Veterinary Parasitology 171 (2010) 106–110

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Veterinary Parasitology

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Efficacy of pyrantel pamoate and ivermectin paste formulationsagainst naturally acquired Oxyuris equi infections in horses

Craig R. Reinemeyera,∗, Julio C. Pradoa, Eric C. Nicholsb, Alan A. Marchiondob,1

a East Tennessee Clinical Research, Inc., 80 Copper Ridge Farm Road, Rockwood, TN 37854, United Statesb Teva Animal Health, Inc., 3915 South 48th Street Terrace, St. Joseph, MO 64503-4711, United States

a r t i c l e i n f o

Article history:Received 30 December 2009Received in revised form 24 February 2010Accepted 24 February 2010

Keywords:EquineAnthelminticOxyuris equiPyrantelIvermectinResistance

a b s t r a c t

In recent years, numerous veterinary practitioners have reported anecdotal episodes inwhich anthelmintic treatment did not appear to deliver the expected efficacy against equinepinworms (Oxyuris equi). Anthelmintic resistance has not been demonstrated formally inequine pinworms, so a clinical study was designed to evaluate the efficacy of paste formu-lations of pyrantel pamoate or ivermectin against naturally acquired infections with O. equi.Twenty-one horses (>4 months to 15 years of age) with patent, naturally acquired pinworminfections were blocked by source of origin and allocated randomly to one of three treat-ment groups: horses (n = 7) assigned to Group 1 were treated orally with pyrantel pamoatepaste at a dosage of 13.2 mg/kg (2× label dosage), Group 2 horses (n = 7) were untreated con-trols, and horses (n = 7) assigned to Group 3 were treated orally with ivermectin paste at adosage of 200 �g/kg. Fourteen days after treatment, horses were euthanatized, necropsied,and large intestinal contents were processed for recovery of adult pinworms. In addition,duplicate 1% aliquots of intestinal contents from the cecum, ventral colon, dorsal colon,and small colon were collected, preserved, and examined for recovery and enumerationof fourth-stage larval O. equi. Anthelmintic efficacy against pinworms was evaluated bycomparing the post-treatment worm counts of Groups 1 and 3 to those of control animals.Mean numbers of O. equi adults recovered postmortem were significantly decreased by

both pyrantel pamoate (P = 0.0366) and ivermectin (P = 0.0137) treatment, with respectiveefficacies of 91.2% and 96.0%. In addition, both products demonstrated >99% efficacy againstfourth-stage O. equi larvae. The current study demonstrated acceptable adulticidal and lar-vicidal efficacy of both pyrantel pamoate and ivermectin paste formulations against O. equiand did not support the existence of macrocyclic lactone or pyrimidine resistance in the

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1. Introduction

In recent years, numerous practitioners have reportedputative failures of anthelmintic treatment to remove adultpinworm (Oxyuris equi) infections. Some perceived failures

∗ Corresponding author. Tel.: +1 865 354 8420; fax: +1 865 354 8421.E-mail address: crr@easttenncr.com (C.R. Reinemeyer).

1 Current address: Pfizer Animal Health, Inc., 7000 Portage Road, Kala-mazoo, MI 49001, United States.

0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.vetpar.2010.02.042

luated.© 2010 Elsevier B.V. All rights reserved.

were based on persistent anal pruritus after anthelmintictreatment, but tail-rubbing can be caused by non-parasiticfactors. Nonetheless, a portion of these reports includedunequivocal evidence of pinworm survival, such as passageof adult worms in feces several weeks after treatment, orappearance of typical egg masses in the perianal region.

