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**Author's personal copy
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Veterinary Parasitology 197 (2013) 614–622

Contents lists available at ScienceDirect

Veterinary Parasitology

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

**Hierarchical model for evaluating pyrantel efﬁcacy 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 efﬁcacy, 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

efﬁcacy of pyrantel pamoate in a study performed in Denmark. The model was then used

to describe two notions of farm efﬁcacy, namely conditional and marginal efﬁcacy. The

median of the lower prediction limits was used to describe a robust classiﬁcation rule. The

performance of the methodology was evaluated using Monte Carlo simulations. The ﬁeld

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 identiﬁcation of Strongylus vulgaris from all horses pre-treatment. Thirty-one

farms (48.4%) were positive for S. vulgaris, but pyrantel efﬁcacy was unaffected by the presence of this parasite in the statistical model. Further, there were no signiﬁcant 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 classiﬁed 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 classiﬁed 68.8%, 17.2%, and 14.1% in

the same categories. Using 10,000 simulated data sets, the methodology provided a classiﬁcation of farms into different efﬁcacy categories with a false discovery of reduced farm

efﬁcacy rate equaling 8.74%. In addition, model-classiﬁcation 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 efﬁcacy 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 efﬁcacy

is needed (Kaplan, 2004; Wolstenholme et al., 2004). For a

precise deﬁnition of efﬁcacy, 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 efﬁcacy. The ultimate gold standard for

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**

M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614–622

**determining efﬁcacy are controlled efﬁcacy 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 difﬁcult 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 difﬁcult 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 efﬁcacy at the farm level is

the fecal egg count reduction test (FECRT). This method has

been applied routinely for measuring anthelmintic efﬁcacy

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 ﬁeld.

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 efﬁcacy 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 efﬁcacies amongst different groups within

a farm making interpretations of farm efﬁcacy complicated.

This paper describes statistical methodologies for interpreting and evaluating factors inﬂuencing drug efﬁcacy

from a multiple farm study. A random effects model is

adopted to identify the role of biological factors affecting anthelmintic efﬁcacy. The results of this model were

used to develop a robust classiﬁcation method. The role

of outliers and their inﬂuence on anthelmintic efﬁcacy is

described, and a mechanism is provided to address this

issue. The methodology is illustrated using data obtained

in a Danish ﬁeld study aiming at evaluating the efﬁcacy 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 (March–May) and in the fall (September–October).

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:

2000–2999: larger Copenhagen and suburbs (17 farms),

2: 3000–3999: Northern Zealand (9 farms), 3: 4000–4999:

Remainder of Zealand and adjacent islands (17 farms), 4:

5000–5999: Funen (14 farms) and 5: 6000 and above:

Jutland (7 farms). These regions are justiﬁed 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 modiﬁed McMaster technique with a detection limit of

20 EPG (Roepstorff and Nansen, 1998). All egg counts were

performed by the ﬁfth 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 ﬁve 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× magniﬁcation. Third stage larvae of Strongylus vulgaris were

identiﬁed according to described criteria (Russell, 1948),

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**

616

M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614–622

and horses were recorded as either being S. vulgaris positive or negative. Farms were classiﬁed 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

efﬁcacy 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 ﬁrst ﬁtted 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 ﬁxed effects and horses, farms and regions

were random effects. The overall variability in observed

efﬁcacy can be described to be due to intra-farm and interfarm variability. The intra-farm variability was captured

using a horse speciﬁc random effect and inter-farm variability was captured using a farm speciﬁc random effect.

The horse speciﬁc 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 speciﬁc random effect was incorporated. All the explanatory

variables and random effects were combined additively to

model the efﬁcacy on a logit scale. All possible interaction

terms were evaluated during model construction, as well as

residual analyses and goodness of ﬁt tests were performed

to check the adequacy of the model ﬁt.

