
Possible explanations for these observations are that:
• Prodelphinidins have more phenolic groups and are thus able to form
more hydrogen bonds with proteins than procyanidins;
• Larger tannins are better able to cross-link different proteins, as shown by
precipitation studies (Kilmister et al., 2015; Zeller et al., 2015b).
It would be interesting to explore whether some of the above features
accounted for the fact that GIN exposed to tzalam (L. latisiliquum) or sainfoin
(O. viciifolia) revealed quite different features of structural damage in electron
microscopic images (Martínez-Ortíz-de-Montellano et al., 2013).
More research will be needed also on the AH effects of galloylated tan-
nins (ie, CTs substituted with gallic acid and also gallotannins). In vitro
studies (Brunet and Hoste, 2006; Molan and Faraj, 2010; Ramsay et al.,
2016) have clearly shown that the presence of gallic acid esters enhanced
the AH activity of flavanol monomers and CTs (Fig. 6). Tannic acid, which
consists of gallotannins, disturbed the larval mobility as assessed using an in
vitro test (Zhong et al., 2014). However, neither carob pods nor Acacia
302 H. Hoste et al.
nilotica leaves (Table 5) reduced H. contortus FECs, or worm numbers or

fecundity (Arroyo-Lopez et al., 2014; Kahiya et al., 2003). As far as we
are aware, these are the only AH trials conducted thus far using feed contain-
ing highly galloylated tannins e although possibly of low molecular weights.
Therefore, other plants with galloylated CTs, or with gallotannins, such as
Rumex sp., grapeseeds, persimmon, and Camellia sinensis, might be worth
studying (Li et al., 2010; Ropiak et al., 2016; Salminen et al., 1999; Spencer
et al., 2007; Tuominen et al., 2013). As only a small number of studies
(Dahmoune et al., 2014; Mancilla-Leyton et al., 2014; Rodriguez-Perez
et al., 2013; Romani et al., 2002) have reported extent of galloylation in
CTs or quantified gallotannins, it is possible that galloylation effects may
have been overlooked in the past. We emphasize this point, because of
the exciting results obtained by feeding goats and sheep on Pistacia lentiscus
(see Azaizeh et al., 2013; Landau et al., 2010; Manolaraki et al., 2010), which
contains low molecular weight gallate esters (ie, galloylated sugars and tara-
tannins) and CTs (Dahmoune et al., 2014; Mancilla-Leyton et al., 2014;
Rodriguez-Perez et al., 2013; Romani et al., 2002).
According to Fakae et al. (2000), substituted phenol-based AH inhibit
helminth glutathione S-transferases, which appear to be important for estab-
lishing chronic infections. Therefore, in addition to tannins, there are several
other polyphenolic compounds that have demonstrated AH activity against
H. contortus (see Kotze et al., 2009). These compounds include flavones
(luteolin, zapotin, 5, 6, 20 -trimethoxyflavone, 5,6,20 ,50 ,60 -pentamethoxy-
30 ,40 -methylenedioxyflavone), flavonols (quercetin; Barrau et al., 2005),
and a flavanone (naringenin; Klongsiriwet et al., 2015). Given that all green
plants contain a wide range of monomeric flavonoids, it is of interest that
synergistic effects were observed between CTs and luteolin or quercetin
on L3s (Klongsiriwet et al., 2015). In contrast, some negative interactions
between tannins and other PSMs have been evoked to explain inconsistent
results obtained with some extracts on H. contortus eggs in a hatch assay
(Vargas-Maga~ na et al., 2014a).
Kumarasingha et al. (2014) reported that combinations of plant extracts
against C. elegans, a free-living nematode, used as a model for H. contortus and
other GINs because of accumulated and extensive knowledge of its biology
and genome, elicited stress responses that differed compared with those
induced by synthetic AH drugs. This interesting result will need evaluation
on H. contortus.
Table 5 is a first attempt to link information from disparate studies on
CT-containing plants with in vitro and in vivo activities against H. contortus.
Table 5 Tannin composition in plants or extracts of forage legumes, browse species, fruit bushes, and agroindustrial by-products
CT (g/100 g Effects on
Plants PC/PD mDP DM ¼ %) Tannin method Haemonchus (*) References

Forage Legumes
a
Desmodium intortum HCleButOH LMIA; feeding Debela et al. (2012),
reduced worm Mbugua et al. (2008)
burdens, female/
male ratio, eggs in
uterus of female
worms
Hedysarum coronarium 11/89 to 27/ 3 to 46 3e13% DM Thiolysis; HCl LMIA, EHA Molan et al. (2004a,b),
73 eButOH Tibe et al. (2011)
Valderrabano et al.
(2010)
Lespedeza cuneata 3/97 33 13% DM Thiolysis see SL section Mechineni et al. (2014)
(grazed)
Lespedeza cuneata 3/97 86 13 Thiolysis see SL section Kommuru et al. (2014)
(pellets)
Lotus corniculatus 34/66 to 60/ 9 to 14 3e5% DM EHA Acharya et al. (2014),
40 Foo et al. (1982,
67/33 to 79/ 1996), Hedqvist et al.
21 (2000), Sivakumaran
73/27 to 50/ et al. (2006)
50
Onobrychis viciifolia 34/66 to 5/95 11 to 84 0.6 to 14 Thiolysis; HCl LEIA; FEC; worm Arroyo-Lopez et al.
eButOH numbers; female (2014), Azuhnwi et al.
fecundity (2013a,b), Quijada
et al. (2015), Stringano
et al. (2012),
303
Theodoridou et al.
(2011)
(Continued)
Table 5 Tannin composition in plants or extracts of forage legumes, browse species, fruit bushes, and agroindustrial by-productsdcont'd
304
Browse
32 different browse Several species had LDA, AMIA Kotze et al. (2009)
species tannins (PVPP)
40 plants (herbs, CT, GT, ET EHA, LMIA Acharya et al. (2014)
shrubs, trees)
Acacia nilotica (fruit) Epigallocatechin AMIA, EHA, LDA, Bachaya et al. (2009),
gallates development, FEC Ncube and Mpofu
(1994)
A. nilotica (AN) Catechin gallates AN less effective than Kahiya et al. (2003), Self
(leaves) AK: FEC, worm et al. (1986)
number
a
A. karroo (AK) (leaves) HCleButOH AK more effective Dube and Ndlovu
than AN: FEC, (1995), Dube et al.
worm number (2001), Kahiya et al.
(2003)
Arachis pintoi, Gliricidia Tannins (PEG) LMIA, LEIA von Son-de Fernex et al.
sepium, Cratylia (2012)
argentea (leaves)
Acacia pennatula, VanillineHCl, LMIA Calderon-Quintal et al.
Lysiloma tannins (PEG) (2010)
latisiliquum, Piscidia
H. Hoste et al.
piscipula
Betula spp. 59/41 4 5% LEIA Quijada et al. (2015),
Ropiak et al. (2016)
a
Ceratonia siliqua pods CT dimers and no significant effects Arroyo-Lopez et al.
trimers, HT, on FEC, worm (2014),

galloylated sugars; numbers, female Papagioannopoulos
flavan-3-ol gallates fecundity et al. (2004)
a
Ficus infectoria/Psidium HCleButOH Worm numbers, FEC Pathak et al. (2013)
guajava (leaf meal
mixture)
Juglans regia 100/0 5 6% Thiolysis LEIA Quijada et al. (2015),
Ropiak et al. (2016)
Leucaena leucocephala 3% HCleButOH Contradictory reports: Nguyen et al. (2005),
1. in vitro L3 toxicity; Osborne and McNeill
2. no effect on LMIA 2001;
1. Lopez et al. (2005),
2. Calderon-Quintal
et al. (2010)
a
Calluna vulgaris, Erica HCleButOH; EHA, LEIA Betts et al. (1967),
cinerea, E. umbellata thiolysis Moreno-Gonzalo
et al. (2012)
Piliostigma thonningii HCleButOH; L3 lethality; inhibition Fakae et al. (2000)
(bark) vanillineHCl of glutathione S-
transferases
Pistacia lentiscus 1. 20% tannins in LEIA; very low FEC 1. Azaizeh et al. (2013),
DM (PEG); Landau et al. (2010),
2. 5% of gallotannins Manolaraki et al.
in DM (galloyl (2010);
derivatives of 2. Rodriguez-Perez
glucose, quinic et al. (2013), Romani
acid and et al. (2002)
myricetin)
305
(Continued)
Table 5 Tannin composition in plants or extracts of forage legumes, browse species, fruit bushes, and agroindustrial by-productsdcont'd
306
3. 3e4% CT (HCl 3. Dahmoune et al.
eButOH) (2014), Mancilla-
Leyton et al. (2014)
Psidium guajava/ HCleButOH FEC Singh et al. (2015)
Eugenia jambolana
Salix spp. 5/95 to 96/4 2 to 18 0.2 to 13 Thiolysis; HCl LEIA, worm numbers, Diaz Lira et al. (2008),
eButOH FEC Falchero et al. (2011),
Klongsiriwet et al.
(2015), Mupeyo et al.
(2011), Orians et al.
(2000), Quijada
(2015), Quijada et al.
(2015), Ramírez-
Restrepo et al. (2010)
a
Sesbania sesban HCleButOH LMIA Debela et al. (2012),
Heering et al. (1996)
a
Veronia amygdalina worm mortality Sirama et al. (2015)
Fruit bushes
Ribes nigrum 5/95 to 9/91 3 to 12 10 to 20 Thiolysis LEIA Klongsiriwet et al.
(2015)
H. Hoste et al.
Ribes rubrum 5/95 to 14/86 5 to 19 e Thiolysis LEIA Klongsiriwet et al.
(2015)
Agroindustrial byproducts
Corylus avellana 80/21 to 82/ 5 to 9 e Thiolysis; CT gallates LEIA Del Rio et al. (2011),
18 Quijada et al. (2015)
Pinus spp. 89/11 to 85/ 2 to 11 10% Thiolysis; HCl LEIA, FEC, worm Min et al. (2015a,b),
15 eButOH numbers Quijada et al. (2015)
Other potential Information on H.
useful plants contortus is
missing
L. pedunculatus 16/84 to 20/ 12 to 44 7% DM Thiolysis, Ostertagia (Te.) Foo et al. (1997), Niezen
80 phloroglucinolysis circumcincta and et al. (1998),
T. colubriformis Sivakumaran et al.
(2006)
Dorycnium spp. 5/95 to 17/83 10 to 15e20% Thiolysis EHA; LDA e Te. Molan and Faraj 2010,
127 DM circumcincta Sivakumaran et al.
(2004)
Herbs
Rumex obtusifolius 100/0 2 to 8 e Thiolysis; 10e29% EHA; LDA e Te. Molan and Faraj 2010,
galloylation; A-type circumcincta Spencer et al. (2007)
CT
*, tests showed effects on Haemonchus contortus (unless otherwise indicated) see Table 2 for the in vitro assays; *, the meaning for in vitro assays are provided in Table 1; PC/PD,
ratio of procyanidins and prodelphinidins; mDP, mean Degree of Polymerisation; CT, condensed tannins.
a
Indicates that presence of condensed tannins has been confirmed.
307
308 H. Hoste et al.
This table provides key information on analytical methods [eg, CTs by thi-
olysis or HCl-butanol assays; hydrolysable tannins by high-performance
liquid chromatography (HPLC) analysis of gallic and ellagic acids; general
tannin methods by PEG or PVPP addition] and on the various parameters
that were assessed on H. contortus. Table 5 includes forage legumes, browse
plants, and agroindustrial by-products, although many other browse plants
and herbal drugs also represent interesting sources of CTs (Ropiak et al.,
2016).
Table 6 shows the tannin composition of selected tree leaves and browse
plants; the procyanidin/prodelphinidin ratios range from 100/0 to 7/93;
tannin sizes, as measured by the mean degree of polymerization from 4 to
34, and CTs content of 3e17% of dry matter. However, Table 5 does
not contain information on the AH activity against H. contortus for three
widely studied plants (L. pedunculatus, Dorycnium rectum, and Rumex obtusifo-
lius) with demonstrated efficacy against other GINs. Such AH effects are also
expected against H. contortus, but still need to be tested.
Structureeactivity studies have shown that larger tannins and also high
proportions of prodelphinidins are linked to high AH efficacy. The presence
of galloylated flavanols and tannins enhances in vitro activity, which may
account for the excellent results observed using P. lentiscus. However,
further research will need to ascertain optimum tannin contents, polymer
Table 6 Browse tannins analysed by thiolysis with benzyl mercaptan. Most results
are still unpublished data except the species indicated by * (see Ropiak et al., 2016)
Plants Plant parts PC/PD mDP CT (g/100 g DM)
Amelanchier canadensis Leaves 100/0 5 4

Arbutus unedo Leaves 60/40 4 5
Corylus avellana Leaves 26/74 10 5
Euonymus alatus Leaves 73/27 8 6
Parrotia persica Leaves 12/88 27 6
Picea abies Leaves 85/15 5 4
Pimenta officinalis Fruits 37/63 14 3
Pinus spp.* Buds 62/38 8 5
Platanus x hybrid Leaves 32/68 13 10
Robinia pseudoacacia Leaves 7/93 34 14
Salix spp.* Bark 100/0 5 15
Taxus baccata Needles 51/49 4 6
Vaccinium vitis-idaea* Leaves 100/0 7 17
Vaccinium myrtillus* Leaves 100/0 7 12
PC/PD, ratio of procyanidins and prodelphinidins; mDP, mean degree of polymerisation; CT,
condensed tannins.
sizes and percentages of prodelphinidin and galloylated tannins, in order to

(1) minimize any antinutritional effect from such feeds, (2) assess what dose is
required, and (3) explain the results from unsuccessful feeding trials with
plant materials that were expected to yield good AH effects (eg, carob
pods and A. nilotica leaves).
3.7 Toward the on-farm use of condensed tannin-containing

nutraceuticals as anthelmintic feeds
The first empirical results obtained in New Zealand reported significant
reduction of FECs in sheep when grazing pastures with a range of different
tannin-containing legumes, that is, birdsfoot trefoil and big trefoil
(L. corniculatus, L. pedunculatus), sulla (Hedysarum coronarium), and sainfoin
(O. viciifolia) (Niezen et al., 1995, 1998, 2002). These findings provided
some directions for future research. However, H. contortus was not assessed
in these studies. These early results have given a strong impetus to seek AH
properties in a wide diversity of plants, characterized by the common pres-
ence of significant concentrations of CTs and polyphenols. The wide distri-
bution of tannins and polyphenols in the Plant Kingdom makes this a
worthy endeavour. The main botanical resources investigated to date
include:
1. Legume forages (Fabaceae) suited to large-scale agronomy;
2. tropical browse resources, which have enabled the exploration of a diver-
sity of forages and leguminous trees, and the complexity of interactions
between PSMs, small ruminants and the local environment;
3. agroindustrial by-products.
3.7.1 Temperate legume forages: sericea lespedeza and sainfoin

In Europe and USA, this recent surge of interest has focussed on two legume
species of the subfamily Faboideae, as potential nutraceutical forages. These
two plants are the Chinese bush clover also known as sericea lespedeza (ie,
SL (L. cuneata)) in the USA and South Africa (Joshi et al., 2011; Kommuru
et al., 2014; Shaik et al., 2006; Terrill et al., 2009, 2012; http://www.acsrpc.
org/) and sainfoin (O. viciifoliae) in Europe (Azuhnwi et al., 2013a,b; Heck-
endorn et al., 2006, 2007; Manolaraki et al., 2010; Paolini et al., 2003c,
2005b; http://sainfoin.eu; www.legumeplus.eu).
The particular focus on these two legumes is explained by: (1) their abil-
ity to produce seeds, to be grown (cultivated) efficiently, and to organize
their production for farm application on a large scale (see the following sec-
tions), and also (2) the fact that many studies with these two plants have
310 H. Hoste et al.
targeted H. contortus infections in both sheep and goats. Some other tannin
containing legumes have been mentioned (eg, birdsfoot and big trefoils
(L. corniculatus, L. pedunculatus), sulla (H. coronarium) (see Tables 2 and 3)
as potential nutraceuticals against GINs. However, in most cases, Teladorsagia
and Trichostrongylus spp. were the dominant nematodes (Marley et al., 2006;
Niezen et al., 1995, 1998). There has been limited in vivo study of the effect
of L. corniculatus or sulla on H. contortus (Heckendorn et al., 2007).
Sericea lespedeza [SL; L. cuneata (Dum-Cours.) G.Don.] is a tannin-
containing warm-season perennial legume that is well adapted to the eastern
and southern USA and other parts of the world, including Asia, Australia and
southern Africa (Mosjidis and Terrill, 2013). It has been an important forage
and soil conservation crop in the USA for >100 years, because of a number
of useful agronomic properties, including drought resistance due to a deep
root system, tolerance of acidic soils high in free Alþ3 levels, low fertilization
requirements (ability to produce its own nitrogen and mine phosphorus
from the soil profile) and limited susceptibility to insects and diseases
(Hoveland et al., 1990). In contrast, it is also worth noting that some
nonnative species exploited for these properties are considered by environ-
mentalists and ecologists to be invasive.
Sainfoin (O. viciifolia) belongs to the family Fabaceae, the tribe Hedysar-
eae. The genus Onobrychis represents >170 species. Two main botanical
forms are identified as O. viciifolia: (1) the single cut (common type), which
is slow establishing and is providing one flowering and one cut per year and
(2) the double cut (giant type), which is faster growing and able to provide
two to three cuts yearly. Sainfoin is cultivated in temperate climatic zones
and used as a perennial legume forage. It is well adapted to basic (chalk)
soil and tolerates drought, cold, and low nutrients in soil. In Europe, sainfoin
was well known until the 1950’s.
Thereafter, its decline was related to the lower yield and persistence
compared with lucerne (Medicago sativa) and different clovers (Trifolium),
which have benefited from genetic selection (Azuhnwi, 2012). Because of
different advantages to ruminant nutrition and health, and for environmental
issues (see reviews by Mueller-Harvey, 2006; Rochfort et al., 2008;
Waghorn, 2008; Wang et al., 2015), sainfoin has been ‘rediscovered’ in the
last 20 years (http://sainfoin.eu; http://www.legumeplus.eu). Its benefits
are (1) a reduced requirement for chemical fertilizers because of biological ni-
trogen fixation; (2) high palatability for ruminants and feeding values; (3) pos-
itive effects by reducing ruminal methane emission and green house gases; (4)
antibloat effects and (5) a switch of nitrogen excretion from urine to faeces.
Results obtained on SL and sainfoin have provided a bulk of information

to address some key issues regarding AH properties related to on-farm
applications. The data obtained for these two models of legume forage
nutraceuticals, which relate to applied objectives, have provided useful
information on other types of resources (tropical browse or by-products).
Exploiting tannin-containing legumes with AH properties
(A) Direct grazing: The most obvious way of exploiting tannin-containing
legumes with AH properties is to allow small ruminants to directly graze or
browse on pastures. This approach corresponds to the early studies of various
GINs (except H. contortus) in New Zealand (Niezen et al., 1995, 1998). In
the rest of this section, we review studies conducted under grazing condi-
tions, where H. contortus was the most prevalent GIN species. These inves-
tigations concerned mainly SL (L. cuneata), and studies of direct grazing on
sainfoin (O. viciifolia) have been far less frequent (Valderrabano et al., 2010).
Studies comparing the AH effects when small ruminants graze on SL as a
CT-containing legume forage versus control (nontannin-containing) for-
ages have been performed either in infected kids and does (Luginbuhl
et al., 2013; Min et al., 2004, 2005) and/or in lambs and ewes (Burke
et al., 2012a,b). The different nontanniniferous plants used as control belong
to different plant species [ie, rye/crab grass (Secale cereale/Digitaria
sanguinalis) (Min et al., 2004); Bermuda grass (Cynodon dactylon) (BG)
(Mechineni et al., 2014) or tall fescue (Festuca arundinacea) (Burke et al.,
2012a,b; Min et al., 2005)], adapted to different regions of the USA.
The first investigation of the antiparasitic properties of SL came from a
crossover design study for two 15-day periods in goats grazing on SL or a
control forage (Min et al., 2004). This early study reported a reduction in
FECs by more than 70% (2500 versus 710 eggs per gram) in the SL versus
control pastures. Moreover, the SL-fed goats had a lower percentage of
GIN eggs developing into L3s in faecal cultures compared with goats grazing
on grass pastures (58% versus 99%, respectively). These first results confirmed
the diversity of impact of a tannin-containing legume on key developmental
stages of GINs (see Section 3.3).
Further studies explored different modes of grazing of SL either (1) by
including rotation between SL and a control forage in a series of experiments,
with does and/or kids (Luginbuhl et al., 2013; Min et al., 2005), or sheep
and lambs (Burke et al., 2012a,b) or (2) by comparing pure SL pasture versus
a control versus a mixed pasture, combining pure SL with a control plant,
again in kids and lambs (Burke et al., 2012a,b; Luginbuhl et al., 2013).
Results of these studies helped to confirm the effects of a CT-containing
312 H. Hoste et al.
nutraceutical on the nematode biology, the dynamics of infection, and the

host responses (summarized in Section 3.3):
• Significant reductions (>70%) in FEC or total faecal output were
shown. For example, the mean FEC for does grazing on pure SL and on
rotation were significantly lower than for the control animals (145, 329
and 894 eggs/g, respectively), while FECs for the SL kids were lower
than for the rotation and the control groups (550, 2757 and 3600 eggs/g,
respectively) (Min et al., 2005). Similar data and FEC reductions were
recorded in a second study, with reduction values reaching almost 90% in
kids grazing on SL compared with controls (Luginbuhl et al., 2013).
Mechineni et al. (2014) also reported reduced FECs in kids grazing SL or
SL mixed with control plant pastures compared with Bermuda control-grass
only (95.4% and 71.5% reduction, respectively) by the end of an eight-week
trial. Such differences in FECs have been associated with significant
decreases in H. contortus numbers determined following slaughter
(Luginbuhl et al., 2013). However, in many studies, the worm counts did
not differ significantly from controls. Data on the fecundity of female H. con-
tortus were usually not available. Moreover, a lower larval development from
faecal cultures was rarely reported in SL versus control animals (Min et al.,
2005).
• By using tracer animals, some of these studies also provided information
on the possible future infectivity of pastures; hence, an overall effect of the
consumption of SL was seen. When grazing on pure SL and in rotation
pastures, tracer kids had 78.3% and 61.0% reduced total worm burdens,
respectively, compared with those grazing on control pastures, while the
numbers of adult H. contortus, Te. circumcincta, and T. colubriformis were
reduced by 89%, 100%, and 50%, respectively, in SL-grazed and 78%,
40%, and 50%, respectively, in animals on rotation compared with control
tracer kids (Min et al., 2005).
• When estimating the effect of the different systems on the host resil-
ience (see Section 3.1.4), an improved anaemia status was usually reflected
in higher PCV values in animals consuming SL, in the rotation or in the
mixed (SLþ a control plant) groups, by comparison with control groups
(Burke et al., 2012a; Luginbuhl et al., 2013; Min et al., 2005). However,
the effects on live-weight gains were usually not significant in a rotation sys-
tem (Luginbuhl et al., 2013; Mechinenni et al., 2014) or when combining
SL and forages without tannins (Burke et al., 2012a,b). Finally, one study
compared lambs grazing only on SL, SL mixed with tall fescue or Bermuda
grass (control). The resilience was evaluated by a reduction in the mean
number of required dewormings per lamb during the grazing season. The
numbers were 0.71, 0.20 and 0.21 in the Bermuda grass, the mixed pastures
and the pure SL groups, respectively (Burke et al., 2012b).
Overall, the conclusions from these grazing studies were that SL, as a
model of a tannin-containing legume nutraceutical, represents a viable plant
for reducing GIN infections in small ruminants, leading to decreased
contamination of pasture with infective larvae. SL has natural AH properties,
but the effects appear to be primarily due to reduced fecundity of GINs
rather than the killing of adult worms. Therefore, producers should exercise
caution when taking sheep and goats off SL pastures.
These results also showed that, despite dilution of tannin-containing
legume over time (by rotation) or space (pasture composed with a mixture
of SL with a plant not containing tannins) (Burke et al., 2012a,b; Luginbuhl
et al., 2013), some antiparasitic effects were maintained, although at a lower
level than pure SL grazing (Burke et al., 2012a,b). In grazing studies of goats
and sheep, the most consistent antiparasitic effects were observed in animals
on pure SL pastures. When goats were switched between SL and non-SL
pastures FECs rapidly decreased while on SL pastures, and then quickly
increased on non-SL pastures, indicating an effect of short-term SL grazing
on worm fecundity, rather than the removal of adult GINs. Longer-term
exposure to CT from grazed SL reduces the numbers of adult H. contortus,
Te. circumcincta, and, to a lesser extent, T. colubriformis (see Min et al.,
2005). Grazing on mixed SL grass pasture has demonstrated positive antipar-
asitic effects in both goats (Mechineni et al., 2014) and sheep (Burke et al.,
2012b), but the effects are often delayed compared with animals on pure SL
pastures. This is particularly true for sheep, as they are more reluctant than
goats to graze on SL in mixed pastures, but they will graze on SL once
they adjust to this plant.
(B) Conserved forms (hay, silage and pellets): For on-farm applications, the
preservation and subsequent processing of tannin-containing legume
forages, such as SL and sainfoin, have several advantages. It gives farmers
greater flexibility to use bioactive forages, and facilitates storage and transport
to areas where these legumes do not grow well or at all. However, processes
such as sun drying, grinding and pelleting can affect tannins, because all pro-
cesses involving heat can affect CT contents, or alter a plant CTs from
extractable to bound forms.
In a study with sun-dried and fresh-frozen high- and low-tannin SL
forage fed to sheep, Terrill et al. (1989) reported that sun-drying decreased
the extractable CT content in SL, and improved intake and digestibility of
314 H. Hoste et al.
the high-CT forage. Subsequent work (Terrill et al., 1992) showed that CT
in dried forages were not reduced, but instead shifted from extractable to
bound forms. The effect of processing SL and sainfoin on the bioactivity
of CT and related polyphenols is still the subject of ongoing research. In
this regard, the different conserved forms of tannin-containing resources
provide a means: (1) to examine the effects of bioactive plants containing
PSMs (CT-containing plants being the model here) under in vivo confined
conditions against GINs (see Section 3.3) and on the host resilience and/or
resistance (Section 3.4) and (2) to better understand the mode of actions of
PSMs on worms (Section 3.6).
• Sainfoin and SL hay
Hay processing appears to have little impact on the antiparasitic proper-
ties of sun-dried sainfoin or SL compared with fresh material. The first results
on the potential use of sainfoin hay were obtained using goats with estab-
lished (natural) H. contortus infection (Paolini et al., 2003c). The authors
reported reductions in FECs by 75% within 3 days following the feeding
of sainfoin hay. These reductions persisted for up to 2 weeks after the goats
were returned to a (negative) control diet. This persistence of effect
suggested a nematocidal effect on worm numbers.
Using SL hay, Shaik et al. (2004) compared isoproteic and isoenergetic
diets of ground SL or Bermuda grass hay (80% of the ration) plus 20% con-
centrates with yearling goats experimentally infected with H. contortus. Egg
counts were 92% and 86% lower than controls in does given the SL ration
on days 21 and 28 of the SL-feeding period, respectively. At the end of the
experimental ration period, after all does were put back on to the control
ration, the FECs did not significantly differ between groups, suggesting
that the main effect related to the fecundity of female H. contortus. These re-
sults were completed with a follow-up study when naturally infected kids
received trickle infection of H. contortus L3s, and were then fed unground
SL or Bermuda grass hay (75% of the ration) plus concentrates for 7 weeks
in pens. FECs in the SL-fed bucks decreased by 80% relative to the control
group, seven days after SL feeding was initiated, and these differences
increased to 88% by the last 2 weeks of the trial. In addition, the SL-fed goats
had reduced abomasal and intestinal GIN numbers. Total reductions in adult
female worms were 77%, 36%, and 50% for H. contortus, Te. circumcincta, and
T. colubriformis, respectively, in the SL group compared with the controls.
The SL-fed group had also a reduced development of infective L3s from
GIN eggs from feces and a higher (average) PCV than the control group
(Shaik et al., 2006).
These results were confirmed in lambs for both legume species (SL and
sainfoin). The effects of feeding unground SL or Bermuda grass hay were
compared for experimental lambs trickle-infected with H. contortus (Lange
et al., 2006). Compared with the control group, FECs of the SL-fed lambs
decreased by 98% after seven days and remained significantly lower
throughout the rest of the trial, with reductions varying from 77% to 86%
during the SL-feeding period, and dropping to 37% and 53% after SL was
discontinued. Worm counts (limited to three lambs per group) indicated a
67% reduction of H. contortus number in the SL group compared with the
control group.
With sainfoin, in lambs experimentally infected with H. contortus and
Cooperia curticei (see Heckendorn et al., 2006), significant reductions in
FECs of 58% and 48%, respectively, for each of the nematode species were
reported. These decreases were associated with a reduction of worm number
for H. contortus. No significant differences were observed for C. curticei.
Hay of tannin-containing legumes has also proved to be efficient at
reducing L3 establishment in the host animal. When sainfoin hay was given
to goats around experimental infection with Haemonchus L3s, a reduction
(not statistically significant) of 38% in worm establishment was reported
compared with a control group (Paolini et al., 2005a). In lambs consuming
SL hay around the infection period, a 26% reduction was seen (Lange et al.,
2006).
• Sainfoin silage
The possibility of exploiting ensiling processes for SL has received little
attention. In contrast, several studies have examined the palatability, the
nutritive and feeding value, and the in vitro digestion of sainfoin silage
in ruminants (Copani et al., 2014; L€ uscher et al., 2014; Theodoridou
et al., 2010; Wang et al., 2015). In regard of the AH effects, some in vitro
studies have compared extracts from fresh, hay, and silage by applying the
in vitro larval exsheathment inhibition assay (LEIA) (cf. Table 1) to
H. contortus. The results revealed a higher in vitro AH activity in hay and
silage samples compared with fresh sainfoin. For silage, these differences
appear to be explained partly by the presence of flavonoid aglycones,
with higher antiparasitic properties than the corresponding glycoside flavo-
nols (Ojeda-Robertos et al., 2010). In experimentally infected lambs,
Heckendorn et al. (2006) also compared the AH effect of sainfoin hay
with silage. They showed that the reduction in FECs and worm counts
were comparable (w50% in both cases) with both conserved forms of
sainfoin.
316 H. Hoste et al.
• Dehydrated pellets of SL or sainfoin

The challenges of feeding hay and/or silage to livestock include the
difficulty of transport and storage, and possible wastage of valuable feed.
Pelleting can help in reducing wastage, improving ease of feed transport
and storing and delivering stable products. However, some high heating,
combined with high pressure, is usually required to produce the dehydrated
pellets. Hence, these harsh treatments during the pelleting processes might
destroy some tannins and related polyphenols and/or increase the amount
of bound CT in plant material (Terrill et al., 1992). These impacts can
seriously affect the bioactivity of the PSMs, in particular the AH activity
against H. contortus.
To determine the effect of pelleting processes on the antiparasitic efficacy
of ground SL whole-plant hay, a study was undertaken with naturally
infected bucks (Terrill et al., 2007). After grazing on a grass pasture contam-
inated with GINs, the bucks were moved into pens and fed a basal control
diet for a three-week adjustment period. Then, they were assigned for
28 days, to different dietary experimental treatments consisting of 75%
hay and 25% commercial diet pellets. The hay treatments were control
ground whole-plant SL, and pelleted whole-plant SL; then all the goats
were switched back to the control ration for seven days. Both SL hay and
pellets significantly reduced FECs compared with control animals by day
seven of the trial, and maintained these differences to the end of the study,
even after switching back to the control ration for 7 days. The reduction in
FECs relative to controls averaged 70% and 54% for goats given the SL pel-
lets and ground SL hay, respectively. A significant effect on the development
of egg to L3 was also less for the goats fed on SL pellets than the animals fed
on Bermuda grass hay, the percentage of H. contortus L3 recovered being
25.0% and 83.4%, respectively. Moreover, the goats fed on SL pellets had
significantly higher PCV values and lower numbers of adult H. contortus
than control goats. By contrast, for the treatment group fed on ground
SL, the PCV values and worm numbers did not differ significantly from
controls. These authors concluded that pelleting SL hay enhanced its AH
efficacy against GINs (Terrill et al., 2007).
Similar results began to be obtained from studies with sainfoin pellets. In
vitro comparisons between hay (dried for two days) and pellets produced
from the same primary material suggested that the pelleting process
maintains AH activity. Moreover, a series of in vivo studies using sainfoin
pellets in ration have confirmed the AH effects against either susceptible
or AH-resistant strain of H. contortus (see Gaudin et al., 2015; Girard
et al., 2013). The results have shown overall reductions in FECs, from 30%
to >50%, depending on the batch of pellets and the tannin content. These
studies with conserved forms of either SL or sainfoin have also helped to
compare and explain different factors associated with variation in results
(see Section 3.5), thus providing useful information for future use under
farm conditions.
• Components and treatment of legume forages
As there are higher levels of CT in SL leaves than stems (12.5% versus
3.3%, respectively; Mechineni et al., 2014), several studies with SL have
focussed on an evaluation of leaves, either as ground leaf meal or as leaf
meal pellets in the diet of sheep and goats, either fed in confinement, or
as a supplement to goats grazing on pasture (Terrill et al., 2012). To deter-
mine whether there was any difference in efficacy between sun-dried,
whole-plant SL and leaves alone, a trial was conducted with kids trickle-
infected with H. contortus L3s (Terrill et al., 2008). After an adjustment
period, with all animals given a control diet, kids were fed either ground
SL whole plant or leaf meal (25% of the ration) and received a supplement
formulated to make the diets isoenergetic/isoproteic. Both diets reduced
FECs to pretrial levels, but FECs of kids on the SL leaf meal diet decreased
more rapidly and were significantly lower than those of goats fed on the
whole-plant SL meal. It was concluded that feeding SL leaves only would
increase the efficacy of this tanniniferous legume against GINs.
• Tannin concentration and dosage
The above studies raise an important question: what dietary tannin con-
centration is needed to affect H. contortus biology and hence improve the
control of infection?
Most results acquired in the different in vitro assays (Table 1), when using
H. contortus as a model (Table 2), have suggested a dose-dependent response
in antiparasitic effects for a whole range of various tannin-containing plants.
The hypotheses that the AH effects are dose-dependent as well as that a
threshold of CT and related polyphenols in the diet is needed to achieve
AH effects have been supported by few in vivo studies, targeting either
the infective L3s or the adult worms of H. contortus.
For instance, a study with sainfoin hay (Brunet et al., 2007) using
cannulated sheep examined the effects of different percentages (0e100%)
of sainfoin in the ration on the exsheathment of Haemonchus L3s. The results
showed that levels of 75% and 100% were needed to achieve significant
reductions in L3 exsheathment when compared with controls (0%). With
SL hay, a confinement feeding study was performed with naturally infected
318 H. Hoste et al.
goats (Terrill et al., 2009). The aim was to examine the effects of different per-
centages of a combination of ground SL (0%, 25%, 50%, and 75%) and
conversely ground BG (control) in hay diet on H. contortus infection. Results
on worm biological traits suggest a proportional relationship between the per-
centage of SL in the hay part of the diet and the effects on the adult H. con-
tortus populations (evaluated by FECs) and egg development into L3. In
addition, FEC measurements suggested that a threshold level of SL hay in
the ration has to be achieved, because repeated significant reductions relative
to control only occurred at levels of 50% and 75% SL in the diet. There was
no effect on blood PCV or adult worm numbers in the 25% and 50% SL hay
groups, but the goats given the 75% SL hay diet tended to have lower PCVs
and had 75% fewer H. contortus worms relative to control animals.
To provide an answer as to how much tannin is needed for an AH effect
is far more complex because:
(1) AH effects depend both on the quantity and the quality of tannins (See
Section 3.5). Although L. cuneata, many browse species and pine bark
extracts have high tannin concentrations, positive effects have also
been recorded with much lower concentrations with other resources
(Tables 5 and 6). Currently, there is insufficient information to specify
what concentrations of which tannin types will deliver AH activity
against H. contortus, or for that matter any other GIN, due to the fact
that feeds are rarely analysed for their quantitative and qualitative tannin
compositions.
(2) Besides tannins, the possible AH effects of some flavonoids (flavanols and
flavonols) have been supported by some in vitro studies (Brunet and
Hoste, 2006; Klongsiriwet et al., 2015; Molan et al., 2003a, 2004b)
(Table 5). In addition, a few studies suggest that some interactions, either
synergistic or antagonistic, can also occur either between polyphenols
(Klongsiriwet et al., 2015) or between different PSMs (Burrit and
Provenza, 2000; Lyman et al., 2008; Vargas-Maga~ na et al., 2014a).
(3) The question of ‘How much?’ also depends on the method used to
measure/quantify the tannins (see Section 3.1.2a). In addition, other
active metabolites in the feed contribute in the AH activity. However,
they are usually not directly measured.
• A time frame for the AH effects in infected ruminants consuming tanninif-
erous nutraceuticals
Studies of goats have shown that the reductions in EPG occurred within
seven days of initiation of grazing SL (Lughinbul et al., 2013; Min et al.,
2005). Longer periods (14e35 days) were mentioned in two lamb trials
(Burke et al., 2012a,b) when grazing mixed pastures with SL and tall fescue.
However, the animals were reluctant to graze the SL in the mixed SL-Tall
fescue pastures. Overall, these data confirm the hypothesis that for any nu-
traceutical plant to be exploited against GINs of ruminants, several days of
consumption are required to reach sufficient concentrations of PSMs in
the different organs of the digestive tract and before any antiparasitic effects
can be detected.
Some indications on the persistence of the effects have also been
obtained in studies of lambs (Burke et al., 2012b), since benefits of SL
grazing on FEC disappeared after 14 days once lambs were moved back
to BG pastures following SL grazing. These data on the period of efficiency
of nutraceuticals need to be compared with results of studies presented in the
following sections using conserved forms of tannin-containing legumes.
A relationship between the duration of consumption and the concentra-
tion of CTs is suggested by the results and conclusions of one study of kids. It
was shown that the FEC reductions were more rapid and significant in kids
grazing on SL only, while FEC reductions in the SL-BG kids were not sig-
nificant compared with control kids (Lugingbuhl et al., 2013).
• When and how?
As previously mentioned, conserved forms of tannin-containing legume
nutraceuticals offer flexibility in use to regulate natural GIN infections in
small ruminants. Studies of hay, silage, or pellets of sainfoin and/or of SL
have illustrated different options.
-Prolonged distribution: A study was completed to determine the AH
potential of pelleted SL leaf meal given as a constant supplement to kids
when grazing grass pasture (predominantly Bermuda grass and
bahiagrass ¼ Paspalum notatum). The kids were assigned to three similar
pastures, and offered either 75% SL leaf meal pellets, 95% SL leaf meal
pellets, or a commercial pellet product at 0.91 kg/head/day using an
automatic feeder for the 77-day study. FECs were similar in all treatment
groups until day 28, after which both SL treatment groups had significantly
lower FECs than the control group through to the end of the trial (days 35e
77). Total numbers of adult H. contortus and Te. circumcincta were lower (94%
and 47%, respectively) in goats given the 95% SL leaf meal pellets as a sup-
plement compared with the commercial pellet product. The authors
concluded that feeding 95% SL and 75% SL leaf meal pellets as a supplement
to goats grazing on grass pasture both reduced effects of GIN infections, with
a greater effect linked to the supplementation with 95% SL pellets.
-Repeated distribution for short-term period: A second option is to offer a
tannin-containing nutraceutical for a few days at regular intervals. A study
320 H. Hoste et al.
was performed with sainfoin hay in naturally infected goats to examine the
effects of such scheme on worm populations and host resilience. H. contortus,
Te. circumcincta, and T. colubriformis were the main GINs. Goats in the exper-
imental group received sainfoin hay indoors for seven days at a monthly
interval. The control goats received ryegrass hay. The two diets were isoe-
nergetic/isoproteic. The distribution of sainfoin was associated with: (1) sig-
nificant reductions of EPG, related to a decrease in worm fertility for the
three parasite species but no significant changes in worm numbers; (2) a bet-
ter host resilience assessed by higher PCV values and the need of salvation
AH treatments for 50% of the animals in the control but not for any goats
in the sainfoin group (Paolini et al., 2005b).
• Targeting PPRI in infected small ruminants
The AH properties of sainfoin silage were exploited in a study (Werne
et al., 2013), aiming at reducing the periparturient rise in FECs, because
of the importance of this biological phenomenon in the epidemiology of
GINs (including H. contortus) (Chartier et al., 1998; Donaldson et al.,
1998). GIN-infected ewes received an experimental infection with H. con-
tortus L3, one month prior to lambing. The study investigated the use of
sainfoin and/or pellets of faba bean (Vicia faba), either as single CT
resource or in combination. The periparturient ewes in late gestation
were fed for 25 days with either (1) a ryegrass-clover forage (control
fed ¼ CF); (2) sainfoin silage (S); (3) a combined CT-feed consisting of sain-
foin forage plus faba bean pellets (SFB) or (4) faba bean pellets and ryegrass-
clover forage (FB). A fifth group composed of ewes in early gestation (EG)
was added to determine the dimension of the PPRI by comparison to the
groups in late gestation (1e4). Compared to the CF group, the differences
in FECs over the feeding period were 55% (S), 40% (SB), þ8% (B), and
41% (EG). These results illustrated that (1) CT originating from sainfoin
can prevent a PPRI effect with possible consequences on GIN epidemi-
ology; (2) the variability in effect depends on the CT resources, since the
faba bean feed did not produce any FEC reduction.
3.7.2 Dissecting the complexity of tropical legumes as nutraceuticals

against H. contortus and other gastrointestinal nematodes
Compared to legume nutraceuticals from temperate regions, a small amount
of data has been acquired for tannin-containing plant materials from the
tropical areas of the world. If we consider the biodiversity of plants that
may contain bioactive PSMs in the different tropical ecosystems such as hu-
mid or subhumid tropical forests, deserts and costal dunes, it is evident that
the work performed so far can be considered very limited. It is only in the
last 10e15 years that tropical plants started to be screened for AH activity
(Alonso-Díaz et al., 2010b). The first in vitro screening studies evaluated
the AH effect of seed extracts against H. contortus including Mangifera indica
(Costa et al., 2002) and Leucaena leucocephala (Ademola et al., 2005, 2006).
Later on, several in vitro screening studies aimed at exploring candidates for
nutraceutical purposes against GIN infections using extracts obtained from
leaves of tannin-rich legumes of the tropical forest. The first candidates
explored were obtained from several browse plants commonly consumed
by sheep and goats and readily available for farmers: Acacia pennatula, Acacia
gaumerii, Brosimum alicastrum, Havardia albicans, L. leucocephala, L. latisiliquum
and Piscidia piscipula (Alonso-Díaz et al., 2008a,b; Hernandez-Ordu~ no et al.,
2008; Calder on-Quintal et al., 2010). These studies showed that most
plants had a promising AH effect against H. contortus. von-Son-de-Fernex
et al. (2012) evaluated extracts of other tropical fodder legumes including
Arachis pintoi, different cultivars of Cratilia argentea and Gliricidia sepium
and showed significant in vitro AH effect against different stages of H. con-
tortus, confirming the role of tannins with the addition of PEG. Other
research groups followed a similar approach, testing for example Manihot
esculenta leaves or Musa paradisiaca leaves which also showed a good AH ac-
tivity against H. contortus (Marie-Magdeleine et al., 2010, 2014). Evidence
obtained so far from the few plant species screened suggests that there could
be a large number of tropical plants with potential AH activity against GIN.
However, the vast quantity of potential candidates without an evaluation
makes it difficult to decide which plants should be screened. Thus, taking
into consideration the interest in nutraceutical materials, meaning that
animals have to eat the plant materials to obtain the AH effect, the first can-
didates to screen should be those that are readily consumed by ruminants
with browsing experience. However, this approach still represents a com-
plex decision because ruminants may eat a large number of plant species
in the tropical forest and those plants are consumed with different levels
of preference (Gonzalez-Pech et al., 2014, 2015). Thus, the next decision
step could be related to the level of preference shown by the animals.
This approach could also help to analyse possible self-medication behaviour
in the ruminant hosts (Villalba et al., 2014).
An entirely different approach consists of evaluating the AH activity of
plant extracts that previously showed activity against other microorganisms.
The in vitro AH activity of Phytolacca icosandra extracts against eggs and larvae
of H. contortus was reported by Hernandez-Villegas et al. (2011) and similar
322 H. Hoste et al.
AH activity was reported for different plant species of the Anonacea family
(Casta~neda-Ramírez et al., 2014).
The in vitro screening of tropical plants has suggested that the PSM
composition of tropical plants is more complex than that of temperate plants.
In vitro studies with a range of tropical tannin-rich legumes and agroindustrial
by-products showed that the AH activity was not totally related to their CT
content as the use of PVPP or PEG failed to block the totality of the AH effect
shown by some of the plant extracts tested (Chan-Pérez et al., 2016; Vargas-
Maga~ na et al., 2014a). In some cases, the AH activity was significantly
enhanced after the tannins were blocked either with PVPP or PEG. The latter
suggests that some plant extracts tested contain certain polyphenols that
perform differently to those already described in temperate tannin-rich plants.
This phenomenon warrants further research. Also, the evidence of such in vi-
tro studies suggests the existence of antagonisms between PSM within a single
plant. If such antagonism exists, then of course it may also be possible that an-
tagonisms between PSM are created when animals consume two or more
plant species. This is especially important when considering the future on-
farm application in ruminants browsing the tropical forest.
Only a handful of pen or field trials have been performed to measure in
vivo the AH activity against GIN of tropical fodders (see reviews by
Alonso-Díaz et al., 2010a and Torres-Acosta et al., 2012). The first evidence
in the literature refers to a trial by Kabasa et al. (2000) reporting an increase in
faecal egg counts in mixed naturally infected goats when regularly receiving
PEG under browsing conditions. Two studies focussing on leaves of Acacia
cyanophylla performed with naturally infected sheep found significant effects
on GIN egg faecal excretion (Akkari et al., 2008a,b). The role of tannins
on the reduction of GIN eggs was confirmed with the use of PEG. However,
the tannins (Mueller-Harvey, 2006) and AH effects of Acacia trees vary be-
tween different species. For instance, A. nilotica fodder failed to offer any
AH effect for goats infected with H. contortus, while A. karoo fodder reduced
faecal egg excretion and worm burden (Kahiya et al., 2003; Marume et al.,
2012). Thus, it would be important to identify the factors affecting the AH
efficacy of different plants of the same species and also the variation between
different species within the same genus.
The in vivo studies using the fodder of L. latisiliquum showed a reduction
in the establishment of H. contortus and T. colubriformis L3 larvae in goats
(Brunet et al., 2008b). A further trial in sheep with established H. contortus
populations reported a reduction of the faecal egg excretion as well as a
reduction of fecundity and size of the female worms for animals consuming
such plant material (Martínez-Ortíz de Montellano et al., 2010). Similar in

vivo studies performed with the foliage of H. albicans, a fodder tree with very
high-tannin content showed nonconsistent effects on the H. contortus faecal
egg excretion (Galicia-Aguilar et al., 2012; Mendez-Ortíz et al., 2012)
although it also reduced both the fecundity and size of female H. contortus
worms (Galicia-Aguilar et al., 2012).
Several early studies aiming at confirming the AH effects of tannin-rich
resources on different GIN stages in sheep (Athanasiadou et al., 2000,
2001) or goats (Paolini et al., 2003a,b, 2005a) were performed by drenching
animals with quebracho, which is a bark extract from south American trees
(Schinopsis spp.). In a similar line, several in vivo studies used bark extracts ob-
tained from tropical plants (Marume et al., 2012; Xhomfulana et al., 2009).
The use of commercial bark extracts from Acacia mearnsi is the most common
example of PSM-rich extracts against GINs, much in the same way as herbal
medicines. The first trial by Cenci et al. (2007) showed that a weekly dose of
18 g/kg for 13 weeks reduced the faecal egg count of sheep with a mixed
GIN infection and even reduced the worm burden. Meanwhile, Minho
et al. (2007) used a dose level of 1.6 g of extract/kg BW for only two days
in sheep and reported a significant reduction in FEC and worm burdens of
H. contortus but without any effect on T. colubriformis. The next studies with
the same material by Max (2010) and Max et al. (2009) failed to find a clear
AH effect on the GIN infection of goats, and the effect was low in the case of
sheep, even when using a larger dose of extract than that applied by Cenci
et al. (2007). The latest report using A. mearnsi extract also failed to show
any clear AH activity against a mixed GIN infection in goats (Costa-J unior
et al., 2014). This lack of consistency in results can be due to factors of vari-
ations exposed under Section 3.5 and Fig. 5. Differences in host species (sheep
vs goats) should also been considered. While the question of dose level re-
mains to be explored for A. mearnsi commercial extracts, other studies evalu-
ated different dose levels for bark extract of Albizia anthelmintica (Gradé et al.,
2008) using naturally infected sheep. They reported that a dose of 0.8 g per
animal resulted in significant effects on FEC. Another type of plant extract
was also tested under in vivo conditions with naturally infected goats.
Hernandez-Villegas et al. (2012) tested a dose of 250 mg of P. icosandra etha-
nolic extract/kg body weight (administered in gel capsules). These authors
showed a reduction in GIN eggs larger than 70% compared to nontreated
controls. Similar to the studies with A. mearnsi, there was no previous infor-
mation leading to determine the correct dose level and/or the duration of the
treatment for goats. Therefore, the dose level was chosen based on previous
324 H. Hoste et al.
reports for crude ethanolic extracts of different plants against natural GIN in-
fections of sheep (Ademola et al., 2004, 2005, 2006, 2007a,b).
To summarize the information from tropical plants, the majority have
shown some evidence of AH effect. However, in most cases the AH effect
is not as strong as it is with the tannin-containing temperate legumes. The
lack of a clear AH effect from feeding bioactive tropical forages could be
explained by different reasons: (1) the structural features of tannins present
in these plants might differ from those in SL and/or sainfoin (see Section
3.1.5); (2) parasites of ruminant species in tropical regions might have adapt-
ed to the bioactive PSMs that are constantly present in the animals’s diet,
becoming less susceptible (Calder on-Quintal et al., 2010); (3) consistent
with some in vitro trials (Chan-Pérez et al., 2016; Vargas-Maga~ na et al.,
2014b), plants possess different PSMs, some of which might have antago-
nistic effects that limit the potential AH effect of other PSMs. Interactions
between PSMs have been previously described (Burrit and Provenza,
2000; Lyman et al., 2008), but are usually overlooked. However, under
the conditions of the tropical forest, interactions between PSMs are highly
probable since browsing animals ingest a ‘cocktail of PSMs’. This complex
question needs further investigations.
Finally, the work with tannin-rich foliage suggests that animals have the
ability to eat large quantities of foliage for several days without any detri-
mental consequences on animal health but there are studies showing a
trade-off that implies a negative effect on the diet digestibility due to the
consumption of tannin-rich foliage (Galicia-Aguilar et al., 2012; Mendez-
Ortíz et al., 2012). The latter is consistent with the antinutritional nature
of some PSM such as CT and saponins at high concentrations. In spite of
such antinutritional evidence, the same studies suggested that sheep artifi-
cially infected with H. contortus might eat significantly more tannin-rich fo-
liage than noninfected animals (Martínez-Ortiz-de-Montellano et al., 2010;
Mendez-Ortíz et al., 2012). The latter is consistent with potential self-
medication behaviour (see reviews by Hutchings et al., 2003; Juhnke
et al., 2012; Villalba and Provenza, 2007; Villalba et al., 2014).
3.7.3 Exploring the value of agroindustrial by-products

Variability in results (see Section 3.6) is one of the key issues affecting the
implementation of tannin-containing nutraceuticals in the integrated con-
trol of GINs worldwide. To pursue basic research on the mode of actions
of tannins and flavonoids against digestive nematodes is a first step toward
addressing this issue. A second important area of research is the continued
exploration of different technological processes aimed at conserving and sta-

bilizing nutraceutical resources, in particular their PSM contents (see Section
3.1.7). In regard to the use of nutraceuticals under farm conditions and in
veterinary sciences, the questions of sustainability and cost of production
have also to be considered.
The definition of plant nutraceuticals in veterinary sciences implies that
the effect depends on the feed intake by animals. The main hypothesis is that
the health effects relate to the presence of PSMs (Hoste et al., 2015). It is
usually assumed that the active PSMs are intrinsic to the botanical resources
exploited as nutraceuticals, such as with tannin-containing legumes.
However, a second option is to add some resources (‘part of the feed’)
with high concentrations of active PSMs. This option explains that, world-
wide, the interest has been growing in exploring the potential of tannin-
containing ‘by-products’ from agroindustries. Until now, these by-products
have generally been considered as ‘waste’. However, they represent a poten-
tial ‘goldmine’ of PSMs that could be added to animal feed.
Regarding the worldwide potential applications for on-farm use, this
approach, which aims at exploiting and/or including ‘waste’ tannin-rich
materials into nutraceutical feeds for antiparasitic properties, has several
potential advantages: (1) by solving the problem of the inherent variability
of PSM contents in nutraceutical plants. The use of stored by-products
would allow measuring active PSMs before use, hence allowing the bioactive
PSM concentration(s) in feeds to be adjusted (Girard et al., 2013), to achieve
positive effects (including antiparasitic activity) without any side-effects caused
by an excess of PSMs; (2) by securing the production of low-cost, tannin-
containing resources and (3) by adding extra value to agroindustrial products,
which until now, have generally been considered as ‘waste’.
Therefore, many tannin-containing by-products from different areas of
the world have been under investigation using in vitro assays, in order to
screen the AH activities. Some examples are mentioned, to illustrate the
potential exploitation of such by-products and the diversity of industrial
by-products in different areas of the world: (1) by-products from the nut
(hazelnut or chestnut) industry in temperate areas (Desrues et al., 2012;
Girard et al., 2013); (2) carob pods and pistacia (Arroyo-Lopez et al.,
2014; Manolaraki et al., 2010) around the Mediterranean Sea and (3)
by-products from tropical resources, including coffee and cocoa fruit husks
and leaves (Covarrubias-Cardenas et al., 2013; Vargas-Maga~ na et al., 2014a),
banana leaves (Musa x paradisiaca) (Marie-Magdeleine et al., 2010), cassava
leaves (M. esculenta) (Marie-Magdeleine et al., 2014; Seng and Rodriguez,
326 H. Hoste et al.
2001, 2003; Sokerya et al., 2009), or shea nut’ meal (Vitellaria paradoxa)
(Ramsay et al., 2016) from tropical resources.
3.7.4 Possible combinations of resources with anthelmintic effects

3.7.4.1 Combining different CT-containing resources?
The rationale for this option relates to the results presented in Section 3.6.
The idea is to improve the AH effects against GINs, either by increasing the
overall diet content of CT and/or by combining resources with different
tannin types (cf. Table 5) (eg, with different prodelphinidin/procyanidin
ratios or mean degrees of polymerization).
A study combining the use of sainfoin silage and faba beans (Werne et al.,
2013) was among the first to explore such combinations (cf. Section 3.1.7).
Another study, examining the possibility of associating sainfoin pellets (mainly
prodelphinidins) and by-products of hazel nuts (mainly procyanidins), was
conducted in lambs experimentally infected with H. contortus (see Girard
et al., 2013). The results suggested that the addition of hazel nut peels to
the sainfoin diet led to lower FECs, toward significant values.
The use of mixtures of L. cuneata (prodelphinidins) plus pine bark pow-
der (procyanidins) and also of Ficus infectoria plus Psidum guajava leaves
resulted in lower worm numbers (Pathak et al., 2013; Wright, 2015).
However, a mixture of P. lentiscus, Arbutus unedo, and Quercus ilex proved
ineffective (Saric et al., 2015), which might be linked to the fact that adult
sheep e rather than lambs e were fed and that they had acquired mixed
natural infections of low intensity. The present concept is attractive, and
seems worthy of further exploration under different epidemiological condi-
tions. However, presently, there is a paucity of information, and no clear
trend has been observed. This is not surprising since the idea relies mainly
on potential interactions between different bioactive compounds; the
complexity of such issues has been explained previously.
3.7.4.2 Combining CT-containing nutraceuticals and synthetic chemical

AH?
The question as to the possible interactions between nutraceuticals and the
synthetic chemical AHs is of considerable practical importance, but has
received little attention until recently. A first in vitro study, measuring the
motility of larvae, suggested that extracts of redberry juniper (Juniperus
pinchotii) could increase the activity of ivermectin against H. contortus
(Armstrong et al., 2013). These first results were then confirmed in an in
vivo study, showing that feeding lambs a diet containing 30% redberry
juniper reduced FECs and increased ivermectin efficacy by 65% (Whitney

et al., 2013). With similar objectives, another study explored the effects of
the consumption of sainfoin pellets on a multiresistant strain of H. contortus.
In contrast to previous data, the FEC results suggested a lower efficacy of
oral ivermectin in lambs consuming sainfoin. These results were confirmed
by changes in pharmacokinetic parameters of the drug in the blood (Gaudin
et al., 2015). It is worth noting that, besides CT and flavonoids, terpenoids
are also present in redberry juniper, which is not the case for sainfoin.
Clearly, further studies are needed to explore these interactions and to better
understand the specific mechanisms associated with these interactions
between the polyphenolic compounds and the pharmacology of macrocy-
clic lactones (Dupuy et al., 2003).
4. CONCLUSIONS
The almost exclusive reliance on treating animals with synthetic AH
drugs is seriously challenged nowadays, because of the high adaptive capacity
of nematodes, illustrated by the constant and rapid development of resistance
to these drugs in worm populations. These facts indicate that a more sustain-
able mode of GIN control is now required, which should not be based on
just one option/principle of control (Hoste and Torres-Acosta, 2011).
Instead, an integrated control of nematodes is currently recommended,
which is based on a combination of different components. The manipula-
tion of host nutrition is one of the key components. However, a better un-
derstanding of grazing management and also animal breeds or lines with
genetic resistance, new synthetic drugs, new modes of application of current
synthetic drugs and, hopefully, vaccines will also be needed and are ex-
pected. These different areas relate to the different chapters of this Thematic
Issue of Advances in Parasitology.
In the current chapter, we illustrated the complexity of interactions
between host nutrition and H. contortus and related GINs in small ruminants.
We also highlighted that a better understanding of these interactions is likely
to offer a range of solutions by exploiting, across the world, the contributions
that host nutrition can make. If you take care of the nutrition of your livestock an-
imals, then they will take care of the parasite threat. This sentence stems from tradi-
tional experience and common sense. It emphasizes the possible benefits that
better nutrition could achieve in animal production and health e and this has
been recognized for a long time. In the case of H. contortus infection in lambs
328 H. Hoste et al.
and kids, improved nutrition can prevent animal deaths, which is essential for
small farmers, because, for them, this is synonymous with animal production.
The first scientific studies that validated quantitative aspects of manipu-
lating host nutrition were initiated by Clunies-Ross (1932). Qualitative
aspects, namely nutraceutical PSMs commenced some 20 years ago. Finally,
it is expected that related studies of self-medication will probably become
more important in the future.
Addressing the basic questions regarding the manipulation of nutrition
for GIN control requires not only parasitological studies, but also multidis-
ciplinary investigations, which will, for example, link research to nutrition,
immunology, phytochemistry and/or pharmacology. Across the world,
basic studies of the exact mechanisms of action will help to identify suitable
solutions for a range of epidemiological and agronomic problems. This focus
will also assist in extending to studies of other animal species, such as cattle,
camelids and exotic ruminants.
ACKNOWLEDGEMENTS
The authors wish to acknowledge the financial support of the European Commission
through the ‘LegumePlus’ project (PITN-GA-2011-289377), the EMIDA ERANET
project CARES, the INRA métaprogramme GISA STREP, the CORE ORGANIC 2
ProPARA, and the PCP FranceeMexico project 2013e2017 (Fondo Institucional CONA-
CYT No. 229330). This research was supported by USDA NIFA Organic Research and
Education Initiative (Project No. 2010-51300-21641) and USDA NIFA Small Business
Innovative Research program (Project No. 2011-33610-30836).
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CHAPTER EIGHT
Immunity to Haemonchus
contortus and Vaccine
Development
A.J. Nisbet*, 1, E.N. Meeusenx, {, J.F. Gonzalezjj, D.M. Piedrafitax, {
*Moredun Research Institute, Edinburgh, United Kingdom
x
Federation University, Churchill, VIC, Australia
{
Monash University, Melbourne, VIC, Australia
jj
Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain
1
Corresponding author: E-mail: Alasdair.Nisbet@moredun.ac.uk
Contents
1. Introduction 354
2. The Immunology of Host Protection Against Haemonchus contortus 355
2.1 Background 355
2.2 Critical role of host immune responses in protection 356
2.2.1 Administration of dexamethasone 357
2.2.2 Lymphocyte involvement in immunity 357
2.3 Host immune protection against larval establishment 358
2.3.1 Mechanisms against L3 358
2.3.2 Rapid rejection 359
2.3.3 Delayed rejection 360
2.3.4 Mechanisms against L4s 361
2.4 Host immune protection against adult worm infection 363
2.5 Unknown factors affecting host immunity 364
3. Development of Vaccines 365
3.1 Background 365
3.2 Gut-derived antigens, H-gal-GP 366
3.3 Gut-derived antigens, H11 368
3.4 Barbervax 369
3.5 Gut-derived antigens, thiol sepharoseeBinding Proteins 370
3.6 Gut-derived antigens, GA1 371
3.7 L3 surface antigen 371
3.8 Excretory/secretory antigens 372
4. Recombinant Subunit Vaccines 374
4.1 Background 374
4.2 Recombinant versions of H-gal-GP components 377
4.2.1 Aspartyl proteinases 377
4.2.2 Zinc metalloproteinases 377
4.2.3 Cystatin 379

j
ISSN 0065-308X
354 A.J. Nisbet et al.
4.3 Recombinant versions of H11 components 379

4.4 Recombinant proteins e others 380
4.4.1 15- and 24-kDa ES proteins 380
4.4.2 Galectins 381
4.4.3 Hc23 381
4.4.4 Enolase 381
4.4.5 Cysteine proteinases 382
4.5 DNA vaccination 383
References 387
Abstract
Sheep are capable of developing protective immunity to Haemonchus contortus
through repeated exposure to this parasite, although this immune protection is the
result of a complex interaction among age, gender, physiological status, pregnancy,
lactation, nutrition and innate and adaptive immunity in the host animal. There are
multiple effectors of the protective immune response, which differ depending on
the developmental stage of the parasite being targeted, and our understanding of
the effector mechanisms has developed considerably in the 2000s. The rational design
of vaccines based on ‘natural’ or ‘exposed’ antigens depends on an understanding of
this exposure-induced immunity. However, the most effective current vaccines rely on
protection via the induction of high circulating antibody levels to ‘hidden’ gut antigens
of H. contortus. The success of this latter strategy has resulted in the launch of a vaccine,
which is based on extracts of the parasite’s gut, to aid in the control of Haemonchus in
Australia. The development of recombinant subunit vaccines based on the compo-
nents of the successful native vaccine has not yet been achieved and most of the
recent successes with recombinant subunit vaccines have focussed on antigens unre-
lated to the gut antigens. The future integration of an understanding of the immunobi-
ology of this parasite with advances in antigen identification, expression (or synthesis)
and presentation is likely to be pivotal to the further development of these recombi-
nant subunit vaccines. Recent progress in each of the components underpinning
this integrated approach is summarized in this review.
1. INTRODUCTION
Haemonchus contortus is the most globally important trichostrongylid
parasite of small ruminants (sheep and goats) in tropical and subtropical areas,
and is a major constraint on ruminant health and production worldwide.
The continued and increasing development of resistance to all chemical
control options (Kotze and Prichard, 2016) and the persistence of chemical
Immunity to Haemonchus contortus and Vaccine Development 355
residues in animal products have highlighted the need for other economi-
cally sustainable control options (Newton and Munn, 1999). The develop-
ment of vaccines against economically important parasitic helminths of
humans and ruminants has been a long-term goal of many immunoparasitol-
ogists, and vaccination which induced sustained protective immunity would
be one of the most cost-effective methods of controlling an infection. For
the optimal design of the most effective vaccines and vaccination strategies,
the immunological control of a helminth via vaccination should be under-
pinned by a thorough knowledge of the hosteparasite interactions, the im-
mune responses involved in protection and the biology of the parasite itself.
While identifying the key antigens and life stages of the parasite that are
targeted by elements of the naturally acquired protective immune response is
possible through an integrated immunoproteomic/glycomic and bioinfor-
matics pipeline (eg, Smith et al., 2009), the remaining, substantial challenge
is to induce the same levels of immunity, through the same mechanisms,
using recombinant or synthetic vaccines. An alternative strategy is to induce
vaccine-mediated protection through mechanisms which are not necessarily
involved in naturally acquired protective immunity (eg, Smith and Smith,
1996) by immunizing with ‘hidden’ antigens. In these cases, it is still essential
to understand the basis of the immune protection induced by the vaccine, to
inform adjuvant selection, immunization strategies, etc.
The aim of this chapter is to review key immunoparasitological studies of
H. contortus and the current state of knowledge of protective immune re-
sponses provoked by infection with this parasite and provide a perspective
on the development of vaccines against H. contortus and the challenges asso-
ciated with developing commercially viable vaccines.
2. THE IMMUNOLOGY OF HOST PROTECTION

AGAINST HAEMONCHUS CONTORTUS
2.1 Background
With repeated parasite exposure, sheep can develop protective immu-
nity to subsequent H. contortus infections (Barger et al., 1985; Christie et al.,
1978; Miller et al., 1983). The ability of sheep to resist the pathogenic effects
of H. contortus infection relies on a complex interplay of multiple factors,
including the level of nutrition, physiological characteristics, pregnancy
status, sex, age and innate and adaptive immunity of the host animal
(Andronicos et al., 2010; Hein et al., 2010; Saddiqi et al., 2011). The
immune system is a critical component, capable of mediating protection

against these parasitic infections and is inherited (Wakelin, 1985) and poly-
genic in nature (Kemper et al., 2009). The basis of immunological protec-
tion has been investigated and several immune mechanisms have been
validated (see Fig. 1). However, recent studies suggest a complex interplay
of immune mechanisms, some previously unknown, which are likely to spe-
cifically target the larval and/or adult parasitic stages and these are high-
lighted in the following discussion.
2.2 Critical role of host immune responses in protection

Infection of sheep with H. contortus is accompanied by pathological changes
in the abomasum associated with flattening and disruption of the mucosa
(Nicholls et al., 1985) and hyperplasia of numerous cell lineages, including
mucus-producing epithelial cells (Scott et al., 1998, 1999), lymphocytes
(Gorrell et al., 1988), mast cells (Miller, 1996), globule leukocytes (GLs;
Stankiewicz et al., 1993) and eosinophils (Balic et al., 2006). Such changes
are associated with the development of immunity in sheep, and early studies,
described briefly later, attempted to demonstrate a critical role for the im-
mune system in acquired immunity to H. contortus infection. These studies
involved the administration of the general immune suppressant dexameth-
asone and the cellular transfer (to naïve recipients) or inhibition of critical
components of the immune system (in the resistant host).
Figure 1 A simple pictorial depiction of the known immune mediators or mechanisms

associated with the development of host resistance against Haemonchus contortus
infection in sheep. The interplays (orange arrows (grey in print verisons)) between
the immune factors to different larval and adult stages (including unidentified factors
to date) are poorly understood.
2.2.1 Administration of dexamethasone

Dexamethasone is a synthetic corticosteroid analogue of the steroid hor-
mone cortisol. Large quantities (eg, 0.5 mg/kg) of corticosteroids exoge-
nously administered to an animal cause non-specific immunosuppression
(Plumb, 2005). Administration of dexamethasone to Merino sheep which
were genetically resistant to gastrointestinal nematode (GIN) infection
demonstrated that immunity to both primary and challenge H. contortus in-
fections was decreased, implicating both innate and adaptive immunity,
respectively (Adams, 1988; Presson et al., 1988). Dexamethasone adminis-
tration also abrogated immune protection in sheep which had been rendered
immune by prior repeated larval challenges for 10e12 weeks (Huntley et al.,
1992). Another study demonstrated that pasture-raised Gulf Coast Native
lambs, a breed considered to have natural immunity to H. contortus infection,
had significantly higher faecal egg counts (FECs) and adult worm counts
than untreated sheep when treated with dexamethasone (Pena et al.,
2004). These early studies on the abrogation of protection against GINs
by using dexamethasone strongly suggested that immunity was an integral
part of the protective response against H. contortus infection; however, the
basis for this immunity was unknown at the time.
2.2.2 Lymphocyte involvement in immunity

Although cortisol has many functions, an effect of large quantities is the non-
specific immunosuppression of all leucocyte subsets (Plumb, 2005). Cell
depletion and transfer studies suggested that lymphocytes may be important
in mediating immune protection against parasites in sheep (Smith et al.,
1984). Sets of twin sheep were used as either donors (of whole lymph or pu-
rified lymphocytes) or recipient sheep (Smith et al., 1984). The donors were
hyperimmunized by repeated infection with H. contortus third larval (L3)
stage, whereas the recipients remained uninfected. Either gastric lymph or
thoracic ductederived lymphocytes were transferred from donor to recip-
ient sheep, resulting in large reductions in FECs in several of the recipient
sheep following a challenge infection (Smith et al., 1984). A further demon-
stration of lymphocyte involvement in immune protection was through the
administration of monoclonal antibodies directed against cell-specific
lymphocyte surface antigen(s): CD4 and CD8 cells were depleted in genet-
ically resistant sheep by injection of specific monoclonal antibodies (Gill
et al., 1993) and CD4, but not CD8, depletion completely abolished the
expression of protection in lambs against H. contortus infection, and reduced
mucosal mast cell hyperplasia and tissue eosinophilia. A similar depletion of
CD4 T cells resulted in a reduction in the natural protection of Gulf Coast

Native sheep against H. contortus infection (Pena et al., 2006). The involve-
ment of CD4 cells in vaccine-induced protection was also investigated in
Dorset cross-breed sheep. Prior to infection, animals were vaccinated by
intramuscular injection with H. contortus gut antigen and, from the time
of parasite challenge, a subset of the sheep was administered anti-CD4 anti-
body daily, resulting in significantly higher levels of FECs in these animals at
21 days post-infection (Karanu et al., 1997).
These studies demonstrated that the immune system is critical in medi-
ating immunity to H. contortus infection and involved CD4 lymphocyte-
mediated mechanism(s). More recently, another lymphocyte subpopulation,
WC1þ gd T cells, has been found to be particularly important in non-
specific immunity in the abomasal mucosa of ruminants, and their associa-
tion with protection against H. contortus infections has been reported (Balic
et al., 2000a,b; Gonzalez et al., 2011; Munoz-Guzman et al., 2012; Pérez
et al., 2008). Further studies, described below, were performed to investigate
the functional aspects of lymphocytes, mediator (cytokine) production, and
to define the cellular basis for the mediation of immune protection. The
infection of ruminants with H. contortus, as for other GINs, is associated
with elevated type 2 immune responses defined by an increased production
of the cytokines interleukin (IL)-4, IL-5 and IL-13; increased parasite-
specific immunoglobulin A (IgA) and IgE; and increased tissue eosinophil,
mast cell and GL numbers (Balic et al., 2000a,b, 2006; Lacroux et al.,
2006). Given the complex nature of the hosteparasite relationship and
the added complexity of the multi-stage parasite life cycle, evidence as to
which parameters are critical in leading to nematode parasite rejection in ru-
minants is limited, and mechanisms are likely to vary among the different
parasite stages. There have been numerous studies investigating the host im-
mune responses to repeated larval infection, particularly those against the L3
stage (which can be readily produced in culture and therefore plentiful),
while immunity against the fourth larval (L4) and adult stages is more chal-
lenging to investigate, which is reflected in the small number of studies pub-
lished to date.
2.3 Host immune protection against larval establishment

2.3.1 Mechanisms against L3
Two major immune-mediated mechanisms (rapid and delayed rejection)
against the incoming infective L3s have been defined through various
studies. Although these two mechanisms can be induced separately in
experimental studies, on pasture these mechanisms are likely to coexist, and

rely on the dynamics of exposure and immune competence of the sheep
(Hein et al., 2010).
2.3.2 Rapid rejection

One well-established form of host immunity to H. contortus infection is the
rapid rejection of infective L3s from sheep that have been hypersensitized
by repeated larval infections over an extended period, and results in the rejec-
tion of a subsequent L3 infection within 48 hours (Balic et al., 2002; Jackson
et al., 1988; Miller et al., 1983). This form of rapid larval rejection, also
termed ‘immune exclusion’, is characterized by an immediate hypersensitiv-
ity (type I) response and associated with the presence of antibody (IgE),
mucosal mast cells and GLs (these latter cells are suggested to be a derivative
of mast cells and are also referred to as intra-epithelial mast cells). Antibody-
mediated activation of these cells results in increased peristalsis of the gut pre-
venting incoming larvae from establishing in the crypts of the abomasal tissues
(Balic et al., 2002; Huntley et al., 1992; Miller et al., 1983; Stankiewicz et al.,
1995). While rapid rejection requires the presence of a specific parasitic stage
(L3) to be initiated, it is considered a non-specific mechanism, as rejection of
unrelated parasites in the same tissue niche or at distal sites further along the
gastrointestinal tract can also occur (Dineen et al., 1977; Emery et al., 1993).
Rapid rejection occurs without significant changes in cellular profiles in
lymph nodes and the mucosa (Balic et al., 2000a,b) or mucin biosynthesis
(Newlands et al., 1990), suggesting that pre-formed mediators, which are
able to inhibit larval establishment, are present within cells/cell granules at
the luminal surface. Architectural changes in abomasal tissue are clearly
visible at the macroscopic level (Kemp et al., 2009). Upon cross-linking
of surface-bound IgE, mucosal mast cells degranulate and release preformed
mediators, such as histamine, proteases, leukotrienes, neutrophils and eosin-
ophil chemotaxins, resulting in abomasal muscle hyper-motility, gastric hy-
per-secretion and hyperplasia of goblet cells, increased mucus production
and recruitment of innate effector cells (Balic et al., 2002; Meeusen, 1999;
Miller et al., 2006). Mucus contains larval inhibitory factors (Douch et al.,
1983, 1996), which can be detected at high levels in the faeces of immune
sheep (Douch et al., 1983), and their production is strongly associated with
GLs (Stankiewicz et al., 1993), suggesting that reduced motility of the L3
aids in the rejection of larvae through peristalsis.
An ex vivo tissue explant model was able to replicate the rapid rejection
of larvae ex situ and provided further understanding of key effector
mechanism (Athanasiadou et al., 2008; Jackson et al., 2004; Kemp et al.,

2009). Large numbers of mucosal mast cells and GLs were observed in
hyper-sensitized tissue, and the ex vivo addition of H. contortus L3s to the
sensitized tissue caused greater degranulation of mast cells measured by
increased levels of histamine (Kemp et al., 2009). Although no difference
in eosinophil numbers was reported in this tissue explant model, a role for
eosinophils in the rapid rejection of larvae was suggested, as an increased
protein level of galectin-14 (a mediator released by eosinophils) was found
in the hyper-sensitized tissue (Kemp et al., 2009). Hypothesized roles for
galectin-14 include the promotion of cellular adhesion and increased mucus
viscosity, which may aid in impeding larval migration and contribute to the
mechanism of rapid rejection (Young et al., 2009), although the precise
role(s) for galectin-14 need to be established.
2.3.3 Delayed rejection

The second identified immune response against the L3 stage, delayed
rejection, is characterized by eosinophil recruitment and a direct role for
eosinophil-mediated larval killing through antibody-dependent cell cyto-
toxicity (ADCC) (Rainbird et al., 1998). Delayed rejection occurs when
ingested L3s penetrate the host tissue either in naïve sheep given a primary
infection, or in sensitized sheep in which immunity has not yet progressed to
the hyper-immune stage or has waned following an extended period of no
infection (Balic et al., 2000a,b, 2002, 2006). In each situation, activation of
gastric lymph nodes and recruitment of lymphocytes to the abomasal mu-
cosa occur, with the up-regulation and activation of CD4 T cells occurring
earlier in immunized compared with naïve animals (Balic et al., 2002). Sig-
nificant increases in gd T cells, CD4þCD25þ T cells, B cells and eosinophils
are detected in the abomasal tissues during the delayed rejection response
(Balic et al., 2000a,b). A time-sequential study of animals hyper-sensitized
to H. contortus, given a 12-week rest period, followed by a challenge infec-
tion to induce a delayed rejection response, revealed elevated blood and tis-
sue eosinophils and gd T cells coinciding with the period of rejection of the
L3 parasitic stage (Robinson et al., 2010).
A consistent association of tissue eosinophils with delayed rejection has
been reported, and eosinophils have been shown to directly kill L3 in vitro,
which was enhanced through antibodies and IL-5, suggesting a mechanism
involving ADCC (Rainbird et al., 1998). Supporting the in vitro mechanism
of killing, eosinophils in vivo are also observed in close proximity to L3
H. contortus in animals exposed to a secondary infection and are associated
with structural damage to the larval parasites (Balic et al., 2006). Further sup-
port for a role of eosinophils in delayed rejection is the release of the eosin-
ophil-specific molecule, galectin-14, into the gut shortly after parasite
challenge, with high galectin-14 expression significantly inversely correlated
with subsequent worm burdens (Dunphy et al., 2000, 2002; Robinson et al.,
2010, 2011; Young et al., 2009).
2.3.4 Mechanisms against L4s

