Sie sind auf Seite 1von 669

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
Haemonchus contortus and
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

AMSTERDAM • BOSTON • HEIDELBERG • LONDON


NEW YORK • OXFORD • PARIS • SAN DIEGO
SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Academic Press is an imprint of Elsevier
Academic Press is an imprint of Elsevier
125 London Wall, London EC2Y 5AS, UK
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, USA
525 B Street, Suite 1800, San Diego, CA 92101-4495, USA

First edition 2016

Copyright © 2016 Elsevier Ltd. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any information storage and retrieval system, without
permission in writing from the publisher. Details on how to seek permission, further information about
the Publisher’s permissions policies and our arrangements with organizations such as the Copyright
Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/
permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher
(other than as may be noted herein).

Notices
Knowledge and best practice in this field are constantly changing. As new research and experience
broaden our understanding, changes in research methods, professional practices, or medical treatment
may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and
using any information, methods, compounds, or experiments described herein. In using such information
or methods they should be mindful of their own safety and the safety of others, including parties for
whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any
liability for any injury and/or damage to persons or property as a matter of products liability, negligence
or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in
the material herein.

ISBN: 978-0-12-810395-1
ISSN: 0065-308X

For information on all Academic Press publications visit our


website at https://www.elsevier.com

Publisher: Zoe Kruze


Acquisition Editor: Mary Ann Zimmerman
Editorial Project Manager: Helene Kabes
Production Project Manager: Vignesh Tamil
Designer: Greg Harris
Typeset by TNQ Books and Journals
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
Wellcome Trust Sanger Institute, Cambridge, United Kingdom
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
The University of Melbourne, Parkville, VIC, Australia
A.C. Kotze
CSIRO Agriculture, Brisbane, QLD, Australia
R. Laing
University of Glasgow, Glasgow, Scotland, United Kingdom
C.E. Lanusse
Laboratorio de Farmacología, Centro de Investigaci
on Veterinaria de Tandil (CIVETAN),
CONICET-CICPBA-UNCPBA, Campus Universitario, Tandil, Argentina
A.L. Lifschitz
Laboratorio de Farmacología, Centro de Investigaci
on Veterinaria de Tandil (CIVETAN),
CONICET-CICPBA-UNCPBA, Campus Universitario, Tandil, Argentina
N.D. Marks
University of Glasgow, Glasgow, United Kingdom
A. Martinelli
Wellcome Trust Sanger Institute, Cambridge, United Kingdom
E.N. Meeusen
Federation University, Churchill, VIC, Australia; Monash University, Melbourne, VIC,
Australia
I. Mueller-Harvey
University of Reading, Reading, United Kingdom
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
University of Calgary, Calgary, AB, Canada
B. Robertsa
University of Glasgow, Glasgow, United Kingdom

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
Fort Valley State University, Fort Valley, GA, United States
J.F.J. Torres-Acosta
Universidad Aut
onoma de Yucatan, Merida, Yucatan, Mexico
A. Tracey
Wellcome Trust Sanger Institute, Cambridge, United Kingdom
W. Tuo
USDA, Agricultural Research Service, Beltsville, MD, United States
J.A. Van Wyk
University of Pretoria, Hatfield, South Africa
N.D. Young
The University of Melbourne, Parkville, VIC, Australia
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
4 E.P. Hoberg and D.S. Zarlenga

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
Evolution and Biogeography of H. contortus 5

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,
6 E.P. Hoberg and D.S. Zarlenga

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
Evolution and Biogeography of H. contortus 7

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
8 E.P. Hoberg and D.S. Zarlenga

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
Evolution and Biogeography of H. contortus 9

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
10 E.P. Hoberg and D.S. Zarlenga

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).
Evolution and Biogeography of H. contortus 11

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
12 E.P. Hoberg and D.S. Zarlenga

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
Evolution and Biogeography of H. contortus 13

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
14 E.P. Hoberg and D.S. Zarlenga

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
Evolution and Biogeography of H. contortus 15

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).
16 E.P. Hoberg and D.S. Zarlenga

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,
Evolution and Biogeography of H. contortus 17

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
18 E.P. Hoberg and D.S. Zarlenga

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
Evolution and Biogeography of H. contortus 19

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
20 E.P. Hoberg and D.S. Zarlenga

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
Evolution and Biogeography of H. contortus 21

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
22 E.P. Hoberg and D.S. Zarlenga

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
Evolution and Biogeography of H. contortus 23

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
24 E.P. Hoberg and D.S. Zarlenga

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
Evolution and Biogeography of H. contortus 25

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.

REFERENCES
Achi, Y.L., Zinsstag, J., Yao, K., Yeo, N., Dorchies, P., Jacquiet, P., 2003. Host specificity of
Haemonchus spp. for domestic ruminants in the savanna in northern Ivory Coast. Vet.
Parasitol. 116, 151e158.
Agosta, S.J., Klemens, J.A., 2008. Ecological fitting by phenotypically flexible genotypes: im-
plications for species associations, community assembly and evolution. Ecol. Lett. 11,
1123e1134.
Agosta, S.J., Janz, N., Brooks, D.R., 2010. How specialists can be generalists: resolving the
“parasite paradox” and implications for emerging infectious disease. Zoologia (Curitiba,
Impresso) 27, 151e162.
Araujo, S.B.L., Braga, M.P., Brooks, D.R., Agosta, S., Hoberg, E.P., von Hathental, F.,
Boeger, W.A., 2015. Understanding host-switching by ecological fitting. PLoS One
10 (10), e0139225. http://dx.doi.org/10.1371/journal.pone.0139225.
Balter, M., 2014. Monumental roots. Science 343, 18e23.
Beck, M.A., Colwell, D.D., Goater, C.P., Kienze, S., 2015. Where’s the risk? Landscape
epidemiology of gastrointestinal parasitism in Alberta beef cattle. Parasites Vectors 8, 434.
Besier, R.R., Kahn, L.P., Sargison, N.D., Van Wyk, J.A., 2016. The pathophysiology, ecol-
ogy and epidemiology of Haemonchus contortus infection in small ruminants. In:
Gasser, R., Samson-Himmelstjerna, G.V. (Eds.), Haemonchus contortus and Haemonchosis
Past, Present and Future Trends. vol. 93, pp. 95e144.
Boomker, J., Horak, I.G., Gibbons, L.M., De Vos, V., 1983. Haemonchus contortus from the
vaal ribbok, Pelea capreolus, and the bontebok, Damaliscus dorcas dorcas, in the Bontebok
National Park. Onderstep. J. Vet. Res. 50, 179e181.
Boomker, J., 1990. A Comparative Study of the Helminth Fauna of Browsing Antelope of
South Africa (Ph.D. dissertation). Medical University of South Africa, Pretoria, South
Africa, p. 297.
Brooks, D.R., Hoberg, E.P., 2000. Triage for the biosphere: the need and rationale for taxo-
nomic inventories and phylogenetic studies of parasites. Comp. Parasitol. 67, 1e25.
Brooks, D.R., Hoberg, E.P., 2007. How will climate change affect host-parasite assemblages?
Trends Parasitol. 23, 571e574.
Brooks, D.R., Hoberg, E.P., 2013. The emerging infectious diseases crisis and pathogen pollu-
tion: a question of ecology and evolution. In: Rohde, K. (Ed.), The Balance of Nature and
Human Impact. Cambridge University Press, Cambridge, UK, pp. 215e229.
Brooks, D.R., McLennan, D.A., 1993. Parascript: Parasites and the Language of Evolution.
Smithsonian Institution Press, Washington, DC, USA, p. 429.
Brooks, D.R., McLennan, D.A., 2002. The Nature of Diversity: An Evolutionary Voyage of
Discovery. University of Chicago Press, Chicago, USA, p. 668.
Brooks, D.R., Hoberg, E.P., Gardner, S.L., Boeger, W., Galbreath, K.E., Herczeg, D.,
Mejía-Madrid, H.H., Racz, E., Tsogtsaikhan Dursahinhan, A., 2014. Finding them
before they find us: informatics, parasites and environments in accelerating climate
change. Comp. Parasitol. 81, 155e164.
Brooks, D.R., 1979. Testing the context and extent of host-parasite coevolution. Syst. Zool.
28, 299e307.
Budischak, S.A., Hoberg, E.P., Abrams, A., Jolles, A.E., Ezenwa, V.O., 2015. A combined
parasitological molecular approach for noninvasive characterization of parasitic nematode
26 E.P. Hoberg and D.S. Zarlenga

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


0998.12382.
Capinha, C., Essel, F., Seebens, H., Moser, D., Miguel Pereira, H., 2015. The dispersal of
alien species redefines biogeography in the Anthropocene. Science 348, 1248e1251.
Caramelli, D., 2006. The origins of domesticated cattle. Hum. Evol. 21, 107e122.
Cerutti, M.C., Citterio, C.V., Bazzochi, C., Epis, S., D’Amelio, S., Ferrari, N., Lanfranchi, P.,
2010. Genetic variability of Haemonchus contortus (Nematoda: Trichostrongyloidea) in
alpine ruminant host species. J. Helminthol. 84, 276e283.
Chaudhry, U., Redman, E.M., Abbas, M., Muthusamy, R., Ashraf, K., Gilleard, J.S., 2015.
Genetic evidence for hybridisation between Haemonchus contortus and Haemonchus placei in
natural field populations and its implications for interspecies transmission of anthelmintic
resistance. Int. J. Parasitol. 45, 149e159.
Chessa, B., Pereira, F., Arnaud, F., Amorim, A., Gyoache, F., Mainland, I., et al., 2009.
Revealing the history of sheep domestication using retrovirus integrations. Science 324,
532e536.
Combes, C., 2001. Parasitism: The Ecology and Evolution of Intimate Interactions. Univer-
sity of Chicago Press, Chicago, USA, p. 728.
Crofton, H.D., Whitlock, J.H., Glazier, R.A., 1965. Ecological and biological plasticity of
sheep nematodes. II. Genetic and environmental plasticity in Haemonchus contortus
(Rud. 1803). Cornell Vet. 55, 251e258.
Dobson, A., 2004. Population dynamics of pathogens with multiple host species. Am. Nat.
164 (Suppl.), S64eS78.
Durette-Desset, M.-C., Hugot, J.P., Darlu, P., Chabaud, A.G., 1999. A cladistic analysis of
the Trichostrongyloidea (Nematoda). Int. J. Parasitol. 29, 1065e1086.
Durette-Desset, M.,C., 1983. Keys to the genera of the superfamily Trichostrongyloidea. In:
Anderson, R.C., Chabaud, A.G. (Eds.), CIH Keys to the Nematode Parasites of
Vertebrates. Commonwealth Agricultural Bureaux, Farnham Royal, pp. 1e86.
Elton, C.S., 1958. The Ecology of Invasions by Animals and Plants. Methuen and Co.
Limited, London, UK, p. 181.
Erwin, T.L., 1985. The taxon pulse: a general pattern of lineage radiation and extinction
among carabid beetles. In: Ball, G.E. (Ed.), Taxonomy, Phylogeny and Biogeography
of Beetles and Ants. Junk, Dordrecht, pp. 437e472.
Fenton, A., Pedersen, A.B., 2005. Community epidemiology framework for classifying dis-
ease threats. Emerg. Inf. Dis. 11, 1815e1821.
Fenton, A., Streicker, D.G., Petchey, O.L., Pedersen, A.B., 2015. Are all hosts created equal?
Partitioning host species contributions to parasite persistence in multihost communities.
Am. Nat. http://dx.doi.org/10.1086/683173.
Galbreath, K.E., Hoberg, E.P., 2015. Host responses to historical climate change
shape parasite communities in North America’s Intermountain West. Folia Zool.
64, 218e232.
Gatesy, J., Arctander, P., 2000. Molecular evidence for the phylogenetic affinities of
Ruminantia. In: Vrba, E.S., Schaller, G.B. (Eds.), Antelopes, Deer and Relatives: Fossil
Record, Behavioral Ecology, Systematics and Conservation. Yale University Press,
pp. 143e155.
Gibbons, L.M., 1979. Revision of the genus Haemonchus Cobb, 1898 (Nematoda;
Trichostrongylidae). Syst. Parasitol. 1, 3e24.
Giudici, C.J., Cabaret, J., Durrette-Desset, M.C., 1999. Description of Haemonchus placei
(Place, 1893) (Nematoda: Trichostrongylidae: Haemonchinae), identification and intra-
specific morphological variability. Parasite 6, 333e342.
Grubb, P., 2005. Order Artiodactyla. In: Wilson, D.E., Reeder, D.M. (Eds.), Mammal Spe-
cies of the World: A Taxonomic and Geographic Reference, third ed. Johns Hopkins
University Press, Baltimore, pp. 637e722.
Evolution and Biogeography of H. contortus 27

Halas, D., Zamparo, D., Brooks, D.R., 2005. A historical biogeographical protocol for study-
ing diversification by taxon pulses. J. Biogeogr. 32, 249e260.
Hanotte, O., Bradley, D.G., Ochieng, J.W., Verjee, Y., Hill, E.W., Rege, E.O., 2002.
African pastoralism: genetic imprints of origins and migrations. Science 296, 336e339.
Harvell, C.D., Mitchell, C.E., Ward, J.R., Altizer, S., Dobson, A.P., Ostfeld, R.S.,
Samuel, M.D., 2002. Climate warming and disease risks for marine and terrestrial biota.
Science 296, 2158e2162.
Hassanin, A., Douzery, E.J.P., 2003. Molecular and morphological phylogenies for the
Ruminantiaand the alternative positions of the Moschidae. Syst. Biol. 52, 206e228.
Haydon, D.T., Cleaveland, S., Taylor, L.H., Laruenson, M.K., 2002. Identifying reservoirs
of infection: a conceptual and practical challenge. Emerg. Infect. Dis. 8, 1468e1473.
Hernandez Fernandez, M., Vrba, E.S., 2005. A complete estimate of the phylogenetic rela-
tionships in Ruminantia: a dated species-level supertree of the extant ruminants. Biol.
Rev. 80, 269e302.
Hoberg, E.P., Brooks, D.R., 2008. A macroevolutionary mosaic: episodic host-switching,
geographic colonization, and diversification in complex host-parasite systems. J. Bio-
geogr. 35, 1533e1550.
Hoberg, E.P., Brooks, D.R., 2010. Beyond vicariance: integrating taxon pulses, ecological
fitting and oscillation in historical biogeography and evolution. In: Morand, S.,
Krasnov, B. (Eds.), The Geography of Host-parasite Interactions. Oxford University
Press, UK, pp. 7e20.
Hoberg, E.P., Brooks, D.R., 2013. Episodic processes, invasion, and faunal mosaics in evolu-
tionary and ecological time. In: Rohde, K. (Ed.), The Balance of Nature and Human
Impact. Cambridge University Press, UK., pp. 199e213
Hoberg, E.P., Brooks, D.R., 2015. Evolution in action: climate change, biodiversity dy-
namics and emerging infectious disease. Phil. Trans. Roy. Soc. B 370, 20130553.
http://dx.doi.org/10.1098/rstb.2013.0553.
Hoberg, E.P., Monsen, K.J., Kutz, S., Blouin, M.S., 1999. Structure, biodiversity and histor-
ical biogeography of nematode faunas in Holarctic ruminants: morphological and molec-
ular diagnoses for Teladorsagia boreoarcticus n. sp. (Nematoda: Ostertagiinae) a dimorphic
cryptic species in muskoxen (Ovibos moschatus). J. Parasitol. 85, 910e934.
Hoberg, E.P., Kocan, A., Rickard, L.G., 2001. Gastrointestinal strongyles in wild ruminants.
In: Samuel, W., Pybus, M., Kocan, A. (Eds.), Parasitic Diseases of Wild Mammals. Iowa
State University Press, pp. 193e227.
Hoberg, E.P., Abrams, A., Carreno, R., Lichtenfels, J.R., 2002. Ashworthius patriciapilittae n.
sp. (Trichostrongyloidea: Haemonchinae), an abomasal nematode in Odocoileus virginia-
nus from Costa Rica, and a first record for the genus in the Western Hemisphere. J. Para-
sitol. 88, 1187e1199.
Hoberg, E.P., Lichtenfels, J.R., Gibbons, L., 2004. Phylogeny for species of the genus Hae-
monchus (Nematoda: Trichostrongyloidea): considerations of their evolutionary history
and global biogeography among Camelidae and Pecora (Artiodactyla). J. Parasitol. 90,
1085e1102.
Hoberg, E.P., Polley, L., Jenkins, E.J., Kutz, S.J., 2008. Pathogens of domestic and free-
ranging ungulates: global climate change in temperate to boreal latitudes across North
America. Rev. Sci. Tech. 27, 511e528.
Hoberg, E.P., Pilitt, P.A., Galbreath, K.E., 2009. Why museums matter: a tale of pinworms
(Oxyuroidea: Heteroxynematidae) among pikas (Ochotona princeps and O. collaris) in the
American west. J. Parasitol. 95, 490e501.
Hoberg, E.P., Galbreath, K.E., Cook, J.A., Kutz, S.J., Polley, L., 2012. Northern host-para-
site assemblages: history and biogeography on the borderlands of episodic climate and
environmental transition. In: Rollinson, D., Hays, S.I. (Eds.), Advances in Parasitology,
79. Elsevier, pp. 1e97.
28 E.P. Hoberg and D.S. Zarlenga

Hoberg, E.P., Agosta, S.J., Boeger, W.A., Brooks, D.R., 2015. An integrated parasi-
tology: revealing the elephant through tradition and invention. Trends Parasitol.
31, 128e133.
Hoberg, E.P., 1997. Phylogeny and historical reconstruction: host parasite systems as key-
stones in biogeography and ecology. In: Reaka-Kudla, M., Wilson, E.O., Wilson, D.
(Eds.), Biodiversity II: Understanding and Protecting Our Resources. Joseph Henry
Press, National Academy of Sciences, Washington, D.C., USA, pp. 243e261.
Hoberg, E.P., 2005. Coevolution and biogeography among Nematodirinae (Nematoda:
Trichostrongylina), Lagomorpha and Artiodactyla (Mammalia): Exploring determinants
of history and structure for the northern fauna across the Holarctic. J. Parasitol. 91,
358e369.
Hoberg, E.P., 2010. Invasive processes, mosaics and the structure of helminth parasite faunas.
Rev. Sci. Tech. 29, 255e272.
Holt, R.D., Dobson, A.P., Begon, M., Bowers, R.G., Schauber, E.M., 2003. Parasite estab-
lishment in host communities. Ecol. Lett. 6, 837e842.
Hulme, P.E., 2014. Invasive species challenge the global response ot emerging diseases.
Trends Parasitol. 30, 267e270.
Hunt, P.W., Knox, M.R., LeJambre, L.F., McNally, J., Andersen, L.J., 2008. Genetic and
phenotypic differences between isolates of Haemonchus contortus in Australia. Int. J. Para-
sitol. 38, 885e900.
IPCC, 2013. Climate Change 2013: The Physical Science Basis. IPCC Working Group I
Contribution to AR5. Intergovernmental Panel on Climate Change (IPCC), Geneva.
Available: https://www.ipcc.ch/report/ar5/wg1.
IPCC, 2014. Climate change 2014: impacts, adaptation, and vulnerability. Part B: regional
aspects. In: Barros, V.R., Field, C.B., Dokken, D.J., Mastrandrea, M.D., Mach, K.J.,
Bilir, T.E., et al. (Eds.), Contribution of Working Group II to the Fifth Assessment
Report of the Intergovernmental Panel on Climate Change. Cambridge University
Press, Cambridge, UK. Available: http://ipcc-wg2.gov/AR5/report/.
Jacquiet, P., Humbert, J.F., Comes, A.M., Cabaret, J., Thiam, A., Cheikh, D., 1995. Ecolog-
ical, morphological and genetic characterization of sympatric Haemonchus spp. parasites of
domestic ruminants in Mauritania. Parasitology 110, 483e492.
Jacquiet, P.F., Cabaret, J., Cheikh, D., Thiam, E., 1997. Identification of Haemonchus species
in domestic ruminants based on morphometrics of spicules. Parasitol. Res. 83, 82e86.
Jacquiet, P.F., Cabaret, J., Thiam, E., Cheikh, D., 1998. Host range and the maintenance of
Haemonchus spp. in an adverse arid climate. Int. J. Parasitol. 28, 253e261.
Janz, N., Nylin, S., 2008. The oscillation hypothesis of host-plant range and speciation. In:
Tilmon, K.J. (Ed.), Specialization, Speciation, and Radiation: The Evolution of Herbiv-
orous Insects. University of California Press, Berkeley, pp. 203e215.
Janzen, D.H., 1985. On ecological fitting. Oikos 45, 308e310.
Klassen, G.J., 1992. Coevolution: a history of the macroevolutionary approach to studying
host parasite associations. J. Parasitol. 78, 573e587.
Leignel, V., Humbert, J.F., 2001. Mitochondrial DNA variation in benzimidazole-resistant
and e susceptible populations of the small ruminant nematode Teladorsagia circumcincta.
J. Hered. 92, 503e506.
Lichtenfels, J.R., Pilitt, P.A., LeJambre, L.F., 1986. Cuticular ridge patterns of Haemonchus
contortus and Haemonchus placei (Nematoda; Trichostrongyloidea). Proc. Helm. Soc.
Wash. 53, 94e101.
Lichtenfels, J.R., Pilitt, P.A., Hoberg, E.P., 1994. New morphological characters for identi-
fying individual specimens of Haemonchus spp. (Nematoda: Trichostrongyloidea) and a
key to species in ruminants of North America. J. Parasitol. 80, 107e119.
Lichtenfels, J.R., Pilitt, P.A., Gibbons, L.M., Boomker, J.D.F., 2001. Haemonchus horaki n. sp.
(Nematoda: Trichostrongyloidea) from the grey rhebuk Pelea capreolus in South Africa.
J. Parasitol. 87, 1095e1103.
Evolution and Biogeography of H. contortus 29

Lichtenfels, J.R., Pilitt, P.A., Gibbons, L.M., Hoberg, E.P., 2002. Redescriptions of Haemon-
chus mitchelli and Haemonchus okapiae (Nematoda: Trichostrongyloidea) and description of
a unique synlophe for the Haemonchinae. J. Parasitol. 88, 947e960.
Loftus, R.T., MacHugh, D.E., Bradley, D.G., Sharp, P.M., 1994. Evidence for two indepen-
dent domestications of cattle. Proc. Natl. Acad. Sci. U.S.A. 91, 2757e2761.
Mas Coma, S., Valero, M.A., Bargues, M.D., 2008. Effects of climate change on animal and
zoonotic helminthiases. Rev. Sci. Tech. 27, 443e452.
Morrison, D.A., H€ oglund, J., 2005. Testing the hypothesis of recent population expansions
in nematode parasites of human-associated hosts. Heredity 94, 426e434.
O’Connor, L.J., Walkden-Brown, S.W., Kahn, L.P., 2006. Ecology of the free-living stages
of major trichostrongylid parasites of sheep. Vet. Parasitol. 142, 1e15.
Poulin, R., Morand, S., 2004. Parasite Biodiversity. Smithsonian Institution Press, Washing-
ton, D.C., USA, p. 216.
Poulin, R., 1998. Evolutionary Ecology of Parasites. Chapman and Hall, London, p. 332.
Prestwood, A.K., Pursglove, S.R., 1981. Gastrointestinal nematodes. In: Davidson, W.R.,
Hayes, F.A., Nettles, V.F., Kellogg, F.E. (Eds.), Diseases and Parasites of White-tailed
Deer, pp. 318e349. Tall Timbers Research Station Miscellaneous Publication No. 7.
Rose, H., Caminade, C., Bashir Bolajoko, M., Phelan, P., van Dijk, J., Baylis, M.,
Williams, D., Morgan, E.R., 2015. Climate driven changes in the spatial-temporal dis-
tribution of the parasitic nematode, Haemonchus contortus, in sheep in Europe. Glob.
Chang. Biol. http://dx.doi.org/10.1111/gcb.13132.
Rosenthal, B.M., 2009. How has agriculture influenced the geography and genetics of animal
parasites? Trends Parasitol. 25, 67e70.
Thompson, J.N., 1994. The Co-evolutionary Process. University of Chicago Press, Chicago,
p. 376.
Thompson, J.N., 2005. The Geographical Mosaic of Coevolution. University of Chicago
Press, Chicago, p. 443.
Troell, K., Waller, P., H€ oglund, P., 2005. The development and overwintering survival of
free-living larvae of Haemonchus contortus in Sweden. J. Helminthol. 79, 373e379.
Troell, K., Engstr€ om, A., Morrison, D.A., Mattson, J.G., H€ oglund, J., 2006. Global patterns
reveal strong population structure in Haemonchus contortus, a nematode parasite of domes-
ticated ruminants. Int. J. Parasitol. 36, 1305e1316.
van Dijk, J., David, G.P., Baird, G., Morgan, E.R., 2008. Back to the future: developing hy-
potheses on the effects of climate change on ovine parasitic gastroenteritis from historical
data. Vet. Parasitol. 158, 73e84.
van Dijk, J., Sargison, N.D., Kenyon, F., Skuce, P.J., 2009. Climate change and infectious
disease: helminthological challenges to farmed ruminants in temperate regions. Animal.
http://dx.doi.org/10.1017/S1751731109990991.
Vrba, E.S., Schaller, G.B., 2000. Phylogeny of Bovidae based on behavior, glands, skulls, and
postcrania. In: Vrba, E.S., Schaller, G.B. (Eds.), Antelopes, Deer and Relatives: Fossil
Record, Behavioral Ecology, Systematics and Conservation. Yale University Press,
pp. 203e222.
Vrba, E.S., 1985. African bovidae; evolutionary events since the Miocene. S. Afr. J. Sci. 81,
263e266.
Vrba, E.S., 1995. The fossil record of African antelopes (Mammalia: bovidae) in relation to
human evolution and paleoclimate. In: Vrba, E.S., Denton, G.S., Partridge, T.C.,
Burckle, L.H. (Eds.), Paleoclimate and Evolution With Emphasis on Human Origins.
Yale University Press, New Haven, USA, pp. 385e424.
Walker, J.G., Morgan, E.R., 2014. Generalists at the interface: nematode transmission be-
tween wild and domestic ungulates. Int. J. Parasitol. Parasites Wildl. 3, 242e250.
Waller, P.J., Rudby-Martin, L., Ljungstr€ om, B.L., Rydzik, A., 2004. The epidemiology of
abomasal nematodes of sheep in Sweden, with particular reference to over-winter sur-
vival strategies. Vet. Parasitol. 122, 207e220.
30 E.P. Hoberg and D.S. Zarlenga

Wang, T., van Wyk, J.A., Morrison, A., Morgan, E.R., 2014. Moisture requirements for the
migration of Haemonchus contortus third stage larvae out of faeces. Vet. Parasitol. 204,
258e264.
Wilson, J.R.U., Dormontt, E.E., Prentis, P.J., Lowe, A.J., Richardson, D.M., 2009. Some-
thing in the way you move: dispersal pathways affect invasion success. Trends Ecol. Evol.
24, 136e144.
Zarlenga, D.S., Hoberg, E.P., Rosenthal, B., Mattiucci, S., Nascetti, G., 2014. Anthropo-
genics: human influence on global and genetic homogenization of parasite
populations. J. Parasitol. 100, 756e772.
Zarlenga, D.S., Hoberg, E.P., Tuo, W., 2016. The identification of Haemonchus species and
diagnosis of Haemonchosis. In: Gasser, R., Samson-Himmelstjerna, G.V. (Eds.), Hae-
monchus contortus and Haemonchosis Past, Present and Future Trends. vol. 93,
pp. 145e180.
CHAPTER TWO

Genetic Diversity and Population


Structure of Haemonchus
contortus
J.S. Gilleard1, E. Redman
University of Calgary, Calgary, AB, Canada
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

Advances in Parasitology, Volume 93


© 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
34 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 35

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
36 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 37

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
38 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 39

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
40 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 41

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
42 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 43

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
44 J.S. Gilleard and E. Redman

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.
Genetic Diversity and Population Structure of H. contortus 45

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
46 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 47

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
48 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 49

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
50 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 51

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
52 J.S. Gilleard and E. Redman

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.
Genetic Diversity and Population Structure of H. contortus 53

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
54 J.S. Gilleard and E. Redman

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,
Genetic Diversity and Population Structure of H. contortus 55

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
56 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 57

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,
58 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 59

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).
60 J.S. Gilleard and E. Redman

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
Genetic Diversity and Population Structure of H. contortus 61

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
62 J.S. Gilleard and E. Redman

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.