Using the ﬁtted statistical model (1.2), the lower limits

of the 95% predicted intervals for pijk were used to assess

reduction in pyrantel efﬁcacy 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

efﬁcacy 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

classiﬁed with three different methods: The model-based

classiﬁcation, 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 ﬁve

regions deﬁned 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 efﬁcacy 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 efﬁcacy was

log

pijk

1 − pijk

= ˇ0 + ˇ1 Xijk + ˇ2 Aijk + ˇ3 Vjk

+

3

ˇ3+l Gijkl + Hijk + Fjk + Rk

(1.2)

l=1

**where ˇ0 was the overall mean efﬁcacy, ˇ1 was the slope
**

associated with pre-treatment egg count, and ˇ2 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 ˇ3 represented

the mean effect of S. vulgaris presence on a farm on the

logit of the efﬁcacy 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 ˇ4 , ˇ5 , and ˇ6 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 justiﬁed, since the group of equine strongyle

parasites comprise more than ﬁfty 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 justiﬁes 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 efﬁcacy

(Levecke et al., 2012a,b). Animals with low to moderate pre-treatment egg count levels tend to have higher

observed efﬁcacies 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 efﬁcacy 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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**

M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614–622

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 classiﬁcation discrepancies occurred between

the model-based classiﬁcation and the arithmetic calculations, data were revisited by removing the one horse with

the lowest arithmetic FECRT on each farm to evaluate the

possible inﬂuence of single outlier horses on classiﬁcation

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 ﬁgure

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 ﬁrst farm, the whole conﬁdence interval

stays above the 92% cutoff, and it is determined having NR. The second

farm moves the LPL into the suspect range of 88–92% 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. Deﬁnitions of farm efﬁcacy

Since pijk deﬁned above is a random variable inﬂuenced

by horse level, farm level, and region level random effect

distributions, the farm efﬁcacy for the jth farm in the kth

region can be deﬁned in two ways: (1) marginal farm

efﬁcacy and (2) conditional farm

njkefﬁcacy. The marginal

farm efﬁcacy is deﬁned to be

E(p )/njk .The condii=1n ijk

jk

p /njk . Ejk is the

tional efﬁcacy is deﬁned to be Ejk =

i=1 ijk

farm efﬁcacy given the random effects as opposed to the

marginal efﬁcacy where we average over all the random

effect distributions.

Assuming that the covariates are centered at the expectations, the marginal efﬁcacy, if deﬁned as an average of

logit of the efﬁcacies, will correspond to the intercept

pij (k) =

exp ˇ0 + ˇ1 Xij + ˇ2 Aij + ˇ3 Vj +

**2.3.4. False discovery of reduced farm efﬁcacy
**

The accuracy of the classiﬁcation method was evaluated by identifying false discovery of reduced farm efﬁcacy

(FDRFE) using Monte Carlo simulations. FDRFE rate is

deﬁned as the percentage of truly no resistance farms

which are incorrectly classiﬁed as resistant by the methodology adopted for classiﬁcation.

2.4. Simulation studies

For the simulation study, we ﬁtted the model (1.2) without region effect and generated data as described below. To

the marginal efﬁcacy for the jth farm, recall that

evaluate

mj

E(p·j ) =

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. Speciﬁcally, for

every farm 10,000 farm speciﬁc normal random variables

and 10,000 horse speciﬁc 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 speciﬁc normal random

variables and 10,000 farm speciﬁc random variables were

generated. Finally E(p.j ) was calculated using the formula

1

E(pj ) =

mj × 10, 000

2.3.3. Farm classiﬁcation

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

k=1

pij (k),

i=1

where

2

ˇ I(Gij = l) + Hij (k) + Fj (k)

l=1 3+l

1 + exp ˇ0 + ˇ1 Xij + ˇ2 Aij + ˇ3 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 efﬁcacy are alternatives to the arithmetic mean. When the horses are homogeneous and the

variability in efﬁcacy 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

efﬁcacy was used for the classiﬁcations made in this study.

10,000 mj

2

.

(2.3)

ˇ 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 farm’s status is NR, otherwise

the farm’s 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 ﬁtting facilitated the model-based

classiﬁcation, and each farm was then classiﬁed as RE, SR,

or NR. The FDRFE rate for this study was then computed by

comparing the farms true resistance status and the classiﬁed status. Speciﬁcally, 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

classiﬁed as resistant.

