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Veterinary Parasitology 197 (2013) 614622

Contents lists available at ScienceDirect

Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Hierarchical model for evaluating pyrantel efcacy against

strongyle parasites in horses
M.K. Nielsen a, , A.N. Vidyashankar b , B.M. Hanlon c , G. Diao b ,
S.L. Petersen d , R.M. Kaplan e
a
b
c
d
e

M.H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA
Department of Statistics, George Mason University, Fairfax, VA, USA
Department of Statistics, University of Wisconsin, Madison, WI, USA
EquiLab Laboratory, Slangerup, Denmark
Department of Infectious Diseases, University of Georgia, Athens, GA, USA

a r t i c l e

i n f o

Article history:
Received 19 September 2012
Received in revised form 15 April 2013
Accepted 19 April 2013
Keywords:
Pyrantel
Anthelmintic resistance
Cyathostomin
Horses
Hierarchical models
Outliers

a b s t r a c t
Anthelmintic resistance is an increasing challenge for the control of equine parasites. The
fecal egg count reduction test (FECRT) is the practical gold standard method for evaluating
reduction in anthelmintic efcacy, but the interpretation is complicated due to high levels of variability. A hierarchical statistical model was described for analysis of FECRT data
from multiple farms to evaluate the role of biological factors in determining the strongyle
efcacy of pyrantel pamoate in a study performed in Denmark. The model was then used
to describe two notions of farm efcacy, namely conditional and marginal efcacy. The
median of the lower prediction limits was used to describe a robust classication rule. The
performance of the methodology was evaluated using Monte Carlo simulations. The eld
study was performed on 64 Danish horse farms of different breeds. Of 1644 horses, 614
had egg counts 200 eggs per gram (EPG) and were treated. Individual coprocultures were
performed for identication of Strongylus vulgaris from all horses pre-treatment. Thirty-one
farms (48.4%) were positive for S. vulgaris, but pyrantel efcacy was unaffected by the presence of this parasite in the statistical model. Further, there were no signicant effects of age,
gender, or interactions between these, while the pre-treatment egg count was negatively
associated with the egg count reduction. The statistical model classied 81.3%, 10.9%, and
7.8% of farms as no signs of resistance (NR), suspect resistance (SR), and resistance (RE),
respectively. In comparison, arithmetic calculations classied 68.8%, 17.2%, and 14.1% in
the same categories. Using 10,000 simulated data sets, the methodology provided a classication of farms into different efcacy categories with a false discovery of reduced farm
efcacy rate equaling 8.74%. In addition, model-classication was unaffected by presence
of single outlier horses in a separate simulation study.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Anthelmintic resistance has become a universal problem in gastrointestinal parasites of horses (Kaplan et al.,
2004), and it is considered an inevitable biological

Corresponding author. Tel.: +1 859 218 1103; fax: +1 859 257 8542.
E-mail address: martin.nielsen@uky.edu (M.K. Nielsen).
0304-4017/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetpar.2013.04.036

consequence of anthelmintic treatment. Because these

reductions in anthelmintic efcacy are known to be a growing problem, it can no longer be assumed that a given drug
is working, and hence a routine monitoring of drug efcacy
is needed (Kaplan, 2004; Wolstenholme et al., 2004). For a
precise denition of efcacy, see Section 2.3.2.
However, in the absence of reliable genetic markers,
other biological measures can be used to evaluate if there
are reductions in efcacy. The ultimate gold standard for

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M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614622