REFERENCES
Angulo-Cubillan, F.J., Garcia-Coiradas, L., Alunda, J.M., Cuquerella, M., De La Fuente, C.,
2010. Biological characterization and pathogenicity of three Haemonchus contortus isolates
in primary infections in lambs. Vet. Parasitol. 171, 99e105.
Attrill, H., Falls, K., Goodman, J.L., Millburn, G.H., Antonazzo, G., Rey, A.J.,
Marygold, S.J., Flybase, C., 2015. Flybase: establishing a gene group resource for
Drosophila melanogaster. Nucleic Acids Res. http://dx.doi.org/10.1093/nar/gkv1046.
Aumont, G., Gruner, L., Hostache, G., 2003. Comparison of the resistance to sympatric and
allopatric isolates of Haemonchus contortus of black belly sheep in Guadeloupe (FWI) and
of INRA 401 sheep in France. Vet. Parasitol. 116, 139e150.
Genetic Diversity and Population Structure of H. contortus 63

Barrere, V., Falzon, L.C., Shakya, K.P., Menzies, P.I., Peregrine, A.S., Prichard, R.K.,
2013. Assessment of benzimidazole resistance in Haemonchus contortus in sheep flocks
in Ontario, Canada: comparison of detection methods for drug resistance. Vet. Para-
sitol. 198, 159e165.
Beech, R.N., Prichard, R.K., Scott, M.E., 1994. Genetic variability of the beta-tubulin genes
in benzimidazole-susceptible and -resistant strains of Haemonchus contortus. Genetics 138,
103e110.
Besier, R.B., Kahn, L.P., Sargison, N.D., Wyk, J.A.V., 2016. The Pathophysiology, Ecology
and Epidemiology of Haemonchus contortus Infection in Small Ruminants. In:
Gasser, R., Samson-Himmelstjerna, G.V. (Eds.). Haemonchus contortus and Haemon-
chosis Past, Present and Future Trends. vol. 93, 95e144.
Blackhall, W.J., Liu, H.Y., Xu, M., Prichard, R.K., Beech, R.N., 1998a. Selection at a
P-glycoprotein gene in ivermectin- and moxidectin-selected strains of Haemonchus
contortus. Mol. Biochem. Parasitol. 95, 193e201.
Blackhall, W.J., Pouliot, J.F., Prichard, R.K., Beech, R.N., 1998b. Haemonchus contortus:
selection at a glutamate-gated chloride channel gene in ivermectin- and moxidectin-
selected strains. Exp. Parasitol. 90, 42e48.
Blouin, M.S., Dame, J.B., Tarrant, C.A., Courtney, C.H., 1992. Unusual population
genetics of a parasitic nematode: mtDNA variation within and among populations.
Evolution 46, 470e476.
Blouin, M.S., Yowell, C.A., Courtney, C.H., Dame, J.B., 1995. Host movement and the
genetic structure of populations of parasitic nematodes. Genetics 141, 1007e1014.
Bremner, K.C., 1954. Cytological polymorphism in the nematode Haemonchus contortus
(Rudolphi 1803) Cobb 1898. Nature 174, 704e705.
Burgess, C.G., Bartley, Y., Redman, E., Skuce, P.J., Nath, M., Whitelaw, F., Tait, A.,
Gilleard, J.S., Jackson, F., 2012. A survey of the trichostrongylid nematode species
present on UK sheep farms and associated anthelmintic control practices. Vet. Parasitol.
189, 299e307.
Cerutti, M.C., Citterio, C.V., Bazzocchi, C., Epis, S., D’amelio, S., Ferrari, N.,
Lanfranchi, P., 2010. Genetic variability of Haemonchus contortus (Nematoda: Trichos-
trongyloidea) in alpine ruminant host species. J. Helminthol. 84, 276e283.
Chapuis, M.P., Estoup, A., 2007. Microsatellite null alleles and estimation of population
differentiation. Mol. Biol. Evol. 24, 621e631.
Charlesworth, B., 2009. Fundamental concepts in genetics: effective population size and
patterns of molecular evolution and variation. Nat. Rev. Genet. 10, 195e205.
Chaudhry, U., Redman, E.M., Abbas, M., Muthusamy, R., Ashraf, K., Gilleard, J.S., 2015a.
Genetic evidence for hybridisation between Haemonchus contortus and Haemonchus placei in
natural field populations and its implications for interspecies transmission of anthelmintic
resistance. Int. J. Parasitol. 45, 149e159.
Chaudhry, U., Redman, E.M., Raman, M., Gilleard, J.S., 2015b. Genetic evidence for the
spread of a benzimidazole resistance mutation across southern India from a single origin
in the parasitic nematode Haemonchus contortus. Int. J. Parasitol. 45, 721e728.
Criscione, C.D., 2013. Genetic epidemiology of Ascaris: cross-transmission between humans
and pigs, focal transmission and effective population size. In: Holland, C. (Ed.), Ascaris:
The Neglected Parasite. Elsevier.
Criscione, C.D., Poulin, R., Blouin, M.S., 2005. Molecular ecology of parasites: elucidating
ecological and microevolutionary processes. Mol. Ecol. 14, 2247e2257.
Dame, J.B., Blouin, M.S., Courtney, C.H., 1993. Genetic structure of populations of
Ostertagia ostertagi. Vet. Parasitol. 46, 55e62.
Das, K.M., Whitlock, J.H., 1960. Subspeciation in Haemonchus contortus (Rudolphi, 1803)
Nemata, Trichostrongyloidea. Cornell Vet. 50, 182e197.
64 J.S. Gilleard and E. Redman

Daskalov, P.B., 1975. Haemonchus contortus: new data on its genetic constitution. Exp.
Parasitol. 37, 341e347.
Davey, J.W., Hohenlohe, P.A., Etter, P.D., Boone, J.Q., Catchen, J.M., Blaxter, M.L., 2011.
Genome-wide genetic marker discovery and genotyping using next-generation
sequencing. Nat. Rev. Genet. 12, 499e510.
Denver, D.R., Dolan, P.C., Wilhelm, L.J., Sung, W., Lucas-Lledo, J.I., Howe, D.K.,
Lewis, S.C., Okamoto, K., Thomas, W.K., Lynch, M., Baer, C.F., 2009. A genome-
wide view of Caenorhabditis elegans base-substitution mutation processes. Proc. Natl.
Acad. Sci. U.S.A. 106, 16310e16314.
Denver, D.R., Morris, K., Lynch, M., Thomas, W.K., 2004. High mutation rate and
predominance of insertions in the Caenorhabditis elegans nuclear genome. Nature 430,
679e682.
Denver, D.R., Morris, K., Lynch, M., Vassilieva, L.L., Thomas, W.K., 2000. High direct
estimate of the mutation rate in the mitochondrial genome of Caenorhabditis elegans.
Science 289, 2342e2344.
Dlugosch, K.M., Anderson, S.R., Braasch, J., Cang, F.A., Gillette, H.D., 2015. The devil is
in the details: genetic variation in introduced populations and its contributions to
invasion. Mol. Ecol. 24, 2095e2111.
Donnelly, M.J., Licht, M.C., Lehmann, T., 2001. Evidence for recent population expansion
in the evolutionary history of the malaria vectors Anopheles arabiensis and Anopheles
gambiae. Mol. Biol. Evol. 18, 1353e1364.
Falzon, L.C., Menzies, P.I., Vanleeuwen, J., Shakya, K.P., Jones-Bitton, A., Avula, J.,
Jansen, J.T., Peregrine, A.S., 2014. Pilot project to investigate over-wintering of free-living
gastrointestinal nematode larvae of sheep in Ontario, Canada. Can. Vet. J. 55, 749e756.
Fleming, M.W., 1988. Size of inoculum dose regulates in part worm burdens, fecundity, and
lengths in ovine Haemonchus contortus infections. J. Parasitol. 74, 975e978.
Gharamah, A.A., Azizah, M.N., Rahman, W.A., 2012. Genetic variation of Haemonchus
contortus (Trichostrongylidae) in sheep and goats from Malaysia and Yemen. Vet. Para-
sitol. 188, 268e276.
Gharamah, A.A., Rahman, W.A., Siti Azizah, M.N., 2014. Morphological variation between
isolates of the nematode Haemonchus contortus from sheep and goat populations in
Malaysia and Yemen. J. Helminthol. 88, 82e88.
Ghisi, M., Kaminsky, R., Maser, P., 2007. Phenotyping and genotyping of Haemonchus
contortus isolates reveals a new putative candidate mutation for benzimidazole resistance
in nematodes. Vet. Parasitol. 144, 313e320.
Gibbons, L.M., 1979. Revision of the genus Haemonchus Cobb 1898 (Nematoda:
Trichostrongylidae). Syst. Parasitol. 1, 3e24.
Gilleard, J.S., 2006. Understanding anthelmintic resistance: the need for genomics and
genetics. Int. J. Parasitol. 36, 1227e1239.
Gilleard, J.S., 2013. Haemonchus contortus as a paradigm and model to study anthelmintic drug
resistance. Parasitology 140, 1506e1522.
Gilleard, J.S., Beech, R.N., 2007. Population genetics of anthelmintic resistance in parasitic
nematodes. Parasitology 134, 1133e1147.
Grillo, V., Jackson, F., Cabaret, J., Gilleard, J.S., 2007. Population genetic analysis of the
ovine parasitic nematode Teladorsagia circumcincta and evidence for a cryptic species. Int.
J. Parasitol. 37, 435e447.
Grillo, V., Jackson, F., Gilleard, J.S., 2006. Characterisation of Teladorsagia circumcincta micro-
satellites and their development as population genetic markers. Mol. Biochem. Parasitol.
148, 181e189.
Gasser, R.B., Schwarz, E.M., Korhonen, P.K., Young, N.D., 2016. Understanding Haemon-
chus contortus better through genomics and ranscriptomics. In: Gasser, R., Samson-
Himmelstjerna, G.V. (Eds.), Haemonchus contortus and Haemonchosis Past, Present and
Future Trends. vol. 93, pp. 519e568.
Genetic Diversity and Population Structure of H. contortus 65

Harris, T.W., Chen, N., Cunningham, F., Tello-Ruiz, M., Antoshechkin, I., Bastiani, C.,
Bieri, T., Blasiar, D., Bradnam, K., Chan, J., Chen, C.K., Chen, W.J., Davis, P.,
Kenny, E., Kishore, R., Lawson, D., Lee, R., Muller, H.M., Nakamura, C.,
Ozersky, P., Petcherski, A., Rogers, A., Sabo, A., Schwarz, E.M., Van Auken, K.,
Wang, Q., Durbin, R., Spieth, J., Sternberg, P.W., Stein, L.D., 2004. WormBase: a
multi-species resource for nematode biology and genomics. Nucleic Acids Res. 32,
D411eD417.
Hart, E.H., Morphew, R.M., Bartley, D.J., Millares, P., Wolf, B.T., Brophy, P.M.,
Hamilton, J.V., 2012. The soluble proteome phenotypes of ivermectin resistant and
ivermectin susceptible Haemonchus contortus females compared. Vet. Parasitol. 190,
104e113.
Hoberg, E.R., Lichtenfels, J.R., Gibbons, L., 2004. Phylogeny for species of Haemonchus
(Nematoda: Trichostrongyloidea): considerations of their evolutionary history and global
biogeography among Camelidae and Pecora (Artiodactyla). J. Parasitol. 90, 1085e1102.
Hoekstra, R., Criado-Fornelio, A., Fakkeldij, J., Bergman, J., Roos, M.H., 1997. Microsa-
tellites of the parasitic nematode Haemonchus contortus: polymorphism and linkage with a
direct repeat. Mol. Biochem. Parasitol. 89, 97e107.
Hoekstra, R., Otsen, M., Tibben, J., Lenstra, J.A., Roos, M.H., 2000a. Non-autonomous
transposable elements in the genome of the parasitic nematode Haemonchus contortus.
Mol. Biochem. Parasitol. 106, 163e168.
Hoekstra, R., Otsen, M., Tibben, J., Lenstra, J.A., Roos, M.H., 2000b. Transposon associ-
ated markers for the parasitic nematode Haemonchus contortus. Mol. Biochem. Parasitol.
105, 127e135.
Hunt, P.W., Knox, M.R., Le Jambre, L.F., Mcnally, J., Anderson, L.J., 2008. Genetic and
phenotypic differences between isolates of Haemonchus contortus in Australia. Int. J. Para-
sitol. 38, 885e900.
Jacquiet, P., Cabaret, J., Cheikh, D., Thiam, E., 1997. Identification of Haemonchus species in
domestic ruminants based on morphometrics of spicules. Parasitol. Res. 83, 82e86.
Jasmer, D.P., Mitreva, M.D., Mccarter, J.P., 2004. mRNA sequences for Haemonchus contortus
intestinal cathepsin B-like cysteine proteases display an extreme in abundance and diver-
sity compared with other adult mammalian parasitic nematodes. Mol. Biochem. Parasi-
tol. 137, 297e305.
Jennions, M.D., Petrie, M., 2000. Why do females mate multiply? A review of the genetic
benefits. Biol. Rev. Camb. Philos. Soc. 75, 21e64.
Johnson, P.C., Webster, L.M., Adam, A., Buckland, R., Dawson, D.A., Keller, L.F., 2006.
Abundant variation in microsatellites of the parasitic nematode Trichostrongylus tenuis and
linkage to a tandem repeat. Mol. Biochem. Parasitol. 148, 210e218.
Karanu, F.N., Rurangirwa, F.R., Mcguire, T.C., Jasmer, D.P., 1993. Haemonchus contortus:
identification of proteases with diverse characteristics in adult worm excretory-secretory
products. Exp. Parasitol. 77, 362e371.
Karanu, F.N., Rurangirwa, F.R., Mcguire, T.C., Jasmer, D.P., 1997. Haemonchus contortus:
inter- and intrageographic isolate heterogeneity of proteases in adult worm excretory-
secretory products. Exp. Parasitol. 86, 89e91.
Koop, J.A., Dematteo, K.E., Parker, P.G., Whiteman, N.K., 2014. Birds are islands for
parasites. Biol. Lett. 10.
Kotze, A.C., Hunt, P.W., Skuce, P., Von Samson-Himmelstjerna, G., Martin, R.J.,
Sager, H., Krucken, J., Hodgkinson, J., Lespine, A., Jex, A.R., Gilleard, J.S.,
Beech, R.N., Wolstenholme, A.J., Demeler, J., Robertson, A.P., Charvet, C.L.,
Neveu, C., Kaminsky, R., Rufener, L., Alberich, M., Menez, C., Prichard, R.K.,
2014. Recent advances in candidate-gene and whole-genome approaches to the discov-
ery of anthelmintic resistance markers and the description of drug/receptor interactions.
Int. J. Parasitol. Drugs Drug Resist. 4, 164e184.
66 J.S. Gilleard and E. Redman

Kotze, A.C., Prichard, R.K., 2016. Anthelmintic resistance in Haemonchus contortus: history,
mechanisms and diagnosis. In: Gasser, R., Samson-Himmelstjerna, G.V. (Eds.), Haemon-
chus contortus and Haemonchosis Past, Present and Future Trends. vol. 93, pp. 397e428.
Kwa, M.S., Kooyman, F.N., Boersema, J.H., Roos, M.H., 1993. Effect of selection for benz-
imidazole resistance in Haemonchus contortus on beta-tubulin isotype 1 and isotype 2
genes. Biochem. Biophys. Res. Commun. 191, 413e419.
Laing, R., Kikuchi, T., Martinelli, A., Tsai, I.J., Beech, R.N., Redman, E., Holroyd, N.,
Bartley, D.J., Beasley, H., Britton, C., Curran, D., Devaney, E., Gilabert, A., Hunt, M.,
Jackson, F., Johnston, S.L., Kryukov, I., Li, K., Morrison, A.A., Reid, A.J.,
Sargison, N., Saunders, G.I., Wasmuth, J.D., Wolstenholme, A., Berriman, M.,
Gilleard, J.S., Cotton, J.A., 2013. The genome and transcriptome of Haemonchus contortus,
a key model parasite for drug and vaccine discovery. Genome Biol. 14, R88.
Laing, R., Martinelli, A., Tracey, A., Holroyd, N., Gilleard, J., Cotton, J.A., 2016. Haemon-
chus contortus: genome structure, organization and comparative genomics. In: Gasser, R.,
Samson-Himmelstjerna, G.V. (Eds.), Haemonchus contortus and Haemonchosis Past, Pre-
sent and Future Trends. vol. 93, pp. 569e598.
Le Jambre, L.F., 1977. Genetics of vulvar morph types in Haemonchus contortus: Haemonchus con-
tortus cayugensis from the Finger Lakes Region of New York. Int. J. Parasitol. 7, 9e14.
Le Jambre, L.F., 1979. Hybridization studies of Haemonchus contortus (Rudolphi, 1803) and H.
placei (place, 1893) (Nematoda: Trichostrongylidae). Int. J. Parasitol. 9, 455e463.
Le Jambre, L.F., Ractliffe, L.H., 1976. Response of Haemonchus contortus cayugensis to a
change in the ratio of smooth to linguiform. Parasitology 73, 213e222.
Le Jambre, L.F., Royal, W.M., 1980. Meiotic abnormalities in backcross lines of hybrid
Haemonchus. Int. J. Parasitol. 10, 281e286.
Le Jambre, L.F., Royal, W.M., Martin, P.J., 1979. The inheritance of thiabendazole resis-
tance in Haemonchus contortus. Parasitology 78, 107e119.
Lichtenfels, J.R., Pilitt, P.A., Hoberg, E.P., 1994. New morphological characters for identi-
fying individual specimens of Haemonchus spp. (Nematoda: Trichostrongyloidea) and a
key to species in ruminants of North America. J. Parasitol. 80, 107e119.
Love, M.I., Huber, W., Anders, S., 2014. Moderated estimation of fold change and disper-
sion for RNA-seq data with DESeq2. Genome Biol. 15, 550.
Lynch, M., Crease, T.J., 1990. The analysis of population survey data on DNA sequence
variation. Mol. Biol. Evol. 7, 377e394.
Mes, T.H., 2003. Demographic expansion of parasitic nematodes of livestock based on
mitochondrial DNA regions that conflict with the infinite-sites model. Mol. Ecol. 12,
1555e1566.
Morrison, D.A., Hoglund, J., 2005. Testing the hypothesis of recent population expansions
in nematode parasites of human-associated hosts. Hered. (Edinb.) 94, 426e434.
Newton, S.E., Morrish, L.E., Martin, P.J., Montague, P.E., Rolph, T.P., 1995. Protection
against multiply drug-resistant and geographically distant strains of Haemonchus contortus
by vaccination with H11, a gut membrane-derived protective antigen. Int. J. Parasitol.
25, 511e521.
Otsen, M., Hoekstra, R., Plas, M.E., Buntjer, J.B., Lenstra, J.A., Roos, M.H., 2001. Ampli-
fied fragment length polymorphism analysis of genetic diversity of Haemonchus contortus
during selection for drug resistance. Int. J. Parasitol. 31, 1138e1143.
Otsen, M., Plas, M.E., Groeneveld, J., Roos, M.H., Lenstra, J.A., Hoekstra, R., 2000a.
Genetic markers for the parasitic nematode Haemonchus contortus based on intron
sequences. Exp. Parasitol. 95, 226e229.
Otsen, M., Plas, M.E., Lenstra, J.A., Roos, M.H., Hoekstra, R., 2000b. Microsatellite diver-
sity of isolates of the parasitic nematode Haemonchus contortus. Mol. Biochem. Parasitol.
110, 69e77.
Genetic Diversity and Population Structure of H. contortus 67

Poeschel, G.P., Todd, A.C., 1972a. Disease-producing capacity of Haemonchus contortus


isolates in sheep. Am. J. Vet. Res. 33, 2207e2213.
Poeschel, G.P., Todd, A.C., 1972b. Selection for variations in pathogenicity of Haemonchus
contortus isolates. Am. J. Vet. Res. 33, 1575e1582.
Prichard, R., 2001. Genetic variability following selection of Haemonchus contortus with
anthelmintics. Trends Parasitol. 17, 445e453.
Redman, E., Grillo, V., Saunders, G., Packard, E., Jackson, F., Berriman, M., Gilleard, J.S.,
2008a. Genetics of mating and sex determination in the parasitic nematode Haemonchus
contortus. Genetics 180, 1877e1887.
Redman, E., Packard, E., Grillo, V., Smith, J., Jackson, F., Gilleard, J.S., 2008b. Microsatel-
lite analysis reveals marked genetic differentiation between Haemonchus contortus labora-
tory isolates and provides a rapid system of genetic fingerprinting. Int. J. Parasitol. 38,
111e122.
Redman, E., Sargison, N., Whitelaw, F., Jackson, F., Morrison, A., Bartley, D.J.,
Gilleard, J.S., 2012. Introgression of ivermectin resistance genes into a susceptible Hae-
monchus contortus strain by multiple backcrossing. PLoS Pathog. 8, e1002534.
Redman, E., Whitelaw, F., Tait, A., Burgess, C., Bartley, Y., Skuce, P.J., Jackson, F.,
Gilleard, J.S., 2015. The emergence of resistance to the benzimidazole anthlemintics
in parasitic nematodes of livestock is characterised by multiple independent hard and
soft selective sweeps. PLoS Negl. Trop. Dis. 9, e0003494.
Redmond, D.L., Windham, R., 2005. Characterization of proteinases in different isolates of
adult Haemonchus contortus. Parasitology 130, 429e435.
Roos, M.H., Hoekstra, R., Plas, M.E., Otsen, M., Lenstra, J.A., 1998. Polymorphic DNA
markers in the genome of parasitic nematodes. J. Helminthol. 72, 291e294.
Roos, M.H., Otsen, M., Hoekstra, R., Veenstra, J.G., Lenstra, J.A., 2004. Genetic analysis of
inbreeding of two strains of the parasitic nematode Haemonchus contortus. Int. J. Parasitol.
34, 109e115.
Rufener, L., Maser, P., Roditi, I., Kaminsky, R., 2009. Haemonchus contortus acetylcholine
receptors of the DEG-3 subfamily and their role in sensitivity to monepantel. PLoS
Pathog. 5, e1000380.
Salgotra, R.K., Gupta, B.B., Stewart JR., C.N., 2014. From genomics to functional markers
in the era of next-generation sequencing. Biotechnol. Lett. 36, 417e426.
Sangster, N.C., Bannan, S.C., Weiss, A.S., Nulf, S.C., Klein, R.D., Geary, T.G., 1999.
Haemonchus contortus: sequence heterogeneity of internucleotide binding domains from
P-glycoproteins. Exp. Parasitol. 91, 250e257.
Sangster, N.C., Redwin, J.M., Bjorn, H., 1998. Inheritance of levamisole and benzimidazole
resistance in an isolate of Haemonchus contortus. Int. J. Parasitol. 28, 503e510.
Sarai, R.S., Kopp, S.R., Coleman, G.T., Kotze, A.C., 2013. Acetylcholine receptor subunit
and P-glycoprotein transcription patterns in levamisole-susceptible and -resistant
Haemonchus contortus. Int. J. Parasitol. Drugs Drug Resist. 3, 51e58.
Sargison, N.D., Wilson, D.J., Bartley, D.J., Penny, C.D., Jackson, F., 2007. Haemonchosis
and teladorsagiosis in a Scottish sheep flock putatively associated with the overwintering
of hypobiotic fourth stage larvae. Vet. Parasitol. 147, 326e331.
Schwarz, E.M., Korhonen, P.K., Campbell, B.E., Young, N.D., Jex, A.R., Jabbar, A.,
Hall, R.S., Mondal, A., Howe, A.C., Pell, J., Hofmann, A., Boag, P.R., Zhu, X.Q.,
Gregory, T., Loukas, A., Williams, B.A., Antoshechkin, I., Brown, C.,
Sternberg, P.W., Gasser, R.B., 2013. The genome and developmental transcriptome
of the strongylid nematode Haemonchus contortus. Genome Biol. 14, R89.
Silvestre, A., Cabaret, J., 2002. Mutation in position 167 of isotype 1 beta-tubulin gene of
Trichostrongylid nematodes: role in benzimidazole resistance? Mol. Biochem. Parasitol.
120, 297e300.
68 J.S. Gilleard and E. Redman

Silvestre, A., Humbert, J.F., 2002. Diversity of benzimidazole-resistance alleles in populations


of small ruminant parasites. Int. J. Parasitol. 32, 921e928.
Silvestre, A., Sauve, C., Cortet, J., Cabaret, J., 2009. Contrasting genetic structures of two
parasitic nematodes, determined on the basis of neutral microsatellite markers and
selected anthelmintic resistance markers. Mol. Ecol. 18, 5086e5100.
Slatkin, M., 1987. Gene flow and the geographic structure of natural populations. Science
236, 787e792.
Tarrant, C.A., Blouin, M.S., Yowell, C.A., Dame, J.B., 1992. Suitability of mitochondrial
DNA for assaying interindividual genetic variation in small helminths. J. Parasitol. 78,
374e378.
Thomas, R.J., Waller, P.J., 1979. Field observations on the epidemiology of abomasal
parasites in young sheep during winter and spring. Res. Vet. Sci. 26, 209e212.
Tregenza, T., Wedell, N., 2000. Genetic compatibility, mate choice and patterns of
parentage: invited review. Mol. Ecol. 9, 1013e1027.
Troell, K., Engstrom, A., Morrison, D.A., Mattsson, J.G., Hoglund, J., 2006a. Global
patterns reveal strong population structure in Haemonchus contortus, a nematode parasite
of domesticated ruminants. Int. J. Parasitol. 36, 1305e1316.
Troell, K., Mattsson, J.G., Alderborn, A., Hoglund, J., 2003. Pyrosequencing analysis
identifies discrete populations of Haemonchus contortus from small ruminants. Int. J. Para-
sitol. 33, 765e771.
Troell, K., Tingstedt, C., Hoglund, J., 2006b. Phenotypic characterization of Haemonchus
contortus: a study of isolates from Sweden and Kenya in experimentally infected sheep.
Parasitology 132, 403e409.
Urdaneta-Marquez, L., Bae, S.H., Janukavicius, P., Beech, R., Dent, J., Prichard, R., 2014.
A dyf-7 haplotype causes sensory neuron defects and is associated with macrocyclic
lactone resistance worldwide in the nematode parasite Haemonchus contortus. Int.
J. Parasitol. 44, 1063e1071.
Van Wyk, J.A., Gerber, H.M., Van Aardt, W.P., 1977. Cryopreservation of the infective
larvae of the common nematodes of ruminants. Onderstepoort J. Vet. Res. 44, 173e194.
Waller, P.J., Rudby-Martin, L., Ljungstrom, B.L., Rydzik, A., 2004. The epidemiology of
abomasal nematodes of sheep in Sweden, with particular reference to over-winter
survival strategies. Vet. Parasitol. 122, 207e220.
Williamson, S.M., Storey, B., Howell, S., Harper, K.M., Kaplan, R.M., Wolstenholme, A.J.,
2011. Candidate anthelmintic resistance-associated gene expression and sequence
polymorphisms in a triple-resistant field isolate of Haemonchus contortus. Mol. Biochem.
Parasitol. 180, 99e105.
Wood, I.B., Amaral, N.K., Bairden, K., Duncan, J.L., Kassai, T., Malone JR., J.B.,
Pankavich, J.A., Reinecke, R.K., Slocombe, O., Taylor, S.M., et al., 1995. World
Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) second
edition of guidelines for evaluating the efficacy of anthelmintics in ruminants (bovine,
ovine, caprine). Vet. Parasitol. 58, 181e213.
Wright, S., 1931. Evolution in mendelian populations. Genetics 16, 97e159.
Xu, M., Molento, M., Blackhall, W., Ribeiro, P., Beech, R., Prichard, R., 1998. Ivermectin
resistance in nematodes may be caused by alteration of P-glycoprotein homolog. Mol.
Biochem. Parasitol. 91, 327e335.
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.
Advances in Parasitology, Volume 93
© 2016 Elsevier Ltd.
j
ISSN 0065-308X
http://dx.doi.org/10.1016/bs.apar.2016.02.010 All rights reserved. 69
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).
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 73

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
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 75

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
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 77

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
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 79

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
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 81

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
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 83

‘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).
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 85

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;
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 87

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
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 89

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
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 91

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.

