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Comparisons of Cyathostome Control and Selection for Benzimidazole Resistance Using Larvicidal Regimens of Moxidectin Gel or Fenbendazole Paste

 Craig R. Reinemeyer, DVM, PhD*

Amy W. Farley, MS*

Bill C. Clymer, PhD†


*East Tennessee Clinical Research, Inc., Knoxville, Tennessee

†Fort Dodge Animal Health, Amarillo, Texas


This study was supported in part by funds from Fort Dodge Animal Health, Overland Park, KS.


KEY WORDS: Anthelmintic, cyathostome, equine, fenbendazole, moxidectin, resistance



Twenty-four 2-year-old male horses with patent cyathostome infections were allocated randomly to 1 of 2 treatment groups. Group 1 was treated with moxidectin oral gel (0.4 mg/kg) on days 0 and 90, and group 2 received fenbendazole paste (10 mg/kg) on days 0, 1, 2, 3, and 4 and again on days 90, 91, 92, 93, and 94. Following initial treatment, the 2 groups were assigned to separate, similar pastures for 181 days. Fecal samples for nematode egg counts and differential coprocultures were collected on days -12, -6, -1, 15, 30, 45, 60, 76, 90, 105, 120, 136, 150, 165, and 181. On day

-1, and at approximately 30-day intervals thereafter, body weights were measured with a certified livestock scale and body condition was evaluated by means of an objective scoring system. Fecal egg count reductions after the first and second treatments with moxidectin were 99.6% and 100%, respectively, compared with 92.9% and 85.6% for fenbendazole at the same intervals. Horses treated with moxidectin had significantly lower cyathostome egg counts (P<0.05) on day 15 and at every interval thereafter for the duration of the trial. Horses treated with moxidectin had significantly higher body condition scores and weight gains (P<0.05) within 60 and 150 days after treatment, respectively. To assess susceptibility to benzimidazole anthelmintics, horses were held on the original, assigned pastures until day 233, when body weights were measured and fecal samples were collected for quantitative egg counts. On day 234, horses in both groups were treated with fenbendazole paste at the adulticidal dosage of 5 mg/kg. Fecal samples were collected on day 244, and pre- and post-treatment egg counts were compared. In the moxidectin-selected group, fenbendazole paste (5 mg/kg) reduced strongylate egg counts by 45.2% at 10 days after treatment. Fenbendazole’s efficacy in the FBZ-selected group was significantly less (P<0.001) at -60.7%. These results indicate that 2 quarterly treatments with a larvicidal regimen of fenbendazole markedly increased the level of benzimidazole resistance in a cyathostome population.


Cyathostomes, also known as small strongyles or cyathostomins,1 are ubiquitous internal parasites of grazing horses. Adult and larval cyathostomes inhabit the equine large intestine, and infections are associated with weight loss, suboptimal growth and performance, colic, and severe diarrhea.2 Cyathostomes are major targets of current equine parasite control measures,3 which consist almost exclusively of anthelmintic treatments to remove worms and reduce transmission.4

All equine anthelmintics developed in the past 50 years have been effective against cyathostomes. Until recently, the spectrum of activity was limited to adult and larval stages in the gut lumen, but larvae in the mucosa were unassailable. Although effective strongyle control can be implemented with dewormers that are strictly adulticidal, it requires rigorous scheduling of treatments to reduce nematode egg production and thus minimize reinfection.5 Arguably, adulticides are suboptimal for cyathostome control because these nematodes inflict their greatest damage before maturity when fourth-stage larvae emerge from fibrous cysts in the lining of the large intestine.2

Larvicidal therapy for cyathostomes became feasible in 1997 with the approval of moxidectin, a milbemycin compound of the macrocyclic lactone class. Moxidectin oral gel was 62.6% to 79.1% effective against encysted, developing (late third stage and fourth stage) cyathostome larvae when administered at 0.4 mg/kg.6 Due in part to its larvicidal activity, moxidectin provided an egg reappearance period (ERP) ranging from 847 to 1688 days after a single treatment. The package insert for moxidectin currently recommends a 90-day interval between successive treatments.