determining efcacy are controlled efcacy and critical test

type studies, where animals are euthanatized and total
worm counts are performed (Drudge and Lyons, 1977).
However, these methods are only practical in research
settings on a limited number of animals and cannot be routinely used at the farm level. In addition, ethical concerns
are growing with respect to studies involving euthanasia of
animals, and these may become difcult to obtain permission to perform in the future. Some in vitro assays have
been validated for usage in sheep, goats, and cattle, but
none have so far proven useful for horses (Matthews et al.,
2012). Furthermore, in vitro assays are technically difcult to perform, and have many other limitations, which
severely restrict their usefulness on a wide scale. Given
the lack of alternative methods, the only practical technique available for evaluating efcacy at the farm level is
the fecal egg count reduction test (FECRT). This method has
been applied routinely for measuring anthelmintic efcacy
in ascarid, trichostrongyle, and strongyle type parasites
in all livestock host species and for all drug classes; and
can be carried out by veterinary practitioners in the eld.
The test is based on comparing fecal egg counts (FECs) in
groups of animals before and after treatment or by comparing FEC in anthelmintic treated and non-treated control
groups. FECRT data are straightforward to obtain, but the
interpretation of data is complicated due to a number of
factors, as recently outlined (Vidyashankar et al., 2012)
and mentioned in the following. Studying equine populations is more complex than ruminant or swine herds,
where animals typically are of the same breed and uniform
age groups. In contrast horse farms can house a variety
of different breeds, and age distributions are often wide
ranging from foals to geriatrics. It is known that younger
animals tend to have higher egg counts (Morris et al., 2004;
Fritzen et al., 2010), and anthelmintic efcacy can potentially vary with age groups. Additionally, a typical horse
farm is comprised of three genders: mares, geldings and
stallions, where stallions are often differently managed.
Indeed, variability in FECRT results between horse genders
has been reported previously (Drudge et al., 1982). This
suggests that on equine farms, it may be appropriate to
account for both gender and age effects when interpreting
FECRT data. Additional possible variables include breed and
use of the horses, and farm management. On the horse level,
over 50 strongyle species have been described infecting
horses (Lichtenfels et al., 2008), and the species composition of the parasite burden in the intestinal tract can vary
between horses. All these factors can in turn lead to heterogeneities in efcacies amongst different groups within
a farm making interpretations of farm efcacy complicated.
This paper describes statistical methodologies for interpreting and evaluating factors inuencing drug efcacy
from a multiple farm study. A random effects model is
adopted to identify the role of biological factors affecting anthelmintic efcacy. The results of this model were
used to develop a robust classication method. The role
of outliers and their inuence on anthelmintic efcacy is
described, and a mechanism is provided to address this
issue. The methodology is illustrated using data obtained
in a Danish eld study aiming at evaluating the efcacy of
pyrantel pamoate.

615

2. Materials and methods

2.1. Field study
The study was carried out as an observational study
amongst farms submitting fecal material for routine
screening to a central laboratory (Equilab Laboratory,
Slangerup, Denmark). Data were collected between March
17 and May 1, 2008.
2.2. Farms
Farms participating in the study were selected among
farms following a parasite control strategy based on selective therapy for at least one year prior to the study. All farms
with at least six horses exceeding 200 strongyle eggs per
gram during the study period were included in the study. In
this parasite control regimen, all the farms submitted fecal
samples from all horses for analyses twice yearly; in the
spring (MarchMay) and in the fall (SeptemberOctober).
On each occasion, horses exceeding the cutoff value for
treatment of 200 strongyle eggs per gram (EPG) were
treated. None of the horses in the study had been treated
for at least four months prior to the sampling for this study.
2.2.1. Regions
Farms represented all geographic regions (counties) of
Denmark. For the statistical analyses, farms were clustered according to their postal zip codes as follows: 1:
20002999: larger Copenhagen and suburbs (17 farms),
2: 30003999: Northern Zealand (9 farms), 3: 40004999:
Remainder of Zealand and adjacent islands (17 farms), 4:
50005999: Funen (14 farms) and 5: 6000 and above:
Jutland (7 farms). These regions are justied by Danish
geography, which is characterized by separate islands and
one peninsula (Jutland).
2.2.2. Fecal egg counts and treatments
Fecal egg counts were performed from all horses using
a modied McMaster technique with a detection limit of
20 EPG (Roepstorff and Nansen, 1998). All egg counts were
performed by the fth author. Horses shedding 200 or more
strongyle EPG were orally treated with pyrantel embonate paste (Banminth vet., Orion Animal Health, Ballerup,
Denmark) at the labeled dosage of 19 mg/kg, which equals
6.6 mg pyrantel base/kg.
Pre- and post-treatment fecal samples were collected
at days 0 and 14, respectively, refrigerated and refrigerated
for up to ve days until egg counts were performed according to general recommendations (Nielsen et al., 2010).
2.2.3. Coprocultures
Coprocultures were performed individually from all
1644 horses on the pre-treatment samples. Ten grams of
feces were moistened with tap water, mixed with vermiculite and set up for culture in a humidity chamber at room
temperature. After 14 days, cultures were baermannized
for 24 h at room temperature, and the whole sediment was
examined under the microscope at 100 and 400 magnication. Third stage larvae of Strongylus vulgaris were
identied according to described criteria (Russell, 1948),

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M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614622