REFERENCES
Amliwala, K., Bull, K., Willson, J., Harder, A., Holden-Dye, L., Walker, R.J., 2004. Emo-
depside, a cyclo-octadepsipeptide anthelmintic with a novel mode of action. Drugs
Future 29, 1015e1024.
Areskog, M., Engstr€ om, A., Tallkvist, J., von Samson-Himmelstjerna, G., H€ oglund, J., 2013.
PGP expression in Cooperia oncophora before and after ivermectin selection. Parasitol. Res.
112, 3005e3012.
Barett, J., 1981. Biochemistry of Parasitic Helminths. MacMillan Publishers Ltd., pp. 72e148
Bargmann, C., 2006. Chemosensation in C. elegans. WormBook 25, 1e29.
Baur, R., Beech, R., Sigel, E., Rufener, L., 2015. Monepantel irreversibly binds to and opens
Haemonchus contortus MPTL-1 and Caenorhabditis elegans ACR-20 receptors. Mol. Phar-
macol. 87, 96e102.
Beech, R.N., Neveu, C., 2015. The evolution of pentameric ligand-gated ion- channels and
the changing family of anthelmintic drug targets. Parasitology. http://dx.doi.org/
10.1017/S003118201400170X.
Beugnet, F., Franc, M., 2012. Insecticide and acaricide molecules and/or combinations to
prevent pet infestation by ectoparasites. Trends Parasitol. 28, 267e279.
Bird, A.F., Zuckerman, B.M., 1989. Studies on the surface coat (glycocalix) of the dauer larva
of Anguina agrotis. Int. J. Parasitol. 19, 235e240.
Bryant, C., Behm, C., 1989. Energy metabolism. In: Biochemical Adaptation in Parasites.
Chapman and Hall, pp. 25e69.
Chen, W., Terada, M., Cheng, J.T., 1996. Characterization of subtypes of gamma-amino-
butyric acid receptors in an Ascaris suum preparation by binding assay and binding of
PF1022A, a new anthelmintic, on the receptors. Parasitol. Res. 82, 97e101.
Crisford, A., Ebbinghaus-Kintscher, U., Schoenhense, E., Harder, A., Raming, K.,
O’Kelly, I.O., Ndukwe, K., O’Connor, V., Walker, R.J., Holden-Dye, L., 2015.
The cyclooctadepsipeptide anthelmintic emodepside differentially modulates nematode,
insect and human calcium-activated potassium (SLO) channel alpha subunits. PLoS
Negl. Trop. Dis. 9 (10), e0004062. http://dx.doi.org/10.1371/oumal.pntd.0004062.
92 A. Harder

Crook, M., 2014. The dauer hypothesis and the evolution of parasitism: 20 years on and still
going strong. Int. J. Parasitol. 44, 1e8.
Dalliere, N., Bhatla, N., Luedtke, Z., Ma, D.K., Woolman, J., Walker, R.J., Holden-
Dye, L., O’Connor, V., October 29, 2015. Multiple excitatory and inhibitory neural sig-
nals converge to fine-tune Caenorhabditis elegans feeding to food availability. FASEB J. pii:
fj.15e279257 (Epub ahead of print).
De Graef, J., Demeler, J., Skuce, P., Mitreva, M., Von Samson-Himmelstjerna, G.,
Vercruysse, J., Claerebout, E., Geldhof, P., 2013. Gene expression analysis of ABC trans-
porters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to
macrocyclic lactones. Parasitology 140, 499e508.
Drogem€ uller, M., Schnieder, T., von Samson-Himmelstjerna, G., 2004. Beta-tubulin
complementary DNA sequence variations observed between cyathostomins from benz-
imidazole-susceptible and -resistant populations. J. Parasitol. 90, 868e870.
Ghisi, M., Kaminsky, R., M€aser, P., 2007. Phenotyping and genotyping of Haemonchus con-
tortus isolates reveals a new putative candidate mutation for benzimidazole resistance in
nematodes. Vet. Parasitol. 144, 313e320.
Godoy, P., Lian, J., Beech, R.N., Prichard, R.K., 2015. Haemonchus contortus P-glycoprotein-
2: in situ localisation and characterisation of macrocyclic lactone transport. Int. J.
Parasitol. 45, 85e93.
Gasser, R.B., Schwarz, E.M., Korhonen, P.K., Young, N.D., 2016. Understanding Haemon-
chus contortus better through genomics and ranscriptomics. In: Gasser, R., Samson-
Himmelstjerna, G.V. (Eds.), Haemonchus contortus and Haemonchosis Past, Present and
Future Trends. vol. 93, pp. 518e568.
Gull, K., Dawson, P.J., Davis, C., Byard, E.H., 1986. 619th Meeting, Cambridge, held at the
University of Cambridge on 2e4 July 1986; Microtubules as target organelles for benz-
imidazole anthelmintic chemotherapy. Biochem. Soc. Trans. 15, 59e60.
Harder, A., Wunderlich, F., 1991. Darmnematoden des Menschen. Biol. Unserer Zeit 21,
37e44.
Harder, A., 2002. Chemotherapeutic approaches to nematodes: current knowledge and
outlook. Parasitol. Res. 88, 271e277.
Hibbs, R.E., Gouaux, E., 2011. Principles of activation and permeation in an anion-selective
Cys loop receptor. Nature 474, 54e60.
Hodgkinson, J.E., Clark, H.J., Kaplan, R.M., Lake, S.L., Matthews, J.B., 2008. The role of
polymorphisms at beta tubulin isotype 1 codons 167 and 200 in benzimidazole resistance
in cyathostomins. Int. J. Parasitol. 38, 1149e1160.
Holden-Dye, L., Crisford, A., Welz, C., von Samson-Himmelstjerna, G., Walker, R.J.,
O’Connor, V., 2012. Worms take to the slo lane: a perspective on the mode of action
of emodepside. Invert. Neurosci. 12, 29e36.
Holden-Dye, L., Joyner, M., O’Connor, V., Walker, R., 2013. Nicotinic acetylcholine re-
ceptors: a comparison of the nAChRs of Caenorhabditis elegans and parasitic nematodes.
Parasitol. Int. 62, 606e615.
Hu, Y., Xiao, S.H., Aroian, R.V., 2009. The new anthelmintic tribendimidine is an L-type
(levamisole and pyrantel). nicotinic acetylcholine receptor agonist. PLoS Negl. Trop.
Dis. 3 (8), e499. http://dx.doi.org/10.1371/journal.pntd.0000499.
Janssen, I.J., Kr€ ucken, J., Demeler, J., Basiaga, M., Kornas, S., von Samson-
Himmelstjerna, G., 2013. Genetic variants and increased expression of Parascaris equorum
P-glycoprotein-11 in populations with decreased ivermectin susceptibility. PLoS One 8
(4), e61635. http://dx.doi.org/10.1371/journal.pone.0061635.
Janssen, I.J., Kr€ucken, J., Demeler, J., von Samson-Himmelstjerna, G., 2015. Transgenically
expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caeno-
rhabditis elegans. Int. J. Parasitol. DDR 5, 44e47.
Biochemistry of Haemonchus contortus and Other Parasitic Nematodes 93

James, C.E., Hudson, A.L., Davey, M.W., 2009. Drug resistance mechanisms in helminthes:
is it survival of the fittest? Trends Parasitol. 25, 328e335.
Joyner, M., 2010. Investigating the Effects of Novel Anthelmintics. Amidantel, Bay d9216
and Tribendimidine. Transfer thesis November 2010.
Kapur, J., Sood, M.L., 1987. Biochemistry of Haemonchus e a review. Angew. Parasitol. 28,
211e228.
Kerboeuf, D., Blackhall, W., Kaminski, R., von Samson-Himmelstjerna, G., 2003. P-glyco-
protein in helminths function and perspectives for anthelmintic treatment and reversal of
resistance. Int. J. Antimicrob. Agents 22, 332e346.
Kita, K., Hirawake, H., Takamiya, S., 1997. Cytochromes in the respiratory chain of hel-
minth mitochondria. Int. J. Parasitol. 27, 617e630.
K€ohler, P., 2006. Stoffwechselphysiologie von Parasiten. In: Hiepe, T., Lucius, R.,
Gottstein, B. (Eds.), Allgemeine Parasitologie. Verlag Parey, pp. 188e218.
Kr€ucken, J., Harder, A., Jeschke, P., Holden-Dye, L., O’Connor, V., Welz, C., von Samson-
Himmelstjerna, G., 2012. Anthelmintic cyclooctadepsipeptides: complex in structure
and mode of action. Trends Parasitol. 28, 385e394.
Kulke, D., von Samson-Himmelstjerna, G., Miltsch, S.M., Wolstenholme, A.J., Jex, A.,
Gasser, R.B., Ballesteros, C., Geary, T.G., Keiser, J., Townson, S., Harder, A.,
Kr€ ucken, J., December 18, 2014. Characterization of the Ca2þ-gated and voltage-
dependent Kþ-channel Slo-1 of nematodes and its interaction with emodepside. PLoS
Negl. Trop. Dis. 8 (12), e3401. http://dx.doi.org/10.1371/journal.pntd.0003401.
Kwa, M.S., Veenstra, J.G., Roos, M.H., 1994a. Benzimidazole resistance in Haemonchus con-
tortus is correlated with a conserved mutation at amino acid 200 in beta-tubulin isotype 1.
Mol. Biochem. Parasitol. 63, 299e303.
Kwa, M.S., Veenstra, J.G., van Dijk, M., Roos, M.H., 1994b. Beta-tubulin genes from the
parasitic nematode Haemonchus contortus modulate drug resistance in Caenorhabditis elegans.
J. Mol. Biol. 246, 500e510.
Laing, R., Kikuchi, T., Martinelli, A., Tsai, I.J., Beech, R.N., Redman, E., Holroyd, N.,
Bartley, D.J., Beasley, H., Britton, C., Curran, D., Devaney, E., Gilabert, A.,
Hunt, M., Jackson, F., Johnston, S.L., Kryukov, I., Li, K., Morrison, A.A., Reid, A.J.,
Sargison, N., Saunders, G.I., Wasmuth, J.D., Wolstenholme, A., Berriman, M.,
Gilleard, J.S., Cotton, J.A., 2013. The genome and transcriptome of Haemonchus contortus,
a key model parasite for drug and vaccine discovery. Genome Biol. 14, R88. http://
dx.doi.org/10.1186/gb-2013-14-8-r88.
Laing, R., Martinelli, A., Tracey, A., Holroyd, N., Gilleard, J., Cotton, J.A., 2016. Haemon-
chus contortus: genome structure, organization and comparative genomics. In: Gasser, R.,
Samson-Himmelstjerna, G.V. (Eds.), Haemonchus contortus and Haemonchosis Past,
Present and Future Trends, 93, pp. 569e598.
Lespine, A., Menez, C., Bourguinat, C., Prichard, R.K., 2012. P-glycoproteins and other
multidrug resistance transporters in the pharmacology of anthelmintics: prospects for
reversing transport-dependent anthelmintic resistance. Int. J. Parasitol. DDR 2, 58e75.
Maizels, R.M., 2013. Toxocara canis: molecular basis of immune recognition and evasion. Vet.
Parasitol. 193, 365e374.
Martin, R.J., Robertson, A.P., Buxton, S.K., Beech, R.N., Charvet, C.L., Neveu, C., 2012.
Levamisole receptors: a second awakening. Trends Parasitol. 28, 289e296.
Mehlhorn, H., 2008a. Encyclopedia of Parasitology, third ed. Springer-Verlag, p. 47.
Mehlhorn, H., 2008b. Encyclopedia of Parasitology, third ed. Springer-Verlag, pp. 950e983.
Osanai, A., Harada, S., Sakamoto, K., Shimizu, H., Inaoka, K., Kita, K., 2009. Crystallization
of mitochondrial rhodoquinolfumarate reductase from the parasitic nematode Ascaris
suum with the specific inhibitor flutolanil. Acta Crystallogr. Sect. F Struct. Biol. Crystal-
logr. Commun. 65, 941e944.
Page, A.P., Johnstone, I.L., 2007. The cuticle. WormBook 19, 1e15.
94 A. Harder

Page, A.P., Rudin, W., Fluri, E., Blaxter, M.L., Maizels, R.M., 1992. Toxocara canis: a labile
antigenic surface coat overlying the epicuticle of infective larvae. Exp. Parasitol. 75, 72e86.
Portillo, V., Jagannathan, S., Wolstenholme, A.J., 2003. Distribution of glutamate-gated
chloride channel subunits in the parasitic nematode Haemonchus contortus. J. Comp.
Neurol. 462, 213e222.
Prichard, R.K., 2001. Genetic variability following selection of Haemonchus contortus with
anthelmintics. Trends Parasitol. 17, 445e453.
Puttachary, S., Trailovic, S.M., Robertson, A.P., Thompson, D.P., Woods, D.J.,
Martin, R.J., 2013. Derqiantel and abamectin: effects and interactions on isolated tissues
of Ascaris suum. Mol. Biochem. Parasitol. 188, 79e86.
Qian, H., Martin, R.J., Robertson, A.P., 2006. Pharmacology of N-, L-, and B-subtypes of
nematode nAChR resolved at the single-channel level in Ascaris suum. FASEB J. 20,
2606e2608.
Rew, R.S., Fetterer, R.H., 1986. Mode of action of antinematodal drugs. In:
Campbell, W.C., Rew, R.S. (Eds.), Chemotherapy of Parasitic Diseases, pp. 321e337
(Chapter 16).
Robinson, M.W., McFerran, N., Trudgett, A., Hoey, L., Faiweather, I., 2004. A possible
model of benzimidazole binding to beta-tubulin disclosed by invoking an inter-domain
movement. J. Mol. Graph. Model. 23, 275e284.
Roos, M.H., 1997. The role of drugs in the control of parasitic nematode infections: must we
do without? Parasitology 114, S137eS144.
Saeger, B., Schmitt-Wrede, H.P., Dehnhardt, M., Benten, W.P., Kr€ ucken, J., Harder, A.,
von Samson-Himmelstjerna, G., Wiegand, H., Wunderlich, F., 2001. Latrophilin-like
receptor from the parasite nematode Haemonchus contortus as target for the anthelmintic
depsipeptide PF 1022A. FASEB J. 15, 1332e1334.
Sakai, C., Tomitsuka, E., Esumi, H., Harada, S., Kita, K., 2012. Mitochondrial fumarate
reductase as a target of chemotherapy: from parasites to cancer cells. Biochim. Biophys.
Acta 1820, 643e651.
Schwarz, E.M., Korhonen, P.K., Campbell, B.E., Young, N.D., Jex, A.R., Jabbar, A.,
Hall, R.S., Mondal, A., Howe, A.C., Pell, J., Hofmann, A., Boag, P.R., Zhu, X.Q.,
Gregory, T.R., Loukas, A., Williams, B.A., Antoshechkin, I., Brown, C.T.,
Sternberg, P.W., Gasser, R.B., 2013. The genome and developmental transcriptome
of the strongyloid nematode Haemonchus contortus. Genome Biol. 14, R89.
Silvestre, A., Cabaret, J., 2002. Mutation in position 167 of isotype 1 beta-tubulin gene of
trichostrongyloid nematodes: role in benzimidazole resistance? Mol. Biochem. Parasitol.
120, 297e300.
Thomas, J.H., Lockerly, S., 1999. Neurobiology. In: Hope, I.A. (Ed.), C. elegans A Practical
Approach, the Practical Approach Series. Oxford University Press, pp. 143e179 (Chap-
ter 8).
Veglia, F., 1916. The anatomy and life history of Haemonchus contortus. In: The Third and
Fourth Reports of the Director of Veterinary Research. Union of South Africa,
pp. 349e500.
Von Samson-Himmelstjerna, G., Blackhall, W.J., McCarthy, J.S., Skuce, P.J., 2007. Single
nucleotide polymorphism (SNP) markers for benzimidazole resistance in veterinary
nematodes. Parasitology 134, 1077e1086.
Walker, R.J., Brooks, H.L., Holden-Dye, L., 1996. Evolution and overview of classical trans-
mitter molecules and their receptors. Parasitology 113, S3eS33.
Wehner, Gehring, 1995a. Zoologie. Thieme-Verlag, p. 538.
Wehner, Gehring, 1995b. Zoologie. Thieme-Verlag, pp. 354e357.
Wolstenholme, A., 2012. Glutamate-gated chloride channels. J. Biol. Chem. 287,
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
University of New England, Armidale, NSW, Australia
{
University of Edinburgh, Roslin, Midlothian, United Kingdom
jj
University of Pretoria, Hatfield, South Africa
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

Advances in Parasitology, Volume 93


© 2016 Elsevier Ltd.
j
ISSN 0065-308X
http://dx.doi.org/10.1016/bs.apar.2016.02.022 All rights reserved. 95
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
98 R.B. Besier et al.

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
Haemonchus contortus Infection in Small Ruminants 99

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
100 R.B. Besier et al.

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
Haemonchus contortus Infection in Small Ruminants 101

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.
102 R.B. Besier et al.

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
Haemonchus contortus Infection in Small Ruminants 103

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
104 R.B. Besier et al.

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
Haemonchus contortus Infection in Small Ruminants 105

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
106 R.B. Besier et al.

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
Haemonchus contortus Infection in Small Ruminants 107

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
110 R.B. Besier et al.

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
Haemonchus contortus Infection in Small Ruminants 111

(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
112 R.B. Besier et al.

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
Haemonchus contortus Infection in Small Ruminants 113

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
114 R.B. Besier et al.

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.
Haemonchus contortus Infection in Small Ruminants 115

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
116 R.B. Besier et al.

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
Haemonchus contortus Infection in Small Ruminants 117

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
118 R.B. Besier et al.

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
Haemonchus contortus Infection in Small Ruminants 119

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.,
120 R.B. Besier 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
Haemonchus contortus Infection in Small Ruminants 121

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
122 R.B. Besier et al.

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:

Haemonchus contortus Infection in Small Ruminants


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
Haemonchus contortus Infection in Small Ruminants
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
126 R.B. Besier et al.

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.
Haemonchus contortus Infection in Small Ruminants 127

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
128 R.B. Besier et al.

(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
Haemonchus contortus Infection in Small Ruminants 129

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.
130 R.B. Besier et al.

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.
Haemonchus contortus Infection in Small Ruminants 131

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.
132 R.B. Besier et al.

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.
Haemonchus contortus Infection in Small Ruminants 133

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.

REFERENCES
Abbott, E.M., Parkins, J.J., Holmes, P.H., 1985a. Influence of dietary protein on parasite
establishment and pathogenesis in Finn Dorset and Scottish Blackface lambs given a
single moderate infection of Haemonchus contortus. Res. Vet. Sci. 38, 6e13.
Abbott, E.M., Parkins, J.J., Holmes, P.H., 1985b. Influence of dietary protein on the
pathophysiology of ovine haemonchosis in Finn Dorset and Scottish Blackface lambs
given a single moderate infection. Res. Vet. Sci. 38, 54e60.
Abbott, E.M., Parkins, J.J., Holmes, P.H., 1986a. The effect of dietary protein on the
pathophysiology of acute ovine haemonchosis. Vet. Parasitol. 20, 291e306.
Abbott, E.M., Parkins, J.J., Holmes, P.H., 1986b. The effect of dietary protein on the path-
ogenesis of acute ovine haemonchosis. Vet. Parasitol. 20, 275e289.
Agyei, A.D., Saponga, D., Probert, A.J., 1991. Periparturient rise in faecal nematode egg
counts in West African Dwarf sheep in Southern Ghana in the absence of arrested stron-
gyle larvae. Vet. Parasitol. 39, 79e88.
Agyei, A.D., 1997. Seasonal changes in the level of infective strongylate nematode larvae on
pasture in the coastal savanna regions of Ghana. Vet. Parasitol. 70, 175e182.
Albers, G.A., Le Jambre, L.F., 1983. Erythrocyte potassium concentration: a simple param-
eter for erythropoiesis in sheep infected with Haemonchus contortus. Res. Vet. Sci. 35,
273e276.
Albers, G.A.A., Gray, G.D., Le Jambre, L.F., Piper, L.R., Barger, I.A., Barker, J.S.F., 1989.
The effect of haemonchus contortus on liveweight gain and wool growth in young Merino
sheep. Aust. J. Agric. Res. 40, 419e432.
Allonby, E.W., Urquhart, G.M., 1975. The epidemiology and pathogenic significance of
haemonchosis in a Merino flock in East Africa. Vet. Parasitol. 1, 129e143.
Altaif, K.I., Issa, W.H., 1983. Seasonal fluctuations and hypobiosis of gastrointestinal nema-
todes of Awassi lambs in Iraq. Parasitology 86, 301e310.
Altaif, K.I., Yakoob, A.Y., 1987. Development and survival of Haemonchus contortus larvae on
pasture in Iraq. Trop. Anim. Health Prod. 19, 88e92.
Amaradasa, B.S., Lane, R.A., Manage, R., 2010. Vertical migration of Haemonchus contortus
infective larvae on Cynodon dactylon and Paspalum notatum pastures in response to climatic
conditions. Vet. Parasitol. 170, 78e87.
Anene, B.M., Onyekwodiri, E.O., Chime, A.B., Anika, S.M., 1994. Gastrointestinal para-
sites in sheep and goats of south eastern Nigeria. Small Rumin. Res. 13, 187e192.
Angulo-Cubillan, F.J., García-Coiradas, L., Alunda, J.M., Cuquerella, M., De la Fuente, C.,
2010. Biological characterization and pathogenicity of three Haemonchus contortus isolates
in primary infections in lambs. Vet. Parasitol. 171, 99e105.
134 R.B. Besier et al.

Aumont, G., Gruner, L., 1989. Population evolution of the free-living stage of goat gastro-
intestinal nematodes on herbage under tropical conditions in Guadeloupe (French West
Indies). Int. J. Parasitol. 19, 539e546.
Ayalew, L., Gibbs, H.C., 1973. Seasonal fluctuations of nematode populations in breeding
ewes and lambs. Can. J. Comp. Med. 37, 79e89.
Bailey, J.N., Kahn, L.P., Walkden-Brown, S.W., 2009. Availability of gastro-intestinal nem-
atode larvae to sheep following winter contamination of pasture with six nematode spe-
cies on the Northern Tablelands of New South Wales. Vet. Parasitol. 160, 89e99.
Banks, D.J.D., Singh, R., Barger, I.A., Pratrap, P.B., Le Jambre, L.E., 1990. Development
and survival of infective larvae of Haemonchus contortus and Trichostrongylus colubriformis
on pasture in a tropical climate. Int. J. Parasitol. 20, 155e160.
Barger, I.A., Benyon, P.R., Southcott, W.H., 1972. Simulation of pasture larval populations
of Haemonchus contortus. Proc. Aust. Soc. Anim. Prod. 9, 38e42.
Barger, I.A., Cox, G.W., 1984. Wool production in sheep chronically infected with Haemon-
chus contortus. Vet. Parasitol. 15, 169e175.
Barger, I.A., Siale, K.I., Banks, D.J.D., Le Jambre, L.F., 1994. Rotational grazing for control
of gastrointestinal nematodes of goats in a wet tropical environment. Vet. Parasitol. 53,
109e116.
Barger, I.A., 1999. The role of epidemiological knowledge and grazing management for hel-
minth control in small ruminants. Int. J. Parasitol. 29, 41e47.
Barnes, E.H., Dobson, R.J., Barger, I.A., 1995. Worm control and anthelmintic resistance:
adventures with a model. Parasitol. Today 11, 53e63.
Barrow, D.B., 1964. The epizootiology of nematode parasites of sheep in the Border area.
Onderstepoort J. Vet. Res. 31, 151e162.
Berberian, J.F., Mizelle, J.D., 1957. Developmental studies on Haemonchus contortus Rudolphi
(1803). Am. Midl. Nat. 57, 421e439.
Berbigier, P., Gruner, L., Mambrini, M., Sophie, S.A., 1990. Faecal water content and egg
survival of goat gastrointestinal strongyles under dry tropical conditions in Guadeloupe.
Parasitol. Res. 76, 379e385.
Beriajaya, Copeman, D.B., 2006. Haemonchus contortus and Trichostrongylus colubriformis in
pen-trials with Javanese thin tail sheep and Kacang cross Etawah goats. Vet. Parasitol.
315e323.
Besier, R.B., 1992. Intraspecific Variation in Haemonchus contortus: Ecological Studies with an
Isolate from Western Australia (Ph.D. thesis). Murdoch University.
Besier, R.B., Dunsmore, J.D., 1993a. The ecology of Haemonchus contortus in a winter rainfall
region of Australia. 1. The development of eggs to infective larvae. Vet. Parasitol. 45,
275e292.
Besier, R.B., Dunsmore, J.D., 1993b. The ecology of Haemonchus contortus in a winter
rainfall region of Australia. 2. The survival of infective larvae on pasture. Vet. Parasitol.
45, 293e306.
Besier, R.B., Kahn, L.P., Sargsion, N.D., Van Wyk, J.A., 2016. Diagnosis, treatment and
management of Haemonchus contortus in small ruminants. In: Gasser, R., Samson-
Himmelstjerna, G.V. (Eds.), Haemonchus contortus and Haemonchosis Past, Present and
Future Trends. vol. 93, pp. 181e238.
Biggs, H.C., Anthonissen, M., 1982. The seasonal incidences of helminth parasites and Oes-
trus ovis in Karakul sheep in the Kalahari region of South West Africa-Namibia. Onder-
stepoort J. Vet. Res. 49, 73e77.
Bisset, S.A., Morris, C.A., 1996. Feasibility and implications of breeding sheep for resilience
to nematode challenge. Int. J. Parasitol. 26, 857e868.
Blackie, S., 2014. A review of the epidemiology of gastrointestinal nematode infections in
sheep and goats in Ghana. J. Agric. Sci. 6, 109e118.
Haemonchus contortus Infection in Small Ruminants 135

Boag, B., Thomas, R.J., 1985. The effect of temperature on the survival of infective larvae of
nematodes. J. Parasitol. 71, 383e384.
Bolajoko, M.B., Morgan, E.R., 2012. Relevance of improved epidemiological knowledge
to sustainable control of Haemonchus contortus in Nigeria. Anim. Health Res. Rev. 13,
196e208.
Bowman, D.D., 2014. Georgi’s Parasitology for Veterinarians, tenth ed. Elsevier Health Sci-
ence Division, Philadelphia, USA.
Brunsdon, R.V., 1970. Seasonal changes in the level and composition of nematode worm
burdens in young sheep. N.Z. Vet. J. 13, 126e148.
Brunsdon, R.V., 1973. Inhibited development of Haemonchus contortus in naturally acquired
infections in sheep. N.Z. Vet. J. 25, 125e126.
Bullick, G.R., Anderson, F.L., 1978. Effect of irrigation on survival of third stage Haemonchus
contortus larvae (Nematoda: Trichostrongylidae). Gt. Basin Nat. 38, 369e378.
Chandrawathani, P., 2004. Problems in the Control of Nematode Parasites of Small Rumi-
nants on Malaysia: Resistance to Anthelmintics and the Biological Control Alternatives
(Doctoral Thesis). Swedish University of Agricultural Sciences, Uppsala, Sweden.
Charles, T.P., 1989. Seasonal prevalence of gastrointestinal nematodes of goats in Pernam-
buco state, Brazil. Vet. Parasitol. 30, 335e343.
Chaudary, F.R., Qayyum, M., Miller, J.E., 2008. Development and survival of Haemonchus
contortus infective larvae derived from sheep faeces under sub-tropical conditions in the
Potohar region of Pakistan. Trop. Anim. Health Prod. 40, 85e92.
Cheah, T.S., Rajamanickam, C., 1997. Epidemiology of gastro-intestinal nematodes of sheep
in wet tropical conditions in Malaysia. Trop. Anim. Health Prod. 29, 165e173.
Chiejina, S.N., Fakae, B.B., Eze, B.O., 1988. Arrested development of gastrointestinal tri-
chostrongylids in goats in Nigeria. Vet. Parasitol. 28, 103e113.
Clark, C.H., Kiesel, G.K., Goby, C.H., 1962. Measurements of blood loss caused by Haemon-
chus contortus infection in sheep. Am. J. Vet. Res. 23, 977e980.
Clunies-Ross, I., Gordon, H.McL., 1936. The Internal Parasites and Parasitic Disease of
Sheep. Angus and Robertson Ltd.
Cobon, D.H., O’Sullivan, B.M., 1992. Effect of Haemonchus contortus on productivity of
ewes, lambs and weaners in a semi-arid environment. J. Agric. Sci. 118, 245e248.
Cohen, R.D.H., Eastoe, R.D., Hotson, I.K., Smeal, M.G., 1972. Effect of anthelmintics and
grazing management on production and helminthosis of Merino wethers. Aust. J. Exp.
Agric. Anim. Husb. 12, 247e251.
Connan, R.M., 1971. The seasonal incidence of inhibition of development in Haemonchus
contortus. Res. Vet. Sci. 12, 271e274.
Conder, G.A., 1978. The effect of UV radiation on survival of free-living stages of Haemon-
chus contortus. Proc. Helminth. Soc. Wash. 45, 230e232.
Crofton, H.D., 1948. The ecology of immature phases of trichostrongyle nematodes. Para-
sitology 39, 17e25.
Crofton, H.D., 1954. The vertical migration of infective larvae of strongyloid nematodes.
J. Helminthol. 28, 35e52.
Crofton, H.D., 1963. Nematode Parasite Population in Sheep and on Pasture. Technical
Communication No. 35, Commonwealth Bureau of Helminthology. Commonwealth
Agricultural Bureaux, St Albans.
Crofton, H.D., Whitlock, J.H., 1965. Ecological and biological plasticity of sheep
nemtatodes.V. The relationship between egg volume and hatching time. Cornell Vet.
55, 275e279.
Dargie, J.D., Allonby, E.W., 1975. Pathophysiology of single challenge infections of Haemon-
chus contortus (In “Merino sheep: studies on red cell kinetics and the ‘self-cure’
phenomenon”). Int. J. Parasitol. 5, 147e157.
136 R.B. Besier et al.