**Author's personal copy
**

618

M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614–622

**Evaluation of the conditional efﬁcacy was performed
**

similarly to the above, but used the conditional efﬁcacy 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 deﬁned using the 0.95 cutoff. Speciﬁcally, a farm’s

true status was NR if Ej ≥ 0.95; otherwise it was deﬁned as

RE.

2.5. Outlier effects

The effect of single outliers on farm classiﬁcation 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 efﬁcacy of the ith

horse. To complete the model speciﬁcation, we modeled

log

pij

1 − pij

= ˇ0 + ˇ1 Xi + ˇ2 Ai +

2

l=1

I(Gi = l) + ˛i

˛i ∼N(0, 12 ),

where ˛i was the horse speciﬁc 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 coefﬁcients were set as ˇ = (ˇ0 , ˇ1 , ˇ2 , ˇ3 , ˇ4 ), where

ˇ0 = 3.5 (intercept), ˇ1 = −0.001 (pre-treatment egg count),

ˇ2 = 0.01 (age), ˇ3 = −0.01 (gender = stallion), and ˇ4 = 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 ﬁxed 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 efﬁcacy of horse 1; = 1 corresponded to the true data, where = X1 /Y1 corresponded to

the case of zero efﬁcacy. 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 signiﬁcant factor to describe variations in efﬁcacies

(P = 0.5981), and there were no signiﬁcant differences in

efﬁcacies 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 signiﬁcant differences in efﬁcacy 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

ﬁxed effects are presented in Tables 1 and 2. Residual analyses and goodness of ﬁt tests (p-values > 0.10) suggested

that the model ﬁt was adequate.

The overall arithmetic mean pyrantel efﬁcacy, obtained

by averaging the FECR from all horses on all farms, was

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**

M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614–622

619

Table 2

Summary of type III tests of ﬁxed 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

1

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 90–95%

range. The LPL-based classiﬁcation of farms into the categories no resistance (NR), suspect resistance (SR) and

resistance (RE) is presented in Fig. 2. For comparison, the

classiﬁcation is presented using LPLs or lower conﬁdence

limits (LCLs) obtained from the model-based classiﬁcation,

the same model without random effects and simple arithmetic calculations without statistical modeling.

Classiﬁcation of each farm with these three methods

is presented as supplementary ﬁles. Fourteen farms (22%)

were differently classiﬁed using the model-based classiﬁcation compared to using arithmetic calculations. On ten of

these farms, the model classiﬁed no resistance (NR), while

the arithmetic approach classiﬁed either suspect resistance

(SR) or resistance (RE). Removing the horse with the lowest

individual FECR changed the classiﬁcation for the arithmetic method in all ten cases. For farms CC, E, L, HH, MM,

MMM, P, WW, and Y the arithmetic classiﬁcation changed

from RE to NR, while farms LLL and MMM both changed

from RE to SR. In each case, the model-based classiﬁcation

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. Classiﬁcation 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 classiﬁcation (black), model without

random effects (gray), arithmetic classiﬁcation (white). The arithmetic

classiﬁcation is based on lower 95% conﬁdence limits.

**Fig. 3. Single farm simulation evaluating the inﬂuence of single outlier
**

horses on the classiﬁcation of the farm into anthelmintic efﬁcacy categories. The farm has 12 horses and one of these is introduced as an outlier

by changing its anthelmintic efﬁcacy as measured by the fecal egg count

reduction test. In the simulation, this horse represented efﬁcacies in the

range of 0.1–1.0. The model-based classiﬁcation is unaffected by this outlier horse, whereas the arithmetic classiﬁcation changes for all efﬁcacy

values of this horse in the range of 0.1–0.9. The horizontal stippled lines

indicate the threshold values for pyrantel efﬁcacy categories used in the

present study.

number is not deﬁned, this would make the model undeﬁned. 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 1–3 and the numerical values were very close to the values presented in these

tables. Further, the classiﬁcations of the 64 farms remained

unchanged (results not included).

3.2. Simulation data: results for false discovery of

reduced farm efﬁcacy rate

The simulations evaluating the marginal efﬁcacy

revealed that amongst 61 farms, 56 farms had an expected

mean efﬁcacy greater than 92%, and 8.74% of them were

wrongly classiﬁed 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 efﬁcacy is evaluated using the conditional deﬁnition.