Although several immune mechanisms have been identified that target the
infective L3 stage, few immune mediators have been identified that specif-
ically target the haematophagous L4 and adult stages of H. contortus.
Following tissue penetration of the host, L3s undergo morphological devel-
opment, exsheath and form the parasitic L4 stage (Veglia, 1915). Immune
protection generated against the L4 stage can result in the induction of
hypobiosis (arrested development) or the regulation of L4 feeding. Regu-
lating L4 feeding is considered a major defence mechanism of local IgA
in sheep against another abomasal GIN, Teladorsagia circumcincta (see Stear
et al., 2004). In T. circumcincta infections of sheep, high levels of secretory
IgA targeting Excretory/Secretory (E/S) products from L4s are consistently
associated with reduced L4 length and are hypothesized to help control
infection until another immune mechanism develops to regulate parasite
numbers (Benothman et al., 2010; Henderson and Stear, 2006; Stear
et al., 1999). L4 length is also strongly associated with adult fecundity
and reduced parasite length results in a reduced egg-laying capacity (Stear
and Bishop, 1999). As such, the mechanism of secretory IgA can also be
classified as an indirect immune mechanism against the adult stage. Corre-
lations between IgA and immunity to H. contortus have not yet been so
firmly established. This may be due to the different nature of parasite
feeding, with T. circumcincta feeding from the mucosa, whereas H. contortus
penetrating the tissue to feed on blood. However, it may also be due to
limited study of the relationship between IgA and H. contortus compared
with the large body of information for T. circumcincta.
Recent studies, described below, have investigated the role of mamma-
lian galectins, a family of evolutionary conserved glycan-binding proteins,
as novel regulators of inflammatory processes, including those induced by
parasite infection (Vasta, 2009). Galectin-14 is an eosinophil-specific galec-
tin in sheep and cattle (Dunphy et al., 2002; Young et al., 2009), which has
been strongly implicated in playing a protective role against the L3 stage of
H. contortus (as discussed above in “Section 2.3.3”). Another host galectin,
galectin-11, has been increasingly reported in ruminants (sheep, goats and

cattle) infected with parasites (Athanasiadou et al., 2008; Dunphy et al.,
2000; Hein et al., 2010; Hoorens et al., 2011; Robinson et al., 2011).
Galectin-11 was initially identified as specifically induced in sheep
following a primary or secondary challenge with H. contortus or Trichostron-
gylus vitrinus (see Dunphy et al., 2000). Galectin-11 was expressed in the nu-
cleus and cytoplasm of the upper epithelial cells lining the gastrointestinal
tract, and secreted into the mucus (Dunphy et al., 2000). A kinetic biopsy
study investigating the delayed rejection of L3 H. contortus showed that
galectin-11 was released into the mucus 3e5 days after infection, coin-
ciding with the development and emergence of L4s from the abomasal
crypts into the lumen, and was observed to correlate with increased mucus
viscosity (Robinson et al., 2010, 2011). The target and role of galectin-11
release are unknown; however, an indirect role in increasing mucus viscos-
ity that impedes parasite establishment and epithelial cell proliferation has
been hypothesized (Hoorens et al., 2011; Robinson et al., 2011; Young
et al., 2009).
The first demonstration of a galectin directly affecting the parasite was in
an in vitro feeding assay, in which galectin-11 inhibited the feeding and
exsheathment of H. contortus L4s and caused significant inhibition of L4
growth (Preston et al., 2015). Galectin-11 was shown to bind to the L4
and adult stages, but not to the L3 H. contortus stage (Preston et al.,
2015). Local secretions of IgA are consistently reported as a protective
mechanism against T. circumcincta by regulating L4 growth (Stear et al.,
2004; Strain and Stear, 1999), and it is possible that glycans present on these
antibodies interact with galectin-11 to mediate protection against H. contor-
tus L4 stage parasites. In addition, a proteomic analysis of sheep gut mucus
following Trichostrongylus colubriformis challenge has also implicated immune
mediators IgA and galectin-11 in the rapid rejection mechanism of immu-
nity (Athanasiadou et al., 2008).
The role of IgA in H. contortus infection is uncertain (Strain and Stear,
2001), as is the relationship between IgA and galectin-11, such that further
investigations are warranted. The recent successful crystallization of
galectin-11 is likely to advance our understanding of ruminant-galectine
parasite interactions and putative parasite ligands for diagnostic and vaccine
development (Sakthivel et al., 2015). These recent findings, as well as the
proposed effects of galectin-11 on mucous viscosity and epithelial prolifer-
ation, suggest that this molecule might have multiple mechanisms of action
that target the parasitic stages of H. contortus.
2.4 Host immune protection against adult worm infection

Manifestations of immune-based protection against the adult stage of H. con-
tortus, as for other GINs, are thought to occur by expulsion, changes in
morphology or a reduction in egg-laying capacity (Balic et al., 2000a). A
reduction in FECs can be a consequence of reduced female worm burdens
and/or reduced egg production in utero. Expulsion of adult parasites usually
occurs after repeated larval infections, and is dose dependent (Barger et al.,
1985; Barnes and Dobson, 1990). Adult expulsion is thought to occur either
through the non-specific mechanism of rapid rejection (see Section 2.3) or
through a specific mechanism of acquired immunity (Balic et al., 2000a).
Rapid rejection of L3s, once initiated, becomes a non-specific mechanism,
which is thought to result in the elimination of residing H. contortus adult
parasites as well as other GINs established within the same or proximal loca-
tion in the gut (Miller, 1984; Rothwell, 1989). Other reports suggest that
the expulsion of adult H. contortus is initiated by a specific immune mecha-
nism, since the rejection of the adult stage does not occur simultaneously
with rapid rejection of L3s (Barger et al., 1985). Further evidence supporting
specific immunity against adult stages of GINs has come from experiments in
which adult T. colubriformis were surgically transplanted into hosts that had
previously generated immunity against L3 challenges (Emery et al., 1992).
Immunity against adult T. colubriformis manifested as inhibition of establish-
ment, damaged cuticle and reduced fecundity (Emery et al., 1992).
The exact cellular basis mediating adult worm damage has usually been
studied in commercial sheep breeds but is poorly understood. Immune
studies of these breeds have shown that adult H. contortus infection evokes
limited change in leucocyte populations compared with larval infections
(Balic et al., 2000b), and suggested that this could be due to the ability of
adult parasites to down-regulate the immune response or due to their min-
imal contact with tissues. In contrast to the intimate contact of the host with
tissue-dwelling larvae, adult H. contortus worms reside in the lumen and tran-
siently feed at different sites within the abomasum (Hein et al., 2010).
Studies of immune function in sheep breeds which are not widely commer-
cially exploited (eg, Rh€ on sheep, Canaria Hair breed sheep) and which have
stronger natural resistance to GIN infections than many of the more wide-
spread commercial breeds, have demonstrated that immunity in these less
common breeds can be generated primarily against the adult worm stage
(Aumont et al., 2003; Gauly and Erhardt, 2001; Gonzalez et al., 2008; Zajak
et al., 1990). The Canaria Hair Breed sheep, which is native to the Canary
Islands, is refractory to H. contortus infection with immunity directed against

adult worms and this immunity is independent of larval establishment
(Gonzalez et al., 2008). Preliminary study (Gonzalez et al., 2011) suggests
that the mechanism may involve gd T cells and eosinophils, cells not previ-
ously implicated in immune mechanisms to the adult stage. The exact role of
gd T lymphocytes in infection with adult H. contortus is unclear. Whether
these cells are involved in natural resistance and/or immunity or if their pres-
ence is only a secondary effect of H. contortus infection is currently unknown.
However, given their association with eosinophils, which have been shown
to mediate cytotoxicity to larvae, it is likely these gd T cells play a functional
role through the activation of eosinophils to directly damage the adult para-
site and/or stimulate mediators (such as toxic granule proteins and galectin-
14), and to reduce viability and enhance the expulsion of H. contortus. In
addition, galectin-11 expression and direct binding of this molecule to adult
H. contortus during parasite establishment is observed both in commercial and
other sheep breeds (Preston et al., 2015; Piedrafita and Gonzalez, unpub-
lished data), and might interfere with parasite feeding as demonstrated for
L4s. Further work, including inhibition studies of key putative effector cells,
such as eosinophils and gd T cells, might help elucidate their role(s) in im-
mune protection.
2.5 Unknown factors affecting host immunity

As there are several mechanisms of immune protection involving different
key effector cells and mediators, it is more than likely that there are multiple
reasons for variation in immunity among individual sheep to GIN infections.
This statement is supported by genomic data (Andronicos et al., 2010),
which report no common mechanisms of immune protection in Merino
sheep bred for genetic resistance, but a common mechanism of susceptibility
relating to interferon-g up-regulation (Andronicos et al., 2010). Genetic
factors affecting physiological mediators and hormones are also likely to
directly or indirectly influence the effectiveness of the immune response
(Andronicos et al., 2010; Diez-Tascon et al., 2005; Hickford et al., 2011;
Ingham et al., 2008; Keane et al., 2006; Kemper et al., 2011; Rowe et al.,
2008, 2009; Sayers and Sweeney, 2005). The preceding sections document
key immune mechanisms involved in the cellular basis of immunity against
some larval stages, but this understanding is incomplete or may not be the
main mechanism of protection across all sheep breeds. The understanding
of immune mechanisms targeting larval and adult stages is currently inade-
quate, and the identification of recent cytotoxic mechanisms directed against
L4 and possibly against adult stages demonstrates the increasing complexity

of the immune mechanisms against GINs. The finding that galectin-11 also
appears to be ruminant-specific, with a major role in parasite immunity,
highlights the limitations of rodent models which underpin much of our
understanding of immunity to GIN infections. Given the complex hoste
parasite interplay and the many mediators involved, more immune mech-
anisms are likely to be identified in the near future, depending on levels of
research investment. Such studies in poorly defined breeds of sheep may
provide fertile ground for new and interesting discoveries of mechanisms
of natural immunity and novel avenues for parasite control (Piedrafita
et al., 2010).
3. DEVELOPMENT OF VACCINES
3.1 Background
The development of commercial vaccines to control parasitic hel-
minths started in the 1950s with the demonstration that infection of calves
with radiation-attenuated cattle lungworm (Dictyocaulus viviparus) larvae
could achieve high levels of protection against challenge infection. This
work led to the development of Dictol (now Bovilis Huskvac, MSD Ani-
mal Health) which was, until very recently, the only commercially available
vaccine for a parasitic nematode of livestock (http://www.worldchanging.
gla.ac.uk/article/?id¼12). By contrast, the administration of irradiation-
attenuated H. contortus infective larvae to mature sheep resulted in vac-
cine-induced protection, but did not give reliable levels of protection in
the target age group of young lambs under field conditions (Urquart
et al., 1966). Thus, the development of vaccines to control haemonchosis
in lambs was focussed on the identification of immunogenic molecules
and complexes in the excretory/secretory products (E/S) from the nema-
tode, as well as on cuticular surface and gut-derived ‘hidden’ antigens (ie,
those not recognized by host immune reactions following infection) from
the blood-feeding stages. For Haemonchus, both E/S and complexes from
the luminal border of the intestine have been successfully employed as
native vaccines (reviewed by Knox et al., 2003; Newton and Meeusen,
2003; Smith and Zarlenga, 2006). These antigens and complexes have
most often been identified as effective vaccines using a pragmatic, iterative
approach of successive enrichment of fractions which initially gave some
level of protection in preliminary protection trials. Ultimately the most
promising vaccine candidates from these trials have then been produced as
recombinant proteins because of the existing paradigm that vaccine
commercialization depends on the scale, quality, uniformity and safety
that recombinant proteins can offer compared with native immunogens
(Geldhof et al., 2007; Knox et al., 2003). This approach, of using recombi-
nant components of native immunogens, has certainly resulted in the devel-
opment of effective vaccines to control cestode parasites (Lightowlers et al.,
2003), but has not yet been successful for an anti-Haemonchus vaccine, as
discussed in Section 4, below.
3.2 Gut-derived antigens, H-gal-GP

H-gal-GP is a gut-derived, galactose-containing glycoprotein complex
which is highly effective when used as a vaccine in lambs, consistently giving
90% reduction in FECs and w70% reduction in worm burdens in
numerous independent experiments in lambs aged 2e10 months (Smith
et al., 1994, 2003a). The major components of H-gal-GP have been iden-
tified as a family of zinc metalloproteinases (MEPs) and pepsinogen-like
aspartyl proteinases (Newlands et al., 2006; Smith et al., 1999, 2003a,b),
with a cystatin, a thrombospondin-like molecule, and a family of galectins
also present in the complex (Newlands et al., 1999; Skuce et al., 2001). A
review of the contributions of the individual components of H-gal-GP to
its protective capacity (Knox et al., 2003) concluded that only one of the
zinc MEPs (MEP 3) and the pepsinogen-like molecules were likely to be
essential for the protective capacity of the complex. Thus, zinc MEPs and
aspartyl proteinases isolated from the complex under non-reducing condi-
tions retained some protective capacity when used as immunogens in lambs,
whereas the isolated galectin component (containing Hco-GAL-2), for
example, did not, in spite of inducing high levels of antigen-specific circu-
lating antibodies (Newlands et al., 1999; Smith et al., 2003a,b). Thrombo-
spondin and cystatin were considered very unlikely to contribute to
protection (Knox et al., 2003), as low amounts of each were present in
the complex and a sub-fraction containing components of <40 kDa
(including cystatin) did not provide protection in vaccine trials in sheep.
High-mass matrix-assisted laser desorption ionization time-of-flight
(MALDI-ToF) analysis has determined the overall molecular weight of
the H-gal-GP complex to be 981 10 kDa, which correlates well with
the estimated molecular mass from electrophoretic analysis (Smith et al.,
1999), and single particle/cryoelectron microscopy revealed the three-
dimensional structure of the H-gal-GP complex (Muench et al., 2008).
Overall, the structure represents an arch formed by the two pepsinogens

over a base formed by the MEPs, and modelling analysis suggested that
the dimensions of the aperture of the arch could accommodate host blood
proteins (eg, haemoglobin and albumin), so that both the aspartyl protein-
ases and the MEPs could access them as substrates (Newlands et al., 2006).
The MEPs have hydrophobic domains and are likely to be embedded in
the luminal surface of the parasite’s intestinal cell membrane (Muench
et al., 2008). Immunolocalization of the proteolytic components of
H-gal-GP demonstrated their presence on the microvillar surface of the in-
testinal cells (Knox et al., 2003; Newlands et al., 1999, 2001), supporting
their roles in the digestion of the blood meal. Therefore, this complex could
form a membrane-bound ‘proteolytic machine’ analogous to bacterial pro-
teosomes in a particle slightly smaller than foot-and-mouth disease virus, and
it has been suggested that it is the inhibition of the proteolytic activity by
vaccine-induced antibodies (Ekoja and Smith, 2010) that is responsible for
vaccine efficacy (Knox et al., 2003).
Glycosylation is a feature of the H-gal-GP complex, which is rich in
b-galactoside sugars (Smith and Smith, 1993), although some elements of
the complex (eg, cystatin) lack N-linked glycosylation sites (Newlands
et al., 2001). Therefore, the H-gal-GP complex is prepared from extracts
of adult H. contortus by successively removing the water-soluble and mem-
brane-associated proteins, solubilizing the integral membrane proteins and
affinity-enriching galactose-containing molecules from the integral mem-
brane proteins by lectin affinity chromatography, followed by gel filtration
(Smith et al., 1994). The protective capacity of H-gal-GP is lost if the confor-
mation is destroyed by incubation in reducing conditions, but not by sodium
dodecyl sulphate (SDS)einduced dissociation alone (Smith and Smith,
1996). This observation gives support to the proposal that although the com-
ponents of H-gal-GP are glycosylated and these glycans may contribute to
protection, it is the contribution of glycans to the overall conformation of
the complex rather than their individual chemical structures that is important
for the retention of vaccine efficacy (Knox et al., 2003; Smith and Smith,
1996). When either of the gut-derived antigens H-gal-GP or H11 (see Sec-
tion 3.3) are used as immunogens, levels of antigen-specific serum IgG are
highly correlated with vaccine efficacy, suggesting that the effect of the vac-
cine is antibody-mediated (Munn et al., 1997; Smith et al., 1999) and a large
proportion of the circulating IgG response in lambs vaccinated with native
H-gal-GP is directed against the glycan components of the complex
(G. Newlands unpublished; cited in Knox et al., 2003).
3.3 Gut-derived antigens, H11

The integral membrane glycoprotein complex, termed H11, is a family of
microsomal aminopeptidases present on the intestinal microvilli of the
blood-feeding stages of H. contortus and, when separated by SDS-polyacryl-
amide gel electrophoresis (PAGE), runs as a doublet with a mean molecular
weight of 110 kDa (Newton and Munn, 1999). Through expressed sequence
tag and, more recently, genome analyses, five isoforms of H11 (termed H11,
H11-1, H11-2, H11-4 and H11-5) have been described (Newton and
Meeusen, 2003; Roberts et al., 2013; Smith et al., 1997). All five isoforms
are tandemly arranged in the H. contortus genome, suggesting that the gene
family has arisen through recent duplication and divergence (Roberts
et al., 2013). The members of the H11 family share 62e75% amino acid
sequence identity and, combined, give the complex both aminopeptidase
A and M-type activities (Newton and Meeusen, 2003; Roberts et al.,
2013; Smith et al., 1997). Proteomic analyses of native H11 showed that
all five isoforms were present in a typical H11 preparation, and RNA-Seq
analyses demonstrated that transcripts representing H11, H11-2 and H11-4
were all highly expressed in the adult gut, whereas H11-5 was expressed
in a female-specific manner (Roberts et al., 2013). H11-1 was the most abun-
dant form in infective L3s (Roberts et al., 2013).
H11 is a highly effective protective immunogen and, when the results of
a large number of protection trials in different breeds of sheep of varying age
and immunocompetence were evaluated, immunization with H11 gave
mean reductions in worm burden of 72% (male worms), 82% (female
worms) and a 91% reduction in FECs (Newton and Munn, 1999). The
potent protective effects of H11 have also been used as a tool to determine
the phenotypic effects of silencing genes encoding vaccine candidate pro-
teins in the parasitic stages of H. contortus (see Samarasinghe et al., 2011).
Thus, exsheathed infective L3s were soaked for 24 hours in double-stranded
RNA representing the H. contortus H11 gene before being used to infect
5-month-old lambs. This treatment led to the silencing of the H11 tran-
scripts and a 57% reduction in FECs, a 40% reduction in total worm burden
and a 64% decrease in aminopeptidase activity in H. contortus recovered from
infected sheep (Samarasinghe et al., 2011).
As for H-gal-GP, correct conformation and, therefore enzymatic activ-
ity, of the H11 complex is important for protection, and the efficacy is
reduced in H11 preparations that have been denatured with SDS or with
SDS plus the reducing agent dithiothreitol (Munn et al., 1997). Levels of
protection in sheep vaccinated with native H11 are highly correlated with
levels of inhibition of H11 aminopeptidase activity by antibodies in the
sera from vaccinated animals (Munn et al., 1997). Each H11 isoform also
contains potential N-linked glycosylation sites and mass spectrometric ana-
lyses of the native material have shown N-glycans with highly fucosylated
core structures including core a1-3 and a1-6 fucosylation which are highly
immunogenic (Haslam et al., 1996).
3.4 Barbervax
Of the antigens tested as vaccines to control haemonchosis, only H-gal-GP
and H11 have consistently conferred protection in experimental trials at
levels that would rival, or exceed, conventional anthelmintic treatment
regimes. In addition, preparations of Haemonchus integral gut membrane
proteins containing both H11 and H-gal-GP, when used as vaccines in
sheep in controlled field trials, reduced anaemia, prevented deaths and
reduced the contamination of pasture with infective L3s (LeJambre et al.,
2008). The demonstration that these antigens could induce protection
when administered in microgram quantities opened the door for the com-
mercial exploitation of a native vaccine: Following the acquisition of a good
manufacturing licence and 12 field trials in Merino sheep in the Northern
Tablelands of New South Wales, in October 2014, the Australian Pesticide
and Veterinary Medicines Authority granted permission to sell Barbervax, a
vaccine containing Haemonchus native integral gut membrane proteins
enriched for H-gal-GP and H11. In order to meet local biosecurity regula-
tions, Barbervax is made from Haemonchus harvested on an industrial scale
from donor sheep at the Albany Laboratory of the Department of Agricul-
ture and Food, Western Australia. The realization of the first vaccine in the
world for a nematode parasite of sheep therefore relied on innovations in
processing and production technology, rather than novel or emerging
biotechnological advances to produce the vaccine in a cost-effective, repro-
ducible and safe manner.
Because the antigens present in Barbervax are hidden antigens, repeated
vaccination is required to stimulate high antigen-specific circulating anti-
body levels but, in areas where haemonchosis is currently controlled by
using repeated anthelmintic drenches throughout the grazing season (eg,
the Northern Tablelands), the required frequency of application of
Barbervax is no higher than that required for drenching (see http://www.
wormboss.com.au). If the first three priming injections happen before the
risk of Haemonchus becomes high in midsummer, two further injections
each at 6-week intervals, should allow protective immunity to be main-

tained in lambs until late autumn when the risk of Haemonchus infection de-
clines (http://barbervax.com.au). A major advantage of the use of the
hidden antigens, which make up Barbervax, is that they can produce pro-
tective effects in situations where natural immunity is either weak or
ineffective, such as in young lambs or in peri-parturient ewes; the effects
in this latter groups of animals is of particular importance in the manage-
ment of transmission of infective L3s to vulnerable lambs early in the
season (Andrews et al., 1995; cf. Smith, 2015 in http://www.vetvaccnet.
ac.uk/sites/vetnet/files/user-files/research-paper/pdf/02-15/Barbevax-%20
Haemonchus%20vaccine.pdf).
3.5 Gut-derived antigens, thiol sepharoseeBinding Proteins

Fractionation of the membrane-bound molecules from adult H. contortus by
peanut lectin affinity chromatography (to remove H-gal-GP) followed by
Thiol Sepharose chromatography resulted in a 24-fold enrichment of
cysteine proteinase activity in the Thiol Sepharose-binding protein
(TSBP) component (Knox et al., 1999). When 10-month-old Greyface/
Suffolk cross lambs were immunized with TSBP in Quil A adjuvant and
challenged with a bolus of infective H. contortus larvae, mean FECs and
worm burdens in the immunized lambs were reduced by 82% and 45%,
respectively (Knox et al., 1999). Immunolocalization experiments demon-
strated that the components of TSBP were located on the intestinal brush
border of the adult parasites (Knox et al., 1999). The cysteine proteinases
in TSBP are encoded by three genes: hmcp1, 4 and 6; possess homology
to cathepsin B-like molecules; and are expressed on the microvillar surface
of the gut cells (Skuce et al., 1999). In addition to the cysteine proteinases,
TSBP contains a glutamate dehydrogenase (GDH) homologue as one of its
principal components, but fractionation of the TSBP by anion exchange
chromatography demonstrated that it is the cysteine proteinases, which
constitute less than 2% of the total protein in TSBP, rather than the GDH
which confer protection (Knox et al., 2005). This finding was supported
by the demonstration that the cysteine proteinase component of TSBP,
when further purified using cystatin affinity chromatography, conferred
protection in Greyface/Suffolk cross lambs aged 5 months (leading to a
28e48% reduction in FECs and a 44e46% worm burden reduction),
whereas the non-cystatin binding component of TSBP gave no protection
in experimental challenge infections (Redmond and Knox, 2004).
3.6 Gut-derived antigens, GA1

Monoclonal antibodies raised against the H. contortus gut surface were used
to isolate a set of three glycosylated proteins of 46, 52 and 100 kDa by
immunoaffinity chromatography (Jasmer et al., 1993). When combined
and co-administered with Freund’s adjuvant to pigmy goat kids, these pro-
teins induced a significant reduction in abomasal worm burden (60% reduc-
tion) (Jasmer et al., 1993). Subsequently, immunoscreening of an H. contortus
adult cDNA library showed that the 46- and 52-kDa proteins were in fact
derived from the 100-kDa polyprotein (Jasmer et al., 1996). The parent
100-kDa molecule was termed gut antigen 1 (GA1), and the two subcom-
ponents named p46GA1 and p52GA1 (Jasmer et al., 1996). The GA1 protein
components were both gut membrane-associated, although only p52GA1
possessed a glycosylinositolphospholipid type anchor to the microvillar sur-
face and, although intimately associated with the gut membrane, GA1 pro-
teins were released in E/S products from adult H. contortus and were detected
in the abomasal mucus from infected goats (Jasmer et al., 1996).
3.7 L3 surface antigen

As the adult stage of H. contortus ingests blood from the host, using the ‘hid-
den antigen’ approach to target gut-derived antigens, as discussed in Sections
3.1e3.4, has been very effective in reducing parasite burdens and patholog-
ical changes. One of the disadvantages of this approach can be the require-
ment for repeated vaccination to stimulate continuous, high antigen-specific
circulating antibody levels. Although our understanding of immune protec-
tion towards H. contortus is still evolving, the ability of some sheep to develop
immunity to H. contortus infection has led to optimism that efficacious vac-
cines are possible using antigens involved in natural immunity. In theory at
least, this approach would obviate the need for the multiple boosting re-
quirements of vaccines based on hidden antigens due to a natural boosting
of the immune system during re-infection on pasture. Hence, another
approach for helminth vaccine development is to target the infective larval
stage of the parasite against which natural immunity is commonly directed.
A 70- to 90-kDa antigen, termed Hc-sL3, which is specifically recog-
nized by the immune system during a rejection response in H. contortus-
immune sheep, was shown to be expressed in a stage-specific manner on
the surface of L3s (Ashman et al., 1995; Bowles et al., 1995; Raleigh and
Meeusen, 1996). Vaccination trials using purified Hc-sL3 antigen have
shown consistently significant levels of reductions (50e70%) in worm
burden and FECs under a range of vaccination regimes and adjuvants

( Jacobs et al., 1999; Piedrafita et al., 2012, 2013). Like Barbervax, the
complexity of this antigen (mucin-like) has precluded recombinant antigen
formulation, but the simple isolation procedure and abundant access to
larval-derived antigens may make this a viable native antigen vaccine.
The importance of adjuvant selection for vaccine-induced immunity
against H. contortus using antigens involved in natural immunity is exempli-
fied by the vaccine trials with the Hc-sL3 antigen. Protection was only
achieved when Th2-inducing adjuvants were used [aluminium hydroxide
and diethylaminoethyldextran (DEAE)] with no protection observed
when Freund’s complete adjuvant (FCA) or Quil A were used in the vaccine
preparations or when Quil A was mixed with aluminium hydroxide ( Jacobs
et al., 1999; Piedrafita et al., 2012, 2013; Turnbull et al., 1992). FCA and
Quil A induce strong cellular and humoural immune responses. The
aluminium-based adjuvants, on the other hand, typically induce much lower
antibody responses than Quil A or FCA, but have been reported to be better
inducers of Th2-type cytokine responses, including eosinophilia (Cox and
Coulter, 1997; Piedrafita et al., 2013). DEAE is a less studied adjuvant but
seems to induce both strong antibody and type 2 responses (Piedrafita
et al., 2013), which may be useful for a combined adult/larval vaccine. It
has been postulated that eosinophil-dependent killing, mediated by
antibodies specific to the Hc-sL3 antigen, may be an important mechanism
in this vaccine-induced immunity, similar to natural immunity, as FECs
were negatively correlated with wheal size and tissue eosinophils for
the DEAE and aluminium-adjuvanted groups, respectively (Piedrafita
et al., 2013).
3.8 Excretory/secretory antigens

Parasite E/S products are a continuous source of natural or exposed antigens
(ie, antigens which provoke an immune response during infection) and may
be responsible for the development of naturally acquired immunity through
continuous exposure and boosting during infection (see Section 2). Nema-
tode E/S products have been investigated as sources of protective antigens
for a number of species (eg, Smith et al., 2009) and, for H. contortus, the
E/S proteins and glycoproteins produced by ex vivo adult worms can confer
significant levels of protection when used in vaccination/challenge experi-
ments (eg, see Vervelde et al., 2003). For vaccine antigen discovery for
H. contortus, the focus has been on two particularly immunogenic E/S pro-
teins of 15 and 24 kDa from a low-molecular-weight subfraction of ex vivo
adult E/S (Schallig and van Leeuwen, 1997; Schallig et al., 1994, 1995;
1997a,b). A preparation of E/S products, highly enriched for the 15 and
24 kDa proteins by anion exchange chromatography, gave substantial levels
of protection in 8-month-old Texel sheep when used as a vaccine formu-
lated with the adjuvant dimethyl dioctadecyl ammonium bromide
(DDA). This preparation gave a 77% reduction in FECs and an 85% reduc-
tion in abomasal worm burden when compared with the adjuvant-only
control group (Schallig et al., 1997a). The protective effects of this vaccine
were further demonstrated in trials using 9- and 6-month-old sheep, but the
vaccine failed to protect younger (3-month-old) lambs against infection
(Vervelde et al., 2001). The function of the 15-kDa protein is, as yet,
unknown and its sequence contains no homology to known functional do-
mains, although it does possess some sequence similarity to 11- and 30-kDa
E/S product vaccine candidate proteins from T. colubriformis (see Schallig
et al., 1997b). The 24-kDa protein contains an SCP domain and possesses
66% amino acid identity across 223 residues to a C-type single domain acti-
vation-associated secreted protein (ASP3) from Ostertagia ostertagi (see Visser
et al., 2008). The transcripts encoding both the 15- and 24-kDa proteins
were only expressed in the parasitic stages of H. contortus, suggesting a critical
role in hosteparasite interactions, which are disrupted by immunization
(Schallig et al., 1997b).
In addition to the 15- and 24-kDa E/S proteins, Thiol Sepharose-
binding components of adult E/S material have been investigated as proto-
type vaccines (Bakker et al., 2004). One subfraction of the thiol-binding
fraction (the DTT-eluted fraction) contained a range of proteins including
MEPs, but no cysteine proteinase activity, and induced reductions of 52%
and 50% in FECs and worm burdens, respectively, when coadministered
to 9-month-old Zwart-Bles lambs with aluminium hydroxide adjuvant in
vaccine/challenge experiments (Bakker et al., 2004). Although these levels
of protection were not statistically significant when compared with the adju-
vant-only controls, the fecundity (numbers of eggs per female) of worms was
significantly reduced when this vaccine was used (Bakker et al., 2004).
The enrichment of the cysteine proteinase components of adult E/S by
cystatin affinity chromatography resulted in the purification of a novel
cathepsin B-like cysteine proteinase, termed AC-5, which induced reduc-
tions of 32% and 36% in FECs and worm burdens, respectively (not signif-
icantly different from the adjuvant-only control) when coadministered to
6-month-old Zwart-Bles lambs with aluminium hydroxide adjuvant in
vaccine/challenge experiments (De Vries et al., 2009). A feature of both
of these latter reports (Bakker et al., 2004 and De Vries et al., 2009) is that
immunization with whole E/S, employing aluminium hydroxide as an
adjuvant, failed to confer protection after parasite challenge. This is in
contrast to previous experiments in which DDA was employed successfully
as the adjuvant for E/S in older lambs (eg, Vervelde et al., 2003) and also in
contrast to the induction of significant levels of protection in 3-month-old
Zwart-Bles lambs (87% reduction in cumulative FECs) when E/S was
coadministered with aluminium hydroxide (Vervelde et al., 2003). In the
protected 3-month-old lambs, immunized with E/S in the context of
aluminium hydroxide adjuvant, there were significant increases in anti-
body levels against GalNacb1,4-(Fuca1,3)GlcNAc, the LDNF glycan
antigen, and high levels of antibody which bound the glycan antigen
Gala1-3GalNAc, suggesting a role for the high glycan-specific antibody
levels in protection in these lambs (van Stijn et al., 2010; Vervelde et al.,
2003). The LDNF glycan is also present on MEP 3, a component of the
H-gal-GP complex (see Sub-section 3.2), but does not contribute to the
protection conferred through the immunization of lambs with H-gal-GP
(Geldhof et al., 2005).
4. RECOMBINANT SUBUNIT VACCINES

4.1 Background
Apart from the potentially high costs associated with vaccine produc-
tion, issues with the use of native antigens for vaccination include quality
control and the risk of contaminants that may prohibit export of a vaccine
to countries around the world. For this reason, there has been a continuous
and concerted effort to produce recombinant or synthetic forms of the an-
tigens. However, in contrast to the successes of native preparations from
H. contortus as vaccine candidates, there have been, until recently, few reports
of success with recombinant vaccines. Where protection has been conferred
by immunization with recombinant antigens, partial protection (at levels
lower than observed for native antigens) is common in most trials under-
taken, and this reduced protective capacity may reflect suboptimal folding
or lack of post-translational modifications of the recombinant proteins.
The results of trials in which recombinant versions of native vaccine compo-
nents were used, and trials in which novel recombinant subunit vaccine can-
didates were employed, are listed in Table 1 and described in the following
subsections:
Table 1 Summary of Vaccine Trials for Haemonchus contortus Using Recombinant Proteins
Effect on
Expression Route of Effect on worm
Antigen system Adjuvant administration FECa burdena Challenge References
H11
H11-1 Baculovirus: None (Sf21 cell Intramuscular Unknown 30%f Bolus 15,000 L3 Reszka et al. (2007)
Spodoptera extract)
frugiperda Sf21
Combined Caenorhabditis QuilA Subcutaneous Nob Nob Bolus 5000 L3 Roberts et al. (2013)
H11-1 elegans
H11-4
Combined C. elegans QuilA Subcutaneous No No Bolus 5000 L3 Roberts et al. (2013)
H11-4
H11-5
H-gal-GP
HcMEP-1 Escherichia coli QuilA Intramuscular No No Bolus 5000 L3 Smith et al. (2003a)
(GST fusion)
HcMEP-3 E. coli (GST QuilA Intramuscular No No Bolus 5000 L3 Smith et al. (2003a)
fusion)
HcPEP1 E. coli Freund’s Intramuscular No No Bolus 5000 L3 Smith et al. (2003b)
HcPEP1 E. coli QuilA Intramuscular No No Bolus 5000 L3 Smith et al. (2003b)
Combined MEPs expressed QuilA Intramuscular No No Bolus 5000 L3 Cachat et al. (2010)
HcMEP-1, in S. frugiperda
HcMEP-3, Sf 9 insect cells;
HcMEP-4 PEP1 in E. coli
HcPEP1
CYS-1 E. coli QuilA Intramuscular No No Bolus 5000 L3 Newlands et al. (2001)
Others
PP2Ac E. coli E. coli Rosetta Intranasal Unknownd No Bolus Fawzi et al. (2013)
2(DE3)pLysS 10000 L3
insoluble fraction mixed speciese
(Continued)
Table 1 Summary of Vaccine Trials for Haemonchus contortus Using Recombinant Proteinsdcont'd
Effect on
Expression Route of Effect on worm
Antigen system Adjuvant administration FECa burdena Challenge References
Combined E. coli Freund’s Intramuscular 37e48% 41e46% Bolus 5000 L3 Yanming et al. (2007)
Hco-gal-m reductionf reductionf
Hco-gal-f
Hc23 E. coli Aluminium Unknown 83% 85% Bolus 15,000 L3 Fawzi et al. (2015)
hydroxide reductiong reductiong
HcENO E.coli Montanide ISA Intramuscular 50% 50% Bolus 5000 L3 Kalyanasud–aram et al. (2015)
(thioredoxin 61VG and reduction reduction
fusion) subcutaneous
Combined E. coli DDA Subcutaneous 0e42% 0e65% Bolus 5000 L3 Vervelde et al. (2002)
Hc15/24 ( Trichoplusia ni reduction reduction
extract)
Combined E.coli (GST QuliA (þ E. coli Intramuscular 0e10% 24e38% Bolus 5000 L3 Redmond and Knox (2004,
hmcp1, 4 and 6 fusion) BL21 extract) reduction reduction 2006)
Hc-CPL-1 C. elegans QuilA Intramuscular No No Bolus 5000 L3 Murray et al. (2007)
All data are from published studies using sheep, unless otherwise stated.
a
Reduction in FEC or worm burden compared with adjuvant-only control
b
No ¼ No significant difference (P < 0.05) when compared with adjuvant-only control group
c
PP2Ar is a 76-amino acid portion of the catalytic region of serine/threonine phosphatase 2A from Angiostrongylus costaricensis
d
Reduction in FEC observed compared to adjuvant-only control at some sampling points post-challenge but species composition of nematode eggs unknown so effect cannot be attributed
to vaccine efficacy against Haemonchus contortus.
e
4000 H. contortus L3; 4000 T. colubriformis L3 and 2000 T. circumcincta L3
f
Trial performed in goats
g
No adjuvant-only control group, comparison is with un-immunized infected group
4.2 Recombinant versions of H-gal-GP components

The success in using the native form of H-gal-GP as a vaccine against hae-
monchosis in lambs stimulated subsequent research to identify and produce
recombinant forms of its components. This initially involved the immuno-
screening of cDNA libraries with sera from lambs successfully immunized
with native H-gal-GP (eg, Newlands et al., 1999, 2001). In addition, the
dissociated complex components were subjected to N-terminal sequencing
followed by polymerase chain reaction approaches (eg, Smith et al., 2003b)
and expression in prokaryotic and, more recently, eukaryotic systems before
vaccine efficacy trials in vivo.
4.2.1 Aspartyl proteinases

Urea-mediated dissociation and subsequent fractionation of the H-gal-GP
complex yielded a pepsinogen-enriched fraction that retained some of the
protective capacity of native H-gal-GP (48% reduction in FECs, compared
with 97% for the undissociated H-gal-GP complex) (Smith et al., 2003b).
However, immunization of sheep with a bacterially expressed, re-folded,
soluble, monomeric, enzymatically inactive recombinant form of the pre-
dominant pepsinogen, HcPEP1, led to the production of high levels of
antigen-specific IgG in sheep, which bound native HcPEP1 but did not
give protection against challenge (Table 1; Smith et al., 2003b). This result
indicated that the correct presentation of the conformational epitopes of the
pepsinogens in H-gal-GP would be required for their protective capacity,
potentially involving their correct glycosylation (Knox et al., 2003; Smith
et al., 2003b).
4.2.2 Zinc metalloproteinases

Under non-reducing conditions, H-gal-GP can be fractionated by SDS-
PAGE into four major zones of 233, 172, 40 and 31 kDa (Smith et al.,
1999). The 233-kDa zone contains three of the MEPS e MEP 1, 2 and
4, whereas MEP 3 is within the 172-kDa zone. Molecular characterization
of the MEPs involved in the H-gal-GP complex showed that they were
members of the M13 zinc metalloendopeptidase family (EC 3.4.24.11).
MEPs 1 and 3 were predicted to possess type II integral membrane protein
structure, whereas MEPs 2 and 4 were predicted to be secreted proteins
(Newlands et al., 2006). MEP3 was present in the complex as a homodimer,
whereas MEP1 and MEP2 formed heterodimers with MEP4. Vaccination of
sheep with a combination of all four MEPs, separated from the H-gal-GP
complex by gel filtration in 8 M urea, resulted in significantly reduced FECs

(45e50% reduction compared to sheep immunized with adjuvant alone)
after parasite challenge, and 26e43% reductions in worm numbers (Smith
et al., 2003a). Following the depletion of MEPs 1, 2 and 4 from H-gal-GP,
protective capacity of the complex was retained (Smith et al., 2000), indi-
cating that MEP 3 was the most important of the MEPs required for vaccine
efficacy. However, when preparations of MEPs 1, 2 and 4, in combination,
were compared with MEP 3 in vaccine trials, the effects on FEC reduction in
immunized sheep were similar (33% reduction for combined MEPs 1, 2 and
4; 34% reduction for MEP 3), indicating a role for each of the MEP prepa-
rations in protection (Smith et al., 2003a). A recombinant version of MEP 1
(rMEP 1) was therefore produced as a glutathione-S-transferase (GST) fusion
protein in Escherichia coli and tested as a protective antigen in vaccination/
challenge experiments in sheep, but no protection was evident (Table 1;
Smith et al., 2003a). H-gal-GP-specific IgG levels in sera from the sheep
immunized with rMEP 1 were significantly lower than those from sheep
immunized with the native H-gal-GP complex (Smith et al., 2003a). A pilot
vaccination/challenge experiment was also performed using GST fusion,
E. coli-expressed versions of 41- and 47-kDa subunits of MEP 3, again
without success (Table 1; Smith et al., 2003a).
The lack of conformational epitopes on the bacterially expressed versions
of MEP 1 and MEP 3, as a result of either incorrect folding or lack of appro-
priate glycosylation, might have contributed to their lack of efficacy in vac-
cine trials (Cachat et al., 2010), so an alternative, eukaryotic, expression
system based on insect cell cultures (Spodoptera frugiperda Sf9 cells) was adop-
ted for the expression of MEPs 1, 3 and 4. A cocktail of these three proteins
along with re-folded E. coli-expressed PEP1 failed to significantly reduce
either FECs or worm burdens (Table 1) when used in vaccination/challenge
experiments in 9-month-old sheep (Cachat et al., 2010). Each of the recom-
binant antigens reacted with IgG from sheep immunized with H-gal-GP
and glycosylation of each of the insect cell expressed recombinant MEPs
was apparent. However, none of the recombinant molecules exhibited
enzymatic activity, suggesting that they were not appropriately folded,
and it seems highly unlikely that the quaternary structures required for the
protective capacity of H-gal-GP were adopted in the recombinant antigen
cocktail (Cachat et al., 2010). The requirement for a combination of correct
epitope presentation from each component and the formation of the
H-gal-GP structure for optimal protective capacity therefore still represents
a huge challenge for molecular, structural and synthetic biology.
4.2.3 Cystatin
Immunoscreening of a cDNA library, prepared from RNA extracted from
H. contortus harvested 11 days after infection, with serum raised against
H-gal-GP, led to the identification of CYS-1, a cysteine proteinase inhibitor
of the type 2 cystatin family (Newlands et al., 2001). The transcript encoding
this protein possessed a similar expression profile to that of the H-gal-GP
galectin Hco-gal-2, as it was only present in significant amounts in the
blood-feeding L4 and adult stages (Newlands et al., 1999, 2001), and these
two molecules were both located in the gut of the parasite, indicating a role
for these H-gal-GP components in the unique physiology of these latter
parasitic stages. A bacterially expressed recombinant version of CYS-1 was
functional as a cysteine proteinase inhibitor, but did not give protection
when used as an immunogen in vaccine trials in 6- to 8-month-old lambs,
despite inducing high antigen-specific circulating antibody levels (Table 1;
Newlands et al., 2001).
4.3 Recombinant versions of H11 components

Initial vaccine trials using combinations of E. coli-expressed isoforms of H11
were unsuccessful (Newton and Munn; cited in Knox et al., 2003). Like-
wise, baculovirus-insect cell expressed, enzymatically active H11 isoforms
also failed to protect sheep in initial immunization/challenge experiments
(Newton and Meeusen, 2003). In later experiments, a baculovirus-insect
cell expressed recombinant version of H11-1 with aminopeptidase A activity
induced a modest level of protection (30% reduction in worm burden;
Table 1) when used in vaccine/challenge studies in 5-month-old Merino
sheep (Reszka et al., 2007), but a GST-fusion version of the same protein
failed to give significant protection (Reszka et al., 2007). The failure of re-
combinant H11 proteins to induce protection has been ascribed to differen-
tial post-translational modifications, incorrect folding, the induction of low
avidity antibodies or the absence of non-H11, contaminating molecules
which may have been critical for the efficacy of the native vaccine (Newton
and Meeusen, 2003). This latter suggestion was endorsed by Smith and
Zarlenga (2006) who suggested that a combination of digestive enzymes
rather than H11 aminopeptidases alone is required for vaccine efficacy.
To address the issues of conformation and post-translational modifica-
tions of recombinant forms of H11 and their effects on vaccine efficacy, a
range of alternative eukaryotic systems has been employed. These ap-
proaches include both DNA vaccination to induce the host cells to produce
H11 and the expression of H11 isoforms in the nematode Caenorhabditis
elegans. A DNA vaccine expressing H11 plus the host cytokine IL-2 was used
to immunize goats (see Section 4.5) and induced high levels of antigen-
specific serum IgG and, following challenge with L3, cumulative mean
FECs and worm burdens were reduced by 57% and 47%, respectively
(Zhao et al., 2012).
The presence of functional a1-3 and a1-6 fucosyltransferases and core
a1-3 and a1-6 fucosylation structures in C. elegans suggested that transgenic
forms of these nematodes, transformed with expression cassettes featuring
H11 isoforms with gut-directed promoters and suitable signal peptides,
should produce recombinant proteins with more appropriate glycosylation
than in other eukaryotic expression systems (Murray et al., 2007; Roberts
et al., 2013). Using this system, Roberts et al. (2013) produced enzymatically
active recombinant forms of H11-1, H11-4 and H11-5, and glycosylation
was confirmed by both lectin binding and MALDI-ToF tandem mass spec-
trometry, which demonstrated the presence of high-mannose structures,
highly fucosylated pauci-mannose like glycans (including a1-3 and a1-6
fucosylation) and evidence of phosphocholine. Serum IgG from sheep
immunized with native H11 preparations bound the C. elegans-expressed re-
combinant forms of H11 and, as is the case for native H11, the majority of
this binding was to glycans (Roberts et al., 2013). In spite of the similarities
in glycosylation between native and recombinant H11 molecules and the
evidence for correct folding of the recombinant H11 molecules, vaccine/
challenge trials in sheep using combinations of rH11-1 and rH11-4 together
or rH11-4 and rH11-5 together did not give any protection. Native H11
appears to be a dimer with a combined mass of around 230 kDa (as deter-
mined by size exclusion chromatography), suggesting the possibility of a
quaternary structural element to protection that may be missing when using
combinations of two or more recombinant versions (Newlands, personal
communication).
4.4 Recombinant proteins e others

4.4.1 15- and 24-kDa ES proteins
Protection studies with combined native 15- and 24-kDa E/S proteins gave
substantial levels of protection in 8-month-old Texel sheep (Schallig et al.,
1997a). Bacterially expressed recombinant versions of both proteins (rHc15
and rHc24; Table 1) initially gave promising results when combined and co-
administered to 7- to 9-month-old sheep with DDA (with and without an
extract of insect cells derived from Trichoplusia ni) as an adjuvant, resulting in
a 38e42% reduction in FECs and a 55e65% reduction in worm burden
compared with the adjuvant-only control (Vervelde et al., 2002). Subse-

quent attempts to repeat these results in 3-month-old lambs and in
9-month-old sheep did not result in reductions in FECs or worm burden
(Vervelde et al., 2002).
4.4.2 Galectins
Although protection trials with recombinant forms of Hco-GAL-2, the H-gal-
GP-associated galectin, have not been performed in sheep, it would seem un-
likely that this recombinant galectin would be protective, as the purified native
molecule did not give protection in vaccine trials (Newlands et al., 1999). In
contrast, recombinant versions of two isoforms (termed Hco-gal-m and Hco-
gal-f because they were derived from male and female worms, respectively) of
a different putative immunomodulatory galectin from H. contortus (Sun et al.,
2007; Wang et al., 2014), induced partial protection in vaccine trials in 9- to
10-month-old goats when administered in Freund’s adjuvant (Yanming et al.,
2007) (Table 1).
4.4.3 Hc23
Hc23 is an exposed antigen of unknown function which constitutes w1.8%
of the total aqueous somatic extract of adult H. contortus (Fawzi et al., 2014).
A pilot study using the native protein suggested that Hc23 may be used as an
effective vaccine antigen when injected into 4- to 5-month-old lambs: re-
ductions in FECs and abomasal worm burdens were 70% and 67%, respec-
tively, when native Hc23 was co-administered with aluminium hydroxide,
and 85% and 87%, respectively, when co-administered with bacterial lipo-
polysaccharide/inactivated bacteria as adjuvant (Fawzi et al., 2014). It should
be noted, however, that these values relate to reductions compared with an
un-immunized challenge group rather than control groups administered the
appropriate adjuvant only (Fawzi et al., 2014). A recombinant form of Hc23,
expressed in E. coli and co-administered with aluminium hydroxide, was also
effective in reducing FECs and worm burden by 83% and 85%, respectively
(Table 1) in vaccination/challenge trials in 4- to 5-month-old Assaf breed
lambs (Fawzi et al., 2015). Again, this trial suffered from a lack of appropriate
(adjuvant-immunized only) controls, but the findings offer some hope for
the use of recombinant proteins as vaccines to control haemonchosis.
4.4.4 Enolase
Enolase (2-phosphoglycerate hydratase; EC 4.2.1.11) is a cytosolic enzyme
performing the penultimate step in glycolysis. In spite of its cytosolic
localization and function, enolase has been detected in E/S of adult H. con-
tortus (see Yatsuda et al., 2003) and antibodies in sera from experimentally
infected goats bound both native and recombinant versions of the enzyme,
indicating exposure of the host to the protein during infection (Han et al.,
2012a). In other helminth species, enolase has been shown to be a compo-
nent of both the E/S material and the tegument of the adult worm (Wang
et al., 2011). In the Chinese liver fluke Clonorchis sinensis, tegumental enolase
is able to bind host plasminogen, potentially facilitating tissue invasion
(Wang et al., 2011). A recombinant form of the H. contortus enolase
HcENO, expressed in E. coli and co-administered as a water-in-oil emulsion
with the adjuvant Montanide ISA 61 VG, was effective in halving both
FECs and worm burden in vaccination/challenge trials in 6-month-old
Mecheri breed lambs (Table 1; Kalyanasundaram et al., 2015).
4.4.5 Cysteine proteinases

Cysteine proteinases purified from TSBP derived from adult H. contortus
confer significant levels of protection against experimental challenge infec-
tion (Redmond and Knox, 2004; Knox et al., 2005), and immunoscreening
of cDNA libraries with sera from protected lambs identified three major
cathepsin B-like cysteine proteinases (hmcp1, 4, and 6) in TSBP (Skuce
et al., 1999). When expressed as GST fusion recombinant proteins in
E. coli and used to immunize Suffolk/Greyface cross sheep, a cocktail of
the three proteins conferred partial protection, with reductions in worm
burden of up to 38% (Table 1; Redmond and Knox, 2004). When the
same cocktail of recombinant proteins was used, but without fusion partners
on the recombinant proteins, reductions of 27% and 29% in FECs and worm
burdens, respectively, were observed (Redmond and Knox, 2006).
In each of the protection trials using hmcp1, 4 and 6, the recombinant
proteins were expressed in an insoluble form, as inactive proteinases. In
contrast, Hc-CPL-1, a functionally active cathepsin L-like cysteine protein-
ase from H. contortus, was expressed in C. elegans and was able to rescue yolk
protein processing in C. elegans loss-of-function mutants, demonstrating cor-
rect folding and activation of the recombinant enzyme (Britton and Murray,
2002). Cysteine proteinase activity of the glycosylated C. elegans-expressed
Hc-CPL-1 was further confirmed using cathepsin L-specific substrates
(Murray et al., 2007) and immunization of 5-month-old Suffolk lambs
with this form of Hc-CPL-1 induced antibodies which bound the native
protein (Murray et al., 2007). In spite of this, immunized lambs were not pro-
tected when infected with H. contortus larvae in an experimental challenge
(Table 1; Murray et al., 2007), although it should be noted that native

Hc-CPL-1 has not been tested in protection trials in sheep, such that this
molecule might not have been a suitable vaccine candidate antigen for
H. contortus.
4.5 DNA vaccination

DNA vaccination as a means to control parasitic disease in production ani-
mals is still in its infancy, and the high levels of antigen-specific antibody
required for successful protection against H. contortus in lambs might be diffi-
cult to reach using this technology (cf. Scheerlinck et al., 2004). Recently,
however, several reports have been published describing partial protection
against Haemonchus in goats following DNA vaccination. Vaccination of
8- to 10-month-old goats with plasmid constructs containing three frag-
ments encoding sections of H11-1 and caprine IL-2 conferred partial, but
statistically significant, protection after challenge (a 57% reduction in
FECs and a 47% reduction in worm burden) compared with goats that
had received only phosphate-buffered saline (PBS) injections (Zhao et al.,
2012). Immunization of goats of the same age with DNA encoding
HC29, an H. contortus glutathione peroxidase (GPX), led to high levels of
HC29-specific serum IgG and IgA and increases in the numbers of CD4þ
T lymphocytes as well as 36% reductions in FECs and worm burdens
compared with goats which had received only PBS (Sun et al., 2011).
Further DNA vaccination trials in goats, employing ubiquitously
expressed proteins as the target antigens, have also shown modest levels of
protection. For instance, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH; a cytosolic enzyme involved in glycolysis) from H. contortus
(HcGAPDH) has been assessed in DNA vaccination studies in 9- to
10-month-old goats (Han et al., 2012b). The administration of HcGAPDH
in plasmid pVAX1 conferred a modest level of protection (35% reduction in
FECs and a 38% reduction in worm burden), accompanied by significantly
increased levels of antigen-specific serum IgG and IgA as well as an increase
in CD4þ T lymphocyte numbers compared with goats that had received
only PBS (Han et al., 2012b). Very similar results were obtained when
DNA encoding H. contortus actin was used as an immunogen instead of
HcGAPDH (Yan et al., 2014), and immunization of goats with DNA
encoding a further structural protein, H. contortus Dim-1, conferred slightly
higher levels of protection (a 46% reduction in FECs and a 51% reduction in
worm burden) compared with goats which had received PBS alone (Yan
et al., 2013).
The success of the hidden antigen approach in controlling H. contortus
has led to the development of a commercially available vaccine, which is
proving effective in the field. The mechanism of action of this vaccine
operates through induction of high antigen-specific circulating IgG levels
that need to be stimulated by repeated immunizations during the risk
period for haemonchosis. As such, the protective mechanism does not
mirror natural immunity, though natural immunity may develop in the
vaccinated hosts from exposure to infective larvae during the growing sea-
son. The future development of further immunoprophylactics to control
H. contortus and other GINs will likely depend on understanding the devel-
opment of immune resistance to underpin both rational vaccine develop-
ment and the enhanced efficacy of some of the prototype vaccines
described herein. This review highlights the complexity of the immune
response in the relationship between the H. contortus and its host animal,
and, as for other helminthehost systems, this complexity is not surprising,
given the multiple developmental stages with distinct niches and antigen
profiles associated with individual developmental stages. The recent recog-
nition of galectins in resistance development as well as sheep breeds with
diverse immune responses against different stages of the parasite (eg, larvae
versus adult) emphasize the challenges for the development of an efficacious
commercial vaccine. In addition, the substantial levels of inter- and intra-
population genetic variation, and thus antigenic diversity, within H. contor-
tus (see Blouin et al., 1995; Hussain et al., 2014; Redman et al., 2008) as
well as the inherent differences in the genetic backgrounds, and thus
immune responses and effectors, of individual definitive ruminant host an-
imals (Bishop, 2012), are also likely to play a role in differential vaccine
responsiveness.
The immune factors affecting host resistance have been investigated in
vaccine trials in which protective efficacy has been correlated with immune
parameters. For the hidden antigen approach, this protection is often
strongly associated with the induction of antibody responses. In contrast,
parallel vaccine studies, investigating cellular changes considered necessary
for the development of natural immunity, have been limited. This has
been partly due to small numbers of protective molecules investigated to
date and partly due to limited immunological analysis carried out in these
experiments, as they are logistically difficult to perform. As such whether
vaccine-induced efficacy and natural immunity are interdependent as would
be expected remains unclear. In addition, selecting the right adjuvant is crit-

ical, but our understanding of immune modulation of adjuvants remains in
its infancy. This highlights the need for further studies to advance our under-
standing of essential requirements for a broader base in vaccine antigen iden-
tification and efficacy.
Subunit recombinant vaccines are highly favoured because of the com-
mercial applicability of such approaches (Section 4). In spite of recent
encouraging advances in the recombinant production of Haemonchus anti-
gens, their appropriate selection and expression remain the major challenges.
If antigen selection is to be based on effective native complexes, it is vital that
the effective component(s) of those complexes are fully identified and un-
derstood. In this regard, it is notable that H11, the most potent native vac-
cine produced thus far (Smith et al., 1993), was originally a very minor
contaminant of the effective contortin-enriched preparation (CEP) that her-
alded Haemonchus vaccine research (Munn, 1977; Munn et al., 1987). While
the potent immunogenicity of H11 in CEP was resolved by immunoblot-
ting (Smith and Munn, 1990), modern proteomic technologies should be
capable of detecting femto- to attomolar quantities of immunogenic com-
ponents in effective native vaccine preparations, though reproducing these
as effective recombinant vaccines remains a challenge. In the early 1990s,
the potential for expressing H. contortus antigens in C. elegans was recognized
(eg, Jasmer et al., 1993) and the subsequent production of enzymically
active, correctly folded, glycosylated Haemonchus antigens in C. elegans in
sufficient quantity for vaccine testing (eg, Murray et al., 2007; Roberts
et al., 2013) might have opened the door for the production of effective re-
combinant versions of the most protective native antigens. However, this
avenue has not yet delivered a successful outcome, potentially because of
the need for quaternary structures in the most complex of the native, effec-
tive vaccine antigens. In contrast, encouraging levels of protection have been
achieved in pilot trials using bacterially expressed versions of single antigens,
such as Hc23 (Fawzi et al., 2015), suggesting that the prospects of developing
recombinant vaccines based on protective native molecules may need to be
focussed on simple antigens rather than complexes such as H-gal-GP until
developments in genome editing, structural and synthetic biology can
enable synthesis of the complex quaternary structures needed for protection
using H11 and H-gal-GP.
Given these challenges, it is important to ask, what do we realistically
expect from a vaccine? It is now prudent to suggest that Haemonchus-specific
vaccines will play an essential role as one tool for parasite control, but it is
also clear that such a vaccine only targets one (albeit highly pathogenic) spe-
cies of GIN infecting sheep in commercial livestock production systems. In
terms of vaccine efficacy required for Barbervax or any other vaccine to con-
trol Haemonchus, there is now acceptance, at least amongst the scientists
involved, that vaccines to control parasitic nematodes will not achieve the
sterile immunity associated with vaccines developed for the control of bac-
terial and viral diseases (Emery, 1996; Knox et al., 2003). It is also highly un-
likely that vaccines will attain the efficacy expected of anthelmintics, but
modelling studies comparing the relative benefits of vaccination against a
conventional anthelmintic control program (Dobson et al., 2011) suggest
that, if vaccines achieve 65% efficacy, they should deliver substantial ben-
efits in the control of haemonchosis in lambs. Thus, 65% vaccine efficacy in
lambs would lead to a mortality rate of 4.5% compared with 27.7% in un-
vaccinated lambs receiving four anthelmintic treatments in regions with high
infection pressure (Dobson et al., 2011; Smith, 2015 in http://www.
vetvaccnet.ac.uk/sites/vetnet/files/user-files/research-paper/pdf/02-15/
Barbevax-%20Haemonchus%20vaccine.pdf).
Issues relating to antigenic diversity in nematodes as well as stage-
specific immune responses and ‘non-responder’ animals suggest that cock-
tail vaccines may be required (eg, Nisbet et al., 2013). The incorporation of
antigens from multiple developmental stages has long been purported as
being essential for the generation of efficacious vaccines. Of course, the
identification of such antigens is challenging, requiring the isolation of
various developmental stages of the parasite from the natural host, the iden-
tification and assessment of a large number of antigens and the financial in-
vestment that is then required for validation. The use of recombinant
subunit cocktail vaccines developed in this way also adds to the cost
and complexity of vaccine manufacture, and is no guarantee of success
(Willadsen, 2008). A vaccine targeting multiple stages of H. contortus based
on native antigens could be an alternative approach. For example, a vaccine
which targets Hc-sL3 (L3 surface antigen) used in combination with the
vaccine targeting hidden gut antigens in the L4 and adult stages of H. con-
tortus (e.g., Barbervax) might provide protection in young lambs early in
the season, while allowing the development of enhanced exposure-related
natural immunity by immunization with the L3 surface antigen, diminish-
ing the requirement for boosting later in the season. Clearly, vaccine devel-
opment against GIN parasites is challenging but is currently looking
brighter for H. contortus than for other species. The pay-off for the success-
ful vaccine development against GINs will be long-term benefits in a world
where increasing animal productivity, rather than increasing animal

numbers, will be essential.
ACKNOWLEDGEMENTS
We are grateful to Prof. Dave Knox and Drs. W. David Smith and George Newlands,
Moredun Research Institute for critical advice on the contents of this manuscript. AJN re-
ceives funding from the Scottish Government’s Rural and Environmental Science and
Analytical Services (RESAS) division.
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CHAPTER NINE
Anthelmintic Resistance in
Haemonchus contortus: History,
Mechanisms and Diagnosis
A.C. Kotze*, 1, R.K. Prichardx
*CSIRO Agriculture, Brisbane, QLD, Australia
x
McGill University, St Anne-de-Bellevue, QC, Canada
1
Corresponding author: E-mail: Andrew.kotze@csiro.au
Contents
1. Introduction 398
2. History of Anthelmintic Resistance 400
3. Mechanisms of Resistance 403
3.1 Benzimidazoles 404
3.2 Imidazothiazoles 406
3.3 Macrocyclic lactones 409
3.4 Closantel 411
3.5 Amino-acetonitrile derivatives 411
3.6 Do different resistance mechanisms have general fitness costs? 412
4. Diagnosis of Resistance 414
4.1 In vivo resistance tests 414
4.2 In vitro bioassay-based tests for Resistance 414
4.2.1 Egg hatch assay 414
4.2.2 Larval development assay 415
4.2.3 Larval motility/migration assay 416
4.3 Molecular-based tests 418
5. Conclusions 419
References 420
Abstract
Haemonchus contortus has shown a great ability to develop resistance to anthelmintic
drugs. In many instances, resistance has appeared less than 10 years after the introduc-
tion of a new drug class. Field populations of this species now show resistance to all
major anthelmintic drug classes, including benzimidazoles (BZs), imidazothiazoles
and macrocyclic lactones. In addition, resistance to the recently introduced amino-
acetonitrile derivative class (monepantel) has already been reported. The existence of
field populations showing resistance to all three major drug classes, and the early
appearance of resistance to monepantel, threatens the sustainability of sheep and
j
ISSN 0065-308X
398 A.C. Kotze and R.K. Prichard
goat production systems worldwide. This chapter reviews the history of the develop-
ment of resistance to the various anthelmintics in H. contortus and examines the mech-
anisms utilized by this species to resist the effects of these drugs. Some of these
mechanisms are well understood, particularly for BZ drugs, while our knowledge and
understanding of others are increasing. Finally, we summarize methods available for
the diagnosis of resistance. While such diagnosis currently relies largely on the faecal
egg count reduction test, which suffers from issues of expense and sensitivity, we
describe past and current efforts to utilize cheaper and less laborious phenotypic assays
with free-living life stages, and then describe progress on the development of molec-
ular assays to provide sensitive resistance-detection tests.
1. INTRODUCTION
Resistance is generally defined as the ability of an organism to survive
doses of drug that would normally kill organisms of the same species and
stage. In practical terms, with respect to anthelmintic resistance in gastroin-
testinal nematode parasites, such as Haemonchus contortus, resistance may be
viewed as a change in response to a drug treatment such that efficacy is
reduced compared to that attained when the drug was released for use in
the field. This definition has been refined somewhat to relate the presence
of resistance to a specific effect of a drug treatment on worm populations
within treated host animals by Coles et al. (1992) who defined resistance ac-
cording to the criteria set by the World Association for the Advancement of
Veterinary Parasitology (WAAVP) as: resistance is considered to be present
in the test population if the percentage reduction in egg count following
drug treatment is <95%, and the lower 95% confidence level is <90%, as
measured in a faecal egg count reduction test (FECRT). Resistance has
been further defined in terms of drug concentrations which inhibit a certain
aspect of development or motility in 50% or 99% of the population in
in vitro doseeresponse experiments with free-living life stages (eg, Coles
et al., 2006; for egg hatch assays for benzimidazole (BZ) drugs), as well as
allele frequencies of genes involved in the resistance (eg, von Samson-
Himmelstjerna et al., 2009b; for b-tubulin and resistance to BZ drugs),
but the overriding definition of resistance in H. contortus and other nematode
parasites lies in the outcome of FECRTs.
While widespread resistance in H. contortus means that the efficacy of
many drugs is now <95%, some of the drugs are still effective at killing a
significant proportion of worm populations in the field. Our ongoing ability
to control this species in the face of resistance is aided by the fact that
Anthelmintic Resistance in Haemonchus contortus 399
resistance mechanisms to the various drug classes generally differ (see Sec-
tion 3) and, hence, a particular drug class may retain activity against the
worms within a field population that are resistant to another drug class.
The exception to this is provided by nonspecific drug efflux pathways
that can confer resistance to multiple drug classes; however, the importance
of these pathways has been considered to be less than for drug classespecific
mechanisms (Section 3). Hence, for at least the period until resistance to
each class becomes absolute (that is, zero efficacy), the use of combinations
of anthelmintics to target the proportion of a population still susceptible to
each of the drugs within the combination will allow for resistant populations
to be controlled to some degree (Bartram et al., 2012). There is evidence,
however, that significant levels of resistance to drug combinations have
developed in field populations of H. contortus; for example, Lyndal-Murphy
et al. (2014) recently detected resistance to drug combinations consisting
of members of each of the three major drug classes (BZs, imidazothiazoles
and macrocyclic lactones (MLs)) in H. contortus in southern Queensland,
Australia.
Haemonchus contortus is the most extensively studied trichostrongylid
nematode with respect to anthelmintic resistance. This is partly because it
has shown such an ability to develop resistance to all the major drug classes;
hence, the resistances shown by this species have had a significant economic
impact on a worldwide scale. However, beyond the economic impact of its
resistances, this species has also been more extensively studied than others
because of aspects of its biology and physiology that make it a good exper-
imental model (Gilleard, 2006, 2013). These factors include its fecundity,
relatively large size of the adult worms (hence providing large amounts of
material for study), the ease of establishing and maintaining large infections
of several thousand worms in host animals, and the availability of simple and
efficient methods for cryopreserving the free-living larval stages. The study
of resistances that are due to changes at drug target sites has also been aided
by the fact that this species has been extensively studied with respect to the
modes of action of anthelmintic drugs. This use of H. contortus as a model is
expected to gain further strength due to the recent sequencing of its genome
(Gasser et al., 2016; Laing et al., 2013; Schwarz et al., 2013) and the impetus
that this should give to whole-genome approaches to the discovery of
markers for anthelmintic resistance in this species (Gilleard, 2013; Kotze
et al., 2014b; Laing et al., 2016).
The present chapter briefly reviews the history of the development of
anthelmintic resistance in H. contortus and then describes the state of
knowledge as to the mechanisms responsible for the resistances to the

different drug classes. Finally, we describe the currently available tests for
resistance and the alternatives that are under development or presently being
sought. The chemical options now available to producers for control of
H. contortus in the face of widespread resistance, and the use of refugia stra-
tegies to maintain anthelmintic-susceptible populations, are covered in
another article in this series (Besier et al., 2016).
2. HISTORY OF ANTHELMINTIC RESISTANCE

Haemonchus contortus has developed resistance to all the anthelmintic
drug classes used against it (Table 1). It is certainly not unique in this regard,
as such resistances have also arisen in all the important gastrointestinal para-
sites of livestock worldwide; however, it has often been the species for
which resistance to a particular drug class has been first reported. The resis-
tance has emerged in most cases within 10 years of the introduction of each
drug group.
The ability of producers to control this parasite was made more difficult
during the 1990s and early 2000s by the emergence of isolates showing
multidrug resistance, that is, resistance to three or more different anthel-
mintic drug classes (Cezar et al., 2010; Echevarria et al., 1991, 1996;
Eddi et al., 1996; Green et al., 1981; Love et al., 2003; Maciel et al.,
1996; Nari et al., 1996; van Wyk and Malan, 1988; van Wyk et al.,
1989, 1997, 1999). Today, most field isolates show some level of resistance
towards BZs, imidazothiazoles and MLs. A number of recent papers have
described the resistance status of H. contortus from different regions of the
world. For example, Papadopoulos et al. (2012) e Europe; Falzon et al.
(2013) e North America; Veríssimo et al. (2012) e South America;
Playford et al. (2014) e Australasia; Tsotetsi et al. (2013) e South Africa;
Chandra et al. (2015) e Asia.
Resistance has also been reported in H. contortus towards the most
recently introduced group, the amino-acetonitrile derivates (AADs) (mon-
epantel) (Mederos et al., 2014; Van de Bron et al., 2015). As the levels of
multidrug resistance become greater (that is, as the efficacy of each individual
drug decreases), and this resistance begins to also include monepantel, the
control of H. contortus will become increasingly difficult.
It is noteworthy that, while a single case of resistance to the organophos-
phate naphthalophos was reported almost 35 years ago (Green et al., 1981),
Anthelmintic Resistance in Haemonchus contortus
Table 1 History of the development of anthelmintic resistance in Haemonchus contortus
Early reports of resistance
Drug class Drug Released in field isolates of H. contortus Country
Benzimidazoles Thiabendazole 1961 Drudge et al. (1964) United States

Smeal et al. (1968) Australia
Berger (1975) South Africa
Le Jambre et al. (1976) Australia
Parbendazole 1966 Berger (1975) South Africa
Fenbendazole 1971 Berger (1975) South Africa
Hogarth-Scott et al. (1976) Australia
Albendazole 1979 Gunawan et al. (1979) Australia
Oxfendazole 1975 Webb and McCully (1979) Australia
Guinan and Kieran (1980) Australia
Mebendazole 1971 Berger (1975) South Africa
Hall et al. (1978) Australia
Salicylanilides Rafoxanide e Van Wyk and Gerber (1980) South Africa
Closantel 1982 in Australia Van Wyk et al. (1982) South Africa
Rolfe et al. (1990) Australia
Organophosphates Naphthalophos 1960s Green et al. (1981) Australia
Imidazothiazoles Levamisole 1965 (1968 in Australia) Green et al. (1981) Australia
Macrocyclic lactones Ivermectin 1981 (1988 in Australia) Carmichael et al. (1987) South Africa
Van Wyk and Malan (1988) South Africa
Eschevarria and Trindade (1989) Brazil
Craig and Miller (1990) United States
Le Jambre (1993) Australia
(Continued)
401
402
Table 1 History of the development of anthelmintic resistance in Haemonchus contortusdcont'd
Early reports of resistance
Drug class Drug Released in field isolates of H. contortus Country
Abamectin 1985 Wooster et al. (2001) Australia
Moxidectin 1992 (1995 in Australia) Vickers et al. (2001) New Zealand
Love et al. (2003) Australia
Doramectin 1993 Terrill et al. (2001) United States
Borgsteede et al. (2007) Netherlands
Eprinomectin 1996 Scheuerle et al. (2009) Switzerland
Amino-acetonitrile Monepantel 2009 in New Zealand; Mederos et al. (2014) Uruguay
derivatives 2010 South America and Van den Brom et al. (2015) Netherlands
Australia; 2011 in Europe
A.C. Kotze and R.K. Prichard

this drug has continued to be effective for the control of H. contortus, at least
in Australia. Its longevity may be the result of its limited use, as it has only a
narrow spectrum of activity, being highly efficacious against H. contortus, but
showing lower activity against the other important gastrointestinal nema-
tode species. However, a recent study from southern Queensland detected
resistance to this compound on one of 16 farms tested (efficacy of 90%)
(Lyndal-Murphy et al., 2014). This finding is a concern, as this drug has
been viewed as maintaining its ability to control H. contortus amidst the
emergence of resistances to the BZs, imidazothiazoles and MLs.
3. MECHANISMS OF RESISTANCE
Haemonchus contortus has been widely studied by researchers looking to
understand the mechanisms by which parasitic nematodes become resistant
to anthelmintic drugs (Fig. 1). These studies have implicated specific
Figure 1 Schematic representation of different factors which could affect the action of
an anthelmintic, leading either to death of the target parasite or to anthelmintic
resistance. Adapted from Prichard, R., Lespine, A., 2013. Genetics and Mechanisms of
Drug Resistance in Nematodes. In: Kennedy, M., Harnett, W. (Eds), Nematodes Molecular
Biology, Biochemistry and Immunology, second ed. CAB International, Wallingford, UK,
pp. 156e183.
molecular mechanisms in resistance to each of the major chemical groups:

BZs, imidazothiazoles and MLs. In some cases, early evidence of the nature
of resistance mechanisms in laboratory-selected parasite isolates has not
translated into explaining the resistance observed in field-derived isolates.
In this context, it is of interest that Sarai et al. (2015) recently reported
that imposition of levamisole selection pressure on free-living larval life
stages produced a life stageespecific resistance phenotype which is quite
different from resistant field isolates. Hence, two aspects may complicate
the elucidation of resistance mechanisms in H. contortus and other species;
firstly, individual isolates, including laboratory-selected isolates, may not
be representative of the range of resistance mechanisms that might arise
in the field. Secondly, differences in the background genetics of the
parasite and the nature of selection pressure imposed on worms in different
farms (under quite different treatment regimes as well as environmental and
management conditions) may cause field-based resistances to single drug
classes to involve different genetic changes and mechanisms (eg, Chaudhry
et al., 2015). Despite this, a great deal of work over recent years has provided
insights into the mechanisms used by H. contortus to develop resistance to the
various drug classes. These mechanisms, and the historical path followed by
researchers to elucidate them, are outlined below.
3.1 Benzimidazoles
Haemonchus contortus was utilized in the early work on the mechanism of
resistance to BZ drugs in parasitic nematodes. Lacey and Prichard (1986)
were the first to draw a direct link between the interaction of BZ drugs
with their target (b-tubulin) and resistance to the drugs. These authors
showed that the binding of tritiated BZ drugs to crude tubulin extracts
was reduced in a resistant isolate compared with a susceptible isolate. Subse-
quently, Lubega and Prichard (1990) used similar experimental methods to
show that resistance in H. contortus was associated with a loss of high-affinity
receptors in resistant worms. The molecular basis of this change in the drug/
receptor interaction was elucidated by Kwa et al. (1994, 1995), using H. con-
tortus and Caenorhabditis elegans. These workers showed that the transfection
of a BZ-resistant C. elegans with an isotype-1 b-tubulin transcript from a
BZ-susceptible H. contortus conferred susceptibility to the drug on the C. ele-
gans worms. The introduction of a Phe to Tyr substitution in the H. contortus
gene at amino acid 200 by in vitro mutagenesis resulted in the loss of the
resistance-reverting activity. This work highlighted the role of this Tyr for
Phe substitution (the F200Y single nucleotide polymorphism, SNP) in
BZ resistance. There is now a great deal of evidence to show that the F200Y
SNP is the major determinant of BZ resistance in H. contortus worldwide
(Fig. 2).
Other mutations in H. contortus isotype-1 b-tubulin have also been
linked to resistance to BZ drugs. Prichard et al. (2000) described a mutation
at amino acid residue 167 (F167Y) in two resistant isolates. However, this
mutation is usually rare in field isolates of this species (Barrere et al.,
2013a; Silvestre and Cabaret, 2002), although Williamson et al. (2011) did
report an unusually high-resistant allele frequency of w40% at codon 167
(alongside a frequency of >95% at codon 200) in a highly BZ-resistant
isolate. Ghisi et al. (2007) described an E198A mutation that conferred
BZ resistance in H. contortus. Although this mutation is not nearly as wide-
spread as the F200Y SNP, Kotze et al. (2012) examined the relative impact
of the 198 and 200 position SNPs in a population possessing a significant
percentage of both and found that the former conferred a higher level of
resistance than the latter. Although mutations in one of F200Y, F167Y or
E198A may confer BZ resistance in H. contortus, examination of b-tubulin
genotypes of individual H. contortus suggests that combinations of two or
three of these genetic changes (eg, F200Y plus F167Y, or F200Y plus
E198A) do not occur in the same b-tubulin isotype 1 allele, suggesting
Figure 2 Mechanism of action and of resistance to benzimidazoles (BZs). (A) BZs act by
binding to b-tubulin, forming a BZ cap at the growth end of microtubules, preventing
further polymerization. (B) b-Tubulin coding sequence in Haemonchus contortus,
showing single nucleotide polymorphic (SNP) sites which can cause resistance.
that multiple mutations at these codons in the same allele may be lethal
(Barrere et al., 2012; Mottier and Prichard, 2008).
Haemonchus contortus possesses a number of b-tubulin genes (Geary et al.,
1992; Saunders et al., 2013), and several studies have implicated changes in
the isotype-2 gene in resistance to BZ drugs in this species, with resistant
populations showing a loss, or at least diminished levels, of this isotype
(Beech et al., 1994; Lubega et al., 1994).
3.2 Imidazothiazoles
An early study of levamisole resistance examined longitudinal contraction in
adult H. contortus worms in vitro and found that resistant worms were
considerably less sensitive to the effects of added acetylcholine (Sangster
et al., 1991), thereby highlighting changes to the nature of the cholinergic
receptor(s) as the likely mechanism of resistance. Subsequent work by this
same group using radioligand binding assays, again with H. contortus, demon-
strated that the binding of levamisole to its nicotinic acetylcholine receptor
(nAChR) target within the worm involved two sites and that levamisole-
resistant worms bound the drug less tightly at the low-affinity site than
susceptible worms (Sangster and Gill, 1999; Sangster et al., 1998b). These
findings indicated that the basis for the resistance was alterations at the
drug target site. A study of the mode of inheritance of levamisole resistance
indicated that the resistance was polygenic (Sangster et al., 1998a).
More recently, molecular studies of a number of parasitic nematode spe-
cies, including H. contortus, have provided insights into mechanisms of this
altered drug/receptor affinity. The studies of H. contortus have described
various molecular changes in the drug target (illustrated in Fig. 3) within
resistant worms:
1. Truncated nAChR subunit genes: Truncated forms of two nAChR subunit
genes, Hco-unc-63 and Hco-acr-8 (truncated forms denoted as Hco-unc63b
and Hco-acr8b, respectively), have been shown to be present in resistant
isolates and absent from susceptible isolates in a number of studies
(Fauvin et al., 2010; Neveu et al., 2007, 2010; Williamson et al.,
2011). Subsequently, Boulin et al. (2011) used a Xenopus oocyte heter-
ologous expression system to examine the impact of the truncated
H. contortus UNC-63 protein on the proper functioning of levamisole
sensitive nAChRs. These authors found that the truncated version
hampered the normal function of the receptors when both forms of
the protein (full length and truncated) were co-expressed, thereby
mimicking a levamisole resistance phenotype that may occur when
Figure 3 Acetylcholine cation channel targets of anthelmintics. (A) Nicotinic acetylcho-

line receptor (nAChR) subunit, (B) receptor and (C) subtypes in parasitic nematode cells.
In (B), channels composed of five similar or different subunits. Changes in the composi-
tion of the subunits may change the properties of the channel. Ancillary proteins and
microtubules anchor receptors in membrane rafts. In (C), the anthelmintics above the
cell schematic are agonist and those below the cell are antagonists. Subunit composition
may change receptor from N-, L- or B-type. M-type channels may be composed of
different subunits to other nAChRs. Different types of nAChRs may be expressed in
different cells. Changes in subunits may cause resistance to AChR anthelmintics.
Adapted from Prichard, R., Lespine, A., 2013. Genetics and Mechanisms of Drug Resis-
tance in Nematodes. In: Kennedy, M., Harnett, W. (Eds), Nematodes Molecular Biology,
Biochemistry and Immunology, second ed. CAB International, Wallingford, UK, pp. 156e183.
both forms are expressed together in nAChRs in resistant worms. Barerre

et al. (2014) examined further the occurrence of the truncated Hco-acr-8b
in resistant isolates from different geographical locations. These authors
identified an indel region of genomic DNA that was responsible for
the generation of the truncated splice variant, with the presence of the
63 bp indel leading to transcription of the full-length ACR8 protein
and the deletion of the 63 bp indel leading to the truncated ACR8b pro-
tein being formed, and an associated loss of levamisole sensitivity.
2. Reduced transcription of nAChR subunit genes: Williamson et al. (2011)

noted a significant decrease in transcription levels of Hco-unc-29.3 and
Hco-unc-63 in a resistant isolate of H. contortus, while Sarai et al. (2013)
noted that Hco-unc-63 was downregulated in resistant isolates. In addi-
tion, the latter authors reported that all four paralogs of Hco-unc-29
were significantly downregulated in adults of one resistant isolate.
Subsequently, Sarai et al. (2014) examined this isolate more closely by
subdividing it into subpopulations, showing increasing levels of resistance
in vitro. A comparison of the nAChR gene expresssion patterns in the
subpopulations showed a significant downregulation of many subunit
genes in the most resistant population (Hco-unc-63a, -63b, -29.2, -29.4,
-26 and -acr-8a).
3. Reduced transcription of ancillary protein genes: A study by Sarai et al. (2014)
(described above) also found that a number of genes associated with the
assembly of nAChRs were significantly downregulated in the most resis-
tant subpopulation (Hco-unc-74, -unc-50, -ric-3.1 and -ric-3.2). Some of
these genes had also been significantly downregulated across various
life stages of resistant isolates in their earlier study (Sarai et al., 2013).
Each of these molecular changes (truncation and reduced transcription of
nAChRs, reduced transcription of ancillary protein genes) could result in a
reduction in the number of functional levamisole receptors in resistant
worms, and hence explain the reduced drug binding in resistant worms
noted earlier by Sangster et al. (Sangster et al., 1998b; Sangster and Gill,
1999). However, none of these changes appear to be universally associated
with observed resistances, suggesting that resistance to levamisole in H. con-
tortus can arise by different mechanisms in different isolates. For example, in
terms of the truncated subunit genes:
• Neveu et al. (2010) detected the truncated Hco-unc-63 transcript in one
resistant H. contortus isolate; however, it was absent from a second resis-
tant isolate.
• Williamson et al. (2011) did not detect the truncated Hco-unc-63 tran-
script in their resistant isolate.
• Sarai et al. (2013) found that truncated Hco-63 was readily detectable in
both a susceptible and three resistant isolates.
• Sarai et al. (2013) found that truncated Hco-acr-8 was readily detectable in
larvae of both susceptible and resistant isolates and was absent from adults
of one resistant isolate.
Furthermore, as an example of inconsistencies of gene expression pat-
terns (not involving truncated forms), Sarai et al. (2013) found that the
Hco-29 paralogs showed unchanged expression relative to a susceptible

isolate, or significant downregulation, in different levamisole-resistant iso-
lates. Hence, it seems likely that H. contortus can utilize a variety of mecha-
nisms to render its nAChRs less sensitive to nicotinic agonist drugs. This
diversity in the specific nature of the resistance mechanism is problematic
for the design of molecular-based tests for resistance for this species (see
Section 4).
3.3 Macrocyclic lactones

Haemonchus contortus has been studied extensively with respect to the
mechanism of resistance to the ML drugs. Rohrer et al. (1994) found no dif-
ferences in ivermectin binding to membrane preparations from single resis-
tant and susceptible isolates, indicating that the resistance shown by this
single resistant isolate was not due to alterations of the target site. However,
the universality of this finding was unknown; hence, the target, namely,
glutamate-gated chloride ion channels (GluCls), was examined more closely
by other groups using molecular techniques. Blackhall et al. (1998a) re-
ported an increased frequency for an allele of an a-subunit GluCl gene in
laboratory-selected ivermectin- and moxidectin-resistant H. contortus iso-
lates, suggesting that a mutation in this gene was associated with the resis-
tance. Subsequently, McCavera et al. (2009) showed that substitutions of
various aromatic amino acid residues for the L256 residue in H. contortus Glu-
Cla3B genes transfected into COS-7 cell membranes caused a reduction in
the binding of ivermectin to membrane preparations, thereby highlighting
mutations at this position as a possible resistance mechanism. However, mu-
tation in the L256 codon has not been found in ML-resistant isolates from
the field. Changes in g-aminobutyric acid (GABA) receptors have also been
implicated in ML resistance in laboratory-selected resistant H. contortus
(Blackhall et al., 2003; Feng et al., 2002).
Recent studies have described the details of the interaction of iver-
mectin with GluCl receptors in nematodes (Hibbs and Gouaux, 2011;
Lynagh and Lynch, 2010, 2012). Haemonchus contortus was found to be
among a group of ivermectin-sensitive species (also including Cooperia onco-
phora and Dirofilaria immitis) that showed the presence of a glycine residue in
the third transmembrane domain of these receptors, in contrast to larger res-
idues at the same position in the receptors from ivermectineinsensitive
trematodes, such as Schistosoma mansoni, Schistosoma japonica and Clonorchis
sinensis (see Lynagh and Lynch, 2012). In addition, mutation of this glycine
residue in the H. contortus GluCla3B protein expressed on HEK293 cells
resulted in the loss of ivermectin sensitivity (Lynagh and Lynch, 2010).