De Chaneet, G.C., Mayberry, C.J., 1978. Ovine Haemonchosis: A Review and Report of
Epizootics in North-west Western Australia and of a Trial at Esperance Western
Australia. Bulletin 41. Department of Agriculture, Western Australia.
Dinaburg, A.G., 1944a. Development and survival under conditions of eggs and larvae of the
common ruminant stomach worm, Haemonchus contortus. J. Agric. Res. 69, 421e433.
Dinaburg, A.G., 1944b. The survival of the common ruminant stomach worm Haemonchus
contortus, on outdoor grass plots. Am. J. Vet. Res. 5, 32e37.
Dinnik, J.A., Dinnik, N.N., 1958. Observations on the development of Haemonchus contortus
larvae under field conditions in the Kenya highlands. Bull. Epiz. Dis. Afr. 9, 11e21.
Dinnik, J.A., Dinnik, N.N., 1961. Observations on the longevity of Haemonchus contortus
larvae on pasture herbage in the Kenya highlands. Bull. Epiz. Dis. Afr. 9, 193e208.
Dobson, R.J., Barnes, E.H., Tyrrell, K.L., Hosking, B.C., Larsen, J.W.A., Besier, R.B.,
Love, S., Rolfe, P.F., Bailey, J.N., 2011. A multi-species model to assess the effect of
refugia on worm control and anthelmintic resistance in sheep grazing systems. Aust.
Vet. J. 89, 200e208.
Donald, A.D., 1968. Ecology of the free-living stages of nematode parasites of sheep. Aust.
Vet. J. 44, 139e144.
Donald, A.D., Morley, F.H.W., Waller, P.J., Axelson, A., Donnelly, J.R., 1978. Availability
to grazing sheep of gastrointestinal nematode infection arising from summer contamina-
tion of pastures. Aust. J. Agric. Res. 29, 180e204.
Domke, A.V.M., Chartier, C., Gjerde, B., Leine, N., Vatn, S., Stuen, S., 2013. Prevalence of
gastrointestinal helminths, lungworms and liver fluke in sheep and goats in Norway. Vet.
Parasitol. 194, 40e48.
Dorny, P., Symoens, C., Jalila, A., Vercruysse, J., Sani, R., 1995. Strongyle infections in
sheep and goats under the traditional husbandry system in peninsular Malaysia. Vet. Para-
sitol. 56, 121e136.
Dunn, A.M., 1978. Veterinary Helminthology, second ed. William Heinemann Medical
Books Ltd, pp. 184e185.
E1-Azazy, O.M.E., 1990. Absence of hypobiosis in abomasal nematodes of sheep and goats in
Egypt. Vet. Parasitol. 37, 55e60.
E1-Azazy, O.M.E., 1995. Seasonal changes and inhibited development of the abomasal nem-
atodes of sheep and goats in Saudi Arabia. Vet. Parasitol. 58, 91e98.
Ellenby, C., 1968. Desiccation survival of the infective larva of Haemonchus contortus. J. Exp.
Biol. 49, 469e475.
Eysker, M., Ogunsusi, R.A., 1980. Observations on epidemiological and clinical aspects of
gastrointestinal helminthiasis of sheep in northern Nigeria during the rainy season.
Res. Vet. Sci. 28, 58e61.
Eysker, M., Bakker, N., Kooyman, F.N.J., Van der Linden, D., Schrama, C.,
Ploeger, H.W., 2005. Consequences of the unusually warm and dry summer of
2003 in The Netherlands: poor development of free living stages, normal survival of
infective larvae and long survival of adult gastrointestinal nematodes of sheep. Vet. Par-
asitol. 133, 313e321.
Fabiyi, J.P., 1973. Seasonal fluctuations of nematode infestations in goats in the savannah belt
of Nigeria. Bull. Epiz. Dis. Afr. 21, 277e285.
Fabiyi, J.P., 1987. Production losses and control of helminths in ruminants of tropical regions.
Int. J. Parasitol. 17, 435e442.
Fakae, B.B., 1990. Seasonal changes and hypobiosis in Haemonchus contortus infections in the
West African Dwarf sheep and goats in the Nigerian derived savannah. Vet. Parasitol. 36,
123e130.
Falzon, L.C., Menzies, P.I., VanLeeuwen, J., Shakya, K.P., Jones-Bitton, A., Avula, J.,
Jansen, J.T., Peregrine, A.S., 2014. Pilot project to investigate over-wintering of free-
living gastrointestinal nematode larvae of sheep in Ontario, Canada. Can. Vet. J. 55,
749e756.
Haemonchus contortus Infection in Small Ruminants 137

Fernandez-Ruvalcaba, M., Vazquez-Prats, V., Liebano-Hernandez, E., 1994. Development


and recovery of Haemonchus contortus first larval stages on experimental plots in Mexico.
Vet. Parasitol. 51, 263e269.
Fox, M.T., 1997. Pathophysiology of infection with gastrointestinal nematodes in domestic
ruminants: recent developments. Vet. Parasitol. 72, 285e308.
Fritsche, T., Kaufman, J., Pfister, K., 1993. Parasite spectrum and seasonal epidemiology of
gastrointestinal nematodes of small ruminants in the Gambia. Vet. Parasitol. 49, 279e283.
Gatongi, P.M., Prichard, R.K., Ranjan, S., Gathuma, J.M., Munyua, W.K., Cheruiyot, H.,
Scott, M.E., 1998. Hypobiosis of Haemonchus contortus in natural infections of sheep and
goats in a semi-arid area of Kenya. Vet. Parasitol. 77, 49e61.
Georgi, J.R., 1974. Parasitology for Veterinarians, 2nd edition. WB Saunders and Company,
pp. 303e304.
Gibbs, H.C., 1986. Hypobiosis in parasitic nematodes e an update. Adv. Parasitol. 25,
129e174.
Gibson, T.E., Everett, G., 1976. The ecology of the free-living stages of Haemonchus contortus.
Br. Vet. J. 132, 50e59.
Githigia, S.M., Thamsborg, S.M., Munyua, W.K., Maingi, N., 2001. Impact of gastrointes-
tinal helminths on production in goats in Kenya. Small Rumin. Res. 42, 21e29.
Gordon, H.M.L., 1948. The epidemiology of parasite diseases, with special reference to
studies with nematode parasites of sheep. Aust. Vet. J. 24, 17e45.
Grant, J.L., 1981. The epizootiology of nematode parasites of sheep in a high rainfall area of
Zimbabwe. J. South Afr. Vet. Med. Assoc. 52, 33e37.
Grosz, D.D., Eljaki, A.A., Holler, L.D., Petersen, D.J., Holler, S.W., Hildreth, M.B., 2013.
Overwintering strategies of a population of anthelmintic-resistant Haemonchus contortus
within a sheep flock from the United States Northern Great Plains. Vet. Parasitol.
196, 143e152.
Gupta, R.R., Singhali, K.C., Saxena, P.N., Kumar, A., 1982. Effect of ultraviolet radiation
on the developmental stages of Haemonchus contortus. Ind. J. Parasitol. 6, 227e229.
Gupta, R.P., Yadav, C.L., Chaudhri, S.S., 1987. Epidemiology of gastrointestinal nematodes
of sheep and goats in Haryana, India. Vet. Parasitol. 24, 117e127.
Helle, O., 1971. The survival of nematodes and cestodes of sheep in the pasture during the
winter in Eastern Norway. Acta Vet. Scand. 12, 504e512.
Holmes, P.H., 1987. Pathophysiology of nematode infections. Int. J. Parasitol. 17, 443e451.
Howlader, M.M.R., Capitan, S.S., Eduardo, S.L., Roxas, N.P., Sevilla, C.C., 1997. Perfor-
mance of growing goats experimentally infected with stomach worm (Haemonchus
contortus). Asian Aust. J. Anim. Sci. 10, 534e539.
Hsu, C., Levine, N.D., 1977. Degree-day concept in development of infective larvae of Hae-
monchus contortus and Trichostrongylus colubriformis under constant and cyclic conditions.
Am. J. Vet. Res. 38, 1115e1119.
Hunt, P.W., Knox, M.R., Le Jambre, L.F., McNally, J., Anderson, L.J., 2008. Genetic and
phenotypic differences between isolates of Haemonchus contortus in Australia. Int. J. Para-
sitol. 38, 885e990.
Hunter, A.R., Mackenzie, G., 1982. The pathogenesis of a single challenge dose of Haemon-
chus contortus in lambs under six months of age. J. Helminthol. 56, 135e144.
Ikeme, M.M., Iskander, F., Chong, L.C., 1987. Seasonal changes in the prevalence of Hae-
monchus contortus and Trichostrongylus colubriformis hypobiotic larvae in tracer goats in
Malaysia. Trop. Anim. Health Prod. 19, 184e190.
Jasmer, D.P., Wescott, R.B., Crane, J.W., 1986. Influence of cold temperatures upon devel-
opment and survival of eggs of Washington isolates of Haemonchus contortus and Ostertagia
circumcincta. Proc. Helm. Soc. Wash. 53, 244e247.
Jasmer, D.P., Wescott, R.B., Crane, J.W., 1987. Survival of third-stage larvae of Washington
isolates of Haemonchus contortus and Ostertagia circumcincta exposed to cold temperatures.
Proc. Helm. Soc. Wash. 54, 48e52.
138 R.B. Besier et al.

Jehan, M., Gupta, V., 1974. The effects of temperature on the survival and development of
the free-living stages of twisted wireworm Haemonchus contortus. Rudolphi, 1803 of sheep
and other ruminants. Z. F. Parasitol. 43, 197e208.
Kahn, L.P., Knox, M., Gray, G., 2003. Enhancing immunity to nematode parasites in
single-bearing Merino ewes through nutrition and genetic selection. Vet. Parasitol.
112, 211e225.
Kates, K.C., 1950. Survival on pasture of free-living stages of some common gastrointestinal
nematodes of sheep. Proc. Soc. Helm. Wash. 17, 39e58.
Kelly, G.A., Kahn, L.P., Walkden-Brown, S.W., 2010. Integrated parasite management for
sheep reduces the effects of gastrointestinal nematodes on the Northern Tablelands of
New South Wales. Anim. Prod. Sci. 50, 1043e1052.
Kerboeuf, D., 1985. Winter survival of trichostrongyle larvae: a study using tracer lambs. Res.
Vet. Sci. 38, 364e367.
Kenyon, F., Sargison, N.D., Skuce, P., Jackson, F., 2009. Sheep helminth parasitic disease in
south eastern Scotland arising as a possible consequence of climate change. Vet. Parasitol.
163, 293e297.
Khadijah, S., Kahn, L.P., Walkden-Brown, S.W., Bailey, J.N., Bowers, S.F., 2013a. Effect of
simulated rainfall timing on faecal moisture and development of Haemonchus contortus and
Trichostrongylus colubriformis eggs to infective larvae. Vet. Parasitol. 192, 199e210.
Khadijah, S., Kahn, L.P., Walkden-Brown, S.W., Bailey, J.N., Bowers, S.F., 2013b. Soil
moisture influences the development of Haemonchus contortus and Trichostrongylus colubri-
formis to third stage larvae. Vet. Parasitol. 196, 161e171.
Kotze, A.C., Prichard, R.K., 2016. Anthelmintic resistance in Haemonchus contortus: history,
mechanisms and diagnosis. In: Gasser, R., Samson-Himmelstjerna, G.V. (Eds.), Haemon-
chus contortus and Haemonchosis Past, Present and Future Trends. vol. 93, pp. 397e428.
Krecek, R.C., Groeneveld, H.T., van Wyk, J.A., 1991. Effects of time of day, season and
stratum on Haemonchus contortus and Haemonchus placei third-stage larvae on irrigated
pasture. Vet. Parasitol. 40, 87e98.
Krecek, R.C., Groeneveld, H.T., Maritz, J.I., 1992. A preliminary study on the effects of
microclimate on Haemonchus contortus and Haemonchus placei third-stage larvae on irrigated
pasture. Int. J. Parasitol. 22, 747e752.
Learmount, J., Taylor, M.A., Smith, G., Morgan, C., 2006. A computer model to simulate
control of parasitic gastroenteritis in sheep on UK farms. Vet. Parasitol. 142, 312e329.
Leathwick, D.M., Barlow, N.D., Vlassof, A., 1992. A model for nematodiasis in New
Zealand lambs. Int. J. Parasitol. 22, 789e799.
Leathwick, D.M., 2013. The influence of temperature on the development and survival of
the pre-infective free-living stages of nematode parasites of sheep. N.Z. Vet. J. 61,
32e40.
Le Jambre, L.F., Whitlock, J.H., 1976. Changes in the hatch rate of Haemonchus contortus eggs
between geographic regions. Parasitology 73, 223e238.
Le Jambre, L.F., 1995. Relationship of blood loss to worm numbers, biomass and egg pro-
duction in Haemonchus contortus infected sheep. Int. J. Parasitol. 25, 269e273.
Le Jambre, L.F., 2006. Eradication of targeted species of internal parasites. Vet. Parasitol. 139,
360e370.
Lettini, S.E., Sukhdeo, M.V.K., 2006. Anhydrobiosis increases survival of trichostrongyle
nematodes. J. Parasitol. 92, 1002e1009.
Levine, N.D., 1963. Weather, climate and the bionomics of ruminant nematode larvae. Adv.
Vet. Sci. 8, 215e269.
Levine, N.D., Todd, K.S., Boatman, P.A., 1974. Development and survival of Haemonchus
contortus on pasture. Am. J. Vet. Res. 35, 1414e1422.
Levine, N.D., 1980. Nematode Parasites of Domestic Animals and of Man, second ed.
Burgess Publishing Company, Minneapolis.
Haemonchus contortus Infection in Small Ruminants 139

Malan, F.S., Van Wyk, J.A., Wessels, C.D., 2001. Clinical evaluation of anaemia in sheep:
early trials. Onderstepoort J. Vet. Res. 68, 165e174.
McArthur, F.A., Kahn, L.P., Windon, R.G., 2013. Immune response of twin-bearing
Merino ewes when infected with Haemonchus contortus: effects of fat score and prepartum
supplementation. Livest. Sci. 157, 568e576.
McCulloch, B., Kasimbala, S., 1968. The incidence of gastrointestinal nematodes of sheep
and goats in Sukumaland, Tanzania. Br. Vet. J. 124, 177e193.
McCulloch, B., Dalbrock, H.G., Kuhn, R.R., 1984. The relation of climate and topography
to worm egg counts of gastro-intestinal nematodes of sheep in the Eastern Cape.
Onderstepoort J. Vet. Res. 51, 223e230.
McKenna, P.B., 1974. The persistence and fate of inhibited Haemonchus contortus larvae in
young sheep. N.Z. Vet. J. 22, 122e127.
McKenna, P.B., 1998. The effect of previous cold storage on the subsequent
recovery of infective third stage nematode larvae from sheep faeces. Vet. Parasitol.
80, 167e172.
McLeod, R.S., 2004. Economic impact of worm infections in small ruminants in South
East Asia, India and Australia. In: Sani, R.A., Gray, G.D., Baker, R.L. (Eds.),
Worm Control of Small Ruminants in Tropical Asia, ACIAR Monograph, 113,
pp. 23e33.
Mederos, A., Fernandez, S., Van Leeuwen, J., Peregrine, A.S., Kelton, D., Menzies, P.,
LeBoeuf, A., Martin, R., 2010. Prevalence and distribution of gastrointestinal nematodes
on 32 organic and conventional commercial sheep farms in Ontario and Quebec, Canada
(2006e2008). Vet. Parasitol. 170, 244e252.
Michel, J.F., 1974. Arrested development of nematodes and some related phenomena. Adv.
Parasitol. 12, 279e366.
Miller, J.E., Bahirathan, M., Lemarie, S.L., Hembry, F.G., Kearney, M.T., Barras, S.R.,
1998. Epidemiology of gastrointestinal nematode parasitism in Suffolk and Gulf Coast
Native sheep with special emphasis on relative susceptibility to Haemonchus contortus
infection. Vet. Parasitol. 74, 55e74.
Misra, S.C., 1978. A note on in vitro effects of temperature on the survival of Haemonchus con-
tortus infective larvae. Ind. J. Anim. Sci. 48, 322e323.
Misra, S.C., Ruprah, N.S., 1972. Survival of Haemonchus contortus infective larvae on exper-
imental grass pots. Ind. Vet. J. 49, 867e873.
Misra, S.C., Ruprah, N.S., 1973a. Effects of temperature, relative humidity and pH on Hae-
monchus contortus eggs. Ind. Vet. J. 50, 136e142.
Misra, S.C., Ruprah, N.S., 1973b. Development of Haemonchus contortus eggs under out-
door conditions. Ind. Vet. J. 50, 231e233.
M€
onnig, H.O., 1930. Studies on the bionomics of free-living stages of Trichostrongylus spp.
and other parasitic nematodes. In: 16th Rept. Dir. Vet. Services and Anim. Ind., Union
of South Afr., pp. 175e198.
M€
onnig, H.O., 1950. Veterinary Helminthology and Entomology, third ed. Balliere, Tindall
& Cox, London. 427 pp.
Morgan, E.R., Van Dijk, J., 2012. Climate and the epidemiology of gastrointestinal nema-
tode infections of sheep in Europe. Vet. Parasitol. 189, 8e14.
Mugambi, J.M., Bain, R.K., Wanyangun, S.W., Murray, M., Stear, M.J., Ihiga, M.A.,
Duncan, J.L., 1997. Resistance of four sheep breeds to natural and subsequent artificial
Haemonchus contortus infection. Vet. Parasitol. 69, 265e273.
Muller, G.L., 1968. The epizootiology of helminth infestations of sheep in the South-
Western districts of the Cape. Onderstepoort J. Vet. Res. 35, 159e194.
Ndamukong, K.J.N., Ngone, M.M., 1996. Development and survival of Haemonchus
contortus and Trichostrongylus sp. on pasture in Cameroon. Trop. Anim. Health Prod.
28, 193e198.
140 R.B. Besier et al.

Nginyi, J.M., Duncan, J.L., Mellor, D.J., Stear, M.J., Wanyangu, S.W., Bain, R.K.,
Gatongi, P.M., 2001. Epidemiology of parasitic gastrointestinal nematode infections of
ruminants on smallholder farms in central Kenya. Res. Vet. Sci. 70, 33e39.
Nnadi, P.A., Kamalu, T.N., Onah, D.N., 2009. The effect of dietary protein on the
productivity of West African Dwarf (WAD) goats infected with Haemonchus contortus.
Vet. Parasitol. 161, 232e238.
Nwosu, C.O., Madu, P.P., Richards, W.S., 2007. Prevalence and seasonal changes in the
population of gastrointestinal nematodes of small ruminants in the semi-arid zone of
north-eastern Nigeria. Vet. Parasitol. 144, 118e124.
O’Connor, L.J., Walkden-Brown, S.W., Kahn, L.P., 2006. Ecology of the free-living stages
of major trichostrongylid parasites of sheep. Vet. Parasitol. 42, 1e15.
O’Connor, L.J., Kahn, L.P., Walkden-Brown, S.W., 2007a. The effects of amount, timing
and distribution of simulated rainfall on the development of Haemonchus contortus to the
infective larval stage. Vet. Parasitol. 146, 90e101.
O’Connor, L.J., Kahn, L.P., Walkden-Brown, S.W., 2007b. Moisture requirements for the
free-living development of Haemonchus contortus: quantitative and temporal effects under
conditions of low evaporation. Vet. Parasitol. 150, 128e138.
O’Connor, L.J., Kahn, L.P., Walkden-Brown, S.W., 2008. Interaction between the effects of
evaporation rate and amount of simulated rainfall on development of the free-living
stages of Haemonchus contortus. Vet. Parasitol. 155, 223e234.
Ogunsusi, R.A., 1979. Pasture infectivity with trichostrongylid larvae in the northern Guinea
savannah of Nigeria. Res. Vet. Sci. 26, 320e323.
Okon, E.D., Enyenihi, U.K., 1977. Development and survival of Haemonchus contortus on
pastures in Ibadan. Trop. Anim. Health Prod. 9, 7e10.
Onyali, I.O., Onwuliri, C.O.E., Ajayi, J.A., 1990. Development and survival of Haemon-
chus contortus larvae on pasture at Vom Plateau State, Nigeria. Vet. Res. Commun. 14,
211e214.
O’Sullivan, B.M., Donald, A.D., 1973. Responses to infection with Haemonchus contortus and
Trichostrongylus colubriformis in ewes of different reproductive status. Int. J. Parasitol. 3,
521e530.
Pandey, V.S., 1990. Haemonchus contortus with low inhibited development in sheep from the
Highveld of Zimbabwe. Vet. Parasitol. 36, 347e351.
Pandey, V.S., Ndao, M., Kumar, V., 1994. Seasonal prevalence of gastrointestinal nematodes
in communal land goats from the Highveld of Zimbabwe. Vet. Parasitol. 51, 241e248.
Perry, B.D., Randolph, R.F., McDermott, J.J., Sones, K.R., Thornton, P.K., 2002. Investing
in Animal Health Research to Alleviate Poverty. International Livestock Research Insti-
tute (ILRI), Nairobi, Kenya, 148 pp.
Polley, L., Hoberg, E., Kutz, S., 2010. Climate change, parasites and shifting boundaries. Acta
Vet. Scand. 52 (Suppl. 1), S1.
Preston, J.M., Allonby, E.W., 1979. The influence of breed on the susceptibility of sheep to
Haemonchus contortus infection in Kenya. Res. Vet. Sci. 26, 134e139.
Pullan, N.B., Megadmi, K., 1983. Haemonchosis in a winter rainfall region in Libya. Trop.
Anim. Health Prod. 15, 237e238.
Qamar, M.F., Maqbool, A., Ahmad, N., 2012. Economic losses due to haemonchosis in
sheep and goats. Sci. Int. 24, 321e324.
Rees, G., 1950. Observations on the vertical migrations of the third-stage larva of Haemonchus
contortus (Rud.) on experimental plots of Lolium perenne S24, in relation to meteorological
and micrometeorological factors. Parasitology 40, 127e143.
Reinecke, R.K., Kirkpatrick, R., Swart, L., Kriel, A.M.D., Frank, F., 1987. Parasites in sheep
grazing on Kikuyu (Pennisetum clandestinum) pastures in the winter rainfall region. Onder-
stepoort J. Vet. Res. 54, 27e38.
Reynecke, D.P., Waghorn, T.S., Oliver, A.M.B., Miller, C.M., Vlassoff, A.,
Leathwick, D.M., 2011. Dynamics of the free-living stages of sheep intestinal parasites
Haemonchus contortus Infection in Small Ruminants 141

on pasture in the North Island of New Zealand. Weather variables associated with
development. N. Z. Vet. J. 59, 287e292.
Rinaldi, L., Catelan, D., Musella, V., Cecconi, L., Hertzberg, H., Torgerson, P.R.,
Mavrot, F., de Waal, T., Selemetas, N., Coll, T., Bosco, A., Biggeri, A., Cringoli, G.,
2015. Haemonchus contortus: spatial risk distribution for infection in sheep in Europe.
Geospatial Health 9, 325e331.
Roberts, J.L., Swan, R.A., 1981. Quantitive studies of ovine haemonchosis. 1. Relationship
between faecal egg counts and total worm counts. Vet. Parasitol. 8, 165e171.
Rose, J.H., 1963. Observations on the free-living stages of the stomach worm Haemonchus
contortus. Parasitology 53, 469e481.
Rose, J.H., 1964. Relationship between environment and the development and migration of
the free-living stages of Haemonchus contortus. J. Comp. Pathol. 74, 163e172.
Rose, J.H., Small, A.J., 1985. The distribution of infective larvae of sheep gastrointestinal
nematodes in soil and on herbage and the vertical migration of Trichostrongylus vitrinus
larvae through the soil. J. Helminthol. 59, 127e135.
Rossanigo, C.E., Gruner, L., 1995. Moisture and temperature requirements in faeces for the
development of free-living stages of gastrointestinal nematodes of sheep, cattle and deer.
J. Helminth. 69, 357e362.
Rossiter, L.W., 1964. The epizootiology of nematode parasites of sheep in the coastal area of
the Eastern Province. Onderstepoort J. Vet. Res. 31, 143e150.
Rowe, J.B., Nolan, J.V., De Chaneet, G., Teleni, E., Holmes, P.H., 1988. The effect of
haemonchosis and blood loss into the abomasum on digestion in sheep. Br. J. Nutr.
59, 125e139.
Sakwa, D.P., Walkden-Brown, S., Dobson, R.J., Kahn, L.P., Lea, J.M., Baillie, N.D.,
2003. Pasture microclimate can influence early development of Haemonchus contortus
in sheep faecal pellets in a cool temperate climate. In: Proc. Aust. Sheep Vet. Soc.,
pp. 33e40.
Santos, M.C., Silva, B.F., Amarante, A.F.T., 2012. Environmental factors influencing the
transmission of Haemonchus contortus. Vet. Parasitol. 188, 277e284.
Sargison, N.D., Wilson, D.J., Bartley, D.J., Penny, C.D., Jackson, F., 2007. Haemonchosis
and teladorsagiosis in a Scottish sheep flock putatively associated with the overwintering
of hypobiotic fourth stage larvae. Vet. Parasitol. 147, 326e331.
Shillhorn Van Veen, T.W., Ogunsusi, R.A.A., 1978. Periparturient and seasonal rise in the
trichostrongylid egg output of infected ewes during the dry season in Northern Nigeria.
Vet. Parasitol. 4, 377e383.
Shillhorn Van Veen, T.W., 1978. Haemonchosis in sheep during the dry season in the Niger-
ian savanna. Vet. Rec. 102, 364e365.
Shorb, D.A., 1944a. Survival on grass plots of eggs and larvae of the stomach worm, Haemon-
chus contortus. J. Agric. Res. 68, 317e324.
Shorb, D.A., 1944b. Factors influencing embryonation and survival of eggs of the stomach
worm, Haemonchus contortus. J. Agric. Res. 69, 279e287.
Silangwa, S.M., Todd, A.C., 1964. Vertical migration of trichostrongylid larvae on grasses.
J. Parasitol. 50, 278e285.
Sissay, M.M., Uggla, A., Waller, P.J., 2007. Epidemiology and seasonal dynamics of gastro-
intestinal nematode infections of sheep in a semi-arid region of eastern Ethiopia. Vet.
Parasitol. 143, 311e321.
Silverman, P.H., Campbell, J.A., 1959. Studies on parasitic worms in Scotland. Embryonic
and larval development of Haemonchus contortus at constant conditions. J. Parasitol. 49,
23e38.
Silverman, P.H., Patterson, J.E., 1960. Histoptrohic (parasitic) stages of Haemonchus contortus.
Nature 185, 54e55.
Skinner, W.D., Todd, K.S., 1980. Lateral migration of Haemonchus contortus larvae on pasture.
Am. J. Vet. Res. 41, 395e398.
142 R.B. Besier et al.