Fenbendazole (FBZ), a substituted benzimidazole (BZD), is also approved as a cyathostome larvicide when administered at 10 mg/kg daily for 5 consecutive days. The multiday regimen of fenbendazole was approximately 92% effective against encysted cyathostomes, and also exhibited 91% to 99% activity against early third stage (EL3) cyathostome larvae.9 The ERP after a larvicidal regimen of FBZ is unknown, but the ERP for the adulticidal dosage of FBZ (5 mg/kg once) was less than 6 weeks when used against BZD-susceptible worms.10

The susceptibility of target cyathostome populations to various anthelmintics is an issue of growing concern. Recent surveys in the southern United States11-13 report that more than 90% of cyathostome populations tested were resistant to the adulticidal dosage of FBZ. However, the efficacy of the larvicidal regimen of FBZ against BZD-resistant cyathostomes has not been evaluated.

An initial trial was designed and implemented to compare the effects of larvicidal regimens of moxidectin and FBZ, administered twice at quarterly intervals, on parasitologic and production parameters in young horses. Subsequently, when parasitologic data suggested a decline in the efficacy of FBZ, a supplemental study was conducted to characterize the levels of benzimidazole resistance in 2 cyathostome populations with different anthelmintic selection histories.

Materials and Methods

Trial 1: Comparison of Parasitologic and Production Parameters

Animals and management: In January, 2001, 24 male Tennessee Walking horses, approximately 2 years of age, were acquired from a single source where they had grazed together for several weeks. Horses were transported to the test facilities and acclimated for 2 weeks before treatment. Facilities consisted of 2 approximately 12-acre, fescue/clover pastures that were similar in forage quality and quantity, exposure, drainage, soil type, and available shade.

Horses were pastured for the duration of the study and had constant access to forage or grass hay and trace-mineralized salt. A commercial horse concentrate (approximately 0.5 lb/cwt body weight) was fed once daily during winter and on alternate days during spring and summer. Potable water was supplied by spring-fed ponds in each pasture.

Allocation and treatment: Horses were eligible for enrollment if they were clinically healthy and had patent cyathostome infections. Candidate horses were ranked by decreasing strongylate egg counts using fecal samples collected on day -1. Each successive pair of horses was considered a replicate, and horses within a replicate were assigned randomly to 1 of 2 treatment groups. Horses in group 1 were treated twice with moxidectin (Quest Gel, Lot No. 744324, Expiry date, July, 2002) (0.4 mg/kg) oral gel on days 0 and 90. Horses in group 2 received larvicidal regimens of FBZ (Panacur Powerpac Paste, Lot No. RCFF, Expiry date, February, 2004) (10 mg/kg daily for 5 consecutive days) on days 0, 1, 2, 3, and 4, and again on days 90, 91, 92, 93, and 94.

Individual doses of moxidectin for horses in group 1 and FBZ for group 2 were based on contemporaneous body weights measured on days -1 or 90. The required doses of each compound were prepared by adjusting commercial syringes to dispense an amount slightly greater than the measured body weight. Gel and paste formulations were administered orally, and syringes were weighed before and after treatment to document the doses of moxidectin and FBZ delivered. After initial treatment, the 2 groups of horses were assigned to separate, similar pastures, where they remained for the duration of the study.

Production parameters: Body weights were measured with a certified livestock scale on days -1, 30, 60, 90, 120, 150, and 181. On the same or proximate days, a technician who was blinded to treatment assignments evaluated the body condition of each horse and assigned an objective score based on a previously described grading system.14

Parasitologic parameters: A fecal sample was collected from each horse on Days -12, -6, -1, 15, 30, 45, 60, 76, 90, 105, 120, 136, 150, 165, and 181. Quantitative egg counts were performed by a modification of the Stoll technique,15 and results were reported as strongylate eggs per gram (EPG) of feces. Laboratory personnel performing egg counts were blinded to treatment assignments.