However, despite these insights into the important determinants of ML
sensitivity, there is no evidence that changes in this amino acid residue
are responsible for field resistances in H. contortus or other worm species.
A study by Williamson et al. (2011) failed to find a link between mutations
in a number of ligand-gated chloride ion channels, including both GluCl
and GABA channels (avr-14B, glc-5, lgc-37 and glc-6) and ML resistance in
field-derived resistant H. contortus.
Resistance to MLs in H. contortus has also been examined with respect to
the possible role of changes in transcription levels of genes coding for iver-
mectin receptors. Williamson et al. (2011) found that transcription of two
GluCls (glc-3 and glc-5) was slightly reduced in a resistant H. contortus isolate;
however, the changes were modest and were not considered to be signifi-
cant enough to explain the observed levels of resistance.
A number of studies have indicated a role for P-glycoprotein (P-gp) drug
efflux pumps in ML resistance in H. contortus and other parasitic nematodes
(reviewed by Lespine et al., 2012). Two lines of evidence exist:
1. Molecular: Some early molecular analyses detected polymorphisms in
H. contortus P-gp genes that may have been associated with resistance
to MLs (Blackhall et al., 1998b; Sangster et al., 1999; Xu et al., 1998).
In addition, a number of studies have shown that the expression of
P-gps is increased in resistant isolates (Williamson et al., 2011; Xu
et al., 1998), while exposure of worm larvae and adults to ML drugs
has resulted in an overexpression of a number of P-gp genes in vitro
(Roulet and Prichard, 2006), and in vivo (Lloberas et al., 2013; Roulet
and Prichard, 2006).
2. Impact of P-gp inhibitors on ML sensitivity: A number of P-gp inhibitors
have been shown to reverse ML resistance in H. contortus, both in vitro
and in vivo (reviewed by Lespine et al., 2012). In vitro studies include
those by Bartley et al. (2009), Heckler et al. (2014) and Raza et al.
(2015). In vivo P-gp inhibitors have been used to increase the efficacy
of ivermectin and moxidetin towards resistant H. contortus in jirds
(Molento and Prichard, 1999; Xu et al., 1998) and ivermectin in sheep
(Lifschitz et al., 2010).
While target site changes and drug efflux pathways have therefore been
investigated with respect to ML resistance in H. contortus, and other species,
and shown to play roles in some isolates, no definitive mechanism that could
explain the observed field resistance was identified in research on these two
biological aspects.
A study by UrdanetaeMarquez et al. (2014), however, has provided

compelling evidence to indicate that another mechanism may be responsible
for ML resistance in H. contortus, namely, changes to the anatomy and/or
function of amphid sensory organs in resistant worms. This study examined
the H. contortus homologue of a C. elegans gene involved in the amphid
dye-filling defects observed in ML-resistant C. elegans worms (Urdanetae
Marquez et al., 2014) (Cel-dyf-7, and its homologue Hco-dyf-7). Polymor-
phisms in the H. contortus gene allowed for the identification of a resistant
haplotype, Hco-dyf-7(r), which was consistently present in resistant isolates
of H. contortus from a range of geographical origins. ML resistance, loss of
the amphid dye-filling phenotype, and the Hco-dyf-7(r) haplotypes all corre-
lated in H. contortus. The finding of this genetic change in ML resistance has
significant implications for the development of resistance-monitoring diag-
nostics (see Section 4).
3.4 Closantel
Closantel is efficacious only against blood-feeding parasites, such as H. con-
tortus and Fasciola hepatica. This narrow spectrum means that far less attention
has been paid to the interaction of the compound with nematodes compared
with other drug classes. However, resistance to the drug has appeared in
several countries (see Table 1) and, hence, there have been some studies
on the mechanisms responsible. Rothwell and Sangster (1997) examined
the uptake and metabolism of the drug in a closantel-resistant and a closan-
tel-susceptible isolate of H. contortus. These authors found that neither isolate
showed an ability to metabolize the drug in vitro or in vivo. They did report,
however, that radiolabelled closantel accumulated at lower levels in resistant
compared with susceptible worms following administration to sheep. The
mechanism responsible remained unknown; however, the authors specu-
lated that it may be due to reduced feeding by resistant worms, a reduced
level of dissociation of the drugealbumin complex in the worm gut, or
increased efflux of the drug from resistant worms. Several later studies failed
to detect polymorphisms in P-gp genes that might have explained the
reduced accumulation of drug in resistant isolates (Kwa et al., 1998; Sangster
et al., 1999). However, these studies were very limited in only examining a
small subset of the P-gp genes in this species; hence, the role of P-gps in clo-
santel resistance remains unknown.
3.5 Amino-acetonitrile derivatives

The study of possible resistance mechanisms that H. contortus may utilize
against the AADs proceeded at an earlier stage than for other drug groups,
as this was one component of the research conducted before the first drug
from this group, monepantel, was released into the market. The paper by
Kaminsky et al. (2008) announcing this new drug class also described exper-
iments in which H. contortus larvae had been selected with AADs over several
generations to produce mutant lines that were resistant to a therapeutic dose
of the drug administered to sheep. The resistant lines showed mutations in
two nAChR subunit genes: Hco-des-2H and Hco-acr-23H (subsequently
renamed Hco-mptl-1). A panel of loss-of-function mutations was identified
in the Hco-mptl-1 gene in the mutant lines. These various mutations included
deletions leading to mis-splicing and insertions and point mutations leading
to premature termination of translation of the protein. However, impor-
tantly, given the artificial nature of the resistance selection method utilized
in the study (larval selection pressure) and reports indicating that the type of
selection regime can have a significant impact on drug resistance phenotypes
and genotypes (Gill and Lacey, 1998; Gill et al., 1998; Sarai et al., 2015), it
remains to be seen whether these mutations in Hco-mptl-1 will also be
responsible for field resistances to this drug class. Monepantel resistance in
H. contortus has been reported from the field (Mederos et al., 2014; Van
de Bron et al., 2015). However, the mechanism responsible has not yet
been described.
3.6 Do different resistance mechanisms have general

fitness costs?
It is difficult to assess any fitness cost that may be associated with anthel-
mintic resistance and which could lead to some level of reversion to sus-
ceptibility, in the absence of selection pressure from a specific class of
anthelmintic. However, this is an important question for how we manage
anthelmintic resistance in the long term. Because studies to attempt to
assess any reversion to susceptibility of nematode populations containing
a proportion of resistant worms might take several years under field con-
ditions, data on this aspect are scarce. Nevertheless, there are some inter-
esting findings. Because the mechanisms of resistance to different drug
classes (and even to different anthelmintic molecules within a drug class)
are likely to vary, the measurement of reversion, as a marker for the fitness
cost of resistance, cannot be extrapolated from one drug class to another or
necessarily from one nematode species to another. Given these limitations,
what can we surmise about the possible fitness cost to different classes of
anthelmintics?
Interesting work has been done on BZ resistance in some nematode spe-

cies. In a field study over a 5-year period, involving sheep with a BZ resistant
strain of H. contortus, Waller et al. (1989) found some evidence of an increase
in the efficacy of thiabendazole (44 mg/kg) from 72% to 91% efficacy when
the BZ-resistant population was left untreated for 5 years. However, alter-
native treatment with only either levamisole or ivermectin over the 5 years
did not lead to an increase in thiabendazole efficacy. Concerning levamisole
resistance, it was noted in the same study that Trichostrongylus colubriformis in
the sheep were highly resistant to levamisole and a dose rate of 7.5 mg/kg
produced an efficacy of only 14% at the beginning of the study. In the group
of sheep left untreated with anthelmintics over the 5 years of the study, this
dose rate of levamisole produced an efficacy of 60% following the removal
of selection pressure for the period of 5 years. This suggests a strong rate of
reversion to susceptibility and a likely significant fitness cost of this type
of resistance, in this species. Furthermore, the use of thiabendazole
(44 mg/kg), eight times per year for 5 years, further increased levamisole ef-
ficacy to 84%. These limited data suggest that there may be some fitness cost
to anthelmintic resistance in some cases. However, usually when resistance
to an anthelmintic is detected, producers would likely switch to using
another class of anthelmintic or switch to combination treatment, often
with the combination still containing the anthelmintic to which some of
the worms have become resistant. In this latter scenario, there would be little
chance for possible reversion. In the case where a different class of anthel-
mintic is used, once resistance is established to the first anthelmintic class,
it may also be difficult to see reversion to susceptibility because of interacting
mechanisms. For example, it has been observed that MLs can select for some of
the genetic changes in b-tubulin (F200Y or F167Y) that cause BZ resistance in
H. contortus (Eng et al., 2006; Mottier and Prichard, 2008). In this context, it is
interesting that ivermectin binds to H. contortus b-tubulin (Ashraf et al., 2015),
such that there could be an effect of repeated ML treatment, limiting possible
reversion to BZ susceptibility. This is of particular interest as MLs have been
very commonly used alone or in combination with BZs when BZ resistance
becomes problematic. Considering the evidence, discussed in Section 3
(Mechanisms of Resistance), it would seem quite likely that there could be
a fitness cost associated with levamisole resistance (defective n-acetylcholine
receptors), and with ML resistance (changes in the Hco-dyf-7 gene which
causes impaired development of dendrites in the sensory amphids). However,
definitive evidence of these likely fitness costs is still lacking.
4. DIAGNOSIS OF RESISTANCE
4.1 In vivo resistance tests
While anthelmintic efficacy can be determined by controlled tests
involving comparison of postmortem worm counts after anthelmintic treat-
ment, with those in comparable untreated animals, the FECRT is most
often used to estimate efficacy of anthelmintics against gastrointestinal nem-
atodes, including H. contortus, by comparing feacal egg counts before a drug
treatment to the counts after treatment. The test has been described in some
detail by Coles et al. (1992, 2006) as it is recommended by the WAAVP for
the routine diagnosis of resistance in nematode parasites. Resistance is
conventionally considered to be present in the test population if the percent-
age reduction in egg count is <95%, and the lower 95% confidence level is
<90%. The test, however, suffers from limitations which affect its wide-
spread use for H. contortus and other species. These limitations include the
costs of conducting the test, its inherent lack of accuracy due to the impre-
cise nature of faecal egg counting techniques and biological variations in egg
output, and a lack of sensitivity (Levecke et al., 2012; Martin et al., 1989;
McKenna, 1990; Miller et al., 2006; Taylor et al., 2002; Waller, 1997).
Despite these shortcomings, it remains the currently used yardstick test for
the detection of resistance in the field.
4.2 In vitro bioassay-based tests for Resistance

Alternatives to the FECRT have been sought for many years. Haemonchus
contortus has been the subject of many of these studies, and in many cases
it has served as the model organism for the development of the assay
methods. One option that has been examined is the use of in vitro bioassays
with the free-living life stages to measure sensitivity to anthelmintics and
hence detect any changes in sensitivity that may indicate that resistance is
emerging. Such bioassays are much cheaper than FECRTs, relatively rapid,
avoid any host animal effects, and avoid some of the inaccuracies associated
with FECRTs (see Section 4.1). In addition, such tests can use a range of
drug concentrations to generate defined values describing the effect of a
drug on the worm population, for example, half maximal inhibitory con-
centrations (IC50 values) and resistance ratios.
4.2.1 Egg hatch assay

An egg hatch assay for detecting resistance to BZ drugs in H. contortus
and Teladorsagia circumcincta was first described by Le Jambre (1976).
Thiabendazole was used for the assay, because of its greater solubility in
aqueous media compared to some other BZ compounds. Subsequently,
Dobson et al. (1986) adapted the assay to detect levamisole resistance. The
use of the assay for BZ resistance detection in field populations was described
by Coles et al. (1992). A population was designated as resistant if the IC50
was >0.1 mg/mL thiabendazole. Coles et al. (2006) added to the interpreta-
tion of assay results by describing this concentration of drug as preventing the
hatching of 99% of eggs from susceptible H. contortus populations (and those
of other species), such that the proportion of eggs that did hatch at this con-
centration could be interpreted as an approximation of the percentage of
resistant individuals within that population. An effort by a number of
European laboratories to standardize the assay for the detection of resistance
to BZs resulted in the publication of a standard operating procedure for con-
ducting and interpreting the test (von Samson-Himmelstjerna et al., 2009a).
The H. contortus component of this study showed that the test was a reliable
indicator of the presence of resistance for this nematode species.
4.2.2 Larval development assay

The larval development assay (LDA) measures the effects of drugs on the
development of eggs to infective third-stage larvae (L3). A number of var-
iations on the basic assay format have been described, although they gener-
ally fall into two groups of either using a liquid medium (Coles et al., 1998;
Hubert and Kerboeuf, 1992; Taylor, 1990), or allowing the development of
the larvae to occur in a thin layer of liquid overlaying agar (Gill et al., 1995).
A number of studies have shown that the assay is able to discriminate be-
tween isolates of H. contortus that are susceptible or resistant to all the three
major drug classes: BZs, imidazothiazoles and MLs. Gill et al. (1995) showed
that ivermectin aglycone and ivermectin monosaccharide were superior to a
number of other ML compounds for resistance detection in H. contortus as
these derivatives discriminated to a greater extent between ML resistant
and susceptible populations. However, more recently, several studies have
also shown that eprinomectin is also useful in such assays (Dolinska et al.,
2013; Kotze et al., 2014a). The LDA is also able to define the dose responses
of larval life stages to the organophosphate naphthalophos (Kotze et al.,
1999), although its ability to detect resistance to this drug (that is, discrimi-
nate between susceptible and resistant isolates) has not been reported. The
assay has recently been shown to clearly discriminate between isolates sus-
cpetible to monepantel and a field-derived isolate showing resistance to
this drug (Raza et al., 2016).
The LDA was developed in the 1990s as a diagnostic method for the eval-
uation of the resistance status of nematodes in Australia (Drenchrite LDA,
Microbial Screening Technologies, Australia). Producers submitted faecal
samples to diagnostic labs and in turn received a report on the resistance status
of the nematodes on their property. The response of the worm larvae to BZ
drugs, levamisole or a BZ/levamisole combination in the LDA could be used
to estimate drug efficacy from a table of assay data correlated with slaughter
trial data. For MLs, the output of the Drenchrite was less clear although still
useful, with the tests able to indicate whether resistance was present rather
than an expected drench efficacy. The Drenchrite test was seen as a much-
needed tool, and the initial response from producers was favourable. How-
ever, it soon became clear that, while the test worked well with BZs and
levamisole for H. contortus and the other important species in Australia,
and was adequate for ML drugs with H. contortus, it performed very poorly
for this drug class for T. circumcincta. Hence, despite its effectiveness for
H. contortus, it was withdrawn from use because it was not applicable to other
species. The test has, however, continued to perform well as a resistance
detection tool where H. contortus is the only species of interest (eg, Dolinska
et al., 2012, 2013; Kaplan et al., 2007; Terrill et al., 2001).
A study by Kotze et al. (2002) reported a single ML-resistant isolate of
H. contortus that showed a different pattern of resistance across the free-living
and parasitic life stages than that normally reported for field isolates. The
adult worms of this isolate were highly resistant, being unaffected by a ther-
apeutic dose of ivermectin in vivo; however, the larval stages showed only a
very low level of resistance in the LDA. The use of the LDA as a field diag-
nostic would depend on isolates of this nature remaining rare and on a better
appreciation of the expression of resistance mechanisms in different stages of
the life cycle.
4.2.3 Larval motility/migration assay

A number of assay formats have been devised for assessing the effects of an-
thelmintics on the movement of parasitic worms, including H. contortus.
These assays have often been developed as drug screening tools for use in
drug discovery programmes; however, in some cases, they have been applied
to the detection of resistance. The methods applied to H. contortus include:
• the use of a micromotility metre to measure movement in H. contortus L3
stage larvae (Folz et al., 1987);
• the scoring of larvae as motile or nonmotile by observation after a period
of exposure to the drug (Gill et al., 1991);
• the separation of drug-affected and nonaffected larvae based on their

ability to migrate through a mesh (Demeler et al., 2010; Rothwell and
Sangster, 1993; Sangster et al., 1988);
• migration out of an agar block (d’Assonville et al., 1996);
• migration through soft agar, followed by migration through a mesh
(Kotze et al., 2006).
The tests have been used as experimental tools for resistance detection,
with limited use in field settings. Hence, many protocol variations have
been described. Demeler et al. (2010) were the first to attempt to develop
a standardized protocol using H. contortus, as well as Ostertagia ostertagia and
C. oncophora. The test was based on the migration of larvae through a filter
mesh following a period of incubation in an aqueous solution containing
ivermectin. The assay was able to distinguish the highly ivermectin-resistant
CAVR isolate (Le Jambre et al., 1995) from a susceptible isolate. However, to
date, the use of this standardized protocol for resistance detection with field-
collected samples has not been reported. Hence, its usefulness for detection of
low levels of resistance remains unknown. A further consideration impacting
on the potential role of such tests for resistance detection is the reported
greater ability of eprinomectin to discriminate between ML-resistant and
ML-susceptible isolates in migration assays compared with ivermectin (Kotze
et al., 2006). Efforts to develop and apply a standardized test should con-
sider using this ML drug to maximize the resistance-detection sensitivity of
the test.
There have been a number of advances in recent years in the development
of automated methods to measure the movement of nematodes (for example,
Buckingham et al., 2014; Buckingham and Sattelle, 2009; Smout et al., 2010;
Storey et al., 2014). Efforts to develop these techniques have been driven by
the need for sensitive, high-throughput methods for anthelmintic discovery
using large chemical compound libraries. However, in some cases these
new techniques have been applied to the detection of resistance in worms,
including H. contortus, to existing anthelmintics (Smout et al., 2010). While
some of these new assays may be highly sensitive with respect to the detection
of anthelmintic effects (low IC50 values), it is important to consider whether
this translates into greater sensitivity for the detection of resistance, that is, an
ability to distinguish between susceptible and resistant isolates. For example,
the assay described by Smout et al. (2010) was more sensitive than a more
traditional migration/motility assay (Kotze et al., 2006) in terms of showing
a lower IC50 for both ivermectin-susceptible and ivermectin-resistant isolates.
However, it was less sensitive than the migration/motility assay in discrimi-

nating between resistant and susceptible isolates.
4.3 Molecular-based tests

The advances in our understanding of the molecular basis of some of the
anthelmintic resistances shown by H. contortus (described in Section 3)
have led to some optimism that molecular-based tests may be possible for
diagnosis of some of these resistances. This area of research has been a partic-
ular focus for the Consortium for Anthelmintic Resistance and Susceptibility
(CARS) group of scientists, and progress towards the development of mo-
lecular markers has been described in various papers from scientists in the
group (eg, Beech et al., 2011; Gilleard and Beech, 2007; Kotze et al.,
2014b; Prichard et al., 2007; von Samson-Himmelstjerna et al., 2007).
The possibilities for developing such molecular tests in the short term are,
however, quite different for the different chemical classes due to the varying
levels of understanding of the molecular nature of the various resistances (see
Section 3). For BZs, the work, to date, to develop molecular tests has been
successful, as this is the best understood of the resistances at the molecular
level. For the other drug groups, the uncertainty about the specific nature
of resistance mechanisms in field isolates, and the fact that molecular changes
reported for some isolates did not occur in other resistant isolates, as
described earlier, has delayed the development of molecular diagnostics.
Coles et al. (2006) described an allele-specific PCR for detection of BZ
resistance-associated F200Y SNP in H. contortus using DNA extracted from
L3 stage larvae. Other molecular methods have also been described for the
detection and/or quantification of this SNP, as well as the other two asso-
ciated with BZ resistance (F167Y, E198A), including restriction fragment
length polymorphism-PCR (Ghisi et al., 2007; Tiwari et al., 2006), real-
time PCR (Alvarez-Sanchez et al., 2005; von Samson-Himmelstjerna
et al., 2009b; Walsh et al., 2007) and pyrosequencing (von Samson-Him-
melstjerna et al., 2009b). This latter study described pyrosequencing assays
for H. contortus codons 167, 198 and 200 of b-tubulin isotypes 1 and 2.
The method proved able to assess the BZ resistance status of a number of
H. contortus isolates, indicating that it may be suitable for routine diagnosis
of resistance in this species. A comparison of molecular data with egg hatch
data showed that the molecular test was not always correlated with the de-
gree of resistance; however, it was able to discriminate well between resistant
and susceptible isolates. The inability to directly correlate with resistance
levels suggests that other resistance mechanisms beside b-tubulin mutations
may also be contributing to resistance in some isolates. The lack of a strong

correlation between the molecular tests and the biological tests, such as the
FECRT, may also reflect on the insensitivity of the biological test used in the
comparison. The ability to detect the presence of resistance in a population
by a molecular test means that the test could be utilized as a diagnostic tool
for detection of resistance, even if not able to produce a quantification of the
level of resistance comparable to FECRT. Two studies (Barrere et al., 2013a,
b) have evaluated the use of the pyrosequencing technique for diagnosis of
BZ resistance in H. contortus under field conditions in Canada. Both studies
showed that the molecular tests were in agreement with more laborious and
expensive FECRTs in diagnosing resistance, but as the level of BZ resistance
was high, it was not possible to calculate a correlation with FECRT results.
Until recently, molecular tests for the other drug classes had not yet been
applied in any field setting due largely to the fact that no suitable molecular
markers have been identified. The identification of a genetic marker for
levamisole resistance in H. contortus (Barrere et al., 2014), based on the pres-
ence or absence of an indel in the Hco-acr-8 gene, may mean that levamisole
resistance caused by this genetic change could be easily detected. However,
the complexity of possible changes to the levamisole receptor, which can
result in loss of levamisole sensitivity (see Section 3.2), may make develop-
ment of a simple and universal molecular assay for the detection of levam-
isole resistance difficult.
The work linking the Hco-dyf-7 gene to ML resistance in H. contortus (see
Urdaneta-Marquez et al., 2014) does, however, raise the possibility of devel-
oping molecular-based tests for detection of ML resistance in the field. The
dyf-7 gene markers have recently been used to assess ivermectin resistance on
numerous sheep farms in Canada and Sweden, and initial evidence suggests a
significant correlation with FECRT data (Prichard, Urdaneta, Barrere,
H€ oglund, unpublished). These markers may prove to be useful for field-
based diagnostic tests. However, the effectiveness of the dyf-7 markers across
H. contortus populations from different regions requires further evaluation.
5. CONCLUSIONS
Haemonchus contortus has shown a history of being able to readily
develop resistance to whatever chemicals are used intensively for its control.
It is too early to judge how long it may take for resistance to the recently
introduced monepantel and the abamectinederquantel combination
(Startect) to have any widespread impact on the effectiveness of these control

options, although, as noted earlier, several reports of resistance to monepan-
tel in H. contortus have already emerged. The expected increase in prevalence
and severity of resistances in field isolates of this species to the available drugs
will make it difficult for producers to control H. contortus on a worldwide
scale. To maintain an ability to control this parasite until further drugs
become available, it is important to develop better diagnostic methods, so
that producers and their advisors may be able to identify drugs or drug com-
binations that remain effective on their farms. Furthermore, to develop bet-
ter diagnostic tests for resistance and to provide good advice on how to limit
resistance development, or deal with it once it is present, it will be important
to gain a much better understanding of the mechanisms and genetics of
anthelmintic resistance.
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CHAPTER TEN
Haemonchus contortus:
Applications in Drug Discovery
T.G. Geary
McGill University, Québec, Canada
E-mail: timothy.g.geary@mcgill.ca
Contents
1. Introduction 430
2. Drug Discovery 431
2.1 Drug discovery: in vitro 431
2.1.1 Larvae 431
2.1.2 Adult stages 436
2.1.3 Mechanism-based assays using H. contortus 437
2.2 Drug discovery: in vivo 439
2.3 Drug discovery: the role of pharmacodynamics 442
2.3.1 Drug efflux 444
2.3.2 Drug metabolism 445
3. Perspectives on H. contortus and Drug Discovery 447
4. Challenges and Research Opportunities 450
References 453
Abstract
Haemonchus contortus is an important pathogen of small ruminants and is therefore a
crucially important target for anthelmintic chemotherapy. Its large size and fecundity
have been exploited for the development of in vitro screens for anthelmintic discovery
that employ larval and adult stages in several formats. The ability of the parasite to
develop to the young adult stage in Mongolian jirds (Meriones unguiculatus) provides
a useful small animal model that can be used to screen compounds prior to their eval-
uation in infected sheep. This chapter summarizes the use of H. contortus for anthel-
mintic discovery, offers a perspective on current strategies in this area and suggests
research challenges that could lead to improvements in the anthelmintic discovery
process.

j
ISSN 0065-308X
430 T.G. Geary
1. INTRODUCTION
Anthelmintic discovery is an endeavour of rare success (Geary et al.,
2004, 2009, 2015; Woods et al., 2007). Historically, discovery of anthelmin-
tics for veterinary use has constituted almost the only exercise in this area;
adaptation of veterinary medicines for human use is rule rather than the
exception in this arena (Geary and Gauvry, 2012). Limited economic returns
compared with drugs for human use and the rare application of species-level
diagnostics for parasitic nematodes of veterinary importance has led to a dis-
covery and development paradigm in which broad-spectrum anthelmintics
are required. Nonetheless, among the possible target pathogens for anthel-
mintic deployment, trichostrongyloid nematodes that parasitize ruminants
are arguably the most important. Although several species in this family
have been used for drug discovery and pharmacological investigations,
Haemonchus contortus has been the most prominent. This status is due to its
significance as a global primary pathogen of small ruminants, the relatively
low cost of its ruminant host (sheep) compared with cattle, its large size
and its fecundity. Infections in young sheep can persist for weeks. Female
H. contortus can produce thousands of eggs per day, which can be harvested
and cultured in abundance from faeces. Development to the infective L3
stage in the laboratory is simple to accomplish, and the larvae can be stored
at 4 C for prolonged periods. The large size of adults allows relatively so-
phisticated, yet straightforward pharmacological studies in vivo and in vitro.
Its robust biology has allowed the use of a drug discovery paradigm that
moves rapidly from target parasite in culture to small animal model to a
target host. As a representative of other ‘clade V’ parasitic nematodes (see
Blaxter, 2003), H. contortus has become an essential model and target organ-
ism in the field of anthelmintic discovery.
The use of H. contortus for drug discovery falls into four main categories:
(1) in vitro screens for anthelmintic discovery, (2) the use of infected animal
models as initial in vivo screens, (3) in vitro studies using adults to charac-
terize drug effects and (4) studies of the identification and of anthelmintic
targets. The material in this chapter has been filtered to include only studies
that specifically employ H. contortus, and only studies that address classical,
small molecule anthelmintics. This choice reflects the topic and not the
value of alternative approaches. Although this chapter is intended to be
comprehensive, in some areas, only representative rather than exhaustive ex-
amples are described.
Haemonchus contortus: Applications in Drug Discovery 431
2. DRUG DISCOVERY
The history of anthelmintic discovery for veterinary applications has
been authoritatively reviewed (Standen, 1963), and has been summarized
(Geary, 2012; Woods et al., 2007). The role of H. contortus in this process
merits special consideration, as this organism has provided a widely used plat-
form and paradigm for modern anthelmintic discovery and development.
2.1 Drug discovery: in vitro

2.1.1 Larvae
Initial systematic attempts to discover novel anthelmintics were based on
testing compounds in infected hosts, using native hostesmall animal models
including rodents or chickens, and so employed model parasites in lieu of the
most important veterinary species. These models were undeniably success-
ful, and led to the discovery of levamisole (Thienpont et al., 1966), pyrantel
(Austin et al., 1966) and ivermectin (see Campbell, 2012). However, the
routine use of infected animals for primary anthelmintic screening was, of
necessity, low throughput and incurred high costs for labour and compound
supply. Many factors worked in concert to divert investment in anthelmintic
discovery towards systems that could accommodate higher throughput with
reduced costs and chemical demand. As medicinal chemistry strategies
changed and led to the routine synthesis of quite small amounts of com-
pounds (<1 mg), there was pressure to minimize the initial amount of
chemical used in any screen. This demand coincided with economic pres-
sures to reduce labour and animal expenses, and with changes in the ethics
of animal use that led to initiatives to minimize the numbers used in research.
The goal thus became to test only the most promising compounds in animal
models. This change in strategy led to the development of whole organism
and, later, mechanism-based primary screens. Since systems which enable
long-term culture of adult parasites (the primary target for chemotherapeutic
control of trichostrongyloid parasites) are not at hand, many strategies that
employed in vitro culture of the readily available larval stages of pathogenic
and model nematode species were investigated. Indeed, the first truly safe
and broad-spectrum anthelmintic, thiabendazole, was discovered in a tri-
chostrongyloid larval assay (Brown et al., 1961).
The use of H. contortus larval cultures for anthelmintic discovery and
characterization was pioneered conceptually in the 1960s (McKern and
Parnell, 1964; Parnell, 1964). More intensive investigations, including
432 T.G. Geary
comparative studies, appeared somewhat later (Boisvenue et al., 1977, 1983;

Ibarra and Jenkins, 1984). Although much of the groundwork was done in
laboratories in animal health companies, which lacked strong motivation to
publish, it is clear that considerable investment was made in developing reli-
able screens using larval stages. These efforts led to the development of
several different screening strategies; Boisvenue et al. (1983) measured
motility and viability in ensheathed L3 stages over 24 h exposures, while
Ibarra and Jenkins (1984) measured drug effects over a 4-day period begin-
ning with eggs in a larval development assay (LDA). This system measured
drug effects on egg hatching, larval development to the L3 stage and, using
microscopy, L3 motility and viability. In each case, visual examination was
used to compare drug-free controls to drug-treated parasites. Although
LDAs had relatively prolonged times to read out, they proved to be much
more sensitive to standard anthelmintics than a 24 h test which directly
measured the effects of anthelmintics on L3 motility and viability. Most
notably, the 24 h test did not identify the majority of benzimidazole anthel-
mintics (Boisvenue et al., 1983), leading to the conclusion that it would not
be as useful as a rapid primary screen of chemical libraries for anthelmintic
discovery.
In contrast, the LDA identified every important class of anthelmintic,
which could be distinguished by differential effects on egg hatching, devel-
opment and motility/viability (Ibarra and Jenkins, 1984). An important
addition to the assay repertoire was a larval migration assay, which measured
the ability of the L3 stage, including that of H. contortus, to migrate out of
agar gels (Douch et al., 1983; Douch and Morum, 1994; see Kotze et al.,
2006a). Variations on these tests became frequently used in anthelmintic dis-
covery operations. The relatively low cost of maintaining the parasite in
lambs and the hardiness of the larval stages have made H. contortus egg hatch
and LDAs globally useful platforms for identifying anthelmintics in collec-
tions of synthetic chemicals and natural products.
Examples of the use of H. contortus larvae for anthelmintic discovery are
floridly abundant; a prominent case is the work done in Australia by Lacey
et al., who explored the use of a modified LDA as a primary screen of diverse
biological materials for natural products with anthelmintic activity. The
screening strategy employed both larval development and larval motility as-
says, which were used to detect or characterize active ‘hits’ (Gill et al., 1991,
1995; Lacey et al., 1990). This work was initiated with streptomycetes and
fungi, known sources of multiple kinds of bioactive molecules, including an-
thelmintics (Anderson et al., 1995; Lacey et al., 1995). Development of a
reliable set of larval assays for relatively high-throughput operation led to a

long-term collaboration with Novartis to screen novel marine biota for
nematocides (Capon et al., 1999, 2001, 2002a,b, 2003, 2004a,b, 2005;
Narkowicz et al., 2002; Ovenden et al., 1999; Vuong et al., 2001). Although
a large number of structurally novel candidate anthelmintics were identified
in this endeavour, none appears to have progressed into full development;
the inability of whole-organism screens to predict new compounds with
activity in animal models is a well-known gap in anthelmintic discovery
(see following sections). The assay employed in this work became commer-
cially available (NemaTox, Microbial Screening Technologies Pty Ltd) for
screening purposes and was used to identify a series of (Z)-2-phenyl-
3-(1H-pyrrol-2-yl)acrylonitrile analogues as potential endectocides (Ali
et al., 2007). This work was followed up with additional medicinal chemis-
try investment and an LDA screen (Gordon et al., 2014), but selectivity
vis-a-vis mammalian cell toxicity was difficult to achieve and a candidate
for clinical development does not seem to have emerged from this series.
The same system was used to identify a different series of ectoparasiticides
that were active against H. contortus larvae (Ali et al., 2008), and a series of
2-phenylimidazo[1,2-b]pyridazines that were highly potent in the Nema-
Tox assay (Ali et al., 2011). None of these series appears to have yet gener-
ated a new anthelmintic candidate.
Larval H. contortus assays have been widely used to screen collections of
synthetic chemicals, either in high-throughput fashion, screening collections
of random compounds, or for more targeted screens that tested compounds
with previously known pharmacology or bioactivity. Indeed, such assays
were very commonly employed in animal health companies with an interest
in the discovery of novel anthelmintics, although much of this effort has not
been published. One such example was the identification of a series of thio-
phenes as anthelmintic candidates in a screen employing a migration assay, in
which L3 stages were exposed to compounds for 2 or 24 h and then allowed
to migrate through a mesh at the bottom of a 96-well plate (Gonzalez et al.,
2004). Again, in vitro activity against the larval stage of a target parasite did
not translate into in vivo activity against adult stages of the same parasite in a
jird model (Gonzalez et al., 2004), for reasons that remain unknown.
Fruitful results were obtained from a screen of compounds active in
agrochemical screens for activity in an H. contortus LDA, which identified
a compound that was the precursor of the novel anthelmintic monepantel
(Ducray et al., 2008; Kaminsky et al., 2008a,b). Targeted screening using
a modified H. contortus LDA (Gill et al., 1995), followed by evaluation in
434 T.G. Geary
jirds infected with H. contortus and Trichostrongylus colubriformis (see Conder

et al., 1991) led to the identification of the amino-acetonitrile class of anthel-
mintics and prioritization of monepantel for more advanced development.
The ease of using larval stages of H. contortus for the identification of
novel compounds with anthelmintic activity has led to the continuing
employment of this model. The most common applications have been in
the area of natural product screening, usually focused on botanical prepara-
tions. This strategy has been carried out in more established laboratories
using various versions of larval assays; examples include a method using
exsheathed L3 larvae exposed for 10 h to plant extracts, with viability moni-
tored by measuring the ability of the parasites to reduce methyl-thiazolyl-
tetrazolium (MTT; H€ ordegen et al., 2006); a screen of Australian shrubs
in a standard LDA (Kotze et al., 2009); and a feeding inhibition assay
employing L1-stage trichostrongyloid larvae, including H. contortus and
Escherichia coli labelled with fluorescein isothiocyanate to screen plant extract

and fungal lectins (Ríos-de Alvarez et al., 2012). The feeding inhibition assay
used a fluorescence microscope to detect ingestion of bacteria after 18 h of
drug exposure. Later work confirmed that fungal lectins are relatively potent
inhibitors of H. contortus larval development (Heim et al., 2015). Even
though these products may not have potential for the treatment of infected
animals, the fungal strains that produce the lections may be able to limit
larval contamination of pasture to reduce parasite burdens. In other exam-
ples, a screen of fungal products measured anthelmintic activity by micro-
scopic evaluation of H. contortus L3 larvae after 72 h exposure (Ayers
et al., 2012) and detected one compound with selective anthelmintic
activity. A small-scale screen of the natural product norcantharadin and its
derivatives revealed that selective inhibition of parasite serineethreonine
phosphatases may have potential for the development of novel anthelmintics
(Campbell et al., 2011a). A similar screen of derivatives of haliclonacycl-
amine marine alkaloids identified analogues active in an LDA (Banwell
et al., 2012), though insufficient material was available for further
characterization.
Of interest is the adaptation of H. contortus larval assays for use in testing
potential anthelmintic botanical preparations in settings with sometimes
limited resources. The simplicity and low cost of running H. contortus egg
hatch and LDAs has been exploited in low-throughput screens in Africa
(Diehl et al., 2004; Eguale et al., 2011; Tadesse et al., 2009) and India
(Kamaraj and Abdul Rahuman, 2011; Kamaraj et al., 2011), and screens
in Mexico, in which fourth-stage larvae (L4) of H. contortus were exposed
to plant extracts, with viability scored after 72 h by detection of motile par-

asites with a microscope (Aguilar et al., 2008; Moreno et al., 2010). A com-
bined egg hatch and larval migration strategy was used to characterize
anthelmintic activity in plants native to the US northern Great Plains
(Acharya et al., 2014). It must be emphasized that these are examples, and
do not comprise an exhaustive list. It is important to recognize that current
efforts to identify anthelmintic active principles in extracts of natural prod-
ucts in nonindustry laboratories overwhelmingly rely on H. contortus larval
assay systems. Finally, it is also worthwhile noting that H. contortus larvae
(and adults) have been used to identify and characterize intriguing bioactive
peptides and proteins, including Bt toxins (Kotze et al., 2005; O’Grady et al.,
2007) and cyclotides (Colgrave et al., 2008a,b, 2010). Further work is
needed to conclude if nonsmall molecule chemotherapeutic strategies can
play an important role in the management of haemonchosis in sheep.
Since the inception of whole-organism anthelmintic screens using larval
stages of H. contortus, many assay modifications have been introduced, some
of which were highlighted above. High-throughput screening strategies rely
on the minimization of labour, compound consumption and time to accel-
erate the discovery process. Standard screens based on larval development
required microscopic evaluation of larval stage and behaviour after several
days of culture, and modifications have emphasized shorter times of evalu-
ation and automatable data acquisition platforms. Examples include assays
based on automated readouts of larval motility after short exposures to test
compounds; although such tests cannot discriminate about agents that paral-
yse parasites versus those that are directly lethal (eg, metabolic poisons), they
provide a rapid readout of overall anthelmintic activity in formats that mini-
mize compound requirements and labour. The first such system was a
micromotility meter (Bennett and Pax, 1986), which was adapted for use
with H. contortus L3s (Folz et al., 1987, 1988; Varady and Corba, 1999).
The micromotility meter employs a light sensor that captures signals from
the meniscus of a solution containing parasites; active movement of parasites
leads to increased deflections of the beam. Paralysed or dead parasites are
detected as reductions in deflections of the beam (Bennett and Pax,
1986). Because of the limitations of the automated platform, which
employed culture tubes rather than multiwell plates, it was used as a second-
ary, low-throughput screen rather than a primary high-throughput opera-
tion. As discussed in the following sections, this instrument also proved to
be useful for the monitoring of the response of adult H. contortus to drugs
in culture.
436 T.G. Geary
Further modifications and improvements of larval assays provide evi-

dence that the use of H. contortus for drug discovery and characterization re-
mains appealing. A higher-throughput, 96-well plate-based larval motility
assay offers a 3-day format compatible with drug screening (Kotze et al.,
2006a). Realization that viability of exsheathed L3 stage H. contortus can
be monitored colorimetrically with MTT formation (H€ ordegen et al.,
2006) has afforded the possibility to discriminate poisons from compounds
that act as paralytics on the neuromuscular system, an important distinction
in pursuing leads; this objective can be realized if the time course of drug
action is followed. This assay may have field applications, as it is easier to
measure colour than motility in some settings (Al-Rofaai et al., 2013).
The development of image analysis platforms for monitoring nematode
motility (eg, Buckingham and Sattelle, 2009) provided new opportunities
for high-throughput approaches to characterize drug effects on H. contortus
larval stages (Buckingham and Sattelle, 2009; Preston et al., 2015a; Smout
et al., 2010). Importantly, these systems are amenable to automation, allow-
ing for a throughput, limited only by the availability of the parasite (a few
100 to a few 1000 per well).
2.1.2 Adult stages

Although several 1000 adult H. contortus may be obtained from a single
infected sheep, the cost of maintaining large numbers of this host species pre-
cludes the use of adult parasites in a primary high-throughput screen. How-
ever, the ability to keep H. contortus adults viable in culture for 2e3 days (see
Geary et al., 1993) enabled the development of secondary screens to prior-
itize hit compounds identified in larval screens or screens using other nem-
atodes. The micromotility meter (Bennett and Pax, 1986) was adapted for
this purpose at the Upjohn Company, in which hits obtained in primary
screens using Caenorhabditis elegans (eg, Conder et al., 1992a; Lee et al.,
2002) were characterized for activity against adult H. contortus (see Bacon
et al., 1998; Conder et al., 1995; Geary et al., 1993; Johnson et al., 2004).
The motivation behind this strategy was to obtain an estimate of the expo-
sure-efficacy profile necessary to cause toxicity (indicated by reduced
motility) against the target stage of a target parasite. Understanding the rela-
tionship between duration of exposure, drug concentration and parasite
viability illuminates the likelihood of attaining efficacy in vivo, and can pre-
clude the advancement of test compounds that have little chance of activity
in infected animal models based on pharmacokinetics achieved in standard
dosing regimens (see following sections). Using groups of five adults,
motility scores were reported at short intervals over 48e72 h in parasites

exposed to various concentrations of test compounds. Datasets obtained
from these studies were useful in understanding anthelmintic pharmacody-
namics (see Ho et al., 1994; Johnson et al., 2004). However, application of
this screen as a filter did not enhance the direct identification of new
compounds that progressed to clinical development (T. Geary, personal
observations).
Similar aims were promoted by O’Grady and Kotze (2004), who devel-
oped a motility assay to detect drug effects on adult H. contortus. Motility was
estimated by visual inspection of groups of 10 parasites 24, 48 or 72 h after
exposure; the authors also measured drug effects on feeding by monitoring
the ingestion of [3H]inulin, which was found not be suitable for routine use
in this context. Of a collection of 200 compounds selected for activity
against H. contortus larvae, only 10 were significantly active in the adult assay.
Whether any of these compounds was active in an infected animal model
was not revealed, but the results illustrate that there may be a high propor-
tion of ‘false negatives’ in screens employing larval stages of H. contortus. It
must be concluded that it remains to be determined whether prioritization
of initial hits based on activity against adult stages in culture can improve the
success rate for whole-organism screens using H. contortus.
2.1.3 Mechanism-based assays using H. contortus

The case for mechanism-based screening for new anthelmintics has been
made repeatedly (eg, Geary, 2012; Geary et al., 1999; Kotze, 2012; Krasky
et al., 2007; Thompson et al., 1996). In contrast to screens that employ
whole organisms and measure survival or behaviour, mechanism-based
screens seek compounds that act on a single protein (typically) under the
premise that interdiction of the function of the protein will have deleterious
consequences for the parasite in the host. In the case of H. contortus, work has
been based on the expression of H. contortus proteins as drug targets in het-
erologous systems that are suitable for high-throughput screening, or
through bioinformatics-based identification of potential drug targets fol-
lowed by whole-organism chemical validation with compounds that are
known to be active against the target in other systems.
Initial examples of mechanism-based screens for anthelmintics were
based on the ability of genes encoding H. contortus metabolic enzymes to
rescue strains of microorganisms (E. coli, Saccharomyces cerevisiae) with null
mutations in the homologous gene. This strategy was used to screen for in-
hibitors of phosphofructokinase (Klein et al., 1991), phosphoenolpyruvate
438 T.G. Geary
carboxykinase (Klein et al., 1992) and ornithine decarboxylase (Klein et al.,

1997). Specific enzyme inhibitors could be identified by growing the recom-
binant strains in defined media that isolated the essential function of the para-
site enzyme (Klein and Geary, 1997; Geary, 2001) in nutrient-dependent
viability assays. Although hits were identified in these screens, none was found
to be suitable for clinical development (T. Geary, personal observations).
An illustrative and successful example of this strategy is found in work done
by scientists at Elanco. This group identified a H. contortus gene encoding a
serotonin (5-HT) G proteinecoupled receptor (GPCR) and functionally
expressed it in a mammalian cell line (Smith et al., 2003). Pharmacological
analysis demonstrated that it had a different ligand profile than mammalian
5-HT receptors and was thus a potential target for anthelmintic discovery.
Screening with the recombinant cell line identified p-amino-phenethyl-m-
trifluoromethylphenyl piperazine (PAPP), a known ligand of monoamine
GPCRs, as an interesting hit (White et al., 2007). Testing in a H. contortus
LDA (Gonzalez et al., 2004) and in a T. colubriformis larval migration assay
showed that PAPP was as potent as levamisole (White et al., 2007). Admin-
istration of PAPP to jirds infected with H. contortus at 100 or 50 mg/kg
resulted in high efficacy, a result that was duplicated in jirds infected with
Teladorsagia circumcincta. However, efficacy was considerably lower against
T. colubriformis in jirds, and dosing subcutaneously or oral administration of
different salts of PAPP failed to improve activity (White et al., 2007). As
nearly universal breadth of spectrum against the trichostrongyloid nematodes
of ruminants is an essential quality for field use, this compound was not pro-
gressed further, and no anthelmintic targeted to monoamine GPCRs has yet
been published as an advanced clinical candidate.
Incorporation of this and related targets in a novel screening approach
was achieved by complementation of the free-living nematode C. elegans
with H. contortus genes encoding 5-HT or tyramine GPCRs (Law et al.,
2015). This strategy employs strains of C. elegans with null mutations in
the genes encoding the 5-HT1 receptor or the tyramine receptor. These
strains were than transformed with cDNAs encoding heterologous mono-
amine GPCRs from H. contortus, Drosophila melanogaster and humans; the re-
ceptors were targeted to be expressed in cholinergic motor neurons or body
wall muscle through the use of tissue-specific promoters. Ligands (including
tyramine, 5-HT and PAPP) induced overt paralysis in animals ectopically
expressing these receptors. Differential sensitivity to PAPP in animals
expressing the H. contortus versus human 5-HT1 receptor documented the
selectivity of this molecule for nematodes. As screens employing C. elegans
were long a staple for anthelmintic discovery in the animal health industry
(see Geary and Thompson, 2001; Geary et al., 1999), adaptation of this plat-
form for differential high-throughput screening would be straightforward.
An alternative approach identifies potential H. contortus drug targets
through bioinformatics searches, using a variety of strategies (eg, Campbell
et al., 2011a,b; Doyle et al., 2010; Kotze, 2012; Krasky et al., 2007; Preston
et al., 2015b; Taylor et al., 2013a,b; Wang et al., 2015a,b). Cases in which
some chemical validation of the target was obtained by assays using H. con-
tortus include the identification of norcantharadin analogues as protein phos-
phatase inhibitors, some of which were active in an LDA, albeit with
relatively low potency (Campbell et al., 2011b). A screen of a set of known
protein kinase inhibitors identified three compounds that were active in an
L3 motility assay (Taylor et al., 2013a), and screening a different set of kinase
inhibitors in a larval motility assay identified two active compounds with
modest potency (Preston et al., 2015b). An analysis of potential target en-
zymes that serve essential choke-point functions revealed that perhexiline,
a carnitine palmitoyl transferase inhibitor, was active and modestly potent
in a larval motility assay (Taylor et al., 2013b). Finally, using the same para-
digm, two lysine deacetylase inhibitors were shown to have potent effects in
this assay (Wang et al., 2015b).
Although none of the hits from these screens has been reported to be
active against adult stages of H. contortus or in infected animal models, these
examples illustrate the potential of bioinformatics-driven approaches to
enhance the success rate of hit identification in screens using H. contortus.
A crucial aspect of using this approach for anthelmintic discovery is the
need to prove on-target effects of the test compounds, most of which
were not remarkably potent against the larval stage of the parasite. Translating
target conservation across the phylogenetic gulf that separates nematodes and
mammals is challenging, and many compounds in collections of inhibitors
may affect multiple targets at high concentrations. Strategies advocated for
H. contortus in this regard have generally taken this into account by incorpo-
rating follow-up assays to validate the on-target nature of the hits. This
confirmation should be an essential step before investing additional medicinal
chemistry or preclinical development resources into a hit compound or series
to avoid chasing compounds that act through unknown mechanisms.
2.2 Drug discovery: in vivo

Anthelmintic discovery originally began with experimental treatment of
infected animals; at least the prototype of almost all anthelmintics available
440 T.G. Geary
on the market was discovered in an infected animal screen (Geary et al.,

2015). For the important trichostrongyloid nematodes of ruminants, lambs
infected with H. contortus were once employed for this purpose (eg, Folz
et al., 1976; Kind et al., 1996) and, indeed, verification of anthelmintic ac-
tivity against this parasite in lambs is often the crucial, final gate through
which a candidate anthelmintic must pass to progress to clinical develop-
ment. However, using infected sheep for screening requires large amounts
of chemical compounds as well as high labour and housing costs, and the
use of multiple sheep per dose of each compound was necessary to achieve
statistical significance, given the large interhost variability in adult worm
numbers. Alternative screens employing nonruminant parasites in rodent
models were available (Coles, 1986, 1990; D€ uwel, 1975; Johnson et al.,
1999). Although these screens had been used to discover anthelmintics
(eg, Austin et al., 1966; Brown et al., 1961; Campbell, 2012; Sasaki et al.,
1991; Thienpont et al., 1966), concerns about their relevance for parasites
of livestock motivated the search for models that employed target trichos-
trongyloid nematode infections.
Switching screening paradigms led to new challenges. Whole-organism
or mechanism-based screens, which operate at high throughput (thousands
to tens of thousands of compounds a day) identify a plethora of hits that
cannot all be validated in costly infected animal models. Nonetheless, assess-
ment of anthelmintic efficacy and potency in an infected animal model
remains the primary go/no-go gate for directing additional chemistry and
preclinical development resources to a particular compound. The use of
standard ‘small animal’eparasite systems as models, particularly rodents
infected with host-specific parasites, was undeniably successful, as discussed
in the previous section, but did not directly address the most important
target parasites (eg, trichostrongyloid nematodes). A major advance in this
regard was the discovery that the Mongolian jird, Meriones unguiculatus,
could be infected with Trichostrongylus axei (see Leland, 1963). This discov-
ery led to studies showing that rodents, especially jirds, infected with T. colu-
briformis constitute a highly useful platform for anthelmintic characterization
(eg, Kates and Thompson, 1967; Ostlind and Cifelli, 1981; Panitz and
Shum, 1981; Williams and Palmer, 1964).
The jird model was developed for use with H. contortus by a team at the
then Upjohn Company (Conder et al., 1990, 1992b). The administration of
hydrocortisone in the food provided modest immunosuppression and
allowed efficient infection with H. contortus L3 stages; exsheathment prior
to infection led to much higher numbers of parasites at necropsy, which
reached the immature adult stage by day 14 post-infection (Conder et al.,

1992b). Importantly, a coinfection model in which both H. contortus and
T. colubriformis could be simultaneously evaluated offered the opportunity
to generate an initial assessment of spectrum of action at a very early stage
(Conder et al., 1991; Ostlind et al., 2006). The jird model provided a robust
and highly reproducible system for the analysis of anthelmintic efficacy and
pharmacology, as discussed in the following section, and greatly enhanced
the throughput of experimental compounds in an early in vivo screen, while
markedly lowering compound requirements and labour costs compared
with sheep models.
Examples of success in anthelmintic discovery based at least in part on the
use of H. contortus in jirds include monepantel and derquantel. Monepantel,
an amino-acetonitrile derivative, was the fruit of an anthelmintic discovery
project at Novartis Animal Health, which focused on testing compounds
previously identified as interesting in screens for agricultural indications.
These test compounds were first evaluated in an LDA employing H. contortus
and T. colubriformis (to test for spectrum at an early stage), with active
compounds then evaluated in a jird model that used coinfections with
H. contortus and T. colubriformis (Kaminsky et al., 2008a,b; Kaminsky and
Rufener, 2012). This process led to the identification of active leads; subse-
quent rounds of medicinal chemistry with iterative testing in this same
scheme led to the selection of highly active analogues, including monepan-
tel, which were then evaluated in sheep and cattle.
Derquantel (2-desoxoparaherquamide) was discovered in a more circular
process. A high-throughput screen of fermentation products in an assay that
measured behaviour, reproduction and viability of C. elegans identified a
bioactive fungal isolate, Penicillium roqueforti. When tested in jirds infected
with H. contortus, the fermentation extract proved to be highly active (Lee
et al., 2002; Woods et al., 2012). Fractionation of the extract led to the iden-
tification of marcfortine A as the active component; interestingly, this mole-
cule is almost inactive against C. elegans, and the activity in the original
extract was found to be due to the coproduction of the mitochondrial toxin
oligomycin (Woods et al., 2012). A considerable medicinal chemistry
program produced analogues that were evaluated in jirds coinfected with
H. contortus and T. colubriformis. Promising compounds were then character-
ized in a sheepeH. contortus model, progressing to clinical development of
derquantel.
Other compounds have also been characterized in the jirdeH. contortus
model, including the progenitor of the cyclodepsipeptide class, PF-1022A.
442 T.G. Geary
This fungal product, like marcfortine A and the paraherquamides, has min-
imal overt effects in the C. elegans screen (T. Geary, unpublished observa-
tions) but is highly potent against H. contortus L4s and adults in culture
and in jirds infected with this parasite, T. colubriformis and Ostertagia ostertagi
(see Conder et al., 1995). The same group used the H. contortusejird
model to characterize the anthelmintic natural product dioxapyrrolomycin
(Conder et al., 1992a), and used this model, along with a larval migration
assay, to show that this compound shared a mechanism of action with clo-
santel; it was not developed further. It was also used as the primary assay to
evaluate anthelmintic activity in a series of b-ketoamides (Lee et al., 1998),
although none of these compounds progressed past evaluation in a H. con-
tortusesheep assay. A series of compounds with insecticidal activity was
assayed in a mixed H. contortuseT. colubriformis jird model, revealing com-
pounds with good activity against the former but not the latter parasite spe-
cies (Wickiser et al., 1998); these compounds do not appear to have
progressed through clinical development. Although the H. contortusejird
model has clearly benefitted the study of anthelmintic pharmacology and
anthelmintic discovery, additional work could enhance its value in identi-
fying promising compounds.
It is essential to realize that the jird, as a monogastric rodent, may not
accurately reflect the pharmacokinetics of anthelmintic candidates in sheep,
which are ruminants with considerably different digestive system anatomy
and physiology prior to the intestinal tract. Even though efficacious dose
levels of orally administered anthelmintics in jirds are quite close to those
in sheep for H. contortus (Conder et al., 1990, 1991), this relationship may
not hold for all compounds. Correlating pharmacokinetic parameters in jirds
and sheep with data on in vitro pharmacodynamics can help to resolve dis-
crepancies in activity between the two in vivo models should they arise.
2.3 Drug discovery: the role of pharmacodynamics

While clinical pharmacology of anthelmintics in the host has been a subject
of considerable interest (see Lanusse et al., 2016), less attention has been paid
to understanding how parasites accumulate and dispose of these drugs and
how these parameters influence intrinsic activity of anthelmintics or test
compounds. The relatively small size of most parasitic nematodes and the
requirement to maintain them in host animals (as opposed to continuous
culture) are challenges to this kind of work. Early studies on drug uptake
and accumulation in H. contortus monitored the influence of pH on the up-
take of thiabendazole (Davis et al., 1969). Subsequent studies described the
physicalechemical characteristics of solute transport across the surface of

parasitic nematodes, with an initial focus on the large species Ascaris suum
(see Ho et al., 1990, 1992; Sims et al., 1994), but later extended to
H. contortus (see Alvarez et al., 2007; Ho et al., 1994; Johnson et al., 2004;
Sims et al., 1996; Thompson et al., 1993).
These studies led to a number of important conclusions about drug accu-
mulation in parasitic nematodes, including H. contortus. The primary source
of drug entry into parasitic nematodes was found to be via transcuticular
diffusion, limited by size and lipophilicity of the solute. The cuticle is
composed of tortuous pores that limit diffusion of molecules with molecular
masses above w3000 Da. These pores are aqueous compartments with pH
w5, based on the excretion of the acidic end products of parasite glycolysis.
Solute entry into the parasite is also limited by the requirement to diffuse
across the lipophilic external surface of the parasite body wall, so that
charged or highly hydrophilic molecules accumulate slowly. In general,
the characteristics of solute absorption by parasitic nematodes are similar
to those which govern absorption from the mammalian intestine. It is
important to note that this may not be the case for the free-living nematode
C. elegans, for which xenobiotic accumulation may be quite distinct from
this process in mammals (see Burns et al., 2010, 2015; Ruiz-Lancheros
et al., 2011) and parasitic species. Importantly, work has shown that bio-
accumulation of compounds in larval stages of H. contortus in culture is gov-
erned by the same general principles that are operative in adult stages (Zhou
et al., 2015), suggesting that this stage is not likely to generate frequent false
negatives based on differential exclusion of xenobiotics.
Further experiments enabled the correlation of intrinsic potency and
accumulation of anthelmintics in adult H. contortus (see Ho et al., 1994;
Johnson et al., 2004; Thompson et al., 1993) and have led to the hypothesis
that anthelmintic accumulation occurs primarily by transcuticular diffusion.
Furthermore, efficacy is predicted by the relationship of intrinsic potency to
equilibrium levels of an anthelmintic inside the parasite. Integrating these
concepts with subsequent research on routes of drug efflux and metabolism,
as discussed below, have led to a better understanding of the fact that internal
concentrations of anthelmintics reflect a balance between rate of entry and
rate of efflux/metabolism until equilibrium is reached. Unfortunately, little
quantitative research on whole-parasite pharmacodynamics has been pub-
lished recently; it remains an area of important research.
As noted, the other determining factor in the kinetics of anthelmintic
accumulation is the ability of nematodes to eliminate xenobiotics, including
444 T.G. Geary
drugs, or to convert them to inactive or less-active derivatives. Recent work,

much of it employing H. contortus, is illuminating this aspect of organism-
level pharmacodynamics of anthelmintics. Two general areas are of interest
in this regard: the ability of nematodes to efflux drugs via P-glycoprotein
(Pgp) pumps and the ability to metabolize them via cytochrome P-450
(CYP450)emediated or conjugative processes.
2.3.1 Drug efflux

Pgps and related transport proteins play important roles in the development
of resistance to chemotherapeutic agents in many settings; their role with re-
gard to this phenomenon is explored elsewhere in this volume (Kotze and
Prichard, 2016) and will not be addressed here. However, to the extent
that endogenous Pgps (or related drug pumps) may influence the accumu-
lation or retention of anthelmintics in H. contortus, it is a relevant topic.
Early studies, using various polymerase chain reaction (PCR)ebased
strategies, revealed the presence of a number of Pgp-like cDNAs in H. con-
tortus (Godoy et al., 2015; Kerboeuf et al., 2003; Lespine et al., 2012;
Prichard and Roulet, 2007; Sangster et al., 1999; Smith and Prichard,
2002; Williamson and Wolstenholme, 2012; Xu et al., 1998), findings
confirmed in the sequenced genome of this organism (Gasser et al., 2016;
Liang et al., 2013; Schwarz et al., 2013). The abundance and tissue distribu-
tion of these proteins is compatible with a potential role in regulating drug
accumulation in parasites. In accord with such a role in other systems, one
could predict that these pumps would facilitate expulsion of xenobiotics
from nematodes. Unfortunately, very few studies have been done to mea-
sure this phenomenon directly. This situation reflects the scarcity of available
supplies of radiolabelled anthelmintics and the relatively small size of parasitic
nematodes, which hampers the use of less-sensitive detection methods.
Although several studies have measured the accumulation of drugs and other
xenobiotics by H. contortus in culture (Bartíkova et al., 2012; Ho et al., 1994;
Johnson et al., 2004; Rothwell and Sangster, 1997; Sims et al., 1996;
Thompson et al., 1993), only one has directly monitored the efflux of an
anthelmintic. Thus, Bartíkova et al. (2012) exposed adult H. contortus to flu-
bendazole (a highly lipophilic anthelmintic) in culture and measured its dis-
tribution and metabolism. The data support a model in which both drug
uptake and elimination after placing exposed parasites in drug-free medium
could be explained by passive diffusion; the addition of the Pgp inhibitor
verapamil had no effect on the elimination kinetics in benzimidazole-
sensitive or -resistant strains.
Indirect data have addressed the possible role of Pgps in limiting drug
accumulation in H. contortus by measuring lethality in the presence or
absence of Pgp inhibitors. A study employing first-stage larvae (L1s) of
H. contortus (and T. circumcincta) showed that several compounds known to
inhibit mammalian Pgps were remarkably effective in increasing sensitivity
to ivermectin in this stage, with an increase in potency of 15- to 70-fold;
this effect was relatively independent of the resistance status, which itself
was not a marked phenotype in this stage (Bartley et al., 2009). These
data suggest that the actively feeding L1 stage may employ Pgps as a protec-
tion against exposure to toxic xenobiotics, such as macrocyclic lactones, in
the environment. In contrast, experiments with nonfeeding H. contortus
L3 larvae in a migration assay and in an LDA (egg to L3) revealed less dra-
matic effects of Pgp inhibitors (Raza et al., 2015). The inhibitors had rela-
tively modest effects on increasing the sensitivity of a susceptible strain to
ivermectin in the LDA, although the effects were more marked for some
of them in the migration assay (which was w10,000-fold less sensitive to
the drug than the LDA). Unfortunately, effects of Pgp inhibitors on adult
stages of this parasite, which is the primary target for chemotherapy, have
not been reported. This study also included thiabendazole and levamisole
for comparison; although some inhibitors affected the potency of these com-
pounds in the assays, it was not possible to conclude that Pgps regulate accu-
mulation of these drugs in H. contortus.
Additional studies with anthelmintics and adult stage parasites are
required to achieve conclusive evidence about the roles that Pgps or other
drug transporters may play in governing the response of H. contortus to these
drugs.
2.3.2 Drug metabolism

Two main pathways account for metabolism of most drugs: CYP450-medi-
ated reactions and conjugations of xenobiotics with water-soluble groups
(glutathione conjugation, glucuronidation and so on). It has been known
for some time that H. contortus has classical CYP50 enzyme reactions,
including aldrin epoxidation and ethoxycoumarin O-deethylation (Kotze,
1997). These activities were much higher in L1 and L3 stages than in adults,
suggesting that encounters with potentially toxic xenobiotics in the environ-
ment may be more frequent than in the stomach of the host. Work at the
genomic level identified a number of candidate CYP450 genes in H. contor-
tus (see Laing et al., 2015). Although function and substrate specificity have
not been confirmed for any of these genes, several are close homologues of
446 T.G. Geary
C. elegans P450s that are involved in xenobiotic metabolism. Many of the

H. contortus CYP450 candidate genes are expressed in the intestine, and
most are much more highly expressed, based on real-time PCR analysis, in
larval stages compared to adults. Little information is available about
the possible roles of CYP450-mediated reactions in H. contortus adults to
the biotransformation of anthelmintics; Alvinerie et al. (2001) reported the
slow production of an unidentified metabolite of moxidectin in homogenates
of adult H. contortus, which was inhibited by CO, a known poison for
CYP450-mediated reactions. Further efforts to identify this metabolite and
define its bioactivity do not seem to have been reported. Similarly, it has
been shown that exposure of larvae and adult H. contortus (and T. colubriformis)
to piperonyl butoxide, a broad-spectrum CYP450 inhibitor, potentiates the
toxicity of the mitochondrial poison rotenone (Kotze et al., 2006b), suggest-
ing a role for CYP450-mediated detoxification of this natural product. How-
ever, metabolism of rotenone was not directly measured in these experiments
to confirm this hypothesis. Although rigorous confirmation of a contribution
of CYP450-mediated reactions to the metabolism of currently available an-
thelmintics has yet to be reported in H. contortus, it must be recognized that
differential expression of these enzymes could lead to differences in drug
sensitivity between larval and adult stages. Importantly, it is possible that
drug screens performed with larval stages may miss active compounds that
would be active against adults due to stage-specific metabolism.
In contrast, non-CYP450emediated reactions that metabolize anthel-
mintics have been more commonly reported in H. contortus and other hel-
minths (Cvilink et al., 2009), and genes predicted to encode homologues
of these kinds of drug-metabolizing enzymes are clearly present in the
H. contortus genome (Laing et al., 2013). For example, investigations with
anthelmintics have shown the adult H. contortus maintained in culture
generate multiple metabolites of the benzimidazoles albendazole and flu-
bendazole (Cvilink et al., 2008a,b; Vokral et al., 2012). These studies
showed that H. contortus can generate albendazole sulphoxide but not alben-
dazole sulphone, and can reduce the ketone group of flubendazole to the
hydroxyl via a carbonyl reductase. Both metabolites are bioactive. In
addition, glucose conjugates (mediated by UDP-glucosyltransferase activity)
of albendazole, flubendazole and reduced flubendazole were detected;
whether the conjugates were more readily released into culture medium
than the parent drugs was not reported. In addition to these drugs, mone-
pantel has been shown to be susceptible to metabolism by adult H. contortus
in culture (Stuchlikova et al., 2014). Nitrile hydrolysis was detected and four
metabolites of the drug were found but not further characterized; no con-
jugates were observed. The extent to which these activities protect H. con-
tortus from the lethal effects of monepantel remains to be determined.
Finally, it has been found that larval stages of H. contortus express UDP-
glucuronosyltransferase activity that is induced by exposure to phenobarbital
and inhibited by classical blockers of these enzymes (Kotze et al., 2014). Glu-
curonidation of the anthelmintic cholinesterase inhibitor naphthalophos was
observed and shown to be relevant for drug toxicity, in keeping with the
results reported for expression of CYP450-mediated reactions. Whether
adult stages exhibit the same phenomena vis-a-vis glucuronide formation
is unknown. Finally, it must also be noted that no metabolites of ivermectin
or closantel were detected upon incubation of adult H. contortus with these
anthelmintics (Rothwell and Sangster, 1997; Vokral et al., 2013), suggesting
that the ability to biotransform drugs is class specific and is more limited than
is the case in mammalian hosts. Clearly, further systematic analyses of the
biotransformational capacities of H. contortus are warranted.
3. PERSPECTIVES ON H. CONTORTUS AND DRUG

DISCOVERY
The drug discovery and development process can be conceptualized as
a series of gates through which compounds must pass to earn additional in-
vestment of chemistry and biology resources. These gates are arranged to
minimize the risk of a ‘false-negative’ result, the failure to detect a com-
pound which is a genuine active (adequate potency, efficacy and specificity
to warrant additional investment). False negatives are unforgiveable in
screening, as a wrongly rejected compound cannot be rescued at a later stage
in the process. To avoid false negatives, screens usually accept any com-
pound with even minimal potency/efficacy, even though the vast majority
of these hits will later prove to be without value. So-called ‘false positives’
are accepted because later assays will remove them from the active file.
Optimal screening processes remove false positives as early as possible,
with as little additional investment as possible. Gates are thus set to ensure
detecting all active compounds and then to quickly winnow down the num-
ber of actives to prioritize those with real apparent value for further study.
The first gate depends on the source of compounds for testing. For high-
throughput screens with H. contortus this includes the testing of random
(unselected) compounds from chemical collections, either synthetic or nat-
ural products, a format employing larval stages; although this is not the target
448 T.G. Geary
stage for anthelmintics, it is not feasible to obtain the large numbers of or-
ganisms required to assay tens of thousands of compounds at a time. The
choice of larval screen strategy and readout (development or motility or
both) is flexible; whether ensheathed or exsheathed L3 stages should be
used in an acute motility screen has not been definitively decided; depend-
ing on the exposure time, ensheathed L3 stages may be more susceptible to
certain anthelmintics (Patel and Campbell, 1997), but extended times of
exposure may remove this difference (eg, Douch and Morum, 1994). In
contrast to high-throughput random screens using larvae, mechanism-based
screens against H. contortus targets will generate fewer hits, as will be the case
for compounds identified in repurposing exercises.
For repurposed compounds selected for activity in nonnematode sys-
tems, an important first step in this approach is to test the hypothesis that
the hit compound acts on the proposed target. Activity against parasites in
culture, especially only if obtained at concentrations of 1 mM, is no guar-
antee of an on-target effect, and resources can be wasted chasing compounds
with higher potency or selectivity if the target is different from that hypoth-
esized. Off-target hits have no more value than those derived from a blind
screen, and must be further evaluated in that context.
Validation of repurposed compounds and hits from high-throughput larval
assays or mechanism-based screens can be done in a low-throughout adult
H. contortus bioassay as a secondary screen. As an alternative, primary screen
hits could be tested immediately in H. contortuseinfected jirds (eg, Conder
et al., 1992b, 1995; Lee et al., 2002). However, inactive compounds are elim-
inated at this gate, with no possibility of understanding the basis for the in vi-
troein vivo discrepancy in activity. This strategy also has a high demand for
animal usage, and reduction in animal use can be achieved by using adult par-
asites isolated from a small number of experimentally infected lambs as a sec-
ondary whole-organism screen prior to enlisting infected jirds as a gate.
An essential aspect of a secondary screening strategy employing adult
H. contortus in culture is to employ an assay readout that can identify the
range of phenotypic alterations that can reflect anthelmintic activity against
adult H. contortus. As discussed in the previous section, the simplest assay is to
monitor motility, either visually or preferably with an automated system
(Bacon et al., 1998; Conder et al., 1995; Geary et al., 1993; Johnson
et al., 2004; Kotze et al., 2012; O’Grady and Kotze, 2004). Additional mea-
surements, such as ATP levels, can distinguish metabolic poisons from para-
lytic agents (Bacon et al., 1998; Geary et al., 1993). Pharyngeal pumping can
be monitored in adult H. contortus by measuring effects on the ingestion of
[3H]inulin (Geary et al., 1993; Kotze, 2000; Paiement et al., 1999) or

another easily detectable substrate which cannot cross the nematode cuticle,
such as dextran labelled with FITC (Kotze et al., 2012). Additional informa-
tion on drug effects can be obtained in less facile assays, including measure-
ments of effects on somatic muscle function using isolated muscle strips
(Atchison et al., 1993) or cannulated intact adult parasites (Demeler et al.,
2014; Marks et al., 1999; Sangster et al., 1991), or a variety of structural
and biochemical effects (reviewed by Kaur and Sood, 1986). Because these
assays are tailored, to some extent, to detect the activities of known anthel-
mintics, it is not clear if they would be plagued by a high frequency of false-
negative results. Additional research, possibly using repurposed compounds
or hits identified in mechanism-based assays, could reveal the extent to
which simple adult H. contortus secondary screens can serve as a useful gate
for prioritizing the admission of test compound to testing in in vivo models.
Information obtained from adult H. contortus bioassays can include
organismal pharmacokinetics for compounds of high value. For example,
mechanism-based assays generate hits with defined affinity (eg, IC50 value)
for the drug target; similar information should be obtained with the parasite
target of repurposed compounds whenever possible. Comparing IC50 values
with potency (EC50 values) in whole-organism bioassays can reveal whether
toxicity is only observed at concentrations far above those needed to affect
the target. Measuring drug levels accumulated in adult parasites can then
reveal if the discrepancy is due to low uptake. If not, the possibility that
the toxicity is due to off-target effects becomes uncomfortably real.
Determining the exposure-efficacy profile of a test compound in adult
H. contortus in culture, as noted in the previous section, could be an impor-
tant step in prioritizing compounds for testing in jirds. It is generally difficult
to sustain drug levels much higher than 1 mM for prolonged periods in
model or target hosts following oral or parenteral dosing of unformulated
compounds. Test compounds that require long-term exposure to concen-
trations >1 mM for >24 h to affect adult H. contortus in culture are, by
this criterion, unlikely to be active in infected jirds. However, this remains
a hypothetical statement; no correlation between potency/speed of action of
test compounds with activity in the jird model has been established. It re-
mains unclear whether adult worms of H. contortus in culture are faithful rep-
resentatives of the parasite in situ.
Exposure-efficacy profiles can also enhance the analysis of data from
infected jird assays. Although the proper host compartment in which to
measure anthelmintic concentrations is not well defined for all parasite
450 T.G. Geary
species (Lanusse et al., 2016), for H. contortus in jirds, blood or stomach con-
tents/mucosa are the compartments in which parasite exposure to a drug oc-
curs. As the ability to conduct preliminary pharmacokinetic measurements
in these compartments becomes more readily available, in vivo efficacy
can be analysed in light of predictions based on in vitro data. If the duration
of exposure to efficacious concentrations of test substances in jirds meet or
exceed the profile established in culture but fail to result in parasite clearance,
it can be concluded that the series is unlikely to produce a compound that
warrants clinical development. This kind of correlation has been established
for several kinds of anthelmintics (Ho et al., 1994; Johnson et al., 2004) and
should provide value for new series as well. Importantly, the jird model is
also capable of detecting time-to-effect (expulsion/removal) of adult H. con-
tortus following drug administration (Johnson et al., 2004; Rothwell et al.,
1993), allowing correlation of time-to-effect in vitro and in vivo as an added
parameter for discriminating among hits.
Defining the exposure-efficacy profile against adult H. contortus in cul-
ture assists in resolving the common gap between the activity of a hit com-
pound in culture compared with its activity in an animal model (eg,
Gonzalez et al., 2004).
Optimal success in drug discovery is achieved when a minimal amount
of resources is invested to identify a compound that is highly effective
against the target parasite in a target host in an acceptable regimen and
with no overt toxicity. Compounds that meet these criteria pass into clinical
development, with the additional investment of resources in determination
of spectrum, optimal formulation, pharmacokinetics, comprehensive toxi-
cology, manufacturing and so on. The costs of clinical development are
high, and it is essential to invest only in compounds with obvious promise.
Challenges include the need to ensure that investment is targeted only at
the most promising compounds and that the decisions to cease investment
are made as quickly as possible, based on data that generate high confidence
in the process. It is useful to build a hypothesis-driven paradigm into the oper-
ation as early as possible; this strategy provides a method to reach high-quality
decisions rapidly, and also may indicate ways to improve the screening funnel.
4. CHALLENGES AND RESEARCH OPPORTUNITIES