Smith, G., Grenfell, B.T., 1994. Modelling of parasite populations: gastrointestinal nematode
models. Vet. Parasitol. 54, 127e143.
Smith-Bujis, C.M.C., Borgesteede, F.H.M., 1986. Effect of cool storage of faecal samples
containing Haemonchus contortus eggs on the results of an in vitro egg development assay
to test anthelmintic resistance. Res. Vet. Sci. 40, 4e7.
Sood, M.L., Kaur, C., 1975. The effects of temperature on the survival and development of
the twisted wireworm, Haemonchus contortus. Rudolphi, 1803. Ind. J. Ecol. 2, 68e74.
Southcott, W.H., Major, G.W., Barger, I.A., 1976. Seasonal pasture contamination and
availability of nematodes for grazing sheep. Aust. J. Agric. Res. 27, 277e286.
Steel, J.W., 2003. Effects of protein supplementation on young sheep on resistance develop-
ment and resilience to parasitic nematodes. Aust. J. Exp. Agric. 12, 1469e1476.
Stromberg, B.E., 1997. Environmental factors influencing transmission. Vet. Parasitol. 72,
247e264.
Swan, R.A., 1970. The epizootiology of haemonchosis in sheep. Aust. Vet. J. 45, 485e492.
Taylor, M.A., Coop, R.L., Wall, R.L., 2007. Veterinary Parasitology, third ed. Blackwell
Publishing, pp. 159e161.
Tembely, S., 1998. Development and survival of infective larvae of nematode parasites of
sheep on pasture in a cool tropical environment. Vet. Parasitol. 79, 81e87.
Thomas, R.J., Waller, P.J., 1979. Field observations on the epidemiology of abomasal
parasites in young sheep during winter and spring. Res. Vet. Sci. 26, 209e212.
Thomas, R.J., 1982. The ecological basis of parasite control: nematodes. Vet. Parasitol. 11,
9e24.
Thomas, R.J., Ali, D.A., 1983. The effect of Haemonchus contortus infection on the pregnant
and lactating ewe. Int. J. Parasitol. 13, 393e398.
Todd, K.S., Levine, N.D., Whiteside, C., 1970. Moisture stress effects on survival of infective
Haemonchus contortus larvae. J. Nematol. 2, 331e333.
Todd, K.S., Levine, N.D., Boatman, P.A., 1976a. Effect of desiccation on the survival
of infective Haemonchus contortus larvae under laboratory conditions. J. Parasit. 62,
247e249.
Todd, K.S., Levine, N.D., Boatman, P.A., 1976b. Effect of temperature on survival of free-
living stages of Haemonchus contortus. Am. J. Vet. Res. 37, 991e992.
Troell, K., Waller, P., H€ oglund, J., 2005. The development and overwintering survival of
free-living larvae of Haemonchus contortus in Sweden. J. Helminthol. 79, 373e379.
Troell, K., Tingstedt, C., H€ oglund, J., 2006. Phenotypic characterisation of Haemonchus
contortus: a study of isolates from Sweden and Kenya in experimentally infected sheep.
J. Parasitol. 132, 403e409.
Uriate, J., Valderrabano, J., 1989. An epidemiological study of parasitic gastroenteritis in
sheep under an intensive grazing system. Vet. Parasitol. 31, 71e81.
Urquhart, G.M., Armour, J., Duncan, J.L., Dunn, A.M., Jennings, F.M., 1996. Veterinary
Parasitology, second ed. Blackwell Science, pp. 19e21.
Van Dijk, J., David, G.O., Baird, G., Morgan, E.R., 2008. Back to the future: developing
hypotheses on the effects of climate change on ovine parasitic gastroenteritis from histor-
ical data. Vet. Parasitol. 158, 73e84.
Van Dijk, J., De Louw, M.D.E., Kalis, L.P.A., Morgan, E.R., 2009. Ultraviolet light in-
creases mortality of nematode larvae and can explain patterns of larval availability at
pasture. Int. J. Parasitol. 39, 1151e1156.
Van Dijk, J., Sargison, N.D., Kenyon, F., Skuce, P.J., 2010. Climate change and infectious
disease: helminthological challenges to farmed ruminants in temperate regions. Anim. 4,
377e392.
Van Dijk, J., Morgan, E.R., 2011. The influence of water on the migration of infective tri-
chostrongyloid larvae onto grass. Parasitology 138, 780e788.
Haemonchus contortus Infection in Small Ruminants 143

Van Wyk, J.A., Bath, G.F., 2002. The FAMACHA© system for managing haemonchosis in
sheep and goats by clinically identifying individual animals for treatment. Vet. Res. 33,
509e529.
Van Wyk, J.A., 2008. Production trials involving use of the FAMACHA© system for hae-
monchosis in sheep: preliminary results. Onderstepoort J. Vet. Res. 75, 331e345.
Van Wyk, J.A., Reynecke, D.P., 2011. Blueprint for an automated specific decision support
system for countering anthelmintic resistance in Haemonchus spp. at farm level. Vet. Para-
sitol. 177, 212e223.
Vatta, A.F., Lindberg, A.L.E., 2006. Managing anthelmintic resistance in small ruminant live-
stock of resource-poor farmers in South Africa, 0038-2809 J. S. Afr. Vet. Assoc. 77, 2e8.
Veglia, F., 1915. The Anatomy and Life History of the Haemonchus contortus (Rud.), pp. 347e
500. Annual Report of the Director Vet. Res., Union S.Afr., 3 and 4.
Vercruysse, J., 1983. A survey of seasonal changes in nematode faecal egg count levels of
sheep and goats in Senegal. Vet. Parasitol. 13, 239e244.
Vercruysse, J., 1985. The seasonal prevalence of inhibited development of Haemonchus contor-
tus in sheep in Senegal. Vet. Parasitol. 17, 159e163. http://www.sciencedirect.com/
science/article/pii/0304401785901025.
Viljoen, J.H., 1969. Further studies on the epizootiology of nematode parasites of sheep in
the Karoo. Onderstepoort J. Vet. Res. 36, 233e264.
Vlassoff, A., 1973. Seasonal availability of infective trichostrongyle larvae on pasture grazed by
lambs. N.Z. J. Exp. Agric. 1, 293e301.
Waghorn, T.S., Reynecke, D.P., Oliver, A.M.B., Miller, C.M., Vlassoff, A., Koolaard, J.P.,
Leathwick, D.M., 2011. Dynamics of the free-living stages of sheep intestinal parasites on
pasture in the North Island of New Zealand. 1. Patterns of seasonal development. N.Z.
Vet. J. 59, 279e286.
Wallace, D.S., Bairden, K., Duncan, J.L., Fishwick, G., Gill, M., Holmes, P.H.,
McKellar, Q.A., Murray, M., Parkins, J.J., Stear, M., 1996. Influence of soyabean
meal supplementation on the resistance of Scottish Blackface lambs to haemonchosis.
Res. Vet. Sci. 60, 138e143.
Waller, P.J., Donald, A.D., 1970. The response to desiccation of eggs of Trichostrongylus colubri-
formis and Haemonchus contortus (Nematoda: Trichostrongylidae). Parasitology 61, 195e204.
Waller, P.J., Rudby-Martin, L., Ljungstr€ om, B.L., Rydzik, A., 2004. The epidemiology of
abomasal nematodes of sheep in Sweden, with particular reference to over-winter sur-
vival strategies. Vet. Parasitol. 122, 207e220.
Waller, P.J., Chandrawathani, P., 2005. Haemonchus contortus: parasite problem No. 1 from
tropics e polar circle. Problems and prospects for control based on epidemiology.
Trop. Biomed. 22, 131e137.
Waller, P.J., Rydzik, A., Ljungstr€ om, B.L., Tornquist, M., 2006. Towards the eradication of
Haemonchus contortus from sheep flocks in Sweden. Vet. Parasitol. 136, 367e372.
Wang, T., Van Wyk, J.A., Morrison, A., Morgan, E.R., 2014. Moisture requirements for the
migration of Haemonchus contortus third stage larvae out of faeces. Vet. Parasitol. 204,
258e264. http://www.sciencedirect.com/science/article/pii/S0304401714002933.
Woolaston, R.R., Baker, R.L., 1996. Prospects of breeding small ruminants for resistance to
internal parasites. Int. J. Parasitol. 26, 845e855.
Yang, Y., Ma, Y., Chen, X., Guo, X., Yan, B., Du, A., 2015. Screening and analysis of Hc-
ubq and Hc-gst related to desiccation survival of infective Haemonchus contortus larvae.
Vet. Parasitol. 210, 179e185.
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

Advances in Parasitology, Volume 93

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
148 D.S. Zarlenga et al.

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
Identification of Haemonchus Species and Diagnosis of Haemonchosis 149

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
150 D.S. Zarlenga et al.

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
Identification of Haemonchus Species and Diagnosis of Haemonchosis 151

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).
152 D.S. Zarlenga et al.

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.
Identification of Haemonchus Species and Diagnosis of Haemonchosis 153

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
154 D.S. Zarlenga et al.

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
Identification of Haemonchus Species and Diagnosis of Haemonchosis 155

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
156 D.S. Zarlenga et al.

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
Identification of Haemonchus Species and Diagnosis of Haemonchosis 157

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
158 D.S. Zarlenga et al.

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
Identification of Haemonchus Species and Diagnosis of Haemonchosis 159

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
160 D.S. Zarlenga et al.

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
Identification of Haemonchus Species and Diagnosis of Haemonchosis 161

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
162 D.S. Zarlenga et al.

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 Haemonchus Species and Diagnosis of Haemonchosis 163

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,
164 D.S. Zarlenga et al.

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
Identification of Haemonchus Species and Diagnosis of Haemonchosis 165

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).
166 D.S. Zarlenga et al.

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
Identification of Haemonchus Species and Diagnosis of Haemonchosis 167

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).
168 D.S. Zarlenga et al.

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
Identification of Haemonchus Species and Diagnosis of Haemonchosis 169

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,
170 D.S. Zarlenga et al.

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
Identification of Haemonchus Species and Diagnosis of Haemonchosis 171

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.

REFERENCES
Achi, Y.L., Zinsstag, J., Yao, K., Yeo, N., Dorchies, P., Jacquiet, P., 2003. Host specificity of
Haemonchus spp. for domestic ruminants in the savanna in northern Ivory Coast. Vet.
Parasitol. 116, 151e158.
Akkari, H., Jebali, J., Gharbi, M., Mhadhbi, M., Awadi, S., Darghouth, M.A., 2013. Epide-
miological study of sympatric Haemonchus species and genetic characterization of Haemon-
chus contortus in domestic ruminants in Tunisia. Vet. Parasitol. 193, 118e125.
Alba-Hurtado, F., Mu~ noz-Guzman, M.A., 2013. Immune responses associated with resis-
tance to haemonchosis in sheep. Biomed. Res. Int. 2013, 162158. http://dx.doi.org/
10.1155/2013/162158.
do Amarante, A.F., 2011. Why is it important to correctly identify Haemonchus species? Rev.
Bras. Parasitol. Vet. 20, 263e268.
Bath, G.F., Malan, F.S., van Wyk, J.A., 1996. The “FAMACHA” ovine anaemia guide to
assist with the control of haemonchosis. In: Proceedings of the 7th Annual Congress
172 D.S. Zarlenga et al.

of the Livestock Health and Production Group of the South African Veterinary Associ-
ation (Port Elizabeth, South Africa).
Beh, K.J., Foley, R.C., Goodwin, E.J., 1989. Restriction fragment length patterns of DNA
from parasitic nematodes of sheep. Res. Vet. Sci. 46, 127e128.
Bendixsen, T., Windon, R.G., Huntley, J.F., MacKellar, A., Davey, R.J., McClure, S.J.,
Emery, D.L., 2004. Development of a new monoclonal antibody to ovine chimeric
IgE and its detection of systemic and local IgE antibody responses to the intestinal nem-
atode Trichostrongylus colubriformis. Vet. Immunol. Immunopathol. 97, 11e24.
Bentounsi, B., Cabaret, J., 1999. Analysis of helminth genetic data: comparative examples
with Haemonchus contortus isozymes using exact tests or resampling procedures. Parasitol.
Res. 85, 855e857.
Bisset, S.A., Knight, J.S., Bouchet, C.L., 2014. A multiplex PCR-based method to identify
strongylid parasite larvae recovered from ovine faecal cultures and/or pasture samples.
Vet. Parasitol. 200, 117e127.
Blitz, N.M., Gibbs, H.C., 1971. Morphological characterization of the stage of arrested
development of Haemonchus contortus in sheep. Can. J. Zool. 49, 991e995.
Blouin, M.S., 2002. Molecular prospecting for cryptic species of nematodes: mitochondrial
DNA versus internal transcribed spacer. Int. J. Parasitol. 32, 527e531.
Blouin, M.S., Yowell, C.A., Courtney, C.H., Dame, J.B., 1997. Haemonchus placei and Hae-
monchus contortus are distinct species based on mtDNA evidence. Int. J. Parasitol. 27,
1383e1387.
Borgsteede, F.H., Hendricks, J., 1974. Identification of infective larvae of gastrointestinal
nematodes in cattle. Tijdschr. Diergeneeskd. 99, 103e113.
Bott, N.J., Campbell, B.E., Beveridge, I., Chilton, N.B., Rees, D., Hunt, P.W.,
Gasser, R.B., 2009. A combined microscopic-molecular method for the diagnosis of
strongylid infections in sheep. Int. J. Parasitol. 39, 1277e1287.
Brasil, B.S., Nunes, R.L., Bastianetto, E., Drummond, M.G., Carvalho, D.C., Leite, R.C.,
Molento, M.B., Oliveira, D.A., 2012. Genetic diversity patterns of Haemonchus placei and
Haemonchus contortus populations isolated from domestic ruminants in Brazil. Int. J. Para-
sitol. 42, 469e479.
Budischak, S.A., Hoberg, E.P., Abrams, A., Jolles, A.E., Ezenwa, V.O., 2015. A combined
parasitological molecular approach for noninvasive characterization of parasitic nematode
communities in wild hosts. Mol. Ecol. Resour. 15, 1112e1119.
Cadman, E.T., Thysse, K.A., Bearder, S., Cheung, A.Y., Johnston, A.C., Lee, J.J.,
Lawrence, R.A., 2014. Eosinophils are important for protection, immunoregulation and
pathology during infection with nematode microfilariae. PLoS Pathog. 10 (3), e1003988.
Carugati, L., Corinaldesi, C., Dell’Anno, A., Danovaro, R., May 7, 2015. Metagenetic tools
for the census of marine meiofaunal biodiversity: an overview. Mar. Genomics pii:
S1874e7787(15)00076-8 (Epub ahead of print).
Cerutti, M.C., Citterio, C.V., Bazzocchi, C., Epis, S., D’Amelio, S., Ferrari, N., Lanfranchi, P.,
2010. Genetic variability of Haemonchus contortus (Nematode: Trichostrongyloidea) in
alpine ruminant host species. J. Helminthol. 84, 276e283.
Chaudhry, U., Miller, M., Yazwinski, T., Kaplan, R., Gilleard, J., 2014. The presence of
benzimidazole resistance mutations in Haemonchus placei from US cattle. Vet. Parasitol.
204, 411e415.
Chaudhry, U., Redman, E.M., Abbas, M., Muthusamy, R., Ashraf, K., Gilleard, J.S., 2015.
Genetic evidence for hybridisation between Haemonchus contortus and Haemonchus placei in
natural field populations and its implications for interspecies transmission of anthelmintic
resistance. Int. J. Parasitol. 45, 149e159.
Chilton, N.B., 2004. The use of nuclear ribosomal DNA markers for the identification of
bursate nematodes (order Strongylida) and for the diagnosis of infections. Anim. Health
Res. Rev. 5, 173e187.
Identification of Haemonchus Species and Diagnosis of Haemonchosis 173

Christensen, C.M., Zarlenga, D.S., Gasbarre, L.C., 1994a. Ostertagia, Haemonchus, Cooperia,
and Oesophagostomum: construction and characterization of genus-specific DNA probes
to differentiate important parasites of cattle. Exp. Parasitol. 78, 93e100.
Christensen, C.M., Zarlenga, D.S., Gasbarre, L.C., 1994b. Identification of a Haemonchus pla-
cei-specific DNA probe. J. Helminthol. Soc. Wash. 61, 249e252.
Christie, M., Jackson, F., 1982. Specific identification of strongyle eggs in small samples of
sheep faeces. Res. Vet. Sci. 32, 113e117.
Colditz, I.G., Le Jambre, L.F., Hosse, R., 2002. Use of lectin binding characteristics to iden-
tify gastrointestinal parasite eggs in faeces. Vet. Parasitol. 105, 219e227.
Cunliffe, G., Crofton, H.D., 1953. Egg sizes and differential egg counts in relation to sheep
nematodes. Parasitology 43, 275e286.
Cuquerella, M., G omez-Mu~ noz, M.T., Alunda, J.M., 1995. Serum IgG response of Man-
chego lambs to infections with Haemonchus contortus and preliminary characterization
of adult antigens. Vet. Parasitol. 38, 131e143.
Demeler, J., Ram€ unke, S., Wolken, S., Ianiello, D., Rinaldi, L., Gahutu, J.B., Cringoli, G.,
von Samson-Himmelstjerna, G., Kr€ ucken, J., 2013. Discrimination of gastrointestinal
nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional
and real-time PCR. PLoS One 8, e61285.
Dikmans, G., Andrews, J.S., 1933. A comparative morphological study of the infective larvae
of the common nematodes parasitic in the alimentary tract of sheep. Trans. Am. Microsc.
Soc. 42, 1e25.
Dobson, R.J., Barnes, E.H., Birclijin, S.D., Gill, J.H., 1992. The survival of Ostertagia circum-
cincta and Trichostrongylus colubriformis in faecal culture as a source of bias in apportioning
egg counts to worm species. Int. J. Parasitol. 22, 1005e1008.
Douvres, F.W., 1957a. The morphogenesis of the parasitic stages of Trichostrongylus axei and
Trichostrongylus colubriformis, nematode parasites of cattle. Proc. Helminthol. Soc. Wash.
24, 4e14.
Douvres, F.W., 1957b. Keys to the identification and differentiation of the immature parasitic
stages of gastrointestinal nematodes of cattle. Am. J. Vet. Res. 18, 81e85.
Dragan, A.I., Pavlovic, R., McGivney, J.B., Casas-Finet, J.R., Bishop, E.S., Strouse, R.J.,
Schenerman, M.A., Geddes, C.D., 2012. SYBR Green I: fluorescence properties and
interaction with DNA. J. Fluoresc. 22, 1189e1199.
Duncan, J.L., Smith, W.D., Dargie, J.D., 1978. Serum IgG response of Manchego lambs to
infections with Haemonchus contortus and preliminary characterization of adult antigens.
Vet. Parasitol. 4, 21e27.
Durette-Desset, M.C., 1983. Keys to the genera of the superfamily Trichostrongyloidea. In:
Anderson, R.C., Chabaud, A.G. (Eds.), CIH Keys to the Nematode Parasites of
Vertebrates. Commonwealth Agricultural Bureaux, Farnham Royal, UK, pp. 1e86.
Echevarria, F.A., Gennari, S.M., Tait, A., 1992. Isoenzyme analysis of Haemonchus contortus
resistant or susceptible to ivermectin. Vet. Parasitol. 44, 87e95.
Emery, D.L., McClure, S.J., Davey, R.J., Bendixsen, T., 1999. Induction of protective im-
munity to Trichostrongylus colubriformis in neonatal merino lambs. Int. J. Parasitol. 29,
1037e1046.
Fawzi, E.M., Gonzalez-Sanchez, M.E., Corral, M.J., Cuquerella, M., Alunda, J.M., 2014.
Vaccination of lambs against Haemonchus contortus infection with a somatic protein
(Hc23) from adult helminths. Int. J. Parasitol. 44, 429e436.
García-Coiradas, L., Angulo-Cubillan, F., Méndez, S., Larraga, V., de la Fuente, C.,
Cuquerella, M., Alunda, J.M., 2009. Isolation and immunolocalization of a putative pro-
tective antigen (p26/23) from adult Haemonchus contortus. Parasitol. Res. 104, 363e369.
Gasser, R.B., Bott, N.J., Chilton, N.B., Hunt, P., Beveridge, I., 2008. Toward practical,
DNA-based diagnostic methods for parasitic nematodes of livestockebionomic and
biotechnological implications. Biotechnol. Adv. 26, 325e334.
174 D.S. Zarlenga et al.

Gasser, R.B., Chilton, N.B., Hoste, H., Stevenson, L.A., 1994. Species identification of tri-
chostrongyle nematodes by PCR-linked RFLP. Int. J. Parasitol. 24, 291e293.
Gasser, R.B., Zhu, X., Chilton, N.B., Newton, L.A., Nedergaard, T., Guldberg, P., 1998.
Analysis of sequence homogenisation in rDNA arrays of Haemonchus contortus by dena-
turing gradient gel electrophoresis. Electrophoresis 19, 2391e2395.
Georgi, J.R., McCulloch, C.E., 1989. Diagnostic morphometry: identification of helminth
eggs by discriminant analysis of morphometric data. Proc. Helminthol. Soc. Wash. 56,
44e57.
Gibbons, L.M., 1979. Revision of the genus Haemonchus Cobb, 1898 (Nematoda:
Trichostrongylidae). Syst. Parasitol. 1, 3e24.
Gill, H.S., Gray, G.D., Watson, D.L., Husband, A.J., 1993. Isotype-specific antibody
responses to Haemonchus contortus in genetically resistant sheep. Parasite Immunol. 15,
61e67.
Gill, H.S., Husband, A.J., Watson, D.L., 1992. Localisation of immunoglobulin-containing
cells in the abomasum of sheep following infection with Haemonchus contortus. Vet.
Immunol. Immunopathol. 31, 179e187.
Giudici, C.J., Cabaret, J., Durette-Desset, M.C., 1999. Description of Haemonchus placei
(Place, 1893) (Nematoda, Trichostrongylidae, Haemonchinae), identification and
intra-specific morphological variability. Parasite 6, 333e342.
Goodey, T., 1922. Observations on the ensheather larvae of some parasitic nematodes. Ann.
Appl. Biol. 9, 33e49.
Gressler, L.T., Da Silva, A.S., Oliveira, C.B., Schafer, A.S., Aires, A.R., Rocha, J.F.,
Tonin, A.A., Schirmbeck, G.H., Casali, E.A., Lopes, S.T., Leal, M.L., Monteiro, S.G.,
2014. Experimental infection by Haemonchus contortus in lambs: influence of disease on
purine levels in serum. Parasitology 141, 898e903.
Gudnason, H., Dufva, M., Bang, D.D., Wolff, A., 2007. Comparison of multiple DNA dyes
for real-time PCR: effects of dye concentration and sequence composition on DNA
amplification and melting temperature. Nucleic Acids Res. 35, e127.
Haig, D.M., Windon, R., Blackie, W., Brown, D., Smith, W.D., 1989. Parasite-specific T
cell responses of sheep following live infection with the gastric nematode Haemonchus
contortus. Parasite Immunol. 11, 463e477.
Hammad, H., Lambrecht, B.N., 2015. Barrier epithelial cells and the control of type 2
immunity. Immunity 43, 29e40.
Hansen, M.F., Shivnani, G.A., 1956. Comparative morphology of infective nematode larvae
of Kansas beef cattle and its use in estimating incidence of nematodiasis in cattle. Trans.
Am. Microsc. Soc. 65, 91e102.
Harmon, A.F., Williams, Z.B., Zarlenga, D.S., Hildreth, M.B., 2007. Real-time PCR for
quantifying Haemonchus contortus eggs and potential limiting factors. Parasitol. Res.
101, 71e76.
Hart, E.H., Morphew, R.M., Bartley, D.J., Millares, P., Wolf, B.T., Brophy, P.M.,
Hamilton, J.V., 2012. The soluble proteome phenotypes of ivermectin resistant and
ivermectin susceptible Haemonchus contortus females compared. Vet. Parasitol. 190,
104e113.
Heise, M., Epe, C., Schnieder, T., 1999. Differences in the second internal transcribed
spacer (ITS-2) of eight species of gastrointestinal nematodes of ruminants. J. Parasitol.
85, 431e435.
Hepworth, M.R., Dani1owicz-Luebert, E., Rausch, S., Metz, M., Klotz, C., Maurer, M.,
Hartmann, S., 2012. Mast cells orchestrate type 2 immunity to helminths through regu-
lation of tissue-derived cytokines. Proc. Natl. Acad. Sci. U. S. A. 109, 6644e6649.
Hillrichs, K., Schnieder, T., Forbes, A.B., Simcock, D.C., Pedley, K.C., Simpson, H.V.,
2012. Use of fluorescent lectin binding to distinguish Teladorsagia circumcincta and Hae-
monchus contortus eggs, third-stage larvae and adult worms. Parasitol. Res. 110, 449e458.
Identification of Haemonchus Species and Diagnosis of Haemonchosis 175

Hindson, B.J., Ness, K.D., Masquelier, D.A., Belgrader, P., Heredia, N.J., Makarewicz, A.J.,
Bright, I.J., Lucero, M.Y., Hiddessen, A.L., Legler, T.C., Kitano, T.K., Hodel, M.R.,
Petersen, J.F., Wyatt, P.W., Steenblock, E.R., Shah, P.H., Bousse, L.J., Troup, C.B.,
Mellen, J.C., Wittmann, D.K., Erndt, N.G., Cauley, T.H., Koehler, R.T., So, A.P.,
Dube, S., Rose, K.A., Montesclaros, L., Wang, S., Stumbo, D.P., Hodges, S.P.,
Romine, S., Milanovich, F.P., White, H.E., Regan, J.F., Karlin-Neumann, G.A.,
Hindson, C.M., Saxonov, S., Colston, B.W., 2011. High-throughput droplet digital
PCR system for absolute quantitation of DNA copy number. Anal. Chem. 83, 8604e8610.
Hoberg, E.P., 2010. Invasive processes, mosaics and the structure of helminth parasite faunas.
Rev. Sci. Tech. 29, 255e272.
Hoberg, E.P., Abrams, A., Carreno, R., Lichtenfels, J.R., 2002. Ashworthius patriciapilittae
n. sp. (Trichostrongyloidea: Haemonchinae), an abomasal nematode in Odocoileus virgin-
ianus from Costa Rica, and a new record for species of the genus in the Western
Hemisphere. J. Parasitol. 88, 1187e1199.
Hoberg, E.P., Kocan, A., Rickard, L.G., 2001. Gastrointestinal strongyles in wild ruminants.
In: Samuel, W., Pybus, M., Kocan, A. (Eds.), Parasitic Diseases of Wild Mammals. Iowa
State University Press, pp. 193e227.
Hoberg, E.P., Lichtenfels, J.R., Gibbons, L., 2004. Phylogeny for species of the genus Hae-
monchus (Nematoda: Trichostrongyloidea): considerations of their evolutionary history
and global biogeography among Camelidae and Pecora (Artiodactyla). J. Parasitol. 90,
1085e1102.
Hoberg, E.P., Polley, L., Jenkins, E.J., Kutz, S.J., 2008. Pathogens of domestic and free-
ranging ungulates: global climate change in temperate to boreal latitudes across North
America. Rev. Sci. Tech. 27, 511e528.
Hoberg, E.P., Zarlenga, D.S., 2016. Evolution and Biogeography of Haemonchus contortus:
Linking Faunal Dynamics in Space and Time. In: Gasser, R.B., von Samson-
Himmelstjerna, G. (Eds.), Haemonchus contortus and haemonchosis past, present and
future trends. vol. 93, pp. 1e30.
Huang, L., Gebreselassie, N.G., Gagliardo, L.F., Ruyechan, M.C., Luber, K.L., Lee, N.A.,
Lee, J.J., Appleton, J.A., 2015. Eosinophils mediate protective immunity against second-
ary nematode infection. J. Immunol. 194, 283e290.
Humbert, J.F., Cabaret, J., 1995. Use of random amplified polymorphic DNA for identifi-
cation of ruminant trichostrongylid nematodes. Parasitol. Res. 81, 1e5.
Huntley, J.F., Gibson, S., Brown, D., Smith, W.D., Jackson, F., Miller, H.R., 1987. Systemic
release of a mast cell proteinase following nematode infections in sheep. Parasite Immu-
nol. 9, 603e614.
Hussain, T., Periasamy, K., Nadeem, A., Babar, M.E., Pichler, R., Diallo, A., 2014. Sympat-
ric species distribution, genetic diversity and population structure of Haemonchus isolates
from domestic ruminants in Pakistan. Vet. Parasitol. 206, 188e199.
Jabbar, A., Cotter, J., Lyon, J., Koehler, A.V., Gasser, R.B., Besier, B., 2014. Unexpected
occurrence of Haemonchus placei in cattle in southern Western Australia. Infect. Genet.
Evol. 21, 252e258.
Jacquiet, P.F., Cabaret, J., Cheikh, D., Thiam, E., 1997. Identification of Haemonchus species
in domestic ruminants based on morphometrics of spicules. Parasitol. Res. 83, 82e86.
Jacquiet, P.F., Humbert, J.F., Comes, A.M., Cabaret, J., Thiam, A., Cheikh, D., 1995.
Ecological, morphological and genetic characterization of sympatric Haemonchus spp.
parasites of domestic ruminants in Mauritania. Parasitology 110, 483e492.
Jasmer, D.P., Lahmers, K.K., Brown, W.C., 2007. Haemonchus contortus intestine: a promi-
nent source of mucosal antigens. Parasite Immunol. 29, 139e151.
Jurasek, M.E., Bishop-Stewart, J.K., Storey, B.E., Kaplan, R.M., Kent, M.L., 2010. Modi-
fication and further evaluation of a fluorescein-labeled peanut agglutinin test for identi-
fication of Haemonchus contortus eggs. Vet. Parasitol. 169, 209e213.
176 D.S. Zarlenga et al.