Individual coprocultures were prepared from fecal samples collected 12 days before initial treatment. Feces were incubated at approximately 78°F for 8 to 10 days, and the resulting larvae were harvested by the Baermann technique.15 Up to 100 third-stage strongylate larvae were identified to subfamily (Cyathostominae), genus (Triodontophorus), or species (Strongylus vulgaris, S. edentatus, or Trichostrongylus axei), and their proportional contributions to fecal contamination were calculated. On days 30, 60, 90, 120, and 181, all positive fecal samples within a treatment group were pooled and processed for coproculture, as described previously.

Calculations and analyses: Analyses were conducted using Statistical Analysis System software (SAS Version 8; SAS Institute, Cary, NC). An individual animal was considered the experimental unit in all analyses, and P<0.05 was selected as the level of significance.

Body weight was analyzed using a model that contained treatment group as a variable. The analysis of day 0 body weight contained only treatment in the model, whereas the analyses of subsequent body weights used initial body weight as a covariate in addition to treatment. The categorical variable of body condition score was analyzed using Chi-square analysis (Friedman).

Egg counts were transformed [log(count + 1)] before analysis by repeated measures analysis of variance. Using egg counts of samples collected at the first interval after each treatment, efficacy was calculated as fecal egg count reduction by Abbott’s formula:


% efficacy =                                                      100.


Trial 2: Selection for
Benzimidazole Resistance

Animals: After completion of the comparison trial, both groups remained on their originally assigned pastures for an additional 7 weeks, pending the resumption of strongyle patency in the majority of horses.

Treatment: Body weights were measured with a certified livestock scale on day 233 and used to calculate doses of FBZ (Panacur Paste, 100 mg/mL; Lot No. NGLM, Expiry date, June, 2002) based on the adulticidal dosage of 5 mg/kg. Individual doses were prepared by transferring a weighed quantity of FBZ paste to a 35-mL Monoject syringe that was labeled with the identification number of the corresponding recipient. The entire dose of paste was administered orally on day 234, and syringes were weighed before and after treatment to document the dose of paste article administered to each animal.

Parasitology: Fecal samples were collected on days 233 and 244, and strongylate eggs were counted using a modification of the Stoll technique.15 Individual coprocultures were prepared from the sample collected on day 244, using methods described previously.

Calculations and analyses: Fecal egg counts were evaluated using analysis of variance with pretreatment egg counts as a covariate. Efficacy (egg count reduction) was calculated as described previously.


Trial 1

The practice of adjusting the commercial syringes to deliver slightly more gel or paste than indicated for a horse’s measured weight resulted in consistent overdosing of both products. Moxidectin gel and FBZ paste were administered at approximately 114% and 118% of the intended doses, respectively.

Mean fecal egg counts were higher in the FBZ group on day -1, but the difference was not significant (Table 1). The lowest mean egg count (20 EPG) of the FBZ group was observed at the first sampling interval after initial treatment, and represented 92.9% efficacy. In comparison, the fecal egg count reduction by moxidectin was 99.6% for the same interval. Efficacies of FBZ and moxidectin on day 105 (15 days after initiation of the second larvicidal regimen) were 85.6% and 100%, respectively. Egg counts of the moxidectin group were significantly lower (P<0.05) than those of FBZ-treated horses from day 15 thru day 181 (Table 1).

Coproculture of individual fecal samples collected on day -12 revealed that the majority of strongylate eggs were produced by cyathostomes and Triodontophorus spp., with varying contributions from S. edentatus and S. vulgaris. On at least 3 occasions after treatment, fecal egg counts of horses treated with moxidectin were uniformly zero and no coprocultures were prepared. Coprocultures of pooled monthly positive fecal samples yielded nearly 100% cyathostomes. During June, however, approximately 2% of the strongylate eggs passed by FBZ-treated horses were Trichostrongylus axei.