The global distribution of strains of trichostrongyloid parasites of sheep
and cattle that are resistant to currently marketed anthelmintics has
intensified the need for new drugs in this arena. Discovery programs depend
on high-quality basic research for support. Unfortunately, discovery pro-
grams aimed at veterinary parasites rank low on the priority list of most
governmental funding bodies, and the continued contraction of the animal
health industry through acquisitions and mergers has severely restricted the
number of industrial laboratories with a deep interest in this area, reducing
opportunities for collaborative research with academic groups. Nonetheless,
it is useful to identify challenges and gaps in knowledge that would benefit
from increased research.
The necessity of maintaining sheep infected with H. contortus as a
source of larvae and adults is a significant impediment to the use of this
parasite for drug discovery. In this regard, development of an egg-to-egg
culture system for this parasite would be of immense benefit. Culture
systems for trichostrongyloid parasites have not been reexplored after
intriguing initial success with some species (eg, Douvres, 1979, 1980;
Douvres and Malakatis, 1977; Leland, 1967; Hansen and Hansen, 1978;
Silverman and Hansen, 1971; Smyth, 1990; Stringfellow, 1986), and the
devotion to this area based on knowledge obtained in other systems in
the past decades, together with advances in analytic platforms, could
have enormous benefits.
There remains a widely acknowledged, if little discussed, gap in anthel-
mintic discovery between activity in whole-organism assays and activity in
infected animal models. Millions of compounds and fermentation extracts
have been screened in primary assays (see Geary et al., 2015), with hit rates
ranging from 0.1% to 10% (T. Geary, unpublished observations). Just a
handful of compounds have been reported to exhibit significant activity
in infected animal models, with only two novel molecules advancing to
the market for control of trichostrongyloid nematodes of livestock in the
past 25 years, neither of which has yet been developed for cattle (a third,
emodepside, is marketed for companion animal parasites).
Several factors may underlie this gap. For instance, it may be that the
demand for broad-spectrum activity with high selectivity and low cost-of-
goods is simply too high a bar to meet, and that many actives which meet
some of these criteria have been discovered but have been ‘dropped’
without publication. It is also possible that the chemical collections screened
for anthelmintic activity are biased in favour of compounds derived from
chemistry exercises focussed around indications relevant for major human
diseases, and are not enriched for anthelmintic templates. It is also possible
that ‘screeners’ have been too lenient in passing compounds with weak or
452 T.G. Geary
unpromising whole-organism activity to infected animal models to avoid

missing any promising hits (fear of false negatives). Finally, it is also possible
that parasites in a host are significantly different from those in a culture well,
and we are failing to screen against the ‘right’ organism; parasites in culture
may simply be so stressed that they are easier to kill than parasites in a host,
leading to the routine discovery of false positives.
Steps to address some of these possibilities are apparent. Application of
mechanism-based screens and bioinformatics-driven compound repurpos-
ing efforts can, at least in theory, generate sets of hits that are more likely
to adversely affect parasites in vivo, assuming adequate pharmacokinetics
and target validation. The application of exposure-efficacy profiling should
enable a better prioritization of compounds for testing in infected animal
models. Finally, the rigorous application of transcriptomics and proteomics
analyses to adult parasites freshly removed from the host, in comparison with
parasites maintained for 24e72 h in culture, could reveal whether short-
term culture causes marked changes in gene expression. Similarly, additional
transcriptomic analysis of L3s in comparison with adults (see Gasser et al.,
2016; Schwarz et al., 2013) could help define how appropriate it is to use
larvae as a primary screening tool. In this regard, it is worthwhile to note
that the transcriptomic profile of in vitroegenerated L4s is similar to that
of adults removed from sheep (Schwarz et al., 2013). It would be valuable
to compare the exposure-efficacy profile of available anthelmintics in L4s
and adult H. contortus; if the profiles are closely related, it should be possible
to substitute the more readily available L4 stages for adults in secondary
screens to eliminate the need to maintain donor sheep (or jirds) as a source
of adult parasites for this purpose.
Additional pharmacodynamic experiments with adult and larval H. con-
tortus are also warranted. In particular, it would be of benefit to directly anal-
yse the ability of this parasite to efflux compounds in both stages, and a more
thorough analysis of drug-metabolizing capacity could identify the likeli-
hood that these processes could lead to a failure to detect valuable templates
in primary or secondary screens.
It is clear that H. contortus remains a valuable tool in anthelmintic discov-
ery programmes. It is equally clear that further improvements in this discov-
ery process are possible. Given the renewed emphasis on anthelmintic
discovery in light of drug-resistant strains of trichostrongyloid nematodes,
it is to be hoped that a renewed emphasis on the basic and applied pharma-
cology of this benchmark species will soon be evident.
ACKNOWLEDGEMENTS
I thank my former colleagues at the Upjohn Company (Pharmacia e Pfizer e now Zoetis) in
Kalamazoo, Michigan, for allowing me to be part of the antiparasitic drug discovery group; it
is in that stimulating environment that I learned how to use H. contortus for anthelmintic dis-
covery. Much of their excellent work is highlighted in this chapter. I acknowledge support
for preparing this review at McGill University from the Canada Research Chairs and the
FRQNT-supported Centre for HosteParasite Interactions.
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CHAPTER ELEVEN
Gaining Insights Into the

Pharmacology of Anthelmintics
Using Haemonchus contortus as a
Model Nematode
C.E. Lanusse1, L.I. Alvarez, A.L. Lifschitz
1
Corresponding author: E-mail: clanusse@vet.unicen.edu.ar
Contents
1. Introduction 466
2. Background on the Pharmacology of Anthelmintic Drugs 467
3. Assessment of Parasite Exposure to Anthelmintic Drugs Using Haemonchus 476
contortus as a Target Nematode
3.1 Haemonchus contortus as a tool to explore ex vivo and in vivo drug 476
accumulation, metabolism and activity in nematode parasites
3.2 Influence of the route of drug administration on parasite exposure 485
3.3 Dosage and relationship between enhanced drug exposure and 491
anthelmintic efficacy
4. Modulation of Drug Efflux Transport: Impact on Clinical Response Against 497
Resistant Haemonchus contortus
4.1 Relevance of cellular efflux transport on systemic exposure and drug action 497
4.2 Modulation on drug transport and efficacy against resistant Haemonchus 500
contortus
5. Pharmacological Evaluation of Drug-Combined Therapy Against Resistant 503
Haemonchus contortus
References 510
Abstract
Progress made in understanding pharmacokinetic behaviour and pharmacodynamic
mechanisms of drug action/resistance has allowed deep insights into the pharma-
cology of the main chemical classes, including some of the few recently discovered
anthelmintics. The integration of pharmaco-parasitological research approaches has
contributed considerably to the optimization of drug activity, which is relevant to
j
ISSN 0065-308X
466 C.E. Lanusse et al.
preserve existing and novel active compounds for parasite control in livestock. A
remarkable amount of pharmacology-based knowledge has been generated using
the sheep abomasal nematode Haemonchus contortus as a model. Relevant funda-
mental information on the relationship among drug influx/efflux balance (accumula-
tion), biotransformation/detoxification and pharmacological effects in parasitic
nematodes for the most traditional anthelmintic chemical families has been obtained
by exploiting the advantages of working with H. contortus under in vitro, ex vivo and in
vivo experimental conditions. The scientific contributions to the pharmacology of
anthelmintic drugs based on the use of H. contortus as a model nematode are summa-
rized in the present chapter.
1. INTRODUCTION
Parasitic nematodes of ruminants represent one of the greatest infec-
tious disease problems in grazing livestock systems worldwide. Despite prom-
ising research results, nonchemical parasite control strategies are not yet
available for routine, commercial use. Thus, nematode control in livestock
still relies on the use of antiparasitic drugs, which comprise the largest sector
of the animal pharmaceutical industry. The integration of available informa-
tion on the host-parasite-environment relationship with an understanding of
the pharmacological properties of traditional antiparasitic molecules has
contributed to more efficient parasite control. However, due to the magni-
tude of the investment in drug medications, it is necessary to acquire scientific
information on how to improve the use of available molecules, and particu-
larly, to avoid/delay the development of anthelmintic resistance. Most fields
of chemotherapy benefit from in vitro test systems that can be used to accu-
rately predict drug concentrations required for efficacy in vivo. However, it
has been difficult to develop a culture system for nematodes to assess the
potency of anthelmintics in vitro. This inconvenience has been a major
limitation to estimating the active drug concentration required to achieve
optimal in vivo activity and has delayed advances in drug development.
However, progress made in understanding pharmacokinetic behaviour and
pharmacodynamic mechanisms of drug action/resistance has allowed deep
insights into the pharmacology of the main chemical classes including some
of the few recently discovered anthelmintics. A remarkable amount of phar-
macology-based knowledge has been acquired using the sheep abomasal
nematode Haemonchus contortus as model. The scientific contributions to the
pharmacology of anthelmintic drugs based on the use of H. contortus as a
model nematode are summarized in the present chapter.
Gaining Insights Into the Pharmacology of Anthelmintics 467
2. BACKGROUND ON THE PHARMACOLOGY

OF ANTHELMINTIC DRUGS
The pharmacokinetics of an anthelmintic drug involves the time
course of drug absorption, distribution, metabolism and elimination from
the host, which, in turn, determines the concentration of the active drug
when it reaches the predilection site of the parasite in the host. Knowledge
of the processes of drug/metabolite diffusion into different target parasites,
together with the available kinetic information, has been relevant to eluci-
date the mechanism of drug penetration and the pharmacological activity of
most anthelmintics. The activity of an anthelmintic drug depends not only
on its binding to the specific receptor (pharmacodynamics), but also on its
ability to reach high and sustained concentrations in the tissue(s) where
the parasite is located, which allows the delivery of effective drug concen-
trations at the receptor within the parasite cells, in sufficient time, to cause
the therapeutic effect (Thompson et al., 1993). There is a close relationship
between pharmacokinetics (which determine drug exposure at the location
site of the parasite) and pharmacodynamics (drug effect). The drug needs to
pass through different ‘barriers’ to reach its specific target receptor within a
target parasite (ie, H. contortus). Dissolution of drug particles in gastrointes-
tinal (GI) fluids is a particularly important phenomenon for drugs adminis-
tered as suspensions by the oral route (such as benzimidazole compounds and
morantel/pyrantel). Dissolution is a crucial step because drug particles must
dissolve in the enteric fluids, to allow absorption through the GI mucosa
and/or penetration through the external surface of GI nematodes. The
undissolved drug particles passing down the GI tract in the luminal content
are excreted in the faeces without exerting anthelmintic action. Anthel-
mintic compounds formulated as drug solutions for parenteral injection in
domestic animals (eg, macrocyclic lactones and levamisole) do not require
dissolution before systemic absorption. In those cases, the GI secretion pro-
cess (ie, abomasal secretion) is an important step to assure drug-nematode
contact. Drug absorption is a main limiting factor that determines the
amount of drug reaching the systemic circulation (systemic exposure).
The reversible exchange between the bloodstream and tissues allows the
drug to achieve concentrations that are anthelmintically active in the tissues
where the parasite is located. The overall pharmacokinetic process, including
drug absorption, tissue distribution and its biotransformation/elimination
pattern, is crucial to allow the drug to reach the target parasites located in
different tissues at sufficient concentrations/time to exert an anthelmintic
effect. Finally, the access of anthelmintic molecules to intracellular sites of

action depends on their ability to penetrate the external cuticular (nema-
todes) or tegumental (cestodes and trematodes) structure of the parasite. Lip-
ophilicity and concentration of the active drug, physicochemical features of
the parasite-surrounding medium, the structure of parasite’s external surface,
are among the factors affecting the transfer (diffusion) and accumulation of
the active drug into the target parasite(s). The particular mode of action of
each compound will affect the onset and the characteristic of the anthel-
mintic effect. Altogether, these different factors will determine the final
anthelmintic activity.
The time that a parasite is exposed to an active drug concentration
determines the efficacy and/or persistence of activity for most of the anthel-
mintics used in ruminants. The characterization of drug concentration
profiles in tissues of parasite location and within target parasites, and its rela-
tionship with the mode of action of each particular molecule provides a basis
for understanding the differences in efficacy observed for the different
chemical families. Benzimidazoles (albendazole, fenbendazole, etc.), imida-
zothiazoles (levamisole), macrocyclic lactones (eg, ivermectin, doramectin
and moxidectin), salicylanilides (closantel), tetrahydropyrimidines (eg, mor-
antel and pyrantel), organophosphates (eg, coumaphos and naphthalophos)
and the novel spiroindoles (derquantel) and aminoacetonitrile derivatives
(AADs, monepantel) are the main chemical families used to control nema-
todes of ruminants, including H. contortus. The potency of most anthelmin-
tics is dependent on their affinity for specific receptors, but also on the
kinetic properties that facilitate the achievement of effective drug concentra-
tions at the site of action (Thompson et al., 1993). The main pharmacolog-
ical properties and the pharmacokineticepharmacodynamic relationships for
the most common drugs used to control H. contortus and other nematodes of
ruminants are described in the following.
Benzimidazoles: Benzimidazole and pro-benzimidazole anthelmintics
are widely used in veterinary medicine. The pro-benzimidazoles (febantel
and netobimin) are inactive prodrugs that are metabolically converted into
anthelmintically active molecules in the host. The benzimidazole methylcar-
bamates are usually most commonly used. Albendazole, fenbendazole and
their sulphoxide derivatives (albendazole sulphoxide or ricobendazole and
oxfendazole, respectively) are currently among the most extensively used
benzimidazole anthelmintics in ruminants. They are indicated for the
removal and control of a broad spectrum of helminth parasites, including
tapeworms, abomasal and intestinal nematodes (adults and L4), lungworms
(adults and larval stages) and have ovicidal activity (w8 h after treatment).
Additionally, albendazole is active against adult (>12 week-old) Fasciola
hepatica (liver fluke). The pharmacological activity of benzimidazoles is based
on the binding to parasite b-tubulin, which produces subsequent disruption
of the tubulin-microtubule dynamic equilibrium (Lacey, 1988). Thus, all
the functions ascribed to microtubules at the cellular level are altered (cell
division, maintenance of cell shape, cell motility, cell secretion, nutrient ab-
sorption and intracellular transport). This particular mode of action requires
a sufficient time of drug-parasite contact to assure that the biochemical
changes following the disruption of the equilibrium of the tubulin-microtu-
bule dynamic result in parasite expulsion. After in vivo administration of
albendazole to artificially H. contortus infected lambs, parasites were recov-
ered viable (with appreciable movement) up to 24 h following treatment
(Alvarez et al., 2000). This and previous observations (Sangster and Prichard,
1985) demonstrate that expulsion from the predilection site is due to an
inability of the parasite to maintain homeostasis. Adults of H. contortus
may be able to survive for a short time after treatment, but if the impairment
of essential functions is extended for a sufficiently long time, the ability of
the parasite to remain in the abomasum is affected. Assisted by the flow of
ingesta, adult parasites are readily expelled from the lumen of the gut, which
helps explain why helminth parasites located outside of the luminal space (ie,
mature F. hepatica, interstitial dwelling and/or arrested larval stages) are less
susceptible to drug treatment compared with adult stages situated within the
gut lumen.
As a chemical class, the benzimidazole methylcarbamates have a limited
solubility in water and are prepared as suspensions for oral or intraruminal
(IR) administration to ruminants. Drug particles must dissolve in the enteric
fluids to facilitate absorption of the benzimidazoles through the GI mucosa.
Benzimidazole dissolution is notably increased by extreme pH values. In
ruminants, benzimidazole dissolution occurs mainly in the abomasum, fav-
oured by a low pH. A drug that does not dissolve in the GI contents passes
down the gut and is excreted in the faeces without exerting its action.
Shortly after administration, benzimidazole compounds are almost
completely adsorbed to particulate matter in the ingesta, reaching an equi-
librium between particulate and fluid portions of the gut content (Hennessy,
1993). The rumen acts as a drug reservoir by slowing the transit time of
ingesta, which results in improved systemic availability of benzimidazole
compounds as a consequence of a greater dissolution of drug particles in
the abomasum at a low pH (Lanusse and Prichard, 1993a). The plasma levels
of the parent sulphides (ie, albendazole, fenbendazole) and/or its active

sulphoxide metabolites (albendazole sulphoxide and oxfendazole) reflect
the amount of drug dissolved in the GI tract. The drug dissolved in the
GI fluid is available for absorption and/or for diffusion through the cuticle
of adult H. contortus located in the abomasum. Thus, H. contortus is largely
exposed to “free” dissolved (not adsorbed to digest a material) drug mole-
cules available in the GI content, and to drug present in gastric secretions,
which recycles between plasma and the abomasum content. Since adult
H. contortus is a blood-feeding parasite, it may also be exposed to drug avail-
able via the bloodstream (Alvarez et al., 2000).
Since benzimidazole anthelmintics are mainly administered by the oral
route, “first pass” metabolism is relevant in the kinetic behaviour of these
compounds. Aromatic benzimidazole derivatives, such as fenbendazole
and oxfendazole, require more extensive hepatic oxidative metabolism
than aliphatic derivatives (albendazole and albendazole sulphoxide) to
achieve sufficient polarity for excretion (Hennessy, 1993). Consequently,
while low fenbendazole concentrations are found in plasma following its
oral/IR administration to sheep and cattle, albendazole is not detected in
the bloodstream after its administration as a parent drug in either species.
Biotransformation takes place predominantly in the liver, although
metabolic activity is apparent in extrahepatic tissues such as lung parenchyma
and small intestinal mucosa (Virkel et al., 2004). Therefore, albendazole
sulphoxide and oxfendazole can be reduced back to their respective thio-
ethers by ruminal and intestinal microflora and may act as a source of alben-
dazole and fenbendazole, respectively, in the GI tract. The high efficacy of
albendazole sulphoxide and oxfendazole against H. contortus depend, in part,
on this bacterial reduction of the sulphoxide to the more pharmacologically
active thioethers (Lanusse and Prichard, 1993b). In fact, although albenda-
zole is not detected in the bloodstream, it has been detected in high concen-
trations in the abomasal mucosa as well as within H. contortus recovered from
treated sheep (Alvarez et al., 2000). The extensive distribution of benzimid-
azole methylcarbamates from the bloodstream to the GI tract and to other
tissues may contribute to achieve good anthelmintic efficacy against parasites
located in peripheral tissues such as the GI mucosa and lungs.
Imidazothiazoles: Levamisole is readily available imidazothiazole
compound for use in veterinary medicine. Levamisole (the L-isomer of tet-
ramisole) is a nematodicidal drug with a broad activity in several host species
against lung and GI nematodes but is inactive against parasitic cestode and
trematodes. Levamisole is active against mature stages of the major GI
nematodes and both mature and larval stages of lungworms. However,

levamisole shows little activity against arrested larval stages (Einstein et al.,
1994). Levamisole is highly effective against adult stages of species of Hae-
monchus, Ostertagia, Cooperia, Nematodirus, Bunostomum, Oesophagostomum,
Chabertia and Dictyocaulus. A major advantage is its formulation flexibility
allowing various routes of administration (oral, parenteral and topical).
Levamisole is a cholinergic receptor agonist and elicits spastic muscle paral-
ysis due to prolonged activation of the excitatory nicotinic acetylcholine
receptors (nAChR) on nematode body wall muscle. Since levamisole affects
the neuromuscular coordination, it causes a spastic paralysis and rapid release
of adult H. contortus from its location in the abomasum. The rate of levam-
isole absorption differs according to the route of administration. The
systemic availability of levamisole in sheep is significantly lower after oral/IR
administration (42e45%) compared with subcutaneous (SC) injection
(Bogan et al., 1982). Levamisole is rapidly and extensively metabolized to
a large number of metabolites in the liver. The main metabolizing pathways
appear to be oxidation, hydrolysis and hydroxylation. Haemonchus contortus is
able to metabolize levamisole into an unidentified compound (Sangster
et al., 1988). Levamisole and metabolites (glucuronyl or S-cysteinyl-glycine
conjugates) are excreted via urine (60%) and faeces (30%). Unchanged
levamisole accounts for 5e10% of the dose in urine and faeces in cattle,
sheep and swine (Nielsen and Rasmussen, 1982). Since levamisole is
short-lived in plasma and GI contents, the peak concentration rather than
the duration of exposure is important for its anthelmintic effect.
Tetrahydropyrimidines: Pyrantel and its methyl analogue morantel
are the two members of this family available in the veterinary market.
Pyrantel is formulated as tartrate, citrate or pamoate (also known as embon-
ate) salts. Pyrantel pamoate is practically insoluble in water, while the tartrate
salt is more water-soluble. Morantel is mainly formulated as a tartrate salt.
Tetrahydropyrimidine compounds act selectively as agonists at synaptic
and extrasynaptic nAChR on nematode muscle and produce contraction
and spastic paralysis (Martin, 1997). These anthelmintics share biologic
properties with acetylcholine and act essentially by mimicking the paralytic
effects of excessive amounts of this natural neurotransmitter. The pyrantel
pamoate salt is poorly absorbed from the GI tract, with the tartrate salt being
more readily absorbed than the pamoate. Peak plasma levels occur at highly
variable times in ruminants. Absorbed drug is rapidly metabolized to inactive
metabolites and excreted into the faeces. Morantel is negligibly absorbed via
the gut in ruminants and is thus largely excreted as the unmetabolized parent
compound in the faeces. Both pyrantel and morantel are highly efficacious
against adult and immature stages of H. contortus in the abomasum and have
minimal activity against arrested larval stages (Einstein et al., 1994). The low
GI absorption and/or efficient metabolism to inactive metabolites explain
the absence of systemic anthelmintic activity against lungworms as well as
arrested tissue larvae.
Organophosphate compounds: Organophosphate compounds have
insecticidal and nematodicidal activities. The main organophosphates used
as anthelmintics in ruminants are haloxon, coumaphos, naphthalophos and
crufomate. Organophosphates’ mode of action related to an irreversible
inhibition of acetylcholinesterase by phosphorylation, which results in
acetylcholine accumulation at cholinergic receptors. The resultant
accumulation of acetylcholine leads to a paralysis and death of the parasite.
Overall, after its oral administration, organophosphates have satisfactory
efficacy against nematode parasites of the abomasum (particularly
Haemonchus) and small intestine, but lack satisfactory efficacy against
nematodes of the large intestine (Oesophagostomum and Chabertia).
Naphthalophos efficacy against adult H. contortus reaches 99e100% even
at a reduced dose of 25 mg/kg (at 75 mg/kg is also active against
H. contortus fifth stage) (Courtney and Roberson, 1995). As described for
levamisole and pyrantel/morantel, the efficacy of the organophosphates
against arrested stages of H. contortus and other abomasal and intestinal
nematodes is rather low. This finding could be explained by the short
time of drug-parasite contact and the reduced exposure of the
larval/immature stages located at the GI mucosa.
Salicylanilides: The most used salicylanilide compound to control H.
contortus is closantel. It is formulated for both, oral and intramuscular admin-
istration. Closantel is highly effective for the treatment of adult flukes and is
also highly effective against H. contortus in sheep. It is used as an alternative
drug for the treatment of ivermectin-, benzimidazole-, levamisole- and
morantel-resistant isolate of this nematode. It is also effective against certain
ectoparasites such as lice, ticks, mites and the nasal bot Oestrus ovis (see
Lanusse et al., 2009). The mode of action of closantel and other salicylani-
lides is mainly based in the uncoupling of oxidative phosphorylation. Addi-
tionally, closantel has been identified as a potent and specific inhibitor of
filarial chitinases (Gloeckner et al., 2010). Closantel pharmacokinetic behav-
iour is characterized by high plasma protein binding, low volume of
distribution (in spite of its high lipophilicity) and long elimination half-
life. Its broad-spectrum activity relies on its long persistence in the systemic
circulation (up to 90 days in sheep), which facilitates drug accumulation into

target parasites, particularly haematophagous parasites. These characteristics
explain the high efficacy against immature and adult H. contortus for up to
28 days after treatment (Lanusse et al., 2009). Following intramuscular
administration of closantel to sheep, the expulsion of susceptible H. contortus
begins at about 8 h following treatment and at 16 h most of the worms are
removed (Rothwell et al., 1993). Additionally, it has been shown under in
vivo conditions that H. contortus expulsion from its predilection site starts
once motility is affected by closantel (Rothwell et al., 1993).
Macrocyclic lactones: The avermectins and milbemycins are closely
related 16-membered macrocyclic lactones, and are active at extremely
low (0.2 mg/kg) dosages against endo- and ectoparasites (Shoop et al.,
1995). The avermectin family includes a series of natural and semisynthetic
molecules, such as abamectin, ivermectin, doramectin, eprinomectin and
selamectin. Ivermectin was the first marketed endectocide molecule.
Nemadectin, moxidectin and milbemycin oxime belong to the milbemycin
family. The macrocyclic lactones induce a reduction in motor activity and
paralysis in both arthropods and nematodes. The paralytic effects are medi-
ated mainly through glutamate-gated chloride channels (GluCl) (Martin,
1997). Furthermore, a study of Caenorhabditis elegans (see Hernando and
Bouzat, 2014) revealed that the muscle levamisole-sensitive receptor
(L-AChR), one of the main excitatory receptors involved in parasite loco-
motion is profoundly inhibited by ivermectin. Macrocyclic lactones are
extremely effective against adult and larval stages of most GI and lung nem-
atodes, exerting a prolonged ‘protective’ effect. The clinical efficacy of the
macrocyclic lactones is closely related to their pharmacokinetic behaviour,
being the time of parasite exposure to active drug concentrations, required
to obtain optimal and persistent antiparasitic activity. The highly lipophilic
macrocyclic lactones are extensively distributed via the bloodstream to
different tissues. The persistence of the broad-spectrum anthelmintic activity
against adult and immature GI parasites is facilitated by their advantageous
pattern of distribution and prolonged residence in the mucosal tissues of
the digestive tract. The long persistence of macrocyclic lactones is mainly
explained by their wide tissue distribution, low metabolism/organic clear-
ance and enterohepatic recycling. As pointed out by Hennessy (2000), the
extent to which the biliary-secreted drug is presented to the gut lumen,
reabsorbed as free compound and participates in the enterohepatic cycle is
a major contributor to parasite exposure. The deposition of macrocyclic
lactones in adipose tissue may represent a drug reservoir that contributes
to the long persistence of these compounds in the bloodstream and in

different sites of parasite location. Following oral administration, ivermectin
reaches a relatively high concentration in plasma (lower than that obtained
after the parenteral treatment), but the concentrations attained in the GI tis-
sue are relevant in terms of efficacy against resistant H. contortus (see specific
comments in Section 3.2).
Monepantel: Monepantel is a novel AAD that is active against larval
and adult stages of GI nematodes of sheep, including H. contortus which
are highly resistant to all the other available anthelmintic chemical families.
Genetic studies of the free-living nematode C. elegans and of H. contortus
have shown that monepantel acts as an agonist on the nAChR, producing
spastic paralysis and death of the worms (Kaminsky et al., 2008; Rufener
et al., 2010). Monepantel exerts its action at a new target as a positive
allosteric modulator of the nematode specific receptor MPTL-1, which
belongs to the DEG-3 subfamily of acetylcholine receptors (Rufener
et al., 2010). The systemic availability of the monepantel parent compound
was significantly lower than that observed for its main metabolite, mone-
pantel sulphone. Furthermore, after the oral administration of monepantel
to sheep, the persistence of monepantel sulphone (9 days) in plasma was
significantly longer than that of monepantel (2 days) (Lifschitz et al.,
2014). An equivalent pharmacological potency of the monepantel parent
drug and its sulphone metabolite against larvae of GI nematodes has
been suggested using in vitro evaluation assays (Karadzovska et al.,
2009). It seems unlikely that monepantel will adequately control lung
nematodes at the dose used for GI nematodes (Hosking, 2010). The
systemic drug availability is relevant for the exposure of lung nematodes
to the active drug/metabolites. Thus, the levels of monepantel/sulphone
systemically available after its oral administration at 2.5 mg/kg to sheep
may be below the critical amount required to reach an optimal efficacy
against lung nematodes (Lifschitz et al., 2014). Although plasma concentra-
tions of monepantel sulphone were detected until 9e12 days after admin-
istration, an efficacy study has confirmed that monepantel is a short-acting
anthelmintic (Hosking, 2010). Thus, monepantel anthelmintic activity
may be based on a substantial drug/metabolite accumulation in the GI tis-
sues and fluid contents in the first 2e3 days following treatment. It is also
likely that the level of drug concentration <0.1 mg/mL measured in plasma
between days 4 and 9 after treatment may not be sufficient to obtain
adequate activity against the different species of nematodes located in
different segments of the digestive tract (Lifschitz et al., 2014).
Derquantel: Derquantel is a semisynthetic derivative of paraherqua-

mide and belongs to the chemical family of spiroindoles. The anthelmintic
activity of derquantel is based on interference with B-subtype nAChR,
acting as an antagonist and leading to a flaccid paralysis in nematodes
(Ruiz-Lancheros et al., 2011). This differential mode of action contributes
to its activity against nematodes that are resistant to other currently available
chemical groups. In an attempt to optimize its anthelmintic activity and to
decrease the selection pressure on resistant nematode isolates, derquantel
has been launched for use in combination with abamectin. Derquantel com-
bined with abamectin has 98.9% efficacy against H. contortus (fourth stage
and adult stages) (Little et al., 2011), and derquantel reaches an absolute
bioavailability of 56% following the oral combined treatment (EMEA,
2010). There is no significant adverse kinetic interaction between derquantel
and abamectin when they are co-administered (Friedlander et al., 2012).
Derquantel undergoes biotransformation to a large number of metabolites
over a short time period (EMEA, 2010).
Although comparative work showed that the derquanteleabamectin
combination failed to reduce burdens of L4s of macrocyclic lactone-
resistant H. contortus and Teladorsagia spp. (George et al., 2012; Kaminsky
et al., 2011), there are no reports or known field cases of anthelmintic resis-
tance to derquantel. There is pharmacology-based evidence of a synergistic
interaction between derquantel and abamectin (Puttachary et al., 2013).
Derquantel may interact additively with the macrocyclic lactone abamec-
tin, and, at higher acetylcholine concentrations, the interaction is synergis-
tic. This interaction is based on the noncompetitive antagonism of
abamectin on the nicotinic receptor that potentiates the competitive antag-
onism produced by derquantel (Puttachary et al., 2013). However, this
combination is not based on drugs with similar pharmacokinetic profiles.
Concerns about matching half-lives of elimination to minimize exposure
to suboptimal concentrations of single constituent actives or their bioactive
metabolites at the tail of the elimination curve may be more relevant for
synergistic combinations compared with combinations that produce
additive effects (Geary et al., 2012). It is expected that the derquantele
abamectin combination achieves adequate parasite drug exposure to ensure
the synergistic effect observed in vitro. However, the different plasma
profile between derquantel and abamectin indicates that the combination
will not prevent abamectin from selecting for resistance during a period of
suboptimal concentrations at the tail of the elimination curve, as would
occur if abamectin is used alone.
3. ASSESSMENT OF PARASITE EXPOSURE TO

ANTHELMINTIC DRUGS USING HAEMONCHUS
CONTORTUS AS A TARGET NEMATODE
3.1 Haemonchus contortus as a tool to explore ex vivo
and in vivo drug accumulation, metabolism and
activity in nematode parasites
The drug-parasite relationship can be characterized using in vitro, ex
vivo and/or in vivo approaches. The transport of different substances has
been investigated in vitro using isolated nematode cuticle, which offers
some advantages over intact organisms, particularly for the interpretation
of permeability data (Thompson et al., 1993). Ex vivo assays involve the
use of intact, live parasites in a closed perfusion system, allowing the study
of the relative contributions of the transcuticular and oral pathways as well
as the influence that underlying tissues, such as somatic muscles and gut,
may have on cuticular drug transport (Ho et al., 1992). Ex vivo character-
ization of drug transfer offers technical advantages and reliable results (Cross
et al., 1998; Ho et al., 1992; Mottier et al., 2006). Additionally, ex vivo ex-
periments have been performed, to deeply understand drug effect on para-
sites exposed to anthelmintics. However, it is clear that the drug-parasite
interaction may differ under in vivo conditions, where worms are exposed
to changing drug concentration over the time in a variable physiological
environment.
The helminth’s external surfaces serve as a barrier which shields the
organism from external conditions, and are vital for nutrient uptake, osmo-
regulation, immunoprotection and structural support. The tegument of
flatworms is a membrane-bound syncytium, allowing the active transport
of nutrients and drugs (Fetterer and Rhoads, 1993). In contrast, a nematode’s
cuticle has been considered to be a barrier limiting the entry of large mole-
cules into the worm (Fetterer and Rhoads, 1993; Ho et al., 1990). Haemonchus
contortus lives in the abomasum attached to the mucosa or free in the abomasal
content and, because of its haematophageous nature, anaemia is often a clin-
ical feature of the infection. Anthelmintic drugs can reach H. contortus either
by oral ingestion of blood or by transcuticular uptake/diffusion (Geary et al.,
1995). Therefore, the concentration of active drug in the bloodstream (oral
ingestion) and/or abomasal fluid/mucosa (transcuticular diffusion) is relevant
to the clinical efficacy against this parasite. Albendazole is not found in the
bloodstream after enteral administration to sheep and cattle, and the active
albendazole sulphoxide metabolite has been postulated to be responsible for
the activity against lungworms and tissue-dwelling parasites (Marriner and

Bogan, 1980). However, higher concentrations of albendazole were
measured in H. contortus recovered from infected treated sheep, compared
with those of its sulphoxide metabolite (Alvarez et al., 2000). Albendazole
and albendazole sulphoxide were rapidly taken up by H. contortus exposed
in vivo to albendazole, being detected in the parasite between 0.5 and 12 h
following administration (Alvarez et al., 2000). Interestingly, the concentra-
tion profiles of albendazole and albendazole sulphoxide during that period
of time were greater in the H. contortus specimens than in abomasal mucosa
and abomasal fluid. Since albendazole was not detected in peripheral plasma,
only drug from the pool found in abomasal fluid and mucosa may be able to
reach the nematode via transcuticular diffusion. The lipoidal hypocuticle tis-
sue is the rate-determining barrier and only allows sufficiently small molecules
to traverse the aqueous-filled, negatively charged collagen matrix of the
cuticle (Ho et al., 1992). Lipophilicity facilitates drug diffusion through the
nematode cuticle (Ho et al., 1992); thus, lipophilic drugs such as
albendazole may have a greater capability to cross the external surface of
the nematode than the more polar albendazole sulphoxide. The exposure
of the nematode to high concentrations of albendazole sulphoxide in the
abomasal lumen may compensate for its lower rate of diffusion across the
cuticle. In spite of the lower concentrations of albendazole in abomasal fluid,
its high lipophilicity may enhance penetration through the external parasite
surface. It should also be pointed out that H. contortus may feed on portal
blood and the relevance of the portal circulation as a source of albendazole
might be considered. However, the low albendazole concentrations found
in portal blood (Alvarez et al., unpublished observations) do not explain
the high concentrations measured in H. contortus (see Alvarez et al., 2000).
These findings confirm the relevance of the transcuticular diffusion process,
even in a blood-feeding parasite, such as H. contortus, where the higher
lipophilicity of albendazole (octanolewater partition coefficient: 3.83) may
have accounted for its greater penetration compared with its sulphoxide
derivative (partition coefficient: 1.24) (Mottier et al., 2003). The greater anti-
parasitic activity of albendazole compared with albendazole sulphoxide has
been demonstrated in vitro by assessing binding to parasite tubulin (Lacey,
1990; Lubega and Prichard, 1990) as well as nematode motility (Petersen
et al., 1997). The higher affinity to parasite tubulin and the greater capacity
to diffuse into the parasite observed for albendazole suggest that the parent
drug may exert greater activity than its metabolite albendazole sulphoxide
against abomasal nematodes.
Available information (Cross et al., 1998; Geary et al., 1995; Ho et al.,

1992; Sims et al., 1996) indicates that oral ingestion does not allow major
drug accumulation in nematodes, particularly for lipophilic molecules
present in large concentrations in tissues and in gut contents. A different
situation might occur with anthelmintics that bind strongly to plasma
proteins. The nematodicidal activity of closantel is almost restricted to
H. contortus. It is likely that the anthelmintic effect of closantel against
blood-feeding parasites depends on its intraparasite accumulation mediated
by oral ingestion. Closantel is a highly lipophilic compound that is exten-
sively bound (>99%) to plasma proteins and has a long half-life
(14.5 days) (Michiels et al., 1987). Closantel-resistant H. contortus accumu-
lated significantly less [14C]closantel than did susceptible worms (Rothwell
and Sangster, 1997). The reduced closantel accumulation in resistant worms
was attributed to different factors including increased efflux from cells,
reduced absorption of closantel across the worm intestine and/or a reduced
feeding rate in the resistant isolate (Rothwell and Sangster, 1997). The mea-
surement of reduced closantel levels in resistant H. contortus provided a clear
in vivo association between drug resistance and decreased drug accumula-
tion (closantel) in a target nematode. Although oral ingestion could be an
important route of closantel entry into H. contortus, ex vivo data indicate
some degree of closantel accumulation by transcuticular transport (Rothwell
and Sangster, 1997).
Drug molecules move across cell membranes either by passive diffusion
or specialized transport mechanisms. In the passive diffusion process, the
membrane behaves as an inert lipid-pore boundary, and drug molecules
traverse this barrier either by diffusion through the lipoprotein region or,
alternatively, by filtering through aqueous pores (channels) without the
expenditure of cellular energy if they are of a sufficiently small size (Ho
et al., 1992). Specialized transport is another potential mechanism of
drug entry into target parasites. This type of transport process is relatively
selective towards the chemical nature of a substance and requires direct
expenditure of energy (Baggot, 1982). If the entry of an anthelmintic
into a target parasite is mediated by specialized transport (a saturable
process), the exposure time is critical to determine the amount of drug
accumulated inside the parasite. On the other hand, if passive diffusion is
the main mechanism of entry of anthelmintics (over active transport),
the concentration and the time will determine the drug concentration
within the worm, and the restrictions imposed by cuticular lipid barriers
will probably be similar to those of standard cellular membranes. The
knowledge of the mechanism of drug entry to parasites is a key to under-

stand drug-parasite relationship.
There is previous evidence that would indicate that different chemical
substances, including anthelmintic drugs, are mainly taken up through the
external surfaces of flatworms (Alvarez et al., 1999, 2000, 2001, 2004;
Bennett and K€ ohler, 1987; Mottier et al., 2003) and nematodes (Alvarez
et al., 2001; Cross et al., 1998; Ho et al., 1990; Sims et al., 1992a) as opposed
to oral ingestion by the parasite. In both cases (tegument/cuticular or
intestinal entry), the anthelmintic molecules need to pass through cell
membranes to reach the biophase around the specific receptor, which
may be influenced by the pharmacokinetic behaviour of the compound,
the concentration gradient and time of drug-parasite contact. In the case
of passive diffusion, as the concentration gradient increases on one side of
the membrane, drug concentrations on the other side will increase in favour
of the concentration gradient. If transporters are involved in the mechanism
of drug absorption, saturation may follow the increase of drug concentration
on one side of a membrane. Therefore, the concentration achieved on the
other side does not necessarily follow a linear relationship with the sur-
rounding concentration in the medium.
Mottier et al. (2006) reported that under ex vivo conditions, benzimid-
azole anthelmintics accumulated inside Moniezia benedeni (fenbendazole),
F. hepatica (triclabendazole sulphoxide) and Ascaris suum (fenbendazole and
oxfendazole) in favour of the concentration gradient. Absorption of benz-
imidazole was linear for the range of concentrations assayed. Additionally,
it is well known that lipid solubility facilitates drug diffusion through the
external surfaces of A. suum (see Ho et al., 1992), M. benedeni (see Mottier
et al., 2003) and F. hepatica (see Mottier et al., 2004). Oxfendazole, the sulph-
oxide metabolite of fenbendazole, is pharmacologically less active and it has a
lower lipid solubility (octanolewater partition coefficient: 2.03) than the
parent compound (octanolewater partition coefficient: 3.93) (Mottier
et al., 2003). Fenbendazole concentrations inside ex vivo incubated M. bene-
deni, for the entire concentration gradient assayed (Mottier et al., 2006), were
greater than those observed for its sulphoxide metabolite. The main way that
a given drug molecule reaches the interior of a cestode parasite is by passing
through its tegument and it is evident that concentration and lipophilicity are
the major factors determining drug penetration (Mottier et al., 2006). Simi-
larly, the higher lipophilicity of albendazole (octanolewater partition coeffi-
cient: 3.83) (Mottier et al., 2003) accounted for its greater penetration
through the external parasite surface, compared with its sulphoxide derivative
(octanolewater partition coefficient: 1.24), observed in M. benedeni under ex

vivo conditions (Mottier et al., 2003). Data reported by Bartíkova et al. (2012)
showed that flubendazole is able to effectively penetrate adult H. contortus
under ex vivo conditions due to its high lipophilicity. Passive diffusion is
probably the only mechanism involved in both flubendazole import and
efflux from H. contortus (see Bartíkova et al., 2012). Additionally, no
differences in flubendazole ex vivo accumulation were found among four
H. contortus isolates with distinct sensitivities to anthelmintics (Bartíkova
et al., 2012). These results relate to those described by Alvarez et al. (2004),
who reported that physicochemical composition of the parasite’s surrounding
environment (in which it is immersed) plays a pivotal role in the process of
drug access to F. hepatica. Since the concentration gradient, drug lipid solubi-
lity, physicochemical characteristics of the incubation medium as well as the
structure and composition of the external surface of a helminth parasite are
critical for the penetration of benzimidazole molecules through these surfaces;
it is clear that passive diffusion is the main mechanism implicated in the entry
of these anthelmintic drugs into nematodes and cestodes. The same is likely to
be true for other anthelmintic drugs.
The small size of H. contortus limits the ex vivo experiments intended to
evaluate the mechanism of drug entry into this nematode parasites. Howev-
er, information has been obtained using other nematodes as models. The
transcuticular transfer appeared to be the main route of passage of ivermectin
into the filarial nematode Onchocerca ochengi (see Cross et al., 1998). The en-
try of ivermectin into adult O. ochengi occurs by the transcuticular route,
where the marked foldings of its cuticle greatly increase the surface area,
favouring ivermectin diffusion. Additionally, in the absence of pharyngeal
pumping, first-stage larvae of C. elegans submerged in ivermectin became
paralysed (Smith and Campbell, 1996), which reinforces the relevance of
ivermectin transcuticular penetration. The in vitro accumulation of [14C]
closantel in adult H. contortus was measured both in the absence and presence
of ivermectin (used to prevent closantel oral uptake) (Rothwell and
Sangster, 1997). Closantel lipophilicity may explain its transcuticular entry
into closantel-susceptible and closantel-resistant H. contortus, even in the
presence of ovine serum albumin and when oral ingestion was abolished
(Rothwell and Sangster, 1997). Nevertheless, the extensive binding to albu-
min may facilitate the oral ingestion of closantel by haematophagous para-
sites such as H. contortus.
The rate of penetration across the H. contortus cuticle depends mainly on
lipophilicity, and, in the case of acidic or basic drugs, on the ionized and
unionized (lipid-permeable) fractions of the drug; this rate is determined by

the relationship between drug pK and pH of the aqueous environment in
the cuticle. The cuticle consists of (1) collagen-like proteins that form the
medial and basal layers; (2) noncollagen proteins that form the epicuticular
and external cortical regions and (3) nonstructural proteins associated with
the external surface (Fetterer and Rhoads, 1993). The lipoidal hypocuticle
tissue is the rate-determining barrier to molecules sufficiently small to
traverse the aqueous-filled negatively charged collagen matrix of the cuticle
(Ho et al., 1992). Lipophilicity facilitates drug diffusion through the nema-
tode hypocuticle (Ho et al., 1992); thus lipophilic drugs, including benz-
imidazole methylcarbamates and macrocyclic lactones, may be better able
to cross the external surface of the nematode than polar compounds. The
water-filled and negatively charged collagenous matrix of the nematode
cuticle permits the passage of molecules by molecular size restricted-
diffusion (Ho et al., 1992). If the molecule is sufficiently small, it could tra-
verse the aqueous-filled negatively charged collagen matrix of the cuticle
(Ho et al., 1992). For example, the external surface of A. suum can be
breached by drugs, and the rate-determining barrier for passive transport is
the lipoidal hypocuticle tissue. The drug lipidewater partition coefficient,
pH/pKa relationship and molecular size influencing the diffusion of
uncharged molecules indicate that the transcuticular transport of weak acids
and bases will be controlled largely by the pH at this surface, since, in the
absence of facilitated transport, only unionized species can partition across
a lipoidal surface (Sims et al., 1992b). Ascaris excretes a number of volatile
fatty acids as well as lower levels of two nonvolatile organic acids (end-
products of carbohydrate metabolism) via the transcuticular route at suffi-
cient rates to establish and maintain a buffered microenvironmental pH of
w5.0 in the aqueous space of the pores in the cuticle (Sims et al., 1992b).
Most drugs are weak organic bases or acids, and exist in solution as both
nonionized and ionized forms. While the poor lipid solubility of ionized
molecules excludes them from passive diffusion, lipophilic, nonionized moi-
eties passively diffuse across cell membranes until an equilibrium is estab-
lished. Accumulated data (Alvarez et al., 2001; Bennett and K€ ohler, 1987;
Cross et al., 1998; Ho et al., 1990; Mottier et al., 2003, 2006; Sims et al.,
1992a) would indicate that passive diffusion over specialized transport is
implicated in drug entry into parasites. However, we cannot exclude the
possible participation of an active entry mechanism in parasitic helminths.
Resistance due to increased drug inactivation is one of the most common
biochemical mechanism accounting for the antibiotic resistance that is
encountered in the clinical treatment of bacterial infections (Pratt, 1990).

The worldwide development of resistance to insecticides is one of the
most common examples of biochemical inactivation as a resistance mecha-
nism. In helminths, mechanisms involved in the development of resistance
to anthelmintics can result from changes in the target molecule, in drug
uptake/efflux mechanisms and also in drug metabolism (Ouellette, 2001).
Metabolism/biotransformation of active anthelmintic drugs by resistant
helminth parasites could account for drug resistance. The sulphoxidation
of triclabendazole to triclabendazole sulphoxide (Alvarez et al., 2005) and
triclabendazole sulphoxide to the triclabendazole sulphone metabolite
(Robinson et al., 2004) are both greater in resistant than in susceptible flukes.
Indeed, triclabendazole resistant flukes have a 39% greater capacity to
metabolize the parent drug (Alvarez et al., 2005).
Haemonchus contortus has also been used as a model to explore potential
implications of drug metabolism as mechanisms of resistance. Rothwell
and Sangster (1997) did not find differences between resistant and suscepti-
ble H. contortus isolates related to closantel metabolism, and concluded that
the contribution of the increased metabolism to closantel resistance between
isolates must be trivial. The metabolism of albendazole and the activities of
selected biotransformation and antioxidant enzymes was investigated in vitro
and ex vivo in three different isolates of H. contortus: susceptible and resistant
to benzimidazoles, and multidrug resistant (Vokral et al., 2013a,b). The in
vitro data showed significant differences between the susceptible and both
resistant isolates regarding the activities of peroxidases, catalase and UDP-
glucosyltransferases. Albendazole sulphoxidation was significantly lower in
benzimidazole resistant than in the susceptible isolate. Under ex vivo condi-
tions, four metabolic products were identified in H. contortus specimens
incubated with albendazole, including the sulphoxide and three glucosides
derivatives. Interestingly, in both resistant isolates, the ex vivo formation
of all albendazole glucosides was significantly higher than in the susceptible
isolate (Vokral et al., 2013a,b). This finding indicates that the altered activ-
ities of particular detoxifying enzymes may partly protect H. contortus against
the toxic effect of albendazole contributing to resistance. Similar experi-
ments were performed to investigate the biotransformation pattern of
flubendazole in different H. contortus isolates (benzimidazole resistant and
susceptible) (Bartíkova et al., 2012). Similar to findings for albendazole
(Vokral et al., 2013a,b), the ex vivo formation of all flubendazole metabo-
lites was significantly higher in the resistant compared with a susceptible
isolate of H. contortus (see Bartíkova et al., 2012). Altogether, these findings
are an indication that resistant nematode isolates may have an increased abil-
ity to deactivate anthelmintics via biotransformation as a mechanism
contributing to drug resistance.
Haemonchus contortus appears to be able to metabolize monepantel under
ex vivo conditions via oxidation and hydrolysis reactions (Stuchlíkova et al.,
2014). The study of monepantel biotransformation in H. contortus revealed
four monepantel metabolites, including monepantel sulphoxide, monepan-
tel sulphone and two monepantel metabolites derived from the hydrolysis of
two different nitrile groups (Stuchlíkova et al., 2014). Unlike sheep liver,
H. contortus adult specimens failed to metabolize monepantel via phase II
biotransformation. Results reported by Alvinerie et al. (2001) demonstrate
that homogenates of adult H. contortus contain enzymes that were able to
metabolize moxidectin. A 24-h incubation period was required for the
production of detectable amounts of the unique metabolite observed, which
did not correspond to the metabolites previously described in vertebrates
(Alvinerie et al., 2001). By contrast, adults H. contortus were not able to deac-
tivate ivermectin through biotransformation, which indicates that this
mechanism does not contribute to the development of ivermectin resistance
(Vokral et al., 2013a,b).
Toxicity of rotenone towards the larvae of H. contortus and Trichostrongy-
lus colubriformis was increased in the presence of piperonyl butoxide, a well-
known cytochrome P450 inhibitor (Kotze et al., 2006). Furthermore, in the
same ex vivo experiment, rotenone activity against adult H. contortus was also
significantly enhanced following pretreatment with piperonyl butoxide.
The synergistic effect observed between rotenone and piperonyl butoxide
indicates that H. contortus and T. colubriformis are able to utilize a cytochrome
P450 enzyme system to detoxify rotenone and indicates that a role may exist
for cytochrome P450 inhibitors to act as synergists for other anthelmintics
that are susceptible to oxidative metabolism within the nematode (Kotze
et al., 2006). Using the ex vivo approach, H. contortus larvae were treated
with phenobarbital, as an inductor of detoxification enzymes (Kotze et al.,
2014). Co-treatment of larvae with phenobarbital and naphthalophos
resulted in a significant increase in the naphthalophos 50% inhibitory
concentration (IC50) compared with the treatment of larvae with the
anthelmintic alone (Kotze et al., 2014). The phenobarbital-induced drug
tolerance was reversed by co-treatment with the UDP-glucuronosyltrans-
ferases inhibitors, demonstrating that H. contortus larvae possess one or
more UDP-glucuronosyltransferase enzymes able to detoxify naphthalo-
phos. In insects, such as houseflies, the typical mechanism of resistance
against chlorinated hydrocarbons (DDT), cyclodienes (dieldrin), organo-

phosphates and carbamates relates to an overproduction of drug-metabo-
lizing enzymes (Pratt, 1990). It is likely that increased biotransformation
activity could relate to a mechanism of drug resistance in helminth parasites.
The in vitro and ex vivo data summarized here, using H. contortus as a nem-
atode model, appear to indicate that at least for some anthelmintic drugs,
metabolism/detoxification are associated with anthelmintic resistance
mechanisms.
On the other hand, H. contortus has been the main nematode of veteri-
nary interest used to interpret different aspects related to the understanding
of drug action. It was established early that, under ex vivo conditions, iver-
mectin inhibits the contraction of pharyngeal muscles responsible for feeding
in nematodes and causes paralysis of their body musculature (Geary et al.,
1993). Inhibition of pharyngeal pumping was more potent than that of
motility in H. contortus (see Geary et al., 1993). It was also established that
the transcuticular uptake of glucose in H. contortus was not altered by iver-
mectin. Thus, it was hypothesized that if oral ingestion of other nutrients
is essential for long-term survival in vivo, disruption of pharyngeal pumping
might represent the primary mechanism of ivermectin action (Geary et al.,
1993). The interruption of a vital function, such as pharyngeal pumping,
which is implicated in nutrient ingestion, excretion, regulation of turgor
pressure, etc., may be critical for worm survival. The effect of ivermectin
on pharyngeal uptake of (3H-inulin) was measured in larvae of both iver-
mectin-susceptible and ivermectin-resistant isolates of H. contortus (see
Kotze, 1998). A higher ivermectin concentration was necessary to reduce
by 50% the feeding by the resistant compared with the susceptible isolate
(Kotze, 1998). This information indicates that susceptible and resistant iso-
lates can be readily distinguished on the basis of the sensitivity of pharyngeal
uptake to macrocyclic lactones. However, the in vivo assessment of iver-
mectin feeding inhibition in H. contortus fails to demonstrate the inhibition
of pharyngeal pumping. Adult H. contortus recovered from sheep treated
with ivermectin 4 h prior to the intravenous [3H]inulin administration
showed equivalent feeding levels (over a 1 h period) to those recovered
from sheep not treated with ivermectin (Sheriff et al., 2005). There was
no difference in the radioactivity in nematodes of an ivermectin-susceptible
and ivermectin-resistant isolate recovered from individual sheep with
concurrent infections after ivermectin treatment (Sheriff et al., 2005). These
results indicate that ivermectin given orally at a dose sufficient to remove
drug-susceptible worms by 8 h did not cause a significant reduction of
[3H]inulin uptake in H. contortus, which questions the significance of feeding

inhibition as in vivo toxic effect of ivermectin on H. contortus (see Sheriff
et al., 2005). In addition, some more recent findings (Kotze et al., 2012)
indicate that, when sensitive worm motility assessment methods are utilized
under ex vivo conditions, worm motility is affected at lower abamectin con-
centrations than worm feeding, suggesting that somatic musculature is a
more important target site for abamectin, and likely also for other macrocy-
clic lactones.
3.2 Influence of the route of drug administration on parasite

exposure
The choice of the administration route for anthelmintic drugs in ruminants is
based on either management factors or influenced by the technical market-
ing of the pharmaceutical companies. The selected administration route is
relevant and relates to the potency of most anthelmintics and is dependent
on their affinity for a specific receptor (site of action) but also on the kinetic
properties that facilitate achieving effective drug concentrations at the site of
action. A remarkable amount of work on the kinetic behaviour of the most
widely used broad-spectrum anthelmintics in ruminants is now available.
The complex connections among route of administration, formulation,
drug physicochemical properties and the resultant kinetic behaviour need
to be understood to optimize drug efficacy. The administration of the
anthelmintic drug by different routes may account for significant differences
in the final parasite drug exposure. The influence of the administration
routes will depend on the physicochemical features of each drug. The
lack of water solubility is an important limitation for the formulation of
benzimidazole compounds, which results in their preparation as suspensions,
pastes or granules for oral or IR administration. Thus, after the oral admin-
istration of benzimidazole compounds, the dissolution of drug particles in
gut contents is a relevant step that has a direct influence on overall anthel-
mintic efficacy.
Different factors may affect the dissolution process and the pharmacoki-
netic behaviour of benzimidazoles. The effect of ruminant oesophageal
groove closure was described as an important modification for the benzimid-
azole absorption process. Occasionally, reduced systemic availability and ef-
ficacy of benzimidazole methylcarbamates have been found after oral
administration compared with IR administration (Hennessy and Prichard,
1981). A portion of the orally administered anthelmintic may bypass the
rumen, rapidly entering the abomasum via the oesophageal groove. As a
consequence, the poor absorption due to insufficient time for dissolution of

benzimidazole suspension’s particles results in a reduced plasma bioavail-
ability of active benzimidazole metabolites. Such an effect may indicate
that the so-called ‘reservoir’ and ‘slow delivery’ effects of the rumen would
be lost and the resultant efficacy significantly reduced. The GI transit time is
another factor that may influence on the benzimidazole dissolution and,
therefore, on its accumulation and efficacy against H. contortus. Feeding
management has been recommended to restore the anthelmintic action of
those benzimidazole compounds whose potency has been compromised
by resistance (Ali and Hennessy, 1995). An enhanced plasma availability
of oxfendazole induced by temporary feed restriction in sheep accounted
for an increased efficacy of the drug against a benzimidazole-resistant nem-
atode isolate (Ali and Hennessy, 1995). Fasting the animals prior to IR treat-
ment resulted in pronounced modifications to the absorption and
disposition kinetics of albendazole metabolites in sheep in which the admin-
istered drug appeared to be absorbed to a greater extent than in fed animal
(Lifschitz et al., 1997). Starvation decreases the flow rate of ingesta. A
delayed GI transit time that decreased the rate of passage of the anthelmintic
drug down the GI tract may have accounted for the enhanced absorption
observed in fasted animals compared with fed animals. The fasting-induced
changes to the kinetic behaviour and quantitative tissue distribution of benz-
imidazole methylcarbamates may have particular relevance for designing
strategies to increase activity against susceptible parasites. However, a fast-
ing-induced improvement of benzimidazole dissolution/absorption may
not be useful when a high level of drug resistance is already established in
the treated nematode population (Alvarez et al., 2010).
In the case of levamisole, the available formulations for ruminants are
prepared as solutions to be administered by oral, SC and topical routes.
The rate of levamisole absorption differs with the route of administration.
The drug is most rapidly absorbed following intramuscular or SC injection
in cattle, and the highest plasma levels (>1 mg/mL) are observed at 0.5e2 h.
Several oral formulations gave similar absorption rates (time to peak concen-
tration [Tmax] ¼ 3 h), and slower absorption was observed after dermal
application (Bogan et al., 1982). Bioavailability differs depending on the
route of administration. In sheep, the highest mean plasma concentrations
were achieved after the SC (3.1 mg/mL) compared with oral (0.7 mg/mL)
administration at a dose rate of 7.5 mg/kg (Bogan et al., 1982). Following
pour-on administration in cattle, plasma and GI concentrations of levamisole
were lower than those measured following parenteral and oral treatments
(Forsyth et al., 1983), which agrees with the limited anthelmintic efficacy
reported for the topical preparation. In small ruminants, the systemic avail-
ability of levamisole has been shown to be 25e33% higher after parenteral
administration compared with the oral route (Fernandez et al., 1998; Saha-
gun et al., 2000, 2001).
The macrocyclic lactones are available to be administered by the paren-
teral, oral and topical routes to ruminants. In the early days, shortly after the
introduction of ivermectin into the market, nematode susceptibility was
high and equivalent efficacy patterns were observed against abomasal para-
sites after parenteral or oral treatment in sheep/goats. A similar pattern was
described later on for other macrocyclic lactones from both the avermectin
(abamectin) and milbemycin (moxidectin) families. A slightly improved
ivermectin efficacy against sheep intestinal nematodes was observed after
oral compared with parenteral treatment (Borgsteede, 1993). However,
when the efficacy was assessed against ivermectin-resistant nematodes, a sig-
nificant greater pharmacological activity was observed after oral administra-
tion of both abamectin and moxidectin compared with their SC
administration to lambs (Alka et al., 2004; Gopal et al., 2001). Thus, the
pharmacological basis underlying the observed differential efficacy patterns
against H. contortus after the macrocyclic lactones administration by both
routes was recently investigated (Lloberas et al., 2012). The simultaneous
measurement of drug concentrations in blood, GI tissues of parasite location
and within resistant target worms was performed on H. contortus-infected
lambs. Enhanced ivermectin plasma concentrations were obtained after
SC treatment compared with oral administration (Lloberas et al., 2012),
which supports the use of the parenteral route to control ectoparasites.
The higher ivermectin plasma profiles observed in the SC-treated iver-
mectin group related to an enhanced systemic availability. The mean iver-
mectin systemic availabilities (measured as AUC) were 129 (SC) and
58.4 ng d/mL (oral treatment). In addition, the described longer mean resi-
dence time and elimination half-life observed for ivermectin after its SC
administration accounted for the persistent antiparasitic activity (over
10 days) against H. contortus in sheep (Borgsteede, 1993). Markedly lower
ivermectin concentrations were recorded in the abomasal contents after
the SC administration of the drug to infected lambs (Lloberas et al.,
2012). The mean ivermectin concentrations achieved (3 days posttreatment)
in the abomasal content were 143 ng/g (oral ivermectin) and 2.53 ng/g (SC
ivermectin). The active secretion of ivermectin (and other macrocyclic lac-
tones, such as doramectin) from the bloodstream to the abomasal lumen is of
little relevance (Hennessy et al., 2000), as opposed to that observed at the

small intestine level. Consistently, early studies with ivermectin showed
that its SC administration at ten times (2 mg/kg) the therapeutic dose to
sheep resulted in very low concentrations in the abomasal content (Bogan
and McKellar, 1988). This phenomenon may indicate that ivermectin con-
centrations achieved in the adult H. contortus after the parenteral treatment
may relate mainly to the drug coming from the bloodstream. The amount
of drug reaching the target parasite is influenced by the drug concentrations
in the tissues where the parasite is located (Lifschitz et al., 2000). The higher
concentrations measured in the abomasal content after the oral administra-
tion of ivermectin accounted for a greater amount of drug being measured
within adult H. contortus recovered from treated sheep. The ivermectin con-
centrations in H. contortus were 74.4 ng/g (oral) and 5.19 (SC). These
enhanced ivermectin concentrations explain the lower number of adult
H. contortus specimens recovered after treatment and the enhanced efficacy
obtained after the oral treatment, despite the high level of resistance
observed. The high concentrations of drug detected in the GI tract during
the first 2e3 days after the oral treatment may have a relevant effect on
the resistant nematodes, being of great importance to induce the pharmaco-
logical action at the target site.
It is also likely that the concentrations of the dissolved drug attained in
the gut lumen after oral administration of an anthelmintic preparation
may be critical to the pharmacological activity against worms in the
abomasum and small intestine, particularly if they have a reduced suscepti-
bility to the drug. Lipophilic drugs, such as ivermectin, may reach the target
parasite from the GI contents (transcuticular route) or from plasma (oral
ingestion) if the nematode (H. contortus) feeds on host blood. Increasing
drug exposure may be a useful strategy for killing heterozygous resistant par-
asites present in the earliest phases of development of resistance. Interest-
ingly, a similar concept was applied to cattle against resistant Cooperia spp.
A recent trial in cattle confirmed these findings (Leathwick and Miller,
2013). The activity of the drug against Cooperia oncophora was significantly
higher after the oral treatment with moxidectin. Furthermore, the clinical
efficacy against ivermectin-resistant nematodes in cattle was significantly
greater after the oral (74%) compared with SC (54%) ivermectin treatment
(Canton et al., 2015). Altogether, these findings show that the administra-
tion of macrocyclic lactones by the SC injection may achieve lower efficacy
against nematodes located at the GI tract compared with oral treatment.
These differences in drug efficacy attributed to the administration route
may only be evidenced if the parasite population has a reduced susceptibility.

The improved efficacy obtained in sheep and cattle after oral administration
of macrocyclic lactones may be based on an enhanced drug exposure of the
worms located at the lumen of the abomasum and/or small intestine. Such a
finding may have direct impact on the practical use of the macrocyclic
lactones in ruminant species accounting for a marked improvement in the
control of macrocyclic lactoneeresistant nematodes in the field, at least in
the early stages of resistance development. The influence of the route of
administration on the systemic exposure and efficacy of macrocyclic lactones
against resistant H. contortus is shown in Fig. 1.
The AADs represent one of the newest anthelmintic classes (Kaminsky
et al., 2008) introduced to veterinary medicine. From many compounds
evaluated, the racemic molecule AAD 96 was selected and the active
s-enantiomer of this molecule, named monepantel, was launched into the
pharmaceutical market for oral administration to sheep in 2009 (Hosking
et al., 2010). The monepantel plasma concentration profiles were deter-
mined after its intravenous and oral administration to sheep at 1, 3 and
10 mg/kg (Karadzovska et al., 2009). The monepantel and monepantel sul-
phone concentrations at the sites of parasites location were also shown to
improve the understanding of its anthelmintic efficacy (Lifschitz et al.,
Routes of macrocyclic lactones administration and efficacy

against resistant Haemonchus contortus
Oral/intraruminal treatment Subcutaneous treatment
Enhanced absorpon, high systemic

Reduced systemic availability.
availability but lower drug levels achieved
Large drug concentraons at the
at the GI luminal contents compared
GI sites of parasites locaon
to oral administraon
Enhanced drug concentrations

recovered within adult H.contortus specimens after the oral treatment
Advantageous pattern of efficacy against resistant worms
Figure 1 Impact of the route of administration on the systemic exposure and efficacy
of the macrocyclic lactones against resistant Haemonchus contortus. GI, gastrointes-
tinal tract.
2014). Higher concentration profiles of the metabolite monepantel

sulphone compared with the parent drug were measured in the sheep
bloodstream (Hosking et al., 2010; Karadzovska et al., 2009; Lifschitz
et al., 2014). The peak concentration (Cmax) of monepantel sulphone
was fourfold higher compared with that measured for the parent compound.
Monepantel is rapidly converted in the liver into different metabolites
(Karadzovska et al., 2009). Under the described circumstances, it is impor-
tant to understand the fate of the drug within the GI tissues and fluid con-
tents. In the abomasum, drug concentrations of the monepantel parent were
much higher than those measured in plasma, but monepantel sulphone was
also recovered. In the abomasal content, monepantel concentrations were
recovered in a range between 2000 and 4000 ng/g during the first 48 h after
treatment. Interestingly, monepantel sulphone was also detected in the
abomasal content, but the concentrations were significantly lower compared
with those of monepantel (P < 0.05) (gastric secretions may be involved in
the appearance of monepantel sulphone in abomasal content, as was demon-
strated for benzimidazole compounds in sheep (Hennessy, 1993)). The char-
acterization of monepantel and monepantel sulphone accumulation in target
digestive tissues provides relevant information on drug exposure for GI
nematode parasites. Such a kinetic pattern may support the well-established
high efficacy of monepantel against H. contortus (see Kaminsky et al., 2009).
Both monepantel and its sulphone metabolite may reach the target parasite
from plasma after oral ingestion. However, considering that they are highly
lipophilic compounds (Karadzovska et al., 2009), the great availability of
monepantel and monepantel sulphone in the abomasal content could facil-
itate an accumulation of both active molecules within the parasite through a
transcuticular diffusion process. There is no published information that cor-
relates the pharmacokinetics of monepantel with clinical efficacy. The con-
centrations of monepantel and monepantel sulphone at the site of the
parasite required to inhibit parasite establishment and/or development
have not been determined. However, the characterization of drug accumu-
lation in target tissues may provide information to predict the drug concen-
tration below which the effectiveness against larval and adult parasites begins
to decrease. AADs act on a nematode-specific acetylcholine receptor and
produce marked effects on the movement, growth and viability of nema-
todes (Kaminsky et al., 2008). In vitro experiments have shown that the
phenotypic effects of these compounds on free-living nematodes and adult
H. contortus are observed at 50e100 ng/mL, but lethality occurs at drug
concentrations of >1000 ng/mL.
The study by Sager et al. (2010) might also provide useful information on
the pharmacokineticepharmacodynamic relationship for this novel drug.
These authors studied the speed at which a reduction of nematode eggs in
the faeces of sheep occurs after monepantel treatment. A significant reduc-
tion of eggs in faeces was obtained 36 h after treatment, and the faecal egg
counts reduced to zero 72 h following treatment. This time-course of
monepantel pharmacological activity correlates to the highest monepantel
concentrations in the abomasum during the first 48 h following treatment.
3.3 Dosage and relationship between enhanced drug

exposure and anthelmintic efficacy
Haemonchus contortus has been used as a nematode model to assess the impact
of increasing the dose rates on the pharmacokinetics and subsequent drug
efficacy against resistant isolates. The therapeutic response to an increased
dosage may depend on the genetic status of the resistant nematode popula-
tion being exposed to the drug. The impact of large increases of dose on sys-
temic concentrations of benzimidazole and on the subsequent efficacy
against benzimidazole-resistant nematodes in ruminants remains unclear.
In the case of benzimidazoles, it has also been demonstrated that their
efficacy relies on the time the parasite is exposed to ‘‘toxic’’ concentrations
and that anthelmintic activity is influenced by the residence time of the drug
in the animal’s body (Lanusse and Prichard, 1993b). The oral absorption of
most drugs follows first-order kinetics, whereby a constant fraction of the
total drug present is absorbed in each equal interval of time (Neubig,
1990). This statement, true for most of the drugs commonly used in
veterinary therapeutics, remains unclear for the benzimidazole compounds
in ruminant species. Moreno et al. (2004) evaluated the clinical efficacy of
albendazole, given at two different dose rates, 3.8 mg/kg (a therapeutic
dose recommended against GI nematodes in some countries) and
7.5 mg/kg against benzimidazole-resistant nematodes in naturally infected
sheep. Significantly higher Cmax and AUC values were observed for
both albendazole metabolites following the treatment at the highest dose
rate (7.5 mg/kg) compared with 3.8 mg/kg. Also the time of residence
(MRT) of albendazole sulphoxide was significantly longer after the albenda-
zole treatment at 7.5 mg/kg. Although parasites were highly resistant to the
treatment with albendazole orally administered at both dose rates, the clin-
ical efficacy against H. contortus tended to be higher after the administration
of albendazole at 7.5 mg/kg compared with that observed at the lowest
investigated dose rate. For instance, clinical efficacy against H. contortus
increased from 5.9% (3.8 mg/kg treatment) up to 46.1% (7.5 mg/kg).