Karanikola, S.N., Kr€ ucken, J., Ram€ unke, S., de Waal, T., H€ oglund, J., Charlier, J.,
Weber, C., M€ uller, E., Kowalczyk, S.J., Kaba, J., von Samson-Himmelstjerna, G.,
Demeler, J., 2015. Development of a multiplex fluorescence immunological assay for
the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus
and Fasciola hepatica in cattle. Parasit. Vectors 8, 335.
Keith, R.K., 1953. The differentiation of the infective larvae of some common nematode
parasites of cattle. Aust. J. Zool. 12, 223e235.
Kerboeuf, D., Aycardi, J., Soubieux, D., 1996. Flow-cytometry analysis of sheep-nematode
egg populations. Parasitol. Res. 82, 358e363.
Knox, D.P., Jones, D.G., 1992. A comparison of superoxide dismutase (SOD, EC:1.15.1.1)
distribution in gastro-intestinal nematodes. Int. J. Parasitol. 22, 209e214.
Kooyman, F.N., Schallig, H.D., Van Leeuwen, M.A., Mackellar, A., Huntley, J.F.,
Cornelissen, A.W., Vervelde, L., 2000. Protection in lambs vaccinated with Haemonchus
contortus antigens is age related, and correlates with IgE rather than IgG1 antibody. Para-
site Immunol. 22, 13e20.
Kooyman, F.N., Van Kooten, P.J., Huntley, J.F., MacKellar, A., Cornelissen, A.W.,
Schallig, H.D., 1997. Production of a monoclonal antibody specific for ovine immuno-
globulin E and its application to monitor serum IgE responses to Haemonchus contortus
infection. Parasitology 114, 395e406.
Learmount, J., Conyers, C., Hird, H., Morgan, C., Craig, B.H., von Samson-
Himmelstjerna, G., Taylor, M., 2009. Development and validation of real-time PCR
methods for diagnosis of Teladorsagia circumcincta and Haemonchus contortus in sheep.
Vet. Parasitol. 166, 268e274.
LeJambre, L.F., Windon, R.G., Smith, W.D., 2008. Vaccination against Haemonchus contor-
tus: performance of native parasite gut membrane glycoproteins in Merino lambs grazing
contaminated pasture. Vet. Parasitol. 153, 302e312.
Lichtenfels, J.R., Pilitt, P.A., 2000. Synlophe patterns of the Haemonchinae of ruminants
(Nematoda: Trichostrongyloidea). J. Parasitol. 86, 1093e1098.
Lichtenfels, J.R., Pilitt, P.A., Gibbons, L.M., Boomker, J.D.F., 2001. Haemonchus horaki n.
sp. (Nematoda: Trichostrongyloidea) from the grey rhebuck Pelea capreolus in South
Africa. J. Parasitol. 87, 1095e1103.
Lichtenfels, J.R., Pilitt, P.A., Gibbons, L.M., Hoberg, E.P., 2002. Redescriptions of Hae-
monchus mitchelli and Haemonchus okapiae (Nematoda: Trichosytrongyloidea) and
description of a unique synlophe for the Haemonchinae. J. Parasitol. 88, 947e960.
Lichtenfels, J.R., Pilitt, P.A., Hoberg, E.P., 1994. New morphological characters for identi-
fying individual specimens of Haemonchus spp. (Nematoda: Trichostrongyloidea) and a
key to species in ruminants of North America. J. Parasitol. 80, 107e119.
Lichtenfels, J.R., Pilitt, P.A., Le Jambre, L.F., 1986. Cuticular ridge patterns of Haemonchus
contortus and Haemonchus placei (Nematoda: Trichostrongyloidea). Proc. Helminthol.
Soc. Wash. 53, 94e101.
Lichtenfels, J.R., Pilitt, P.A., Le Jambre, L.F., 1988. Spicule lengths of the ruminant stomach
nematodes Haemonchus contortus, Haemonchus placei, and their hybrids. Proc. Helminthol.
Soc. Wash. 55, 97e100.
Maitra, R.D., Kim, J., Dunbar, W.B., 2012. Recent advances in nanopore sequencing.
Electrophoresis 33, 3418e3428.
McNally, J., Callan, D., Andronicos, N., Bott, N., Hunt, P.W., 2013. DNA-based method-
ology for the quantification of gastrointestinal nematode eggs in sheep faeces. Vet.
Parasitol. 198, 325e335.
Melville, L., Kenyon, F., Javed, S., McElarney, I., Demeler, J., Skuce, P., 2014. Develop-
ment of a loop-mediated isothermal amplification (LAMP) assay for the sensitive detec-
tion of Haemonchus contortus eggs in ovine faecal samples. Vet. Parasitol. 206, 308e312.
Michel, J.F., 1963. The phenomenon of host resistance and the course of infection of Oster-
tagia ostertagi in calves. Parasitology 53, 63e84.
Identification of Haemonchus Species and Diagnosis of Haemonchosis 177

Michel, J.F., 1974. Arrested development of nematodes and some related phenomena. Adv.
Parasitol. 12, 279e366.
Miller, H.R., 1996. Prospects for the immunological control of ruminant gastrointestinal
nematodes: natural immunity, can it be harnessed? Int. J. Parasitol. 26, 801e811.
Miller, H.R., Pemberton, A.D., 2002. Tissue-specific expression of mast cell granule serine pro-
teinases and their role in inflammation in the lung and gut. Immunology 105, 375e390.
Miller, J.E., Horohov, D.W., 2006. Immunological aspects of nematode parasite control in
sheep. J. Anim. Sci. 84, E124eE132.
Ministry of Agriculture, Fisheries and Food (MAFF), 1986. Manual of Veterinary Parasitolog-
ical Techniques, third ed. Ministry of Agriculture, Fisheries and Food, London, UK. 160 p.
Mobegi, V.A., Duffy, C.W., Amambua-Ngwa, A., Loua, K.M., Laman, E., Nwakanma, D.C.,
MacInnis, B., Aspeling-Jones, H., Murray, L., Clark, T.G., Kwiatkowski, D.P.,
Conway, D.J., 2014. Genome-wide analysis of selection on the malaria parasite Plasmodium
falciparum in West African populations of differing infection endemicity. Mol. Biol. Evol.
31, 1490e1499.
M€onnig, H.O., 1931. The specific diagnosis of nematode infestation in sheep. In: 17th
Annual Report of the Director of Veterinary Services and Animal Industry. Onderste-
poort, Pretoria, Union of South Africa, pp. 255e264.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T.,
2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28 (12), E63.
Nwachukwu, M.A., Mackenzie, C.D., Litchfield, T.M., Howard, G., 1987. Lectin binding
to the developing forms of Onchocerca gutturosa microfilariae. Trop. Med. Parasitol. 38,
131e134.
Ochkur, S.I., Kim, J.D., Protheroe, C.A., Colbert, D., Condjella, R.M., Bersoux, S.,
Helmers, R.A., Moqbel, R., Lacy, P., Kelly, E.A., Jarjour, N.N., Kern, R., Peters, A.,
Schleimer, R.P., Furuta, G.T., Nair, P., Lee, J.J., Lee, N.A., 2012. A sensitive high
throughput ELISA for human eosinophil peroxidase: a specific assay to quantify eosino-
phil degranulation from patient-derived sources. J. Immunol. Methods 384, 10e20.
Pallen, M.J., 2014. Diagnostic metagenomics: potential applications to bacterial, viral and
parasitic infections. Parasitology 141, 1856e1862.
Palmer, D.G., McCombe, I.L., 1996. Lectin staining of trichostrongylid nematode eggs of
sheep: rapid identification of Haemonchus contortus eggs with peanut agglutinin. Int. J.
Parasitol. 26, 447e450.
Pemberton, A.D., Belham, C.M., Huntley, J.F., Plevin, R., Miller, H.R., 1997. Sheep mast
cell proteinase-1, a serine proteinase with both tryptase- and chymase-like properties, is
inhibited by plasma proteinase inhibitors and is mitogenic for bovine pulmonary artery
fibroblasts. Biochem. J. 323, 719e725.
Pemberton, A.D., McAleese, S.M., Huntley, J.F., Collie, D.D., Scudamore, C.L.,
McEuen, A.R., Walls, A.F., Miller, H.R., 2000. cDNA sequence of two sheep mast
cell tryptases and the differential expression of tryptase and sheep mast cell proteinase-
1 in lung, dermis and gastrointestinal tract. Clin. Exp. Allergy 30, 818e832.
na, M.T., Miller, J.E., Horohov, D.W., 2006. Effect of CD4þ T lymphocyte depletion on
Pe~
resistance of Gulf Coast Native lambs to Haemonchus contortus infection. Vet. Parasitol.
138, 240e246.
Porazinska, D.L., Morgan, M.J., Gaspar, J.M., Court, L.N., Hardy, C.M., Hodda, M., 2014.
Discrimination of plant-parasitic nematodes from complex soil communities using
ecometagenetics. Phytopathology 104, 749e761.
Preston, S.J., Sandeman, M., Gonzalez, J., Piedrafita, D., 2014. Current status for gastroin-
testinal nematode diagnosis in small ruminants: where are we and where are we
going? J. Immunol. Res. 2014, 210350. http://dx.doi.org/10.1155/2014/210350.
Rabouam, C., Comes, A.M., Bretagnolle, V.V., Humbert, J.F., Periquet, G., Bigot, Y.,
1999. Features of DNA fragments obtained by random amplified polymorphic DNA
(RAPD) assays. Mol. Ecol. 8, 493e503.
178 D.S. Zarlenga et al.

Raleigh, J.M., Meeusen, E.N., 1996. Developmentally regulated expression of a Haemonchus


contortus surface antigen. Int. J. Parasitol. 26, 673e675.
Ramalingam, T., Porte, P., Lee, J., Rajan, T.V., 2005. Eosinophils, but not eosinophil perox-
idase or major basic protein, are important for host protection in experimental Brugia
pahangi infection. Infect. Immun. 73, 8442e8443.
Ransom, B.H., 1906. The Life History of the Twisted Wire-Worm (Haemonchus contortus) of
Sheep and Other Ruminants. (Preliminary Report). Circular 93. Bureau of Animal in-
dustry, US Department of Agriculture, Washington, D.C, pp. 1e7.
Rathore, D.K., Suchitra, S., Saini, M., Singh, B.P., Joshi, P., 2006. Identification of a 66 kDa
Haemonchus contortus excretory/secretory antigen that inhibits host monocytes. Vet. Para-
sitol. 138, 291e300.
Redmond, D.L., Knox, D.P., 2004. Protection studies in sheep using affinity-purified and
recombinant cysteine proteinases of adult Haemonchus contortus. Vaccine 22, 4252e4261.
Reinhardt, S., Scott, I., Simpson, H.V., 2011. Neutrophil and eosinophil chemotactic factors
in the excretory/secretory products of sheep abomasal nematode parasites: NCF and
ECF in abomasal nematodes. Parasitol. Res. 109, 627e635.
Roeber, F., Jex, A.R., Campbell, A.J., Campbell, B.E., Anderson, G.A., Gasser, R.B., 2011.
Evaluation and application of a molecular method to assess the composition of strongylid
nematode populations in sheep with naturally acquired infections. Infect. Genet. Evol.
11, 849e854.
Roeber, F., Jex, A.R., Campbell, A.J., Nielsen, R., Anderson, G.A., Stanley, K.K.,
Gasser, R.B., 2012a. Establishment of a robotic, high-throughput platform for the specific
diagnosis of gastrointestinal nematode infections in sheep. Int. J. Parasitol. 42, 1151e1158.
Roeber, F., Jex, A.R., Gasser, R.B., 2013a. Advances in the diagnosis of key gastrointestinal
nematode infections of livestock, with an emphasis on small ruminants. Biotechnol. Adv.
31, 1135e1152.
Roeber, F., Jex, A.R., Gasser, R.B., 2013b. Next-generation molecular-diagnostic tools for
gastrointestinal nematodes of livestock, with an emphasis on small ruminants: a turning
point? Adv. Parasitol. 83, 267e333.
Roeber, F., Larsen, J.W., Anderson, N., Campbell, A.J., Anderson, G.A., Gasser, R.B.,
Jex, A.R., 2012b. A molecular diagnostic tool to replace larval culture in conventional
faecal egg count reduction testing in sheep. PLoS One 7, e37327.
Roos, M.H., Grant, W.N., 1993. Species-specific PCR for the parasitic nematodes Haemon-
chus contortus and Trichostrongylus colubriformis. Int. J. Parasitol. 23, 419e421.
Roos, M.H., Boersema, J.H., Borgsteede, F.H., Cornelissen, J., Taylor, M., Ruitenberg, E.J.,
1990. Molecular analysis of selection for benzimidazole resistance in the sheep parasite
Haemonchus contortus. Mol. Biochem. Parasitol. 43, 77e88.
Rosenthal, B.M., 2009. How has agriculture influenced the geography and genetics of animal
parasites? Trends Parasitol. 25, 67e70.
von Samson-Himmelstjerna, G., Harder, A., Schnieder, T., 2002. Quantitative analysis of
ITS2 sequences in trichostrongyle parasites. Int. J. Parasitol. 32, 1529e1535.
Santos, M.C., Amarante, M.R., Silva, M.R., Amarante, A.F., 2014a. Differentiation of Hae-
monchus placei from Haemonchus contortus by PCR and by morphometrics of adult parasites
and third stage larvae. Rev. Bras. Parasitol. Vet. 23, 495e500.
Santos, M.C., Xavier, J.K., Amarante, M.R., Bassetto, C.C., Amarante, A.F., 2014b. Im-
mune response to Haemonchus contortus and Haemonchus placei in sheep and its role on
parasite specificity. Vet. Parasitol. 203, 127e138.
Schallig, H.D., 2000. Immunological responses of sheep to Haemonchus contortus. Parasitology
120, S63eS72.
Schallig, H.D., van Leeuwen, M.A., Cornelissen, A.W., 1997. Protective immunity induced
by vaccination with two Haemonchus contortus excretory secretory proteins in sheep. Para-
site Immunol. 19, 447e453.
Identification of Haemonchus Species and Diagnosis of Haemonchosis 179

Schallig, H.D., van Leeuwen, M.A., Hendrikx, W.M., 1995. Isotype-specific serum antibody
responses of sheep to Haemonchus contortus antigens. Vet. Parasitol. 56, 149e162.
Schnieder, T., Heise, M., Epe, C., 1999. Genus-specific PCR for the differentiation of eggs
or larvae from gastrointestinal nematodes of ruminants. Parasitol. Res. 85, 895e898.
Schwartz, C., Turqueti-Neves, A., Hartmann, S., Yu, P., Nimmerjahn, F., Voehringer, D.,
2014. Basophil-mediated protection against gastrointestinal helminths requires IgE-
induced cytokine secretion. Proc. Natl. Acad. Sci. U. S. A. 111, E5169eE5177.
Schwartz, L.B., 2006. Diagnostic value of tryptase in anaphylaxis and mastocytosis. Immunol.
Allergy Clin. North Am. 26, 451e463.
Shakya, K.P., Miller, J.E., Lomax, L.G., Burnett, D.D., 2011. Evaluation of immune
response to artificial infections of Haemonchus contortus in Gulf Coast Native compared
with Suffolk lambs. Vet. Parasitol. 181, 239e247.
Shaw, R.J., Grimmett, D.J., Donaghy, M.J., Gatehouse, T.K., Shirer, C.L., Douch, P.G.,
1996. Production and characterisation of monoclonal antibodies recognising ovine
IgE. Vet. Immunol. Immunopathol. 51, 235e251.
Shorb, D.A., 1939. Differentiation of Eggs of Various Genera of Nematodes Parasitic in
Domestic Ruminants in the United States. Tech. Bull. U.S. D.A. No. 694.
Siedek, E.M., Burden, D., von Samson-Himmelstjerna, G., 2006. Feasibility of genus-spe-
cific real-time PCR for the differentiation of larvae from gastrointestinal nematodes of
naturally infected sheep. Berl. Munch. Tierarztl. Wochenschr. 119, 303e307.
Smith, W.D., 1977. Serum and mucus antibodies in sheep immunised with larval antigens of
Haemonchus contortus. Res. Vet. Sci. 22, 128e129.
Sommer, C., 1996. Digital image analysis and identification of eggs from bovine parasitic
nematodes. J. Helminthol. 70, 143e151.
Sommer, C., 1998. Quantitative characterization of texture used for identification of eggs of
bovine parasitic nematodes. J. Helminthol. 72, 179e182.
Stevenson, L.A., Chilton, N.B., Gasser, R.B., 1995. Differentiation of Haemonchus placei
from H. contortus (Nematoda: Trichostrongylidae) by the ribosomal DNA second inter-
nal transcribed spacer. Int. J. Parasitol. 25, 483e488.
Sweeny, J.P., Robertson, I.D., Ryan, U.M., Jacobson, C., Woodgate, R.G., 2011. Compar-
ison of molecular and McMaster microscopy techniques to confirm the presence of natu-
rally acquired strongylid nematode infections in sheep. Mol. Biochem. Parasitol. 180,
62e67.
Sweeny, J.P., Ryan, U.M., Robertson, I.D., Niemeyer, D., Hunt, P.W., 2012. Develop-
ment of a modified molecular diagnostic procedure for the identification and quantifica-
tion of naturally occurring strongylid larvae on pastures. Vet. Parasitol. 190, 467e481.
Tanaka, R., Hino, A., Tsai, I.J., Palomares-Rius, J.E., Yoshida, A., Ogura, Y., Hayashi, T.,
Maruyama, H., Kikuchi, T., Oct 23, 2014. Assessment of helminth biodiversity in wild
rats using 18S rDNA based metagenomics. PLoS One 9 (10), e110769.
Tetley, J.H., 1941. Haemonchus contortus eggs: comparison of those in utero with those recov-
ered from faeces, and a statistical method for identifying H. contortus eggs in mixed
infections. J. Parasitol. 27, 453e464.
Torgerson, P.R., Lloyd, S., 1993. The B cell dependence of Haemonchus contortus antigen-
induced lymphocyte proliferation. Int. J. Parasitol. 23, 925e930.
Troell, K., Engstr€om, A., Morrison, D.A., Mattson, J.G., H€ oglund, J., 2006. Global patterns
reveal strong population structure in Haemonchus contortus, a nematode parasite of domes-
ticated ruminants. Int. J. Parasitol. 36, 1305e1316.
Tuo, W., Bazer, F.W., Davis, W.C., Zhu, D., Brown, W.C., 1999. Differential effects of type
I IFNs on the growth of WC1CD8þ gamma delta T cells and WC1þ CD8gamma
delta T cells in vitro. J. Immunol. 162, 245e253.
Vande Walle, L., Kanneganti, T.D., Lamkanfi, M., 2011. HMGB1 release by inflammasomes.
Virulence 2, 162e165.
180 D.S. Zarlenga et al.

Veglia, F., 1915. The Anatomy and Life History of Haemonchus contortus (Rud.), 3rd and 4th
Reports of the Director of Veterinary Research. Onderstepoort, Pretoria, Union of
South Africa, pp. 349e500.
Veglia, F., 1926. The oviposition of some Strongylidae: laboratory observations and practical
deductions. S. Afr. J. Sci. 23, 713e715.
Wang, W., Wang, S., Zhang, H., Yuan, C., Yan, R., Song, X., Xu, L., Li, X., 2014a. Galec-
tin Hco-gal-m from Haemonchus contortus modulates goat monocytes and T cell function
in different patterns. Parasit. Vectors 7, 342.
Wang, W., Yuan, C., Wang, S., Song, X., Xu, L., Yan, R., Hasson, I.A., Li, X., 2014b.
Transcriptional and proteomic analysis reveal recombinant galectins of Haemonchus con-
tortus down-regulated functions of goat PBMC and modulation of several signaling cas-
cades in vitro. J. Proteomics 98, 123e137.
Wilson, M., Glaser, K.C., Adams-Fish, D., Boley, M., Mayda, M., Molestina, R.E., 2015.
Development of droplet digital PCR for the detection of Babesia microti and Babesia
duncani. Exp. Parasitol. 149, 24e31.
Wu, T., Ayres, E., Bardgett, R.D., Wall, D.H., Garey, J.R., 2011. Molecular study of world-
wide distribution and diversity of soil animals. Proc. Natl. Acad. Sci. U. S. A. 108,
17720e17725.
van Wyk, J.A., Mayhew, E., 2013. Morphological identification of parasitic nematode infec-
tive larvae of small ruminants and cattle: a practical lab guide. Onderstepoort J. Vet. Res.
80, 1e14.
Yan, F., Xu, L., Liu, L., Yan, R., Song, X., Li, X., 2010. Immunoproteomic analysis of
whole proteins from male and female adult Haemonchus contortus. Vet. J. 185, 174e179.
Yang, R., Paparini, A., Monis, P., Ryan, U., 2014. Comparison of next-generation droplet
digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptospo-
ridium oocysts in faecal samples. Int. J. Parasitol. 44, 1105e1113.
Yin, F., Gasser, R.B., Li, F., Bao, M., Huang, W., Zou, F., Zhao, G., Wang, C., Yang, X.,
Zhou, Y., Zhao, J., Fang, R., Hu, M., 2013. Genetic variability within and among Hae-
monchus contortus isolates from goats and sheep in China. Parasit. Vectors 6, 279.
Yuan, C., Zhang, H., Wang, W., Li, Y., Yan, R., Xu, L., Song, X., Li, X., 2015. Transmem-
brane protein 63A is a partner protein of Haemonchus contortus galectin in the regulation of
goat peripheral blood mononuclear cells. Parasit. Vectors 8, 211.
Zarlenga, D.S., Chute, M.B., Gasbarre, L.C., Boyd, P.C., 2001. A multiplex PCR assay for
differentiating economically important gastrointestinal nematodes of cattle. Vet. Parasi-
tol. 97, 199e209.
Zarlenga, D.S., Chute, M.B., Martin, A., Kapel, C.M., 1999. A multiplex PCR for unequiv-
ocal differentiation of all encapsulated and non-encapsulated genotypes of Trichinella. Int.
J. Parasitol. 29, 1859e1867.
Zarlenga, D.S., Hoberg, E., Rosenthal, B., Mattiucci, S., Nascetti, G., 2014. Anthropogenics:
human influence on global and genetic homogenization of parasite populations. J. Para-
sitol. 100, 756e772.
Zarlenga, D.S., Stringfellow, F., Nobary, M., Lichtenfels, J.R., 1994. Cloning and character-
ization of ribosomal RNA genes from three species of Haemonchus (Nematoda: Trichos-
trongyloidea) and identification of PCR primers for rapid differentiation. Exp. Parasitol.
78, 28e36.
Zaros, L.G., Neves, M.R., Benvenuti, C.L., Navarro, A.M., Sider, L.H., Coutinho, L.L.,
Vieira, L.S., 2014. Response of resistant and susceptible Brazilian Somalis crossbreed
sheep naturally infected by Haemonchus contortus. Parasitol. Res. 113, 1155e1161.
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
University of New England, Armidale, NSW, Australia
{
University of Edinburgh, Roslin, Midlothian, United Kingdom
jj
University of Pretoria, Hatfield, South Africa
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

Advances in Parasitology, Volume 93


© 2016 Elsevier Ltd.
j
ISSN 0065-308X
http://dx.doi.org/10.1016/bs.apar.2016.02.024 All rights reserved. 181
182 R.B. Besier et al.

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
184 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 185

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
186 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 187

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
188 R.B. Besier et al.

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.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 189

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
190 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 191

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
192 R.B. Besier et al.

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,
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 193

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
194 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 195

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
196 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 197

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.
198 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 199

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).
200 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 201

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
202 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 203

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
204 R.B. Besier et al.

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.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 205

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
206 R.B. Besier et al.

(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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 207

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
208 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 209

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
210 R.B. Besier et al.

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,
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 211

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
212 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 213

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’.
214 R.B. Besier et al.

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
218 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 219

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
220 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 221

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
222 R.B. Besier et al.

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
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 223

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
224 R.B. Besier et al.

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.

REFERENCES
Abbott, E.M., Parkins, J.J., Holmes, P.H., 1985. Influence of dietary protein on parasite
establishment and pathogenesis in Finn Dorset and Scottish Blackface lambs given a sin-
gle moderate infection of Haemonchus contortus. Res. Vet. Sci. 38, 6e13.
Abbott, E.M., Parkins, J.J., Holmes, P.H., 1986. The effect of dietary protein on the path-
ogenesis of acute ovine haemonchosis. Vet. Parasitol. 20, 275e289.
Albers, G.A.A., Gray, G.D., Le Jambre, L.F., Piper, L.R., Barger, I.A., Barker, J.S.F., 1989.
The effect of haemonchus contortus on liveweight gain and wool growth in young Merino
sheep. Aust. J. Agric. Res. 40, 419e432.
Allonby, E.W., Urquhart, G.M., 1975. The epidemiology and pathogenic significance of
haemonchosis in a Merino flock in East Africa. Vet. Parasitol. 1, 129e143.
Amarante, A.F.T., Craig, T.M., Ramsey, W.S., El-Sayed, N.M., Desouki, A.Y.,
Bazer, F.W., 1999. Comparison of naturally acquired parasite burdens among Florida
Native, Rambouillet and crossbreed ewes. Vet. Parasitol. 85, 61e69.
Amarante, A.F., Bricarello, P.A., Huntley, J.F., Mazzolin, L.P., Gomes, J.C., 2005. Relation-
ship of abomasal histology and parasite-specific immunoglobulin A with the resistance to
Haemonchus contortus infection in three breeds of sheep. Vet. Parasitol. 128, 99e107.
Anderson, N., Martin, P.J., Jarrett, R.G., 1988. Mixtures of anthelmintics: a strategy against
resistance. Aust. Vet. J. 65, 62e64.
Andronicos, N.M., Henshall, J.M., Le Jambre, L.F., Hunt, P.W., Ingham, A.B., 2014. A
one-shot blood phenotype can identify sheep that resist Haemonchus contortus
challenge. Vet. Parasitol. 205, 595e605.
Athanasiadou, S., Tzamaloukas, O., Kyriazakis, I., Jackson, F., Coop, R.L., 2005. Testing for
direct anthelmintic effects of bioactive forages against Trichostrongylus colubriformis in graz-
ing sheep. Vet. Parasitol. 127, 233e243.
Athanasiadou, S., Githiori, J., Kyriazakis, I., 2007. Medicinal plants for helminth parasite con-
trol: facts and fiction. Animal 1, 1392e1400.
Aumont, G., Gruner, L., Hostache, G., 2003. Comparison of the resistance to sympatric and
allopatric isolates of Haemonchus contortus of Black Belly sheep in Guadeloupe (FWI) and
of INRA 401 sheep in France. Vet. Parasitol. 116, 139e150.
Bailey, J.N., Walkden-Brown, S.W., Kahn, L.P., 2009. Comparison of strategies to provide
lambing paddocks of low gastrointestinal nematode infectivity in a summer rainfall
region of Australia. Vet. Parasitol. 161, 218e231.
Baldock, F.C., Lyndal-Murphy, M., Pearse, B., 1990. An assessment of a composite sampling
method for counting strongyle eggs in sheep faeces. Aust. Vet. J. 67, 165e167.
Bang, K.S., Familton, A.S., Sykes, A.R., 1990. Effect of copper oxide wire particle treatment
on establishment of major gastrointestinal nematodes in lambs. Res. Vet. Sci. 49, 132e137.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 225

Banks, D.J.D., Singh, R., Barger, I.A., Pratap, B., Le Jambre, L.F., 1990. Development and
survival of infective larvae of Haemonchus contortus and Trichostrongylus colubriformis in a
tropical environment. Int. J. Parasitol. 20, 155e160.
Barger, I.A., Dash, K.M., 1987. Repeatability of ovine faecal egg counts and blood packed
cell volumes in Haemonchus contortus infections. Int. J. Parasitol. 17, 977e980.
Barger, I.A., Siale, K., Banks, D.J.D., Le Jambre, L.F., 1994. Rotational grazing control of
gastrointestinal nematodes of goats in a wet tropical environment. Vet. Parasitol. 53,
109e116.
Barger, I.A., 1985. The statistical distribution of trichostrongylid nematodes in grazing lambs.
Int. J. Parasitol. 15, 645e649.
Barger, I.A., 1997. Control by management. Vet. Parasitol. 72, 493e506.
Barnes, E.H., Dobson, R.J., Barger, I.A., 1995. Worm control and anthelmintic resistance:
adventures with a model. Parasitol. Today 11, 56e63.
Barnes, E.H., Dobson, R.J., Stein, P.A., Le Jambre, L.F., Lenane, I.J., 2001. Selection of
different genotype larvae and adult worms for anthelmintic resistance by persistent and
short-acting avermectin/milbemycins. Int. J. Parasitol. 31, 720e727.
Bartram, D.J., Leathwick, D.M., Taylor, M.A., Geurden, T., Maeder, S.J., 2012. The role of
combination anthelmintic formulations in the sustainable control of sheep nematodes.
Vet. Parasitol. 186, 151e158.
Bassetto, C.C., Picharillo, M.E., Newlands, G.F.J., Smith, W.D., Fernandes, S., Siqueira, E.R.,
Amarante, A.F.T., 2014. Attempts to vaccinate ewes and their lambs against natural infec-
tion with Haemonchus contortus in a tropical environment. Int. J. Parasitol. 44, 1049e1054.
Bath, G.F., 2006. Practical implementation of holistic internal parasite management in sheep.
Small Rumin. Res. 62, 13e18.
Besier, R.B., Dunsmore, J.D., 1993. The ecology of Haemonchus contortus in a winter rainfall re-
gion of Australia. 2. The survival of infective larvae on pasture. Vet. Parasitol. 45, 293e306.
Besier, R.B., Hopkins, D.L., 1988. Anthelmintic dose selection by farmers. Aust. Vet. J. 65,
193e194.
Besier, R.B., Smith, W.D., 2014. A new approach to the control of barbers pole worm. In:
Proc. Conf. Aust. Sheep Veterinarians (Perth, Australia, May 2014), pp. 11e16.
Besier, R.B., Love, R.J., Lyon, J., van Burgel, A.J., 2010. A targeted selective treatment
approach for effective and sustainable sheep worm management: investigations in West-
ern Australia. Anim. Prod. Sci. 50, 1034e1042.
Besier, B., Kahn, L., Dobson, R., Smith, D., 2015. Barbervax e a new strategy for Haemon-
chus management. In: Proc. Conf. Aust. Sheep Veterinarians (Brisbane, Australia, May
2015), pp. 373e377.
Besier, R.B., Kahn, L.P., Sargsion, N.D., Van Wyk, J.A., 2016. The pathophysiology, ecol-
ogy and epidemiology of Haemonchus contortus infections in small ruminants. In:
Gasser, R., Samson-Himmelstjerna, G.V. (Eds.), Haemonchus contortus and Haemonchosis
Past, Present and Future Trends. vol. 93, pp. 95e144.
Besier, R.B., 2008. Targeted treatment strategies for sustainable worm control in small
ruminants. Trop. Biomed. 25 (Suppl.), 9e17. Proc. 5th Int. workshop; Novel
approaches to the control of helminth parasites of livestock, 2008, Ipoh, Malaysia.
Besier, R.B., 2012. Refugia-based strategies for sustainable worm control: factors affecting
the acceptability to sheep and goat owners. Vet. Parasitol. 186, 2e9.
Bisset, S.A., Morris, C.A., 1996. Feasibility and implications of breeding sheep for resilience
to nematode challenge. Int. J. Parasitol. 26, 857e868.
Bisset, S.A., Knight, J.S., Bouchet, C.L.G., 2014. A multiplex PCR-based method to identify
strongylid parasite larvae recovered from ovine faecal cultures and/or pasture samples.
Vet. Parasitol. 200, 117e127.
Bolajoko, M.B., Morgan, E.R., 2012. Relevance of improved epidemiological knowledge
to sustainable control of Haemonchus contortus in Nigeria. Anim. Health Res. Rev. 13,
196e208.
226 R.B. Besier et al.