and horses were recorded as either being S. vulgaris positive or negative. Farms were classied as S. vulgaris present
if any of the horses tested positive for the presence of S.
vulgaris on the coproculture.
2.3. Statistical analyses
All statistical analyses were performed using the glimmix procedure with SAS version 9.3 (SAS Institute Inc., Cary,
NC, USA). Six horses with post-treatment egg count larger
than the pre-treatment egg count were removed from the
data set prior to the analysis. Biologically, anthelmintic
treatment is expected to reduce egg counts, even if some
efcacy is lost, and thus it was assumed that these six
horses were either incorrectly treated or accidentally not
treated at all. All remaining horses had egg count reductions of 40% or higher.
Data were rst tted to a hierarchical generalized linear mixed logistic regression model where pre-treatment
egg count, age, gender, and the presence of S. vulgaris on
farms were xed effects and horses, farms and regions
were random effects. The overall variability in observed
efcacy can be described to be due to intra-farm and interfarm variability. The intra-farm variability was captured
using a horse specic random effect and inter-farm variability was captured using a farm specic random effect.
The horse specic random effect was introduced to account
for possible differences in the species composition of the
parasitic burden between horses. To account for common
characteristics between farms within a region, a region specic random effect was incorporated. All the explanatory
variables and random effects were combined additively to
model the efcacy on a logit scale. All possible interaction
terms were evaluated during model construction, as well as
residual analyses and goodness of t tests were performed
to check the adequacy of the model t.
Using the tted statistical model (1.2), the lower limits
of the 95% predicted intervals for pijk were used to assess
reduction in pyrantel efcacy and classify the farms into
signs of resistance (RE), suspect resistance (SR), and no
resistance (NR) categories. Since the prediction intervals
depend on the covariates, either the mean or the median of
these lower limits could be used as a summary measure of
efcacy for each farm. The median was chosen as this summary measure since it is a robust estimator of the center of
the distribution. Using the proposed methods, farms were
classied with three different methods: The model-based
classication, the same model without random effects and
simple arithmetic calculations without statistical modeling.
2.3.1. Statistical model
Let the indices i, j and k represent the horse, farm, and
region respectively. The farms were grouped into the ve
regions dened in Section 2.2.2 and the possible values for
k were 1, 2, 3, 4, and 5.
The number of farms within a region changed with the
region and number of horses within a farm changed with
the farm. nk denoted the number of farms in region k, while
njk denoted the number of horses on farm j of region k. Let
(Xijk , Yijk ) represent the pre-treatment and post-treatment

egg count of the ith horse, in the jth farm which belonged
to the kth region. It was assumed that,
Yijk |pijk , Xijk Bin(Xijk , 1 pijk ),

(1.1)

where pijk represented the pyrantel efcacy on horse i, on

farm j, and belonging to region k. Let Aijk denote the age of
the ith horse in the jth farm which belongs to the kth region.
The regression model to assess reduction in efcacy was

log

pijk
1 pijk

= 0 + 1 Xijk + 2 Aijk + 3 Vjk

3


3+l Gijkl + Hijk + Fjk + Rk

(1.2)

l=1

where 0 was the overall mean efcacy, 1 was the slope

associated with pre-treatment egg count, and 2 was the
slope associated with the age. The variable Vjk was used as
an indicator variable representing the presence or absence
of S. vulgaris in each farm. Thus, the effect 3 represented
the mean effect of S. vulgaris presence on a farm on the
logit of the efcacy of the pyrantel paste. In general, Gijk
represented the gender variable and took values 1, 2, 3 and
4, where 1 represented male horses, 2 represents female
horses, 3 represented neutered males and 4 for horses
whose gender information was unknown. Thus,
Gijkl = I[Gijk=l ], for l = 1, 2, 3, 4.

(1.3)

The parameters 4 , 5 , and 6 represented mean effects

of each gender group. Hijk represented the horse effect, Fjk
represented the farm effect, and Rk represented the region
effect. It was assumed that Hijk , Fjk , and Rk were latent
effects. These latent effects are assumed to be mutually
independent normal random variables with expectations
0 and variances H2 , F2 , and R2 respectively. This formulation implies that the, variances of farms were all the same
across regions and the correlations between farms within
a region were all the same.
We notice that, conditioned on pre-treatment egg count
values, the post-treatment values have a binomial distribution, whose success probabilities are random and drawn
from a normal distribution on the logit scale. This is biologically justied, since the group of equine strongyle
parasites comprise more than fty species, existing in complex mixed-species infections (Lichtenfels et al., 2008), but
the eggs of these species cannot be differentiated using traditional methods. The composition of the strongyle fauna
could potentially be different between individual horses.
This justies the use of binomial distribution with random
success probabilities.
It has been observed that pre-treatment egg count levels cause bias in the calculated values for drug efcacy
(Levecke et al., 2012a,b). Animals with low to moderate pre-treatment egg count levels tend to have higher
observed efcacies than horses with high pre-treatment
egg counts, and this appears to be explained by the detection limit of the egg counting technique (Levecke et al.,
2012a). Thus, a high measurement of efcacy is biased
toward horses with lower pre-treatment egg count levels.
We note that a typical farm contains a mix of horses belonging to three basic categories: low egg shedders, moderate

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617

and 0.92, with LPLs between the two values falling in the SR
category. The principles behind this are outlined in Fig. 1.
Where classication discrepancies occurred between
the model-based classication and the arithmetic calculations, data were revisited by removing the one horse with
the lowest arithmetic FECRT on each farm to evaluate the
possible inuence of single outlier horses on classication
using the two methods.