Albendazole molecules adsorbed to ruminal particulate material gradually
reached the abomasum and small intestine, the main sites of dissolution
and absorption, respectively (Hennessy, 1993). Within a therapeutic dose
range, when a higher oral dose is administered, a greater amount of the
drug suspension will gradually pass to the abomasum being available to be
absorbed in the duodenum over time following treatment. The lower
plasma profiles of both albendazole metabolites observed at the 3.8 mg/kg
treatment may be due to a lower total amount of albendazole being available
to be absorbed, which explains the positive relationship between the
amount of drug administered and the systemically available concentrations
of both metabolites in the treated animals. Furthermore, a high correlation
between the concentration profiles of albendazole metabolites measured in
the bloodstream and those in tissues of parasite location and within target
parasites collected from treated sheep has been demonstrated (Alvarez
et al., 1999, 2000). These previous pharmacokinetic findings clearly indicate
that the enhanced drug concentrations and the prolonged plasma detection
of the drug observed for a higher dose may account for greater drug expo-
sure of the target nematodes, which would also explain the tendency
observed in the anthelmintic efficacy against resistant parasites. Further
studies evaluated the pharmacokinetic and efficacy of albendazole against
resistant H. contortus administered at 5, 15 and 45 mg/kg (Alvarez et al.,
2012). The dose level affected the plasma disposition kinetics of albendazole
sulphoxide in treated lambs. The time of albendazole sulphoxide detection
in plasma increased from 1e48 h (ABZ5 mg/kg) to 1e96 h (ABZ15 mg/kg)
and 1e120 h (ABZ45 mg/kg). The AUC of the active albendazole sul-
phoxide metabolite increased from 21.0 6.4 mg h/mL (ABZ5 mg/kg)
up to 158.6 19.4 mg h/mL (ABZ15 mg/kg) and 389.7 98.2 mg h/mL
(ABZ45 mg/kg). Furthermore, when the mean AUC values were adjusted
by the dosages, the albendazole sulphoxide plasma exposure after the admin-
istration of 15 and 45 mg/kg doses was 78% and 43% higher, compared with
the therapeutic dose (5 mg/kg). These AUC values show a lack of propor-
tionality in the relationship between dose and systemic availability. Addi-
tionally, a significantly longer MRT value was observed for albendazole
sulphoxide in both ABZ15 mg/kg and ABZ45 mg/kg treated groups. Thus,
the higher dose-normalized AUC (ABZ15 mg/kg) and the longer MRT
values (ABZ15 mg/kg and ABZ45 mg/kg) obtained for albendazole sulphoxide
reflect some type of nonproportionality in the doseeplasma concentrations
relationship. The lack of dose proportionality observed for albendazole
(estimated as the albendazole sulphoxide systemic exposure) may be associ-

ated with a saturation of the enzymatic pathways involved in biotransforma-
tion. It has been demonstrated that the FMO-mediated sulphoxidation
accounted for up to 60% of the albendazole sulphoxide production from
albendazole, while CYP contributed the remainder (40%) in sheep liver
microsomes (Virkel et al., 2004). Thus, a decreased metabolic rate for the
conversion of albendazole sulphoxide into albendazole sulphone may
have accounted for the marked changes observed in the albendazole
sulphoxide disposition kinetics after the administration of three times
(ABZ15 mg/kg) the therapeutic dose. As expected, there was a highly negative
correlation between the albendazole sulphoxide AUC and Cmax values,
and the number of adult H. contortus recovered from treated lambs. The
enhanced systemic exposure achieved after albendazole treatments at the
highest dosages correlated with a significant increment in drug efficacy
against a resistant H. contortus isolate. In fact, the efficacies against resistant
H. contortus were 16% (ABZ5 mg/kg), 59% (ABZ15 mg/kg) and 94%
(ABZ45 mg/kg) (Barrere et al., 2012). Benzimidazole resistance has been
correlated with genetic changes associated with the b-tubulin gene (Lubega
and Prichard, 1990). These changes, mainly at positions 200 and 167 of the
b-tubulin gene, determine a reduced binding affinity, which explains the
development of anthelmintic resistance to benzimidazole compounds
(Beech et al., 1994; Kwa et al., 1994). A loss of receptor affinity increases
the concentration of drug needed for a given degree of response. These find-
ings indicate that higher active drug/metabolite concentrations associated
with the highest albendazole dosages resulted in significant increased drug
efficacy. As benzimidazole metabolites are reversibly exchanged between
the bloodstream and the GI tract (Lanusse et al., 1993), the enhanced
drug concentrations associated with the increasing doses administered may
account for GI nematodes being exposed to toxic drug concentrations for
an extended period of time. This finding helps to explain the reversion of
the drug resistance phenomenon observed after administration of albenda-
zole at a high dose level. In addition, Barrere et al. (2012) evaluated the pres-
ence of single nucleotide polymorphisms (SNPs) in the b-tubulin gene of
H. contortus after the albendazole treatment at three dose rates (5, 15 and
45 mg/kg). This investigation corroborated a strong association between
the presence of SNPs at codons 167 and 200 in isotype 1 of b-tubulin
and the survival of H. contortus individuals at a high dose rate of albendazole.
The genotype (Phe/Phe)167-(Tyr/Tyr)200 was consistently observed in
worms exhibiting high levels of resistance. Heterozygosity at both codons
167 and 200 conferred resistance with treatments of up to three times the
recommended albendazole dose rate (Barrere et al., 2012). Routine tests
for the evaluation of benzimidazole resistance, using analysis of the b-tubulin
gene, should include the assessment of both the 167 and 200 codons to assess
heterozygosity at both of these codons.
From a pharmacological point of view, anthelmintic drugs need to have
the best opportunity to act on the specific target nematode site of action
(Hennessy, 1997). This concept applies to the different strategies addressed
to increase parasite drug exposure. For macrocyclic lactones, the impact of
increasing dosage levels was evaluated in a high-resistance scenario, allowing
the prediction of the consequences of rational dose adjustments. The com-
parison of the pharmacokinetics, distribution and efficacy against resistant
H. contortus of single and double doses of ivermectin and moxidectin was
recently evaluated in lambs (Lloberas et al., 2015). The plasma concentration
profiles were related to the dose-rate administered for both drugs. For both
ivermectin and moxidectin, the mean Cmax and AUC were higher for the
0.4 compared with 0.2 mg/kg treatments (P < 0.05), but there were no dif-
ferences for other parameters. Ivermectin and moxidectin concentrations in
the GI target tissues and in H. contortus were much higher compared with
those measured in plasma. Mean drug concentration levels in abomasal
contents were 26e47 (ivermectin) and 26e36 (moxidectin) fold higher
than those achieved in plasma at day 1 following administration of both
compounds at 0.2 and 0.4 mg/kg, respectively. Ivermectin and moxidectin
concentrations in the GI target tissues were significantly higher after their
administration at 0.4 mg/kg. The exposure of H. contortus to ivermectin
and moxidectin was related to the level of the administered dose. Concen-
trations of the macrocyclic lactones measured in H. contortus correlated to
those recovered from abomasal content. The increment of drug concentra-
tions at the tissue sites of parasite location accounted for an enhancement on
drug levels measured within the worm. Based on pharmacological princi-
ples, all the strategies that maximize drug availability (exposure) at the
hosteparasite interface may increase the nematodicidal effect. As drug
concentrations at the GI target tissues/contents during the first 2e3 days af-
ter treatment are relevant for the effectiveness of the macrocyclic lactones
against resident worms in sheep (Lloberas et al., 2013), the higher drug accu-
mulation observed within the nematode after the administration of iver-
mectin and moxidectin at double doses (0.4 mg/kg) may be useful to
increase the efficacy against resistant worms. Despite the high concentrations
recovered from the abomasal content and within the worm, ivermectin
failed to control a highly resistant isolate of H. contortus (see Lloberas et al.,

2013). Moxidectin showed a higher performance at both doses assayed.
Ivermectin had no efficacy at both dose rates. In contrast, the efficacy of
moxidectin against this ivermectin-resistant H. contortus isolate was 85.1%
(0.2 mg/kg) and 98.1% (0.4 mg/kg). Interestingly, whereas the double
dose of ivermectin remained ineffective against H. contortus, high efficacy
(98.2%) was achieved after the administration of moxidectin at
0.4 mg/kg. Particular pharmacodynamic features for each macrocyclic
lactone may play a relevant role on the activity against resistant nematodes.
A differential pattern of interaction at the glutamated-gated chloride channel
may support the higher efficacy of moxidectin (Hibbs and Gouaux, 2011;
Prichard et al., 2012). The way a drug enters into the nematodes is relevant
to the efficacy of the anthelmintics compounds. To corroborate the in vivo
results, the accumulation of both compounds was also evaluated ex vivo.
The incubation of adult nematodes in 0.5 mM of ivermectin and moxidectin
reflects the in vivo concentrations of both drugs measured in the abomasal
contents after their administration at 0.2 mg/kg to sheep. There was a con-
centration and time influence in the accumulation of ivermectin and mox-
idectin in H. contortus. A significantly higher amount of both drugs was
measured within H. contortus after the incubation with 5 mM of ivermectin
and moxidectin. In addition, the incubation for 3 h resulted in a higher
accumulation of both drugs (five- to sevenfold) compared with the accumu-
lation assay performed over 15 min. The ex vivo uptake of benzimidazole
drugs by nematodes, trematodes and cestodes has been extensively studied
(Alvarez et al., 1999, 2001; Mottier et al., 2006). Drug lipophilicity and
the permeability of helminth external surfaces determine the effective con-
centrations that reach the site of action. These findings confirm that the
increment on drug exposure for both ivermectin and moxidectin accounted
for an enhanced amount of drug being recovered from the target parasite,
which was observed in both, the ex vivo and in vivo experiments. The
degree of susceptibility of a nematode population is a relevant topic to deter-
mine the impact of the increment of the local drug exposure. If the response
of a drug reaches 80% of its maximum, the final effect will be insensitive to
further changes in drug concentrations (Holford and Sheiner, 1981). If a
clinical study is conducted with animals infected with a susceptible parasite
isolate, a dose-dependant increased therapeutic effect will not be detected.
However, if animals are infected with nematodes displaying reduced suscep-
tibility, the clinical response to the increment of the drug exposure at the site
of action may be increased (Martinez, 2014). The administration of
ivermectin and moxidectin at 0.4 mg/kg accounted for enhanced drug

exposure in the target tissues as well as for higher drug concentrations within
the resistant nematodes. Given the extremely high degree of resistance of the
H. contortus isolate being tested, the administration of a double-dose treat-
ment was only effective for moxidectin.
Alvarez et al. (2015) assessed the disposition kinetics and efficacy of iver-
mectin against resistant H. contortus after SC and IR administrations at one,
five and ten times the therapeutic dose to lambs. The ivermectin systemic
exposure increased from 41.9 20.1 ng d/mL (IVMSCx1) up to
221 55.9 ng d/mL (IVMSCx5) and 287 100.4 ng d/mL (IVMSCx10).
The higher dose levels (five and ten times) were also correlated to a signif-
icant enhancement of the ivermectin peak plasma concentrations, which
were obtained at the same time after treatment. Furthermore, when the
mean AUC and Cmax values were normalized based on dose, differences
among groups did not reach statistical significance to show a dose-propor-
tional relationship. The plasma concentrations of ivermectin obtained after
IR administration were also related to the level of the different doses. A
higher AUC value was measured for the IVMIRx10 group
(AUC ¼ 323 ng d/mL) compared with that obtained after the IVMIRx1
treatment (AUC ¼ 20.8 ng d/mL). As observed following the SC treat-
ment, when the AUC and Cmax values were normalized by the dose,
similar absorption rates were obtained for the treatments at different doses.
The low efficacy level (42e50%) obtained at the therapeutic dose
(0.2 mg/kg) after both routes of administration confirmed the high iver-
mectin-resistant status of the worms. The efficacy of ivermectin against adult
H. contortus increased with the increment of the dose both, after the SC and
IR treatment. After the SC treatment, an anthelmintic efficacy of 75% was
observed for the administration of the five and ten times the doses. After the
IR administration of ivermectin, these higher doses resulted in a significant
(P < 0.05) reduction in adult H. contortus counts, compared with that
observed both in the group treated at the therapeutic dose and the untreated
control. A high efficacy was observed in both the IVMIRx5 (96%) and
IVMIRx10 (98%) groups. Since drug accumulation in GI nematodes appears
to be mainly related to drug diffusion from the surrounding medium (GI
fluids), the enhanced ivermectin concentration in abomasal content after
the IR administration may have accounted for an increased drug accumula-
tion in H. contortus, explaining the improved efficacy observed after IR treat-
ment at the highest doses. Within a population of H. contortus, individual
parasites do not respond uniformly to treatment. Some were killed by a
therapeutic ivermectin dose, other tolerated this dose, but were eliminated
by treatment at the higher doses (five and ten times), and some particular in-
dividuals survived even ten times the therapeutic dose. This ‘dose-related
behaviour’ may be explained by genetic diversity in the parasite population
(Prichard, 2001). High genetic diversity has been described for different
H. contortus populations, also relating to genes encoding b-tubulins (Beech
et al., 1994; Kwa et al., 1994), P-gp (Blackhall et al., 1998a; Sangster and
Gill, 1999) and glutamate-gated chloride channel (GluClR) subunits (Black-
hall et al., 1998b). Macrocyclic lactones resistance is quite complex, with
mechanisms varying both within and between species (Gill and Lacey,
1998). The development of resistance to ivermectin requires of the simulta-
neous mutation of several genes to develop a high level of resistance (Martin
et al., 2002). In this context, the variation in response according to the
dosage administered may be explained by genetic diversity within the
isolate. In conclusion, both the genetic variability and the potential differ-
ences on drug accumulation, according to the location of H. contortus within
the abomasum, may have accounted for the observed differences in efficacy
related to the dose level of ivermectin. These data shown the ivermectin
resistance may be overcome by increasing the amount of the active drug
at the biophase. Indeed, under experimental conditions, an IR ivermectin
dose as high as 5- to 10-fold the therapeutic dosage was necessary to reach
an acceptable efficacy level against resistant H. contortus. From a clinical point
of view, the inconvenience of recommending high dose rates may be asso-
ciated with the selection of highly resistant nematodes, in addition to the
impact on drug residues, withdrawal times, etc., which would preclude its
use as a ‘practical’ strategy when resistant parasite populations are present.
4. MODULATION OF DRUG EFFLUX TRANSPORT:

IMPACT ON CLINICAL RESPONSE AGAINST RESISTANT
4.1 Relevance of cellular efflux transport on systemic
exposure and drug action
The influence of cell transporter systems on the pharmacokinetic
behaviour of different drug compounds has been profoundly studied, and
the interaction between drug compounds at the transport proteins levels is
now considered as a key pharmacological issue with a variety of potential
therapeutic implications. For instance, the interactions of macrocyclic lac-
tones with different ATP-binding cassette (ABC) transporters have been
thoroughly investigated. Of all identified cell transporters, P-gp has been the
most studied. P-gp was initially described due to its capacity of preventing
the intracellular accumulation and cytotoxic effects of antineoplastic drugs
by actively removing them from the cell membrane before they reach their
intracellular target. Besides tumour cells, P-gp has also been identified in
several healthy tissues and particularly in organs actively involved in drug
pharmacokinetics (Schinkel, 1997). P-gp is located in tissues and particularly
in organs involved in the processes of drug absorption (eg, mucosa of the
small and large intestine), distribution (eg, braineblood barrier) and elimina-
tion (luminal surface of hepatocytes and ducts cells, kidney tubules and
enterocytes) (Lin, 2003). The interaction of macrocyclic lactones with
different P-gp has been well demonstrated (Lespine et al., 2007), and
different aspects of this interaction recently reviewed in the literature
(Lespine et al., 2012; Lifschitz et al., 2012). Considering the wide use of
the macrocyclic lactones in different animal species, it is likely that some
kind of drugedrug interactions may occur after their co-administration
with a large variety of drug compounds. The induction of the activity of
this transport system, for example, at the intestinal level, will lead to the
reduction of the bioavailability of orally administered P-gp substrates, while
an increment of the bioavailability would be observed when an inhibitor is
co-administered with a P-gp substrate. The interaction between macrocy-
clic lactones and cell transporters was characterized by in vitro and in vivo
trails. Ivermectin is actively secreted by cells transfected with gene encoding
P-gp in mice (Schinkel et al., 1995). The interaction of moxidectin with the
ABC transporters was demonstrated in cultured rat hepatocytes. Ketocona-
zole, quercetin and fumagillin increased the quantity of 14C-moxidectin in
hepatocytes (Dupuy et al., 2003, 2006). Recently, it has been shown that
affinity by P-gp may differ among different macrocyclic lactone molecules
(Lespine et al., 2007). The different macrocyclic lactones were tested for
their ability to inhibit the P-gp mediated rhodamine 123 (Rho123) transport
function in recombinant cell lines over-expressing P-gp. The different aver-
mectins (ivermectin, eprinomectin, abamectin, doramectin and selamectin)
increased the intracellular Rho123 accumulation with a similar potency. It is
interesting to note that moxidectin appears to have different P-gp efflux
potential, with a half-maximal inhibitory effect (IC50) approximately ten
times higher than that reported for ivermectin (Griffin et al., 2005; Lespine
et al., 2007). Using the everted sac technique, the ivermectin accumulation
rate in the intestinal wall was significantly higher after its incubation with the
P-gp inhibitors itraconazole and PSC833 than that obtained after its
incubation alone (Ballent et al., 2006). The strong ivermectin interaction

with sheep intestinal P-gps was demonstrated employing an Ussing chamber
system (Ballent et al., 2012). The in vivo involvement of multiple trans-
porters suggests a complex interplay between macrocyclic lactones and the
different ABC transporter proteins, which could affect their systemic
disposition.
In vivo trials performed on different animal species provided information
on the action of different P-gp modulators on the macrocyclic lactones
pharmacokinetic disposition. Important changes to the plasma disposition
of the macrocyclic lactones have been observed when these compounds
were co-administered with P-gp modulating agents. The effect of verapamil
(a P-gp modulator) on ivermectin plasma disposition kinetics after pour-on
treatment in rats (Alvinerie et al., 1999) and after oral administration to sheep
(Molento et al., 2004) has been demonstrated. Significantly higher moxidec-
tin plasma concentrations were observed after its co-administration with
loperamide (an opioid derivative acting as P-gp substrate) compared with
those measured after moxidectin given alone (Lifschitz et al., 2002). In
lambs, quercetin (Dupuy et al., 2003), itraconazole (Ballent et al., 2007)
and ketoconazole (Alvinerie et al., 2008) produced a significant increase
on moxidectin and/or ivermectin systemic exposure.
The differential affinities of macrocyclic lactones by P-gp were recently
assayed in vivo using the knockout mouse model (Kiki-Mvouaka et al.,
2010). P-gp deficiency led to a significant increase in the systemic availability
of ivermectin (1.5-fold) and eprinomectin (3.3-fold), whereas the moxidec-
tin availability remained unchanged. Ivermectin and to a greater extent
eprinomectin were both excreted by the intestine via a P-gp-dependent
pathway, whereas moxidectin excretion was less compared to the avermec-
tin-type macrocyclic lactones (Kiki-Mvouaka et al., 2010).
Most studies of drug interactions mediated by cell transporters have been
conducted to modulate/inhibit their activity, and thus, to increase the
absorption or delay the elimination of therapeutically relevant drugs.
However, the effect of potential inducers of the transport proteins on the
kinetic behaviour of macrocyclic lactones is not fully understood. Recent
work reported that ivermectin can also induce P-gp expression and function
through mRNA stabilization in murine hepatic cells (Ménez et al., 2012).
Although numerous therapeutic agents can induce P-gp expression under
in vitro conditions, the relevance of these observations in relation to P-gp
induction in vivo is not entirely clear. The experimental effect of the inducer
agent phenobarbital on both plasma and GI disposition of ivermectin was
examined in our laboratory (Ballent et al., 2010). The ivermectin AUC

values measured were significantly lower in the plasma, intestine and liver
tissue of rats pretreated with phenobarbital. On the other hand, the chronic
administration of dexamethasone in sheep produced a decrease in P-gp
expression along the small intestine compared with untreated control
animals (Ballent et al., 2013). Thus, a better understanding of the factors
regulating P-gp and other cell transporters expression is needed to elucidate
the clinical implications of drugedrug interactions in pharmacotherapy in
livestock animals. More specifically, this is an open field for the future of
the antiparasitic drugs that needs to be addressed if the combination of
anthelmintic molecules turns into an alternative for parasite control in resis-
tant populations.
4.2 Modulation on drug transport and efficacy against

resistant Haemonchus contortus
Currently, resistance to antiparasitic drugs is recognized as a problem in small
ruminant and bovine production systems (Kaplan and Vidyashankar, 2012;
Prichard, 1994). The drugedrug interactions at the efflux transporter pro-
tein level are not only important in the host but also at the target nematodes.
P-gp has been described not only in mammals but also in parasites, such as
Onchocerca volvulus (see Kwa et al., 1998) and H. contortus (see Prichard and
Roulet, 2007). Drug efflux mediated by P-gps in different parasites has
been proposed as a potential resistance mechanism for different drugs (Xu
et al., 1998). Increased scientific evidence supporting this concept has
been reported during the last few years. Modifications of the pattern of P-
gp expression have been observed in resistant nematodes recovered from
lambs treated with macrocyclic lactones (Prichard and Roulet, 2007). An
upregulation of P-gp in H. contortus recovered one day after ivermectin
treatment has been reported, and also in a lesser degree, after the moxidectin
administration (Prichard and Roulet, 2007). Recent work demonstrates that
ivermectin treatment significantly increases P-gp2 expression in resistant
H. contortus recovered from treated lambs 0.5 and 1 day after treatment
compared with those parasites recovered from untreated animals (Lloberas
et al., 2013). However, treatment with moxidectin did not induce any
significant modification on the pattern of the drug transporter expression
in the nematode (Lloberas et al., 2013). The chemical structure differences
between moxidectin and ivermectin may account for their differential
pharmacokinetic, pharmacodynamic and P-gp interaction patterns (Prichard
et al., 2012).
To evaluate the impact of P-gp in the parasites, the inhibition of the ac-
tivity of P-gp has been assayed as a pharmacology-based strategy not only to
increase the systemic availability of the macrocyclic lactones in the host an-
imal, but also to improve their clinical efficacy. The combinations of VRP
and CL347099 with either ivermectin or moxidectin significantly reduced
worm counts of resistant isolates of H. contortus (see Molento and Prichard,
1999). In addition, the modulation of P-gp increased the in vitro activity of
ivermectin against ivermectin-sensitive and resistant larvae of H. contortus.
The presence of the P-gp modulators PSC833, VRP, ketoconazole and
pluronic 85 in the larval feeding inhibition test enhanced the sensitivity of
larvae (H. contortus resistant isolates) to ivermectin by between 15- and
57-fold (Bartley et al., 2009). Another study has shown that macrocyclic
lactones anthelmintics, which inhibit P-gp-mediated efflux in mammals,
activate transport activity in nematodes, suggesting a complex interaction
of macrocyclic lactones with P-gps in the parasites (Kerboeuf and Guégnard,
2011).
Although a modification of macrocyclic lactone activity after P-gp
modulation was confirmed in vitro, in vivo trials performed under field
conditions are necessary to evaluate the clinical impact of the P-gp inhibi-
tion. The enhanced sensitivity of resistant larvae to ivermectin obtained after
its co-incubation with pluronic 85 did not correlate with their in vivo
co-administration to sheep (Bartley et al., 2009). In the in vivo trial, the pres-
ence of pluronic 85 did not improve the efficacy against resistant H. contortus
(see Bartley et al., 2012). However, significant increment on ivermectin ef-
ficacy against resistant nematodes of sheep together with an enhancement on
ivermectin systemic availability was obtained in the presence of loperamide
(Lifschitz et al., 2010). The faecal egg count reduction increased from 78.6%
(ivermectin alone treatment) to 96% after the co-administration with loper-
amide. A nematode population highly resistant to ivermectin was identified.
The efficacy of ivermectin against H. contortus was 0%, and the percentage of
reduction against intestinal nematodes, such as T. colubriformis and Nematodi-
rus spp. was 77.9% and 85.5%, respectively. The clinical efficacy against the
resistant nematodes was enhanced in the presence of loperamide, with per-
centages of reduction of 72.5% (H. contortus), 96.3% (T. colubriformis) and
93.0% (Nematodirus spp.) (Lifschitz et al., 2010). Thus, there is evidence
that the P-gp-mediated drugedrug interaction increases the ivermectin sys-
temic exposure in the host, and it may also decrease the P-gp-mediated
efflux transport over-expressed in target resistant nematodes. The interac-
tion at the parasite tissue-level was specifically investigated using C. elegans
as a model system. It was confirmed that different P-gp isoforms protect

C. elegans from ivermectin toxicity and the interaction of P-gp with different
modulator agents enhances susceptibility to ivermectin, depending on the
drug concentration used (Ardelli and Prichard, 2013). Recently, the locali-
zation of P-gp2 was studied in H. contortus. P-gp2 was expressed in the phar-
ynx, the first portion of the worm’s intestine and perhaps in adjacent nervous
tissue, suggesting a role for this gene in regulating the uptake of avermectins
and in protecting nematode tissues from the effects of macrocyclic lactone
anthelmintic drugs (Godoy et al., 2015). The impact of P-gp modulation
on drug pharmacokinetics in the host and the efficacy against resistant
H. contortus is shown in Fig. 2.
It is evident that a P-gp-mediated drugedrug interaction increases the
systemic exposure of macrocyclic lactones in the host. However, such an
interaction might also occur in the target worm, which would decrease
the P-gp-mediated efflux transport over-expressed in target resistant nema-
todes. Different pharmacological approaches to delay the bile/intestinal se-
cretions and to extend the plasma-intestine recycling time of macrocyclic
lactones in the host have been investigated. The involvement of the
efflux-transport protein P-gp (and perhaps, other drug transporters) on
both the pharmacokinetic disposition (host) and resistance mechanisms
P-glycoprotein and therapeutic response

in resistant Haemonchus contortus
In vivo co-administraon of
Macrocyclic lactones and P-gp modulang agents
Enhanced target parasite exposure

Loperamide, Verapamil and Itraconazole- induced changes on
ivermecn disposion kinecs in sheep
(Molento et al., 2004, Ballent et al., 2007; Lifschitz et al., 2010)
Increased efficacy against resistant H.contortus

Loperamide restored ivermecn efficacy from complete
therapeuc failure up to 73 %
(Lifschitz et al., 2010)
Figure 2 Influence of the modulation of the efflux transport protein P-glycoprotein

(P-gp) on both drug pharmacokinetics in the host and efficacy against resistant Hae-
monchus contortus.
(target parasites) to different anthelmintic chemical groups has been out-

lined. The potential side effects and changes to the pattern of tissue residues
induced by the P-gp modulating agents should be carefully investigated
(Lespine et al., 2008). However, the search for specific P-gp modulators
with high affinity to parasite transport proteins may identify useful pharma-
cological tools to extend the life span of the macrocyclic lactones in veter-
inary medicine. The effect of the most-recently developed group of P-gp
inhibitors (the so-called ‘third generation’ of inhibitors, such as tariquidar,
zosuquidar and elacridar) was evaluated (Raza et al., 2015). Zosuquidar
restored the sensitivity of resistant worms to levels observed in susceptible
worms. On the other hand, the ability of tariquidar to increase the sensitivity
of ivermectin to the susceptible isolate raised the possibility of reducing the
recommended dose of an anthelmintic, while maintaining 100% efficacy
against susceptible worms (Raza et al., 2015). Further work is required to
assess the practical pharmaco-parasitological implications of the chemical
modulation of these cell efflux pump systems on antiparasitic therapy.
5. PHARMACOLOGICAL EVALUATION OF DRUG-

COMBINED THERAPY AGAINST RESISTANT
There is a long history of the use of drug combination for treating the
most dreadful diseases. For instance, the use of drug combination in cancer
chemotherapy was reviewed early (Goldin and Mantel, 1957). In 1959, it
was shown that the administration of both streptomycin and isoniazid to
tuberculosis patients markedly reduced the emergence of a drug-resistant
strain of the tubercle bacillus (Conn et al., 1959). Concerning parasite con-
trol, a drug combination (ie, ivermectin-clorsulon) has been successfully
used as a strategy to expand the efficacy spectrum (Geary et al., 2012). Addi-
tionally, in an attempt to overcome/manage anthelmintic resistance in rumi-
nants, combinations of two or more anthelmintics are primarily being used
(Geary et al., 2012). Haemonchus contortus has been extensively used as a
model nematode to test different strategies/approaches related to drug com-
binations. The combination of nematodicidal drugs could be achieved using
two or more pharmaceutical formulations containing a different active prin-
ciple each, or, alternatively, through the use of combined veterinary medic-
inal products (two or more active substances in the same preparation) named
as ‘fixed combination products’ (EMEA, 2006). The main goal of the use of
two or more drugs with different modes of action is to increase the treatment
efficacy. Other possible favourable outcomes after the use of drug combina-
tions include (1) the use of lower doses to avoid toxicity (reaching equivalent
or even higher efficacy) and (2) minimizing or slowing down the develop-
ment of drug resistance. The probability of selecting resistant parasites is
decreased if two drugs with different mechanism of action and different
biochemical pathways of resistance are administered together in combina-
tion chemotherapy. Thus, individual worms may have a lower degree of
resistance to a multiple component formulation (each chemical with
different mode of action) compared with that observed when a single
anthelmintic compound is used (Anderson et al., 1988; Barnes et al.,
1995; Leathwick et al., 2009). Thus, several pharmaceutical formulations,
combining either two or three chemical entities, have been developed.
Preparations combining the actives from the main available chemical groups
have been introduced into the veterinary pharmaceutical market in many
countries. However, potential pharmacokinetic and/or pharmacodynamic
interactions between drug components may occur.
A potential drug interaction refers to the possibility that one drug may
alter the intensity of the pharmacological effects of another drug when given
concurrently (Nies and Spielberg, 1996). The modified effect may result
from a change in the concentration of either one or both drugs in the organ-
ism (pharmacokinetic interaction) or from a change in the relationship
between drug concentration and response of the organism to the drug (phar-
macodynamic interaction). Pharmacodynamic drug-to-drug interactions
may occur at three different levels: (1) at the receptor site, (2) at the signalling
(ie, second messenger) or (3) at the effector level. They can lead to both
enhanced (additive or synergic effect) and diminished (antagonism) drug
responses. Overall, an additive effect is present when the combined activity
of two drugs equals the sum of their independent activities measured
separately. On the other hand, a synergistic effect is achieved when the com-
bined effects of the drugs are significantly greater than the independent
effects (Prescott, 2000). The presence of a pharmacological synergism im-
plies a drug effect is greater than that of additive effect. The effect achieved
in the presence of antagonism is less than additive (Chou, 2010). Synergism
normally occurs between drugs that exert the same effect (ie, anthelmintic)
via different modes of action. Since levamisole, albendazole, monepantel,
derquantel, organophosphates, closantel and ivermectin are chemical entities
that differ in their intrinsic anthelmintic mode of action, their co-adminis-
tration may potentially induce a synergistic effect. On the other hand,
when multiple resistance relates to different worm genera, each one resistant
to a unique chemical group, worms surviving one compound could be

killed by the other. In this case, the ‘additive’ effect exerted by the combined
product may allow the control of resistant nematodes.
In an effort to improve the control of highly resistant nematode (ie, H.
contortus) different drug combinations have been assessed. Pharmacodynamic
interactions resulting in synergistic effects can be clinically relevant and
would represent an ideal situation to deal with resistant parasites. For
example, a worm resistant to two different anthelmintics (bi-resistant
worm) could be killed by the combined effect of the same two drugs acting
synergistically. Due to the increasing anthelmintic resistance problem in the
ruminant livestock production systems, as well as in horses, nematodicidal
drug combinations appear to be potentially useful in veterinary medicine,
particularly in delaying the emergence and spread of resistance, and/or con-
trolling parasite populations with existing resistance (Geary et al., 2012). In
an ideal situation, if an anthelmintic treatment reaches 100% efficacy, selec-
tion of anthelmintic resistance will not occur. To achieve the highest effi-
cacy in treated animals, while the few surviving parasites are diluted into a
susceptible untreated nematode population, is a key principle for slowing
the emergence of anthelmintic resistance in a real field situation (Dobson
et al., 2001). Consequently, in farms where multiple-resistant nematode
populations are present, the use of drug combinations may be an alternative
to improving chemical control.
The development of anthelmintic resistance in H. contortus is a major
global problem, which has motivated the development and use of nemato-
dicidal combinations of two or more anthelmintics in several countries such
as Australia, New Zealand (Sutherland and Leathwick, 2011) and Uruguay
(Suarez et al., 2014). Early work investigated the potential additive or syn-
ergistic effect of different nematodicidal drugs used in combination against
multiple resistant nematodes, particularly H. contortus. Evidence of synergism
between fenbendazole and levamisole against H. contortus has been reported
(Miller and Craig, 1996). In a goats flock naturally infected with a resistant
isolate of H. contortus, the efficacy of fenbendazole, levamisole, fenbenda-
zoleelevamisole, ivermectin, albendazole and albendazoleeivermectin
was evaluated using a faecal egg count reduction test (FECRT). The results
showed no significant reduction for either fenbendazole (1%) or levamisole
(23%), whereas the combination reduced egg counts up to 62%. When the
animals were treated with albendazole or ivermectin alone, faecal egg count
reductions of 72% (albendazole) and 0% (ivermectin) were observed. An
enhanced efficacy (97%) was observed following the combined treatment
(Miller and Craig, 1996). These reductions in egg counts indicate an additive
action, which was not enough to be clinically effective in the case of the fen-
bendazoleericobendazole combination. Furthermore, a synergistic interac-
tion between derquantel and abamectin occurs under in vitro laboratory
conditions (Puttachary et al., 2013). In this study, the effects of derquantel,
abamectin and their combination on somatic muscle nAChR and pharyn-
geal muscle glutamate-gated chloride receptor channels of A. suum was
assessed. The study demonstrated that abamectin and derquantel interact
at the nAChR on the somatic muscle. At this level, the effect of the com-
bination was significantly greater than the predicted by an additive effect
of both drugs, suggesting a synergistic effect of the combination (Puttachary
et al., 2013).
A faecal egg count reduction of 92% was reported in naturally infected
lambs after the intravenous co-administration of albendazole and ivermectin,
in comparison to 73% (albendazole) and 79% (ivermectin) (Entrocasso et al.,
2008). The enhanced efficacy value observed for the albendazoleeivermectin
treatment, in comparison to each drug administered alone, was related to an
additive effect of both anthelmintic molecules acting via different modes on
different parasite genus/species. Albendazole and ivermectin each adminis-
tered alone by the intravenous route demonstrated high efficacies against
Haemonchus spp. (95.1% and 99.3%, respectively). Furthermore, the highest
reduction in Haemonchus spp. counts was observed with the albendazolee
ivermectin combination (99.9%) (Entrocasso et al., 2008). In the same study,
the combination of both chemicals administered by the IR (albendazole) or
SC (ivermectin) route did not have a positive effect on eggs count reduction.
In fact, the albendazoleeivermectin co-administration reached an efficacy of
71% in comparison to egg reductions of 44% (albendazole) and 80% (iver-
mectin). The faecal cultures showed Haemonchus spp. as the main parasite
resistant to albendazole and ivermectin (Entrocasso et al., 2008). The com-
bined action of albendazole and ivermectin on GI nematodes did not improve
the efficacy of ivermectin administered alone by the SC route. Similar to these
results, a recent field study undertaken in Uruguay (Suarez et al., 2014a)
showed an equivalent efficacy against multiple resistant H. contortus, following
the use of either a triple-combined treatment (levamisoleealbendazolee
ivermectin) or ivermectin alone. The observed anthelmintic efficacies were
87% (combined treatment), 80% (ivermectin treatment), 72% (albendazole
treatment) and 52% (levamisole treatment), indicating no advantageous effect
of the triple-combined preparation. However, the situation appears to be
different when a combined treatment is designed to control H. contortus
with low/moderate pre-existing levels of resistance against the drugs included

in the combination. The clinical efficacy of closantel and moxidectin admin-
istered each drug alone or in combination by the SC or the oral route was
assessed in lambs naturally parasitized with resistant nematodes (mainly
H. contortus) (Suarez et al., 2013). The results obtained showed that the
administration of closantel and moxidectin as a single active principle reached
efficacy levels (estimated as the FECRT) ranging from 80% (moxidectin oral),
84% (closantel oral) up to 85% (closantel SC) and 92% (moxidectin SC).
However, the combined treatments given both orally and subcutaneously
reached 100% efficacy. The combination permitted a restoration to
maximum efficacy levels, which were not reached by the individual active
ingredients. The above situation illustrates the performance of drug combina-
tions when the efficacy of the individual ingredients is relatively high.
However, when the individual components included in the combined
product demonstrate a low efficacy, a different situation may occur. Suarez
et al. (2014b) evaluated the efficacy of levamisole, albendazole, ivermectin
and their combinations (levamisoleealbendazole, levamisoleeivermectin,
albendazoleeivermectin, levamisoleealbendazoleeivermectin) against
multiple resistant H. contortus in naturally infected lambs. The observed
efficacies of the single drug treatments were 45% (levamisole), 68%
(albendazole) and 0% (ivermectin). For the combined treatments, efficacies
of 73% (levamisoleealbendazole), 35% (levamisoleeivermectin), 63%
(albendazoleeivermectin) and 71% (levamisoleealbendazoleeivermectin)
were observed. Thus, albendazole was the active compound with the highest
individual efficacy, and only the combinations containing albendazole
reached efficacies 60%. A ‘worse’ situation was described in a Brazilian
sheep flock in which low efficacy against the nematode genera Haemonchus
spp., Trichostrongylus spp. and Ostertagia spp. was observed for most of the
anthelmintic groups commonly used to control nematodes (Cezar et al.,
2010). The observed efficacies, evaluated by the FECRT, were 54%
(moxidectin), 32% (nitroxynil), 26% (disophenol), 23% (levamisole) and 0%
(albendazole sulphoxide, ivermectin, trichlorphon and closantel). The combi-
nation of levamisole, albendazole and ivermectin reached an efficacy of 68%,
showing that, in this scenario of compromised chemical control, the combi-
nation (with its limitations) demonstrated some utility to control GI nema-
todes. The enhanced anthelmintic efficacy obtained after the use of
nematodicidal combinations against resistant H. contortus derived from the
occurrence of positive pharmacokinetic and/or pharmacodynamic interac-
tions between combined molecules is illustrated in Fig. 3. The sustainability
of drug combinations in this scenario is unlikely. In conclusion, when a high

resistance status is observed, the combined treatments can offer a slight in-
crease in efficacy against multiple-resistant nematodes (including H. contortus),
but it seems likely that it will become ineffective with long-term use.
In cattle production systems, where individual active ingredients still
maintain their highest efficacy, the use of anthelmintic combinations could
be a useful tool to delay the development of resistance. Recent work
(Canton et al., 2014) evaluated the clinical efficacy (FECRT) observed after
the SC administration of ivermectin and ricobendazole given either sepa-
rately or co-administered to calves naturally parasitized with GI nematodes
resistant to ivermectin (H. contortus L3 represents 85% in faecal cultures). The
observed efficacies were 48% (ivermectin), 94% (ricobendazole) and 98%
(ivermectinericobendazole). Since no significant differences in the egg
Figure 3 Increased anthelmintic efficacy may be obtained after the use of nematodi-
cidal combinations against resistant Haemonchus contortus upon the occurrence of
positive pharmacokinetic (PK) and/or pharmacodynamic (PD) interactions between
combined molecules. The increased drug exposure (Alvarez et al., 2008; Suarez et al.,
2014), additive (Anderson et al., 1988; Entrocasso et al., 2008) or synergistic PD effects
(Miller and Craig, 1996) observed after the administration of a drug-combined product
may help to control resistant H. contortus. The main concerns related to the use of
‘potentially favourable’ combinations are associated with a limited sustainability if
the efficacy of each active included in the combined product is low (presence of highly
multiple-resistant parasites) and modifications of the safety profile and/or the pattern
of tissue residues depletion of a drug and/or its metabolites if large positive changes on
drug exposure occurs. Further details on the pharmacological basis of nematodicidal
combinations can be obtained from Lanusse, C., Alvarez, L., Lifschitz, A., 2014. Pharma-
cological knowledge and sustainable anthelmintic therapy in ruminants. Vet. Parasitol.
204, 18e33.
counts were observed between groups treated with ricobendazole alone and
the combined treatment (Canton et al., 2014), no therapeutic advantage was
observed for the combination. Preliminary results indicate that the combi-
nation of macrocyclic lactones (parenteral) and levamisole (oral) used in
combination was highly effective in minimizing the survival of macrocyclic
lactoneeresistant nematodes and the subsequent transport of parasites be-
tween farms (Smith, 2014). Since a key factor for an ‘optimal result’ of a
combined nematodicidal treatment is that the individual molecules reached
their highest efficacy (Geary et al., 2012), the use of anthelmintic combina-
tions in cattle production systems may be an important tool to delay resis-
tance (Lanusse et al., 2014). However, in vivo data obtained from sheep
production systems indicate that the use of anthelmintic combinations
may have limited success and sustainability, particularly in situations where
H. contortus populations with multiple drug resistance patterns are exten-
sively disseminated.
Anthelmintic resistance in human and animal pathogenic helminths
has been spreading in prevalence and severity. Multidrug resistance is
becoming a widespread problem in livestock animals. The use of pharma-
cology-based information for existing and novel molecules is critical to
the design of successful strategies for parasite control in the future. Modern
technologies will likely contribute to some leading products in the field of
diagnostic or drug discovery. Meanwhile, further pharmaco-parasitological
integrated work, supported by the substantial progress achieved in parasite
genomics, is required to generate the basic scientific knowledge necessary
to optimize drug action and to preserve available and novel active ingredi-
ents as useful tools for parasite control in livestock animals. The remarkable
amount of knowledge acquired using H. contortus as a target nematode has
markedly contributed to the overall understanding of the pharmacology
of anthelmintic drugs as well as of the comprehension of the mechanisms
of drug resistance. Overall, this knowledge has had a favourable impact on
parasite control both in veterinary and human medicine. Relevant, funda-
mental knowledge of the relationship among drug influx/efflux, biotrans-
formation, accumulation and pharmacological effect in parasitic
nematodes has been obtained exploiting the advantages of working with
H. contortus under in vitro, ex vivo and in vivo experimental conditions.
ACKNOWLEDGEMENTS
The authors appreciate the contribution of their co-workers to the outline and execution of
the research whose results are summarised here. Research at the Laboratorio de Farmacología,
CIVETAN, UNCPBA-CICPBA-CONICET, is supported by the Agencia Nacional de
Promoci on Científica y Tecnol ogica, Universidad Nacional del Centro Pcia. de Buenos Aires
and Consejo Nacional de Investigaciones Científicas y Técnicas (all from Argentina).
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CHAPTER TWELVE
Understanding Haemonchus
contortus Better Through
Genomics and Transcriptomics
R.B. Gasser*, 1, E.M. Schwarz*, x, P.K. Korhonen*, N.D. Young*
*The University of Melbourne, Parkville, VIC, Australia
x
Cornell University, Ithaca, NY, United States
1
Corresponding author: E-mail: robinbg@unimelb.edu.au
Contents
1. Introduction 520
2. Background on Haemonchus and Haemonchosis 521
2.1 Biology and disease 521
2.2 Immune responses 522
2.3 Anthelmintics and drug resistance issues 522
2.4 Vaccine research 524
3. Caenorhabditis elegans and WormBase: Key Resources for Understanding the 525
Molecular Biology of H. contortus
4. Pregenomic Transcriptomic Studies of H. contortus 528
4.1 Using conventional techniques 528
4.2 Using 454 sequencing technology 530
4.3 Challenges and the need for a genome of H. contortus 531
5. Genomes and Transcriptomes of H. contortus e a Window to Understanding 532
the Molecular Biology of the Worm
5.1 Genome characteristics and annotation 532
5.2 Transcriptional alterations in the life cycle or between tissues 535
5.3 Immunobiology 537
5.4 Predicting targets for new anthelmintics 539
5.5 Xenobiotic detoxification 541
5.6 Immunogens 541
6. Prospects 543
References 547

j
ISSN 0065-308X
520 R.B. Gasser et al.
Abstract
Parasitic roundworms (nematodes) cause substantial mortality and morbidity in ani-
mals globally. The barber’s pole worm, Haemonchus contortus, is one of the most
economically significant parasitic nematodes of small ruminants worldwide. Although
this and related nematodes can be controlled relatively well using anthelmintics, resis-
tance against most drugs in common use has become a major problem. Until recently,
almost nothing was known about the molecular biology of H. contortus on a global
scale. This chapter gives a brief background on H. contortus and haemonchosis, im-
mune responses, vaccine research, chemotherapeutics and current problems associ-
ated with drug resistance. It also describes progress in transcriptomics before the
availability of H. contortus genomes and the challenges associated with such work. It
then reviews major progress on the two draft genomes and developmental transcrip-
tomes of H. contortus, and summarizes their implications for the molecular biology of
this worm in both the free-living and the parasitic stages of its life cycle. The chapter
concludes by considering how genomics and transcriptomics can accelerate research
on Haemonchus and related parasites, and can enable the development of new
interventions against haemonchosis.
1. INTRODUCTION
Parasitic nematodes (roundworms) of humans and other animals are of
major significance as pathogens (Anderson, 2000). In particular, parasitic nem-
atodes of livestock, including species of Haemonchus, Ostertagia and Trichostron-
gylus (Strongylida: Trichostrongyloidea) cause substantial economic losses due
to reduced growth, poor productivity, costs of anthelmintic treatment and
deaths (Kaplan and Vidyashankar, 2012; Wolstenholme et al., 2004). In addi-
tion to their economic impact, anthelmintic resistance in nematodes of live-
stock (Gilleard, 2006; Wolstenholme and Kaplan, 2012) has stimulated
research to develop alternative intervention and control strategies against these
parasites. Despite current knowledge of many aspects concerning Haemonchus
contortus (see Anderson, 2000; chapter: The pathophysiology, ecology and
epidemiology of Haemonchus contortus infection in small ruminants by Besier
et al., 2016 e in this issue; Sutherland and Scott, 2010), little has been under-
stood about the molecular biology and genetics of this worm, the interaction
that it has with its host animals and the disease (haemonchosis) that it causes at
the molecular and biochemical levels. Gaining an improved understanding of
these areas could offer a possible pathway to discover new methods of
diagnosis, treatment and control. Advanced genomic and bioinformatic tech-
nologies provide some opportunities to explore, for example, basic develop-
mental and reproductive processes in nematodes. In particular, genomic and
transcriptomic studies of parasites have become instrumental in various areas,
such as gene discovery and characterization, and for gaining insights into
Haemonchus contortus Genomics and Transcriptomics 521
aspects of gene expression, regulation and function (Bird et al., 2015; Blaxter
and Koutsovoulos, 2015; Lv et al., 2015; Weiberg et al., 2015; Zarowiecki
and Berriman, 2014). The purpose of this chapter is to (1) give a brief back-
ground on H. contortus and haemonchosis, immune responses, vaccine
research, anthelmintics and problems associated with drug resistance; (2)
describe progress in transcriptomics prior to the availability of genomic
information for H. contortus and the challenges associated with this work;
(3) review the characterization of two draft genomes of H. contortus and pro-
vide an account of the insights that annotation and analyses of these genomes
have given into the biology of this worm, its relationship with its host and dis-
ease as well as identify prospects for designing new approaches to combat
haemonchosis.
2. BACKGROUND ON HAEMONCHUS AND

HAEMONCHOSIS
2.1 Biology and disease
Globally, H. contortus is one of the most important representatives of
nematodes of the order Strongylida infecting small ruminants. This parasite
is of greatest importance in tropical and subtropical (summer rainfall) areas
(chapter: The pathophysiology, ecology and epidemiology of Haemonchus con-
tortus infection in small ruminants by Besier et al., 2016 e in this issue.),
whereas some other trichostrongyle species are often dominant in winter rain-
fall areas due to their ability to develop and survive at lower temperatures than
H. contortus does (Anderson et al., 1978). The life cycle of H. contortus is direct,
with eggs being laid by females in the abomasum (Veglia, 1915). Under suit-
able environmental conditions, first-stage larvae (L1s) hatch from eggs to
develop, via the second-stage larvae (L2s), to infective, third-stage larvae
(L3s). The cuticle of the L2 is retained as a sheath around the L3 and protects
it from desiccation (Anderson, 2000; Veglia, 1915). Small ruminants become
infected by ingesting L3s from contaminated pastures. These (infective) larvae
pass through the forestomachs and exsheath, to then establish, via the parasitic
fourth-stage larvae (L4s), as adult males and females in the abomasum within
w3 weeks (Veglia, 1915). The exsheathment process is triggered mainly by
dissolved gaseous CO2 and undissociated carbonic acid in the stomach (Davey
et al., 1982; Petronijevic and Rogers, 1983); the L3s respond to these stimuli
by producing an exsheathment fluid, which enables the detachment of the
sheath from the larvae (Rogers and Sommerville, 1963, 1968;
Sommerville, 1957). The adults of H. contortus feed on blood from vessels
in the gastric wall. Consequently, the main clinical signs of acute hae-
monchosis are anaemia, variable degrees of oedema, lethargy, decreased
live-weight gain, impaired wool/milk production and decreased reproductive

performance, often leading to death, especially in young animals with heavy
worm burdens (Noble and Noble, 1982; Sutherland and Scott, 2010).
2.2 Immune responses

Various studies have described cells and molecules implicated in host immune
responses against parasitic nematodes (Allen and Maizels, 2011; Artis, 2006;
Hewitson et al., 2009; Maizels et al., 2004; Maizels and Yazdanbakhsh,
2003; chapter: Immunity to Haemonchus contortus and Vaccine Development
by Nisbet et al., 2016 e in this issue). The primary immunological responses
induced by nematodes are dependent on the processes and mechanisms of
invasion of and establishment in the host (Artis, 2006). For H. contortus,
the invasion of the abomasum by larvae leads to a localized IgE-mediated
immune response (Miller and Horohov, 2006). Adult stages appear to stim-
ulate similar immunological responses in their host. These responses include
(1) increased production of mucus by the gastrointestinal epithelium of the
host, (2) eosinophilia and increased presence of mast cells and leukocytes at
the infection site and (3) production of specific antibodies (cf. Artis, 2006;
Balic et al., 2000). Responses against primary infections by gastrointestinal
parasitic nematodes are reported to be linked to a T helper (Th) 2-type im-
mune response which, in turn, relates to the secretion of multiple types of
cytokines, including interleukin (IL)-4, IL-5, IL-9 and IL-13 (cf. Artis,
2006; Maizels and Yazdanbakhsh, 2003). In contrast, immunological re-
sponses in hosts with chronic infections appear to be regulated mainly by
a Th1-type immune response, characterized by the production of IL-2,
IL-18 and interferon-g (Allen and Maizels, 2011; Maizel et al., 2004).
2.3 Anthelmintics and drug resistance issues

The control of H. contortus and related gastrointestinal nematodes has relied
heavily on the use of a range of anthelmintic drugs (chapter: Haemonchus con-
tortus: applications in drug discovery by Geary, 2016 e in this issue; Holden-
Dye and Walker, 2014). Such drugs include aminoacetonitrile derivatives
(eg, monepantel), aminophenylamidines (eg, tribendimidine), benzimidazoles
(eg, albendazole), imidazothiazoles (eg, levamisole), macrocyclic lactones (eg,
ivermectin and moxidectin), spiroindoles (eg, derquantel) and tetrahydropyr-
imidines (eg, morantel, oxantel and pyrantel). Levamisole and pyrantel act by
binding to a subgroup of nematode acetylcholine receptor ion channels in
parasite nerves and muscles of parasitic nematodes, resulting in a spastic muscle
contraction (Robertson et al., 2010) or paralysis of the worms; the parasites are
unable to move in the intestinal tract and are removed by peristalsis. Benz-
imidazoles are active against a range of species of nematodes (Keiser and
Utzinger, 2008); by binding to tubulin (cytoskeletal protein), they block
microtubular matrix formation, which is essential for various biological
processes in the cell, including chromosome movement and cell division
(Holden-Dye and Walker, 2014; Stepek et al., 2006; Wolstenholme et al.,
2004). Macrocyclic lactones act by opening glutamate-gated chloride chan-
nels, increasing the flow of chloride ions, leading to defects in neurotransmis-
sion and flaccid paralysis (Stepek et al., 2006). Recently, new classes of
anthelmintics, ie, the cyclooctadepsipeptides (eg, emodepside), the aminoace-
tonitrile derivatives (eg, monepantel) and the spiroindoles (eg, derquantel)
have become available commercially (Epe and Kaminsky, 2013; Holden-
Dye and Walker, 2014). These chemicals bind G proteinecoupled latrophi-
lin-like receptors and voltage-/calcium-dependent potassium channels
(emodepside), some acetylcholine receptors (monepantel) or B-subtype nico-
tinic acetylcholine receptors (derquantel), leading to spastic (monepantel) or
flaccid (emodepside and derquantel) paralysis of some parasitic nematodes
and their subsequent death (Epe and Kaminsky, 2013; Kr€ ucken et al., 2012).
The relatively low cost and ease of administration of anthelmintic drugs
against gastrointestinal parasitic nematodes of animals has led to their extensive
use and, consequently, to the emergence of resistance (Gilleard, 2006; Kaplan
and Vidyashankar, 2012; chapter: Anthelmintic resistance in Haemonchus con-
tortus: history, mechanisms and diagnosis by Kotze and Prichard, 2016 e in
this issue; Wolstenholme et al., 2004). Indeed, resistance in nematodes of an-
imals to benzimidazoles, imidazothiazoles/tetrahydropyrimidines and macro-
cyclic lactones has been reported in many parts of the world and on all
continents (eg, Becerra-Nava et al., 2014; Bourguinat et al., 2015;
Byaruhanga and Okwee-Acai, 2013; Chagas et al., 2013; Demeler et al.,
2009; Falzon et al., 2013; Gasbarre, 2014; Hughes et al., 2007; Keane
et al., 2014; Kudo et al., 2014; Mahieu et al., 2014; Mu~ niz-Lagunes et al.,
2015; Nabukenya et al., 2014; Papadopoulos et al., 2012; Playford et al.,
2014; Rialch et al., 2013; Rose et al., 2015; Tsotetsi et al., 2013; Verissimo
et al., 2012). Ominously, resistance even to the newly developed anthelmintic
monepantel has been recently detected in Europe (Van den Brom et al.,
2015). Three main mutations in the gene encoding the beta-tubulin isotype
1 in H. contortus (Hco-tbb-iso-1) have been linked to benzimidazole resistance
(Ghisi et al., 2007; Kwa et al., 1994, 1995; Rufener et al., 2009), and there is
some evidence that mutation of its paralogue, Hco-tbb-iso-2, might increase
such resistance as well (Beech et al., 1994; Kwa et al., 1993a,b; Saunders
et al., 2013). Given the incomplete knowledge of the molecular mechanisms

associated with resistance in parasitic nematodes (Gilleard, 2006, 2013), geno-
mics and transcriptomics will be crucial tools for identifying new drug targets,
developing new anthelmintics and possibly devising strategies to prevent drug
resistance (Geary et al., 2015; Kotze et al., 2014; Morgan and Coles, 2010).
2.4 Vaccine research

Over the years, considerable research has focused on developing vaccines
against H. contortus (see Bassetto and Amarante, 2015; Hewitson and Maizels,
2014; Knox, 2011; chapter: Immunity to Haemonchus contortus and Vaccine
Development by Nisbet et al., 2016 e in this issue), in order to circumvent
anthelmintic resistance issues. For instance, irradiated larvae were used as the
basis for a vaccine against H. contortus infection in sheep (Jarrett et al., 1959,
1961; Smith and Angus, 1980). More recently, various proteins of the
epithelial cell surface membrane of the digestive tract of some gastrointes-
tinal nematodes have been evaluated as vaccine candidates in livestock.
For example, a 110-kDa integral membrane aminopeptidase of H. contortus,
which is heavily glycosylated and localized in the brush border of the epithe-
lial cells of the gut of the adult worm, was shown to be effective in reducing
the intensity of H. contortus infection in different breeds and ages of sheep
(Munn et al., 1993; Newton, 1995; Newton et al., 1995; Smith et al.,
2001). However, protection is limited to native proteins, administered mul-
tiple times, usually in Freund’s adjuvant (Knox, 2011; Knox and Smith,
2001). Another peptidase complex (P1), separated from the membrane
aminopeptidase H11 by chromatography, was identified (Smith et al.,
1993) and shown to represent a ubiquitous component of the microvillar
membrane of the intestinal cells of H. contortus (see Smith et al., 1993).
Although vaccination with this protein complex resulted in a significant
reduction (69%) in the number of H. contortus eggs in the faeces from
vaccinated sheep following H. contortus challenge infection, P1 led only to
a w22e38% reduction of infection intensity (Newton, 1995). On the other
hand, vaccination with the glucose-binding glycoprotein complex (H-gal-
GP), separated by lectin affinity chromatography from other integral
membrane proteins from the gut of adult H. contortus achieved w53e72%
protection and a >90% reduction in the number of eggs in the faeces
from vaccinated sheep (Smith et al., 1994). However, the vaccination of
lambs (9 months of age) with prokaryotically expressed recombinant H-
gal-GP failed to induce protective immunity against challenge infection
with H. contortus L3s (Cachat et al., 2010). It appears that the same is true
for recombinant H11 tested in a commercial setting.
Proteases in excretory/secretory (ES) products from parasitic nematodes

have also been an important focus for vaccine development, given their
inferred roles in the digestion of nutrients acquired from the host tissues
during invasion, establishment and infection (Knox, 2011). Metalloproteases
and aspartic and cysteine proteases have received considerable attention for
blood-feeding nematodes, such as H. contortus (see Redmond and Knox,
2004; Skuce et al., 1999; Smith et al., 2003a,b). For instance, vaccination
with a cysteine proteaseeenriched fraction from membrane extracts from
the microvillar surface of intestinal cells from adult H. contortus (see Knox
et al., 2005) was shown to reduce infection intensity by 47% and the number
of eggs in faeces by 77% in sheep following a single challenge infection
(Knox et al., 1999).
Although success with recombinant H. contortus vaccines has been limited
(Knox, 2011), a protective vaccine (Barbervax) derived from extracts from
live H. contortus parasites has been developed and commercialized (chapter:
Immunity to Haemonchus contortus and Vaccine Development by Nisbet
et al., 2016 e in this issue). To replace this effective vaccine with recombinant
H. contortus vaccines may require using a mixture of several different antigens,
targeting several different parasite functions at once (cf. Nisbet et al., 2013).
This strategy, in turn, might require a much better understanding of para-
siteehost interactions at the molecular level. Genome-wide analysis of the
transcriptome of H. contortus and its dynamic activity throughout infection
and disease could facilitate the discovery of new intervention strategies.
3. CAENORHABDITIS ELEGANS AND WORMBASE: KEY

RESOURCES FOR UNDERSTANDING THE MOLECULAR
BIOLOGY OF H. CONTORTUS
Deciphering the genomes and transcriptomes of parasitic nematodes
relies heavily on our understanding of the free-living model nematode
Caenorhabditis elegans, which is extensively annotated in WormBase
(www.wormbase.org; Harris et al., 2014). C. elegans has a simple anatomy
(959 somatic cells in the hermaphrodite and 1031 in the male), has a short
life cycle (3 days at 20 C) and is easy to culture in vitro (Brenner, 1974;
Lewis and Fleming, 1995). The genome of C. elegans is 100.3 Mb in size
(C. elegans Consortium, 1998; Hillier et al., 2005); although this genome
is substantially more compact than that of H. contortus (w340 Mb), the
two species have extensive orthology and synteny (Laing et al., 2013;
Schwarz et al., 2013). As of release WS248 (June 2015), WormBase contains
detailed information on 20,391 protein-coding and 24,718 noncoding

RNA (ncRNA)-coding C. elegans genes, along with associated data on tran-
scription/expression profiles in different developmental stages, tissues and
cells, mutants and their phenotypes, genetic and physical maps, single-
nucleotide polymorphisms (SNPs), information on geneegene and
proteineprotein interactions as well as literature pertaining to C. elegans.
Importantly, WormBase has precomputed homologies between C. elegans
and genes of key nematode parasites, including H. contortus, and is linked
to the ParaSite database (parasite.wormbase.org), which contains genomic
data for more than 60 parasitic nematode species.
RNA interference (RNAi; Fire et al., 1998) has revolutionized the study
of gene function in metazoan organisms and elucidated the functions of
most genes in C. elegans (see Barstead, 2001; Kamath and Ahringer, 2003;
Simmer et al., 2003; Sonnichsen et al., 2005; Sugimoto, 2004). RNAi relies
on the introduction of double-stranded RNA (dsRNA) into the cells of a
living organism, which induces the degradation of the homologous (target)
mRNA (Fire et al., 1998). The dsRNA can be introduced directly into
C. elegans by injection (Fire et al., 1998), by soaking worms in solution
(Tabara et al., 1998) or by feeding worms Escherichia coli expressing a dsRNA
fragment of a target gene (Timmons et al., 2001); it can also be introduced
using a transgene expressing dsRNA (Tabara et al., 1999; Tavernarakis et al.,
2000). This gene silencing approach opened up avenues for large-scale
studies of molecular function in C. elegans (see Ashrafi et al., 2003; Barstead,
2001; Kamath and Ahringer, 2003; Maeda et al., 2001; Simmer et al., 2003;
Sugimoto, 2004; Tabara et al., 1999).
Transgenesis of C. elegans has also been widely used for assessing gene
function (Frokjaer-Jensen et al., 2014; Praitis and Maduro, 2011; Sarov
et al., 2012). Transgenes can be introduced to C. elegans through microinjec-
tion of the mitotically active gonadal syncytium, or through biolistic micro-
bombardment with DNA-coated gold or tungsten particles; in either case,
expression constructs (which can be plasmid, cosmid or linear polymerase
chain reaction (PCR)-amplified DNA) typically use fluorescent proteins as
markers for gene expression, along with selectable markers for body
morphology or drug resistance (Hutter, 2012; Praitis and Maduro, 2011; Sha-
ner et al., 2005); the most commonly used marker is green fluorescent protein
(GFP; Chalfie et al., 1994). GFP allows the study of many biological pro-
cesses, including gene expression, protein localization and dynamics, pro-
teineprotein interactions, cell division, chromosome replication and
organization, intracellular transport pathways, organelle inheritance and
biogenesis (Hobert and Loria, 2006). In addition to single-gene assays through

transgenesis, gene activity in C. elegans has also been extensively characterized
genome-wide, first using microarrays (Jiang et al., 2001; Kim et al., 2001;
Reinke et al., 2000), and then using RNA-seq (Gerstein et al., 2010, 2014;
Li et al., 2014b; Spencer et al., 2011, 2014).
Tools for functional analysis and computational annotation of C. elegans
genes have aided the analysis of H. contortus genes in two different ways.
First, many of these technologies, having been initially developed in the
experimentally facile C. elegans, have then been adapted (with various
degrees of success) to H. contortus and other parasites: RNAi (Britton and
Murray, 2006; chapter: Functional Genomics Tools for Haemonchus contortus
and Lessons From Other Helminths by Britton et al., 2016 e in this issue;
Geldhof et al., 2006, 2007; Zawadzki et al., 2006; Rosso et al., 2009; Selkirk
et al., 2012), transgenesis (Lok, 2009, 2012; Lok and Artis, 2008) and RNA-
seq (Laing et al., 2013; Schwarz et al., 2013). Second, many of the results for
C. elegans genes can be used to infer likely functions for their H. contortus ho-
mologues. H. contortus belongs to a class of parasitic nematodes, strongylids,
that are more closely related to the free-living C. elegans than are most other
known nematode species, including free-living species, such as Oscheius tipu-
lae and Pristionchus pacificus which are very similar to C. elegans (see Blaxter,
2011; Chilton et al., 2006; Kiontke et al., 2011; van Megen et al., 2009). In
other words, despite H. contortus and other strongylids having evolved a
parasitic life cycle very different from that of C. elegans, this evolution ap-
pears to have occurred relatively recently, and must have occurred on a
genomic and transcriptomic substrate of functions conserved between
H. contortus and C. elegans (see B€ urglin et al., 1998; Nikolaou and Gasser,
2006; Parkinson et al., 2004). It is this relatively close relationship between
H. contortus and C. elegans (both members of the nematode ‘clade V’; Blaxter
et al., 1998) that makes evolutionary inference reasonably reliable. For
instance, genes with preferential expression in C. elegans germline were
identified by Reinke et al. (2000); many of these genes were subsequently
found to have homologues in parasitic nematodes whose sex-enriched tran-
scriptional profiles indicated conserved germline function (Campbell et al.,
2008; Cottee et al., 2006; Nisbet and Gasser, 2004). Moreover, in H. contor-
tus, at least some protein-coding genes (Britton and Murray, 2002; Couthier
et al., 2004; Glendinning et al., 2011; Hu et al., 2010a; Kwa et al., 1995; Li
et al., 2014a) and cis-regulatory elements (Britton et al., 1999; Hu et al.,
2010b; Kwa et al., 1995; Li et al., 2014a) as well as various neuronal recep-
tors (eg, Glendinning et al., 2011; Miltsch et al., 2012; Welz et al., 2011)
exhibit conserved functions when transgenically assayed in C. elegans. The

ability to map functional data and homologous genomic regions between
C. elegans and H. contortus via WormBase is therefore crucial for dissecting
the molecular biology and transcriptomics of the latter species.
4. PREGENOMIC TRANSCRIPTOMIC STUDIES OF

H. CONTORTUS
4.1 Using conventional techniques
The techniques of RNA (Northern) blotting (Alwine et al., 1977),
differential gene display (Liang and Pardee, 1992) and quantitative real-
time, reverse transcription PCR (qRT-PCR; Higuchi et al., 1993) have
been used to define patterns of transcription for single or small numbers
of genes in H. contortus and related strongylid nematodes (eg, Boag et al.,
2000; Hartman et al., 2003; Moore et al., 2000; Nikolaou et al., 2002; Pratt
et al., 1990; Sangster et al., 1999; Savin et al., 1990). Another approach used
was serial analysis of gene expression (SAGE; Velculescu et al., 1995), which
assays gene activity by constructing libraries of short (w14 nt) complemen-
tary DNA (cDNA) tags from mRNAs and then sequencing them. Despite its
demonstrated utility in studies of yeast and humans (Velculescu et al., 1997;
Boon et al., 2002; Liang, 2002), the application of SAGE for investigations
of transcription in parasitic nematodes was limited until RNA-seq (which,
essentially, does the same thing, but with larger tags and on a much larger
scale) superseded it. A single study (Skuce et al., 2005) used SAGE to
sequence and analyze w3000 transcripts from adult H. contortus, of which
w60% had homologues in public databases.
In contrast to SAGE, expressed sequence tag (EST) sequencing has
been widely used to analyze transcriptomes of parasitic nematodes.
ESTs generally consist of single-pass Sanger (1977a,b) sequencing reads
derived from cloned cDNAs (Adams et al., 1991; McCombie et al.,
1992). Crucially, these Sanger sequencing reads are long enough to allow
partial open reading frames of the cDNAs to be identified and classified by
their similarity to full-length proteins (Wasmuth and Blaxter, 2009). Until
the cost of next-generation sequencing of both genomes and transcrip-
tomes reduced substantially, ESTs allowed a rapid and cost-effective way
to probe both protein-coding genes and their developmental expression
in both H. contortus and many other parasitic helminths, with the results
being analyzed and archived in databases such as NEMBASE4 and
Helminth.net (Elsworth et al., 2011; Martin et al., 2015). Accordingly,

ESTs have been used in various studies aimed at investigating fundamental
processes in parasitic nematodes as well as drug and vaccine target discovery
(Blaxter et al., 1996; Clifton and Mitreva, 2009; Daub et al., 2000; Geldhof
et al., 2005; Hoekstra et al., 2000; McCarter et al., 2000; Parkinson and
Blaxter, 2009; Parkinson et al., 2001, 2004; Williams et al., 2000). For
parasitic nematodes of animal hosts, applications have ranged from
analyses of stage- and sex-specific transcripts (eg, Boag et al., 2000;
Campbell et al., 2008; Cottee et al., 2006; Hartman et al., 2001; Hoekstra
et al., 2000) to more global analyses of gene transcription (eg, Daub et al.,
2000; Parkinson et al., 2001; Rabelo et al., 2009; Ranjit et al., 2006; Yin
et al., 2008).
Microarrays (DeRisi et al., 1996; Stoughton, 2005) were another major
advance for large-scale analysis of the transcriptomes of parasitic nema-
todes. In such microarrays, thousands of oligonucleotides (such as cDNAs,
EST clones or fragments of PCR products) are spotted on to glass slides or
chips in precise positions; the sequences of the oligonucleotides are chosen
to be complementary (by DNAeRNA hybridization) to known or sus-
pected genes or transcripts. The mRNAs from different stages or tissues
can then be labelled (typically with fluorescent markers) and hybridized
to the spots on the array. The relative abundance of hybridization for
each mRNA population can then be determined by comparing the relative
signal intensity of each marker (DeRisi et al., 1996). An advantage of array
analysis, which has still not been fully superseded in all applications of
RNA-seq, is that it allows bulk hybridization of whole RNA (without
an effort being made to purify poly(A)þ RNA) without the resultant signal
being swamped by rRNA (which will typically comprise 97% of the total
RNA). Microarray technology has allowed large-scale transcriptional
analyses of different tissues, developmental stages and sexes of parasitic
nematodes, revealing molecules likely to be crucial for parasite survival,
development and reproduction (eg, Gasser et al., 2007; Newton and
Meeusen, 2003). In particular, microarrays greatly expanded knowledge
of the transcriptomes of H. contortus and related strongylid nematodes,
especially when coupled to suppressive-subtractive hybridization to
rapidly compare transcriptomes between different life cycle stages, sexes
or species of parasitic nematodes (Campbell et al., 2008; Cantacessi
et al., 2012; Cottee et al., 2006; Datu et al., 2008; Huang et al., 2008;
Liu et al., 2007; Moser et al., 2005; Nisbet and Gasser, 2004; Nisbet
et al., 2008).
4.2 Using 454 sequencing technology

Advances in sequencing technologies (Bentley et al., 2008; Harris et al.,
2008; Koboldt et al., 2013; Mardis, 2008, 2013; Margulies et al., 2005;
Pandey et al., 2008) provided the opportunity to perform de novo analyses
of whole transcriptomes. Initially, the 454/Roche platform (Margulies et al.,
2005; www.454.com) was applied to H. contortus (see Cantacessi et al., 2010).
This technology uses a sequencing-by-synthesis approach. For transcrip-
tomic studies, cDNA is randomly fragmented (by ‘nebulization’) into frag-
ments of variable size; adaptors are ligated to each end of these fragments,
which are then mixed with agarose beads that have on their surface anchor
oligonucleotides complementary to the 454-specific adapter sequence, such
that each bead is associated with a single fragment. Each of these complexes
is transferred into individual oilewater micelles containing amplification
reagents, and is then subjected to an emulsion PCR step, during which
w10 million copies of each cDNA are produced and bound to individual
beads. Subsequently, in the sequencing phase, the beads anchoring the
cDNAs are deposited on a pico-titre plate, together with other enzymes
required for the pyrophosphate sequencing reaction (ie, ATP sulfurylase
and luciferase) and the sequencing is carried out by flowing sequencing
reagents (nucleotide and buffers) over a plate (Mardis, 2008).
Cantacessi et al. (2010) used 454 sequencing and bioinformatics to
explore functional differences in gene transcription between the (free-living)
ensheathed third-stage larvae (L3s) and (parasitic) exsheathed third-stage
larvae (xL3s) of H. contortus. These analyses showed that transthyretin-like
(TTL) proteins and calcium-binding proteins were highly represented in
the transcriptomes of both H. contortus L3 and xL3, whereas selected tran-
scripts encoding collagens and neuropeptides were present exclusively in
L3, and proteases only in xL3 (Cantacessi et al., 2010). In nematodes, the
synthesis of collagens has been observed to increase significantly prior to a
moult (Kim et al., 2013), whereas proteins involved in the function of the
nervous system are more highly expressed in L3 or earlier larval stages
than in later stages (Kim et al., 2013; Schwarz et al., 2015; Tang et al.,
2014). Therefore, decreased transcription of neuropeptide genes after the
L3 stage of H. contortus (see Cantacessi et al., 2010) is consistent with a gen-
eral downregulation of ‘neurological’ genes that may be conserved in both
parasitic and nonparasitic nematodes (Schwarz et al., 2015). In parasitic nem-
atodes, this pattern is likely to be under direct environmental control: in
H. contortus, the transition from the free-living to the parasitic L3 is triggered
by gaseous CO2, detected by chemosensory neurons of amphids, located in

the anterior end of the L3 stage, ultimately leading to the secretion of the
neurotransmitter noradrenaline (Nikolaou and Gasser, 2006). Conversely,
a large number of C. elegans orthologues of H. contortus xL3-specific tran-
scripts encoded peptidases and other enzymes involved in protein catabo-
lism, supporting previous evidence that cysteine proteases have a crucial
role in the catabolism of globin, as is the case for hookworms (Pratt et al.,
1990; Ranjit et al., 2008, 2009; Williamson et al., 2004). A similar spectrum
of proteases and other molecules linked to catalytic activity had been shown
to be highly represented in the transcriptomes of activated xL3 stages of
both H. contortus and Ancylostoma caninum by comparison with their L3s
(Cantacessi et al., 2009b). This finding, for two haematophagous bursate
nematodes with differing life histories, indicates key roles that these mole-
cules have in host tissue invasion, degradation and/or digestion.
4.3 Challenges and the need for a genome of H. contortus

Although ESTs, microarrays and 454 sequencing all enabled significant tran-
scriptomic analyses of H. contortus, they also had several limitations, including
incomplete or ambiguous assembly, transcript redundancy, incomplete
annotations, inability to quantitatively assess differential transcription (due
to normalized cDNA libraries for this technology) and an inability to predict
alternative splicing. To partially overcome these limitations, the nuclear
genome of H. contortus was sequenced between 2009 and 2012 using
Illumina technology (Bentley et al., 2008; www.illumina.com), at the same
time that this ‘second-generation’ technology was also allowing the direct
sequencing of many other helminth genomes (parasitic and otherwise) for
the first time (Zarowiecki and Berriman, 2014), and that Illumina technol-
ogy was also revolutionizing cDNA analysis through RNA-seq (Wang et al.,
2009). Consequently, 454 technology became obsolete, and Illumina
sequencing has supplanted most other technologies, although ‘third-
generation’ sequencing technologies, which obtain very long reads from
single DNA molecules, are on the horizon (Berlin et al., 2015; Madoui
et al., 2015; Sharon et al., 2015). Illumina technology involves the fragmen-
tation of a DNA sample (either from genomic DNA or cDNA) and
construction of a shotgun library, followed by the in vitro ligation of
Illumina-specific adaptors to each DNA template; the termini of the tem-
plate are covalently attached to the surface of a glass slide (or flow cell).
Attached to the flow cell are primers complementary to the other end of
the template, which bend the DNAs to form bridge-like structures. In an
amplification step (bridge-PCR), clonal clusters, each consisting of >1000

amplicons, are generated; subsequently, the DNAs are linearized, and the
sequencing reagents are directly added to the flow cell, with four fluores-
cently labelled nucleotides. After the incorporation of a fluorescent base,
the flow cell is interrogated with a laser in several locations, which results
in several image acquisitions at the end of a single synthesis cycle (Mardis,
2008). This technology is readily applicable to both de novo assembly and
resequencing projects, targeted sequencing, SNP analyses and gene
transcription studies. Applying this technology to sequence the draft genome
and transcriptome of H. contortus should thus provide the scientific commu-
nity with important molecular resources for a plethora of fundamental and
applied studies of this highly significant parasite.
5. GENOMES AND TRANSCRIPTOMES OF

H. CONTORTUS e A WINDOW TO UNDERSTANDING
THE MOLECULAR BIOLOGY OF THE WORM
In 2013, two genomic assemblies and transcriptomes for H. contortus
were published simultaneously, derived from two different strains:
MHco3(ISE).N1 from the UK (Laing et al., 2013) and McMaster from
Australia (Schwarz et al., 2013). These two studies used different methods
to achieve assembly of the highly repetitive and polymorphic H. contortus
genome, and also focused on complementary biological implications (Laing
et al., 2013; Schwarz et al., 2013). Here, we review the implications of these
genomes, and, in particular, their expressed gene content as detected in their
associated transcriptomes, for H. contortus development, immunobiology,
host interactions, drug resistance (both through mutation and through
detoxification) and possible targets for anthelmintic drugs and vaccines.
5.1 Genome characteristics and annotation

Both genomes of H. contortus were sequenced at w185-fold coverage,
producing draft assemblies of 320 Mb (McMaster strain; Schwarz et al.,
2013) to 370 Mb (MHco3(ISE).N1 strain; Laing et al., 2013; Table 1).
Both of these draft genome sizes are similar to a physical estimate of
315 Mb, made through Feulgen staining of H. contortus nuclei (cf. Hardie
et al., 2002; Schwarz et al., 2013). Both assemblies had mean GC nucleotide
contents of 41.3e42.4%, a somewhat less skewed fraction than that seen in
C. elegans (35%). Most (92e93%) of 248 core single-copy eukaryotic genes
were detected within the two assemblies using the program CEGMA (Parra
Table 1 Comparison of features of two published draft genomes for Haemonchus

contortus
a
Sources
Description of features Laing et al. (2013) Schwarz et al. (2013)
Total number of base 369,852,402 319,640,208

pairs (bp) within
assembled scaffolds
Total number of 23,862; 62,121 14,419; 930,981
scaffolds; contigs
N50 length in bp; total 83,287; 10,735 56,328; 11,000
number >2 kilobases
(kb) in length
N90 length in bp; total 11,529; 5505 13,105; 6085
number > N90
length
GC content of the 43.1 42.4
whole genome (%)
Repetitive sequences 29.0 13.4
(%)
Proportion of genome 8.2; 36.3 8.1; 47.0
that is coding (exonic;
incl. introns; %)
Number of putative 21,903 23,610
coding genes
Gene size (mean bp) 5897 6377
Average coding domain 1379 865
length (mean bp)
Average exon number 9.0 7.8
per gene (mean)
Gene exon length 127 142
(mean bp)
Gene intron length 593 775
(mean bp)
GC content in coding 46.8 46.4
regions (%)
a
For direct comparison, values were calculated from original datasets (referenced).
et al., 2009), indicating that they both represented w92.5% of the H. contortus
genome. It needs to be noted, however, that this estimate of assembly
completeness ignores possible strain-specific differences in genomic content;
in distinct strains of C. elegans, such genomic variations can be substantial
(Stewart et al., 2005; Thompson et al., 2015; Vergara et al., 2014), and genetic
data indicate that they should be similarly large for H. contortus (see Hunt et al.,
2008; Redman et al., 2008). The repetitive content of the Laing et al. (2013)
assembly was 29%, which might be more accurate than the 13% estimate of
Schwarz et al. (2013), since the latter assembly used data filtering of overrep-
resented sequences to aid genomic assembly (Pell et al., 2012); an estimate of
29% equates to 93e107 Mb of repetitive DNA. Considerable challenges
were encountered in the assembly of the genomes due to substantial sequence
heterogeneity (Hunt et al., 2008; Redman et al., 2008).
The draft genome sizes for H. contortus were noticeably larger than those
for its free-living relatives C. elegans and P. pacificus (100 Mb and 172 Mb,
respectively; Dieterich et al., 2008; Hillier et al., 2005). As such, they defy
the expectation that parasitic genomes will inevitably be smaller and more
reduced than those of free-living nematodes. This counter-intuitive pattern
of larger genomes in parasites is also seen for several strongylid nematodes
other than H. contortus: 244 and 313 Mb, respectively, for the hookworms
Necator americanus and Ancylostoma ceylanicum, 260 Mb for the rat lungworm
Angiostrongylus cantonensis and 443 Mb for the pig nodule worm Oesophagos-
tomum dentatum (see Schwarz et al., 2015; Tang et al., 2014; Tyagi et al.,
2015a; Yong et al., 2015). It is not clear whether this pattern of large
genomes in strongylid parasites is due to reduced natural selection, which
might permit genome sizes to expand (Lynch, 2007), or due to increased
natural selection for genomic diversity, which might permit parasites to
survive variations in their hosts (Raffaele and Kamoun, 2012). One piece
of evidence suggesting that strongylid parasites may live under relaxed con-
ditions of natural selection is that parasitic nematodes have a consistent
pattern of growing to larger sizes than their free-living relatives (Yeates
and Boag, 2006). On the other hand, the larger genomes of H. contortus
and other strongylid nematodes do contain gene families that have few
members in free-living nematodes, but many members in other strongylids,
and that exhibit prominent patterns of stage-specific expression during in-
fections of their hosts (Cantacessi et al., 2012; Laing et al., 2013; Schwarz
et al., 2013, 2015; Tang et al., 2014). It is therefore possible both that the
relative leniency of parasitic life permits genomic expansion and that this
genomic expansion, in turn, fosters the diversification of gene families that
enable parasitism.
Using transcriptomic data from the main life cycle stages, de novo
predictions and homology-based searching, 21,799e23,610 protein-coding
genes were predicted to exist (Table 1; Laing et al., 2013; Schwarz et al.,
2013). Most of these genes had homologues in other nematodes (w70%),
including C. elegans, Ascaris suum, Brugia malayi, P. pacificus and Trichinella
spiralis (cf. Laing et al., 2013; Schwarz et al., 2013). Conversely, w30% of
these genes appeared to be unique to H. contortus relative to other species.
More than 80% of H. contortus genes that had one-to-one orthologues
with C. elegans genes, and that mapped to a single H. contortus genomic scaf-
fold, had their C. elegans orthologues on the same C. elegans chromosome
(Laing et al., 2013), indicating that both the gene content and general syn-
teny of individual chromosomes were conserved between the two species.
However, there was a noticeable minority of genes that violated
chromosomal synteny and gene order, as had been previously observed in
comparisons of C. elegans with other nematodes (Ghedin et al., 2007;
Guiliano et al., 2002).
5.2 Transcriptional alterations in the life cycle or between

tissues
H. contortus development involves various tightly regulated and timed
biological processes (Veglia, 1915). Development takes place in the environ-
ment for L1 to L3 stages (free-living) or in the host animal for L4 and adult
(parasitic) stages. Each of these developmental stages has distinct require-
ments, in terms of metabolism, motility, sensory perception and chemosen-
sation as well as regulation of the endocrine system. L3, which is the
infective stage in the environment, is a transitional stage from free-living
to parasitic. When ingested by the host animal, it receives a signal (mainly
CO2) to exsheath, develop and reproduce. Therefore, the complexity of
the life cycle of H. contortus coincides with developmental changes in the
nematode that are associated with rapid transcriptional alterations.
Schwarz et al. (2013) explored changes in transcription (from one devel-
opmental stage to the next) as H. contortus developed from egg to adult.
From egg to L1, there was a significant increase in transcription of around
1600 genes: these principally encoded protein kinases and phosphatases, G
proteinecoupled receptors (classes A and SR), transcription factors and
channels, such as ligand-gated ion channels, and other proteins predicted
to be excreted or secreted (thus were termed ES proteins). The upregulation
of genes encoding ES proteins (including peptidases and their inhibitors)
might reflect the L1’s search for microbial food sources as it adapts to its
external environment. The upregulation of some other classes of genes in
this stage could reflect the requirements of young, free-living H. contortus
for chemo-, touch-, osmosensation and/or proprioception; still other upre-
gulated genes are likely to be associated with mitosis, organelle biogenesis,
apoptosis and gene expression during the rapid growth and development
phase of L1s. In the transition from L1 to L2, there was a 50% decrease in the
number of upregulated genes, which might reflect an adaptation to the envi-
ronment and less stress for finding food. In the subsequent transition from
L2s to L3s, there was a w90% decrease in the number of differentially tran-
scribed genes. This probably reflected the developmentally arrested, but
motile state of the L3 stage; following ensheathment, the L3 is unable to
feed and lives on accumulated reserves, at a reduced metabolic rate, to sur-
vive in the external environment (Nikolaou and Gasser, 2006). Following
ingestion of L3s by the host animal, there was a massive surge in the number
of differentially transcribed genes, including some structural proteins (eg,
collagens). However, there was very limited alteration between the L4
and adult stages, with the exception of molecules linked to reproductive
processes in the adult (Schwarz et al., 2013).
In both haematophagous (parasitic) stages, there was an upregulation of
transcription for genes encoding >120 peptidases, including MA (metallo-
peptidases; M12A, M01, M13, M12A, M10A), AA (aspartic peptidases; all
A01A) and CA (cysteine peptidases; mostly CA01A; Schwarz et al.,
2013), many of which are likely involved in the degradation of blood and
other tissues in the abomasal wall, and critical for the parasite’s development
and survival in the host. There was also an upregulation of transcription of
genes involved in redox balance (glutamate dehydrogenase and succinate
dehydrogenase subunit B), oxygen uptake, osmotic regulation, iron storage
and/or detoxification (haemoglobin-like proteins; glutathione S-transferase,
cytoplasmic Cu/Zn superoxide dismutase, catalase, glutathione peroxidase
and/or peroxiredoxin; Schwarz et al., 2013).
In H. contortus adults, numerous genes exhibited sex-enriched transcrip-
tion, some of which were associated with genital, germline and embryonic
development as well as growth and reproduction (Schwarz et al., 2013).
More than a third of sex-enriched genes (977 of 2813) in H. contortus had
no homologues in other organisms. In males, w1600 gene clusters related
to sperm/spermatogenesis (eg, vab-1, vpr-1, spe-15, hsp-12.2, hsp-12.3, fer-
1, cyk-4 and alg-4) were identified. In females, w400 gene clusters were
associated predominantly with germline (eg, rpn-1, plk-2, glp-1 and cdc-
25.2), oogenesis or egg laying (eg, sos-1, rme-2, ptp-2, mpk-1, ima-3, epi-1,
daf-4 and car-1), embryogenesis (eg, let-767, let-92, spk-1, unc-130, unc-6,
rab-7 and nhr-25) and vulval development (eg, rab-8, lin-11 and let-60).
In addition to investigating differential transcription between develop-
mental stages, with similar results to Schwarz et al. (2013), Laing et al.
(2013) studied transcription in the intestine of H. contortus by comparing
gut tissue from the adult female with the whole worm, and found abundant
transcription for genes encoding protein kinases, cysteine-type peptidases
and their inhibitors, consistent with some previous findings (Yin et al.,
2008). In addition, genes associated with cobalamin and sugar binding
were significantly upregulated in the gut, as were those linked to molecular
transport (oligopeptides, cations and anions). Increased oxidoreductase gene
transcription likely reflects detoxification activities (Chakrapani et al., 2008).
5.3 Immunobiology
H. contortus and other parasitic nematodes can inhabit their mammalian hosts
for months or sometimes years and yet avoid being destroyed by the im-
mune systems of their hosts (McNeilly and Nisbet, 2014). It has long
been clear that this feat is achieved by tricking or blocking the host’s im-
mune system; but how this occurs is far from settled, and understanding
the parasiteehost interplay is a key goal of characterizing the H. contortus
transcriptome during infection. It is likely that H. contortus uses a vast array
of different ES proteins to support initial infection, its establishment as a
parasite and modulation or evasion of the host immune system. Schwarz
et al. (2013) compiled a list of ES genes whose homologies to proteins
known to have immunomodulatory or immunogenic functions (Hewitson
et al., 2009) made them key candidates for enabling infection; these genes
comprised about 6% of the H. contortus secretome. Such ES molecules
included homologues of N-acetylglycosaminyltransferase and leucyl amino-
peptidase ES-62 of the filarioid nematode Acanthocheilonema vitae; ES-62,
which is known to inhibit B-cell, T-cell and mast cell proliferation/responses,
induces a Th2 response through the inhibition of IL-12p70 production by
dendritic cells, and promotes alternative activation of the host macrophages
via the inhibition of Toll-like receptor signalling. Other secreted H. contortus
proteins that might dampen the host immune system included homologues of
another B-cell inhibitor (CYS-1), neutrophil inhibitory factors (NIFs) and
serpins. Still other putative H. contortus ES proteins that might mimic mole-
cules involved in immune responses, and thus might help evade the immune
system rather than dampen it, included C-type lectins, concanavalin A and
galectins. Detailed background on these homologues has been given else-
where (Hewitson et al., 2009). Schwarz et al. (2013) also described genes
encoding peptidases, sperm-coating protein (SCP)-like/Tpx-1/Ag5/PR-1/
Sc7 (SCP/TAPS) extracellular proteins (Cantacessi et al., 2009a), lectins,
TTL proteins (cf. Jacob et al., 2007), peptidase inhibitors and fatty acid
retinoid-binding proteins, which were collectively upregulated in the
haematophagous stages of H. contortus; some of these genes are likely to be

important for parasiteehost interactions and parasitism.
Both the SCP/TAPS and the TTL gene families encode proteins that are
expanded in parasitic nematodes, but whose exact functions are unclear.
Although two genes encoding the SCP/TAPS proteins Hc24 and Hc40 had
been identified previously in ES products of adult H. contortus (see Schallig
et al., 1997; Rehman and Jasmer, 1998), many more such SCP/TAPS genes
were found in the complete H. contortus genome and transcriptome. SCP/
TAPS genes encode a diverse set of secreted cysteine-rich proteins, known
by an equally diverse set of names, including Ancylostoma or activation-asso-
ciated secreted proteins (ASPs) and cysteine-rich secretory proteins, antigen
5, and pathogenesis related 1 proteins; their functions in parasitic nematodes
may include blocking immune responses and blood clotting (Cantacessi et al.,
2009a; Tribolet et al., 2014), but might also include binding or transporting
sterols, and regulating body size (Choudhary and Schneiter, 2012; Maduzia
et al., 2002; Morita et al., 2002). Similarly, TTLs comprise a large nema-
tode-specific gene family, found in both H. contortus and other animal- and
plant-parasitic nematodes (Furlanetto et al., 2005; Gao et al., 2003; Hewitson
et al., 2008; Jacob et al., 2007; McCarter et al., 2004; Mulvenna et al., 2009;
Parkinson et al., 2004; Saverwyns et al., 2008), within a larger superfamily of
transthyretin homologues (including 5-hydroxyisourate hydrolase, which
converts 5-hydroxyisourate to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimida-
zoline in purine catabolism), which is conserved across prokaryotes and
eukaryotes (Hennebry et al., 2006; Lee et al., 2005; Ramazzina et al.,
2006; Zanotti et al., 2009). In vertebrates, transthyretin can bind the hormone
thyroxine (T4) directly, and can also bind vitamin A (retinol) indirectly by
forming a complex with retinol-loaded retinol-binding protein (Li and
Buxbaum, 2011). In C. elegans, other members of this superfamily regulate
lifespan (Hansen et al., 2005; Kim and Sun, 2007) or enable cell corpse
engulfment by binding surface-exposed phosphatidylserine on apoptotic cells
(Wang et al., 2010). In H. contortus, a TTL isolated from ES products of adult
worms has been shown to be immunogenic in the host (Yatsuda et al., 2003).
Taken together, current evidence indicates that H. contortus has an arsenal of
ES proteins that might be involved in the modulation, evasion and/or block-
ing of host responses. Of these, SCP/TAPS proteins are of particular interest
because some of them, when expressed as recombinant proteins and used as
vaccines, can induce significant levels of immunological protection against
infections of animals by the strongylid nematodes Cooperia oncophora and
Heligmosomoides polygyrus (see Hewitson et al., 2015; Vlaminck et al., 2015).
5.4 Predicting targets for new anthelmintics