Baseline body weights of both groups were similar on day -1 (Table 2). Horses treated with moxidectin weighed significantly more (P<0.05) than FBZ-treated animals on days 150 and 181, and exhibited a 34-lb advantage in body weight at the conclusion of the trial.

Mean body condition scores on day 0 were similar in both treatment groups (Table 3). By day 60, however, moxidectin-treated horses earned significantly higher body condition scores (P<0.05) than those treated with FBZ. The body condition advantage in the moxidectin group remained significantly different for the duration of the study.

Trial 2

Although all 24 horses were treated on day 234, data from only 11 members of each group were used for analysis. One horse in group 1 had 0 EPG on day 233, and no fecal sample could be collected on day 244 from one horse in group 2. Calculations based on pre- and post-treatment syringe weights determined that each horse received at least 5 mg FBZ per kg body weight.

Mean fecal egg counts were higher in group 1 horses on day 233, but the difference was not significant (Table 4). In the moxidectin-selected group, FBZ paste (5 mg/kg) reduced strongylate fecal egg counts by 45.2% at 10 days after treatment. In comparison, the adulticidal efficacy in the FBZ-selected group was significantly less (P<0.001) at -60.7%.

Coprocultures of individual fecal samples collected on day 244 revealed that cyathostomes produced 100% of the strongylate eggs passed after treatment.


Differences in anthelmintic performance between treatment groups were attributed to BZD resistance in the target cyathostome populations. Evidence of resistance was apparent as early as day 15, when fecal egg count reduction by FBZ was 92.9%. Although this level of efficacy exceeds the accepted standard of 90% for differentiating susceptible from resistant populations,16 it was achieved with a larvicidal regimen incorporating 10 times more FBZ than the adulticidal dosage of 5 mg/kg administered once. The diminished efficacy of FBZ (85.6%) after the second quarterly regimen suggested an increase in the level of BZD resistance in group 2.

Genetic combinations that impart resistance are initially rare within a population, but their frequency increases whenever resistant worms are able to reproduce in the absence of competition from susceptible strains. Such intervals of reproductive advantage follow each use of a resistance-prone dewormer, so selection for resistance is intensified and accelerated by repeated and frequent treatments.17

The proportion of a parasite population that is not exposed to anthelmintic treatment constitutes a reservoir of susceptible genes and is described as being “in refugia.”17,18 Selection for resistance is facilitated by minimizing the refugia. Theoretically, larvicidal regimens may select more intensively for anthelmintic resistance because they exhibit activity against more parasitic stages than adulticidal doses, and thereby effectively reduce the refugia.

Studies with nematodes of sheep have demonstrated that resistant worms can be overwhelmed temporarily by increasing the dosage or frequency of BZD treatments.19 However, this is a short-lived strategy that ultimately selects for higher levels of resistance, as confirmed in our second study by the significantly lower egg count reduction in the FBZ-selected population.

Improved body condition scores and weight gains were observed in horses treated with moxidectin, putatively through superior removal of adult and larval small strongyle populations. The typical cyathostome life cycle presents opportunities for at least 2 different pathologic events. Major harm results when fourth-stage larvae emerge from mucosal cysts in the cecum and colon. Larval emergence causes mechanical damage, but also releases waste materials sequestered within the cyst, resulting in focal or general typhlitis and colitis.2 Because larvicides kill encysted larvae in situ, moxidectin treatment could obviate the inflammatory consequences of larval emergence.

The other pathologic event occurs in grazing horses when recently ingested third-stage larvae invade the mucosa of the large intestine.2 Moxidectin at quarterly intervals could ameliorate this condition by suppressing egg production for up to 3 months after treatment, thereby reducing the numbers of infective larvae acquired from pasture. Quarterly moxidectin represents a suppressive treatment strategy, and similar performance benefits were observed when ivermectin was administered at 60-day intervals to approximate its ERP.20

Assuming that both mucosal invasion and larval emergence affect equine performance adversely, grazing horses would experience optimal benefits from moxidectin if treatments were timed to minimize pasture contamination with worm eggs.