Fig. 1. Illustration of the model-based method for classifying farms as

no resistance (NR), suspect resistance (SR), or resistance (RE). The gure
shows the means and prediction intervals (error bars) for three theoretical
farms. The two lower prediction limit (LPL) cutoff values used here are
92% and 88% (bold lines). For the rst farm, the whole condence interval
stays above the 92% cutoff, and it is determined having NR. The second
farm moves the LPL into the suspect range of 8892% and is determined
SR. The third farm extends the LPL well below the 88% cutoff value and is
determined RE.

egg shedders, and high egg shedders. For these reasons,

we include pre-treatment egg count as a covariate in the
model.
2.3.2. Denitions of farm efcacy
Since pijk dened above is a random variable inuenced
by horse level, farm level, and region level random effect
distributions, the farm efcacy for the jth farm in the kth
region can be dened in two ways: (1) marginal farm
efcacy and (2) conditional farm
njkefcacy. The marginal
farm efcacy is dened to be
E(p )/njk .The condii=1n ijk
jk
p /njk . Ejk is the
tional efcacy is dened to be Ejk =
i=1 ijk
farm efcacy given the random effects as opposed to the
marginal efcacy where we average over all the random
effect distributions.
Assuming that the covariates are centered at the expectations, the marginal efcacy, if dened as an average of
logit of the efcacies, will correspond to the intercept

pij (k) =

exp 0 + 1 Xij + 2 Aij + 3 Vj +

2.3.4. False discovery of reduced farm efcacy

The accuracy of the classication method was evaluated by identifying false discovery of reduced farm efcacy
(FDRFE) using Monte Carlo simulations. FDRFE rate is
dened as the percentage of truly no resistance farms
which are incorrectly classied as resistant by the methodology adopted for classication.

2.4. Simulation studies

For the simulation study, we tted the model (1.2) without region effect and generated data as described below. To
the marginal efcacy for the jth farm, recall that
evaluate
mj
E(pj ) =
E(pij )/mj , where mj is the number of horses on
i=1
the jth farm. Since E(p.j ) does not have a closed form expression, a Monte Carlo approach was adopted. Specically, for
every farm 10,000 farm specic normal random variables
and 10,000 horse specic normal random variables were
generated for each horse on that farm. For the kth simulation, we denote the realization of pij as pij (k). For example, if
a farm had six horses, 60,000 horse specic normal random
variables and 10,000 farm specic random variables were
generated. Finally E(p.j ) was calculated using the formula

1
 E(pj ) =
mj 10, 000

2.3.3. Farm classication

Cutoff lower prediction limit (LPL) values for the classication of farms into RE, SR, and NR categories were 0.88


k=1

pij (k),

i=1

where

2

I(Gij = l) + Hij (k) + Fj (k)

l=1 3+l

1 + exp 0 + 1 Xij + 2 Aij + 3 Vj +

term of the statistical model (1.2). However, in the probability metric studied in this manuscript, this is not the case.
These two notions of efcacy are alternatives to the arithmetic mean. When the horses are homogeneous and the
variability in efcacy between horses is negligible, these
two notions reduce to the arithmetic mean and hence they
can be viewed as generalizations of arithmetic mean to the
non-homogeneous case. Due to multiple sources of variability and outliers in various farms, the conditional farm
efcacy was used for the classications made in this study.

10,000 mj

2

.

(2.3)

I(Gij = l) + Hij (k) + Fj (k)

l=1 3+l

The true status of the farm was then determined using the
0.95 cutoff: if E(p.j ) 0.95, the farms status is NR, otherwise
the farms status is RE. The parameter values were chosen
to be similar to the values obtained from the analysis of the
equine data set.
Now each simulated data set was analyzed using a variant of model (1.2). This tting facilitated the model-based
classication, and each farm was then classied as RE, SR,
or NR. The FDRFE rate for this study was then computed by
comparing the farms true resistance status and the classied status. Specically, 10,000 independent data sets were
generated, and the FDRFE was computed as the proportion
of non-resistant farms (in these 10,000 data sets) wrongly
classied as resistant.

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M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614622

Evaluation of the conditional efcacy was performed

similarly to the above, but used the conditional efcacy for
farm j, namely Ej , given by
Ej =

mj
1 

mj

Fixed effects

pij ,

i=1

and mj is the number of horses in farm j. Note that the

predicted values of the random effects were used in the
calculation. The true status of the farm (RE or NR) was
again dened using the 0.95 cutoff. Specically, a farms
true status was NR if Ej 0.95; otherwise it was dened as
RE.
2.5. Outlier effects
The effect of single outliers on farm classication was
evaluated using simulated data from a single farm with
12 horses. The data were generated using a regression
model with age, gender, and pre-treatment egg count as
covariates. Let Xi and Yi denote the pre-treatment and posttreatment egg count of the ith horse. The gender of the ith
horse was coded as Gi = 1, 2, and 3 for stallion, mare and
gelding, respectively. Then,
Yi |Xi , pi Bin(Xi , 1 pi ),
where pi represented the anthelmintic efcacy of the ith
horse. To complete the model specication, we modeled