Most anecdotal reports of treatment failure haveinvolved the repeated use of ivermectin in mature horses.The possibility of macrocyclic lactone resistance in O. equihas not been evaluated formally, but adult pinworms havebeen reported to survive treatment with ivermectin paste

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C.R. Reinemeyer et al. / Veterin

dministered at the label dosage of 200 �g/kg (Reinemeyert al., 2006a). Pinworms surviving ivermectin treatmentere subsequently removed by pyrantel pamoate paste

6.6 mg/kg), suggesting that some individual nematodesithin the population were not uniformly susceptible toacrocyclic lactone anthelmintics. Other researchers have

ecently reported the survival of ivermectin treatment bydult and larval pinworms (Lyons et al., 2009).

Anthelmintic resistance in strongyles and ascarids oforses can be detected and monitored by the use of Fecalgg Count Reduction Testing (Coles et al., 1992). However,uantitative egg counting techniques are not applicable to. equi because pinworm eggs are not routinely passed in

eces. Rather, gravid females protrude from the anus andeposit eggs in a sticky film directly onto the anus anderianal skin. Aided by the host’s body temperature, eggs

arvate and become infective within approximately fiveays. Eggs flake off into the environment, and transmis-ion is accomplished via inadvertent ingestion. The usualrepatent period of O. equi is 5 months (Urquhart et al.,996).

To investigate the possibility of anthelmintic resistancen equine pinworms, a clinical study was designed to eval-ate the efficacy of pyrantel pamoate or ivermectin pasteormulations against naturally acquired infections with O.qui.

. Materials and methods

.1. Animals and husbandry

Twenty-one light saddle breed horses, >4 months to 15ears of age, were either born and weaned at the testingacility during 2007 and 2008, or acquired from commer-ial sources during 2008. Foals and mature horses weredentified by uniquely numbered, plastic, leg or neck bands,espectively. Horses were housed individually in 4 m × 4 mtalls constructed of tubular metal panels. Stall flooring wasacked limestone bedded with pine or hardwood shavingsnd sawdust. Horses were offered hay and grain daily at1.5% and 0.5% of body weight, respectively, divided into

imilar portions offered during a.m. and p.m. hours. Drink-ng water was provided ad libitum in 14-L buckets, and waseplenished at least twice daily. Feces were removed daily,nd stall bedding was removed and replaced at approxi-ately weekly intervals.

.2. Experimental design

A blinded, controlled efficacy study was implementedo determine the susceptibility of naturally acquired O. equinfections to pyrantel pamoate or ivermectin, administerednce orally. When three or more horses were concurrentlydentified with patent O. equi infections, candidates werecclimated to experimental conditions for seven days. Ahysical examination of each horse was conducted by a vet-

rinarian at the onset of acclimation (Day −7), and clinicalbservations of general health were conducted by trainedersonnel once daily throughout the acclimation and post-reatment phases of the study. On Day −1, candidates werelocked by source of origin, ranked by ascending identi-

sitology 171 (2010) 106–110 107

fication number, and allocated randomly to one of threetreatment groups. Anthelmintic treatments were admin-istered orally on Day 0 by unblinded personnel. Enrolledhorses were euthanatized 14 days after treatment, andappropriate alimentary samples were collected for enu-meration of adult and larval stages of O. equi.

Masking was achieved by complete separation of duties.Personnel with knowledge of treatment assignments didnot determine any outcome measures (e.g., physical exam-inations, health observations, worm counts), and personnelwho determined outcome measures were unaware oftreatment assignments.

2.3. Treatments

Horses assigned to Group 1 (n = 7) were treated witha 19.13% paste formulation of pyrantel pamoate (Pyran-tel Pamoate Paste®)2 once orally on Day 0 at a targetdosage of 13.2 mg/kg. This dosage was selected because themanufacturer of the pyrantel formulation is systematicallyevaluating its efficacy against various nematode targets inhorses (Reinemeyer et al., 2008). Horses in Group 2 (n = 7)served as untreated controls, and Group 3 horses (n = 7)were treated with a 1.87% paste formulation of ivermectin(Eqvalan Paste®)3 once orally on Day 0 at a target dosageof 200 �g/kg. Individual doses of both products were pre-pared by weight to the nearest 0.1 g, and were based onbody weights measured with a certified livestock scale onthe day before treatment (Day −1). Prior to administrationof paste formulation, the oral cavity of each intended recipi-ent was flushed with tap water to remove any residual hayor feed. Paste was delivered orally, over the back of thetongue, by unmasked personnel.