The excessive and uncontrolled use of a small number of drug classes for the
treatment of haemonchosis has led to major problems of drug resistance in
H. contortus and related bursate nematodes (Kaplan and Vidyashankar, 2012;
see Section 2.4; chapter: Anthelmintic resistance in Haemonchus contortus: his-
tory, mechanisms and diagnosis by Kotze and Prichard, 2016 e in this issue).
With only a few new anthelmintics (ie, cyclooctodepsipeptides and aminoa-
cetylnitriles) having been commercialized in the past two decades (Kaminsky
et al., 2008; Harder et al., 2003), genomics might identify new targets for
mechanism-based drug design (Eder et al., 2014; Shanmugam et al., 2012).
Ideally, a genomics-based strategy will identify gene products that can be
inactivated by a drug (ie, are ‘druggable’; Griffith et al., 2013; Russ and
Lampel, 2005) without affecting the host, either because the host’s homolo-
gous proteins are too divergent to be affected by the drug or because the host’s
genome is completely devoid of homologues (as is the case for macrocyclic
lactones, which mainly target inhibitory glutamate-gated chloride channels,
a class of ion channels present in nematodes and arthropods but absent
from vertebrates; Wolstenholme, 2012). For nematodes whose life cycle
cannot be fully maintained in vitro and for which no gene perturbation
method exists to study gene function, genetic essentiality can be inferred
from functional information for other organisms, particularly C. elegans (see
Doyle et al., 2010).
In their paper on the draft genome, Schwarz et al. (2013) predicted 641
H. contortus protein-coding genes that had single-copy homologues in
C. elegans and whose C. elegans homologues exhibited lethal phenotypes
upon gene silencing by RNAi. These authors also scanned for enzymatic
chokepoints in biological pathways of H. contortus. Such chokepoints repre-
sent reactions that consume or uniquely produce a molecular compound;
the disruption of such enzymes should lead to a toxic accumulation (ie,
for unique substrates) or a starvation (ie, for unique products) of metabolites
within cells (cf. Berriman et al., 2009; Taylor et al., 2013; Yeh et al., 2004).
Using this approach, Schwarz et al. (2013) predicted 260 genes encoding
druggable proteins in H. contortus, 106 of which had ligands that met gener-
ally accepted chemical criteria (the ‘rule of five’) for being possible drugs
(Lipinski, 2004). Conspicuous among these 107 gene products were 17
channels or transporters representing known targets for anthelmintics,
including macrocyclic lactones, levamisole and aminoacetonitrile derivatives
(Campbell et al., 1983; Kaminsky et al., 2008; Qian et al., 2008), and 38
other candidates including protein kinases and phosphatases known to be
specific targets for norcantharidin analogues (Campbell et al., 2011).
By comparison, in their chokepoint analysis, Laing et al. (2013) predicted

362 enzymes, five of which lacked isoenzymes and were divergent from
those of humans (and thus likely to be absent from any mammalian host
of H. contortus; see Yeh et al., 2004). Two of these five enzymes were
already known to be drug targets based on information in the Therapeutic
Target Database: dTDP-4-dehydrorhamnose 3,5-epimerase (EC 5.1.3.13;
Kantardjieff et al., 2004) and trehalose-6-phosphatase (EC 3.1.3.12;
Kushwaha et al., 2011). These findings indicate the need to experimentally
assess these target candidates in vitro and in vivo.
Laing et al. (2013) emphasized the importance of ligand-gated ion chan-
nel subunit genes (LGICs) as neuromuscular drug targets, since they encode
one of most targeted protein classes in parasitic nematodes: imidazothiazoles
(eg, levamisole), monepantel, tribendimidine and spiroindoles act against
nicotinic acetylcholine receptors, while macrocyclic lactones (eg, iver-
mectin) act against glutamate-gated chloride channels (Holden-Dye and
Walker, 2014). The authors noted that LGICs of H. contortus are partly
conserved in C. elegans (ie, many LGIC genes have direct orthologues in
C. elegans). However, they also noted significant disparities: two genes
encoding macrocyclic lactone targets in H. contortus (Hco-glc-5 and Hco-
glc-6) had been lost from the C. elegans genome; a well-characterized target
of ivermectin in C. elegans, Cel-glc-1, proved to be a species-specific
paralogue; Cel-acr-13, encoding a subunit of a multimeric receptor and lev-
imasole target, was absent from H. contortus; and the gene Hco-mptl-1, encod-
ing a monepantel target in H. contortus, was most closely related to two
paralogous genes in C. elegans, acr-20 and acr-23. Considering this mosaic
of similarities and dissimilarities, Laing et al. (2013) noted that, while C. ele-
gans is likely to be a useful model for investigating the neuromusculature of
H. contortus in drug screens, significant differences probably exist in the ef-
fects of particular anthelmintics. One way in which this problem might be
addressed, so that the experimental convenience of C. elegans is combined
with the native biological function of drug targets encoded by the H. contor-
tus genome, would be to express H. contortus genes (such as LGICs) in
C. elegans and then observe what responses to anthelmintics the transgenes
conferred. Indeed, this strategy was successfully used for the H. contortus
tubulin gene Hco-tbb-iso-1, which encodes a target of albendazole, long
before the H. contortus genomes and transcriptomes were determined
(Kwa et al., 1995; Saunders et al., 2013); recently, it has also been shown
to work for the glutamate-gated chloride channel gene Hco-glc-6 (which en-
codes a target of ivermectin) (Glendinning et al., 2011), and for both
serotonin and tyramine receptors of H. contortus, for which agonists may

have anthelmintic effects (Law et al., 2015).
5.5 Xenobiotic detoxification

Laing et al. (2013) undertook a detailed analysis to predict the repertoire of
inducible transporters and metabolizing enzymes likely to protect
H. contortus against toxins. Based on evidence for C. elegans (see Lindblom
and Dodd, 2006), xenobiotic detoxification takes place in three phases:
modification, involving short-chain dehydrogenase/reductases (SDRs) and
the cytochrome P450s (CYPs); conjugation, involving glutathione S-
transferases (GSTs) and UDP-glucoronosyl transferases (UGTs); and cellular
excretion involving P-glycoproteins (PGPs), a subtype of ATP-binding
cassette (ABC) transporters. In H. contortus, Laing et al. (2013) identified
44 SDR genes, 42 CYPs, 28 GSTs, 34 UGTs and 10 PGPs, suggesting a
wide capacity for detoxification. Of these genes, PGPs were of particular
interest because there already existed evidence that they could mediate
anthelmintic resistance in H. contortus and other nematodes (Lespine et al.,
2012). As with LGIC genes, the H. contortus set of PGP genes were
evolutionarily divergent from those of C. elegans, with both gains and losses
of genes in each lineage. Subsequent work has demonstrated that chemically
inhibiting PGPs in H. contortus can synergistically increase the effectiveness of
anthelmintics, validating the potential importance of these transporters as
drug targets (Raza et al., 2015).
5.6 Immunogens
As indicated previously (Section 2.4), to circumvent problems with
anthelmintic resistance, a number of research groups have worked to devise
vaccines against haemonchosis (Knox, 2011). Most effort was directed at
inducing immunity in sheep against proteins expressed in or excreted/
secreted from the H. contortus gut, with the aim of disrupting or inhibiting
the parasite’s digestion of host blood. To date, the two most effective immu-
nogens assessed have been the aminopeptidase family H11 (Munn, 1977;
Newton and Munn, 1999; Knox, 2011) and the H. contortus galactose-
containing glycoprotein complex (H-gal-GP; Smith and Smith, 1996).
Both of these molecular complexes contain integral membrane proteins
with haemoglobinase activity, and are expressed mainly in the microvillar
surface of the parasite’s gut; each of them induces >70e90% protection
against infection in distinct breeds of sheep (Knox, 2011).
Using the genomic and transcriptomic data, Schwarz et al. (2013) were
able to predict the molecular variants within each of these two complexes.
They reported that H11 appears to represent tens of different metallopepti-
dases (clan MA; family M01; Table S15), which are upregulated 6- to
210-fold in the parasitic over the free-living stages of H. contortus. Key
components of H-gal-GP, representing mainly metallopeptidases (eg,
MEPs 1e4; Redmond et al., 1997; Newlands et al., 2006), aspartyl-peptidases
(eg, HcPEPs 1 and 2; Longbottom et al., 1997; Smith et al., 2003a) or cysteine
peptidases (eg, AC-1 to AC-5; HMCP-1 to HMCP-6; Cox et al., 1990;
Longbottom et al., 1997; Pratt et al., 1990, 1992; Skuce et al., 1999) were
also identified using sequence data from previous proteomic studies.
All three classes of peptidases were significantly upregulated in the
haematophagous stages (Schwarz et al., 2013). A diversity of cysteine pepti-
dase genes (n ¼ 81) was seen, some of whose products have been under
study as vaccine candidates (Knox, 2011). Many of them (14) represent
clan C01A (cathepsin B-like peptidases), and 34.6% were represented in
the ES degradome (Schwarz et al., 2013). There were also identified 11
legumain genes (clan CD; family C13), whose products might activate
key family C01A peptidases through cleavage of the peptide backbone
between the prosegment and mature enzyme domains (Dalton et al.,
2009). In addition, the serine peptidase complex contortin has received
attention as a vaccine candidate, since it is an efficient anticoagulant (with
dipeptidyl IV activity) in parasitic stages of H. contortus (see Geldhof and
Knox, 2008). Contortin is inferred to belong to clan SC serine peptidases
(family S28). Among more than 100 serine peptidases predicted, Schwarz
et al. (2013) found 13 representatives of the family S28 (lysosomal Pro-Xaa
carboxypeptidases), all of which were upregulated in parasitic stages. Nine of
these carboxypeptidases were represented in the ES degradome, supporting
the hypothesis that contortin is also immobilized (Dalton et al., 2009). Inter-
estingly, H. contortus shares many of these distinct classes of peptidases with
other haematophagous parasites, such as hookworms, indicating relative
conservation in sequence and function, associated mainly with feeding
(anticoagulation and digestion). To date, it has not been possible to achieve
high levels of protection using selected recombinant proteins representing
H11 or H-gal-GP, and carbohydrate moieties alone do not protect either
(Knox, 2011). Assisted by present genomic and transcriptomic resources
for H. contortus (see Laing et al., 2013; Schwarz et al., 2013), future work
might focus on exploring immunogens in more detail using proteomic, gly-
comic and structural biological tools.
6. PROSPECTS
The initial analyses of the expressed portion of the H. contortus
genome, ie, the transcriptome, were necessarily a first round of effort rather
than a final achievement. Building on their implications will be crucial for
realizing the promise of genomics and transcriptomics in this parasite. There
are many different ways in which such progress might occur.
The first is simply to do genomic sequencing over again, but better. The
H. contortus genome and transcriptomes published in 2013, although crucial at
the time, were not perfect; both assemblies were missing w7% of the haploid
genome, and were in thousands of pieces. New techniques for ‘third-gener-
ation’ genome sequencing and assembly of large sets of long but error-prone
DNA reads have made it possible to generate de novo genome sequences of
far higher quality than the existing H. contortus assemblies (Berlin et al., 2015;
Madoui et al., 2015; Sharon et al., 2015). For instance, a third-generation
reassembly of the genome of Drosophila melanogaster generated an entire chro-
mosome arm of unbroken sequence, exceeding the quality that had been
achieved through over a decade of first- and second-generation sequencing
(Berlin et al., 2015). At the same time, it has become clear that many species
in nature for which genomic assemblies are desirable are highly diploid and
polymorphic, like H. contortus, unlike the highly inbred model organisms
such as C. elegans towards which genome assembly software was first directed
(Cutter et al., 2013). This has motivated the development of new assemblers
and post-assembly programs aimed at producing valid quasi-haploid genome
assemblies from polymorphic, diploid sequencing data (Bodily et al., 2015;
Huang et al., 2012; Kajitani et al., 2014; Safonova et al., 2015). The combi-
nation of third-generation and diploid assembly techniques should allow the
resequencing of various key strains of H. contortus to chromosome-scale con-
tiguity. Such resequencing should resolve many regions of tandem multigene
families that are readily lost from second-generation sequencing with short
Illumina reads (Alkan et al., 2011), but that are important for understanding
species- and strain-specific traits of biological importance (Kondrashov,
2012; Qian and Zhang, 2014).
A second avenue of progress could be to compare the genome and tran-
scriptome of H. contortus to those of many other nematodes. Such compar-
ison is feasible, because the successful sequencing of key parasitic nematodes
(Zarowiecki and Berriman, 2014) has motivated initiatives by major genome
centres to sequence the genomes of a great many parasitic helminths (both
nematodes and trematodes); as of June 2015, the database
parasite.wormbase.org (Harris et al., 2014) contained unpublished draft

genomic sequences for 39 parasitic nematode species, of which 10 species
were close relatives of H. contortus (clade V strongylids). Genomic compar-
isons between both parasitic and nonparasitic nematode species afford the
possibility of identifying genes that undergoing positive selection or gene
family amplification within parasitic lineages, and may, therefore, be partic-
ularly important for parasitism (Blaxter and Koutsovoulos, 2015; Zarowiecki
and Berriman, 2014). It may also be possible to elucidate how the transcrip-
tome of H. contortus is regulated, by large-scale comparisons of conserved
noncoding DNA with possible cis-regulatory function (Shlyueva et al.,
2014; Zhao et al., 2012), which, in turn, may provide a means to block
expression of co-regulated gene families (such as those encoding SCP/
TAPS or TTL proteins) whose collective activity may promote infection,
parasitism and/or disease. Perhaps most powerfully, it should become
possible to apply phylogenetic profiling to the protein-coding gene set of
H. contortus. Phylogenetic profiling consists of determining orthologies
(direct homologies) between genes in many (w100) phylogenetically
well-distributed genomes within a given class of organism (such as unicellu-
lar microbes or multicellular eukaryotes), and then classifying each gene by
the vector of its presence or absence in each of the individual species being
characterized (Pellegrini, 2012; Skunca and Dessimoz, 2015). The phyloge-
netic vectors are then clustered, so that genes with statistically significant
correlations in their presence or absence within given organisms are placed
in a single phylogenetic group. The power of such an analysis is that some of
these genes can have well-known functions, while other genes that have no
known function, but that do have strongly correlated patterns of
evolutionary presence or absence, can be identified as being likely to share
function with their well-characterized group members. Phylogenetic
profiling was first successfully used on bacterial genomes at the end of the
1990s; however, there have only recently arisen enough genomic
sequences (Blaxter and Koutsovoulos, 2015; Zarowiecki and Berriman,
2014) and efficient algorithms (Dey et al., 2015; Li et al., 2014c; Tabach
et al., 2013) for phylogenetic profiling to be applied to multicellular eukary-
otes. The recent growth of genomic sequences for parasitic nematodes
should thus make it relatively straightforward to analyze the entire proteome
of H. contortus and predict which of its over 20,000 protein-coding genes act
specifically in parasitism, as opposed to other genes effecting core metazoan
functions (Moroz et al., 2014; Richter and King, 2013; Ryan et al., 2013),
and still other genes shared by C. elegans that may have ecological functions
outside of the host during the free-living phase of H. contortus’ life cycle (cf.
Frezal and Felix, 2015; Hodgkin, 2001; Petersen et al., 2015).
Third, the noncoding RNA genes of H. contortus can be identified, and
their detailed transcriptomics can be determined. This area is of central
importance to understanding parasitism, because it has recently been
demonstrated that a wide phylogenetic range of parasitic nematodes secrete
micro RNAs (miRNAs), and that these miRNAs can exert immunomodu-
latory effects on their mammalian hosts (Buck et al., 2014; Hansen et al.,
2015; Quintana et al., 2015; Tritten et al., 2014a,b). Both of the published
draft genome analyses for H. contortus focused on protein-coding genes;
neither attempted to predict ncRNA genes, perhaps because such predic-
tions were very computationally intensive at the time. However, initial
RNA-seq analysis of small RNAs independently identified 192 H. contortus
miRNAs, of which 138 (72%) were species-specific rather than being visibly
conserved between H. contortus and other species such as C. elegans (see
Winter et al., 2012). Moreover, Nawrocki and Eddy (2013) have recently
developed a version of INFERNAL, the leading program for ncRNA
detection in genomes, that is rapid enough to scan an entire metazoan
genome in roughly a day (previous versions were w100 times slower). At
the same time, the RFAM database for ncRNA families has been steadily
growing; the most recent version (24.0, released in July 2014), contains
2450 families that include 415 distinct miRNAs (Nawrocki et al., 2015).
Therefore, it should be possible to define the comprehensive set of miRNAs
and other ncRNAs encoded by the H. contortus genome, and to determine
what subset of those ncRNAs contribute to parasitism.
Fourth, the transcriptome of H. contortus can be used to model its
metabolism. In a limited but important way, both initial genome analyses
attempted to do just that, by predicting key points of vulnerability in this
worm’s biochemistry that might be effective drug targets (chapter: The
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes by
Harder, 2016 e in this issue). However, such analyses are relatively naïve
compared with new approaches for modelling an entire organism’s meta-
bolism, or, more ambitiously, its full cell biology, that has been successfully
developed for single-celled bacteria or unicellular eukaryotes (O’Brien et al.,
2015). As with phylogenetic profiling, metabolic and cellular modelling was
necessarily first developed on very simple organisms (Bordbar et al., 2014;
Macklin et al., 2014), but should be of great value when it is extended to
H. contortus and other multicellular parasites. Pioneering analyses of parasitic
nematode metabolism have recently been published by Mitreva and
coworkers (Taylor et al., 2013; Tyagi et al., 2015b); one can hope for more
such work in the next few years.
Fifth, H. contortus genes can be identified that are specifically transcribed in
tissues specifically required for effective parasitism, and which are therefore
more likely than other genes to enable infection. Laing et al. (2013) have
already done this work for the intestine, and these results can be usefully
related to work conducted by Mitreva and coworkers (Rosa et al., 2015)
on intestinally expressed genes of A. suum. What remains to be done are an-
alyses of small sets of cells, such as the oesophageal glands of the pharynx (cf.
Veglia, 1915), or of sensory neurons that regulate developmental transitions
during infection (cf. Davey et al., 1982). In cases where a tissue of interest
consists of one or a few cells, microdissection, followed by RNA amplifica-
tion and RNA-seq should be possible, as has been done for single cells in
C. elegans (see Schwarz et al., 2012). Ideally, one could instead express a trans-
gene in the cell type of interest and use transgenic expression to harvest large
numbers of specific cell types from dissociated larvae or adults, through cell
sorting (Spencer et al., 2014). The latter technology should work if transgen-
esis in H. contortus can itself be achieved in the future; until then, microdissec-
tion is likely to be necessary for cell-specific transcriptomics.
Sixth, both existing transcriptomic data and results from future work
(such as that proposed earlier) can be used to annotate a reference H. contortus
genome in WormBase, so that the available data are easily usable both for
biologically focused, small-scale queries by individual researchers and for
large-scale data mining by system biologists. Fortunately, this can be done
efficiently, because the computational substructure of WormBase has been
intensively developed for 15 years in such a way that all of the tools con-
structed for bioinformatics in C. elegans (such as highly flexible genome
browsing, customizable data queries through the EnsMart interface,
genome-wide orthologies and paralogies, motif scanning of noncoding
DNA regions, etc.) can have H. contortus or other species slotted into
them computationally as user demand entails (Harris et al., 2014).
Finally, H. contortus can be converted from a passive object of study
(‘nonmodel organism’) to an active subject of experimental perturbation
(‘emerging model organism’), by adapting existing technologies for trans-
genesis, gene inactivation and genomic modification from C. elegans to
H. contortus (see Rowan et al., 2011). The existence of usable draft genomes
and permanently frozen isogenic H. contortus strains (Chylinski et al., 2015)
was a first and crucial step towards this goal. Even before the H. contortus
genomes were completed and published, efforts had been made to apply
RNAi to silencing H. contortus genes (Samarasinghe et al., 2011; Zawadzki

et al., 2012); it is to be hoped that the draft genomes will allow such efforts
to become more effective. Transgenesis in parasitic nematodes has also been
performed by a few pioneering laboratories, although not yet in H. contortus
itself (Hagen et al., 2012; Lok, 2012). Most excitingly and recently,
CRISPR/Cas9 technologies have been developed that allow efficient
site-specific mutagenesis of C. elegans and its free-living relative P. pacificus,
a form of genetic modification that had been previously achieved in yeast
and mammalian cells, but not in nematodes (chapter: Functional Genomics
Tools for Haemonchus contortus and Lessons From Other Helminths by
Britton et al., 2016 e in this issue; Frokjaer-Jensen, 2013; Sternberg and
Doudna, 2015; Witte et al., 2015). When it is possible to take two otherwise
isogenic strains of H. contortus, with one of them having a specific mutation
inactivating one or more genes suspected to mediate successful infection,
and observe them in vivo in a sheep host, we should finally be in a position
to test many of the hypotheses implied, but not proven, by the two draft ge-
nomes and transcriptomes that currently exist.
ACKNOWLEDGEMENTS
R.B.G thanks all current and past Lab members, and numerous collaborators for their
contributions to some of the research described in this article. Funding from the Australian
Research Council (ARC) and the National Health and Medical Research Council (NHMRC)
is gratefully acknowledged (R.B.G.), as is support from the Victorian Life Sciences Computa-
tion Initiative (grant number VR0007) on its Peak Computing Facility at the University of
Melbourne, an initiative of the Victorian Government. Other support from the Australian
Academy of Science, the Australian-American Fulbright Commission, Alexander von Hum-
boldt Foundation and Melbourne Water Corporation is gratefully acknowledged. E.M.S.
gratefully acknowledges support from the Moore Foundation, the US National Institutes of
Health and Cornell University. N.D.Y. holds an NHMRC Career Development Fellowship,
and P.K.K. a STRAPA scholarship from The University of Melbourne.
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CHAPTER THIRTEEN
Haemonchus contortus: Genome

Structure, Organization and
Comparative Genomics
R. Laing*, A. Martinellix, A. Traceyx, N. Holroydx, J.S. Gilleard{, 1,
J.A. Cottonx
*University of Glasgow, Glasgow, Scotland, United Kingdom
x
{
1
Corresponding author: E-mail: jsgillea@ucalgary.ca
Contents
1. Introduction 570
1.1 Overview and historical context 570
1.2 The need for a high-quality ‘finished’ reference genome 571
2. From Draft Assembly to Reference Genome 573
2.1 Progress so far 573
2.2 Future and ongoing work 577
3. Genome Structure 579
3.1 Genome size, repetitive sequence and Guanine and Cytosine (GC) content 580
3.2 Chromosomal synteny 580
4. Genome Content 581
4.1 Gene number, size and structure 581
4.2 Orthology and gene family evolution 582
4.3 Operons and trans-splicing 587
4.4 Repetitive sequence and mobile elements 588
5. Future Directions 589
References 591
Abstract
One of the first genome sequencing projects for a parasitic nematode was that for
Haemonchus contortus. The open access data from the Wellcome Trust Sanger Institute
provided a valuable early resource for the research community, particularly for the iden-
tification of specific genes and genetic markers. Later, a second sequencing project was
initiated by the University of Melbourne, and the two draft genome sequences for
H. contortus were published back-to-back in 2013. There is a pressing need for long-
range genomic information for genetic mapping, population genetics and functional
j
ISSN 0065-308X
570 R. Laing et al.
genomic studies, so we are continuing to improve the Wellcome Trust Sanger Institute
assembly to provide a finished reference genome for H. contortus. This review describes
this process, compares the H. contortus genome assemblies with draft genomes from
other members of the strongylid group and discusses future directions for parasite
genomics using the H. contortus model.
1. INTRODUCTION
1.1 Overview and historical context
The first metazoan organism to have a full genome sequence pub-
lished was the free-living nematode Caenorhabditis elegans in 1998; even
now, the genome of this model organism is still one of the few truly ‘com-
plete’ metazoan genomes (C. elegans Sequencing Consortium, 1998). The
associated database WormBase (https://www.wormbase.org) was one of
the first to integrate genomic, genetic and phenotypic data and published
literature and to have ongoing curation. Although the C. elegans Sequencing
Consortium played a central role in the development of whole genome
sequencing approaches, until recently, parasitic nematodes have been largely
left out of these endeavours. Indeed, whilst research groups working on bac-
terial and protozoan pathogens have rapidly advanced their genomic re-
sources, the progress in producing high-quality reference genomes for the
major parasitic helminths has been slower. There are a number of reasons
for this, including the large size and complexity of helminth genomes rela-
tive to other classes of pathogens and the lower priority given to parasitic
helminths by the major genome centres due to the relatively small size of
the research communities. However, over the last 5e6 years, advances in
massively parallel sequencing technologies have dramatically reduced raw
sequencing costs which, together with related developments in informatics,
has resulted in significant progress in sequencing helminth genomes (Ghedin
et al., 2007; Jex et al., 2011; Mitreva et al., 2011; Protasio et al., 2012; Tsai
et al., 2013). For example, a recent initiative at the Wellcome Trust Sanger
Institute aims to provide draft reference genomes for 50 parasitic helminths
of major importance to human and veterinary medicine and agricultural pro-
duction https://www.sanger.ac.uk/research/initiatives/globalhealth/research/
helminthgenomes/.
One of the first parasitic nematode genome sequencing projects to be
initiated at one of the major genome centres was that for Haemonchus contortus
(http://www.sanger.ac.uk/resources/downloads/helminths/haemonchus
contortus.html). This project was started in 2004 using the standard
The Haemonchus contortus Genome 571
approach available at the time; a combination of capillary sequencing and

physical mapping with Bacterial Artificial Chromosome (BAC) or cos-
mid/fosmid libraries. MHco3.ISE was chosen as the reference strain for
this project. This strain is a version of the ISE isolate that has been passaged
and maintained at the Moredun Research Institute since 2002 (Redman
et al., 2008b). It was chosen because it is susceptible to all the major anthel-
mintic drugs and had been previously inbred by multiple rounds of serial
passage infections with progeny from a single female worm (Roos et al.,
2004). The draft genome sequence produced at this time provided a valu-
able, early resource for researchers in the field, particularly for the identifi-
cation of specific individual genes and genetic markers (Kaminsky et al.,
2008; McCavera et al., 2007; Redman et al., 2008b). This strain was further
inbred using a single pair mating approach to produce the more highly
inbred strain MHco3(ISE).N1 that was used to produce DNA template
for subsequent sequencing in the genome project (Sargison, Redman,
Naghra, Cotton and Gilleard, unpublished data). However, the relatively
large genome size (w 300 Mb) and high levels of sequence polymorphism,
even in this partially inbred strain, made completion of a high-quality refer-
ence genome challenging and prohibitively expensive at that time. It was
not until the availability and application of ‘second-generation’ sequencing
approaches that significant progress on the assembly and annotation of a
good quality ‘draft’ reference genome was made. Subsequently, another
genome project was initiated to sequence the H. contortus McMaster strain
which was derived from an Australian isolate. In 2013, the draft genome se-
quences of both the MHco3.ISE and McMaster strains were published in
complementary papers (Laing et al., 2013; Schwarz et al., 2013). These draft
genomes were of a quality such that >90% of genes are believed to be at least
partially represented and mostly in scaffolds that contain multiple genes. The
MHco3.ISE reference genome was 346.2 Mb in length, with a scaffold N50
of 83,287 bp and with 21,799 predicted protein-coding genes (Laing et al.,
2013). The McMaster reference genome was 320 Mb in length, with a scaf-
fold N50 of 56,300 bp and with 23,610 predicted protein-coding genes
(Schwarz et al., 2013). Although these can be considered to be good quality
draft sequences, and so valuable resources, they are still far from ‘finished’
genomes.
1.2 The need for a high-quality ‘finished’ reference genome

The rapid development of next-generation sequencing technologies has
made large-scale sequencing faster and cheaper than ever before and has
572 R. Laing et al.
resulted in a large number of parasitic helminth genome projects (Kumar

et al., 2012). The strategy for most of these projects has been to use short-
read technologies to sequence genomes at great depth and then undertake
shotgun assembly, to avoid the labour-intensive process of physical mapping
of large-insert genomic clones. Although mate pair libraries are often used to
aid the assembly of contigs into larger scaffolds, most of the current helminth
genome projects are producing draft genomes that are still highly frag-
mented and do not have funding to progress these to chromosome-level
assemblies (Fig. 1). Although these draft genomes are extremely valuable
for identifying ‘genes of interest’ and for some comparative and evolutionary
studies, chromosome-level assemblies will greatly improve the power of
future functional genomic and genetic studies.
Consequently, one of the major challenges facing the research commu-
nity working on parasitic helminths at present is how to ensure that we
continue to improve these genome assemblies and annotations for those par-
asites of high medical, veterinary or agricultural importance, particularly
those species that can serve as model experimental systems. Unfortunately,
there is a disproportionate cost and effort of finishing a draft reference
genome and a poor return, in terms of peer-review publication, acts as a ma-
jor disincentive. Haemonchus contortus is a good example of this, in that it is
the best available model system for studying the biology of strongylid nem-
atodes, for anthelmintic drug discovery and for anthelmintic resistance
research (Burns et al., 2015; Kaminsky et al., 2008). For this latter research
area, in particular, H. contortus provides an opportunity to apply both classical
genetic mapping and population genomic studies to identify anthelmintic
resistance loci (Gilleard and Beech, 2007; Redman et al., 2012). However,
this work is extremely difficult with the current draft genomes and so we are
continuing to improve the MHco3.ISE assembly to provide a finished refer-
ence genome for H. contortus.
Figure 1 Limitations of draft genome assemblies.

2. FROM DRAFT ASSEMBLY TO REFERENCE GENOME

2.1 Progress so far
To generate the draft genomes (Laing et al., 2013; Schwarz et al.,
2013), a combination of short and long insert Illumina libraries were gener-
ated, with the addition of 454 libraries for the MHco3.ISE assembly (see
Mardis, 2008 for review of these sequencing technologies). Standard de
novo assembly software (Velvet: Zerbino and Birney, 2008; Celera: Myers
et al., 2000) was used together with HaploMerger (Huang et al., 2012) to
reduce the amount of haplotypic expansion. Illumina and 454 are both
short-read sequencing technologies, with particularly Illumina sequencing
being highly cost-effective, in terms of generating large amounts of sequence
data, and highly accurate, in terms of the correct identification of sequenced
bases. Assembly involves finding overlaps between sequence reads, so that
longer strings of contiguous known bases (contigs) can be reconstructed.
Two aspects make this process difficult and have led to an enormous diver-
sity of algorithms and software packages for genome assembly (eg, Bradnam
et al., 2013; Earl et al., 2011; Nagarajan and Pop, 2013; Schatz et al., 2010
for reviews and comparison). First, the sheer number of reads produced by
modern sequencing technologies means that algorithms and their software
implementation need to be extremely efficient in finding overlaps between
reads (eg, Simpson and Durbin, 2010). More fundamentally, repetitive
strings occurring in DNA or the presence of polymorphism can make the
reconstruction of the genome ambiguous and lead to fragmented assemblies.
Sequencing bases from both ends of a range of different sizes of DNA frag-
ments can show that certain contigs belong close to each other, with some
unknown sequence in-between being represented by a gap in between two
contigs. Strings of contigs joined in this way are known as scaffolds (Fig. 2).
The presence of such gaps, and of sets of scaffolds where the order and orien-
tation between them is unknown, is the hallmark of a draft assembly, and
generally short-read technologies can only produce draft assemblies for all
but the very smallest and simplest genomes. Each of the Haemonchus genome
assemblies published in 2013 (Laing et al., 2013; Schwarz et al., 2013) was in
over 10,000 scaffolds.
Since publication of the draft sequences, we have continued to improve
the MHco3.ISE genome with the aim of assembling the six chromosomes
that are predicted by karyotype analysis (Redman et al., 2008a), and so
provide a true reference-quality genome assembly (Chain et al., 2009)
574 R. Laing et al.
Figure 2 Sequence reads and assembly. A paired-end sequencing library is generated

by randomly fragmenting a genome and some additional molecular biology steps
before sequencing stretches of nucleotides from both ends of each fragment. The as-
sembly process identifies stretches of overlapping nucleotides between reads to form
contigs. Two contigs that contain reads from the same pair are united into the same
scaffold, with unsequenced or ambiguous bases in-between leading to a gap between
adjacent contigs.
for H. contortus. One key approach is to generate additional data that

allow long-range scaffolding and contiguation of the existing assembly.
We have generated long-read sequence data using the PacBio sequencing
platform (Korlach et al., 2010) and used contigs from a de novo assembly of
these data to bridge gaps and identify joins between scaffolds in the previ-
ous assembly. Longer-range scaffolding is improved by optical mapping e a
technology in which individual DNA fragments are immobilized and
digested with restriction enzymes or labelled at sequence-specific nicks
in the DNA molecules (Lam et al., 2012; Samad et al., 1995). The distri-
bution of these cuts or nicks is visualized microscopically to produce an or-
dered map of the relative location of short-sequence motifs along a
molecule. Combining data from many thousands of molecules allows the
reconstruction of very long maps, up to whole chromosomes for smaller,
less-repetitive genomes. Aligning sequence data against these maps or using
the maps to identify adjacent sequence contigs in other ways (eg, Dong
et al., 2013) can help build long scaffolds with extensive sequence gaps
that can then be filled using other methods.
A second component of the genome improvement work has been a pro-
gramme of manual curation and genome improvement, initially based on
the inspection of short reads mapped to the entire genome to allow correc-
tion of the assembly where necessary, and latterly also making use of longer
PacBio reads. This is achieved using the genome viewing and editing tool
Gap5 (Bonfield and Whitwham, 2010). Using this approach, it is possible
to identify places throughout the genome where the sequence is too diver-
gent, or where pairs of reads do not lie in the correct relative orientation or
spacing, and these features give clues to places where the sequence is
incorrect, and how to correct the problems. For example, if many of the
reads have their ‘mate’ e the other read of the pair e elsewhere in the as-
sembly, it might indicate that the existing sequence needs to be broken, and
an additional contig needs to be inserted. This read pair information,
together with unusual patterns in coverage, can also identify regions where
haplotypes have been too dissimilar to assemble and the contigs end prema-
turely e an issue that seems to have particularly plagued the automatic as-
semblies of H. contortus. Recently, significant progress in improving the
contiguation of the genome assembly has been achieved by taking contigs
de novo assembled from PacBio data, validating and assembling them
with other data and then using these sequences e representing longer
stretches of a single haplotype e to replace gaps or misassembled regions
of the assembly.
This ‘manual’ improvement process is somewhat automated e Gap5 it-
self contains many features to help automatically find potential joins and
identify potentially misassembled regions, and other software has also been
developed to aid genome improvement. Algorithmic approaches to identi-
fying problematic regions eg, REAPR (Hunt et al., 2013) are used to help
locate possible misassemblies for manual improvement. Small errors can be
automatically corrected (Otto et al., 2010), and automated approaches to
close gaps (Tsai et al., 2010) and improve scaffolding using data from existing
assemblies (Assefa et al., 2009) are all available. These approaches have
already provided a much improved genome assembly, referred to here as
MHco3.ISE.2015.
Table 1 shows a comparison of assembly statistics for the two published
H. contortus genomes and the current H. contortus MHco3.ISE.2015
genome. While the published H. contortus genomes have comparable
assembly metrics to other draft strongylid genomes (Table 2, Section 3),
Table 1 Assembly statistics for Haemonchus contortus

H. contortus H. contortus H. contortus
MHco3.ISE McMaster MHco3.ISE.2015
Assembly size (Mb) 370 320 340

Number of scaffolds 26,044 14,419 5051
N50 count 1151 1684 16
N50 (kb) 83 56 7446
N90 count 5518 6085 552
N90 (kb) 11 13 32
Largest scaffold (kb) 947 345 25,917
Table 2 Genome statistics for Haemonchus contortus and other clade V nematodes
576
H. contortus Oesophago-
MHco3.ISE H. contortus Necator Ancylostoma stomum Dictyocaulus Angiostrongylus Caenorhabditis
(MHco3.ISE.2015) McMaster americanus ceylanicum dentatum viviparus cantonensisa elegans WS248
Assembly size 370 (340) 320 244 313 443 169 261 100
(Mb)
Number of 26,044 (5051) 14,419 11,713 1737 64,258 17,715b 17,280 6
scaffolds
Scaffold N50 83 (7446) 56 213 668 26 22.6b 42 17,493
(kb)
GC content 43 42 40 43 33 34.5 41 35
(%)
Repetitive 29 13 24 41 31 Not reported 25 16
sequence
(%)
Number of 21,869 23,610 19,151 30,738 25,291 14,306 17,482 20,388
genes
Gene density 59 74 79 98 57 85 67 200
(per Mb)
Mean gene 6524 6167 4289 4560 2171 Not reported Not reported 3037
footprint
(bp)
Mean number 8.8 6 5.4 11 4.3 Not reported Not reported 5.1
introns per
gene
Mean intron 601 832 642 319 352 Not reported Not reported 320
size (bp)
Figures are all as reported in the genome paper for each species cited in the text, except gene density figures calculated from number of genes and assembly size where necessary.
Angiostrongylus cantonensis figures are for the Yong et al. (2015) assembly.
R. Laing et al.
a
b
Dictyocaulus viviparus assembly statistics are for contigs, not scaffolds.
the MHco3.ISE.2015 assembly is significantly better. Fifty percent of the

genome is now assembled into 16 scaffolds (N50 ¼ 7.4 Mb), and 80% of
the genome is assembled into 66 scaffolds. The largest scaffold is
25.7 Mb in length. To visually display the scale of improvement, synteny
plots against the C. elegans reference genome annotation (WS235) were
generated using the 20 largest scaffolds for the three H. contortus assemblies
(Fig. 3). The scaffolds and chromosomes are shown to scale, and for
MHco3.ISE.2015, these 20 scaffolds comprise more than half of the
genome. As expected, MHco3.ISE.2015 and the published MHco3.ISE
assembly are highly collinear, although a number of corrected misassem-
blies interrupt this pattern. A more detailed investigation of synteny
between the two H. contortus isolates is consistent with the general pattern
of colinearity of the two assemblies: most McMaster scaffolds that show any
orthology to MHco3.ISE.2015 scaffold 5 show orthology only to this scaf-
fold (Fig. 4). Scaffold 5, which is approximately 6 Mb in size and syntenic
with C. elegans chromosome I was chosen as an arbitrary representative
scaffold close to the N50 of the improved assembly.
2.2 Future and ongoing work

The presence of extensive haplotypic sequence diversity in Haemonchus e
even in the inbred MHco3.ISE isolate e means manual intervention is
currently essential in improving the genome assembly, and much of the
work described in Section 2.1 is still in progress. If the combination of
approaches being used fails to produce a finished assembly of H. contortus
chromosomes, other strategies may be required. One possibility may be to
exploit the significant degree of synteny with C. elegans for an assisted
chromosomal assembly, if necessary. Work to generate a genetic linkage
map for H. contortus is also underway, consisting of a cross between the
MHco3.ISE isolate and a genetically divergent multidrug-resistant isolate
MHco18.UGA2004 originally isolated from a sheep at the University of
Georgia in 2004 (Williamson et al., 2011). The genetic linkage map will
aid final assembly and provide the first quantitative data on the rates of meiotic
crossing over and gene conversion in this group of nematodes, which may be
important in interpreting data from the forward genetic and population
genetic studies currently underway or being planned in Haemonchus. Once
genome improvement work is complete, the ‘finished’ genome will be
coupled to a verified de novo annotation, and all data will be released via
WormBase ParaSite http://parasite.wormbase.org/index.html and the
578 R. Laing et al.
Figure 3 (A) Synteny between the 20 largest scaffolds in the published H. contortus
McMaster and MHco3.ISE assemblies and the WormBase C. elegans genome (from
WS235). The longest McMaster and MHco3.ISE scaffolds are marked, with the long
contiguous bars representing the rest of the genome for each draft assembly to the
same scale, showing how much of the genome is assembled only into small fragments.
Lines link genomic locations of single-copy orthologs shared between the three
genomes, shaded by chromosome location of the C. elegans ortholog. (B) Synteny be-
tween 20 largest scaffolds in the H. contortus McMaster and improved MHco3.ISE.2015
assemblies and C. elegans. Large McMaster scaffolds and MHco3.ISE.2015 scaffolds are
marked, with the long contiguous bars representing the rest of the genome for each
assembly. Lines link genomic locations of single-copy orthologs shared between the
three genomes, shaded by chromosome location of the C. elegans ortholog. (C) Synteny
between McMaster scaffolds and scaffold 5 of the MHco3.ISE.2015 assembly. McMaster
scaffolds are shaded (only scaffolds with at least one single-copy ortholog on
MHco3.ISE.2015 scaffold 5 are shown), MHco3.ISE.2015 scaffold 5 is shaded and out-
lines and other MHco3.ISE.2015 scaffolds with orthologs to any of the McMaster scaf-
folds shown are outlined. Single-copy orthology from McMaster scaffolds to
MHco3.ISE.2015 scaffold 5 are linked by red (black in print versions) lines, all other links
are yellow (grey in print versions).
Figure 4 Overlap of predicted proteomes between two Haemonchus contortus

genomes and Caenorhabditis elegans. In (A) genes are considered shared between
genomes if they are members of a gene family present in both, while in (B) genes
are considered shared between genomes only if they are one-to-one orthologs.
Wellcome Trust Sanger Institute ftp site ftp://ftp.sanger.ac.uk/pub/

pathogens/Haemonchus/contortus.
3. GENOME STRUCTURE
Clade V nematodes share a conserved genetic core, which facilitates
powerful comparative and functional genomics using a C. elegans model
(Gilleard, 2013). However, nematode genomes are notoriously dynamic
in structure and organization (Blaxter, 2003), and each species may be as
readily defined by its unique adaptations to a particular niche as by its
common ancestry. Since the publication of the H. contortus genomes in
2013, draft assemblies have been published for five more members of the
strongylid group, the hookworms Necator americanus (Tang et al., 2014)
and Ancylostoma ceylanicum (Schwarz et al., 2015), the nodule worm Oesopha-
gostomum dentatum (Tyagi et al., 2015a), the bovine lungworm Dictyocaulus
viviparus (Koutsovoulos et al., 2014) and two different assemblies for the
zoonotic pathogen rat lungworm, Angiostrongylus cantonensis (Morassutti
et al., 2013; Yong et al., 2015). We therefore have the opportunity to
explore aspects of the H. contortus genome that are broadly conserved in
clade V nematodes, that are a general feature of the strongylid group, and
that are unique to this species. Vertebrate parasites are hypothesized to
have evolved from a free-living ancestor though ‘preadaptation’ in insect
hosts, so the publication of the genome of Heterohabditis bacteriophora (Bai
et al., 2013), an insect parasite in a sister group to the strongylids, may pro-
vide insight to their evolution as vertebrate parasites. Further, we have the
580 R. Laing et al.
opportunity to compare independent genome assemblies of two geograph-

ically isolated H. contortus strains.
3.1 Genome size, repetitive sequence and Guanine and

Cytosine (GC) content
Strongylid genomes are significantly expanded relative to C. elegans; with
the exception of D. viviparus, all are at least double the size (Table 2). A
large proportion of this expansion appears to be repetitive sequences:
24% of the N. americanus and A. cantonensis genome assemblies are repeti-
tive as is an incredible 41% of the A. ceylanicum genome. Strikingly, the
H. contortus MHco3.ISE, A. ceylanicum and O. dentatum genomes all contain
over 100 Mb of repeats (equivalent to the entire the C. elegans genome).
The disparity in genome size between H. contortus MHco3.ISE and
McMaster assemblies is largely due to a difference in repeat content, which
is most likely of technical note rather than biological interest, as repeats are
difficult to represent correctly in draft genome assemblies. Similarly, the
presence of haplotypic sequence will also contribute to differences in sizes
of draft assemblies. GC content is slightly higher in H. contortus and the
hookworms than O. dentatum and C. elegans but this difference does not
appear to correlate with a bias in base composition of the repetitive
sequence, as the GC content is comparable in both MHco3.ISE and
McMaster assemblies.
3.2 Chromosomal synteny

As shown in Section 2 (Fig. 2), the degree of chromosomal synteny
between H. contortus and C. elegans is striking. However, gene order is
generally poorly conserved, other than in small regions of microsynteny
(Laing et al., 2011). This is consistent with comparison of the C. elegans
and Caenorhabditis briggsae genomes, which found high rates of intrachro-
mosomal rearrangements (Coghlan and Wolfe, 2002; Guiliano et al.,
2002; Stein et al., 2003). Such rearrangements are suggested to occur
more commonly than interchromosomal rearrangements because they
require fewer DNA breakpoints and because conformation of the nuclear
scaffold could maintain associations between local regions. Stein et al.
(2003) also found that rearrangements were more common in the auto-
somes than in the X chromosome, and more frequent in the chromosomal
arms than centres. The finished genome will reveal whether a similar
pattern is true for H. contortus.
4. GENOME CONTENT
4.1 Gene number, size and structure
The apparent differences in genome content between H. contortus iso-
lates (Table 2) are expected to be largely due to technical differences in
genome assembly and annotation, although there may be some between-
isolate and between-individual biological variation. Both assemblies were
relatively fragmented at the time of publication and are known to contain
some split genes (ie, single genes spanning unassembled contigs annotated
as multiple genes) and some haplotypic sequences. Coverage of the coding
portion of the genome is comprehensive in both H. contortus assemblies,
with 91e92% of core eukaryotic genes (Parra et al., 2007) represented, so
the number of predicted genes is expected to be an upper limit and may
decrease slightly as assembly improves. The MHco3.ISE genome encodes
a smaller number of genes, with longer transcripts (of a comparable size to
C. elegans), so it will be used for between-species comparisons of gene struc-
ture in this section.
The H. contortus genome encodes a remarkably similar number of genes
to C. elegans, despite the striking difference in genome size. Other strongylid
genomes encode more widely varying numbers of genes (eg, 30% difference
between hookworms; although this may be at least partly due to different
methods of gene prediction). Gene number does not appear to correlate
with genome size in the Strongylida, but may do so within genera (this is
the case in members of the Caenorhabditis genus; Fierst et al., 2015). It is
notable that both lungworm genome assemblies (D. viviparus and A. canto-
nensis) are smaller than those for other strongylids and have fewer gene
models, although both of these assemblies are relatively fragmented, so it
is unclear whether this represents a biological difference between strongylid
groups or a technical artefact. However, relative to C. elegans, gene density is
consistently lower (less than half) in all strongylids.
While the average transcript length is similar in H. contortus and C. elegans,
the average gene length is more than double in the parasite. Comparison of a
subset of one-to-one orthologs suggests that this is due to longer introns and a
higher number of introns per gene. Although less so than in H. contortus, the
average gene length in the hookworm genomes is significantly increased
relative to C. elegans, again due to an expansion of intronic sequences;
A. ceylanicum has more introns per gene, while N. americanus has larger introns.
Notably, O. dentatum has a smaller average gene length than C. elegans,
582 R. Laing et al.
resulting from a smaller number of similarly sized introns. While differences in

gene content, such as expansion of particular genes and gene families with
long introns, may affect these genome-wide averages e and these metrics
in draft genomes are sensitive to technical variation e there is clearly a trend
for intronic expansion in the hookworms as well as H. contortus.
The significance of this intronic expansion is unknown. Although a
greater number of introns might promote protein diversity through alterna-
tive splicing, it is not clear what advantage large introns would confer on H.
contortus, particularly if they result in greater time and energy costs for tran-
scription (Castillo-Davis et al., 2002). Introns do play an integral role in the
regulation of gene expression in other organisms and often contain small
regulatory RNAs or binding sites for functional elements (Hube and
Francastel, 2015), but the link between gene expression and intron size or
structure in Haemonchus (or any other parasitic nematode) has not been
explored. However, a large fraction of transposable elements (TEs) and
repetitive DNA are also found in introns (Palazzo and Gregory, 2014)
and, thus, intron expansion could equally reflect relatively unconstrained
evolution.
4.2 Orthology and gene family evolution

Nematodes share a core set of highly conserved genes, enriched for roles in
developmental processes and signalling pathways. However, despite their
closer phylogenetic relationship and parasitic lifestyles, the number of
one-to-one orthologs shared by H. contortus and the strongylid parasites is
comparable with those shared with C. elegans. For example, Schwarz et al.
(2015) report that H. contortus shares 5268 strict orthologs with A.
ceylanicum and 4576 with C. elegans, although the precise numbers vary
with the dataset and analysis method used. These differences may partly
reflect the draft nature of the parasite genome assemblies, confounding
the detection of single-copy orthologs that can be more easily identified
in C. elegans, but it is also indicative of a significant degree of gene family
diversity among members of the Strongylida. This diversity is highlighted
by the marked difference in gene number between the two hookworm
genomes and is consistent with reports of large-scale duplication events,
lineage-specific amplifications and gene losses that appear to be widespread
elsewhere in the phylum Nematoda (Markov et al., 2015). Such differences
are even apparent between the annotated gene models for the two different
H. contortus isolates (Fig. 4) e while most genes can be assigned to gene
families shared by the two gene sets (and with C. elegans), few of them are
single-copy orthologs, suggesting that many gene families are differently

annotated in the two assemblies. At the current state of completion of the
two published assemblies, it is difficult to assess how much of the variation
in gene content might be due to genuine genetic differences between
isolates, to chance assembly of different haplotypic alleles in the two assem-
blies or to differences in the gene-finding approaches used.
Recent analysis of metabolic pathways and enzyme abundance in free-
living and parasitic nematodes across the phylum did not find any
completely conserved parasitism pathways, suggesting substantial divergence
and adaptation to particular lifestyles and niches (Tyagi et al., 2015b).
Clearly, similar life traits can be underpinned by different genomic adapta-
tions, and it appears that such ‘convergent’ rather than orthologous evolu-
tion (Zarowiecki and Berriman, 2015) may be common in nematodes,
particularly when comparing related groups of species that are adapted to
live in different hosts, such as H. contortus and the hookworms.
Parasitic lifestyles theoretically afford the opportunity for genome
simplification, due to metabolic provision by the host. In nematodes
without free-living life stages, such as Trichinella spiralis and Brugia malayi,
as well as in cestodes and trematodes, host metabolic provision appears to
have resulted in a considerable loss of biosynthetic pathways (Tyagi et al.,
2015b; Zarowiecki and Berriman, 2015). However, this does not appear
to be a feature of the H. contortus genome. While the complement of genes
involved in amino acid and carbohydrate metabolism appears to differ
between H. contortus and C. elegans (see Laing et al., 2013), no large-scale
losses of biosynthetic pathways are apparent. As proposed by Blaxter
(2003), nematodes with both free-living and parasitic life stages probably
cannot simplify their genomes. Further, as they require additional capabil-
ities for host infection, migration, nutrient salvage and immune avoidance,
their genomes may need to be more complex than their pre-parasitic
ancestors.
For example, the H. contortus proteome is significantly enriched for
proteases and aminopeptidases, which are required for tissue invasion, hae-
moglobin digestion, anticoagulation and modulation of host immune
response. Large expansions are seen in the cathepsin protease families,
involving both the cathepsin D aspartic proteases (83 genes) and the
cathepsin B cysteine proteases (63 genes). Phylogenetic analysis with other
clade V nematodes shows that these expansions form large monophyletic
groups, including six distinct clusters of cathepsin D proteases and three
distinct clusters of cathepsin B proteases (Laing et al., 2013). The magnitude
584 R. Laing et al.
of the expansion in H. contortus is striking, even relative to the hookworms,

and may reflect a requirement to digest particularly large volumes of host
blood. However, the substantial number of cathepsin B cysteine (113) and
cathepsin D aspartic (54) proteases in the non-blood-feeding strongylid
O. dentatum highlights their diversity of function and suggests a role for other
selective forces in driving protease gene family evolution, such as host im-
mune selection.
Other gene families that have expanded in strongylid nematodes are less
well characterized, such as the ASPs (activation-associated secreted proteins,
also known as SCP/TAPs proteins). These encode cysteine-rich products of
unknown function, but their expression is upregulated on host infection
and, in the hookworms, on exposure to serum and 37 C (Hawdon et al.,
1996, 1999). The C. elegans genome encodes 35 ASPs, but the family is
significantly expanded in the parasites with 161 ASPs in H. contortus, 137
in N. americanus, 284 in O. dentatum and a remarkable 432 in A. ceylanicum
(Schwarz et al., 2015; Tyagi et al., 2015a). Schwarz et al. (2015) describe two
other intriguing gene families: one group of 24 strongylid-specific genes
termed SL4Ps, and another (SCVPs) which is greatly expanded in strongyl-
ids; both sets of genes are predicted to encode secreted proteins of unknown
function.
However, the H. contortus genome also encodes a number of gene fam-
ilies that are notably conserved relative to C. elegans. These include various
chemoreceptors and detoxification enzyme systems. For example, the
C. elegans genome is significantly enriched for cytochrome P450s (CYPs),
a large group of enzymes that metabolize endogenous and exogenous
toxins. This gene family has been characterized in H. contortus, due to its po-
tential role in anthelmintic metabolism (Laing et al., 2015). Although the
majority of CYPs with endogenous roles appear to have one-to-one ortho-
logs, H. contortus lacks the characteristic large clusters of tandem gene dupli-
cations seen in CYP families with exogenous roles in C. elegans.
Comparative analysis with the CYP family in other strongylid nematodes
suggests a similar trend (Fig. 5), although small clusters of strongylid-specific
CYPs are apparent. These include a small group of parasite CYPs (four in
H. contortus and O. dentatum, six in A. ceylanicum and two in N. americanus)
branching with the C. elegans CYP34 and CYP35 families, members of
which have a putative role in metabolizing anthelmintic compounds (Laing
et al., 2010). The necromenic clade V insect parasite Pristionchus pacificus has
an extremely expanded family of detoxification enzymes, including 198
CYPs, which had been hypothesized to represent a preadaptation to
Figure 5 The cytochrome P450 family in Caenorhabditis elegans, Haemonchus contor-

tus, Necator americanus, Ancylostoma ceylanicum and Oesophagostomum dentatum.
Neighbour-joining tree of predicted polypeptide sequences encoding Interpro P450
domain (IPR001128), with Ascaris suum CYP as an out-group.
parasitism (Dieterich et al., 2008). However, in the CYP family at least, the
relatively small numbers in H. contortus, the strongylid parasites of humans
and the more closely related insect parasite H. bacteriophora suggest that
this is not the case.
Major aims of sequencing the H. contortus genome were to better
understand anthelmintic resistance and to develop novel methods of parasite
control. Most of our understanding of drug targets and modes of action have
come from studies in C. elegans, but important differences in gene families
encoding anthelmintic targets were identified in the H. contortus genome.
Notably, H. contortus lacks an ortholog of the major target of ivermectin
in C. elegans, glc-1, which encodes a glutamate-gated chloride channel
586 R. Laing et al.
(GluCl) subunit (Dent et al., 2000; Ghosh et al., 2012). However, the
H. contortus genome encodes two alternative subunits, glc-5 and glc-6, which
are absent from C. elegans, in addition to orthologs of glc-2, glc-3, glc-4, avr-14
and avr-15. Interestingly, heterologous expression of H. contortus glc-6 can
rescue the ivermectin resistance phenotype of a C. elegans GluCl mutant
strain, suggesting that this gene not only encodes a GluCl that responds to
the drug but can replace the function of nonorthologous C. elegans genes
(Glendinning et al., 2011). This appears to be a remarkably divergent
gene family in strongylid nematodes, given its role in nervous system func-
tion, with only 3 family members reported in N. americanus (homologues of
glc-2, glc-3 and avr-14) and 10 family members described in A. ceylanicum
(including homologues of glc-2, avr-14 and avr-15). However, there are
also clear between-species differences in the nicotinic acetylcholine receptor
family (Neveu et al., 2010), which are targets of levamisole and monepantel,
and in the b-tubulin family (Saunders et al., 2013), which includes the major
target of the benzimidazoles.
The P-glycoproteins (PGPs) are members of the ATP-binding cassette
(ABC) transporter family and have been implicated in resistance in
H. contortus and related parasitic nematodes to various classes of anthelmintic
(reviewed by Ardelli, 2013; Kotze et al., 2014). The H. contortus pgp family
differs significantly from C. elegans; there are 10 pgp genes in the parasite
relative to 14 in C. elegans, and species-specific gene duplications appear
to have occurred frequently (Laing et al., 2013). For example, gene dupli-
cations in C. elegans (pgp-3 and pgp-4; pgp-12, pgp-13 and pgp-14) correspond
to single genes in H. contortus, while two paralogous copies of C. elegans pgp-
9 are present in the parasite genome. Caenorhabditis elegans pgp-5, pgp-6, pgp-7
and pgp-8 are clustered in the genome and have no orthologs in H. contortus,
while the parasite genome encodes two genes, Hco-pgp-16 and Hco-pgp-17,
which are absent from C. elegans (Fig. 6).
While the pgp families in A. ceylanicum, N. americanus and O. dentatum are
less well characterized than in H. contortus, preliminary analysis reveals some
clear trends. First, these pgp genes tend to branch with individual members of
the C. elegans pgp family, but form distinct strongylid clusters, including
H. contortus pgps. In A. ceylanicum and N. americanus, single-copy orthologs
could be detected for almost all H. contortus pgps, including Hco-pgp-16
and Hco-pgp-17, which are absent from C. elegans. The ABC family genes
of O. dentatum were very fragmented and only two partial length pgps
were sufficiently complete for analysis, but these fit the same pattern,
branching with H. contortus Hco-pgp-3 and Hco-pgp-10. As is the case for
Figure 6 The P-glycoprotein family in Caenorhabditis elegans, Haemonchus contortus,

Necator americanus, Ancylostoma ceylanicum and Oesophagostomum dentatum. Neigh-
bour-joining tree of predicted polypeptide sequences, with Ascaris suum PGP as an out-
group.
H. contortus, no A. ceylanicum, N. americanus or O. dentatum orthologs were

detected for the C. elegans pgp-5, pgp-6, pgp-7 and pgp-8 cluster. The H. con-
tortus tandem duplication of Hco-pgp-9 was not detected in the other parasite
draft genomes, but N. americanus has a tandem duplication of pgp-10. The
A. ceylanicum and N. americanus genomes encode a pgp gene that is not present
in H. contortus or C. elegans, but branches with pgp-12, pgp-13 and pgp-14.
4.3 Operons and trans-splicing

Operons are clusters of 2e8 genes, which are co-transcribed from the same
promoter. Around 17% of C. elegans genes are in operons (Allen et al.,
588 R. Laing et al.
2011), and 23% of them appear to be conserved in H. contortus (Laing et al.,

2013). A further 10% appear to be partially conserved, ie, at least two ortho-
logs are present in the correct order and orientation, but one or more of the
genes appear in a different order or in the opposite orientation in H. contortus
compared with C. elegans, or are missing. This is similar to findings in N.
americanus, where 28% of C. elegans operons appear to be conserved (Tang
et al., 2014).
In C. elegans, functional constraints are thought to maintain operonic
intergenic distance at around 100 bp, although a small number of operons
with much larger intergenic distances exist (Morton and Blumenthal,
2011). However, the average intergenic distance in putative H. contortus
operons is nearly 10-fold greater (Laing et al., 2013). For this analysis,
nine putative H. contortus operons with intergenic distances of >10 kb
were excluded on the assumption that they represented conservation of
microsynteny only, but a more recent comparison with putative operons
in N. americanus (average intergenic distance of 9141 bp; Tang et al.,
2014) suggests they should not be ruled out on this basis.
Polycistronic mRNAs transcribed from operons are resolved by 50 trans-
splicing with spliced leader sequences (SL1 and SL2); usually, the first gene is
trans-spliced with SL1, and the downstream genes are trans-spliced with SL2.
In H. contortus, the first genes in all putative operons are SL1 trans-spliced,
but less than half of the downstream genes appear to be SL2 trans-spliced;
of these genes, the majority are also SL1 trans-spliced. In C. elegans, operons
with downstream SL1 trans-splicing and longer intergenic distances often
contain an extra promoter in the intercistronic region (Blumenthal et al.,
2015), but the degree to which this is true of H. contortus operons is currently
unknown.
4.4 Repetitive sequence and mobile elements

As described in Section 3, strongylid genomes contain large amounts of
repetitive sequence, which greatly influences genome size. Repetitive
sequences include TEs, such as retrotransposons and DNA transposons,
and various inverted, tandem and dispersed repeats. The H. contortus genome
contains 2e2.5% retrotransposons and 2.1e5% DNA transposons (McMas-
ter and MHco3.ISE, respectively) and transcriptomic data suggest that active
transposition is occurring. Many TE insertions will have no impact on fitness
and may accumulate unnoticed, but the large size and fecundity of
H. contortus populations should promote rapid selection for any TE insertions
that are beneficial and against any that are deleterious. In light of the major
expansions seen in various gene families, and the propensity of H. contortus to

rapidly adapt to different hosts and environments (Gilleard, 2013), it is
tempting to speculate that active transposition could be a significant driver
of genetic diversity in this species.
Numerous tandem repeats are found throughout the H. contortus
genome. Although their function and derivation are largely unknown, their
high mutation rate makes them particularly useful for resolving relationships
among different populations. Microsatellites are repetitive sequences
composed of short tandem repeats and have provided extremely valuable
tools for population-genetics studies and fingerprinting of laboratory isolates
(Redman et al., 2008b). In H. contortus, microsatellites are often found to be
associated with one or more copies of a downstream repeat element,
HcRep, which is transcribed, but is of unknown function (Callaghan and
Beh, 1994; Hoekstra et al., 1997; Laing et al., 2011).
5. FUTURE DIRECTIONS
In recent years, there has been an exponential increase in the availability
of draft nematode genomes. For example, the Helminth Genomes Initiative is
a joint venture by the Wellcome Trust Sanger Institute and the Genome Insti-
tute at Washington University, aiming to generate genome sequences for the
parasitic helminths that have the greatest impact on human health and are
responsible for diseases of veterinary and agricultural importance. The inten-
tion is to rapidly produce draft data for 50 helminths, to complement efforts
to generate high-quality reference genomes for exemplars of major parasite
groups and for the most important pathogens. These data are all available on
WormBase ParaSite. Meanwhile, there have been major advances in the func-
tional annotation of genomes, with comprehensive transcriptome data sets
now available for many helminth species (http://nematode.net). Together,
the increasing richness of both genomic and transcriptomic resources for
strongylid nematodes provides an unprecedented opportunity to better un-
derstand the evolution and genetic basis of important traits such as pathoge-
nicity and drug resistance in H. contortus and related parasites.
The falling price and increasing throughput of sequencing technology
has driven the availability of draft genomes for an increasing number of
strongylid species, and is also opening up the prospects of sequencing of
multiple individuals and undertaking genomic analysis of H. contortus popu-
lations. There is some evidence that H. contortus shows particularly high
590 R. Laing et al.
levels of genetic diversity, with a high density of SNP and small indel vari-
ants (Redman et al., 2008a), presumably driven by high population sizes.
Genome diversity data, combined with the improved genome assembly,
should shed light on this, and on other aspects of the diversity, epidemiology
and genetics of H. contortus. This aspect is further explored in Chapter
“Genetic Diversity and Population Structure of Haemonchus contortus” by
Gilleard and Redman (2016), of this volume.
In both H. contortus draft genome papers, and the majority of parasitic
nematode genomes published since, the focus lies almost entirely on the
protein-coding fraction of the genome. However, there is increasing interest
in the important roles performed by nonprotein-coding sequences (Gerstein
et al., 2010), including those that are transcribed into functional RNAs (such
as tRNAs, rRNAs, small noncoding RNAs and long noncoding RNAs) and
regulatory sequences such as transcription start sites and transcription factor
binding sites. For example, microRNAs are small noncoding RNAs, which
regulate post-transcriptional gene expression. Nearly 200 microRNAs have
been identified in the H. contortus genome, many of which appear to be
unique to this species and may reflect adaptations to different environments
and lifestyles (Winter et al., 2012). Their differential expression between the
L3 and adult life stages suggests involvement in developmental gene regula-
tion, a function that is well characterized in C. elegans (Karp et al., 2011), and
a recent study highlights the upregulation of a single H. contortus miRNA in
two ivermectin-resistant laboratory isolates and two ivermectin-resistant
backcross isolates (Gillan and Devaney, in preparation).
The wealth of genomic, transcriptomic and epigenomic data now avail-
able for C. elegans via modENCODE (Brown and Celniker, 2015) provides
a compelling argument for a broader view of functional content in the
H. contortus genome and in related parasites with reference genomes.
Comparative analysis with the C. elegans data may identify many conserved
aspects of genome organization and function, but it should also be possible
to apply similar experimental approaches (eg, CHIP-seq for genome-wide
profiling of histone modification or of proteins interacting with DNA
(Landt et al., 2012)), albeit on a smaller scale, to study parasites directly.
One epigenetic process for which C. elegans data cannot provide a model
is DNA methylation, as C. elegans apparently completely lacks 5-methyl
cytosine (Simpson et al., 1986). However, this methylation mark present
in the clade I parasite T. spiralis varies through the life cycle (Gao et al.,
2012) and is directed by small RNA molecules (Sarkies et al., 2015). The
presence of genes encoding the cytosine methylation machinery appears
to vary across the nematode phylum (Gutierrez and Sommer, 2004; Was-
muth et al., 2008), but the pathway is conserved in another (distantly related)
parasite of clade I, Romanomermis culcivorax (see Schiffer et al., 2013). The
discovery of adenine methylation in C. elegans (see Greer et al., 2015) raises
the possibility of an alternative epigenetic mechanism in parasitic nematodes,
but the role of DNA methylation and its distribution in other nematodes is
currently unknown.
The available genomes of H. contortus are already enabling a wide range
of exciting molecular research on this species, and a ‘finished’ reference
H. contortus genome is now imminent. Although species-specific adaptations
must be considered, H. contortus is well placed as a parasite model for mem-
bers of the order Strongylida. Therefore, the availability of a reference
genome, combined with draft sequences for related species of human and
veterinary importance, will allow researchers to undertake more detailed
genetic and genomic analyses than have been possible previously. This
comparative approach is likely to be crucial for our understanding of impor-
tant traits, such as anthelmintic resistance, which, in light of the size and
complexity of strongylid genomes, may require whole genome and genetic
mapping approaches to solve.
ACKNOWLEDGEMENTS
JAC, AM, NH and AT are funded by the Wellcome Trust through their core support of the
Wellcome Trust Sanger Institute (grant 098051). Improvement of the H. contortus genome is
funded by Wellcome Trust grant 098051 and a BBRSC Strategic Lola grant (BB/M003949/
1). RL acknowledges funding from the same BBSRC Strategic Lola and from the Scottish
Government through the Strategic Partnership for Animal Science Excellence (SPASE).
JG acknowledges support from the Canadian Institutes of Health Research (CIHR) (grant
230927). The authors thank Robin Beech for manual curation of the H. contortus P-
glycoprotein family; Guillaume Sallé, Stephen Doyle, Hayley Bennett and Collette Britton
for comments on this manuscript and Matthew Berriman for his continued support of the H.
contortus genome project.
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CHAPTER FOURTEEN
Functional Genomics Tools for

Lessons From Other Helminths
C. Britton1, B. Robertsa, N.D. Marks
1
Corresponding author: E-mail: Collette.Britton@glasgow.ac.uk
Contents
1. Introduction 600
2. Gene Silencing by RNAi 601
2.1 RNAi studies in H. contortus 601
2.2 RNAi pathway genes 603
2.3 Successful RNAi in plant parasitic nematodes and in trematodes 605
3. How to Progress RNAi as a Functional Genomics Tool for H. contortus 606
3.1 Optimizing delivery of RNA 607
3.2 Developmental stages suitable for RNAi 609
4. Transgenic Approaches to Identify Essential Gene Function 611
5. Functional Genomics Using CRISPR/CAS Genome Editing 613
References 617
Abstract
The availability of genome and transcriptome data for parasitic nematodes, including
Haemonchus contortus, has highlighted the need to develop functional genomics tools.
Comparative genomic analysis, particularly using data from the free-living nematode
Caenorhabditis elegans, can help predict gene function. Reliable approaches to study
function directly in parasitic nematodes are currently lacking. However, gene knock-
down by RNA interference (RNAi) is being successfully used in schistosome and
planarian species to define gene functions. Lessons from these systems may be applied
to improve RNAi in H. contortus. Previous studies in H. contortus and related nematodes
demonstrated reliable RNAi-mediated silencing of some genes, but not others. Current
a
Present address: Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow,
United Kingdom
j
ISSN 0065-308X
600 C. Britton et al.
data suggest that susceptibility to RNAi in these nematodes is limited to genes

expressed in sites accessible to the environment, such as the gut, amphids and excre-
tory cell. Therefore, RNAi is functional in H. contortus, but improvements are needed to
develop this system as a functional genomics platform. Here, we summarize RNAi
studies on H. contortus and discuss the optimization of RNA delivery and improvements
to culture methods to enhance larval development, protein turnover and the induction
of phenotypic effects in vitro. The transgenic delivery of RNA or dominant-negative gene
constructs and the recently developed CRISPR/Cas genome-editing technique are
considered as potential alternative approaches for gene knockout. This is a key time
to devote greater effort in progressing from genome to function, to improve our under-
standing of the biology of Haemonchus and identify novel targets for parasite control.
1. INTRODUCTION
Genome and transcriptome data are available for an increasing number
of nematode species, including Haemonchus contortus (see Laing et al., 2013;
Schwarz et al., 2013). These data have significantly advanced our knowledge
of nematodes at the molecular level, and also aid genome assembly and
annotation for related species. The full complement of genes and their
expression patterns should soon be known for most major parasitic
helminths. However, there is an urgent need to develop effective tools to
fully exploit these data, in order to better understand parasite development
and hosteparasite interactions. In addition, by identifying essential genes,
functional genomic tools will provide a rational approach for development
of novel control strategies. For parasites such as H. contortus, where drug
resistance is a serious threat, identifying alternative drug targets and potential
vaccine antigens is an increasing priority.
The importance of sequence data in progressing parasite research and un-
derstanding parasite biology has been debated (Viney, 2014; Wasmuth,
2014). Comparative analysis across both distant and closely related species
is a very useful first step in determining potential gene function. Where
genes are not widely conserved and have no characterized functional
domains, identifying their presence in related species with similar life cycles
or similar niches within the host can help give an indication of possible func-
tion. Transcriptome information can identify sets of genes switched on in
particular life-cycle stages and, combined with proteomic data and identifi-
cation of predicted signal peptides or transmembrane domains, can add to
our understanding of potential gene function. However, as discussed by
Wasmuth (2014), genomics is only part of the solution; what is required
to significantly advance new therapeutic design is the development of
Functional Genomics Tools for H. contortus 601
reliable functional genomics platforms. Reports of identifying essential gene

function in schistosome parasites using RNA interference (RNAi) (Guidi
et al., 2015; Patocka et al., 2014) are very encouraging and should stimulate
a concerted effort to improve gene silencing approaches for H. contortus and
other nematodes. This chapter reviews RNAi studies on H. contortus and
considers current limitations and possible improvements to this technique.
In addition, progress on alternative and stable gene-editing approaches using
viral integration and the CRISPR/Cas system are discussed.
2. GENE SILENCING BY RNAi