log

pij

1 pij

= 0 + 1 Xi + 2 Ai +

2
l=1

I(Gi = l) + i

i N(0, 12 ),
where i was the horse specic random effect, Ai is the
age of the ith horse. The values of the covariates were
randomly chosen from a farm with 12 horses. The regression coefcients were set as = (0 , 1 , 2 , 3 , 4 ), where
0 = 3.5 (intercept), 1 = 0.001 (pre-treatment egg count),
2 = 0.01 (age), 3 = 0.01 (gender = stallion), and 4 = 0.01
(gender = mare). An outlier effect was induced by altering
the post-treatment egg count for horse 1. The observed
post-treatment egg count for horse 1 was replaced with
the altered value Y1 , given by

Table 1
Summary of parameter estimates for xed and random effects from the
analysis of the observational farm data.

Y1 = Y1 ,
for different values of  in the range [1,X1 /Y1 ]. Increasing
 decreased the observed efcacy of horse 1;  = 1 corresponded to the true data, where  = X1 /Y1 corresponded to
the case of zero efcacy. Note that the symbol x denoted
the integer part of x.
3. Results
3.1. Farm data
Sixty-four farms with at least six horses exceeding
200 EPG were included in the study. On each farm, all
horses exceeding this threshold were included in the data
set. Farm size ranged from six to 68 horses (mean = 25.7,
median = 24), with 1644 horses being included in the

Estimate

Intercept
Pre-treatment egg count
Age
Gender (no information)
Gender (mare)
Gender (gelding)
Gender (stallion)
Presence of Strongylus vulgaris 0
Presence of Strongylus vulgaris 1

5.96880
0.00095
0.01128
0.44720
0.60130
0.78310
0
0.02592
0

Standard error
0.8434
0.0002
0.0214
0.8772
0.7936
0.7916
0.3253

Random effects

Estimate

Standard error

Cluster
Farm
Horse

0.5154
0.1705
6.4137

0.3451
0.2137
0.5244

The residual variance was 3.29.

study. A total of 614 out of these horses were treated

during the time period of this study. On the individual
farms, the number of treated horses ranged from 6 to 22
(mean = 9.6, median = 9). Horses aged between 1 and 33
years (mean = 6.9, median = 5) with 49% being geldings,
48% mares and 3% stallions. Horses were primarily mixed
type riding horses, such as Danish Warmbloods, Icelandic
horses, Norwegian Fjordhorse, and other pony breeds. S.
vulgaris was detected in 78 horses (4.75%), with 31 farms
(48.4%) having at least one horse positive.
All statistical estimates provided below are given on the
logit scale, unless otherwise stated. The variance estimates
and standard errors for random effects (horse, farm, and
region) in the model are presented in Table 1. The analyses did not reveal an effect of region. An analysis without
the region as a random effect did not change the conclusions (results not shown). Therefore, region was left in the
model to emphasize the usefulness of this strategy. The
estimate of the variance (standard error) of the horse, the
farm, and the region were estimated to be 6.4137(0.5244),
0.5154(0.3451) and 0.1705(0.2137), respectively. The proportion of variance explained by the horse effect was
61.73% while that by the farm was 4.96%. The proportion
of variance explained by the region was 1.64%.
The estimate of the slope of the pre-treatment egg
count was determined to be 0.0010 (SE = 0.0002), which
was statistically different from 0 (P < 0.0001). The slope for
the age was determined to 0.0113 (SE = 0.0214). Age was
not a signicant factor to describe variations in efcacies
(P = 0.5981), and there were no signicant differences in
efcacies of horses between genders (P = 0.6838). The data
was also analyzed by removing all horses with missing gender (16%), and results were similar (not shown). Finally,
there were no signicant differences in efcacy between
farms with and without S. vulgaris detected (P = 0.9403). No
apparent interactions were found between the parameters
mentioned above. Summaries of parameter estimates of
xed effects are presented in Tables 1 and 2. Residual analyses and goodness of t tests (p-values > 0.10) suggested
that the model t was adequate.
The overall arithmetic mean pyrantel efcacy, obtained
by averaging the FECR from all horses on all farms, was

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619

Table 2
Summary of type III tests of xed effects from analyzing the observational
farm data set.
Effect

Num DF

Den DF

F value

P>F

Pre-treatment egg
count
Age
Gender
Presence of Strongylus
vulgaris

515

22.27

<0.0001

1
3
1

515
11
4

0.28
0.51
0.01

0.5981
0.6838
0.9403

Num DF: degrees of freedom of the numerator; Den DF: degrees of freedom of the denominator.