2.4. Parasitologic parameters

Individual fecal samples were collected from each can-didate on one or more occasions and processed by means ofa sucrose centrifugation flotation technique (Cox and Todd,1962). If O. equi infection could not be confirmed by fecalexamination, each candidate’s perianal region was scrapedwith a tongue depressor coated with lubricant. Capturedskin detritus was transferred to a glass slide and exam-ined microscopically. Eligibility for enrollment was basedon antemortem evidence of patent O. equi infection, usingone or both laboratory methods.

Horses were euthanatized and necropsied 14 days aftertreatment. At necropsy, the cecum, ventral colon, dorsalcolon, and small colon were removed and separated. Eachsegment of gut was opened separately, and the contentswere collected in a suitable container. The mucosal surfaceof each organ was rinsed with water, and the washings

2 Pyrantel Pamoate Paste, Teva Animal Health, Inc. (previously IVX Ani-mal Health, Inc.).

3 Eqvalan, Merial Ltd., Duluth, GA, USA.

ary Parasitology 171 (2010) 106–110

Table 1Counts of adult and fourth-stage larval Oxyuris equi specimens recoveredat necropsy, by treatment group and animal.

Horse I.D. Block GroupA Adults 4th stage larvae

113 1 1 72 2800115 1 1 0 2400141 1 1 0 800116 1 2 222 5000142 1 2 502 5200143 1 2 70 4700117 1 3 0 800144 1 3 505 3500147 1 3 0 1200145 2 1 2 1000276 2 1 88 1300146 2 2 264 7200248 2 2 4 700114 2 3 0 0946 2 3 0 0367 3 1 7 0352 3 2 0 500382 3 3 0 0174 4 1 0 0178 4 2 99 60,000

nificantly lower than the control group mean (51.2), andefficacies were 91.2% and 96.0%, respectively.

Although both anthelmintics demonstrated >99% effi-cacy against fourth-stage larval O. equi, the geometric meanlarval counts of the pyrantel pamoate (38.3; P = 0.0501) and

Table 2Geometric mean counts and percent reduction of worm counts for totaladult and fourth-stage larval Oxyuris equi.

GroupA

1 2 3

Total adults 4.5 51.2 2.1P-value 0.0366 N/A 0.0137PRWCB—Ad 91.2% N/A 96.0%

108 C.R. Reinemeyer et al. / Veterin

in separate, labeled containers and preserved with a simi-lar volume of 10% formalin. The remaining contents wererinsed over #10 (2 mm aperture) sieves and visible adultpinworms were recovered and placed in labeled contain-ers. Formalin solution (10%) was added for preservation,and individual O. equi specimens were examined micro-scopically and counted.

The 1% aliquots of organ contents were examined with30× magnification and fourth-stage larval pinworms wererecovered, counted, and transferred to a labeled containerand preserved in 10% formalin. Numbers of adult and larvalpinworms were recorded and tabulated for analysis.

2.5. Calculations and statistical analysis

Counts of adult and larval O. equi were transformed (i.e.,[loge(count + 1)] prior to analysis of variance. Necropsy dateand necropsy date by group interactions were includedin the model as random effects. The likelihood ratiotest was used to test the assumption of equality ofgroup variances. For each treated group, a one-sided testwas performed with an alternate hypothesis of a largerloge(count + 1) value for controls compared to treatmentgroups. Kenward–Rogers degrees of freedom were used.PROC MIXED of the SAS System, version 9.1.3, was used toperform the analysis. All statistical tests were performed atan �-level of 0.05, using a commercially available softwarepackage (SAS System).4

A conclusion of anthelmintic efficacy was reached ifthe mean, log-transformed worm count was significantlygreater for the control group (Group 2) than for Groups 1or 3, and if percentage efficacy for treated groups was >90%.Percentage efficacy was calculated as

% Efficacy = 100 × GMcontrol − GMtreated

GMcontrol

where GM = geometric mean calculated by back-transforming the least squares means of thelog-transformed counts.