2.1 RNAi studies in H. contortus
RNAi-mediated gene silencing was first characterized in the free-
living nematode Caenorhabditis elegans (see Fire et al., 1998). It is thought
to act as a defence mechanism to control transposons and double-stranded
RNA (dsRNA) viral infections, a theory supported by the observation that
C. elegans RNAi mutants show increased susceptibility to Orsay virus
infection (Felix et al., 2011). RNAi in C. elegans was initially achieved
following injection of dsRNA into different regions of adult hermaphro-
dite worms and monitoring effects on developing F1 progeny (Fire
et al., 1998). Subsequent studies showed that soaking in dsRNA solution
(Tabara et al., 1998) or feeding worms with bacteria expressing dsRNA
(Fraser et al., 2000) were also effective. The success of RNAi in C. elegans
led to adaptation of this approach to parasitic nematodes. The first demon-
stration of RNAi-mediated gene silencing in a parasitic nematode was in
the gastrointestinal (GI) nematode Nippostrongylus brasiliensis. This was
achieved by soaking adult worms in dsRNA to acetylcholinesterase
(AChE) genes, which resulted in a specific reduction in AChE activity
(Hussein et al., 2002). Applying RNAi to other nematodes followed this
initial work and, encouragingly, phenotypic effects were reported in the
filarial nematodes Brugia malayi (see Aboobaker and Blaxter, 2003) and
Onchocerca volvulus (see Lustigman et al., 2004) and the GI nematode Ascaris
suum (see Islam et al., 2005), all achieved by soaking worms in dsRNA
solution.
Subsequently, RNAi was tested in H. contortus. The first study showed
successful knockdown of two b-tubulin genes (tub 8e9, also referred to as iso-
type-1, and tub 12e16, isotype-2) in third stage larvae (L3s) and adult worms,
following soaking in dsRNA for up to 6 days (Kotze and Bagnall, 2006).
Treated L3s showed a specific decrease in the level of target gene transcript,
demonstrated by qRT-PCR, and a reduced ability to migrate through a filter
(20 mm) and to develop to the fourth-stage larvae (L4s) in vitro. We aimed to
determine whether gene knockdown as well as phenotypic effects could be
observed in H. contortus by selecting a number of genes, the homologues of
which gave obvious RNAi phenotypes in C. elegans. Of 11 genes targeted
in the L3 stage by soaking in dsRNA, only two were effectively silenced
(Geldhof et al., 2006). These were b-tubulin isotype-1, consistent with the
findings of Kotze and Bagnall (2006), and sec-23, a secretory pathway gene.
No other genes showed a reduction in mRNA level, and no phenotypes
were observed. This was the first indication that H. contortus was not as
amenable to RNAi as C. elegans. A study of N. brasiliensis using various deliv-
ery methods (Selkirk et al., 2012) also reported a failure to silence genes, other
than the originally tested AChE transcripts. It is now recognized that even
among Caenorhabditis species, C. elegans is unique in its sensitivity to RNAi
(Felix, 2008). This information highlights the challenge in trying to apply
RNAi to define gene function in H. contortus and other nematodes.
We examined why some genes in H. contortus could be silenced, while
others were seemingly refractory (Samarasinghe et al., 2011). Genes were
selected for RNAi targeting based on their level of expression, from available
expressed sequence tag (EST) data, or on site of expression, from published
data or location of homologous genes in C. elegans. By soaking exsheathed
L3s in dsRNA, 11 genes were targeted and transcript knockdown examined
72 h later by semiquantitative PCR. This study showed that four of six genes
putatively expressed in the intestine, amphids or excretory cell were
effectively and reproducibly silenced. In contrast, genes selected based on
transcript abundance were not susceptible to silencing. Although only a
small number of genes were tested, the data indicated that genes expressed
in tissues accessible to the external environment were more likely to be
silenced, which most likely reflects better uptake of dsRNA into these cells.
In an extensive study by Zawadzki et al. (2012) reduced levels of a num-
ber of H. contortus transcripts were reported following RNAi, including b-
tubulin, tropomyosin and ubiquitin. This study compared different routes to
deliver RNA and is discussed further in Section 3.1. It was concluded that
the feeding of pre-parasitic stages on bacteria expressing dsRNA was the
most effective route for inducing phenotypic effects in H. contortus.
A number of other factors may influence RNAi success. In a detailed
study of RNAi in Ostertagia ostertagi, a GI nematode of cattle, variability be-
tween different batches of dsRNA was a contributory factor (Visser et al.,
2006). Work on a related nematode, Teladorsagia circumcincta, identified that

the age of infective L3s used for soaking influenced their RNAi sensitivity.
Tzelos et al. (2015) demonstrated the effective silencing of two genes encod-
ing activation-associated secreted proteins (ASPs) using freshly collected
T. circumcincta L3s. However, larvae stored for several months were refrac-
tory to RNAi. Whether larval age influences sensitivity of H. contortus to
RNAi has not been examined in any detail. However, using different
batches of dsRNA and larvae stored for different times, transcripts encoding
b-tubulin and H11 aminopeptidase were significantly reduced in all studies
that we carried out (Geldhof et al., 2006; Samarasinghe et al., 2011).
Despite the apparent limited uptake of dsRNA by H. contortus infective
L3s and lack of observable phenotypes in vitro, we were able to demonstrate
a significant detrimental effect of RNAi on H. contortus development in
sheep. Importantly, soaking exsheathed L3s in dsRNA to a gene encoding
aminopeptidase H11 (a key vaccine component for H. contortus; see Smith
et al., 1993) prior to infection resulted in a 57% reduction in faecal egg count
and a 40% reduction in infection intensity compared with L3s soaked in
control C. elegans dsRNA (Samarasinghe et al., 2011). This study demon-
strated that at least for H11, sufficient RNA was taken up to cause stable
gene knockdown, the effects of which were evident in vivo. It also provided
proof of principle that RNAi is a valid approach to identify essential gene
function. While effects of gene knockdown can be monitored in vivo,
the aim is to build on data from H. contortus and other helminths to improve
RNAi and develop this technique as a reliable in vitro screening platform.
2.2 RNAi pathway genes

The genes required for effective RNAi have been extensively studied in
C. elegans (see Grishok et al., 2001). In C. elegans, dsRNA enters cells via
the SID-1 transmembrane transporter and is bound by a protein complex
containing the dsRNA-binding protein RDE-4, an Argonaute-related pro-
tein RDE-1, DRH-1 helicase and Dicer ribonuclease which cleaves dsRNA
into small inhibitory RNAs (siRNAs). These primary siRNAs are loaded on
to the RISC (RNA-induced silencing complex) to trigger target mRNA
degradation through homologous base pairing. RNA-dependent RNA
polymerases (RdRP) RRF-1 and EGO-1 are important for amplifying
the response through the generation of secondary siRNAs. These are
referred to as 22G secondary siRNA, and are characterized by being 22 nu-
cleotides in length, beginning with a guanine and having a 50 triphosphate
(Pak and Fire, 2007).
Homology analyses (by BLAST) have been used to identify RNAi

pathway genes in genomes of parasitic nematodes, including H. contortus.
For most genes in the pathway, putative homologues can be identified,
suggesting conservation of at least some functions (Dalzell et al., 2011;
Geldhof et al., 2007). From the available genome assemblies for H. contortus,
it appears that only two components of the pathway, sid-2 and rde-2, seem to
be absent or are not well conserved (Schwarz et al., 2013). C. elegans SID-2
(systemic interference defective 2) is a membrane transporter required for
dsRNA uptake from the intestinal lumen. Even among Caenorhabditis spe-
cies, SID-2 shows functional differences. In Caenorhabditis briggsae, which
is not sensitive to RNAi by feeding or soaking (referred to as environmental
RNAi), transgenic expression of C. elegans sid-2 enabled environmental
RNAi. However, SID-2 may not be unique in allowing dsRNA uptake,
as has been demonstrated in Drosophila cells, in which components of the
endocytic pathways function in this role (Saleh et al., 2006). Thus, in the
absence of SID-2, alternative endocytic transporters may mediate RNA up-
take in nematodes. We have speculated that this alternative transport system
may occur in H. contortus and might explain the limited repertoire of genes
amenable to silencing (Samarasinghe et al., 2011). RNA may be taken up at
a sufficient level only by cells accessible to the environment, such as the gut,
amphids or excretory cell.
RDE-2 (RNAi defective) is a novel protein that is identified only in
Caenorhabditis species and is involved in RNAi silencing of germline-
expressed genes. RDE-2 functions together with MUT-7 in a large protein
complex and both genes are proposed to be involved in siRNA amplifica-
tion (Tops et al., 2005). As well as a role in the RNAi pathway, RDE-2 and
MUT-7 regulate chromosome integrity and function, with mutations in
either gene resulting in chromosome instability, as indicated by a Him
phenotype (high incidence of males). Mutants of rde-2 and mut-7 also
show high rates of germline transposon mobilization and a lack of
transgene silencing in the germline, processes that are often influenced
by environmental conditions, such as heat or stress (Sundaram et al.,
2008). It is possible that the role of RDE-2 in the C. elegans germline is
either not required in parasitic species or is carried out by another
mechanism. Relevant to this, Piwi-interacting RNAs (piRNAs) are
involved in germline transposon silencing and have been identified in
strongylid (bursate) nematodes, including H. contortus (Winter et al.,
2012). Whether these can compensate for the apparent absence of RDE-
2 is currently unknown.
2.3 Successful RNAi in plant parasitic nematodes and in

trematodes
RNAi has been demonstrated to be effective in plant parasitic nematodes,
including the economically important root-knot nematode Meloidogyne
and the cyst nematodes Globodera and Heterodera (reviewed in Lilley et al.,
2012); dsRNA is delivered by soaking of the juvenile J2 stage, and effects
of gene knockdown are examined following plant infection. Phenotypic
effects, such as reductions in migration, reproduction or male:female ratio
have been reported for a number of genes, including neuronally expressed
flp genes (FMRF-like peptide neurotransmitter) (Kimber et al., 2007).
Similar to our findings for H. contortus, target genes expressed in accessible
sites, such as the amphids, oesophageal gland cells and intestine, seem to
be most affected by RNAi via soaking (Rosso et al., 2005, 2009). Consistent
with this, incubation of J2 larvae with FITC-labelled dsRNA showed signif-
icant uptake into these cells (Urwin et al., 2002). In planta delivery of hairpin
dsRNA expressed under the control of a plant promoter is being increas-
ingly used to identify essential gene function in parasitic nematodes of plants.
An advantage of this approach is the sustained delivery of RNAi and, while
the technology is in the early stages of development, it has potential to
control specific nematode infections in crops (Dutta et al., 2014).
Significant progress has been made in RNAi-mediated gene silencing in
the parasitic trematodes Schistosoma and Fasciola. Combined with the avail-
ability of genome and transcriptome data for these species (Berriman
et al., 2009; Cwiklinski et al., 2015; The Schistosoma japonicum Genome
Sequencing and Functional Analysis Consortium, 2009), RNAi is beginning
to be more widely applied to characterize gene function. Several studies of
schistosome species have demonstrated specific and robust phenotypes
following RNAi silencing of genes encoding known drug targets. The
motility of Schistosoma mansoni schistosomula and adult worms was signifi-
cantly reduced following soaking in dsRNA targeting G proteinecoupled
receptor or nicotinic acetylcholine receptor genes (MacDonald et al.,
2014; Patocka et al., 2014). Similarly, detrimental effects on schistosomula
viability were observed following RNAi of several protein kinase genes
(Guidi et al., 2015). Importantly, the phenotypes produced could be
replicated by chemical inhibition of target activity. The demonstration
that RNAi can replicate the effects of chemical inhibition provides proof
of principle that RNAi screening is a potentially powerful and valid
approach to identify novel drug targets. While RNAi can clearly identify
essential gene function in schistosomes, there remain some challenges in

applying this approach to high-throughput analyses (Guidi et al., 2015; de
Moraes Mourao et al., 2009). Effects were generally not seen until 2e
3 weeks after RNAi treatment, and, therefore, suitable in vitro culture
conditions are required to maintain parasite viability during screening. In
addition, while culture supplementation with red blood cells induces schis-
tosomula development in vitro, worms vary in their rate of development,
and those not feeding are unaffected by RNAi, which makes phenotypic
analysis difficult (Guidi et al., 2015). The optimization of culture conditions
for the induction and monitoring of RNAi effects in flukes and nematodes
should therefore be an important priority.
In the liver fluke Fasciola hepatica, significant, rapid (within 4 h) and stable
(up to 28 days) gene knockdown has been demonstrated for a number of
genes in the newly excysted juvenile (NEJ) stage following RNAi by soak-
ing (McVeigh et al., 2014). However, no obvious phenotypes were
observed for most genes. Detailed analysis using antibodies to encoded target
proteins demonstrated a significant lag time between loss of RNA, indicated
by qRT-PCR, and a reduction in protein levels by immunoblot analysis
(McVeigh et al., 2014). This study indicates the difficulty in identifying
phenotypic effects in Fasciola. It may be possible to overcome this issue using
more physiological culture conditions, to stimulate parasite development
and protein turnover. RNAi targeting of genes switched on during develop-
ment or in response to stress or signalling, where little or no premade protein
is present, may be more likely to result in phenotypic effects. This approach
has been used in copepods to demonstrate the essential role of a heat shock
protein gene (hspb1) in response to increased temperature (Barreto et al.,
2015). Despite the difficulties reported in identifying phenotypic effects,
the demonstration of successful knockdown of a number of genes in trem-
atodes encourages further efforts to optimize RNAi for use in parasitic
nematodes.
3. HOW TO PROGRESS RNAi AS A FUNCTIONAL

GENOMICS TOOL FOR H. CONTORTUS
To date, studies of H. contortus indicate that the RNAi pathway is
functional. However, improvements are required to develop RNAi
technology as a reliable functional genomics platform. Most studies have
identified factors important for successful knockdown in nematodes, such
as site of target gene expression or parasite age or stage, but further effort
is needed to develop a robust system for phenotypic analysis. Lessons learnt

from studies of H. contortus and other helminths can help optimize delivery
of RNA, identify which developmental stages might be best to target and
how phenotypic effects may be induced.
3.1 Optimizing delivery of RNA

RNAi in the schistosomula stage of schistosomes can be achieved by soaking
in RNA solution for 16e24 h or by electroporation. The latter causes some
damage to the parasites, and soaking is therefore favoured. For adult worms,
electroporation is the most effective delivery route for dsRNA or siRNAs
(Krautz-Peterson et al., 2010). For H. contortus, we found that electropora-
tion had a detrimental effect on the viability of larval stages, while soaking in
dsRNA with lipofectin was effective in silencing a number of genes in larval
or adult stages (Geldhof et al., 2006; Kotze and Bagnall, 2006). Surprisingly,
a similar level of b-tubulin gene knockdown was observed when adults of
H. contortus were incubated with ivermectin to reduce pharyngeal uptake,
suggesting that an alternative route, such as transcuticular delivery, may
also be involved (Kotze and Bagnall, 2006). A study of A. suum tested the
injection of dsRNA into the pseudocoelomic cavity of adult worms and
achieved effective knockdown of all eight genes targeted (McCoy et al.,
2015). Target genes expressed in different tissue types, including neuronal
cells, were silenced, and the response spread to different parts of the
worm from the injection site. Injection of dsRNA is feasible in Ascaris
due to the large size of the worm and its capacity for introduced solution.
We have attempted the microinjection of H. contortus L3 and L4 stages as
a possible route to ensure direct delivery into the worms. However, the
difficulty of manipulating these small larvae and the damage caused by injec-
tion, most likely due to high internal pressure, limit the application of this
approach.
In a detailed study by Zawadzki et al. (2012), it was concluded that
feeding H. contortus free-living (L1, L2 and L3) stages on bacteria expressing
dsRNA was effective in inducing phenotypes. Larvae could be maintained
for 6e10 days during which time they were monitored for motility,
development and viability. RNAi targeting of a number of genes affected
viability, which in some cases, but not all, correlated with transcript
reduction. In contrast, electroporation of free-living stages or soaking of
adult worms in dsRNA solution reduced expression of target genes, but
no phenotypes were observed.
Recently, we have examined the potential of bacteria to deliver dsRNA

into the L4 stage of H. contortus. L4 larvae developing in vitro feed and ingest
bacteria, as indicated by uptake of green fluorescent protein (GFP)elabelled
OP50 Escherichia coli. However, it is difficult to control the amount of
dsRNA delivered to worms and the presence of too much bacterial culture
can be detrimental to larval survival, thus hampering phenotypic interpreta-
tion (Britton and Roberts, unpublished data; Zawadzki et al., 2012).
Feeding on bacterially expressed RNA is amenable to genome-wide analysis
and is widely used in C. elegans (see Kamath and Ahringer, 2003). This
technique has also been used successfully to silence genes in the free-living
planarian Schmidtea mediterannea. RNAi by feeding has identified genes
essential for S. mediterannea intestinal function, morphology and viability
(Forsthoefel et al., 2012), and for stem cell regulation and regeneration
(Wagner et al., 2012). However, the difficulties of assessing the quality
and quantity of dsRNA delivered by bacterial feeding led to development
of an improved method, whereby in vitroesynthesized dsRNA is incorpo-
rated into liver extract and fed to planarians (Rouhana et al., 2013). The use
of this approach resulted in robust knockdown with effects observed earlier
than with the previous bacterial feeding method. We and other researchers
have shown that dsRNA is ingested by H. contortus larvae and adult stages,
and is effective at silencing some genes (Geldhof et al., 2006; Kotze and
Bagnall, 2006). Optimization of this delivery method, for example, by
combining it with improved in vitroeculture systems to encourage parasite
feeding and protein turnover, may make this a feasible approach for large-
scale functional analysis.
The incorporation of RNA within nanoparticles has been shown to
enhance uptake by mammalian cells (reviewed in Miele et al., 2012). The
majority of nanoparticles are liposome or polymer based, and several are
being tested in clinical trials for siRNA delivery for tumour therapy (Lee
et al., 2013). Polystyrene-based nanoparticles are taken up by the intestine,
pharynx and gonad of C. elegans (see Pluskota et al., 2009), but have not yet
been tested in H. contortus. Microparticle bombardment may provide an
alternative route for dsRNA delivery. Bombardment with gold particles
coated with DNA is applied to C. elegans to generate transgenic embryos,
in which the delivered DNA is integrated into the genome (Praitis et al.,
2001). Particle bombardment has also been employed to deliver DNA
and RNA into embryos of parasitic nematodes, including Ascaris and Brugia
(Davis et al., 1999; Higazi et al., 2002). In our initial studies (Britton, unpub-
lished data), bombardment of the exsheathed L3 stage of H. contortus with
gold particles proved unsuccessful, most likely due to the difficulty of pene-
trating the L3 cuticle. However, further studies should be carried out to test
whether other stages, such as L4 or adult worms, are more amenable to par-
ticle bombardment.
3.2 Developmental stages suitable for RNAi

Previous studies showed that the pre-parasitic stages of H. contortus and the
related strongylid nematode Trichostrongylus colubriformis are amenable to
RNAi, with phenotypic effects being observed following bacterial feeding
(Issa et al., 2005; Zawadzki et al., 2012). However, identification of genes
with essential roles in the parasitic L3, L4 or adult worms would be prefer-
able for drug target discovery or vaccine design. The L4 and adult worms,
which feed in vitro, may therefore be the most suitable stages for the
optimization of RNAi technology. Relevant to this is the wealth of tran-
scriptomic data now available for H. contortus L3, L4 and adult stages (Laing
et al., 2013; Schwarz et al., 2013). These data can be exploited to identify
genes that are up-regulated during infection and may be essential for feeding
and development. Such genes are likely to be key targets for identifying
RNAi effects. In addition, genes expressed in the gut may be more amenable
to RNAi silencing by soaking or feeding and can be selected from the avail-
able gut transcriptome data (Laing et al., 2013). Genes encoding metabolic
chokepoints, enzymes that catalyse unique reactions, have also been identi-
fied from genome data and are relevant targets for testing induction of
RNAi phenotypes, given their proposed essential roles (Laing et al.,
2013). We have been focussing on a subset of metabolic chokepoint-encod-
ing genes of H. contortus, with the aim of silencing those showing increased
expression from the L3 to L4 stage, based on transcriptome data. However,
no obvious phenotypes have yet been observed.
Adult H. contortus survive for only 2e3 days in vitro, which may limit
identification of gene function. However L4s, collected 7 days post-infec-
tion maintain viability for up to a week. This time frame may be sufficient
for phenotypic effects to be monitored, although it should be considered
that in S. mansoni it took 2e3 weeks for knockdown phenotypes to be
observed (Guidi et al., 2015).
We have been exploring the potential to develop H. contortus L4 in vitro.
This would provide a readily available source from stored L3s without the
immediate need for infected sheep. We have observed significant uptake
of labelled RNA by L4s developing in vitro (Fig. 1B). In addition, initial
findings indicate that in vitro coculture of CO2-exsheathed L3 with
(A) (B)
(C) (D)
Figure 1 Feeding and development of the H. contortus L4 stage maintained in vitro.

(A and B) Uptake of fluorescently labelled siRNA by L4s in vitro. L3 larvae were
exsheathed and incubated in Earle’s balanced salt solution (EBSS)/5% CO2 at 37 C in
the presence of 0.5 mM Cy3-labelled siRNA (Ambion). After 96 h, L3 and L4 stages
were washed three times in PBS and pipetted on to 2% agarose pads on microscope
slides and viewed by bright field (A) or fluorescent (B) microscopy at 40 magnification.
Images were collected using an Axioskop 2 Plus microscope (Zeiss), ORCA-ER digital
camera (Hamamatsu) and Openlab (Improvision) software. Fluorescence was observed
throughout the intestine of L4s. No fluorescence was observed in the L3 stage or in any
stage in the absence of Cy3-labelled siRNA. Larvae are about 700 mM in length. (Repro-
duced from Britton, C., Winter, A.D., Marks, N.D., Gu, H., McNeilly, T.N., Gillan, V., Devaney,
E., 2015. Applications of small RNA technology for improved control of parasitic helminths.
Vet. Parasitol. 212(1e2):47e53. Under Creative Commons license CC-BY). (C and D)
Enhanced development of L4 larvae co-cultured with Caco2 gut epithelial cell line.
(C) Exsheathed L3 larvae were incubated for 7 days in Dulbecco’s modified Eagle me-
dium (DMEM)/10% foetal calf serum/5% CO2 at 37 C in the presence of Caco2 cells. (D)
Exsheathed L3 cultured for 7 days in EBSS/5% CO2 at 37 C. Gut development is evident
in (C) (shown by an arrow).
Caco2 cells and foetal calf serum improves the rate of development from L3
to L4. The developing L4 larvae were viable for up to 3 weeks, although
none developed to adult worms (Fig. 1C). Caco2 cells are a transformed
human gut epithelial cell line and have been used previously to promote
in vitro development and moulting of Trichinella spiralis L1 larvae, with a
small percentage of these developing to adult worms (Gagliardo et al.,
2002). The apparent feeding and growth of H. contortus L4s developing in
vitro suggest that monitoring detrimental effects of gene silencing may be

feasible, although, as observed for F. hepatica, this will depend on protein
turnover. Guidi et al. (2015) noted that RNAi phenotypic effects in
schistosomula were significantly enhanced when red blood cells were added
to in vitro cultures, which enhanced larval activation. Whether red blood
cells or alternative stimuli can enhance H. contortus L4 activation is currently
being examined (Britton, unpublished data). The development of in vitro
culture conditions for nematodes has proved challenging, and progress in
maintaining parasitic stages in more physiological conditions would signifi-
cantly advance the application of RNAi in identifying phenotypic effects.
4. TRANSGENIC APPROACHES TO IDENTIFY

ESSENTIAL GENE FUNCTION
Efficient delivery of RNA into H. contortus, maintaining gene knock-
down and stimulating larvae to develop and turnover protein are currently
key hurdles in the optimization of RNAi. An alternative approach has been
applied in S. mansoni. This study introduced plasmid DNA into cells, not to
silence gene expression but to act as a dominant-negative gene (Liang et al.,
2013). The advantage of this approach is that it does not depend on protein
turnover to observe phenotypes, but on a loss of function of endogenous
proteins through competition for binding partners, target sites or regulators.
Liang et al. (2013) focused on Sm-Mef-2 (myocyte enhancer factor 2), which
encodes a DNA-binding transcriptional activator. These authors showed
that the transfection and expression of an Sm-Mef-2 deletion construct,
which removed the N-terminal transactivation domain, resulted in a
decrease in expression of two wingless-type (wnt) genes, speculated to be
targets of the activator. A decrease in expression of Sm-Mef-2 itself was
also measured, suggesting auto-regulation. Despite the reduced wnt expres-
sion, no phenotypes were observed in schistosomula maintained in vitro.
Nevertheless, this study provided proof of principle that dominant-negative
forms of genes could be introduced and expressed in parasitic helminths to
examine function.
Dominant-negative gene constructs have been used to identify gene
function in protozoan parasites and can also be applied to nematodes
through transgenic expression of mutated genes. The parasitic nematode
systems most amenable to transgenesis are Strongyloides (Li et al., 2006)
and Parastrongyloides (Grant et al., 2006). These species undergo free-living
and parasitic cycles, and transfection is achieved by microinjection of the
free-living adult female worms and monitoring transgene expression in the

F1 progeny and beyond. A significant effort has been made to enhance
transgene expression in these species by incorporating parasite-specific
promoters and 30 -untranslated regions (30 -UTR) to regulate transgene
expression (Grant et al., 2006; Junio et al., 2008; Li et al., 2006). This
approach has been successful in identifying the spatial expression of genes
of interest from promoter-GFP reporter constructs (Junio et al., 2008).
However, using this transient transfection system, silencing of the intro-
duced repetitive episomal arrays seems to be a limiting factor in maintaining
transformed lines. This issue also occurs in other nematodes, including C.
elegans and Pristionchus pacificus. The addition of linearized genomic DNA
to the injection mix increases the frequency of stable expressing lines (Kelly
et al., 1997; Schlager et al., 2009), but this approach has not been extensively
tested in Strongyloides (Lok, 2009).
To study the function and regulation of encoded proteins in Strongyloides
stercoralis, the introduction of mutated gene constructs has also been applied
successfully. Castelletto et al. (2009) demonstrated the importance of phos-
phorylation in regulating the activity and localization of S. stercoralis forkhead
transcription factor-1 (FKTF-1), similar to the regulation of the homologous
DAF-16 transcription factor in C. elegans. Importantly, S. stercoralis larvae
expressing a phospho-null and C-terminal transactivation domain-deleted
version of FKTF-1 failed to undergo pharyngeal and intestinal tissue remod-
elling in preparation for infection. This result suggested competition
between the mutated and endogenous proteins, and demonstrated the value
of dominant-negative gene constructs in defining important functional roles
for proteins such as FKTF-1.
Further applications of transgene injection in Strongyloides have been pro-
posed and could potentially be adapted to other parasitic nematodes (Lok,
2012). They include the introduction of short-hairpin RNA (shRNA)
expression vectors to mediate gene silencing. By combining transgenesis
and RNAi, sustained gene knockdown may be achieved and might allow
the examination of phenotypes when an existing protein is depleted. In
addition, using tissue-specific promoters to drive transgene expression, siR-
NAs can be produced locally to silence genes in specific cell types. This
approach has been used in C. elegans to examine tissue-specific RNAi and
to identify the mechanisms of RNAi silencing and spreading (Timmons
et al., 2003). While being an effective approach for transient analysis, the
lack of stable expression and inheritance of episomally expressed transgenes
in Strongyloides limits extensive functional analysis studies.
The generation of stable, chromosomally integrated transgenes would

permit examination of gene function in more detail over several generations.
Microparticle bombardment has been shown to result in chromosomal inte-
gration in C. elegans. However, the efficiency of transformation by this route
is very low (0.05%) and requires a large number of worms (Praitis et al.,
2001). Transgene integration mediated by lentiviruses, retroviruses or trans-
posons (eg, piggyBac), presents a more efficient delivery route for stable
transgene expression. This approach has been used effectively in schistosome
species for almost a decade (reviewed in Beckmann and Grevelding, 2012;
Morales et al., 2007; Suttiprapa et al., 2012) and has also been demonstrated
in Strongyloides ratti (see Shao et al., 2012).
In a study by Hagen et al. (2014), a novel approach was used to examine
the function of genes highly expressed in the egg stage of S. mansoni. A lenti-
virus construct encoding microRNA-adapted short hairpin RNAs
(shRNAmirs) under the control of the cytomegalovirus promoter was
employed to silence the omega-1 gene. Omega-1 knockdown had no effect
on egg viability, but resulted in a significant reduction in the infiltration of
dendritic cells, T helper cells and B cells and, importantly, a reduction in
granuloma size in a mouse model system. This study reinforced the role
of omega-1 as a key factor in the initiation of granuloma formation. The ad-
vantages of this approach are the increased persistence of gene knockdown,
particularly important for studying effects in vivo and the apparent lack of
off-target effects. The application of stable transgenes to induce sustained
gene silencing throughout the life cycle of H. contortus would provide a
powerful route to identify key molecules involved in parasite survival,
reproduction, pathology and immunosuppression.
5. FUNCTIONAL GENOMICS USING CRISPR/CAS

GENOME EDITING
Since its discovery in 1998, RNAi-mediated gene silencing has been
applied to a wide range of organisms. However, the lack of complete
knockout and potential problem of off-target effects, particularly with
siRNAs in mammalian cells, has encouraged the development of stable
genome-editing approaches. These methods use nucleases to induce
double-stranded DNA breaks that are incorrectly repaired, resulting in small
insertions and deletions at the target site (reviewed in Kim and Kim, 2014).
Zinc finger nucleases and transcription activatorelike effector nucleases
(TALENs) have been applied to a range of organisms. However, in 2013, a
more versatile RNA-guided nuclease system was developed, referred to as the

clustered regularly interspaced short palindromic repeat (CRISPR)/Cas
(CRISPR-associated) system (Gilbert et al., 2013; Mali et al., 2013). The
CRISPR/Cas mechanism was identified in bacteria and archaea as an adap-
tive immune response to foreign DNA (Wiedenheft et al., 2012). DNA from
invading viruses or plasmids is processed into small ‘spacer’ fragments that are
incorporated into a CRISPR site between short repeat sequences in the
genome. Transcription and processing of the repeat-spacer array result in
the production of small CRISPR RNAs (crRNAs) that contain full or partial
spacer sequences which function as single-guide RNA (sgRNA). These
sgRNAs direct Cas ribonucleoprotein complexes to recognize and cleave
the foreign DNA. Although of bacterial origin, the mechanism of this
sequence-specific cleavage works effectively in eukaryotic cells. This
genome-editing mechanism has now been adapted to induce double-
stranded DNA breaks in human cells (Cho et al., 2013a; Mali et al., 2013),
mice (Wang et al., 2013), Drosophila (see Bassett et al., 2013), zebrafish (Chang
et al., 2013), Arabidopsis (Feng et al., 2014) and the nematodes C. elegans (see
Chen et al., 2013; Waaijers et al., 2013) and P. pacificus (see Witte et al., 2015).
With its versatility and increasing use, improvements to CRISPR/Cas meth-
odology, efficiency and specificity are continuing to be reported.
In C. elegans, CRISPR/Cas-induced mutations have been generated
predominantly by injection of plasmids expressing sgRNA and Cas9
nuclease, together with a marker plasmid (GFP or mCherry) into the adult
hermaphrodite gonad using standard injection procedure (Chen et al., 2013;
Waaijers et al., 2013). Target sites are selected within exonic gene sequence
and are required to contain the sequence NGG (protospacer adjacent motif
[PAM] recognized by Cas9) at the 30 end preceded by 19 bases and G at the
50 end (50 -GN19NGG-30 ) (see Fig. 2). Target sites can be selected using on-
line CRISPR design tools (eg, http://crispr.mit.edu) and sgRNA synthe-
sized. Following germline injection, transgenic F1 progeny are maintained
and loss-of-function phenotypes in the developing F2 generation are char-
acterized. Mutation of the specific target gene is confirmed by PCR and
sequencing or restriction enzyme digest analysis (outlined in Fig. 2). While
off-target mutation is reported to be minimal, the use of sgRNAs designed
to different exons of the same gene and backcrossing of mutated strains with
the wild-type C. elegans N2 strain is recommended to confirm phenotypic
specificity.
While transgenic expression of sgRNA and Cas9 can be achieved in C.
elegans using well-characterized promoter sequences, this is more difficult in
(A) (B)
(C)
Figure 2 Outline of CRISPR-Cas9-mediated gene editing in nematodes. (A) Microinjec-

tion of transgenes expressing Cas9, sgRNA to the target gene (eg, dpy-11) and a GFP
marker plasmid. Transgenic F1 progeny expressing GFP are predominantly heterozy-
gous for target gene disruption. Homozygous deletion in the dpy-11 target gene is
identified by the dumpy body morphology in the F2 generation. PCR and sequencing
confirm target gene disruption. Biallelic targeting in somatic cells occurs at a low rate,
with observable phenotypes in the F1 progeny. (B) Gene disruption in germline or
somatic cells is also feasible by direct injection of Cas9 protein and sgRNA, and identi-
fied by phenotype selection and PCR. (C) Example of CRISPR-Cas9 target sequence
showing the protospacer adjacent motif (PAM) (sequence NGG) in red (light grey in
print versions) and the target gene recognition sequence in blue (grey in print versions)
(conforming to the sequence 50 -GN19NGG-30 ). The gene-specific portion of the sgRNA is
identical to the target region, but does not include the PAM sequence. The arrow indi-
cates a possible cleavage site and resultant deletion. Adapted from Kim, H., Ishidate, T.,
Ghanta, K.S., Seth, M., Conte, D. Jr., Shirayama, M., Mello, C.C., 2014. A co-CRISPR strategy
for efficient genome editing in Caenorhabditis elegans. Genetics 197, 1069e1080 and
Farboud, B., Meyer, B.J., 2015. Dramatic enhancement of genome editing by CRISPR/Cas9
through improved guide RNA design. Genetics 199, 959e971.
other nematodes and organisms. To overcome this issue, direct injection of

pre-made sgRNA and Cas9 protein into worms is feasible and has been
shown to be successful in C. elegans (see Cho et al., 2013b). There are several
advantages to this approach that make it attractive for testing in parasitic nem-
atodes: firstly, it does not require generation of new promoter-transgene
constructs and the characterization of active promoters and 30 -UTRs for each
species; secondly, the introduced riboprotein complex acts rapidly, without
the need for transcription. Direct injection of sgRNA and Cas9 protein
was applied to the necromenic nematode P. pacificus to generate a targeted
deletion in the identified dpy-1 gene. Following germline injection, F2 prog-
eny showed the dumpy (Dpy) phenotype associated with loss-of-function
mutation in this gene, and specific gene deletion was confirmed by PCR
and DNA sequencing (Witte et al., 2015). Some differences in outcome
were observed between P. pacificus and C. elegans CRISPR/Cas9 mutation.
For example, co-mutating two genes or two exons of the same gene is feasible
in C. elegans (see Kim et al., 2014), but is far less effective in P. pacificus (see
Witte et al., 2015), suggesting that there is species-specific variation in the
mechanism. Nevertheless, this represents a significant advancement in gener-
ating stable and specific gene deletions in parasitic nematodes. Mutation in
germline cells generated by gonadal injection requires the observation of
F2 progeny to identify homozygous mutants. However, biallelic gene editing
of somatic cells has also been reported in C. elegans (see Cho et al., 2013b) and
in Drosophila (Port et al., 2014). Studies in C. elegans have employed heat-
shock or tissue-specific promoters to induce expression of Cas9 in specific
developmental stages or tissues (http://www.cell.com/cms/attachment/
2018172252/2038345857/mmc2.pdf), allowing the examination of muta-
tion effects in somatic cells. As the complete life cycle of obligate parasitic
nematodes cannot currently be maintained in vitro, the ability to mutate
both gene copies in somatic cells and examine any resultant phenotypes
would be a huge step forward in identifying, characterizing and/or defining
gene function. While significant efforts will be needed to test and optimize
CRISPR/Cas technology, including how best to deliver sgRNA and Cas9,
this approach may revolutionize functional genomics in Haemonchus and other
parasitic species.
It is clear that RNAi-mediated gene silencing is possible in Haemonchus
and related parasitic nematodes. Significant and reproducible knockdown of
specific mRNA transcripts is evident from a number of studies. However,
the application of RNAi as a functional genomics approach to exploit the
genome data and progress understanding of Haemonchus biology requires im-
provements. These enhancements should include a better delivery of RNA
to enable targeting of all genes, not only those expressed in accessible sites,
and enhanced in vitro culture methods to allow parasite development and
protein turnover. Optimization studies are likely to be most applicable to
L4 and adult stages, and initial studies suggest that cell coculture and addition
of activating factors, such as serum or red blood cells, as used for schisto-
somes, may enhance the development of Haemonchus L4s. Identifying
RNAi-mediated phenotypes in vitro is a major hurdle, not only for Haemon-
chus but also for many parasitic helminths. Efforts to establish more physio-
logical conditions for parasite maintenance should be a priority. Importantly,
reductions in worm burden and egg output following RNAi of H. contortus
H11 have been demonstrated in vivo, indicating that once induced, gene
knockdown is sustained and can identify essential gene function.
Better in vitro conditions for larval development would also allow the
exploration of transgenic approaches for RNAi using viral or transposon-
mediated introduction of shRNA expression constructs. Although we
have focussed on RNAi silencing, the introduction of dominant-negative
gene constructs to compete with endogenous gene function and genome
editing via CRISPR/Cas may prove to be effective alternatives.
CRISPR/Cas was only adapted to C. elegans in 2013, and, already there
have been significant advancements made to its efficacy and ease of use.
The successes and lessons learnt from other systems, such as C. elegans and
schistosomes, should enable further efforts to develop functional genomic
tools for Haemonchus. This is an exciting time in parasitic nematode research,
with a wealth of genomic and transcriptomic data available. We need to
progress to the next level and identify gene function as a rational approach
for the design of new intervention strategies.
ACKNOWLEDGEMENTS
Some of the work described here was funded by the European Union FP7 PARAVAC
consortium and by a PhD studentship award from BBSRC/Knowledge Transfer Network
(KTN)/Zoetis. The authors thank Dr Alan Winter (University of Glasgow) for helpful
discussion on the CRISPR/Cas9 mechanism, Dr Roz Laing (University of Glasgow) for
help with transcriptome data and Prof. Dave Knox (Moredun Research Institute) for
many stimulating discussions on RNAi.
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INDEX
‘Note: Page numbers followed by “f” indicate figures, “t” indicate tables, and “b” indicate
boxes’.
A Allele-specific PCR, 418–419

AAD. See Amino-acetonitrile derivatives Alpine chamois (Rupicapra r. rupicapra), 45
(AAD) Alpine ibex (Capra ibex ibex), 45
ABC transporters. See ATP-binding Alternative anthelmintic compounds,
cassette transporters (ABC 210–211
transporters) Amino acid metabolism, 78. See also
AC-5, 373–374 Energy metabolism in nematodes;
Acetate, 75 Nucleic acid metabolism
Acetylcholine (ACh), 83 nematodes, 78
Acetylcholinesterase genes (AChE genes), polyamines, 78–79
601 Amino acids pathophysiological studies,
ACh. See Acetylcholine (ACh) 257
AChE genes. See Acetylcholinesterase Amino-acetonitrile derivatives (AAD),
genes (AChE genes) 196, 400, 411–412, 468
Activation-associated secreted proteins g-Aminobutyric acid (GABA), 71, 83, 409
(ASPs), 538, 584, 602–603 g-Aminobutyric acid-A receptors, 85
ASP3, 372–373 Ammonia excretion, 78–79
ADCC. See Antibody-dependent cell Amplified fragment length polymorphism
cytotoxicity (ADCC) (AFLP), 35–36, 40–41
Additive effect, 504–505 Anaemia assessment, FAMACHA system
Adenosine-tri-phosphate (ATP), 76 for, 186–187
Adult nematodes, 72 Ancylostoma spp., 247
energy metabolism in, 75 A. ceylanicum, 579–580
ATP, 76 Angiostrongylus cantonensis. See Rat
blood of mammals, 75 lungworm (Angiostrongylus
C. curticei, 76–77 cantonensis)
malate dismutation, 75–76 Animal(s)
Pyruvate kinase/PEP carboxykinase age of, 257
ratio, 77 on excellent nutritional level, 269
respiratory chains, 76 on good nutritional level, 269
rhodoquinone, 77 management factors, 184
T. colubriformis, 76–77 on poor nutritional level, 268–269
Adult stages, 436–437 production level, 259–261
Adult worms, 274 Anthelmintic laboratory strains, 53–54
AFLP. See Amplified fragment length AH properties, 244
polymorphism (AFLP) combinations of resources with AH effects
Agroindustrial by-products, exploring combining CT-containing
value of, 324–326 nutraceuticals and synthetic chemical
AH. See Anthelmintic(s) (AH) AH, 326–327
Albendazole, 468–469 combining different CT-containing
Albendazole sulphoxidation, 482–483 resources, 326
625 j
626 Index
Anthelmintic resistance, 399–400, Antibody-dependent cell cytotoxicity

505–506. See also Drug resistance (ADCC), 360
diagnosis of resistance Antibody-secreting cells identification,
molecular-based tests, 418–419 170
in vitro bioassay-based tests for Antigen-presenting cells (APCs), 168–169
resistance, 414–418 Antigen-specific antibodies, 165–166
in vivo resistance tests, 414 Antiparasitic drugs, 466
history, 400–403, 401t–402t APCs. See Antigen-presenting cells (APCs)
mechanisms of resistance, 403–404 Arid climates, 129
AAD, 411–412 Arid regions, 104–105, 121
BZ, 404–406 Arid zones, 220
Closantel, 411 Aromatic benzimidazole derivatives, 470
factors, 403f Ascaris lumbricoides (A. lumbricoides), 39–40
general fitness costs, 412–413 Ascaris suum (A. suum), 39–40, 75–76
Imidazothiazoles, 406–409 Aspartyl proteinases, 377
ML, 409–411 ASPs. See Activation-associated secreted
Anthelmintic treatment, 193 proteins (ASPs)
anthelmintic groups, 194 ATP. See Adenosine-tri-phosphate (ATP)
amino-acetonitrile derivatives, 196 ATP-binding cassette transporters (ABC
benzimidazoles, 194–195 transporters), 89, 497–499, 541,
combination anthelmintics, 197 586
imidazothiazoles, 195 Avermectin family, 473–474
ML, 195–196
organophosphates, 197 B
salicylanilides, 196 Barbervax, 369–370
spiroindoles, 196–197 Benzimidazoles (BZ), 194–195, 398,
substituted phenols, 196 404–405, 468–469. See also
tetrahydropyrimidines, 195 Imidazothiazoles
anthelmintic-resistance management, 198 anthelmintics, 470
anthelmintic choice, 201–204 H. contortus isotype-1 b-tubulin, 405–406
minimizing resistance-selection mechanism of action and of resistance to,
treatment practices, 198–199 405f
prevention of resistant nematodes methylcarbamates, 468–470
introduction, 204–205 resistance, 82–83, 88–89
refugia strategies, 199–201 b-Tubulin genes, 406
Anthelmintic(s) (AH), 437–438, 522–524 Bermuda grass (BG), 311
benzimidazoles, 82–83 Biochemistry of H. contortus and parasitic
choice, 201–202 nematodes, 71–72
broad-spectrum, 202–203 amino acid metabolism, 78–79
combination anthelmintics, 204 drug resistance biochemistry, 87–90
detection of resistance, 202 ecosystems of H. contortus life cycle stages,
long-acting anthelmintics, 203–204 72
and resistance management, 221–222 energy metabolism in nematodes, 74–77
discovery, 430 gene expression in parasitic life cycle
drugs targeting energy, 77 stages, 73–74
selection effect, 46–47 lipid metabolsim, 79–80
underdosing with, 198–199 nervous system in nematodes, 83–87
Index 627
nucleic acid metabolism, 79 cDNA. See complementary DNA (cDNA)

structure and biochemical composition of Cell soma, 81–82
cuticle, 80–81 Census population size, 38
tubulin, 81–83 CF. See Control fed (CF)
Bioclimatographs, 130 Chicory (Cichorium intybus), 270
Biodiversity information, 2–3 Chokepoint
Biogeography, 5 analysis, 540
Bioinformatics functions, 439
bioinformatics-driven approaches, 439 Choline, 84
searches, 439 Chromosomal synteny, 580
Biological control, 208–210 Chromosomally integrated transgenes, 613
Biotransformation, 470 Chronic haemonchosis, 107
Blood-feeding parasites, 411 Cichorium intybus. See Chicory (Cichorium
Bovine lungworm (Dictyocaulus viviparus), intybus)
579–580 Clade V nematodes, 579–580, 583–584
Brugia malayi (B. malayi), 583 Climate change potential effects, 131
BZ. See Benzimidazoles (BZ) Climate impacts integrating historical
perspectives, 19
C behavioural patterns of parasites, 20–21
C. elegans Genome Consortium, 570 perturbation, 22
Caco2 cells, 609–611 seasonal effects, 21
Caenorhabditis briggsae (C. briggsae), 580, Clinical signs of disease, 108, 184–186
604 haemonchosis, 110–111
Caenorhabditis elegans (C. elegans), 33, host animals, 108–109
37–38, 51–52, 525–528, 570, 580 hyperacute cases, 109
gene duplications, 586 Closantel, 411, 472–473, 478
Calcium-activated voltage-gated lipophilicity, 480
potassium channel SLO-1, 87 Clustered regularly interspaced short
Calcium-binding proteins, 530–531 palindromic repeat (CRISPR),
Canarias Hair Breed sheep, 363–364 613–614
Capra hircus. See Domestic goat CRISPR-Cas9-mediated gene, 614–616,
(Capra hircus) 615f
Capra ibex ibex. See Alpine ibex (Capra ibex CRISPR/Cas9 technologies, 546–547
ibex) CRISPR/Cas-induced mutations, 614
Capreolus capreolus. See Roe deer (Capreolus CRISPR/Cas mechanism, 613–614
capreolus) Cobalamin, 73–74
Carbohydrates, 75 Cold temperate zones, 219–220
metabolism, 77 Combination anthelmintics, 197, 204
CARS. See Consortium for Anthelmintic Commercial vaccines, 365–366
Resistance and Susceptibility complementary DNA (cDNA), 528
(CARS) Computer simulation models, 131
Cathepsin B cysteine protease gene Condensed tannins (CTs), 270, 299
families, 56–57 CTs-containing nutraceuticals, 309
Cathepsin D aspartic protease gene combinations of resources with AH
families, 56–57 effects, 326–327
CD4 cells, 357–358 dissecting complexity of tropical
CD8 cells, 357–358 legumes, 320–324
628 Index
Condensed tannins (CTs) (Continued ) Cysteine proteinases, 370, 382–383

exploring value of agroindustrial Cytochrome P450 (CYP450), 73–74,
by-products, 324–326 443–444, 541, 584–585, 585f
temperate legume forages, 309–320 Cytokine, 169–170
Consortium for Anthelmintic Resistance Cytoskeleton, 81–82
and Susceptibility (CARS), 418 DAMA. See Documentation-Assessment-
Contortin, 542–543 Monitoring-Action (DAMA)
Control fed (CF), 320 DAMP. See Danger-associated molecular
Controlled environment studies, 112 pattern (DAMP)
intraspecific differences in critical Danger-associated molecular pattern
requirements, 115–116 (DAMP), 169
moisture requirements DDA. See Dioctadecyl ammonium
for egg development and survival, bromide (DDA)
112–113
for survival of infective larvae, 113 D
other environmental factors, 116 ddPCR. See Droplet digital PCR
temperature requirements (ddPCR)
for development of eggs, 113–114 DEAE. See Diethylaminoethyldextran
for survival of infective larvae, 114–115 (DEAE)
Controlled infections. See Pen studies Delayed rejection, 360–361
Conventional techniques, 528 Deprotonation, 88–89
EST sequencing, 528–529 Derquantel, 196–197, 441
microarrays, 529–530 Derquantel–abamectin combination, 475
Cool temperate 2-Desoxoparaherquamide. See Derquantel
climates, 120–121, 128–129 Detection of resistance, 202
regions, 104 Dexamethasone administration, 357
Cooperia curticei (C. curticei), 76–77 Diagnosis and disease monitoring,
Cooperia oncophora (C. oncophora), 488–489 183–184
Copper OxideWire Particles (COWPs), animal management factors, 184
267 clinical signs, 184–186
Cortisol, 357–358 FAMACHA system for anaemia
Cosmopolitan distribution, 7 assessment, 186–187
COWPs. See Copper OxideWire Particles haematology, 192–193
(COWPs) laboratory diagnosis
CRISPR. See Clustered regularly FWECs, 187–190
interspaced short palindromic laboratory identification of eggs,
repeat (CRISPR) 190–191
CRISPR RNAs (crRNAs), 613–614 molecular techniques, 191–192
CTs. See Condensed tannins (CTs) postmortem examination, 187
Cuticle, 480–481 Dictol, 365–366
structure and biochemical composition of, Dictyocaulus viviparus. See Bovine
80–81 lungworm (Dictyocaulus viviparus)
Cynodon dactylon. See Bermuda grass (BG) Dictyocaulus viviparus (D. viviparus),
CYP450. See Cytochrome P450 365–366
(CYP450) Dietary energy, supplementation with,
Cystatin, 366, 379 263–264
Cysteine proteases, 530–531 I-NS, 265–266
Index 629
MP requirements, 264–265 role of pharmacodynamics, 442–443

Diethylaminoethyldextran (DEAE), 372 drug efflux, 444–445
“Dinaburg Line”, 121 drug metabolism, 445–447
Dioctadecyl ammonium bromide (DDA), kinetics of anthelmintic accumulation,
372–373 443–444
Direct grazing, 311 parasitic nematodes, 443
Direct hypothesis, 298–299 in vitro
Dissolution, 467–468 adult stages, 436–437
Diversification, 7–8 larvae, 431–436
Diversity, 22–24 mechanism-based assays using H.
DNA vaccination, 383 contortus, 437–439
Documentation-Assessment-Monitoring-Action in vivo, 439–440
(DAMA), 23–24 jird model, 440–442
Dolichol biosynthesis, 80 marcfortine A and the
Domestic goat (Capra hircus), 45 paraherquamides, 441–442
Dominant-negative gene constructs, Novartis Animal Health, 441
611–612 switching screening paradigms, 440
Dose-related behaviour, 496–497 Drug efflux, 444
double-stranded RNA (dsRNA), 526, 601 additional studies, 445
Dpy phenotype. See Dumpy phenotype indirect data, 445
(Dpy phenotype) PCR, 444
Draft assembly to reference genome Drug efflux transport modulation
future and ongoing work, 577–579 on drug transport and efficacy against
overlap of predicted proteomes, 579f resistant H. contortus, 500
progress, 573–577 impact of P-gp, 501
assembly statistics of H. contortus, 575t P-gp-mediated drug–drug interaction,
genome statistics of H. contortus, 576t 502–503, 502f
sequence reads and assembly, 574f pluronic 85, 501–502
synteny between scaffolds, 578f relevance of cellular efflux transport,
Droplet digital PCR (ddPCR), 162 497–499
Drosophila cells, 604 ivermectin, 499–500
Drosophila melanogaster (D. melanogaster), P-gp deficiency, 499
37–38, 51–52 in vivo trials, 499
Drug absorption, 467–468 Drug metabolism, 445–447
Drug discovery, 431 Drug resistance. See also Anthelmintic
challenges and research opportunities, resistance
450–452 biochemistry, 87–90
perspectives on H. contortus and, 447 issues, 522–524
exposure-efficacy profile of test nonspecific resistance mechanisms, 89–90
compound, 449 specific resistance mechanisms
jird model, 449–450 Benzimidazole resistance, 88–89
larval screen strategy and readout, Levamisole resistance, 89
447–448 Drug-combined therapy, pharmacological
optimal success in drug discovery, 450 evaluation of, 503–504
primary screen hits, 448 anthelmintic resistance, 505–506
repurposed compounds, 448 cattle production systems, 508–509
secondary screening strategy, 448–449 faecal egg count reduction, 506–508
630 Index
Drug-combined therapy, pharmacological EG. See Early gestation (EG)

evaluation of (Continued ) Egg development and survival, moisture
increased anthelmintic efficacy, 508f requirements for, 112–113
pharmacodynamic interactions, 505 Egg hatch assay, 414–415
potential drug interaction, 504–505 Eggs identification, 152–155
Drug-parasite relationship, 476 Eggs laboratory identification, 190–191
dsRNA. See double-stranded RNA eggs per gram (epg), 160–161, 188
(dsRNA) Electrophysiology, 85
DTT-eluted fraction, 373 ELISA, 165–166
Dumpy phenotype (Dpy phenotype), ELISPOT. See Enzyme-linked
614–616 immunospot assay (ELISPOT)
Duodenal fluid, 72 Embonate salts, 471–472
Emodepside, 87
E Endoparasites, 80
E/S products. See Excretory/secretory Energy metabolism in nematodes, 74–77.
products (E/S products) See also Amino acid metabolism;
Early gestation (EG), 320 Lipid metabolsim
EAT-2, 85 adult nematodes, 75–77
Ecological fitting, 12–14 anthelmintic drugs targeting energy and/
Ecology of H. contortus, 111 or carbohydrate metabolism, 77
controlled environment studies, 112 larval nematodes, 74–75
intraspecific differences in critical Enolase, 381–382
requirements, 115–116 Environmental factors, 116
moisture requirements for egg environmental RNAi, 604
development and survival, 112–113 Enzyme-linked immunospot assay
moisture requirements for survival of (ELISPOT), 170
infective larvae, 113 Eosinophil peroxidase assays, 167–168
other environmental factors, 116 Eosinophil-specific peroxidase (EPX),
temperature requirements for 167–168
development of eggs, 113–114 Eosinophilia, 168
temperature requirements for survival Eosinophils, 167–168
of infective larvae, 114–115 epg. See eggs per gram (epg)
ecological investigations in field, 116–117 Epidemiologically based preventative
arid regions, 121 programmes, 214
cool, temperate climates, 120–121 arid zones, 220
lateral and vertical migration of cold temperate zones, 219–220
infective larvae, 123–125 Mediterranean climatic zones, 219
effect of microclimatic factors on larval subtropical zones, 218
development, 122–123 summer rainfall temperate zones,
subtropical climates, 117–119 218–219
tropical climates, 117–119 wet tropical zones, 214–218
warm, temperate and Mediterranean Epidemiology of H. contortus, 125
climates, 119–120 arid climates, 129
Economic significance, 105–106 cool temperate climates, 128–129
Ecosystems of H. contortus life cycle stages, subtropical regions, 125–126
72 tropical regions, 125–126
Effective population size (Ne), 37–40 warm, temperate climates, 127–128
Index 631
Eprinomectin, 415 Fasciola hepatica (F. hepatica), 606

EPX. See Eosinophil-specific peroxidase Fatty acids, 75, 80
(EPX) FCA. See Freund complete adjuvant (FCA)
Essential amino acids, 78 FECRT. See Faecal egg count reduction
EST sequencing. See Expressed sequence test (FECRT)
tag sequencing (EST sequencing) FECs. See Faecal egg counts (FECs)
Evolution and biogeography of H. Feeds, 242b–243b
contortus, 3 Feedstuff, 242b–243b
domestication, geographical expansion Female vulva, 57–58
and invasion, 7–9 Fenbendazole, 468–470, 479–480
multispecies infections, 4 Field studies, 254. See also Pen studies
phylogeny 50% inhibitory concentration (IC50),
and biogeography, 4–7 483–484
perspective for host-group distribution Filamentous proteins, 81–82
and coevolutionary, 6f “First pass” metabolism, 470
Ex vivo tissue explant model, 359–360 First-stage larvae (L1), 72
Excessive treatment frequency, 199 Fixed combination products, 503–504
Excretory/secretory products (E/S FKTF-1. See Forkhead transcription
products), 56, 365–366, 372–374, factor-1 (FKTF-1)
525 Flaccid paralysis, 85
Exposure-efficacy profile of test flp genes. See FMRF-like peptide
compound, 449 neurotransmitter (flp genes)
Expressed sequence tag sequencing (EST FMC. See Faecal moisture content (FMC)
sequencing), 528–529, 602 FMRF-like peptide neurotransmitter (flp
Exsheathed third stage larvae (xL3s), genes), 605
530–531 Forkhead transcription factor-1 (FKTF-1),
Exsheathment process, 521–522 612
454 sequencing technology, 530–531
F Fourth-stage larvae (L4s), 72, 74, 151–152,
Faecal egg count reduction test (FECRT), 434–435, 601–602. See also Third-
161–162, 202, 398, 505–506 stage larvae (L3)
Faecal egg counts (FECs), 59, 164, 255, Free-living stages, 72
357 Freund complete adjuvant (FCA), 372
Faecal moisture content (FMC), 112 a1–3 Fucosyltransferases, 380
Faecal worm egg counts (FWECs), a1–6 Fucosyltransferases, 380
106–107, 184, 187–188 Fumarate/succinate system, 77
degree of pasture contamination, 189 Functional genomics tool
diagnostic toolkit, 190 using CRISPR/CAS genome editing,
diagnostic value, 188 613–616
McMaster procedure, 189–190 RNAi for H. contortus, 606–607
FAffa MAlan CHArt (FAMACHA), 109, developmental stages suitable for
152 RNAi, 609–611
system for anaemia assessment, 186–187 feeding and development of H.
False negatives, 447 contortus L4 stage, 610f
False positives, 447 optimizing delivery of RNA, 607–609
FAMACHA. See FAffa MAlan CHArt functional RNAs, 590
(FAMACHA) Fungal species, 208–209
632 Index
Fuzzy coat, 81 genome characteristics and annotation,

FWECs. See Faecal worm egg counts 532–534
(FWECs) chromosomal synteny and gene order,
534–535
G draft genome sizes, 534
G protein–coupled receptor (GPCR), 438 immunobiology, 537–539
22G secondary siRNA, 603 immunogens, 541–543
GA1. See Gut antigen 1 (GA1) predicting targets for new anthelmintics,
GABA. See g-Aminobutyric acid (GABA) 539
Galectins, 381 chokepoint analysis, 540
Galectin-11, 361–364 LGICs, 540–541
Galectin-14, 359–362 single-copy homologues,
Gala1–3GalNAc, 373–374 539–540
Gap5, 574–575 transcriptional alterations in life cycle or
GAPDH. See Glyceraldehyde-3- between tissues, 535
phosphate dehydrogenase changes in transcription, 535–536
(GAPDH) haematophagous stages, 536
Gastrointestinal nematodes (GIN), 241, sex-enriched transcription, 536
357, 601–603 transcription in intestine of H. contortus,
biology of key stages, 272–274 537
Gastrointestinal secretion process (GI xenobiotic detoxification, 541
secretion process), 467–468 Geographical distribution, 97
GDH. See Glutamate dehydrogenase arid regions, 104–105
(GDH) cool temperate regions, 104
Gene expression in parasitic life cycle subtropical climates, 100
stages, 73–74 tropical climates, 100
Gene silencing, 526 warm temperate regions, 100–104
RNAi GFP. See Green fluorescent protein (GFP)
pathway genes, 603–604 GI secretion process. See Gastrointestinal
in plant parasitic nematodes and in secretion process (GI secretion
trematodes, 605–606 process)
studies in H. contortus, 601–603 GIN. See Gastrointestinal nematodes
Generalists, 14–17 (GIN)
Genetic diversity in population, glc-1, 585–586
37–38 glc-5, 86
Genetic factors, 261 glc-6, 86
Genetic selection against H. contortus, Globodera, 605
207–208 Globule leukocytes (GLs), 356
Genome Institute at Washington GLs. See Globule leukocytes (GLs)
University, 589 GluCl. See Glutamate-gated chloride
Genome-wide analysis, 37–38 channels (GluCl)
Genome-wide SNPs, 44 GluClR. See Glutamate-gated chloride
Genomes and transcriptomes of H. channels (GluCl)
contortus, 532. See also Pregenomic GluCla, 86–87
transcriptomic studies of H. GluClb, 86–87
contortus Glutamate, 85–86
comparison of features, 533t Glutamate dehydrogenase (GDH), 370
Index 633
Glutamate-gated chloride channels H. contortus galactose-containing

(GluCl), 85–87, 409, 473–474, glycoprotein complex (H-gal-GP
496–497, 585–586 complex), 541–542
Glutathione peroxidase (GPX), 383 H. contortus Glyceraldehyde-3-phosphate
Glutathione S-transferases (GSTs), 89, dehydrogenase (HcGAPDH), 383
302, 377–378, 541 H. contortus infection, control
Glyceraldehyde-3-phosphate improvement of, 267
dehydrogenase (GAPDH), 383 animals
Glycocalyx, 81 on excellent nutritional level, 269
Glycolipids, 81 on good nutritional level, 269
Glycoproteins, 81 on poor nutritional level, 268–269
Glycosylation, 367 outcome of supplementary feeding for,
Gold standard, 147–148 268f
adult worms identification, 148–150 [3H]inulin, 437, 448–449
eggs identification, 152–155 H11 components, recombinant versions
identification of infective L3, of, 379–380
150–151 H11 gut-derived antigens, 368–369
parasitic fourth-stage larvae identification, Haematology, 192–193
151–152 Haemoglobin (Hb), 246–247
GPCR. See G protein–coupled receptor Haemonchines, 4–5
(GPCR) Haemonchosis
GPX. See Glutathione peroxidase (GPX) risk assessment, 213
Grazing management, 205–206 Haemonchosis, 105, 127, 183, 185,
Grazing system, 241 214–218, 220–221, 520–522, 539,
Green fluorescent protein (GFP), 541–542
526–527, 608 antibody assays
GSTs. See Glutathione S-transferases diagnosis of haemonchosis, 165–166
(GSTs) as research tools to studying
Gut antigen 1 (GA1), 371 haemonchosis, 166–170
Gut-derived antigens immunological methods for diagnosing,
GA1, 371 164
H-gal-GP, 366–367 Haemonchus, 3
H11, 368–369 eggs, 73
TSBP, 370 species-level, 3–4
Haemonchus contortus (H. contortus), 32,
H 70–71, 96, 146–147, 155–157,
H-gal-GP complex. See H. contortus 183, 243–244, 354–355, 399,
galactose-containing glycoprotein 570–572, 600
complex (H-gal-GP complex) anthelmintics and drug resistance issues,
H-gal-GP components, recombinant 522–524
versions of, 377 background information on reproduction
aspartyl proteinases, 377 and genetics, 33–34
cystatin, 379 biology and disease, 521–522
zinc metalloproteinases, 377–378 consequences
H-gal-GP gut-derived antigens, high genetic diversity, 47–48
366–367 low population structure between
H. contortus enolase (HcENO), 381–382 hosts, 50
634 Index
Haemonchus contortus (H. contortus) genome structure, 579–580

(Continued ) chromosomal synteny, 580
low regional population structure genome size, repetitive sequence and
within country, 48–49 GC content, 580
substantial global population structure, immune responses, 522
49–50 importance of sequence data, 600–601
draft assembly to reference genome isotype-1 b-tubulin, 405–406
future and ongoing work, 577–579 low but discernable regional population
progress, 573–577 structure within countries, 41–43
ecological features and epidemiology, genetic markers, 44
101t–103t population bottlenecks, 44
effects of environmental factors on free- T. circumcincta, 43–44
living stages, 98t–99t phenotypic variation in laboratory strains,
exploring ex vivo and in vivo, 476 50
albendazole sulphoxidation, 482–483 within and between laboratory strains,
benzimidazole anthelmintics, 479–480 54–61
chemical substances, 479 variation in gene expression and
closantel, 478 function, 55–57
closantel lipophilicity, 480 variation in life history traits and
drug molecules, 478–479 pathogenicity, 59–61
helminth’s external surfaces, 476–477 variation in morphological traits, 57–59
monepantel, 483 progressing RNAi as functional genomics
pharyngeal pumping, 484–485 tool, 606–611
rate of penetration, 480–481 proteome, 583–584
resistance, 481–482 transgenic approaches to identify essential
rotenone, 483–484 gene function, 611–613
future directions, 589–591 vaccine research, 524–525
genetic diversity and population structure Haemonchus placei (H. placei), 33, 35–36,
anthelmintic selection effect, 46–47 146–147, 155–157
extremely high levels of genetic Haemonchus species, 147, 155–156
diversity, 35–37 molecular methods for identification
factors influencing, 34–35 H. contortus, 155–157
genetic differentiation, 45–46 H. placei, 155–157
large population size, 37–40 next generation, 162–164
substantial global population structure, real-time PCR, 159–162
40–41 traditional PCR, 157–159
genetic variation in laboratory strains, 50 Hb. See Haemoglobin (Hb)
within and between laboratory strains, Hc-CPL-1, 382–383
52–54 Hc-sL3 antigen, 371–372
genome content Hc23, 381
gene number, size and structure, HcENO. See H. contortus enolase
581–582 (HcENO)
operons and trans-splicing, 587–588 HcGAPDH. See H. contortus
orthology and gene family evolution, Glyceraldehyde-3-phosphate
582–587 dehydrogenase (HcGAPDH)
repetitive sequence and mobile HcGluCla, 85–86
elements, 588–589 HcGluCla3A, 85–86
Index 635
HcGluCla3B, 85–86 phylogenetic perspective of host-group

HcGluClb, 85–86 distribution, 11f
Hco-29 paralogs, 408–409 sloppy fitness space, 12–14
Hco-acr-8 gene, 406–407, 419 specialists, 14–17
Hco-dyf-7 gene, 419 Host resilience, impact on, 274–294
Hco-pgp-16, 586–587 HPLC. See High-performance liquid
Hco-pgp-17, 586–587 chromatography (HPLC)
Hco-unc-63, 406–407 hspb1. See Heat shock protein gene (hspb1)
Hco4(WRS) strain, 55–56 5-HT. See Serotonin (5-HT)
HcRep, 589 5-Hydroxyisourate hydrolase, 538
Heat shock protein gene (hspb1), 606 Hyperacute haemonchosis, 184–185
Helminth Genomes Initiative, 589 Hypobiosis, 128
Heterodera, 605 Hypoproteinaemia, 185
Heterohabditis bacteriophora (H. bacteriophora),
579–580 I
‘Hidden antigen’ approach, 371 I-NS. See Infected not supplemented
Hidden antigens, 369–370 (I-NS)
High mobility group box 1 (HMGB1), IC50. See 50% inhibitory concentration
169 (IC50)
High-mass matrix-assisted laser desorption IgA. See Immunoglobulin A (IgA)
ionization time-of-flight analysis IL. See Interleukin (IL)
(MALDI-ToF analysis), 366–367 Image analysis platforms development, 436
High-performance liquid chromatography Imidazothiazoles, 195, 406, 470–471
(HPLC), 302–308 acetylcholine cation channel targets of
High-quality ‘finished’ reference genome, anthelmintics, 407f
571–572 inconsistencies of gene expression
limitations of draft genome assemblies, patterns, 408–409
572f molecular changes, 408
HMGB1. See High mobility group box 1 molecular studies, 406–408
(HMGB1) Immune exclusion. See Rapid larval
Host alarmin assays, 169–170 rejection
Host and geographical colonization in Immune responses, 522
faunal assembly, 17–19 Immune-based hypothesis. See Indirect
Host colonization, 12–14 hypothesis
Host distribution, 5 Immunogens, 541–543
Host nutrition to controlling H. contortus Immunoglobulin A (IgA), 358
infection Immunohistochemistry, 170
nutrition, 251–252 Immunology of host protection against H.
targeted dietary supplementation for contortus, 355–356
nutritional components, 252–253 critical role of host immune responses in
theoretical framework, 252f protection, 356
Host range for H. contortus, 9–10 dexamethasone administration, 357
ecological fitting, 12–14 lymphocyte involvement in immunity,
generalists, 14–17 357–358
host colonization, 12–14 host immune protection against adult
host groups, 12 worm infection, 363–364
636 Index
Immunology of host protection against H. Intra-epithelial mast cells, 359

contortus (Continued ) Intraruminal administration (IR
host immune protection against larval administration), 469–470
establishment IPM. See Integrated parasite management
delayed rejection, 360–361 (IPM)
mechanisms against L3, 358–359 IR administration. See Intraruminal
mechanisms against L4s, 361–362 administration (IR administration)
rapid rejection, 359–360 ISE strain, 51–52
immune mediators or mechanisms, 356f Isolates, 50–51
recombinant subunit vaccines, 374–383 Isotype-1See b-tubulin genes (tub 8–9)
unknown factors affecting host immunity, Isotype-2. See tub 12–16
364–365 ITS-2. See Internal transcribed spacer 2
vaccines development, 365–374 (ITS-2)
In vitro bioassay-based tests for resistance, Ivermectin (IVM), 85–86
414 Ivermectin, 473–474, 488–489, 497–499
egg hatch assay, 414–415 aglycone, 415
larval motility/migration assay, 416–418 monosaccharide, 415
LDA, 415–416 IVM. See Ivermectin (IVM)
In vivo drug discovery, 439–440
adult stages, 436–437 J
jird model, 440–442 Jird model, 440–442, 449–450
larvae, 431–436
marcfortine A and the paraherquamides, K
441–442 15-kDa protein, 372–373, 380–381
mechanism-based assays using H. contortus, 24-kDa protein, 372–373, 380–381
437–439
Novartis Animal Health, 441 L
switching screening paradigms, 440 L1. See First-stage larvae (L1)
In vivo resistance tests, 414 L2. See Second-stage larvae (L2)
‘In-the-field’ assays, 162–163 L3 surface antigen, 371–372
Indirect hypothesis, 299–300 L3. See Third-stage larvae (L3)
Infected not supplemented (I-NS), L4s. See Fourth-stage larvae (L4s)
265–266 Laboratory diagnosis
Infective larvae, lateral and vertical FWECs, 187–190
migration, 123–125 laboratory identification of eggs, 190–191
Inhibitory neurotransmitters in nematodes, molecular techniques, 191–192
85–87 Laboratory strains, 50–51
g-Aminobutyric acid-A receptors, 85 LAMP. See Loop-mediated isothermal
calcium-activated voltage-gated amplification (LAMP)
potassium channel SLO-1, 87 Larvae, 431
GluCl, 85–87 adaptation of H. contortus larval assays,
ML, 85–87 434–435
Integrated parasite management (IPM), anthelmintic discovery and
184 characterization, 431–432
Interbreeding parasites, 50 fruitful results, 433–434
Interleukin (IL), 358, 522 high-throughput screening strategies,
Internal transcribed spacer 2 (ITS-2), 156 435
Index 637
image analysis platforms development, M

436 Macrocyclic lactones (ML), 85–87,
Larval H. contortus assays, 433 195–196, 398–399, 409, 473–474,
larval migration assay, 432 487–488
modifications and improvements, ivermectin interaction, 409–410
436 P-gp, 410
MTT, 434 resistance to, 410–411
(Z)-2-phenyl-3-(1H-pyrrol-2-yl) target site changes and drug efflux
acrylonitrile analogues, 432–433 pathways, 410
Larval development assay (LDA), 415–416, Malate dismutation, 75–76
431–432 MALDI-ToF analysis. See High-mass
Larval exsheathment inhibition assay matrix-assisted laser desorption
(LEIA), 315–316 ionization time-of-flight analysis
Larval H. contortus assays, 433 (MALDI-ToF analysis)
Larval migration assay, 432 Male bursa, 57–58
Larval motility/migration assay, Manual curation and genome
416–418 improvement, 574–575
Larval nematodes, energy metabolism in, Manual improvement process, 575
74–75 Marcfortine A, 441–442
LAT-1. See Latrophilin-1 (LAT-1) ‘Martinik’ Black Belly breed, 59
Latrophilin-1 (LAT-1), 87 Mast cell, 168
LDA. See Larval development assay Mastocytosis assays, 168
(LDA) Mazamastrongylus odocoilei (M. odocoilei),
LDNF glycan antigen, 373–374 35–36
LEIA. See Larval exsheathment inhibition McMaster, 51–52
assay (LEIA) H. contortus strain, 570–571
LEV-1, 85 Mechanism-based assays using H. contortus,
LEV-8, 85 437
Levamisole, 470–471, 486–487 anthelmintics, 437–438
receptor, 419 bioinformatics searches, 439
resistance, 89 bioinformatics-driven approaches, 439
LGICs. See Ligand-gated ion channel novel screening approach, 438–439
subunit genes (LGICs) PAPP, 438
Ligand-gated ion channel subunit genes Mediterranean climates, 119–120
(LGICs), 540–541 climatic zones, 219
Linear discrimination analysis, Mef-2. See Myocyte enhancer factor 2
153–154 (Mef-2)
Lipid metabolsim, 79–80 Meloidogyne, 605
Lipophilic drugs, 488–489 MEPs. See Metalloproteinases (MEPs)
Long-acting anthelmintics, 203–204 Metabolizable protein (MP), 254
Long-read sequence data, 573–574 Metagenomics, 163–164
Loop-mediated isothermal amplification Metalloproteinases (MEPs), 366, 542
(LAMP), 162–163, 192 Methyl-thiazolyl-tetrazolium (MTT), 434
Lymphocyte involvement in immunity, MHco10(CAVR) strain, 51–52, 60–61
357–358 MHco3.ISE, 570–571
Lysiloma latisiliquum. See Tzalam (Lysiloma assembly, 573, 575–577
latisiliquum) MHco3. ISE. 2015 assembly, 575–577
638 Index
MHco3.ISE (Continued ) identification of infective L3, 150–151

MHco3(ISE). N1 strain, 570–571 parasitic fourth-stage larvae
MHco3(ISE) strain, 60–61 identification, 151–152
MHco3(ISE)/MHco10(CAVR) strain, Mosaic structure, 5–7
56–57 MOSI strain, 56
MHco3(ISE)/MHco4(WRS) strain, 56–57 Motility, 437
MHco4(WRS) strain, 60–61 Moxidectin, 473–474
MHco4(WRS)/MHco10(CAVR) strain, MP. See Metabolizable protein (MP)
56–57 MPTL. See Monepantel (MPTL)
micro RNAs (miRNAs), 545 MPTL-1, 474
Microarrays, 529–530 MRI isolates. See Moredun Research
Microclimatic factors, 122–124 Institute isolates (MRI isolates)
Microparticle bombardment, 613 mtDNA sequences. See
microRNA-adapted short hairpin RNAs mitochondrialDNA sequences
(shRNAmirs), 613 (mtDNA sequences)
Microsatellites, 589 MTT. See Methyl-thiazolyl-tetrazolium
Microtubules, 71 (MTT)
microtubular functions, 81–82 Mucus, 359
microtubular matrix formation, 522–523 Mutation rate (m), 37–38
microtubuli, 82 Myocyte enhancer factor 2 (Mef-2), 611
Milbemycin oxime, 473–474
Mineral metabolism, 266 N
Mineral micronutrients, supplementation nAChR. See nicotinic acetylcholine
with, 266–267 receptor (nAChR)
miRNAs. See micro RNAs (miRNAs) nad4 gene. See Nicotinamide adenine
mitochondrialDNA sequences (mtDNA dinucleotide dehydrogenase
sequences), 34 subunit 4 gene (nad4 gene)
ML. See Macrocyclic lactones (ML) NADH, 79–80
Mobile elements, 588–589 NADH–fumarate–reductase, 76
modENCODE, 590–591 Natural infections. See Field studies
Moisture requirements ncRNA. See noncoding RNA (ncRNA)
for egg development and survival, Necator americanus (N. americanus), 579–580
112–113 Necropsy, 440–441
for survival of infective larvae, 113 NEJ. See Newly excysted juvenile (NEJ)
Molecular markers, 418 Nemadectin, 473–474
Molecular techniques, 191–192 Nematodes, 78
Molecular-based tests, 418–419 Nervous system in nematodes, 83
Monepantel (MPTL), 84, 441, 474 inhibitory neurotransmitters in
Monitoring of H. contortus burdens, 221 nematodes, 85–87
Morantel, 471–472 nicotinic AChRs in H. contortus, 83–85
Moredun Research Institute isolates (MRI Neurohormones, 78
isolates), 60 Neuromuscular system, 71
Morphological approaches for identifying Neurotransmitters, 78
H. contortus Newly excysted juvenile (NEJ), 606
gold standard, 147–148 Next generation
adult worms identification, 148–150 LAMP, 162–163
eggs identification, 152–155 metagenomics, 163–164
Index 639
pyrosequencing, 163–164 Nutrition and infection interactions

Nicotinamide adenine dinucleotide AH properties, 244
dehydrogenase subunit 4 gene GIN parasites, 245
(nad4 gene), 35–36 Haemonchus infection model, 246–247
nicotinic acetylcholine receptor (nAChR), interaction between host nutrition and
406, 470–471 GIN infections, 247
Nicotinic AChRs in Haemonchus contortus, nutrition–parasite interactions, 245
83–85 qualitative aspects, 269
Nippostrongylus brasiliensis (N. brasiliensis), biology of key stages of GINs, 272–274
601 chemistry of tannins and related
Nitrile hydrolysis, 446–447 polyphenols, 271
Nitrogen resources, supplementation with, in vitro anthelmintic effects of tannin-
253 containing resources, 275t–280t
in controlled pen studies, 254–261 in vivo anthelmintic efficacy of tannin-
dietary protein supplementation, 254 containing resources, 281t–293t
supplementary feeding in grazing animals, impact on host resilience, 274–294
261–263 methodological issues, 271–272
Nodule worm (Oesophagostomum modes of action of tannin-containing
dentatum), 579–582 plants, 298–309
Non-CYP450–mediated reactions, on-farm use of CTs-containing
446–447 nutraceuticals, 309–327
Nonchemical control, 205 variability in effects of PSMs, 294–298
alternative anthelmintic compounds, in vitro methods, 273t
210–211 quantitative aspects, 247–248
biological control, 208–210 host nutrition to controlling H.
genetic selection against H. contortus, contortus infection, 251–253
207–208 improving control of H. contortus
grazing management, 205–206 infection, 267–269
nutritional management, 206–207 pathophysiological and nutritional
vaccines, 211–213 consequences of H. contortus
Nonchemical strategies, 220–221 infections, 248–251, 248f
noncoding RNA (ncRNA), 525–526 supplementation with dietary energy,
Nonprotein nitrogen (NPN), 256 263–266
Nonspecific resistance mechanisms, 89–90. supplementation with mineral
See also Specific resistance micronutrients and trace elements,
mechanisms 266–267
Novartis Animal Health, 441 supplementation with nitrogen
Novel screening approach, 438–439 resources, 253–263
NPN. See Nonprotein nitrogen (NPN) relations between host nutrition and
Nucleic acid metabolism, 79. See also pathological and pathophysiological
Amino acid metabolism changes, 243f
purine metabolism, 79
Nutraceuticals, 242b–243b, 270, 294 O
Nutrients, 242b–243b Oesophagostomum dentatum. See Nodule
Nutrition, 251–252 worm (Oesophagostomum dentatum)
nutritional management, 206–207 Onobrychis viciifolia. See Sainfoin (Onobrychis
nutritional status, 107–108 viciifolia)
640 Index
Onobrychis viciifolia (O. viciifolia). See lipophilic drugs, 488–489

Sainfoin macrocyclic lactones, 487–488
Operons, 587–588 pharmacokinetic–pharmacodynamic
Optimal nutrition, 220–221 relationship, 491
Optimal screening processes, 447 Parasite–host interplay, 537–538
Organophosphate(s), 197 Parasitic fourth-stage larvae identification,
compounds, 472 151–152
naphthalophos, 400–403 Parasitic lifestyles, 583
Oscillation Hypothesis, 16 Parasitic nematodes, 79, 89, 520–521
Ostertagia ostertagi (O. ostertagi), 35–36, of ruminants, 466
39–40 variability in effects of PSMs, 294
Ovis aries. See Sheep (Ovis aries) anthelmintic flavonoids, 297f
Oxaloacetate, 75–76 flavonoids and tannins, 296f
Oxfendazole, 470, 479–480 variation in plant tannin composition,
Oxidative deamination, 78–79 295–298
Oxidoreductase activity, 74 variations from nematode species and
life cycle stages, 294–295
P Parastrongyloides, 611–612
p-amino-phenethyl-m- Particle bombardment, 608–609
trifluoromethylphenyl piperazine Passive diffusion process, 478–479
(PAPP), 438 Pasture-raised Gulf Coast Native lambs,
P-glycoproteins (PGPs), 89–90, 410, 357
443–444, 541, 586, 587f Pathogenesis, H. contortus, 106–108
Packed cell volume (PCV), 106–107, Pathophysiology
246–247 H. contortus, 106–108
PAGE. See Polyacrylamide gel and nutritional consequences of H.
electrophoresis (PAGE) contortus infections, 248–251,
Paired-end sequencing library, 574f 248f
PAPP. See p-amino-phenethyl-m- PBMC. See Peripheral blood mononuclear
trifluoromethylphenyl piperazine cells (PBMC)
(PAPP) PBS. See Phosphate-buffered saline (PBS)
Paraherquamides, 441–442 PC mixtures. See Procyanidin mixtures
ParaSite database, 525–526 (PC mixtures)
Parasite E/S products, 372–373 PCoA. See Principal coordinate analysis
Parasite exposure assessment to (PCoA)
anthelmintic drugs PCR. See Polymerase chain reaction
dosage and relationship, 491–494 (PCR)
disposition kinetics and efficacy of PCV. See Packed cell volume (PCV)
ivermectin, 496–497 PD mixtures. See Prodelphinidin mixtures
plasma concentration profiles, 494–496 (PD mixtures)
H. contortus exploring ex vivo and in vivo, Peanut agglutinin (PNA), 154
476–485 PEG. See Polyethylene glycol (PEG)
influence of route of drug administration Pen studies, 253–254
on parasite exposure, 485, 489f in New South Wales, 109–110
AADs, 489–490 supplementation with dietary nitrogen
GI transit time, 485–486 sources in, 254
levamisole, 486–487 age of animals, 257
Index 641
amino acids pathophysiological studies, Phenylmethylsulfonyl fluoride (PMSF), 56