92.93%. Twelve farms (19%) had arithmetic means below

90%, while another 18 farms (28%) were within the 9095%
range. The LPL-based classication of farms into the categories no resistance (NR), suspect resistance (SR) and
resistance (RE) is presented in Fig. 2. For comparison, the
classication is presented using LPLs or lower condence
limits (LCLs) obtained from the model-based classication,
the same model without random effects and simple arithmetic calculations without statistical modeling.
Classication of each farm with these three methods
is presented as supplementary les. Fourteen farms (22%)
were differently classied using the model-based classication compared to using arithmetic calculations. On ten of
these farms, the model classied no resistance (NR), while
the arithmetic approach classied either suspect resistance
(SR) or resistance (RE). Removing the horse with the lowest
individual FECR changed the classication for the arithmetic method in all ten cases. For farms CC, E, L, HH, MM,
MMM, P, WW, and Y the arithmetic classication changed
from RE to NR, while farms LLL and MMM both changed
from RE to SR. In each case, the model-based classication
was unaffected by removing the horses with the lowest
FECR.
As mentioned in Section 2.3, we initially removed six
horses from the data set. It is worthwhile to mention here
that if these horses were included, then (pre-post)/pre
would become negative. As the logarithm of any negative

Fig. 2. Classication of farms into the three categories no resistance (NR),

suspect resistance (SR) and resistance (RE) to pyrantel embonate. The
outcomes are presented comparing three different methods for analyzing FECRT data: full model-based classication (black), model without
random effects (gray), arithmetic classication (white). The arithmetic
classication is based on lower 95% condence limits.

Fig. 3. Single farm simulation evaluating the inuence of single outlier

horses on the classication of the farm into anthelmintic efcacy categories. The farm has 12 horses and one of these is introduced as an outlier
by changing its anthelmintic efcacy as measured by the fecal egg count
reduction test. In the simulation, this horse represented efcacies in the
range of 0.11.0. The model-based classication is unaffected by this outlier horse, whereas the arithmetic classication changes for all efcacy
values of this horse in the range of 0.10.9. The horizontal stippled lines
indicate the threshold values for pyrantel efcacy categories used in the
present study.

number is not dened, this would make the model undened. Alternatively, we performed the same analyses, with
the entire data set by setting the post-treatment values to
equal the pre-treatment values. This did not change any of
the conclusions described in Tables 13 and the numerical values were very close to the values presented in these
tables. Further, the classications of the 64 farms remained
unchanged (results not included).
3.2. Simulation data: results for false discovery of
reduced farm efcacy rate
The simulations evaluating the marginal efcacy
revealed that amongst 61 farms, 56 farms had an expected
mean efcacy greater than 92%, and 8.74% of them were
wrongly classied as resistant or belonging to the suspect
resistant category. This then determined the FDRFE rate for
this study.
Table 3 presents the results of the simulations study
where efcacy is evaluated using the conditional denition.
The results are displayed for each of the twelve farms and
then aggregated across all of the farms. The table compares
the performance of the model with the arithmetic classication. In this study, of the 12,000 total simulated farms,
3161 were resistant and 8839 non-resistant. Both methods
had low rates of FDRFE; the model-based classication had
an FDRFE of 0 compared to an FDRFE of 0.0547 for the arithmetic classication. The model outperformed arithmetic
classication in terms of power in detecting truly resistant
farms: 0.7062 versus 0.5135.
3.2.1. Outlier effects
The inuence of a single outlier horse on a single
farm is illustrated in Fig. 3. The gure clearly shows that

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M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614622

Table 3
Classication of farms according to their anthelmintic efcacy status based on 1000 simulated data sets, each based on 12 farms. Farms are classied using
the model-based and the arithmetic classications. Classication results are presented both separated out on each farm, and aggregated across farms.
Farm

True status

NR
RE
NR
RE
NR
RE
NR
RE
NR
RE
NR
RE
NR
RE
NR
RE
NR
RE
NR
RE
NR
RE
NR
RE

Model-based
NR

2
3
4
5
6
7
8
9
10
11
12

Data aggregated across farms

NR
RE

Arithmetic
SR

RE

NR

SR

RE

25
9
40
7
26
17
41
10
283
69
187
53
406
28
346
51
280
99
265
101
537
75
723
32

0
75
0
69
0
79
0
108
1
204
0
203
0
231
0
225
0
261
0
246
1
225
0
120

0
891
0
884
0
878
0
841
0
443
0
557
0
335
0
378
0
360
0
388
0
162
0
125

24
57
31
51
26
69
41
79
251
257
82
337
404
166
345
205
107
495
155
499
396
304
723
107

1
129
9
135
0
99
0
127
33
175
55
183
2
194
1
206
93
122
91
121
118
87
0
96

0
789
0
774
0
806
0
753
0
284
50
293
0
234
0
243
80
103
19
115
24
71
0
74

3159
551

2
2046

0
6242

2585
2626

403
1674

173
4539

NR = no resistance; SR = suspect resistance; RE = resistance.