To be consistent with regulatory guidelines foranthelmintic label claims, 90% was stipulated as the lowerrange of acceptable efficacy (Anonymous, 2002). A conclu-sion of anthelmintic resistance was reached if the mean,log-transformed worm counts of Groups 1 or 3 were notsignificantly less than that of the control group and if per-centage efficacy was <80%. Although efficacy <90% wouldnot warrant a label claim, a mean worm count reduction<80% was considered unequivocal evidence of resistance.

Similar analyses were conducted for fourth-stage lar-vae (L4) of O. equi as supporting evidence of anthelminticefficacy or resistance.

3. Results

Twenty-one eligible candidates were enrolled in thestudy, which was conducted as four separate cohorts intime between December, 2007 and November, 2008. Treat-ment records documented that all horses allocated to

4 SAS System, version 9.1.3, SAS Institute Inc., Cary, NC, USA.

176 4 3 4 2700

A Group 1 horses were treated with pyrantel pamoate paste(13.2 mg/kg) on Day 0; Group 2 horses were untreated controls; Group3 horses were treated with Ivermectin (200 �g/kg) on Day 0.

Group 1 received doses of pyrantel pamoate that providedat least 100.4% (range: 100.4–103%; X = 101.5%) of the tar-get dosage, and those in Group 3 received at least 100%(range: 100–111.1%; X = 104.8%) of the target dosage ofivermectin. No adverse health events were noted subse-quent to treatment with either anthelmintic compound.

Total counts of O. equi adults and fourth-stage larvae col-lected at necropsy are presented by block, treatment group,and individual horse in Table 1. Back-transformed, groupgeometric mean worm counts, P-values, and percentageefficacy are presented in Table 2.

The back-transformed, geometric mean counts of adultworms in the pyrantel pamoate- (4.5; P = 0.0366) andivermectin-treated (2.1; P = 0.0137) groups were both sig-

4th stage larvae 38.3 4241.7 43.8P-value 0.0501 N/A 0.0541PRWC—L4 99.1% N/A 99.0%

A Group 1 horses were treated with Pyrantel Pamoate Paste(13.2 mg/kg) on Day 0; Group 2 horses were untreated controls; Group3 horses were treated with Ivermectin (200 �g/kg) on Day 0.

B PRWC = percent reduction of worm counts (Ad, adults; L4, fourth-stagelarvae).

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vermectin (43.8; P = 0.0541) groups did not differ signifi-antly from the control group (4241.7).

Adequate infections with O. equi are not defined in reg-latory guidelines for equine anthelmintic efficacy testingAnonymous, 2002), but 6/7 control horses harbored 4 or

ore adult pinworms, and 7/7 had burdens of >500 fourth-tage larvae (Table 1).

. Discussion

This study determined that both pyrantel pamoate13.2 mg/kg) and ivermectin (200 �g/kg) demonstratedatisfactory adulticidal and larvicidal efficacy against theaturally acquired O. equi populations evaluated. Thisvidence does not support a conclusion of anthelminticesistance.

Pyrantel pamoate demonstrated marginally higher effi-acy against adult pinworms (91.2%) than in a prior report82%) (Lyons et al., 2001), but its activity against larvae>99%) was far superior in comparison to the same study42%). In the referenced trial, pyrantel pamoate was eval-ated at a dosage of 6.6 mg/kg, so it is probable that theuperior performance reported herein was a direct con-equence of increased dosage (13.2 mg/kg). The specificroduct used in this study2 is the only pyrantel pamoateormulation in the U.S. that is labeled for use at 13.2 mg/kg,nd the relevant label indication is specifically for removalnd control of equine cestode infections (Anoplocephalaerfoliata) (Marchiondo et al., 2006; Reinemeyer et al.,006b).