Some authorities have recommended that susceptibility to BZDs should be shown before using this drug class against cyathostomes.11,12 The results reported here support extending that caveat to larvicidal uses of BZDs as well.


Horses treated twice with moxidectin (0.4 mg/kg) at 90-day intervals had significantly lower egg counts, higher body condition scores, and greater average weight gains than horses treated at the same intervals with FBZ (10 mg/kg) administered daily for 5 consecutive days. Consecutive quarterly treatments with a larvicidal regimen of FBZ markedly increased the level of BZD resistance in a cyathostome population.


1. Lichtenfels JR, Gibbons LM, Krecek RC: Recommended terminology and advances in the systematics of the Cyathostominea (Nematoda:Strongyloidea) of horses. Vet Parasitol 107:337–342, 2002.

2. Love S, Murphy D, Mellor D: Pathogenicity of cyathostome infection. Vet Parasitol 85:113–122, 1999.

3. Herd RP: The changing world of worms: The rise of the cyathostomes and the decline of Strongylus vulgaris. Compend Contin Ed Practic Vet 12:732–736, 1990.

4. Kaplan RM: Anthelmintic resistance in nematodes of horses. Vet Res 33:491–507, 2002.

5. Drudge JH, Lyons ET: Control of internal parasites of the horse. J Am Vet Med Assoc 148:378–383, 1966.

6. Xiao LH, Herd RP, Majewski GA: Comparative efficacy of moxidectin and ivermectin against hypobiotic and encysted cyathostomes and other equine parasites. Vet Parasitol 53:83–90, 1994.

7. DiPietro JA, Hutchens DE, Lock TF, et al: Effect of moxidectin oral gel on post–treatment strongyle egg count suppression in horses, in Proceedings, American Association of Veterinary Parasitologists. 41st Annual Meeting, Louisville, KY, p. 32, 1996.

8. Jacobs DE, Hutchinson MJ, Parker L, Gibbons LM: Equine cyathostome infection: suppression of faecal egg output with moxidectin. Vet Rec 137:545, 1995.

9. DiPietro JA, Klei TR, Reinemeyer CR: Efficacy of fenbendazole against encysted small strongyle larvae, in Proceedings, American Association of Equine Practitioners. Phoenix, AZ, pp. 343–344, 1997.

10. Herd RP, Miller TB, Gabel AA: A field evaluation of pro-benzimidazole, benzimidazole, and non-benzimidazole anthelmintics in horses. J Am Vet Med Assoc 179:686–691, 1981.

11. Woods TF, Lane TJ, Zeng QY, Courtney CH: Anthelmintic resistance on horse farms in north central Florida. Equine Pract 20:14–17, 1998.

12. Tarigo-Martinie JL, Wyatt AR, Kaplan RM: Prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses. J Am Vet Med Assoc 218:1957–1960, 2001.

13. Kaplan RM, Klei TR, Lyons ET, et al: Prevalence of anthelmintic resistance on horse farms in the southern United States, in Proceedings. 47th Annual Meeting, American Association of Veterinary Parasitologists, Nashville, TN, July 13–16, 2002. 

14. Henneke DR, Potter GD, Kreider JL, Yeates BF: Relationship between condition score, physical measurement and body fat percentage in mares. Equine Vet J 15:371–372, 1983.

15. Klei TR: Laboratory diagnosis. Vet Clin N Am, 2:381–393, 1986.

16. Coles G, Bauer C, Borgsteede F, et al: World Association for the Advancement of Veterinary Parasitology (WAAVP). Methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol 44:35–44, 1992.