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 classiﬁcation. 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 classiﬁcation had

an FDRFE of 0 compared to an FDRFE of 0.0547 for the arithmetic classiﬁcation. The model outperformed arithmetic

classiﬁcation in terms of power in detecting truly resistant

farms: 0.7062 versus 0.5135.

3.2.1. Outlier effects

The inﬂuence of a single outlier horse on a single

farm is illustrated in Fig. 3. The ﬁgure clearly shows that

**Author's personal copy
**

620

M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614–622

Table 3

Classiﬁcation of farms according to their anthelmintic efﬁcacy status based on 1000 simulated data sets, each based on 12 farms. Farms are classiﬁed using

the model-based and the arithmetic classiﬁcations. Classiﬁcation results are presented both separated out on each farm, and aggregated across farms.

Farm

True status

1

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 classiﬁcation using the median was unaffected by the outlier, whereas the arithmetic classiﬁcation

is highly driven by the FECR value of the outlier.

4. Discussion

This study provides a ﬂexible model for combining

horse-speciﬁc covariates and random effects on multiple

farms within different regions, and a statistical methodology for evaluating anthelmintic efﬁcacy, which is robust to

the presence of outliers in farms. As a summary statistic

following the model ﬁt, we introduce two new notions of

efﬁcacy, namely, marginal and conditional efﬁcacies. 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 deﬁnitions (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 efﬁcacy 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 94–100% 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 efﬁcacy 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 efﬁcacies. 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 reﬂects both the mean efﬁcacy 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 conﬁdence 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

sufﬁcient, 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

**Author's personal copy
**

M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614–622

**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,

signiﬁcant 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 efﬁcacy 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% efﬁcacy 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 efﬁcacy. 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 signiﬁcantly, meaning that pyrantel efﬁcacy 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 efﬁcacy will tend to

increase with age. There are experimental data suggesting

that anthelmintic efﬁcacy 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 classiﬁcation discrepancies between the modelbased classiﬁcation and the simple arithmetic calculations,

it was clear that removing the horse with the lowest FECR

strongly affected the arithmetic classiﬁcation, while the

model-based classiﬁcation was unaffected. This suggests

that any anthelmintic efﬁcacy classiﬁcation 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

efﬁcacy 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 classiﬁcation provides a

reliable and stable classiﬁcation 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 efﬁcacy should be handled

621

**with care. The overall arithmetic mean efﬁcacy 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 efﬁcacy 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 ﬁrst 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 efﬁcacy (Craven et al., 1998). In Germany,

von Samson-Himmelstjerna et al. (2007) found pyrantel

efﬁcacies in the range of 92–100% and concluded four farms

to be suspect resistant due to mean efﬁcacies below 95%.

A Swedish study reported pyrantel efﬁcacies 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 efﬁcacies below 90% on farms in Italy, United Kingdom and Germany (Traversa et al., 2009). Altogether, farm

pyrantel efﬁcacy levels reported in other European studies

were often in the 80–100% range, and it is a challenge to

delineate between reduced efﬁcacy and chance variations

of FECRT data.

It is often assumed that presence of large strongyle

species can affect the efﬁcacy 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 efﬁcacy 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 efﬁcacies. 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 efﬁcacy estimates would have been

biased toward a higher level. However, further studies are

needed to conﬁrm this notion. Further, other factors possibly affecting parasite populations could be recorded in

**Author's personal copy
**

622

M.K. Nielsen et al. / Veterinary Parasitology 197 (2013) 614–622

**future studies and subsequently included in the statistical
**

model. These could include farm size, stocking rate, breed,

use, level of horse trafﬁc, 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 classiﬁcation 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 efﬁcacy 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 speciﬁc farm.

The methodology used here is not host, worm species, or

drug speciﬁc. Therefore, a similar approach can be adapted

to analyze data from other host species. For instance, in

cattle the efﬁcacy of avermectin/milbemycin anthelmintics

against Cooperia spp. is variable even in the absence of

reduced anthelmintic efﬁcacy (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 efﬁcacy

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 efﬁcacy, 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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