Bott, N.J., Campbell, B.E., Beveridge, I., Chilton, N.B., Rees, D., Hunt, P.W.,
Gasser, R.B., 2009. A combined microscopic-molecular method for the diagnosis of
strongylid infections in sheep. Int. J. Parasitol. 39, 1277e1287.
Bowdridge, S., MacKinnon, K., McCann, J.C., Zajac, A.M., Notter, D., 2013. Hair-type
sheep generate an accelerated and longer-lived humoral immune response to Haemonchus
contortus infection. Vet. Parasitol. 196, 172e178.
Bowman, D.D., 2014. Georgi’s Parasitology for Veterinarians, tenth ed. Elsevier Saunders, St
Louis, Missouri.
Britton, C., Roberts, B., Marks, N.D., 2016. Functional genomics tools for Haemonchus con-
tortus and lessons from other helminths. In: Gasser, R., Samson-Himmelstjerna, G.V.
(Eds.), Haemonchus contortus and Haemonchosis Past, Present and Future Trends. vol.
93, pp. 599e617.
Brundson, R.V., 1970. Within-flock variations in strongyle worm infections in sheep: the
need for adequate diagnostic samples. N.Z. Vet. J. 18, 185e188.
Burke, J.M., Miller, J.E., 2002. Relative resistance of Dorper crossbred ewes to gastrointes-
tinal nematode infection compared with St. Croix and Katahdin ewes in the southeastern
United States. Vet. Parasitol. 109, 265e275.
Burke, J.M., Miller, J.E., 2006. Control of Haemonchus contortus in goats with a sustained-
release multi-trace element/vitamin ruminal bolus containing copper. Vet. Parasitol.
141, 132e137.
Burke, J.M., Miller, J.E., Larsen, M., Terrill, T.H., 2005. Interaction between copper oxide
wire particles and Duddingtonia flagrans in lambs. Vet. Parasitol. 134, 141e146.
Burke, J.M., Kaplan, R.M., Miller, J.E., Terrill, T.H., Getz, W.R., Mobini, S., Valencia, E.,
Williams, M.J., Williamson, L.H., Vatta, A.F., 2007. Accuracy of the FAMACHA sys-
tem for on-farm use by sheep and goat producers in the southeastern United States.
Vet. Parasitol. 147, 89e95.
Burke, J.M., Wells, A., Casey, P., Miller, J.E., 2009a. Garlic and papaya lack control over
gastrointestinal nematodes in goats and lambs. Vet. Parasitol. 159, 171e174.
Burke, J.M., Miller, J.E., Terrill, T.H., 2009b. Impact of rotational grazing on management
of gastrointestinal nematodes in weaned lambs. Vet. Parasitol. 163, 52e56.
Burke, J.M., Miller, J.E., Mosjidis, J.A., Terrill, T.H., 2012. Use of a mixed Sericea lespedeza
and grass pasture system for control of gastrointestinal nematodes in lambs and kids. Vet.
Parasitol. 186, 328e336.
Burke, J.M., Miller, J.E., Terrill, T.H., Mosjidis, J.A., 2014. The effects of supplemental ser-
icea lespedeza pellets in lambs and kids on growth rate. Livest. Sci. 159, 29e36.
Cachat, E., Newlands, G.F.J., Ekoja, S.E., McAllister, H., Smith, W.D., 2010. Attempts
to immunize sheep against Haemonchus contortus using a cocktail of recombinant
proteases derived from the protective antigen, H-gal-GP. Parasite Immunol. 32,
414e419.
Campbell, N.J., Hall, C.A., Kelly, J.D., Martin, I.C., 1978. The anthelmintic efficacy of non-
benzimidazole anthelmintics against benzimidazole resistant strains of Haemonchus contor-
tus and Trichostrongylus colubriformis in sheep. Aust. Vet. J. 54, 23e25.
Campbell, W.C., Fisher, M.H., Stapley, E.O., Albers-Schonberg, G., Jacobs, T.A., 1983.
Ivermectin: a potent new anthelmintic agent. Science 221, 823e828.
Castillo, F.A.J., Méndez, Villalobos, J.M.B., Gayosso-Vazquez, A., Ulloa-Arvízu, R.,
Rodríguez, R.A., Ramírez, H.P., Morales, A., Rogelio, A., 2011. Association between
major histocompatibility complex microsatellites, fecal egg count, blood packed cell vol-
ume and blood eosinophilia in Pelibuey sheep infected with Haemonchus contortus. Vet.
Parasitol. 177, 339e344.
Cezar, A.S., Toscan, G., Camillo, G., Sangioni, L.A., Riba, H.O., Vogel, F.S.F., 2010. Mul-
tiple resistance of gastrointestinal nematodes to nine different drugs in a sheep flock in
southern Brazil. Vet. Parasitol. 173, 157e160.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 227

Chagas, A.C.S., Vieira, L.S., Freitas, A.R., Ara ujo, M.R.A., Ara ujo-Filho, J.A.,
Aragu~ao, W.R., Navarro, A.M.C., 2008. Anthelmintic efficacy of neem (Azadirachta ind-
ica A. Juss) and the homeopathic product Fator VermesÒ in Morada Nova sheep. Vet.
Parasitol. 151, 68e73.
Chandrawathani, P., Yussof, N., Waller, P.J., 2004a. Total anthelmintic failure to control
nematode parasites of small ruminants on government breeding farms in Sabah, East
Malaysia. Vet. Res. Commun. 28, 479e489.
Chandrawathani, P., Jamnah, O., Adnan, M., Waller, P.J., Larsen, M., Gillespie, A.T.,
2004b. Field studies on the biological control of nematode parasites of sheep in the tro-
pics, using the microfungus Duddingtonia flagrans. Vet. Parasitol. 120, 177e187.
Chandrawathani, P., Chang, K.W., Nurulaini, R., Waller, P., Adnan, M., Zaini, C.M.,
Jamnah, O., Khadijah, S., Vincent, N., 2006. Daily feeding of fresh neem leaves (Aza-
dirachta indica) for worm control in sheep. Trop. Biomed. 23, 23e26.
Chiejina, S.N., Behnke, J.M., Musongong, G.A., Nnadia, P.A., Ngongeh, L.A., 2010. Resis-
tance and resilience of West African Dwarf goats of the Nigerian savanna zone exposed
to experimental escalating primary and challenge infections with Haemonchus contortus.
Vet. Parasitol. 171, 81e90.
Christie, M., Jackson, F., 1982. Specific identification of strongyle eggs in small samples of
sheep faeces. Res. Vet. Sci. 32, 113e117.
Coadwell, W.J., Ward, P.F., 1982. The use of faecal egg counts for estimating worm burdens
in sheep infected with Haemonchus contortus. Parasitology 85, 251e256.
Colditz, I.G., le Jambre, L.F., 2008. Development of a faecal occult blood test to determine
the severity of Haemonchus contortus infections in sheep. Vet. Parasitol. 153, 93e99.
Colditz, I.G., le Jambre, L.F., Hosse, R., 2002. Use of lectin binding characteristics to identify
gastrointestinal parasite eggs in faeces. Vet. Parasitol. 105, 219e227.
Colvin, A.F., Walkden-Brown, S.W., Knox, M.R., Scott, M.J., 2008. Intensive rotational
grazing assists control of gastrointestinal nematodosis of sheep in a cool temperate envi-
ronment with summer-dominant rainfall. Vet. Parasitol. 153, 108e120.
Coop, R.L., Holmes, P.H., 1996. Nutrition and parasite interaction. Int. J. Parasitol. 26,
951e962.
Cornelius, M.P., Jacobson, C., Besier, R.B., 2014. Body condition score as a selection tool
for targeted selective treatment-based nematode control strategies in Merino ewes. Vet.
Parasitol. 206, 173e181.
Costa, C.T.C., Bevilaqua, C.M.L., Maciel, M.V., Camurça-Vasconcelos, A.L.F.,
Morais, S.M., Monteiro, M.V.B., Farias, V.M., da Silva, M.V., Souza, M.M.C., 2006.
Anthelmintic activity of Azadirachta indica A. Juss against sheep gastrointestinal
nematodes. Vet. Parasitol. 137, 306e310.
Courtney, C.H., Parker, C.F., McClure, K.E., Herd, R.P., 1985. Resistance of exotic and
domestic lambs to experimental infections with Haemonchus contortus. Int. J. Parasitol.
15, 101e109.
Dargie, J.D., Allonby, E.W., 1975. Pathopbysiology of single challenge infections of Haemon-
chus contortus in Merino sheep: studies on red cell kinetics and the ‘self-cure’
phenomenon. Int. J. Parasitol. 5, 147e157.
Dash, K.M., 1986. Control of helminthosis in lambs by strategic treatment with closantel and
broad-spectrum anthelmintics. Aust. Vet. J. 63, 4e8.
De Chaneet, G.C., Mayberry, C.J., 1978. Ovine Haemonchosis: A Review and Report of
Epizootics in North-west Western Australia and of a Trial at Esperance Western
Australia. Bulletin 41. Department of Agriculture, Western Australia.
De la Chevrotiere, C.C., Bishop, S., Arquet, R., Bambou, J.C., Schibler, L., Amigues, Y.,
Moreno, C., Mandonnet, N., 2012. Detection of quantitative trait loci for
resistance to gastrointestinal nematode infections in Creole goats. Anim. Genet. 43,
768e775.
228 R.B. Besier et al.

De Montellano, C.M.O., Vargas-Maga~ na, J.J., Aguilar-Caballero, A.J., Sandoval-


Castro, C.A., Cob-Galera, L., May-Martínez, M., Miranda-Soberanis, R., Hoste, H.,
Camara Sarmiento, R., Torres-Acosta, J.F.J., 2007. Combining the effects of supplemen-
tary feeding and copper oxide needles for the control of gastrointestinal nematodes in
browsing goats. Vet. Parasitol. 146, 66e76.
Demeler, J., Ramunke, S., Wolken, S., Ianiello, D., Rinaldi, L., Bosco Gahutu, J.,
Cringoli, G., von Samson-Himmelstjerna, G., Krucken, J., 2013. Discrimination of
gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant
conventional and real-time PCR. PLoS One 8, e61285, 1e13.
Dikmans, G., Andrews, J.S., 1933. A comparative morphological study of the infective larvae
of the common nematodes parasitic in the alimentary tract of sheep. Trans. Am. Microsc.
Soc. 52, 1e25.
Dobson, R.J., Barnes, E.H., Birclijin, S.D., Gill, J.H., 1992. The survival of Ostertagia circum-
cincta and Trichostrongylus colubriformis in faecal culture as a source of bias in apportioning
egg counts to worm species. Int. J. Parasitol. 22, 1005e1008.
Dobson, R.J., le Jambre, L.F., Gill, J.H., 1996. Management of anthelmintic resistance: in-
heritance of resistance and selection with persistent drugs. Int. J. Parasitol. 26, 993e1000.
Dobson, R.J., Hosking, B., Besier, R.B., Love, S.C.J., Larsen, J., Rolfe, P.F., Bailey, J.N.,
2011. Minimising the development of anthelmintic resistance, and optimising the use
of the novel anthelmintic monepantel, for the sustainable control of nematode parasites
in Australian sheep grazing systems. Aust. Vet. J. 89, 160e166.
Doligalska, M., Moskwa, B., Niznikowski, R., 1997. The repeatability of faecal egg counts in
Polish Wrzosowka sheep. Vet. Parasitol. 70, 241e246.
Dorny, P., Symoens, C., Jalila, A., Vercruysse, J., Sani, R., 1995. Strongyle infections in
sheep and goats under the traditional husbandry system in peninsular Malaysia. Vet. Para-
sitol. 56, 121e136.
Dunn, A.M., 1978. Veterinary Helminthology, second ed. William Heinemann Medical
Books Ltd., pp. 184e185
Edwards, G.T., Sian, E., Mitchell, E., Hardwood, D.G., 2007. Anthelmintic use in goats.
Vet. Rec. 161, 763e764.
Eysker, M., Bakker, N., Kooyman, F.N.J., Van der Linden, D., Schrama, C., Ploeger, H.W.,
2005. Consequences of the unusually warm and dry summer of 2003 in The Netherlands:
poor development of free living stages, normal survival of infective larvae and long survival
of adult gastrointestinal nematodes of sheep. Vet. Parasitol. 133, 313e321.
Fabiyi, J.P., 1987. Production losses and control of helminths in ruminants of tropical regions.
Int. J. Parasitol. 17, 435e442.
Fawzi, E.M., Gonzalez-Sanchez, M.E., Corral, M.J., Alunda, J.M., Cuquerella, M., 2015.
Vaccination of lambs with the recombinant protein rHc23 elicits significant protection
against Haemonchus contortus challenge. Vet. Parasitol. 211, 54e59.
Fiel, C., Guzman, M., Steffan, P., Rodriguez, E., Prieto, O., Bhushan, C., 2011. The efficacy
of trichlorphon and naphthalophos against multiple anthelmintic-resistant nematodes of
naturally infected sheep in Argentina. Parasitol. Res. 109, 139e148.
Fontenot, M.E., Miller, J.E., Pe~ na, M.T., Larsen, M., Gillespie, A., 2003. Efficiency of
feeding Duddingtonia flagrans chlamydospores to grazing ewes on reducing availability
of parasitic nematode larvae on pasture. Vet. Parasitol. 118, 203e213.
Galindo-Barboza, A.J., Torres-Acosta, J.F.J., Camara-Sarmiento, R., Sandoval-Castro, C.A.,
Aguilar-Caballero, A.J., Ojeda-Robertos, N.F., Reyes-Ramírez, R., Espa~ na-Espa~
na, E.,
2011. Persistence of the efficacy of copper oxide wire particles against Haemonchus contor-
tus in sheep. Vet. Parasitol. 176, 201e207.
Gamble, H.R., Zajac, A.M., 1992. Resistance of St. Croix lambs to Haemonchus contortus in
experimentally and naturally acquired infections. Vet. Parasitol. 41, 211e225.
Gasser, R.B., Chilton, N.B., Hoste, H., Beveridge, I., 1993. Rapid sequencing of rDNA
from single worms and eggs of parasitic helminths. Nucleic Acids Res. 21, 2525e2526.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 229

Gasser, R.B., Bott, N.J., Chilton, N.B., Hunt, P., Beveridge, I., 2008. Toward practical,
DNA-based diagnostic methods for parasitic nematodes of livestock e bionomic and
biotechnological implications. Biotechnol. Adv. 26, 325e334.
Gasser, R.B., Schwarz, E.M., Korhonen, P.K., Young, N.D., 2016. Understanding Haemon-
chus contortus better through genomics and transcriptomics. In: Gasser, R., Samson-
Himmelstjerna, G.V. (Eds.), Haemonchus contortus and Haemonchosis Past, Present and
Future Trends. vol. 93, pp. 519e568.
Gasser, R.B., 2006. Molecular tools e advances, opportunities and prospects. Vet. Parasitol.
136, 69e89.
Geary, T.G., Hosking, B.C., Skuce, P.J., von Samson-Himmelstjerna, G., Maeder, S.,
Holdsworth, P., Pomroy, W., Vercruysse, J., 2012. World Association for the Advance-
ment of Veterinary Parasitology (W.A.A.V.P.) guideline: anthelmintic combination prod-
ucts targeting nematode infections of ruminants and horses. Vet. Parasitol. 190, 306e316.
Gibbs, H.C., 1986. Hypobiosis in parasitic nematodes e an update. Adv. Parasitol. 25,
129e174.
Gillespie, R.-A.M., Williamson, L.H., Terrill, T.H., Kaplan, R.M., 2010. Efficacy of anthel-
mintics on South American camelid (llama and alpaca) farms in Georgia. Vet. Parasitol.
172, 168e171.
Githiori, J.B., Athanasiadou, S., Thamsborg, S.M., 2006. Use of plants in novel approaches
for control of gastrointestinal helminths in livestock with emphasis on small ruminants.
Vet. Parasitol. 139, 308e320.
Gonzalez-Canga, A., Belmar-Liberato, R., Escribano, M., 2012. Extra-label use of iver-
mectin in some minor ruminant species: pharmacokinetic aspects. Curr. Pharm. Bio-
technol. 13, 924e935.
Gordon, H.McL., 1948. The epidemiology of parasite diseases, with special reference to
studies with nematode parasites of sheep. Aust. Vet. J. 24, 17e45.
Gordon, H.McL., 1961. Thiabendazole: a highly effective anthelmintic for sheep. Nature
191, 1409e1410.
Greer, A.W., Kenyon, F., Bartley, D.J., Jackson, E.B., Gordon, Y., Donnan, A.A.,
McBean, D.W., Jackson, F., 2009. Development and field evaluation of a decision sup-
port model for anthelmintic treatments as part of a targeted selective treatment (TST)
regime in lambs. Vet. Parasitol. 164, 12e20.
Grosz, D.D., Eljaki, A.A., Holler, L.D., Petersen, D.J., Holler, S.W., Hildreth, M.B., 2013.
Overwintering strategies of a population of anthelmintic-resistant Haemonchus contortus
within a sheep flock from the United States Northern Great Plains. Vet. Parasitol.
196, 143e152.
Gruner, L., Cabaret, J., Sauve, C., Pailhories, R., 1986. Comparative susceptibility of roma-
nov and lacaune sheep to gastrointestinal nematodes and small lungworms. Vet. Parasitol.
19, 85e93.
Hall, C.A., Kelly, J.D., Whitlock, H.V., Ritchie, L., 1981. Prolonged anthelmintic effect of
closantel and disophenol against a thiabendazole selected resistant strain of Haemonchus
contortus in sheep. Res. Vet. Sci. 31, 104e106.
Harmon, A.F., Williams, Z.B., Zarlenga, D.S., Hildreth, M.B., 2007. Real-time PCR for
quantifying Haemonchus contortus eggs and potential limiting factors. Parasitol. Res.
101, 71e76.
Heckendorn, F., H€aring, D.A., Maurer, V., Zinsstag, J., Langhans, W., Hertzberg, H., 2006.
Effect of sainfoin (Onobrychis viciifolia) silage and hay on established populations of Hae-
monchus contortus and Cooperia curticei in lambs. Vet. Parasitol. 142, 293e300.
Hillrichs, K., Schnieder, T., Forbes, A.B., Simcock, D.C., Pedley, K.C., Simpson, H.V., 2012.
Use of fluorescent lectin binding to distinguish Teladorsagia circumcincta and Haemonchus con-
tortus eggs, third-stage larvae and adult worms. Parasitol. Res. 110, 449e458.
H€
oglund, J., Engstr€ om, A., von Samson-Himmelstjerna, G., Demeler, J., Tydén, E., 2013.
Real-time PCR detection for quantification of infection levels with Ostertagia ostertagi
and Cooperia oncophora in cattle faeces. Vet. Parasitol. 197, 251e257.
230 R.B. Besier et al.

Hosking, B.C., Kaminsky, R., Sager, H., Rolfe, P.F., Seewald, W., 2010. A pooled analysis
of the efficacy of monepantel, an amino-acetonitrile derivative against gastrointestinal
nematodes of sheep. Parasitol. Res. 106, 529e532.
Hoste, H., Jackson, F., Athanasiadou, S., Stig, M., Thamsborg, S.M., Hoskin, S.O., 2006.
The effects of tannin-rich plants on parasitic nematodes in ruminants. Trends Parasitol.
22, 253e261.
Hoste, H., Torres-Acosta, J.F.J., Quijada, J., Chan-Perez, I., Dakheel, M.M.,
Kommuru, D.S., Harvey, I.M., Terrill, T.H., 2016. Interactions between nutrition
and infections with Haemonchus contortus and related gastrointestinal nematodes in small
ruminants. In: Gasser, R., Samson-Himmelstjerna, G.V. (Eds.), Haemonchus contortus
and Haemonchosis Past, Present and Future Trends. vol. 93, pp. 239e352.
Hoste, H., Martínez-Ortiz-De-Montellano, C., Manolaraki, F., Brunet, S., Ojeda-
Robertos, N., Fourquaux, I., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., 2012. Direct
and indirect effects of bioactive tannin-rich tropical and temperate legumes against nem-
atode infections. Vet. Parasitol. 186, 18e27.
Houdijk, J.G.M., Kyriazakis, I., Kidanea, A., Athanasiadou, S., 2012. Manipulating small
ruminant parasite epidemiology through the combination of nutritional strategies. Vet.
Parasitol. 186, 38e50.
Houdijk, J.G.M., 2008. Influence of periparturient nutritional demand on resistance to par-
asites in livestock. Parasite Immunol. 30, 113e121.
Hughes, P.L., Dowling, A.F., Callinan, A.P.L., 2007. Resistance to macrocyclic lactone an-
thelmintics and associated risk factors on sheep farms in the lower North Island of New
Zealand. N. Z. Vet. J. 55, 177e183.
Ikeme, M.M., Iskander, F., Chong, L.C., 1987. Seasonal changes in the prevalence of Hae-
monchus contortus and Trichostrongylus colubriformis hypobiotic larvae in tracer goats in
Malaysia. Trop. Anim. Health Prod. 19, 184e190.
Irum, S., Ahmed, H., Mukhtar, M., Mushtaq, M., Mirza, B., Donskow- qysoniewska, K.,
Qayyum, M., Simsek, S., 2015. Anthelmintic activity of Artemisia vestita Wall ex DC. and
Artemisia maritima L. against Haemonchus contortus from sheep. Vet. Parasitol. 212, 451e455.
Jabbar, A., Campbell, A.J.D., Charles, J.A., Gasser, R.B., 2013. First report of anthelmintic
resistance in Haemonchus contortus in alpacas in Australia. Parasites Vectors 6, 243.
Jackson, F., Miller, J., 2006. Alternative approaches to control e Quo vadit? Vet. Prasitol.
139, 371e384.
Jackson, F., Waller, P.J., 2008. Managing refugia. Trop. Biomed. 25 (Suppl.), 34e40. Proc.
5th Int. workshop; novel approaches to the control of helminth parasites of livestock.
2008, Ipoh, Malaysia.
Jackson, F., Varady, M., Bartley, D.J., 2012. Managing anthelmintic resistance in goats e can
we learn lessons from sheep? Small Rumin. Res. 103, 3e9.
Jurasek, M.E., Bishop-Stewart, J.K., Storey, B.E., Kaplan, R.M., Kent, M.L., 2010.
Modification and further evaluation of a fluorescein-labelled peanut agglutinin test for
identification of Haemonchus contortus eggs. Vet. Parasitol. 169, 209e213.
Kahn, L.P., Woodgate, R.G., 2012. Integrated parasite management: products for adoption
by the Australian sheep industry. Vet. Parasitol. 186, 58e64.
Kahn, L.P., Knox, M., Gray, G., 2003. Enhancing immunity to nematode parasites in sin-
gle-bearing Merino ewes through nutrition and genetic selection. Vet. Parasitol. 112,
211e225.
Kaminsky, R., Gauvry, N., Schorderet Weber, S., Skripsky, T., Bouvier, J., Wenger, A.,
Schroeder, F., Desaules, Y., Hotz, R., Goebel, T., Hosking, B.C., Pautrat, F.,
Wieland-Berghausen, S., Ducray, P., 2008. Identification of the amino-acetonitrile de-
rivative monepantel (AAD 1566) as a new anthelmintic drug development candidate.
Parasitol. Res. 103, 931e939.
Kaplan, R.M., Vidyashankar, A.N., 2012. An inconvenient truth: global worming and
anthelmintic resistance. Vet. Parasitol. 186, 70e78.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 231

Kathiravan, P., Pichler, R., Poli, M., Cristel, S., Cetra, B., Medus, D., Basar, M.,
Thiruvenkadan, A.K., Ramasamy, S., Babbar Ellahi, M.B., Mohammed, F.,
Teneva, A., Shamsuddin, M., Garcia Podesta, M.G., Diallo, A., 2014. Candidate gene
approach for parasite resistance in sheep e variation in immune pathway genes and as-
sociation with fecal egg count. PLoS One 9, e88337.
Kelly, P., Good, B., Hanrahan, J.P., Fitzpatrick, R., de Waal, T., 2009. Screening for the
presence of nematophagous fungi collected from Irish sheep pastures. Vet. Parasitol.
165, 345e349.
Kelly, G.A., Kahn, L.P., Walkden-Brown, S.W., 2013. Measurement of phenotypic resil-
ience to gastro-intestinal nematodes in Merino sheep and association with resistance
and production variables. Vet. Parasitol. 193, 111e117.
Kemper, K.E., Elwin, R.L., Bishop, S.C., Goddard, M.E., Woolaston, R., 2009. Haemonchus con-
tortus and Trichostrongylus colubriformis did not adapt to long-term exposure to sheep that were
genetically resistant or susceptible to nematode infections. Int. J. Parasitol. 39, 607e614.
Kenyon, F., Jackson, F., 2012. Targeted flock/herd and individual ruminant treatment
approaches. Vet. Parasitol. 186, 10e17.
Kenyon, F., Greer, A.W., Coles, G.C., Cringoli, G., Papadopoulos, E., Cabaret, J.,
Berrag, B., Varady, M., Van Wyk, J.A., Thomas, E., Vercruysse, J., Jackson, F., 2009.
The role of targeted selective treatments in the development of refugia-based approaches
to the control of gastrointestinal nematodes of small ruminants. Vet. Parasitol. 164, 3e11.
Ketzis, J.K., Vercruysse, J., Stromberg, B.E., Larsen, M., Athanasiadou, S., Houdijk, J.G.M.,
2006. Evaluation of efficacy expectations for novel and non-chemical helminth control
strategies in ruminants. Vet. Parasitol. 139, 321e335.
Knox, M.R., 2002. Effectiveness of copper oxide wire particles for Haemonchus contortus con-
trol in sheep. Aust. Vet. J. 80, 224e227.
Knox, D., 2013. A vaccine against Haemonchus contortus: current status and future possibilities.
In: Kennedy, M.W., Harnett, W. (Eds.), Parasitic Nematodes: Molecular Biology,
Biochemistry and Immunology. CAB E-books, pp. 245e260. Agriculture and the
applied life sciences. (Chapter 13).
Kotze, A.C., Prichard, R.K., 2016. Anthelmintic resistance in Haemonchus contortus: history,
mechanisms and diagnosis. In: Gasser, R., Samson-Himmelstjerna, G.V. (Eds.), Haemon-
chus contortus and Haemonchosis Past, Present and Future Trends. vol. 93, pp. 397e428.
Krawczyk, A., Slota, E., 2009. Genetic markers to gastrointestinal nematode resistance in
sheep: a review. Helminthologia 46, 3e8.
Krecek, R.C., Waller, P.J., 2006. Towards the implementation of the “basket of best op-
tions” approach to helminth parasite control of livestock: emphasis on the tropics/
subtropics. Vet. Parasitol. 139, 270e282.
Lacey, E., 1988. The role of the cytoskeletal protein tubulin in the mode of action and mech-
anism of drug resistance to benzimidazoles. Int. J. Parasitol. 18, 855e936.
Laing, R., Martinelli, A., Tracey, A., Holroyd, N., Gilleard, J., Cotton, J.A., 2016. Haemon-
chus contortus: genome structure, organization and comparative genomics. In: Gasser, R.,
Samson-Himmelstjerna, G.V. (Eds.), Haemonchus contortus and Haemonchosis Past, Pre-
sent and Future Trends. vol. 93, pp. 569e598.
Lawrence, K.E., Rhodes, A.P., Jackson, R., Leathwick, D.M., Heuer, C., Pomroy, W.E.,
West, D.M., Waghorn, T.S., Moffat, J.R., 2006. Farm management practices associated
with macrocyclic lactone resistance on sheep farms in New Zealand. N. Z. Vet. J. 54,
283e288.
Lawrence, K.E., Leathwick, D.M., Rhodes, A.P., Jackson, R., Heuer, C., Pomroy, W.E.,
West, D.M., Waghorn, T.S., Moffat, J.R., 2007. Management of gastrointestinal nem-
atode parasites on sheep farms in New Zealand. N. Z. Vet. J. 55, 228e234.
Le Jambre, L.F., Dobson, R.J., Lenane, I.J., Barnes, E.H., 1999. Selection for anthel-
mintic resistance by macrocyclic lactones in Haemonchus contortus. Int. J. Parasitol.
29, 1101e1111.
232 R.B. Besier et al.