17. Sangster NC: Pharmacology of anthelmintic resistance in cyathostomes: Will it occur with the avermectin/milbemycins? Vet Parasitol 85:189–204, 1999.

18. Van Wyk JA: Refugia: Overlooked as perhaps the most potent factor concerning the development of anthelmintic resistance. Onderstepoort J Vet Res 68:55–67, 2001.

19. Hennessy DR: Modifying the formulation or delivery mechanism to increase the activity of anthelmintic compounds. Vet Parasitol 72:367–382, 1997.

20. Reinemeyer CR, Clymer BC: Comparative efficiency of moxidectin gel or ivermectin paste for cyathostome control in young horses. J Equine Vet Sci 22:33–36, 2002.


 (Pretreatment EPG) - (Post-treatment EPG)


(Pretreatment EPG)



Table 1. Mean Fecal Egg Counts (EPG) by Treatment Group and Sampling Day


  Study     Moxidectin     Fenbendazole

  Day             Gel                 Paste            P level


  -1               222                  281             0.6090

  15               0.8*                  20†             0.0166

  30                0*                   58†             0.0019

  45               2.5*                  94†             0.0005

  60               11*                 568†           <0.0001

  76               21*                 860†           <0.0001

  90               18*                 737†           <0.0001

  105              0*                  106†            0.0002

  120              0*                   93†            <0.0001

  135             0.8*                 197†           <0.0001

  150              0*                  418†           <0.0001

  165             4.2*                 374†           <0.0001

  181             34*                 516†            0.0004


  *†Within a row, means with dissimilar symbols were significantly different at P<0.05.




The International Journal of Applied Research in Veterinary Medicine • Vol. 1, No. 1, Winter 2003


Table 2. Mean Body Weights of Horses by Treatment Group and Study Day


Table 3. Mean Body Condition Scores by Treatment Group and Study Day


  Study     Moxidectin     Fenbendazole

  Day             Gel                 Paste            P level


  -1               738                  732             0.8348

  30               744                  751             0.5948

  60               773                  760             0.2986

  90               791                  781             0.2489

  120             842                  824             0.1268

  150            881*                850†            0.0040

  181            895*                861†            0.0128


  *†Within a row, means with dissimilar symbols were significantly different at P<0.05.


  Study     Moxidectin     Fenbendazole

  Day             Gel                 Paste            P level


  0                  4.3                   4.5             0.4575

  30                4.2                   3.8             0.3813

  60               4.7*                 4.2†            0.0358

  90               4.7*                 4.2†            0.0358

  120             5.1*                 4.2†            0.0035

  150             5.3*                 4.6†            0.0051

  181             5.1*                 4.6†            0.0223


  *†Within a row, means with dissimilar symbols were significantly different at P<0.05.


Table 4. Pre- and Post-Treatment Fecal Egg Counts


          Moxidectin-Selected (Group 1)                                       Fenbendazole-Selected (Group 2)

  Horse     Day 233        Day 244          Percent                   Horse       Day 233       Day 244        Percent

    ID           EPG              EPG             Efficacy                     ID             EPG             EPG          Efficacy


  12              230               140                39.1                        10               60               120            -100.0

  13              340                60                 82.4                        18              550              170              69.1

  15              430               210                51.2                        19              360              290              19.4

  16              560               110                80.4                        21              110              200             -81.8

  17              120               100                16.7                        24              180              330             -83.3

  22              290               300                -3.4                        25              160              310             -93.8

  27              120               100                16.7                        29              360              220              38.9

  31              370               190                48.6                        32              230              550            -139.1

  34              180               100                44.4                        33               70               290            -314.3

  157            620               490                21.0                        35              470              450               4.3

  162             60                  0                 100.0                       37              230              200              13.0

  Mean        301.8            163.6            45.2%*                   Mean          252.7           284.5         -60.7%†


  *†Within a row, means with dissimilar symbols were significantly different at P<0.001. 

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