model-based classication using the median was unaffected by the outlier, whereas the arithmetic classication
is highly driven by the FECR value of the outlier.
4. Discussion
This study provides a exible model for combining
horse-specic covariates and random effects on multiple
farms within different regions, and a statistical methodology for evaluating anthelmintic efcacy, which is robust to
the presence of outliers in farms. As a summary statistic
following the model t, we introduce two new notions of
efcacy, namely, marginal and conditional efcacies. These
arise due to the introduction of random effects into the statistical model. Finally, we validated our methodology using
simulation.
The choice of cutoff values for determining anthelmintic
resistance deserves more critical attention. Although
guidelines have been published, these are primarily
focused on sheep (Coles et al., 1992), and no recommendations exist for horses. As a result, there is no consensus
for the choice of any of these cutoff values, and each study
tends to use its own denitions (Ihler, 1995; Craven et al.,
1998; von Samson-Himmelstjerna et al., 2007; Lind et al.,
2007; Traversa et al., 2009). As the performance of a diagnostic test critically depends on the choice of cutoff, this
constitutes an underlying problem in assessing reduction
in efcacy of a drug using FECRT data. Clearly, the cutoffs
chosen in this study were arbitrary as well. However, we
based these on historical data available, where pyrantel

was originally reported to be in the range of 94100% with

substantial levels of variability within and between farms
(Lyons et al., 1975; Drudge et al., 1982). This is much different compared to other anthelmintics such as ivermectin,
where the expected efcacy is 99.9% (Lyons et al., 1980).
Given this information, a lower prediction limit of 92%
appears as a reasonable choice, while it should be higher
for a drug like ivermectin. As pointed out in Vidyashankar
et al. (2012), there are several technical, biological, and
sampling factors which are typically not well-controlled
leading to biased estimates of empirical farm efcacies. To
address this issue, we found it prudent to introduce a category called suspect resistance (SR) category to encourage
people to collect more data before a conclusion is reached.
We choose to classify farms to belong to this category if the
LPL was between 88% and 92%, but other cutoffs may apply.
The concept of using lower prediction limits is new. We
chose this as it reects both the mean efcacy as well as represents the variability of the data in just one number. This
could be seen as an alternative to the traditional approach
with evaluating a combination of means and lower condence limits (Coles et al., 1992). However, further work is
needed for identifying useful guideline cutoff criteria for
classifying anthelmintic resistance.
Fig. 2 illustrates the need for including the random
effects in the model. Only accounting for covariates is not
sufcient, since in many cases the sample size will be too
small to make an inference. Thus, combining the information through random effects in individual animals and their
farms is a useful approach. Other authors have addressed

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M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614622

sources of variability by assuming a Poisson distribution of

eggs in a sample and incorporating this into a statistical
approach (Morgan et al., 2005). However, variation arising
from the FEC methodology is just one of the many sources of
variation that impede interpretation of FECRT. By considering some of the important biological sources of variability,
signicant progress can be made.
Understanding the slope estimates provides useful
information about the covariates used in the model. For
instance, a negative slope for the pre-treatment egg count
means that as the pre-treatment egg count increases, the
pyrantel efcacy decreases. There are several possible biological explanations for this relationship. In fact, we believe
the explanation is technical, not biological, for the following reasons. Using a technique with a detection limit of 20
EPG, a horse with a 200 EPG count treated with a drug with
a 90% efcacy will be likely to exhibit 0 EPG post-treatment,
because the low level egg count is likely to go undetected.
If a 1000 EPG horse is treated with the same drug, however,
eggs are likely to be detected post-treatment leading to a
lower observed efcacy. Since the level of pre-treatment
egg count is highly variable within and between farms, one
way to account for this relationship is to include it in the
model. Similar observations regarding pre-treatment egg
count levels and detection limit of egg counting techniques
have recently been made by other authors (Levecke et al.,
2012a,b).
The slope for age was positive, although not signicantly, meaning that pyrantel efcacy tends to increase
with increasing age. This makes sense, because pretreatment egg counts decrease with age, and young horses
tend to exhibit higher egg counts. Understanding the role
of the pre-treatment egg count level outlined above, it
can be deduced that the observed efcacy will tend to
increase with age. There are experimental data suggesting
that anthelmintic efcacy is lower in younger horses (Herd
and Gabel, 1990). It remains plausible that such observations could be fully or partially explained by the higher
pre-treatment egg counts in young horses.
An important question is whether a single observation
on any of these farms can affect the conclusions of the
data analysis if arithmetic methods are used. On ten of the
farms with classication discrepancies between the modelbased classication and the simple arithmetic calculations,
it was clear that removing the horse with the lowest FECR
strongly affected the arithmetic classication, while the
model-based classication was unaffected. This suggests
that any anthelmintic efcacy classication using arithmetic data may be unstable because of the large variability
between horses on each farm and the small group sizes.
Removing just one horse can change the results substantially, which makes the inference concerning anthelmintic
efcacy questionable. In Fig. 3, we show that using farm
median LPLs the outlier effect can be reduced considerably. Altogether, these discrepancies and our simulation
results illustrate that the model classication provides a
reliable and stable classication by taking into account
pre-treatment egg counts and the different sources of variability in FECRT data.
The experience with this data set clearly illustrates that
data sets evaluating pyrantel efcacy should be handled