257 Phosphate-buffered saline (PBS), 383
genetic factors, 261 Phosphoenolpyruvate (PEP), 75–76
H. contortus infection, 255–256 2-Phosphoglycerate hydratase. See Enolase
host immune response, 256–257 Phosphoribosyltranserases (PRTases), 79
level of animal production, 259–261 Phosphorylation, 472
protein supplementation, 255 Phylogeny, 5
reproduction, 258–259 Phytotherapeutic drugs, 270
PEP. See Phosphoenolpyruvate (PEP) Piperazine, 85
Pepsinogen-like aspartyl proteinases, 366 Piwi-interacting RNAs (piRNAs), 604
Peptidases, 542–543 Plant metabolites, 271
Periparturient relaxation of immunity Plant secondary metabolites (PSMs), 241
(PPRI), 258 variability in PSM effects, 294
Peripheral blood mononuclear cells anthelmintic flavonoids, 297f
(PBMC), 168–169 flavonoids and tannins, 296f
PGPs. See P-glycoproteins (PGPs) variation in plant tannin composition,
Pharmacodynamic drug-to-drug 295–298
interactions, 504–505 variations from nematode species and
Pharmacological-like hypothesis. See life cycle stages, 294–295
Direct hypothesis Pluronic 85, 501–502
Pharmacology of anthelmintic drugs, PMSF. See Phenylmethylsulfonyl fluoride
467–468 (PMSF)
benzimidazole methylcarbamates, PNA. See Peanut agglutinin (PNA)
469–470 Point-of-care diagnostic systems, 192
benzimidazoles, 468–469 Polyacrylamide gel electrophoresis
characterization of drug concentration (PAGE), 368
profiles, 468 Polyamines, 78–79
Derquantel, 475 Polyandry, 33
derquantel–abamectin combination, 475 Polycistronic mRNAs, 588
drug efflux transport modulation, Polyethylene glycol (PEG), 271–272
497–503 Polymerase chain reaction (PCR), 444,
“first pass” metabolism, 470 526–527
imidazothiazoles, 470–471 Polystyrene-based nanoparticles,
Macrocyclic lactones, 473–474 608–609
Monepantel, 474 Polyvinyl poly pyrrolidone (PVPP),
Organophosphate compounds, 472 271–272
parasite exposure assessment to Population genetic structure, 34
anthelmintic drugs, 476–497 Postmortem examination, 187
pharmacological evaluation of drug- PPRI. See Periparturient relaxation of
combined therapy, 503–509 immunity (PPRI)
Salicylanilides, 472–473 Pre-formed mediators, 359
tetrahydropyrimidines, 471–472 Preadaptation, 579–580
Pharyngeal pumping, 448–449, 484–485 Prediction of occurrence of H. contortus
(Z)-2-Phenyl-3-(1H-pyrrol-2-yl) potential effects of climate change,
acrylonitrile analogues, 432–433 131
2-Phenylimidazo[1,2-b]pyridazines, predictive models, 130
432–433 Predictive models, 130
642 Index
Pregenomic transcriptomic studies of H. Q

contortus. See also Genomes and qPCR. See quantify PCR (qPCR)
transcriptomes of H. contortus qRT-PCR. See Quantitative real-time,
challenges and need for genome of H. reverse transcription PCR
contortus, 531–532 (qRT-PCR)
conventional techniques, 528 QTLs. See Quantitative trait loci (QTLs)
EST sequencing, 528–529 Qualitative aspects, 269
microarrays, 529–530 biology of key stages of GINs, 272–274
454 sequencing technology, 530–531 chemistry of tannins and related
Preventative programmes, 213 polyphenols, 271
anthelmintic choice and resistance impact on host resilience, 274–294
management, 221–222 methodological issues
epidemiologically based preventative AH properties of tannins and related
programmes, 214 polyphenols, 272
arid zones, 220 analytical tools for tannins, 271–272
cold temperate zones, 219–220 modes of action of tannin-containing
Mediterranean climatic zones, 219 plants, 298
subtropical zones, 218 direct hypothesis, 298–299
summer rainfall temperate zones, indirect hypothesis, 299–300
218–219 possible mechanisms of action,
wet tropical zones, 214–218 300–309
haemonchosis risk assessment, 213 SAR studies, 300–309
monitoring of H. contortus burdens, 221 tannin analysing by thiolysis, 308t
nonchemical strategies, 220–221 tannin composition in plants,
relative risk of occurrence of 303t–307t
haemonchosis and management on-farm use of CTs-containing
strategies, 215t–217t nutraceuticals, 309
Primary screen hits, 448 combinations of resources with AH
Principal coordinate analysis (PCoA), 41, effects, 326–327
42f dissecting complexity of tropical
Pro-benzimidazoles, 468–469 legumes, 320–324
Procyanidin mixtures (PC mixtures), exploring value of agroindustrial
300–301 by-products, 324–326
Prodelphinidin mixtures (PD mixtures), temperate legume forages, 309–320
300–301 tannin-containing resources
Protonation, 88–89 comparing in vitro anthelmintic effects,
PRTases. See Phosphoribosyltranserases 275t–280t
(PRTases) comparing in vivo anthelmintic
PSMs. See Plant secondary metabolites efficacy, 281t–293t
(PSMs) variability in PSMs effects, 294
Purine metabolism, 79 anthelmintic flavonoids, 297f
PVPP. See Polyvinyl poly pyrrolidone flavonoids and tannins, 296f
(PVPP) variation in plant tannin composition,
Pyrantel, 471–472 295–298
Pyrosequencing, 163–164 variations from nematode species and
Pyruvate kinase/PEP carboxykinase ratio, life cycle stages, 294–295
77 quantify PCR (qPCR), 160–161
Index 643
Quantitative aspects, 247–248 Recombinant version of MEP 1 (rMEP 1),

host nutrition to controlling H. contortus 377–378
infection, 251–253 Refugia strategies, 199–201
improving control of H. contortus Repetitive sequence, 588–589
infection, 267–269 Reproduction, 258–259
pathophysiological and nutritional Reservoir, 485–486
consequences of H. contortus Resistance, 71–72, 242b–243b, 398
infections, 248–251, 248f Resistance-selection treatment practices,
supplementation minimizing
with dietary energy, 263–266 excessive treatment frequency, 199
with mineral micronutrients and trace underdosing with anthelmintics,
elements, 266–267 198–199
with nitrogen resources, 253–263 Resistant nematodes introduction,
Quantitative real-time, reverse prevention of, 204–205
transcription PCR (qRT-PCR), Respiratory chains, 76
528 Restriction fragment length
Quantitative trait loci (QTLs), 208 polymorphism (RFLP), 159
Quebracho (Schinopsis spp), 298–299 RFLP. See Restriction fragment length
Quil A, 372 polymorphism (RFLP)
Rhodoquinone, 77
R RIA. See Radio-immunoassay (RIA)
Radio-immunoassay (RIA), 165 Ribosomal DNA (rDNA), 156
Rapid larval rejection, 359 RISC. See RNA-induced silencing
Rapid rejection, 359–360 complex (RISC)
Rat lungworm (Angiostrongylus cantonensis), rMEP 1. See Recombinant version of MEP
579–580 1 (rMEP 1)
RDE-2 protein, 604 RNA interference (RNAi), 526, 600–601
rDNA. See Ribosomal DNA (rDNA) gene silencing, 526
RdRP. See RNA-dependent RNA pathway genes, 603–604
polymerases (RdRP) in plant parasitic nematodes and in
Real-time PCR, 159–162 trematodes, 605–606
Recombinant proteins studies in H. contortus, 601–603
Cysteine proteinases, 382–383 pathway genes, 603–604
Enolase, 381–382 in plant parasitic nematodes and in
Galectins, 381 trematodes, 605–606
Hc23, 381 screening, 605–606
15-and 24-kDa ES proteins, 380–381 studies in H. contortus, 601–603
Recombinant subunit vaccines, 374. RNA-dependent RNA polymerases
See also Vaccines development (RdRP), 603
DNA vaccination, 383 RNA-induced silencing complex (RISC),
H-gal-GP components, recombinant 603
versions of, 377–379 RNAi. See RNA interference (RNAi)
recombinant proteins, 380–383 Roe deer (Capreolus capreolus), 45
recombinant versions of H11 Romanomermis culcivorax (R. culcivorax),
components, 379–380 590–591
summary of vaccine trials for Haemonchus Rotational grazing, 218
contortus using, 375t–376t Rotenone, 483–484
644 Index
Rupicapra r. rupicapra. See Alpine chamois Simulation models, 130

(Rupicapra r. rupicapra) Single nucleotide polymorphisms (SNPs),
35–36, 88, 156, 404–405, 491–494
S single-guide RNA (sgRNA), 613–614
Saccharomyces cerevisiae (S. cerevisiae), 37–38 Single-nucleotide polymorphisms (SNPs),
SAGE. See Serial analysis of gene 525–526
expression (SAGE) siRNAs. See small inhibitory RNAs
Sainfoin (Onobrychis viciifolia), 298–299, (siRNAs)
309–320 SL. See Sericea lespedeza (SL)
Salicylanilides, 196, 472–473 SL4Ps, 584
SAR studies. See Structure–activity SLO-1 receptor, 71, 83
relationship studies (SAR studies) Sloppy fitness space, 12–14
SC injection. See Subcutaneous injection Slow delivery effects of rumen, 485–486
(SC injection) Sm-Mef-2, 611
Scaffolds, 573 small inhibitory RNAs (siRNAs), 603
Scanning electron microscopy (SEM), 298 SMCP. See Sheep mast cell proteinase
Schinopsis spp. See Quebracho (Schinopsis (SMCP)
spp) SNPs. See Single nucleotide
Schistosoma, 605–606 polymorphisms (SNPs); Single-
SCP. See Sperm-coating protein (SCP) nucleotide polymorphisms (SNPs)
SCP/TAPs proteins. See Activation- Sodium dodecyl sulphate (SDS), 367
associated secreted proteins (ASPs) Specialists, 14–17
SCVPs, 584 Specific resistance mechanisms. See also
SDRs. See Short-chain dehydrogenase/ Nonspecific resistance mechanisms
reductases (SDRs) Benzimidazole resistance, 88–89
SDS. See Sodium dodecyl sulphate (SDS) Levamisole resistance, 89
Second-generation technology, 531–532 Sperm-coating protein (SCP), 537–538
Second-stage larvae (L2), 72 Spiroindoles, 196–197
Secondary screening strategy, 448–449 Stockholm Paradigm, 17–18
Self-medication, 242b–243b Strain contamination, 51–52
SEM. See Scanning electron microscopy Strongylid genomes, 580
(SEM) Strongyloides, 611–612
Sequencing-by-synthesis approach, 530 Structure–activity relationship studies
Serial analysis of gene expression (SAGE), (SAR studies), 300–309
528 Subcutaneous injection (SC injection),
Sericea lespedeza (SL), 297–298, 309–320 470–471
Serotonin (5-HT), 438 Substituted phenols, 196
sgRNA. See single-guide RNA (sgRNA) Subtropical climates, 100, 117–119
Sheep (Ovis aries), 45 Subtropical regions, 125–126
Sheep mast cell proteinase (SMCP), 168 Subtropical zones, 218
Short-chain dehydrogenase/reductases Sulphoxide derivatives, 468–469
(SDRs), 541 Summer rainfall temperate zones, 218–219
short-hairpin RNA (shRNA), 612 Supplementary feeding in grazing animals,
shRNAmirs. See microRNA-adapted 261–262
short hairpin RNAs (shRNAmirs) on adult goats, 263
SID-2. See Systemic interference defective during parasite infection, 262
2 (SID-2) Supplementation
Index 645
with dietary energy, 263–264 Targeted dietary supplementation for

I-NS, 265–266 nutritional components, 252–253
MP requirements, 264–265 Targeted selective treatment (TST),
with mineral micronutrients and trace 200–201
elements, 266–267 Targeted treatment, 200–201
with nitrogen resources, 253 Taxon Pulse, 18
in controlled pen studies, 254–261 TCA cycle. See Tricarboxylic acid cycle
dietary protein supplementation, 254 (TCA cycle)
supplementary feeding in grazing Teladorsagia circumcincta (T. circumcincta), 33,
animals, 261–263 35–36, 43–44, 361, 414–415,
Survival, 113 602–603
Switching screening paradigms, 440 TEM. See Transmission electron
Synergistic effect, 504–505 microscopy (TEM)
Synlophe, 57–58 Temperate climates, 119–120
Systemic interference defective 2 (SID-2), Temperate legume forages, 309
604 AH effect, 318
components and treatment, 317
T dehydrated pellets of SL, 316
T cell proliferation assay, 168–169 hay processing, 314
gd T cells, 363–364 on-farm applications, 313
T helper 2-type immune response prolonged distribution, 319
(Th 2-type immune response), 522 with sainfoin, 315
gd T lymphocytes, 363–364 sainfoin AH properties, 320
TALENs. See Transcription activator–like sainfoin silage, 315
effector nucleases (TALENs) SL, 310
Tandem repeats, 589 tannin-containing legumes with AH
Tannic acid, 299–302 properties, 311
Tannin-containing plants tracer animals, 312
modes of action, 298 Temperature requirements
direct hypothesis, 298–299 for development of eggs, 113–114
indirect hypothesis, 299–300 for survival of infective larvae, 114–115
possible mechanisms of action, TEs. See Transposable elements (TEs)
300–309 Tetrahydropyrimidines, 195, 471–472
SAR studies, 300–309 Th 2-type immune response. See T helper
tannin analysing by thiolysis, 308t 2-type immune response
tannin composition in plants, (Th 2-type immune response)
303t–307t Th2-inducing adjuvants, 372
Tannin(s) Thiol Sepharose-binding protein (TSBP),
AH properties and related polyphenols, 370
272 Thiol-binding fraction, 373
analysing by thiolysis, 308t Third generation of inhibitors, 502–503
analytical tools for, 271–272 Third-stage larvae (L3), 50–51, 72,
chemistry and related polyphenols, 271 149–150, 242b–243b, 274, 415
flavonoids and, 296f identification of infective, 150–151
tannin-neutralizing compounds, 271–272 Thrombospondin, 366
variation in plant composition, 295–298 Thymic stromal lymphopoietin (TSLP),
TAPS. See Tpx-1/Ag5/PR-1/Sc7 (TAPS) 169
646 Index
Toxocara canis (T. canis), 81 b-Tubulin, 468–469, 601–602

Tpx-1/Ag5/PR-1/Sc7 (TAPS), 537–538 anthelmintic benzimidazoles, 82–83
Trace elements, supplementation with, microtubuli, 82
266–267 a-Tubulin dimers, 71, 82–83
Traditional PCR, 157–159 b-Tubulin dimers, 71, 82–83, 88
Trans-splicing, 587–588 Two-dimensional gels (2-D gels), 55–56
Transcription activator–like effector Tzalam (Lysiloma latisiliquum), 298–299
nucleases (TALENs), 613–614
Transcriptome information, 600–601 U
Transgenes, 526–527 Ubiquinone, 77
Transmission electron microscopy (TEM), UDP-glucuronosyl transferases (UGTs),
298 89, 541
Transposable elements (TEs), 582, UGTs. See UDP-glucuronosyl transferases
588–589 (UGTs)
Transthyretin-like proteins (TTL Ultra-performance liquid chromatography–
proteins), 530–531 tandem mass spectrometry (UPLC/
Tribendimidine, 84 MS/MS), 271–272
Tricarboxylic acid cycle (TCA cycle), Ultraviolet (UV), 116
70–71 UMBs. See Urea-molasses blocks (UMBs)
Trichinella spp., 147 UNC-63, 85, 89
T. spiralis, 583 UNC-68, 85
Trichostrongylus colubriformis Underdosing with anthelmintics, 198–199
(T. colubriformis), 76–77, 362, 609 30 -Untranslated regions (30 -UTR),
Trichostrongylus tenuis (T. tenuis), 35–36 611–612
Tropical climates, 100, 117–119 UPLC/MS/MS. See Ultra-performance
Tropical legumes, dissecting complexity liquid chromatography–tandem
of, 320–321 mass spectrometry (UPLC/MS/
GIN infection, 323–324 MS)
tannin-rich foliage, 324 Urea-molasses blocks (UMBs), 256
in vitro AH activity of P. icosandra, 30 -UTR. See 30 -Untranslated regions
321–322 (30 -UTR)
in vivo studies, 322–323 UV. See Ultraviolet (UV)
Tropical regions, 125–126
Truncated nAChR subunit genes, V
406–407 Vaccines, 211–213
Tryptases. See Tryptic peptidases development, 365–366
Tryptic peptidases, 168 Barbervax, 369–370
TSBP. See Thiol Sepharose-binding E/S antigens, 372–374
protein (TSBP) GA1, 371
TSLP. See Thymic stromal lymphopoietin H-gal-GP, 366–367
(TSLP) H11, 368–369
TST. See Targeted selective treatment L3 surface antigen, 371–372
(TST) TSBP, 370
TTL proteins. See Transthyretin-like research, 524–525
proteins (TTL proteins) Vertebrate parasites, 579–580
tub 12–16, 601–602 Veterinary helminthology, 270
Tubulin, 81–82 Voluntary feed intake (VFI), 244–245
Index 647
W Worm-resistant animals, 220–221

WAAVP. See World Association for the WormBase, 525–528
Advancement of Veterinary WormBase ParaSite, 589
Parasitology (WAAVP) WRS strain, 56
Warm
climates, 119–120 X
temperate climates, 127–128 Xenobiotic detoxification, 541
temperate regions, 100–104 Xenobiotics, 443–444
Wellcome Trust Sanger Institute, 589 xL3s. See Exsheathed third stage larvae
Western blotting, 165–166 (xL3s)
Wet tropical zones, 214–218
Whole-organism or mechanism-based Z
screens, 440 Zinc metalloproteinases, 377–378
Wingless-type genes (wnt genes), 611
World Association for the Advancement of
Veterinary Parasitology
(WAAVP), 398
CONTENTS OF VOLUMES IN THIS SERIES
Volume 41 Volume 43
Drug Resistance in Malaria Parasites of Genetic Exchange in the Trypanosomatidae
Animals and Man W. Gibson and J. Stevens
W. Peters
The Host-Parasite Relationship in Neosporosis
Molecular Pathobiology and Antigenic A. Hemphill
Variation of Pneumocystis carinii
Y. Nakamura and M. Wada Proteases of Protozoan Parasites
P.J. Rosenthal
Ascariasis in China
P. Weidono, Z. Xianmin and Proteinases and Associated Genes of Parasitic
D.W.T. Crompton Helminths
J. Tort, P.J. Brindley, D. Knox,
The Generation and Expression of Immunity K.H. Wolfe, and J.P. Dalton
to Trichinella spiralis in Laboratory Rodents
R.G. Bell Parasitic Fungi and their Interaction with the
Insect Immune System
Population Biology of Parasitic Nematodes: A. Vilcinskas and P. G€otz
Application of Genetic Markers
T.J.C. Anderson, M.S. Blouin Volume 44
and R.M. Brech
Cell Biology of Leishmania
Schistosomiasis in Cattle B. Handman
J. De Bont and J. Vercruysse
Immunity and Vaccine Development in the
Volume 42 Bovine Theilerioses
N. Boulter and R. Hall
The Southern Cone Initiative Against Chagas
The Distribution of Schistosoma bovis Sonaino,
Disease
1876 in Relation to Intermediate Host
C. J. Schofield and J.C.P. Dias
Mollusc-Parasite Relationships
Phytomonas and Other Trypanosomatid H. Moné, G. Mouahid, and S. Morand
Parasites of Plants and Fruit
The Larvae of Monogenea (Platyhelminthes)
E.P. Camargo
H.D. Whittington, L.A. Chisholm, and
Paragonimiasis and the Genus Paragonimus K. Rohde
D. Blair, Z.-B. Xu, and T. Agatsuma
Sealice on Salmonids: Their Biology and
Immunology and Biochemistry of Hymenolepis Control
diminuta A.W. Pike and S.L. Wadsworth
J. Anreassen, E.M. Bennet-Jenkins, and
C. Bryant Volume 45
Control Strategies for Human Intestinal The Biology of some Intraerythrocytic
Nematode Infections Parasites of Fishes, Amphibia and Reptiles
M. Albonico, D.W.T. Cromption, and A.J. Davies and M.R.L. Johnston
L. Savioli
The Range and Biological Activity of FMR
DNA Vaocines: Technology and Applications Famide-related Peptides and Classical
as Anti-parasite and Anti-microbial Agents Neurotransmitters in Nematodes
J.B. Alarcon, G.W. Wainem and D. Brownlee, L. Holden-Dye,
D.P. McManus and R. Walker
649 j
650 Contents of Volumes in This Series
The Immunobiology of Gastrointestinal Forecasting Diseases Risk for Increased

Nematode Infections in Ruminants Epidemic Preparedness in Public Health
A. Balic, V.M. Bowles, and E.N.T. Meeusen M.F. Myers, D.J. Rogers, J. Cox, A. Flauhalt,
and S.I. Hay
Volume 46 Education, Outreach and the Future of

Remote Sensing in Human Health
Host-Parasite Interactions in Acanthocephala: B.L. Woods, L.R. Beck, B.M. Lobitz, and
A Morphological Approach M.R. Bobo
H. Taraschewski
Eicosanoids in Parasites and Parasitic Infections Volume 48
A. Daugschies and A. Joachim The Molecular Evolution of
Trypanosomatidae
Volume 47 J.R. Stevens, H.A. Noyes, C.J. Schofield, and
W. Gibson
An Overview of Remote Sensing and Geodesy
for Epidemiology and Public Health Transovarial Transmission in the Microsporidia
Application A.M. Dunn, R.S. Terry, and J.E. Smith
S.I. Hay Adhesive Secretions in the Platyhelminthes
Linking Remote Sensing, Land Cover and I.D. Whittington and B.W. Cribb
Disease The Use of Ultrasound in Schistosomiasis
P.J. Curran, P.M. Atkinson, G.M. Foody, and C.F.R. Hatz
E.J. Milton
Ascaris and Ascariasis
Spatial Statistics and Geographic Information D.W.T. Crompton
Systems in Epidemiology and Public
Health Volume 49
T.P. Robinson
Antigenic Variation in Trypanosomes:
Satellites, Space, Time and the African Enhanced Phenotypic Variation in a
Trypanosomiases Eukaryotic Parasite
D.J. Rogers H.D. Barry and R. McCulloch
Earth Observation, Geographic Information The Epidemiology and Control of Human
Systems and Plasmodium falciparum Malaria African Trypanosomiasis
in Sub-Saharan Africa J. Pépin and H.A. Méda
S.I. Hay, J. Omumbo, M. Craig, and
R.W. Snow Apoptosis and Parasitism: from the Parasite to
the Host Immune Response
Ticks and Tick-borne Disease Systems in Space G.A. DosReis and M.A. Barcinski
and from Space
S.E. Randolph Biology of Echinostomes Except Echinostoma
B. Fried
The Potential of Geographical Information
Systems (GIS) and Remote Sensing in the
Epidemiology and Control of Human
Volume 50
Helminth Infections The Malaria-Infected Red Blood Cell:
S. Brooker and E. Michael Structural and Functional Changes
B.M. Cooke, N. Mohandas, and R.L. Coppel
Advances in Satellite Remote Sensing
of Environmental Variables for Schistosomiasis in the Mekong Region:
Epidemiological Applications Epidemiology and Phytogeography
S.J. Goetz, S.D. Prince, and J. Small S.W. Attwood
Contents of Volumes in This Series 651
Molecular Aspects of Sexual Development Enzymes Involved in the Biogenesis of the

and Reproduction in Nematodes and Nematode Cuticle
Schistosomes A.P. Page and A.D. Winter
P.R. Boag, S.E. Newton, and R.B. Gasser
Diagnosis of Human Filariases (Except
Antiparasitic Properties of Medicinal Plants and Onchocerciasis)
Other Naturally Occurring Products M. Walther and R. Muller
S. Tagboto and S. Townson
Volume 54
Volume 51 Introduction d Phylogenies, Phylogenetics,
Aspects of Human Parasites in which Surgical Parasites and the Evolution of Parasitism
Intervention May Be Important D.T.J. Littlewood
D.A. Meyer and B. Fried
Cryptic Organelles in Parasitic Protists and Fungi
Electron-transfer Complexes in Ascaris B.A.P. Williams and P.J. Keeling
Mitochondria
K. Kita and S. Takamiya Phylogenetic Insights into the Evolution
of Parasitism in Hymenoptera
Cestode Parasites: Application of In Vivo and J.B. Whitfield
In Vitro Models for Studies of the
Host-Parasite Relationship Nematoda: Genes, Genomes and the
M. Siles-Lucas and A. Hemphill Evolution of Parasitism
M.L. Blaxter
Volume 52 Life Cycle Evolution in the Digenea: A New

Perspective from Phylogeny
The Ecology of Fish Parasites with Particular T.H. Cribb, R.A. Bray, P.D. Olson, and
Reference to Helminth Parasites and their D.T. J. Littlewood
Salmonid Fish Hosts in Welsh Rivers:
Progress in Malaria Research: The Case for
A Review of Some of the Central
Phylogenetics
Questions
S.M. Rich and F.J. Ayala
J.D. Thomas
Phylogenies, the Comparative Method and
Biology of the Schistosome Genus
Parasite Evolutionary Ecology
Trichobilharzia
S. Morand and R. Poulin
P. Horak, L. Kolarova, and C.M. Adema
Recent Results in Cophylogeny Mapping
The Consequences of Reducing Transmission
M.A. Charleston
of Plasmodium falciparum in Africa
R.W. Snow and K. Marsh Inference of Viral Evolutionary Rates from
Molecular Sequences
Cytokine-Mediated Host Responses during
A. Drummond, O.G. Pybus, and A. Rambaut
Schistosome Infections: Walking the Fine
Line Between Immunological Control Detecting Adaptive Molecular Evolution:
and Immunopathology Additional Tools for the Parasitologist
K.F. Hoffmann, T.A. Wynn, and J.O. Mclnerney, D.T.J. Littlewood, and
D.W. Dunne C.J. Creevey
Volume 53 Volume 55
Interactions between Tsetse and Contents of Volumes 28–52
Trypanosomes with Implications for the Cumulative Subject Indexes for Volumes
Control of Trypanosomiasis 28–52
S. Aksoy, W.C. Gibson, and M.J. Lehane Contributors to Volumes 28–52
Volume 56 Variation in Giardia: Implications for

Taxonomy and Epidemiology
Glycoinositolphospholipid from Trypanosoma R.C.A. Thompson and P.T. Monis
cruzi: Structure, Biosynthesis and
Immunobiology Recent Advances in the Biology of Echinostoma
J.O. Previato, R. Wait, C. Jones, species in the “revolutum” Group
G.A. DosReis, A.R. Todeschini, N. Heise B. Fried and T.K. Graczyk
and L.M. Previata Human Hookworm Infection in the
Biodiversity and Evolution of the Myxozoa 21st Century
E.U. Canning and B. Okamura S. Brooker, J. Bethony, and P.J. Hotez
The Mitochondrial Genomics of Parasitic The Curious Life-Style of the Parasitic Stages
Nematodes of Socio-Economic of Gnathiid Isopods
Importance: Recent Progress, and N.J. Smit and A.J. Davies
Implications for Population Genetics
and Systematics
M. Hu, N.B. Chilton, and R.B. Gasser
Volume 59
Genes and Susceptibility to Leishmaniasis
The Cytoskeleton and Motility in
Emanuela Handman, Colleen Elso, and Simon
Apicomplexan Invasion
Foote
R.E. Fowler, G. Margos, and G.H. Mitchell
Cryptosporidium and Cryptosporidiosis
Volume 57 R.C.A. Thompson, M.E. Olson, G. Zhu,
S. Enomoto, Mitchell S. Abrahamsen and
Canine Leishmaniasis N.S. Hijjawi
J. Alvar, C. Ca~navate, R. Molina, J. Moreno,
and J. Nieto Ichthyophthirius multifiliis Fouquet and
Ichthyophthiriosis in Freshwater Teleosts
Sexual Biology of Schistosomes R.A. Matthews
H. Moné and J. Boissier
Biology of the Phylum Nematomorpha
Review of the Trematode Genus Ribeiroia B. Hanelt, F. Thomas, and A. Schmidt-Rhaesa
(Psilostomidae): Ecology, Life History,
and Pathogenesis with Special Emphasis
on the Amphibian Malformation Problem Volume 60
P.T.J. Johnson, D.R. Sutherland, J.M. Kinsella Sulfur-Containing Amino Acid Metabolism
and K.B. Lunde in Parasitic Protozoa
The Trichuris muris System: A Paradigm of Tomoyoshi Nozaki, Vahab Ali, and Masaharu
Resistance and Susceptibility to Intestinal Tokoro
Nematode Infection The Use and Implications of Ribosomal DNA
L.J. Cliffe and R.K. Grencis Sequencing for the Discrimination of
Scabies: New Future for a Neglected Disease Digenean Species
S.F. Walton, D.C. Holt, B.J. Currie, Matthew J. Nolan and Thomas H. Cribb
and D.J. Kemp Advances and Trends in the Molecular
Systematics of the Parasitic
Volume 58 Platyhelminthes
Peter D. Olson and Vasyl V. Tkach
Leishmania spp.: On the Interactions they
Establish with Antigen-Presenting Cells Wolbachia Bacterial Endosymbionts of Filarial
of their Mammalian Hosts Nematodes
J.-C. Antoine, E. Prina, N. Courret, and Mark J. Taylor, Claudio Bandi, and
T. Lang Achim Hoerauf
The Biology of Avian Eimeria with an Emphasis Implementation of Human Schistosomiasis

on their Control by Vaccination Control: Challenges and Prospects
Martin W. Shirley, Adrian L. Smith, and Alan Fenwick, David Rollinson, and
Fiona M. Tomley Vaughan Southgate
Volume 62
Volume 61
Models for Vectors and Vector-Borne Diseases
Control of Human Parasitic Diseases: Context D.J. Rogers
and Overview
David H. Molyneux Global Environmental Data for Mapping
Infectious Disease Distribution
Malaria Chemotherapy S.I. Hay, A.J. Tatem, A.J. Graham,
Peter Winstanley and Stephen Ward S.J. Goetz, and D.J. Rogers
Insecticide-Treated Nets Issues of Scale and Uncertainty in the Global
Jenny Hill, Jo Lines, and Mark Rowland Remote Sensing of Disease
Control of Chagas Disease P.M. Atkinson and A.J. Graham
Yoichi Yamagata and Jun Nakagawa Determining Global Population Distribution:
Human African Trypanosomiasis: Methods, Applications and Data
Epidemiology and Control D.L. Balk, U. Deichmann, G. Yetman,
E.M. Févre, K. Picozzi, J. Jannin, F. Pozzi, S.I. Hay, and A. Nelson
S.C. Welburn and I. Maudlin Defining the Global Spatial Limits of Malaria
Chemotherapy in the Treatment and Control Transmission in 2005
of Leishmaniasis C.A. Guerra, R.W. Snow and
Jorge Alvar, Simon Croft, and Piero Olliaro S.I. Hay
Dracunculiasis (Guinea Worm Disease) The Global Distribution of Yellow Fever and
Eradication Dengue
Ernesto Ruiz-Tiben and Donald R. Hopkins D.J. Rogers, A.J. Wilson, S.I. Hay, and
A.J. Graham
Intervention for the Control of
Soil-Transmitted Helminthiasis in Global Epidemiology, Ecology and Control
the Community of Soil-Transmitted Helminth Infections
Marco Albonico, Antonio Montresor, S. Brooker, A.C.A. Clements and
D.W.T. Crompton, and Lorenzo D.A.P. Bundy
Savioli Tick-borne Disease Systems: Mapping
Control of Onchocerciasis Geographic and Phylogenetic Space
Boakye A. Boatin and S.E. Randolph and D.J. Rogers
Frank O. Richards, Jr. Global Transport Networks and Infectious
Lymphatic Filariasis: Treatment, Control and Disease Spread
Elimination A.J. Tatem, D.J. Rogers and S.I. Hay
Eric A. Ottesen Climate Change and Vector-Borne Diseases
Control of Cystic Echinococcosis/Hydatidosis: D.J. Rogers and S.E. Randolph
1863-2002
P.S. Craig and E. Larrieu Volume 63
Control of Taenia solium Cysticercosis/ Phylogenetic Analyses of Parasites in the New
Taeniosis Millennium
Arve Lee Willingham III and Dirk Engels David A. Morrison
Targeting of Toxic Compounds to the T. Sicheritz-Ponten, P. G. Foster,

Trypanosome’s Interior J. Samuelson, C.J. Noël, R.P. Hirt,
Michael P. Barrett and Ian H. Gilbert T.M. Embley, C. A. Gilchrist,
B.J. Mann, U. Singh, J.P. Ackers,
Making Sense of the Schistosome Surface S. Bhattacharya, A. Bhattacharya,
Patrick J. Skelly and R. Alan Wilson A. Lohia, N. Guillén, M. Duchene,
Immunology and Pathology of Intestinal T. Nozaki, and N. Hall
Trematodes in Their Definitive Hosts Epidemiological Modelling for Monitoring
Rafael Toledo, José-Guillermo Esteban, and and Evaluation of Lymphatic Filariasis
Bernard Fried Control
Systematics and Epidemiology of Trichinella Edwin Michael, Mwele N. Malecela-Lazaro,
Edoardo Pozio and K. Darwin Murrell and James W. Kazura
The Role of Helminth Infections in
Volume 64 Carcinogenesis
David A. Mayer and Bernard Fried
Leishmania and the Leishmaniases: A Parasite
A Review of the Biology of the
Genetic Update and Advances in
Parasitic Copepod Lernaeocera
Taxonomy, Epidemiology and
branchialis (L., 1767)(Copepoda:
Pathogenicity in Humans
Pennellidae
Anne-Laure Ba~nuls, Mallorie Hide and
Adam J. Brooker, Andrew P. Shinn, and
Franck Prugnolle
James E. Bron
Human Waterborne Trematode and
Protozoan Infections Volume 66
Thaddeus K. Graczyk and Bernard Fried
Strain Theory of Malaria: The First 50 Years
The Biology of Gyrodctylid Monogeneans: F. Ellis McKenzie,* David L. Smith,
The “Russian-Doll Killers” Wendy P. O’Meara, and
T.A. Bakke, J. Cable, and P.D. Harris Eleanor M. Riley
Human Genetic Diversity and the Advances and Trends in the Molecular
Epidemiology of Parasitic and Other Systematics of Anisakid Nematodes, with
Transmissible Diseases Implications for their Evolutionary
Michel Tibayrenc Ecology and HostParasite
Co-evolutionary Processes
Simonetta Mattiucci and Giuseppe Nascetti
Volume 65
Atopic Disorders and Parasitic Infections
ABO Blood Group Phenotypes and Aditya Reddy and Bernard Fried
Plasmodium falciparum Malaria: Unlocking
a Pivotal Mechanism Heartworm Disease in Animals and Humans
María-Paz Loscertales, Stephen Owens, John W. McCall, Claudio Genchi, Laura
James O’Donnell, James Bunn, H. Kramer, Jorge Guerrero, and
Xavier Bosch-Capblanch, and Luigi Venco
Bernard J. Brabin
Structure and Content of the Entamoeba Volume 67
histolytica Genome Introduction
C.G. Clark, U.C.M. Alsmark, Irwin W. Sherman
M. Tazreiter, Y. Saito-Nakano, V. Ali,
S. Marion, C. Weber, C. Mukherjee, An Introduction to Malaria Parasites
I. Bruchhaus, E. Tannich, M. Leippe, Irwin. W. Sherman
The Early Years Polyamines

Irwin W. Sherman Irwin W. Sherman
Show Me the Money New Permeability Pathways and Transport
In Vivo and In Vitro Models Hemoglobinases
Malaria Pigment Erythrocyte Surface Membrane Proteins
Chloroquine and Hemozoin Trafficking
Invasion of Erythrocytes Erythrocyte Membrane Lipids
Vitamins and Anti-Oxidant Defenses
Irwin W. Sherman
Volume 68
HLA-Mediated Control of HIV and HIV
Shocks and Clocks
Adaptation to HLA
Irwin W. Sherman
Rebecca P. Payne, Philippa C. Matthews,
Transcriptomes, Proteomes and Data Julia G. Prado, and Philip J.R. Goulder
Mining
An Evolutionary Perspective on Parasitism as a
Irwin W. Sherman
Cause of Cancer
Mosquito Interactions Paul W. Ewald
Irwin W. Sherman
Invasion of the Body Snatchers: The Diversity
Isoenzymes and Evolution of Manipulative Strategies
Irwin W. Sherman in HostParasite Interactions
Thierry Lefévre, Shelley A. Adamo, David G.
The Road to the Plasmodium falciparum Biron, Dorothée Missé , David Hughes, and
Genome Frédéric Thomas
Irwin W. Sherman
Evolutionary Drivers of Parasite-Induced
Carbohydrate Metabolism Changes in Insect Life-History Traits:
Irwin W. Sherman From Theory to Underlying Mechanisms
Pyrimidines and the Mitochondrion Hilary Hurd
Irwin W. Sherman Ecological Immunology of a Tapeworms’
The Road to Atovaquone Interaction with its Two Consecutive
Irwin W. Sherman Hosts
Katrin Hammerschmidt and
The Ring Road to the Apicoplast Joachim Kurtz
Irwin W. Sherman
Tracking Transmission of the Zoonosis
Ribosomes and Ribosomal Ribonucleic Acid Toxoplasma gondii
Synthesis Judith E. Smith
Irwin W. Sherman
Parasites and Biological Invasions
De Novo Synthesis of Pyrimidines and Folates Alison M. Dunn
Irwin W. Sherman
Zoonoses in Wildlife: Integrating Ecology into
Salvage of Purines Management
Irwin W. Sherman Fiona Mathews
Understanding the Interaction Between an Volume 70

Obligate Hyperparasitic Bacterium,
Pasteuria penetrans and its Obligate Ecology and Life History Evolution of
Plant-Parasitic Nematode Host, Frugivorous Drosophila Parasitoids
Meloidogyne spp. Frédéric Fleury, Patricia Gibert, Nicolas Ris, and
Keith G. Davies Roland Allemand
HostParasite Relations and Implications for Decision-Making Dynamics in Parasitoids of

Control Drosophil
Alan Fenwick Andra Thiel and Thomas S. Hoffmeister
OnchocercaSimulium Interactions and the Dynamic Use of Fruit Odours to Locate Host
Population and Evolutionary Biology of Larvae: Individual Learning, Physiological
Onchocerca volvulus State and Genetic Variability as Adaptive
María-Gloria Basa~nez, Thomas S. Churcher, Mechanisms
and María-Eugenia Grillet Laure Kaiser, Aude Couty, and
Raquel Perez-Maluf
Microsporidians as Evolution-Proof Agents of
Malaria Control? The Role of Melanization and Cytotoxic
Jacob C. Koella, Lena Lorenz, and By-Products in the Cellular Immune
Irka Bargielowski Responses of Drosophila Against Parasitic
Wasps
A. Nappi, M. Poirié, and Y. Carton
Virulence Factors and Strategies of Leptopilina
Volume 69 spp.: Selective Responses in Drosophila
Hosts
The Biology of the Caecal Trematode
Mark J. Lee, Marta E. Kalamarz,
Zygocotyle lunata
Indira Paddibhatla, Chiyedza Small,
Bernard Fried, Jane E. Huffman, Shamus Keeler,
Roma Rajwani, and Shubha Govind
and Robert C. Peoples
Variation of Leptopilina boulardi Success in
Fasciola, Lymnaeids and Human Fascioliasis,
Drosophila Hosts: What is Inside the Black
with a Global Overview on Disease
Box?
Transmission, Epidemiology,
A. Dubuffet, D. Colinet, C. Anselme,
Evolutionary Genetics, Molecular
S. Dupas, Y. Carton, and M. Poirié
Epidemiology and Control
Santiago Mas-Coma, María Adela Valero, and Immune Resistance of Drosophila Hosts Against
María Dolores Bargues Asobara Parasitoids: Cellular Aspects
Patrice Eslin, Genevieve Prévost, Sebastien
Recent Advances in the Biology of
Havard, and Géraldine Doury
Echinostomes
Rafael Toledo, José-Guillermo Esteban, and Components of Asobara Venoms and their
Bernard Fried Effects on Hosts
Sébastien J.M. Moreau, Sophie Vinchon, Anas
Peptidases of Trematodes
Cherqui, and Genevieve Prévost
Martin Kasný, Libor Mikes, Vladimír Hampl,
Jan Dvorak, Conor R. Caffrey, John P. Strategies of Avoidance of Host Immune
Dalton, and Petr Horak Defenses in Asobara Species
Geneviéve Prevost, Géraldine Doury, Alix D.N.
Potential Contribution of
Mabiala-Moundoungou, Anas Cherqui, and
Sero-Epidemiological Analysis for
Patrice Eslin
Monitoring Malaria Control and
Elimination: Historical and Current Evolution of Host Resistance and Parasitoid
Perspectives Counter-Resistance
Chris Drakeley and Jackie Cook Alex R. Kraaijeveld and H. Charles J. Godfray
Local, Geographic and Phylogenetic Scales of Coordinating Research on Neglected Parasitic

Coevolution in DrosophilaParasitoid Diseases in Southeast Asia Through
Interactions Networking
S. Dupas, A. Dubuffet, Y. Carton, and Remi Olveda, Lydia Leonardo, Feng Zheng,
M. Poirié Banchob Sripa, Robert Bergquist, and
Xiao-Nong Zhou
DrosophilaParasitoid Communities as
Model Systems for HostWolbachia Neglected Diseases and Ethnic Minorities in
Interactions the Western Pacific Region: Exploring
Fabrice Vavre, Laurence Mouton, and Bart the Links
A. Pannebakker Alexander Schratz, Martha
Fernanda Pineda, Liberty G. Reforma,
A Virus-Shaping Reproductive Strategy in a Nicole M. Fox, Tuan Le Anh,
Drosophila Parasitoid L. Tommaso Cavalli-Sforza,
Julien Varaldi, Sabine Patot, Mackenzie K. Henderson,
Maxime Nardin, and Raymond Mendoza, J€urg Utzinger,
Sylvain Gandon John P. Ehrenberg, and
Ah Sian Tee
Volume 71 Controlling Schistosomiasis in Southeast Asia:
A Tale of Two Countries
Cryptosporidiosis in Southeast Robert Bergquist and Marcel Tanner
Asia: What’s out There?
Yvonne A.L. Lim, Aaron R. Jex, Schistosomiasis Japonica: Control and
Huw V. Smith, and Robin B. Gasser Research Needs
Xiao-Nong Zhou, Robert Bergquist,
Human Schistosomiasis in the Economic Lydia Leonardo, Guo-Jing Yang,
Community of West African States: Kun Yang, M. Sudomo, and
Epidemiology and Control Remigio Olveda
Hélené Moné, Moudachirou Ibikounlé,
Achille Massougbodji, Schistosoma mekongi in Cambodia and Lao
and Gabriel Mouahid People’s Democratic Republic
Sinuon Muth, Somphou Sayasone,
The Rise and Fall of Human Sophie Odermatt-Biays,
Oesophagostomiasis Samlane Phompida, Socheat Duong, and
A.M. Polderman, M. Eberhard, S. Baeta, Peter Odermatt
Robin B. Gasser, L. van Lieshout,
P. Magnussen, A. Olsen, N. Spannbrucker, Elimination of Lymphatic Filariasis in
J. Ziem, and J. Horton Southeast Asia
Mohammad Sudomo, Sombat
Chayabejara, Duong Socheat,
Volume 72 Leda Hernandez, Wei-Ping Wu, and
Robert Bergquist
Important Helminth Infections in Southeast
Asia: Diversity, Potential for Control and Combating Taenia solium Cysticercosis in
Prospects for Elimination Southeast Asia: An Opportunity for
J€urg Utzinger, Robert Bergquist, Remigio Improving Human Health and Livestock
Olveda, and Xiao-Nong Zhou Production Links
A. Lee Willingham III, Hai-Wei Wu,
Escalating the Global Fight Against James Conlan, and Fadjar Satrija
Neglected Tropical Diseases Through
Interventions in the Asia Pacific Echinococcosis with Particular Reference to
Region Southeast Asia
Peter J. Hotez and John P. Ehrenberg Donald P. McManus
Food-Borne Trematodiases in Southeast Asia: Towards Improved Diagnosis of

Epidemiology, Pathology, Clinical Zoonotic Trematode Infections in
Manifestation and Control Southeast Asia
Banchob Sripa, Sasithorn Kaewkes, Pewpan M. Maria Vang Johansen, Paiboon
Intapan, Wanchai Maleewong, and Sithithaworn, Robert Bergquist, and
Paul J. Brindley J€urg Utzinger
Helminth Infections of the Central Nervous The Drugs We Have and the Drugs
System Occurring in Southeast Asia and We Need Against Major Helminth
the Far East Infections
Shan Lv, Yi Zhang, Peter Steinmann, Jennifer Keiser and J€urg Utzinger
Xiao-Nong Zhou, and J€urg Utzinger
Research and Development of
Less Common Parasitic Infections in Southeast Antischistosomal Drugs in the People’s
Asia that can Produce Outbreaks Republic of China: A 60-Year Review
Peter Odermatt, Shan Lv, and Somphou Sayasone Shu-Hua Xiao, Jennifer Keiser,
Ming-Gang Chen, Marcel Tanner,
and J€urg Utzinger
Volume 73
Control of Important Helminthic Infections:
Concepts in Research Capabilities Vaccine Development as Part of the
Strengthening: Positive Experiences of Solution
Network Approaches by TDR in the Robert Bergquist and Sara Lustigman
People’s Republic of China and
Eastern Asia Our Wormy World: Genomics, Proteomics
Xiao-Nong Zhou, Steven Wayling, and and Transcriptomics in East and Southeast
Robert Bergquist Asia
Jun Chuan, Zheng Feng,
Multiparasitism: A Neglected Reality on Paul J. Brindley, Donald P. McManus,
Global, Regional and Local Scale Zeguang Han, Peng Jianxin, and Wei Hu
Peter Steinmann, J€urg Utzinger,
Zun-Wei Du, and Xiao-Nong Zhou Advances in Metabolic Profiling of
Experimental Nematode and Trematode
Health Metrics for Helminthic Infections Infections
Charles H. King Yulan Wang, Jia V. Li, Jasmina Saric, Jennifer
Implementing a Geospatial Health Data Keiser, Junfang Wu, J€urg Utzinger, and
Infrastructure for Control of Asian Elaine Holmes
Schistosomiasis in the People’s Republic Studies on the Parasitology, Phylogeography
of China and the Philippines and the Evolution of HostParasite
John B. Malone, Guo-Jing Yang, Lydia Interactions for the Snail Intermediate
Leonardo, and Xiao-Nong Zhou Hosts of Medically Important Trematode
The Regional Network for Asian Genera in Southeast Asia
Schistosomiasis and Other Helminth Stephen W. Attwood
Zoonoses (RNAS+): Target Diseases
in Face of Climate Change
Guo-Jing Yang, J€urg Utzinger, Shan Lv,
Volume 74
Ying-Jun Qian, Shi-Zhu Li, Qiang Wang, The Many Roads to Parasitism: A Tale of
Robert Bergquist, Penelope Vounatsou, Convergence
Wei Li, Kun Yang, and Xiao-Nong Zhou Robert Poulin
Social Science Implications for Control of Malaria Distribution, Prevalence, Drug
Helminth Infections in Southeast Asia Resistance and Control in Indonesia
Lisa M. Vandemark, Tie-Wu Jia, and Iqbal R.F. Elyazar, Simon I. Hay, and
Xiao-Nong Zhou J. Kevin Baird
Cytogenetics and Chromosomes of Advances in Imaging of Animal Models of

Tapeworms (Platyhelminthes, Cestoda) Chagas Disease
Marta Spakulova, Martina Orosova, and Linda A. Jelicks and Herbert B. Tanowitz
John S. Mackiewicz
The Genome and Its Implications
Soil-Transmitted Helminths of Humans in Santuza M. Teixeira, Najib M. El-Sayed,
Southeast AsiadTowards Integrated and Patrícia R. Araujo
Control
Aaron R. Jex, Yvonne A.L. Lim, Jeffrey Genetic Techniques in Trypanosoma cruzi
Bethony, Peter J. Hotez, Martin C. Taylor, Huan Huang, and
Neil D. Young, and Robin B. Gasser John M. Kelly
The Applications of Model-Based Geostatistics Nuclear Structure of Trypanosoma cruzi

in Helminth Epidemiology and Control Sergio Schenkman, Bruno dos Santos
Ricardo J. Soares Magalh~aes, Archie C.A. Pascoalino, and Sheila C. Nardelli
Clements, Anand P. Patil, Aspects of Trypanosoma cruzi Stage
Peter W. Gething, and Simon Brooker Differentiation
Samuel Goldenberg and Andrea
Volume 75
Rodrigues Avila
Epidemiology of American Trypanosomiasis The Role of Acidocalcisomes in the Stress

(Chagas Disease) Response of Trypanosoma cruzi
Louis V. Kirchhoff Roberto Docampo, Veronica Jimenez,
Sharon King-Keller, Zhu-hong Li, and
Acute and Congenital Chagas Disease Silvia N.J. Moreno
Caryn Bern, Diana L. Martin, and
Robert H. Gilman Signal Transduction in Trypanosoma cruzi
Huan Huang
Cell-Based Therapy in Chagas Disease
Antonio C. Campos de Carvalho,
Adriana B. Carvalho, and Regina Volume 76
C.S. Goldenberg Bioactive Lipids in Trypanosoma cruzi Infection
Targeting Trypanosoma cruzi Sterol Fabiana S. Machado, Shankar Mukherjee,
14a-Demethylase (CYP51) Louis M. Weiss, Herbert B. Tanowitz, and
Galina I. Lepesheva, Fernando Villalta, Anthony W. Ashton
and Michael R. Waterman Mechanisms of Host Cell Invasion by
Experimental Chemotherapy and Approaches Trypanosoma cruzi
to Drug Discovery for Trypanosoma cruzi Kacey L. Caradonna and Barbara
Infection A. Burleigh
Frederick S. Buckner Gap Junctions and Chagas Disease
Vaccine Development Against Trypanosoma Daniel Adesse, Regina Coeli Goldenberg, Fabio
cruzi and Chagas Disease S. Fortes, Jasmin, Dumitru A. Iacobas, Sanda
Juan C. Vazquez-Chagoyan, Iacobas, Antonio Carlos Campos de
Shivali Gupta, and Nisha Jain Garg Carvalho, Maria de Narareth
Meirelles, Huan Huang, Milena B. Soares,
Genetic Epidemiology of Chagas Disease Herbert B. Tanowitz, Luciana Ribeiro
Sarah Williams-Blangero, John L. VandeBerg, Garzoni, and David C. Spray
John Blangero, and Rodrigo Corrêa-Oliveira
The Vasculature in Chagas Disease
Kissing Bugs. The Vectors of Chagas Cibele M. Prado, Linda A. Jelicks,
Lori Stevens, Patricia L. Dorn, Justin O. Schmidt, Louis M. Weiss, Stephen M. Factor,
John H. Klotz, David Lucero, and Herbert B. Tanowitz, and
Stephen A. Klotz Marcos A. Rossi
Infection-Associated Vasculopathy in Assessment and Monitoring of Onchocerciasis

Experimental Chagas Disease: in Latin America
Pathogenic Roles of Endothelin and Mario A. Rodríguez-Pérez,
Kinin Pathways Thomas R. Unnasch, and
Julio Scharfstein and Daniele Andrade Olga Real-Najarro
Autoimmunity
Edecio Cunha-Neto, Priscila Camillo Volume 78
Teixeira, Luciana Gabriel Nogueira, Gene Silencing in Parasites: Current Status and
and Jorge Kalil Future Prospects
ROS Signalling of Inflammatory Cytokines Raul Manzano-Roman, Ana Oleaga,
During Trypanosoma cruzi Infection Ricardo Pérez-Sanchez, and
Shivali Gupta, Monisha Dhiman, Jian-jun Mar Siles-Lucas
Wen, and Nisha Jain Garg GiardiadFrom Genome to Proteome
Inflammation and Chagas Disease: Some R.C. Andrew Thompson and Paul Monis
Mechanisms and Relevance Malaria Ecotypes and Stratification
André Talvani and Mauro M. Teixeira Allan Schapira and Konstantina Boutsika
Neurodegeneration and Neuroregeneration in The Changing Limits and Incidence of Malaria
Chagas Disease in Africa: 19392009
Marina V. Chuenkova and Mercio Robert W. Snow, Punam Amratia,
PereiraPerrin Caroline W. Kabaria, Abdisalan M. Noor,
Adipose Tissue, Diabetes and Chagas Disease and Kevin Marsh
Herbert B. Tanowitz, Linda A. Jelicks,
Fabiana S. Machado, Lisia Esper, Volume 79
Xiaohua Qi, Mahalia S. Desruisseaux,
Streamson C. Chua, Philipp E. Scherer, Northern Host Parasite Assemblages:
and Fnu Nagajyothi History and Biogeography on the
Borderlands of Episodic Climate and
Environmental Transition
Volume 77 Eric P. Hoberg, Kurt E. Galbreath,
Joseph A. Cook, Susan J. Kutz, and
Coinfection of Schistosoma (Trematoda) with Lydden Polley
Bacteria, Protozoa and Helminths
Amy Abruzzi and Bernard Fried Parasites in Ungulates of Arctic North America
and Greenland: A View of Contemporary
Trichomonas vaginalis Pathobiology: New Diversity, Ecology and Impact in a World
Insights from the Genome Sequence Under Change
Robert P. Hirt, Natalia de Miguel, Susan J. Kutz, Julie Ducrocq, Guilherme
Sirintra Nakjang, Daniele Dessi, G. Verocai, Bryanne M. Hoar, Doug D.
Yuk-Chien Liu, Nicia Diaz, Colwell, Kimberlee B. Beckmen,
Paola Rappelli, Alvaro Acosta-Serrano, Lydden Polley, Brett T. Elkin, and
Pier-Luigi Fiori, and Jeremy C. Mottram Eric P. Hoberg
Cryptic Parasite Revealed: Improved Prospects Neorickettsial Endosymbionts of the Digenea:
for Treatment and Control of Human Diversity, Transmission and Distribution
Cryptosporidiosis Through Advanced Jefferson A. Vaughan, Vasyl V. Tkach, and
Technologies Stephen E. Greiman
Aaron R. Jex, Huw V. Smith, Matthew
J. Nolan, Bronwyn E. Campbell, Priorities for the Elimination of Sleeping
Neil D. Young, Cinzia Cantacessi, and Sickness
Robin B. Gasser Susan C. Welburn and Ian Maudlin
Scabies: Important Clinical Consequences Natural Acquisition of Immunity to

Explained by New Molecular Studies Plasmodium vivax: Epidemiological
Katja Fischer, Deborah Holt, Bart Currie, and Observations and Potential Targets
David Kemp Ivo Mueller, Mary R. Galinski, Takafumi
Tsuboi, Myriam Arevalo-Herrera,
Review: Surveillance of Chagas Disease William E. Collins, and
Ken Hashimoto and Kota Yoshioka Christopher L. King
Volume 80 G6PD Deficiency: Global Distribution,
Genetic Variants and Primaquine Therapy
The Global Public Health Significance of Rosalind E. Howes, Katherine E. Battle,
Plasmodium vivax Ari W. Satyagraha, J. Kevin Baird, and
Katherine E. Battle, Peter W. Gething, Simon I. Hay
Iqbal R.F. Elyazar,
Catherine L. Moyes, Maríanne E. Sinka, Genomics, Population Genetics and
Rosalind E. Howes, Carlos A. Guerra, Evolutionary History of Plasmodium vivax
Ric N. Price, J. Kevin Baird, and Jane M. Carlton, Aparup Das, and
Simon I. Hay Ananias A. Escalante
Relapse Malariotherapy Insanity at the Service of
Nicholas J. White and Mallika Imwong Malariology
Georges Snounou and Jean-Louis Pérignon
Plasmodium vivax: Clinical Spectrum, Risk
Factors and Pathogenesis
Nicholas M. Anstey, Nicholas M. Douglas, Volume 82
Jeanne R. Poespoprodjo, and
Ric N. Price Recent Developments in Blastocystis Research
C. Graham Clark, Mark van der Giezen,
Diagnosis and Treatment of Plasmodium vivax Mohammed A. Alfellani, and
Malaria C. Rune Stensvold
J. Kevin Baird, Jason D. Maguire, and
Ric N. Price Tradition and Transition: Parasitic Zoonoses of
People and Animals in Alaska, Northern
Chemotherapeutic Strategies for Canada, and Greenland
Reducing Transmission of Plasmodium Emily J. Jenkins, Louisa J. Castrodale,
vivax Malaria Simone J.C. de Rosemond, Brent R. Dixon,
Nicholas M. Douglas, George K. John, Stacey A. Elmore, Karen M. Gesy,
Lorenz von Seidlein, Nicholas M. Anstey, Eric P. Hoberg, Lydden Polley,
and Ric N. Price Janna M. Schurer, Manon Simard, and
R.C. Andrew Thompson
Control and Elimination of Plasmodium vivax
G. Dennis Shanks The Malaria Transition on the Arabian
Peninsula: Progress toward a Malaria-Free
Volume 81 Region between 1960-2010
Robert W. Snow, Punam Amratia, Ghasem
Plasmodium vivax: Modern Strategies Zamani, Clara W. Mundia, Abdisalan M.
to Study a Persistent Parasite’s Noor, Ziad A. Memish, Mohammad H. Al
Life Cycle Zahrani, Adel AlJasari, Mahmoud Fikri, and
Mary R. Galinski, Esmeralda V.S. Meyer, and Hoda Atta
John W. Barnwell
Microsporidia and ‘The Art of Living
Red Blood Cell Polymorphism and Together’
Susceptibility to Plasmodium vivax Jir í Vavra and Julius Lukes
Peter A. Zimmerman, Marcelo U. Ferreira,
Rosalind E. Howes, and Odile Patterns and Processes in Parasite Co-Infection
Mercereau-Puijalon Mark E. Viney and Andrea L. Graham
Volume 83 Volume 85
IronSulphur Clusters, Their Biosynthesis, Diversity and Ancestry of Flatworms Infecting
and Biological Functions in Protozoan Blood of Nontetrapod Craniates “Fishes”
Parasites Raphael Orélis-Ribeiro, Cova R. Arias,
Vahab Ali and Tomoyoshi Nozaki Kenneth M. Halanych,Thomas H. Cribb,
and Stephen A. Bullard
A Selective Review of Advances in Coccidiosis
Research Techniques for the Diagnosis of Fasciola
H. David Chapman, John R. Barta, Infections in Animals: Room for
Damer Blake, Arthur Gruber, Mark Jenkins, Improvement
Nicholas C. Smith, Xun Suo, and Cristian A. Alvarez Rojas, Aaron R. Jex,
Fiona M. Tomley Robin B. Gasser, and
Jean-Pierre Y. Scheerlinck
The Distribution and Bionomics of
Anopheles Malaria Vector Mosquitoes in Reevaluating the Evidence for Toxoplasma
Indonesia gondii-Induced Behavioural Changes in
Iqbal R.F. Elyazar, Marianne E. Sinka, Rodents
Peter W. Gething, Siti N. Tarmidzi, Amanda R. Worth, R.C. Andrew Thompson,
Asik Surya, Rita Kusriastuti, Winarno, and Alan J. Lymbery
J. Kevin Baird, Simon I. Hay, and
Michael J. Bangs Volume 86
Next-Generation Molecular-Diagnostic Tools Historical Patterns of Malaria Transmission in
for Gastrointestinal Nematodes of China
Livestock, with an Emphasis on Small Jian-Hai Yin, Shui-Sen Zhou, Zhi-Gui Xia,
Ruminants: A Turning Point? Ru-Bo Wang, Ying-Jun Qian, Wei-Zhong
Florian Roeber, Aaron R. Jex, and Yang, and Xiao-Nong Zhou
Robin B. Gasser
Feasibility and Roadmap Analysis for Malaria
Elimination in China
Volume 84 Xiao-Nong Zhou, Zhi-Gui Xia, Ru-Bo Wang,
Joint Infectious Causation of Human Ying-Jun Qian, Shui-Sen Zhou, J€urg
Cancers Utzinger, Marcel Tanner, Randall Kramer,
Paul W. Ewald and Holly A. Swain Ewald and Wei-Zhong Yang
Neurological and Ocular Fascioliasis in Lessons from Malaria Control to Elimination:

Humans Case Study in Hainan and Yunnan
Santiago Mas-Coma, Veronica H. Agramunt, Provinces
and María Adela Valero Zhi-Gui Xia, Li Zhang, Jun Feng, Mei Li,
Xin-Yu Feng, Lin-Hua Tang, Shan-Qing
Measuring Changes in Plasmodium falciparum Wang, Heng-Lin Yang, Qi Gao, Randall
Transmission: Precision, Accuracy and Kramer, Tambo Ernest, Peiling Yap, and
Costs of Metrics Xiao-Nong Zhou
Lucy S. Tusting, Teun Bousema, David
L. Smith, and Chris Drakeley Surveillance and Response to Drive the
National Malaria Elimination Programme
A Review of Molecular Approaches for Xin-Yu Feng, Zhi-Gui Xia, Sirenda Vong,
Investigating Patterns of Coevolution in Wei-Zhong Yang, and Shui-Sen Zhou
Marine HostParasite Relationships
G€otz Froeschke and Sophie von der Heyden Operational Research Needs Toward Malaria
Elimination in China
New Insights into Clonality and Panmixia in Shen-Bo Chen, Chuan Ju, Jun-Hu Chen,
Plasmodium and Toxoplasma Bin Zheng, Fang Huang, Ning Xiao, Xia
Michel Tibayrenc and Francisco J. Ayala Zhou, Tambo Ernest, and Xiao-Nong Zhou
Approaches to the Evaluation of Malaria Andrea Bosman, Robert David Newman,

Elimination at County Level: Case Study Tambo Ernest, Michael O’leary, and
in the Yangtze River Delta Region Ning Xiao
Min Zhu, Wei Ruan, Sheng-Jun Fei,
Jian-Qiang Song, Yu Zhang, Xiao-Gang
Mou, Qi-Chao Pan, Ling-Ling Zhang, Volume 87
Xiao-Qin Guo, Jun-Hua Xu, Tian-Ming The Allee Effect and Elimination of Neglected
Chen, Bin Zhou, Peiling Yap, Li-Nong Tropical Diseases: A Mathematical
Yao, and Li Cai Modelling Study
Surveillance and Response Strategy in the Manoj Gambhir, Brajendra K. Singh, and
Malaria Post-elimination Stage: Case Edwin Michael
Study of Fujian Province Mathematical Modelling of Leprosy and Its
Fa-Zhu Yang, Peiling Yap, Shan-Ying Zhang, Control
Han-Guo Xie, Rong Ouyang, Yao-Ying David J. Blok, Sake J. de Vlas,
Lin, and Zhu-Yun Chen Egil A.J. Fischer, and Jan Hendrik Richardus
Preparation of Malaria Resurgence in China: Mathematical Models of Human African
Case Study of Vivax Malaria Trypanosomiasis Epidemiology
Re-emergence and Outbreak in Kat S. Rock, Chris M. Stone,
Huang-Huai Plain in 2006 Ian M. Hastings, Matt J. Keeling,
Hong-Wei Zhang, Ying Liu, Shao-Sen Zhang, Steve J. Torr, and Nakul Chitnis
Bian-Li Xu, Wei-Dong Li, Ji-Hai Tang,
Shui-Sen Zhou, and Fang Huang Ecology, Evolution and Control of Chagas
Disease: A Century of Neglected
Preparedness for Malaria Resurgence in Modelling and a Promising Future
China: Case Study on Imported Cases in Pierre Nouvellet, Zulma M. Cucunuba,
2000–2012 and Sébastien Gourbiere
Jun Feng, Zhi-Gui Xia, Sirenda Vong,
Wei-Zhong Yang, Shui-Sen Zhou, and Mathematical Inference on Helminth Egg
Ning Xiao Counts in Stool and Its Applications in
Mass Drug Administration Programmes
Preparation for Malaria Resurgence in China: to Control Soil-Transmitted
Approach in Risk Assessment and Rapid Helminthiasis in Public Health
Response Bruno Levecke, Roy M. Anderson,
Ying-Jun Qian, Li Zhang, Zhi-Gui Xia, Dirk Berkvens, Johannes Charlier,
Sirenda Vong, Wei-Zhong Yang, Brecht Devleesschauwer, Niko Speybroeck,
Duo-Quan Wang, and Ning Xiao Jozef Vercruysse, and Stefan Van Aelst
Transition from Control to Elimination: Modelling Lymphatic Filariasis Transmission
Impact of the 10-Year Global Fund and Control: Modelling Frameworks,
Project on Malaria Control and Lessons Learned and Future Directions
Elimination in China Wilma A. Stolk, Chris Stone, and Sake J. de Vlas
Ru-Bo Wang, Qing-Feng Zhang, Bin Zheng,
Zhi-Gui Xia, Shui-Sen Zhou, Lin-Hua Modelling the Effects of Mass Drug
Tang, Qi Gao, Li-Ying Wang, and Administration on the Molecular
Rong-Rong Wang Epidemiology of Schistosomes
Poppy H.L. Lamberton, Thomas Crellen,
China–Africa Cooperation Initiatives in James A. Cotton, and Joanne P. Webster
Malaria Control and Elimination
Zhi-Gui Xia, Ru-Bo Wang, Duo-Quan Economic and Financial Evaluation of
Wang, Jun Feng, Qi Zheng, Chang-Sheng Neglected Tropical Diseases
Deng, Salim Abdulla, Ya-Yi Guan, Wei Bruce Y. Lee, Sarah M. Bartsch, and
Ding, Jia-Wen Yao, Ying-Jun Qian, Katrin M. Gorham
Volume 88 Volume 90
Recent Developments in Malaria The Importance of Fossils in Understanding
Vaccinology the Evolution of Parasites and Their
Benedict R. Halbroth and Vectors
Simon J. Draper Kenneth De Baets and D. Timothy J. Littlewood
PfEMP1 – A Parasite Protein Family of The Geological Record of Parasitic Nematode
Key Importance in Plasmodium Evolution
falciparum Malaria Immunity and George O. Poinar, Jr.
Pathogenesis
Lars Hviid and Anja TR. Jensen Constraining the Deep Origin of Parasitic
Flatworms and Host-Interactions with
Prospects for Vector-Based Gene Silencing to Fossil Evidence
Explore Immunobiological Features of Kenneth De Baets, Paula Dentzien-Dias,
Schistosoma mansoni Ieva Upeniece, Olivier Verneau, and
Jana Hagen, Jean-Pierre Y. Scheerlinck, Philip C.J. Donoghue
Neil D. Young, Robin B. Gasser, and
Bernd H. Kalinna From Fossil Parasitoids to Vectors: Insects as
Parasites and Hosts
Chronobiology of Trematode Cercarial Christina Nagler and Joachim T. Haug
Emergence: from Data Recovery to
Epidemiological, Ecological and Trace Fossil Evidence of Trematode–Bivalve
Evolutionary Implications Parasite–Host Interactions in Deep Time
André Théron John Warren Huntley and Kenneth De Baets
Strongyloidiasis with Emphasis on Human Fossil Crustaceans as Parasites and Hosts

Infections and Its Different Clinical Adiël A. Klompmaker and Geoff A. Boxshall
Forms A Prejudiced Review of Ancient Parasites and
Rafael Toledo, Carla Mu~noz-Antoli, and Their Host Echinoderms: CSI Fossil
José-Guillermo Esteban Record or Just an Excuse for Speculation?
A Perspective on Cryptosporidium and Giardia, Stephen K. Donovan
with an Emphasis on Bovines and Recent Differentiating Parasitism and Other
Epidemiological Findings Interactions in Fossilized Colonial
Harshanie Abeywardena, Aaron R. Jex, and Organisms
Robin B. Gasser Paul D. Taylor
Palaeoparasitology – Human Parasites in
Volume 89 Ancient Material
Ecology of Free-Living Metacercariae Adauto Araujo, Karl Reinhard, and
(Trematoda) Luiz Fernando Ferreira
Neil J. Morley Human Parasites in Medieval Europe:
Cross-Border Malaria: A Major Obstacle for Lifestyle, Sanitation and Medical
Malaria Elimination Treatment
Kinley Wangdi, Michelle L. Gatton, Gerard C. Piers D. Mitchell
Kelly, Archie CA. Clements
Development of Malaria Transmission-
Volume 91
Blocking Vaccines: From Concept to Malaria Parasite Proteins and Their Role in
Product Alteration of the Structure and Function
Yimin Wu, Robert E. Sinden, Thomas S. of Red Blood Cells
Churcher, Takafumi Tsuboi, Vidadi Nicholas I. Proellocks, Ross L. Coppel, Narla
Yusibov Mohandas, and Brian M. Cooke
Harnessing the Toxocara Genome to Underpin Epidemiological Features and Control

Toxocariasis Research and New Progress of Schistosomiasis in Waterway-
Interventions Network Region in P.R. China
Robin B. Gasser, Pasi K. Korhonen, L. Shi, W. Li, F. Wu, J.-F. Zhang, K. Yang,
Xing-Quan Zhu, and Neil D. Young and X.-N. Zhou
Coinfection of Schistosoma Species with The Establishment and Function of
Hepatitis B or Hepatitis C Viruses Schistosomiasis Surveillance System
Amy Abruzzi, Bernard Fried, and Towards Elimination in People’s
Sukaina B. Alikhan Republic of China
L.-J. Zhang, S.-Z. Li, L.-Y. Wen,
Recent Advances in Elucidating Nematode D.-D. Lin, E.M. Abe, R. Zhu, Y. Du,
Moulting – Prospects of Using S. Lv, J. Xu, B.L. Webster, D. Rollinson,
Oesophagostomum dentatum as a Model and X.-N. Zhou
Martina Ondrovics, Robin B. Gasser, and
Anja Joachim Applications of Spatial Technology in
Schistosomiasis Control Programme in
A Population Biology Perspective on the China
Stepwise Infection Process of the X.-Y. Wang, J. He, K. Yang, and S. Liang
Bacterial Pathogen Pasteuria ramosa in
Daphnia Reaching the Surveillance-Response Stage of
Dieter Ebert, David Duneau, Matthew D. Hall, Schistosomiasis Control in the People’s
Pepijn Luijckx, Jason P. Andras, Louis Du Republic of China: A Modelling
Pasquier, and Frida Ben-Ami Approach
Y. Feng, L. Liu, S. Xia, J.-F. Xu,
The Increase of Exotic Zoonotic Helminth R. Bergquist, and G.-J. Yang
Infections: The Impact of Urbanization,
Climate Change and Globalization Biology and Control of Snail Intermediate Host
Catherine A. Gordon, Donald P. McManus, of Schistosoma japonicum in P.R. China
Malcolm K. Jones, Darren J. Gray, and Z.-J. Li, J. Ge, J.-R. Dai, L.-Y. Wen,
Geoffrey N. Gobert D.-D. Lin, H. Madsen, X.-N. Zhou,
and S. Lv
Volume 92 Integrated Control Strategy of Schistosomiasis

in China: Projects Involving Agriculture,
Evolution of the National Schistosomiasis Water Conservancy, Forestry, Sanitation
Control Programmes in the People’s and Environmental Modification
Republic of China Y. Yang, Y.-B. Zhou, X.-X. Song, S.-Z. Li,
J. Xu, P. Steinman, D. Maybe, X.-N. Zhou, B. Zhong, T.-P. Wang, R. Bergquist,
S. Lv, S.-Z. Li, and R. Peeling X.-N. Zhou, and Q.-W. Jiang
Epidemiological Features and Effectiveness of Towards the Elimination of Schistosomiasis
Schistosomiasis Control Programme in Japonica through Control of the Disease
Lake and Marshland Region in China in Domestic Animals in China: A Tale of
S.-Q. Zhang, C.-S. Sun, M. Wang, over 60 Years
D.-D. Lin, X.-N. Zhou, and Z.-G. Cao, Y.-E. Zhao, A. Lee Willingham,
T.-P. Wang and T.-P. Wang
Epidemiological Features and Effectiveness Health Education as an Important Component
of Schistosomiasis Control Programme in the National Schistosomiasis Control
in Mountainous and Hilly Region of Programme in the People’s Republic of
China China
Y. Liu, Y.-B. Zhou, R.-Z. Li, J.-J. Wan, L. Chen, B. Zhong, J. Xu, R.-Z. Li, and
Y. Yang, D.-C. Qiu, and B. Zhong C.-L. Cao
Policy Support and Resources Mobilization Development and Application of Diagnostics

for the National Schistosomiasis Control in the National Schistosomiasis Control
Programme in the People’s Republic of Programme in the People’s Republic of
China China
H. Zhu, P. Yap, J. Utzinger, T.-W. Jia, J.-F. Zhang, J. Xu, R. Bergquist, L.-L. Yu,
S.-Z. Li, X.-B. Huang, and S.-X. Cai X.-L. Yan, H.-Q. Zhu, and L.-Y. Wen
New Anti-Schistosoma Approaches in the China–Africa and China–Asia Collaboration
People’s Republic of China: on Schistosomiasis Control: A SWOT
Development of Diagnostics, Vaccines Analysis
and Other New Techniques Belonging to J. Xu, R. Bergquist, Y.-J. Qian, Q. Wang,
the ‘Omics’ Group Q. Yu, R. Peeling, S. Croft, J.-G. Guo, and
S.-B. Chen, L. Ai, W. Hu, J. Xu, X.-N. Zhou
R. Bergquist, Z.-Q. Qin, and J.-H. Chen

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VOLUME NINETY THREE

GEORG VON SAMSON-HIMMELSTJERNA

AMSTERDAM • BOSTON • HEIDELBERG • LONDON

First edition 2016

Copyright © 2016 Elsevier Ltd. All rights reserved.

For information on all Academic Press publications visit our

Publisher: Zoe Kruze

The emergence of anthelmintic resistance in H. contortus and related

Robin B. Gasser and Georg von Samson-Himmelstjerna

Evolution and Biogeography of

biogeography (with phylogeographical approaches) provide a foundation to

2. HAEMONCHUS: HISTORY AND BIODIVERSITY

3. PHYLOGENY AND BIOGEOGRAPHY: OUT OF AFRICA

and African ungulates during the Miocene (Durette-Desset et al., 1999),

Figure 1 Phylogenetic perspective for host-group distribution and coevolutionary his-

be substantial determinants in the potential outcomes for widespread

4. DOMESTICATION, GEOGRAPHICAL EXPANSION

white-tailed deer, O. virginianus (Zimmermann), across the southern lati-

5. HOST RANGE FOR HAEMONCHUS CONTORTUS

Figure 2 Phylogenetic perspective of host-group distribution for H. contortus among

Further, H. longistipes occurs in six species of ungulates, including camelids,

5.1 Host colonization, ecological ﬁtting and sloppy

5.2 Generalists and specialists: an obsolete nomenclature

multi-host associations, including species of Haemonchus (Brooks and

Alternating trends for generalization and specialization emerge in the

perturbation and episodic events that have structured faunal assemblages.

6. HOST AND GEOGRAPHICAL COLONIZATION IN

with processes deﬁned in the Stockholm Paradigm, which constitutes a synthe-

perturbation and ecological ﬁtting as drivers of faunal assembly across all

7. CLIMATE IMPACTS INTEGRATING HISTORICAL

and emergence (chapter: The Pathophysiology, Ecology and Epidemiology

could not be recognized (Troell et al., 2005). In these environments, char-

such population mosaics has implications for patterns of potential emergence

8. UNDERSTANDING DIVERSITY: SOME

pathways results from patchy information about the genetic structure of

communities in wild hosts. Mol. Ecol. Resour. http://dx.doi.org/10.1111/1755-

Genetic Diversity and Population

Advances in Parasitology, Volume 93

2. BACKGROUND INFORMATION ON REPRODUCTION

of anthelmintic resistance loci utilizing the rapidly improving H. contortus

3. GENETIC DIVERSITY AND POPULATION STRUCTURE

arid regions (chapter: The Pathophysiology, Ecology and Epidemiology of

3.2 Extremely high levels of genetic diversity are seen within

example, a study of laboratory strains of H. contortus reported the diversity

some indication of the level of sequence polymorphism between laboratory

3.3 Large population size is a major determinant of the high

et al., 2009). Consequently although it is possible that the mutation rate in

populations of M. odocoilei, in spite of there being substantial partitioning of

3.4 Haemonchus contortus has substantial global population

3.5 Haemonchus contortus has a low but discernable

0.16 based on the mtDNA nad4 data. In addition, in the minimum-

3.6 Current evidence regarding genetic differentiation

Yemen (Gharamah et al., 2012). In this study, marked clustering of

3.7 Effect of anthelmintic selection on the overall genetic

ruminant) farms in Pakistan with a history of intense benzimidazole drug se-

4. CONSEQUENCES OF HAEMONCHUS CONTORTUS

volume). For the common F200Y (TAC) mutation, several studies

4.2 Consequence of low regional population structure

4.3 Consequence of substantial global population structure

such populations, even at neutral genetic loci, and, therefore, it is critical to

4.4 Consequence of low population structure between hosts

5. GENETIC AND PHENOTYPIC VARIATION IN

contaminated with other species. Although these can be removed by trans-

5.1 Genetic variation within and between laboratory strains

(Advances in Parasitology 92) Xiao-Nong Zhou, Shi-Zhu Li, Jürg Utzinger and Robert Bergquist (Eds.)-Schistosomiasis in the People's Republic of China From Control to Elimination-Academic Press (2016)

(Advances in Parasitology 92) Xiao-Nong Zhou, Shi-Zhu Li, Jürg Utzinger and Robert Bergquist (Eds.)-Schistosomiasis in the People's Republic of China From Control to Elimination-Academic Press (2016)