Le Jambre, L.F., Dominik, S., Eady, S.J., Henshall, J.M., Colditz, I.G., 2007. Adjusting worm
egg counts for faecal moisture. Vet. Parasitol. 145, 108e115.
Le Jambre, L.F., Windon, R.G., Smith, W.D., 2008. Vaccination against Haemonchus contor-
tus: performance of native parasite gut membrane glycoproteins in Merino lambs grazing
contaminated pasture. Vet. Parasitol. 153, 302e312.
Le Jambre, L.F., Martin, P.J., Johnston, A., 2010. Efficacy of combination anthelmintics
against multiple resistant strains of sheep nematodes. Prod. Sci. 50, 946e952.
Le Jambre, L.F., 1995. Relationship of blood loss to worm numbers, biomass and egg pro-
duction in Haemonchus infected sheep. Int. J. Parasitol. 25, 269e273.
Le Jambre, L.F., 2006. Eradication of targeted species of internal parasites. Vet. Parasitol. 139,
360e370.
Learmount, J., Conyers, C., Hird, H., Morgan, C., Craig, B.H., von Samson-
Himmelstjerna, G., Taylor, M., 2009. Development and validation of real-time PCR
methods for diagnosis of Teladorsagia circumcincta and Haemonchus contortus in sheep.
Vet. Parasitol. 166, 268e274.
Leathwick, D.M., Besier, R.B., 2014. The management of anthelmintic resistance in grazing
ruminants in Australasia e strategies and experiences. Vet. Parasitol. 204, 44e54.
Leathwick, D.M., Moen, I.C., Miller, C.M., Sutherland, I.A., 2000. Ivermectin-resistant
Ostertagia circumcincta from sheep in the lower North Island and their susceptibility to
other macrocyclic lactone anthelmintics. N.Z. Vet. J. 48, 151e154.
Leathwick, D.M., Miller, C.M., Atkinson, D.S., Haack, N.A., Alexander, R.A.,
Oliver, A.-M., Waghorn, T.S., Potter, J.F., Sutherland, I.A., 2006. Drenching adult
ewes: implications of anthelmintic treatments pre- and post-lambing on the develop-
ment of anthelmintic resistance. N.Z. Vet. J. 54, 297e304.
Leathwick, D.M., Hosking, B.C., Bisset, S.A., McKay, C.H., 2009. Managing anthelmintic
resistance: is it feasible in New Zealand to delay the emergence of resistance to a new
anthelmintic class? N.Z. Vet. J. 57, 181e192.
Leathwick, D.M., Waghorn, T.S., Miller, C.M., Candy, P.M., Oliver, A.-M.B., 2012. Man-
aging anthelmintic resistance e use of a combination anthelmintic and leaving some
lambs untreated to slow the development of resistance to ivermectin. Vet. Parasitol.
187, 285e294.
Levine, N.D., 1980. Nematode Parasites of Domestic Animals and of Man, second ed.
Burgess Publishing Company, Minneapolis.
Little, P.R., Hodge, A., Watson, T.G., Seed, J.A., Maeder, S.J., 2010. Field efficacy and
safety of an oral formulation of the novel combination anthelmintic, derquantel-abamec-
tin, in sheep in New Zealand. N.Z. Vet. J. 58, 121e129.
Little, P.R., Hodge, A., Maeder, S.J., Wirtherle, N.C., Nicholas, D.R., Cox, G.C.,
Conder, G.A., 2011. Efficacy of a combined oral formulation of derquantel-abamectin
against the adult and larval stages of nematodes in sheep, including anthelmintic-resistant
strains. Vet. Parasitol. 181, 180e193.
Lloberas, M., Alvarez, L., Entrocasso, C., Virkel, G., Ballent, M., Mate, M., Lanusse, C.,
Lifschitz, A., 2013. Comparative tissue pharmacokinetics and efficacy of moxidectin, aba-
mectin and ivermectin in lambs infected with resistant nematodes: impact of drug treat-
ments on parasite P-glycoprotein expression. Int. J. Parasitol. Drugs Drug Resist. 3, 20e27.
Macarthur, F.A., Kahn, L.P., Windon, R.G., 2013. Immune response of twin-bearing
Merino ewes when infected with Haemonchus contortus: effects of fat score and prepartum
supplementation. Livest. Sci. 157 (2e3), 568e576.
Mackintosh, C.G., Mason, P.C., Manley, T., Baker, K., Littlejohn, R., 1985. Efficacy and
pharmacokinetics of febantel and ivermectin in red deer (Cervus elaphus). N. Z. Vet. J.
33, 127e131.
Mahieu, M., Aumont, G., 2009. Effects of sheep and cattle alternate grazing on sheep para-
sitism and production. Trop. Anim. Health Prod. 41, 229e239.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 233

Mahieu, M., Arquet, R., Kandassamy, T., Mandonnet, N., Hoste, H., 2007. Evaluation of
targeted drenching using Famacha method in Creole goat: reduction of anthelmintic use,
and effects on kid production and pasture contamination. Vet. Parasitol. 146, 135e147.
Maia, D., Rosalinski-Moraes, F., Van Wyk, J.A., Weber, S., Sotomaior, C.S., 2014. Assessment
of a hands-on method for FAMACHA© system training. Vet. Parasitol. 200, 165e171.
Maingi, N., Krecek, R.C., Van Biljon, N., 2006. Control of gastrointestinal nematodes in
goats on pastures in South Africa using nematophagous fungi Duddingtonia flagrans and
selective anthelmintic treatments. Vet. Parasitol. 138, 328e336.
Malan, F.S., Van Wyk, J.A., Wessels, C.D., 2001. Clinical evaluation of anaemia in sheep:
early trials. Onderstepoort J. Vet. Res. 68, 165e174.
Mariadass, B., McArthur, M., McKenna, P.B., Wrigley, J., 2006. Resistance to a triple
combination of broad-spectrum anthelmintics in naturally-acquired Ostertagia circumcincta
infections in sheep. N.Z. Vet. J. 4, 47e49.
Marshall, K., Mugambi, J.M., Nagda, S., Sonstegard, T.S., Tassell, C.P., Baker, R.L.,
Gibson, J.P., 2013. Quantitative trait loci for resistance to Haemonchus contortus artificial
challenge in Red Maasai and Dorper sheep of East Africa. Anim. Genet. 44, 285e295.
Martin, R., Pennington, A.J., 1988. Effect of dihydroavermectin B1a on chloride-single-
channel currents in Ascaris muscle. Pestic. Sci. 24, 90e91.
Martin, R.J., 1997. Modes of action of anthelmintic drugs. Vet. J. 154, 11e34.
McCoy, M.A., Edgar, H.W.J., Kenny, J., Gordon, W., Dawson, L.E.R., Carson, A.F., 2005.
Evaluation of on-farm faecal worm egg counting in sheep. Vet. Rec. 156, 21e23.
McKellar, Q.A., Jackson, F., 2004. Veterinary anthelmintics: old and new. Trends Parasitol.
20, 461e465.
McKenna, P.B., 1998. The effect of previous cold storage on the subsequent recovery of
infective third stage nematode larvae from sheep faeces. Vet. Parasitol. 80, 167e172.
McKenna, P.B., 2010. Update on the prevalence of anthelmintic resistance in gastrointestinal
nematodes of sheep in New Zealand. N.Z. Vet. J. 58, 172e173.
McNally, J., Callan, D., Andronicos, N., Bott, N., Hunt, P.W., 2013. DNA-based method-
ology for the quantification of gastrointestinal nematode eggs in sheep faeces. Vet. Para-
sitol. 198, 325e335.
McRae, K.M., McEwan, J.C., Dodds, K.G., Gemmell, N.J., 2014. Signatures of selection in
sheep bred for resistance or susceptibility to gastrointestinal nematodes. BMC Genomics
15, 637e649.
Mederos, A., Fernandez, S., Van Leeuwen, J., Peregrine, A.S., Kelton, D., Menzies, P.,
LeBoeuf, A., Martin, R., 2010. Prevalence and distribution of gastrointestinal nematodes
on 32 organic and conventional commercial sheep farms in Ontario and Quebec, Canada
(2006e2008). Vet. Parasitol. 170, 244e252.
Mederos, A.E., Ramos, Z., Banchero, G.E., 2014. First report of monepantel Haemonchus
contortus resistance on sheep farms in Uruguay. Parasites Vectors 7, 598.
Melville, L., Kenyon, F., Javed, S., McElarney, I., Demeler, J., Skuce, P., 2014. Development
of a loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of
Haemonchus contortus eggs in ovine faecal samples. Vet. Parasitol. 206, 308e312.
Min, B.R., Pomroy, W.E., Hart, S.P., Sahlu, T., 2004. The effect of short term consumption
of forage containing condensed tannins on gastrointestinal nematode parasite infections
in grazing wether goats. Small Rumin. Res. 51, 279e283.
Moors, E., Gauly, M., 2009. Is the FAMACHA© chart suitable for every breed? Correlations
between FAMACHA© scores and different traits of mucosa colour in naturally parasite
infected sheep breeds. Vet. Parasitol. 166, 108e111.
Moreno, F.C., Gordon, I.J., Knox, M.R., Summer, P.M., Skerrat, L.F., Benvenutti, M.A.,
Saumell, C.A., 2012. Anthelmintic efficacy of five tropical native Australian plants
against Haemonchus contortus and Trichostrongylus colubriformis in experimentally infected
goats (Capra hircus). Vet. Parasitol. 187, 237e243.
234 R.B. Besier et al.

Morgan, E.R., Van Dijk, J., 2012. Climate and the epidemiology of gastrointestinal nema-
tode infections of sheep in Europe. Vet. Parasitol. 189, 8e14.
Morgan, E.R., Cavill, L., Curry, G.E., Wood, R.M., Mitchell, E.S.E., 2005. Effects of
aggregation and sample size on composite faecal egg counts in sheep. Vet. Parasitol.
131, 79e87.
Morgan, E.R., Hosking, B.C., Burston, S., Carder, K.M., Hyslop, A.C., Pritchard, L.J.,
Whitmarsh, A.K., Coles, G.C., 2012. A survey of helminth control practices on sheep
farms in Great Britain and Ireland. Vet. J. 192, 390e397.
Morley, F.H.W., Donald, A.D., 1980. Farm management and systems of helminth control.
Vet. Parasitol. 6, 105e134.
Munn, E.A., Smith, T.S., Graham, M., Tavernor, A.S., Greenwood, C.A., 1993. The poten-
tial value of integral membrane proteins in the vaccination of lambs against Haemonchus
contortus. Int. J. Parasitol. 23, 261e269.
Mylrea, G.E., Mulley, R.C., English, A.W., 1991. Gastrointestinal helminthosis in fallow
deer (Dama dama) and their response to treatment with anthelmintics. Aust. Vet. J. 68,
74e75.
Newton, S.E., Meeusen, E.N., 2003. Progress and new technologies for developing vaccines
against gastrointestinal nematode parasites of sheep. Parasite Immunol. 25, 283e296.
Nicholls, J., Obendorf, D.L., 1994. Application of a composite faecal egg count procedure in
diagnostic parasitology. Vet. Parasitol. 52, 337e342.
Nieuwoudt, S.W., Theron, H.E., Kruger, L.P., 2012. Genetic parameters for resistance to
Haemonchus contortus in Merino sheep in South Africa. J. S. Afr. Vet. Assoc. 73, 4e7.
Niezen, J.H., Robertson, H.A., Waghorn, G.C., Charleston, W.A.G., 1998. Production,
faecal egg counts and worm burdens of ewe lambs which grazed six contrasting
forages. Vet. Parasitol. 80, 15e27.
Nisbet, A.J., Meeusen, E.N., Gonzalez, J.F., Piedrafita, D.M., 2016. Immunity of Haemonchus
contortus and vaccine development. In: Gasser, R., Samson-Himmelstjerna, G.V. (Eds.),
Haemonchus contortus and Haemonchosis Past, Present and Future Trends. vol. 93,
pp. 353e396.
Nnadi, P.A., Kamalu, T.N., Onah, D.N., 2009. The effect of dietary protein on the
productivity of West African Dwarf (WAD) goats infected with Haemonchus contortus.
Vet. Parasitol. 161, 232e238.
O’Connor, L.J., Walkden-Brown, S.W., Kahn, L.P., 2006. Ecology of the free-living stages
of major trichostrongylid parasites of sheep. Vet. Parasitol. 42, 1e15.
Palmer, D.G., McCombe, I.L., 1996. Lectin staining of trichostrongylid nematode eggs of
sheep: rapid identification of Haemonchus contortus eggs with peanut lectin. Int. J. Parasitol.
26, 447e450.
Paolini, V., Bergeaud, J.P., Grisez, C., Prevot, F., Dorchies, P., Hoste, H., 2003. Effects of
condensed tannins on goats experimentally infected with Haemonchus contortus. Vet.
Parasitol. 113, 253e261.
Playford, M.C., Smith, A.N., Love, S.C.J., Besier, R.B., Kluver, P., Bailey, J.N., 2014.
Prevalence and severity of anthelmintic resistance in ovine nematodes in Australia
(2009-2012). Aust. Vet. J. 92, 464e471.
Preston, J.M., Allonby, E.W., 1979. The influence of breed on the susceptibility of sheep to
Haemonchus contortus infection in Kenya. Res. Vet. Sci. 26, 134e139.
Prichard, R.K., Hall, C.A., Kelly, J.D., Martin, I.C.A., Donald, A.D., 1980. The problem of
anthelmintic resistance in nematodes. Aust. Vet. J. 56, 239e251.
Prichard, R., Ménez, C., Lespine, A., 2012. Moxidectin and the avermectins: consanguinity
but not identity. Int. J. Parasitol. Drugs Drug Resist. 2, 134e153.
Reynecke, D.P., Van Wyk, J.A., Gummow, B., Dorny, P., Boomker, J., 2011a. Validation
of the FAMACHA eye colour chart using sensitivity/specificity analysis on two South
African sheep farms. Vet. Parasitol. 177, 203e211.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 235

Reynecke, D.P., Van Wyk, J.A., Gummow, B., Dorny, P., Boomker, J., 2011b. A stochastic
model accommodating the FAMACHA© system for estimating worm burdens and asso-
ciated risk factors in sheep naturally infected with Haemonchus contortus. Vet. Parasitol.
177, 231e241.
Riley, D.G., Van Wyk, J.A., 2009. Genetic parameters for FAMACHA© score and related
traits for host resistance/resilience and production at differing severities of worm chal-
lenge in a Merino flock in South Africa. Vet. Parasitol. 164, 44e52.
Riley, D.G., Van Wyk, J.A., 2011. The effect of penalization of FAMACHA© scores of
lambs treated for internal parasites on the estimation of genetic parameters and prediction
of breeding values. Small Rumin. Res. 99, 122e129.
Rinaldi, L., Veneziano, V., Morgoglione, M.E., Pennacchio, S., Santaniello, M.,
Schioppi, M., Musella, V., Fedele, V., Cringoli, G., 2009. Is gastrointestinal strongyle
faecal egg count influenced by hour of sample collection and worm burden in goats?
Vet. Parasitol. 163, 81e86.
Rinaldi, L., Coles, G.C., Maurelli, M.P., Musella, V., Cringoli, G., 2011. Calibration and
diagnostic accuracy of simple flotation, McMaster and FLOTAC for parasite egg counts
in sheep. Vet. Parasitol. 177, 345e352.
Roberts, J.L., Swan, R.A., 1981. Quantitive studies of ovine haemonchosis. 1. Relationship
between faecal egg counts and total worm counts. Vet. Parasitol. 8, 165e171.
Roberts, J.L., Swan, R.A., 1982a. Quantitative studies of ovine haemonchosis 3. The inter-
pretation and diagnostic significance of the changes in serial egg counts of Haemonchus
contortus in a sheep flock. Vet. Parasitol. 9, 211e216.
Roberts, J.L., Swan, R.A., 1982b. Quantitative studies of ovine haemonchosis. 2. Relation-
ship between total worm counts of Haemonchus contortus, haemoglobin values and
bodyweight. Vet. Parasitol. 9, 217e222.
Robertson, S.J., Martin, R.J., 1993. Levamisole-activated single channel currents from mus-
cle of the nematode parasite Ascaris suum. Br. J. Pharmacol. 108, 170e178.
Roeber, F., Kahn, L., 2014. The specific diagnosis of gastrointestinal nematode infections in
livestock: larval culture technique, its limitations and alternative DNA-based approaches.
Vet. Parasitol. 205, 619e628.
Roeber, F., Jex, A.R., Campbell, A.J.D., Nielsen, R., Anderson, G.A., Stanley, K.K.,
Gasser, R.B., 2012. Establishment of a robotic, high-throughput platform for the specific
diagnosis of gastrointestinal nematode infections in sheep. Int. J. Parasitol. 42, 1151e1158.
Roeber, F., Jex, A.R., Gasser, R.B., 2013. Next-generation molecular-diagnostic tools for
gastrointestinal nematodes of livestock, with an emphasis on small ruminants: a turning
point? Adv. Parasitol. 31, 1135e1152.
Roeber, F., Jex, A.R., Gasser, R.B., 2015. A real-time PCR for the diagnosis of gastrointes-
tinal nematode infections of small ruminants. Methods Mol. Biol. 1247, 145e152.
Rolfe, P.F., 1990. Resistance of Haemonchus contortus to broad and narrow spectrum drugs. In:
Boray, J.C., Martin, P.J., Rousch, R.T. (Eds.), Resistance to Parasites to Anti-parasitic
Drugs. MSD Agvet, Rahway, New Jersey USA, pp. 115e122.
Rossanigo, C.E., Gruner, L., 1996. The length of strongylid nematode infective larvae as a
reflection of developmental conditions in faeces and consequence on their viability. Para-
sitol. Res. 82, 304e311.
Sager, H., Bapst, B., Strehlau, G.A., Kaminsky, R., 2012. Efficacy of monepantel, derquantel
and abamectin against adult stages of a multi-resistant Haemonchus contortus isolate. Para-
sitol. Res. 111, 2205e2207.
Sargison, N.D., Wilson, D.J., Bartley, D.J., Penny, C.D., Jackson, F., 2007. Haemonchosis
and teladorsagiosis in a Scottish sheep flock putatively associated with the overwintering
of hypobiotic fourth stage larvae. Vet. Parasitol. 147, 326e331.
Sargison, N.D., 2012. Pharmaceutical treatments of gastrointestinal nematode infections of
sheep - future of anthelmintic drugs. Vet. Parasitol. 189, 79e84.
236 R.B. Besier et al.

Shaik, S.A., Terrill, T.H., Miller, J.E., Kouakou, B., Kannan, G., Kaplan, R.M., Burke, J.M.,
Mosjidis, J.A., 2006. Sericea lespedeza hay as a natural deworming agent against gastroin-
testinal nematode infection in goats. Vet. Parasitol. 139, 150e157.
Shakya, K.P., Miller, J.E., Lomax, L.G., Burnett, D.D., 2011. Evaluation of immune
response to artificial infections of Haemonchus contortus in Gulf Coast Native compared
with Suffolk lambs. Vet. Parasitol. 181, 239e247.
Shaw, R.J., Morris, C.A., Wheeler, M., Tate, M., Sutherland, I.A., 2012. Salivary IgA: a suit-
able measure of immunity to gastrointestinal nematodes in sheep. Vet. Parasitol. 186,
109e117.
Smith, G., Grenfell, B.T., Isham, V., Cornell, S., 1999. Anthelmintic resistance revisited:
under-dosing, chemoprophylactic strategies, and mating probabilities. Int. J. Parasitol.
29, 77e91.
Smith, W.D., Van Wyk, J.A., Van Striip, M.F., 2001. Preliminary observations on the
potential of gut membrane proteins of Haemonchus contortus as candidate vaccine antigens
in sheep on naturally infected pasture. Vet. Parasitol. 98, 285e297.
Smith, W.D., 1993. Protection in lambs immunised with Haemonchus contortus gut membrane
proteins. Res. Vet. Sci. 54, 94e101.
Sotomaior, C.S., Rosalinski-Moraes, F., Barbosa da Costa, A.R., Maia, D.,
Monteiro, A.L.G., Van Wyk, J.A., 2012. Sensitivity and specificity of the
FAMACHA© system in Suffolk sheep and crossbred Boer goats. Vet. Parasitol. 190,
114e119.
Southcott, W.H., Barger, I.A., 1975. Control of nematode parasites by grazing management-
II. decontamination of sheep and cattle pastures by varying periods of grazing with the
alternate host. Int. J. Parasitol. 5, 45e48.
Spickett, A., de Villiers, J.F., Boomker, J., Githiori, J.B., Medley, G.F., Stenson, M.O.,
Waller, P.J., Calitz, F.J., Vatta, A.F., 2012. Tactical treatment with copper oxide wire
particles and symptomatic levamisole treatment using the FAMACHA© system in indig-
enous goats in South Africa. Vet. Parasitol. 184, 48e58.
Steel, J.W., 2003. Effects of protein supplementation on young sheep on resistance develop-
ment and resilience to parasitic nematodes. Aust. J. Exp. Agric. 12, 1469e1476.
Suter, R.J., Besier, R.B., Perkins, N.R., Robertson, I.D., Chapman, H.M., 2004. Sheep-
farm risk factors for ivermectin resistance in Ostertagia circumcincta in Western Australia.
Prev. Vet. Med. 63, 257e269.
Taylor, M.A., Coop, R.L., Wall, R.L., 2007. Vet. Parasitol., third ed. Blackwell Publishing,
pp. 159e161.
Terrill, T.H., Miller, J.E., Burke, J.M., Mosjidis, J.A., Kaplan, R.M., 2012. Experiences with
integrated concepts for the control of Haemonchus contortus in sheep and goats in the
United States. Vet. Parasitol. 186, 28e37.
Thomas, R.J., Ali, D.A., 1983. The effect of Haemonchus contortus infection on the pregnant
and lactating ewe. Int. J. Parasitol. 13, 393e398.
Torgerson, M., Schnyder, H., Hertzberg, H., 2005. Detection of anthelmintic resistance: a
comparison of mathematical techniques P.R. Vet. Parasitol. 128, 291e298.
Torgerson, P.R., Paul, M., Lewis, F.I., 2012. The contribution of simple random sampling to
observed variations in faecal egg counts. Vet. Parasitol. 188, 397e401.
Torres-Acosta, J.F.J., Hoste, H., 2008. Alternative or improved methods to limit gastro-in-
testinal parasitism in grazing sheep and goats. Small Rumin. Res. 77, 159e173.
Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., Aguilar-Caballero, A.J., Camara-
Sarmiento, R., Alonso-Díaz, M.A., 2012. Nutritional manipulation of sheep and goats
for the control of gastrointestinal nematodes under hot humid and subhumid tropical
conditions. Small Rumin. Res. 103, 28e40.
Urquhart, G.M., Armour, J., Duncan, J.L., Dunn, A.M., Jennings, F.M., 1996. Vet. Parasi-
tol., second ed. Blackwell Science, pp. 19e21.
Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants 237

Vadlejch, J., Petrtýl, M., Zaichenko, I., Cadkov  a, Z., Jankovska, I., Langrova, I.,
Moravec, M., 2011. Which McMaster egg counting technique is the most reliable? Para-
sitol. Res. 109, 1387e1394.
Van Burgel, A.J., Lyon, J., Besier, R.B., Palmer, D.G., 2014. Proficiency testing assess-
ments for nematode worm egg counting based on Poisson variation. Vet. Parasitol.
205, 385e388.
Van den Brom, R., Moll, L., Kappert, C., Vellema, P., 2015. Haemonchus contortus resistance
to monepantel in sheep. Vet. Parasitol. 209, 278e280.
Van Wyk, J.A., Bath, G.F., 2002. The FAMACHA© system for managing haemonchosis in
sheep and goats by clinically identifying individual animals for treatment. Vet. Res. 33,
509e529.
Van Wyk, J.A., Mayhew, E., 2013. Morphological identification of parasitic nematode infec-
tive larvae of small ruminants and cattle: a practical lab guide. Onderstepoort J. Vet. Res.
80, 1e14.
Van Wyk, J.A., Reynecke, D.P., 2011. Blueprint for an automated specific decision support
system for countering anthelmintic resistance in Haemonchus spp. at farm level. Vet. Para-
sitol. 177, 212e223.
Van Wyk, J.A., Riley, D.G., 2009. Genetic parameters for FAMACHA© score and related
traits for host resistance/resilience and production at differing severities of worm chal-
lenge in a Merino flock in South Africa. Vet. Parasitol. 64, 44e52.
Van Wyk, J.A., Gerber, H.M., Alves, R.M.R., 1982. Slight resistance to the residual effect of
closantel in a field strain of Haemonchus contortus which showed an increased resistance
after one selection in the laboratory. Onderstepoort J. Vet. Res. 49, 257e262.
Van Wyk, J.A., Malan, F.S., Randles, J.L., 1997a. How long before resistance makes it
impossible to control some field strains of Haemonchus contortus in South Africa with
any of the modern anthelmintics? Vet. Parasitol. 70, 11e122.
Van Wyk, J.A., Malan, F.S., Van Rensburg, L.J., Oberem, P.T., Allan, M.J., 1997b. Quality
control in generic anthemintics: is it adequate. Vet. Parasitol. 72, 157e165.
Van Wyk, J.A., Cabaret, J., Michael, L.M., 2004. Morphological identification of nematodes
of small ruminants and cattle simplified. Vet. Parasitol. 119, 277e306.
Van Wyk, J.A., Hoste, H., Kaplan, R.M., Besier, R.B., 2006. Targeted selective treatment
for worm managementdhow do we sell rational programs to farmers? Vet. Parasitol.
139, 336e346.
Van Wyk, J.A., 2001. Refugia e overlooked as perhaps the most important factor concerning
the development of anthelmintic resistance. Onderstepoort J. Vet. Res. 68, 55e67.
Vatta, A.F., Letty, B.A., van der Linde, M.J., Van Wijk, E.F., Hansen, J.W., Krecek, R.C.,
2001. Testing for clinical anaemia caused by Haemonchus spp. in goats farmed under
resource-poor conditions in South Africa using an eye colour chart developed for
sheep. Vet. Parasitol. 99, 1e14.
Vatta, A.F., Waller, P.J., Githiori, J.B., Medley, G.F., 2009. The potential to control Haemon-
chus contortus in indigenous South African goats with copper oxide wire particles. Vet.
Parasitol. 162, 306e313.
Vatta, A.F., Waller, P.J., Githiori, J.B., Medley, G.F., 2012. Persistence of the efficacy of
copper oxide wire particles against Haemonchus contortus in grazing South African goats.
Vet. Parasitol. 190, 159e166.
Waghorn, G., 2008. Beneficial and detrimental effects of dietary condensed tannins for sus-
tainable sheep and goat production - progress and challenges. Anim. Feed Sci. Technol.
147, 116e139.
Wallace, D.S., Bairden, K., Duncan, J.L., Fishwick, G., Gill, M., Holmes, P.H.,
McKellar, Q.A., Murray, M., Parkins, J.J., Stear, M., 1996. Influence of soyabean
meal supplementation on the resistance of Scottish Blackface lambs to haemonchosis.
Res. Vet. Sci. 60, 138e143.
238 R.B. Besier et al.

Waller, P.J., Larsen, M., 1993. The role of nematophagous fungi in the biological control of
nematode parasites of livestock. Int. J. Parasitol. 23, 539e546.
Waller, P.J., Echevarria, F., Eddi, C., Maciel, S., Nari, A., Hansen, J.W., 1996. The preva-
lence of anthelmintic resistance in nematode parasites of sheep in Southern Latin Amer-
ica: general overview. Vet. Parasitol. 62, 181e187.
Waller, P.J., Knox, M.R., Faedo, M., 2001. The potential of nematophagous fungi to control
the free-living stages of nematodes of sheep: feeding and block studies with Duddingtonia
flagrans. Vet. Parasitol. 102, 321e330.
Waller, P.J., Rudby-Martin, L., Ljungstr€ om, B.L., Rydzik, A., 2004. The epidemiology of
abomasal nematodes of sheep in Sweden, with particular reference to overwinter survival
strategies. Vet. Parasitol. 122, 207e220.
Waller, P.J., Rydzik, A., Ljungstr€ om, B.L., Tornquist, M., 2006. Towards the eradication of
Haemonchus contortus from sheep flocks in Sweden. Vet. Parasitol. 136, 367e372.
Waller, P.J., 1986. Anthelmintic resistance in Australia. Parasitol. Today 7, S16eS18.
Waller, P.J., 1997. Nematode parasite control of livestock in the tropics/subtropics: the need
for novel approaches. Int. J. Parasitol. 27, 1193e1201.
Waller, P.J., 2006. Sustainable nematode parasite control strategies for ruminant livestock by
grazing management and biological control. Anim. Feed Sci. Technol. 126, 277e289.
Whitlock, J.H., Crofton, H.D., Georgi, J.R., 1972. Characteristics of parasite populations in
endemic trichostrongylidosis. Parasitology 64, 413e417.
Whitlock, H.V., 1948. Some modifications of the McMaster helminth egg counting tech-
nique and apparatus. J. Counc. Sci. Ind. Res. 21, 177e180.
Wimmer, B., Craig, B.H., Pilkington, J.G., Pemberton, J.M., 2004. Non-invasive assessment
of parasitic nematode species diversity in wild Soay sheep using molecular markers. Int. J.
Parasitol. 34, 625e631.
Woodgate, R.G., Love, S., 2012. WormKill to WormBoss e can we sell sustainable sheep
worm control? Vet. Parasitol. 186, 51e57.
Woolaston, R.R., Baker, R.L., 1996. Prospects of breeding small ruminants for resistance to
internal parasites. Int. J. Parasitol. 26, 845e855.
Woolaston, R.R., Elwin, R.L., Barger, I.A., 1992. No adaptation of Haemonchus contortus to
genetically resistant sheep. Int. J. Parasitol. 22, 377e380.
Wooster, M.J., Woodgate, R.G., Chick, B.F., 2008. Reduced efficacy of ivermectin, aba-
mectin and moxidectin against field isolates of Haemonchus contortus. Aust. Vet. J. 79,
840e842.
Yan, R., Sun, W., Song, X., Xu, L., Li, X., 2013. Vaccination of goats with DNA vaccine
encoding Dim-1 induced partial protection against Haemonchus contortus: a preliminary
experimental study. Res. Vet. Sci. 95, 189e199.
Zarlenga, D.S., Hoberg, E.P., Tuo, W., 2016. The identification of Haemonchus species and
diagnosis of haemonchosis. In: Gasser, R., Samson-Himmelstjerna, G.V. (Eds.), Haemon-
chus contortus and Haemonchosis Past, Present and Future Trends. vol. 93, pp. 145e180.
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
University of Reading, Reading, United Kingdom
#
Fort Valley State University, Fort Valley, GA, United States
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

Advances in Parasitology, Volume 93


© 2016 Elsevier Ltd.
j
ISSN 0065-308X
http://dx.doi.org/10.1016/bs.apar.2016.02.025 All rights reserved. 239
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.
Interactions Between Nutrition and Haemonchus contortus & Related Nematodes 243

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
Interactions Between Nutrition and Haemonchus contortus & Related Nematodes 245

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 V