621

with care. The overall arithmetic mean efcacy was high

(92.93%), but the large variability complicated the interpretation of data. Overall, it appears that pyrantel would be
useful on most of the studied farms. A direct comparison
with other studies reporting pyrantel efcacy is not possible since different methods were used. Given the higher
variability of pyrantel data, interpretation is further complicated. For instance, the study by Ihler (1995) is often
being reported as the rst documentation of pyrantel paste
resistance in cyathostomins based on a 94% mean FECRT
found on one farm. Similarly, a Danish study suggested
early signs of pyrantel resistant cyathostomins on two
farms based on a 95% mean FECRT cutoff value for determining reduced efcacy (Craven et al., 1998). In Germany,
von Samson-Himmelstjerna et al. (2007) found pyrantel
efcacies in the range of 92100% and concluded four farms
to be suspect resistant due to mean efcacies below 95%.
A Swedish study reported pyrantel efcacies above 95% for
all 23 farms, but a large variability on nine farms led the
authors to conclude suspect resistance (Lind et al., 2007).
Similarly, a recent multinational European study reported
pyrantel efcacies below 90% on farms in Italy, United Kingdom and Germany (Traversa et al., 2009). Altogether, farm
pyrantel efcacy levels reported in other European studies
were often in the 80100% range, and it is a challenge to
delineate between reduced efcacy and chance variations
of FECRT data.
It is often assumed that presence of large strongyle
species can affect the efcacy data measured for cyathostomins, since Strongylus spp. have not yet shown signs of
developing anthelmintic resistance (Craven et al., 1998;
von Samson-Himmelstjerna et al., 2007). However, this
could not be supported in our analyses. There may be several reasons for this, but in practice large strongyles, when
present, will never dominate the egg output, and typically
more than 95% of the eggs will be of cyathostomin origin. In addition, there are 50 different cyathostomin species
described as infecting equids, and hence this group cannot
be treated as uniform either. With coprocultures, it may be
possible to account for only a handful of the many strongyle
species potentially present. Using the molecular tools that
were recently developed for detection of 21 of the most
common cyathostomin species (Hodgkinson et al., 2003;
Traversa et al., 2007; Cwiklinski et al., 2012.) there is now
a possibility to further account for the species composition
in strongyle burdens and to connect this information with
the efcacy data. This information could then be built into
the statistical model, which would be worthwhile in future
studies.
It should be noted that in this data set, FECRTs were
only performed on horses exceeding 200 EPG. This threshold was chosen because of the detection limit of the
egg counting method used (20 EPG), making the method
unsuitable for detecting minor reductions in pyrantel efcacies. Given the clear role of the pre-treatment egg count
level in the FECRT illustrated with this study, it is realistic
to assume that if horses with egg counts below 200 EPG
had been included, the efcacy estimates would have been
biased toward a higher level. However, further studies are
needed to conrm this notion. Further, other factors possibly affecting parasite populations could be recorded in

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M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614622

future studies and subsequently included in the statistical

model. These could include farm size, stocking rate, breed,
use, level of horse trafc, and anthelmintic treatment regimen employed on the farm. This might allow for accounting
for more of the variability observed in the data set, which
could potentially improve the classication model further.
Ultimately, different egg counting techniques are likely to
perform differently, and more sensitive methods appear to
be preferable (Levecke et al., 2012b).
The proposed statistical methodology is useful to identify beginning stages of reduction in efcacy when data
from multiple farms are available. However, this approach
may not be easily applicable for routine use especially by
veterinary practitioners when dealing with a specic farm.
The methodology used here is not host, worm species, or
drug specic. Therefore, a similar approach can be adapted
to analyze data from other host species. For instance, in
cattle the efcacy of avermectin/milbemycin anthelmintics
against Cooperia spp. is variable even in the absence of
reduced anthelmintic efcacy (Vermunt et al., 1996), and
hence FECRT data are subject to high levels of variability.
In conclusion, we developed a hierarchical model for
evaluating occurrence of reduced anthelmintic efcacy
using observational data from multiple farms. Using the
random effects in the statistical model, we combined information among farms to distinguish between variability and
genuine reduction in efcacy, and thus provide a robust
method to determine which farms are likely to be resistant,
suspect resistant, or not resistant to a given anthelmintic
drug. Alternatively, count models can be used to model the
egg counts and these can be extended to include the random effects. It will be interesting to compare our results
with these alternative approaches.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.vetpar.2013.04.036.
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