The efficacy of ivermectin against adult or larval pin-orms (96.0% and >99%, respectively) was similar to prior

eports of 99% or greater for both life cycle stages (Kleit al., 2001; Costa et al., 1998). The absence of a signifi-ant difference between mean larval counts of treated andontrol groups was attributed to large variances in wormumbers. Larval populations were evaluated in this studyecause biochemically based resistance should be mani-ested as decreased efficacy against all life cycle stages forhich a label claim has been approved.

Although this study was not conducted with artificialnfections using a characterized isolate of O. equi, nine foalsn Block 1 and three foals in Block 4 were raised in sub-equent years on a premise where incomplete removal ofinworms by ivermectin had been demonstrated. Thus, the2 animals in these two cohorts were likely to harbor sim-

lar isolates.It is difficult to explain perceived anthelmintic failures

hen no evidence of pinworm resistance was demon-trated by the present study. Some of the putative failuresay have been based on non-specific criteria, such as

ersistent anal pruritus after anthelmintic treatment.lternatively, the clinical problems might be attributed tohanges in the biology of Oxyuris infections during recentecades. Historically, pinworms have infected juvenile and

mmature horses almost exclusively. This pattern was so

onsistent that the recovery of one and 22 adult pinwormsrom two geriatric horses (23 and 21 years old, respec-ively) was described as “the most unexpected finding” inne postmortem survey conducted in 2005 (Lyons et al.,006). This may not remain a rare observation, because

sitology 171 (2010) 106–110 109

eight horses with confirmed, patent pinworm infectionsin 2006 averaged 14.0 years of age (range 5–25 years)(Reinemeyer et al., 2006a), and the majority of recent,anecdotal treatment failures have involved adult horses.Acquired immunity is the most obvious explanation whennematode infections are more prevalent in young animals.Thus, altered immunogenicity of Oxyuris populations is onefeasible hypothesis for the apparent expansion of the agedistribution of pinworm infections in recent years.

Additional, putative biological changes for O. equiinclude abbreviation of the life cycle. Classic sources listthe prepatent period of Oxyuris as five months (Urquhartet al., 1996), but one author (CRR) has observed pinwormegg patches in the perianal regions of several foals thatwere only 3.5 months of age. Cyathostomin nematodesof horses have also exhibited temporal modifications oftheir life cycles, with 50% or greater reduction of egg reap-pearance periods in recent years (Lyons et al., 2008, 2009;Molento et al., 2008; von Samson-Himmelstjerna et al.,2007). Presumably, these changes were a direct conse-quence of anthelmintic selection pressure.

It is unrealistic to expect 100% efficacy against mostnematode populations, so equine practitioners may needto adjust their expectations regarding anthelmintic treat-ment. Therapeutic elimination of O. equi may not bepossible, given the apparent, incomplete efficacy of all drugclasses against pinworms. Despite the lack of supportingevidence in the present study, future vigilance for emerginganthelmintic resistance in equine pinworms is warranted.

5. Conclusions and relevance

This study demonstrated that pyrantel pamoate(13.2 mg/kg) and ivermectin (200 �g/kg) paste formula-tions were respectively 91.2% and 96.0% effective againstO. equi adults, and >99% effective against fourth-stage pin-worm larvae. No evidence of resistance to macrocycliclactone or pyrimidine anthelmintics was observed in theO. equi populations evaluated.

Conflict of interest

The work reported herein was funded by Teva AnimalHealth (formerly IVX Animal Health). Two authors are cur-rent (ECN) or former (AAM) employees of Teva AnimalHealth, and assisted with study design and review of themanuscript.

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