Journal of the American Association for Laboratory Animal Science Vol 45, No 3 Copyright 2006 May 2006 by the American Association for Laboratory Animal Science Pages 23-26

Efficacy and Safety of Topical Selamectin to Eradicate Pinworm (Syphacia spp.) Infections in Rats (Rattus norvegicus) and Mice (Mus musculus)

William Allen Hill,1,* Mildred Montgomery Randolph,1 Sharon Jean Lokey,1 Ernestine Hayes,1 Kelli Lynn Boyd,2 and Timothy David Mandrell1

We evaluated the efficacy and safety of topical selamectin, a novel avermectin, in eliminating naturally acquiredSyphacia muris infections in rats and S. obvelata infections in mice. S. muris-positive rats were assigned randomly to 4 groups: selamectin (0.6 mg/kg), selamectin (6.0 mg/kg), fenbendazole-medicated (150 ppm) chow, and untreated. S. obvelata-positive mice were allocated into 4 groups similar to those for rats. Animals not exposed to pinworm-contaminated bedding were designated as negative controls. Treatment success was assessed weekly by anal tape impressions and by necropsy examinations at the end of week 9. Evaluations of intestinal contents at necropsy revealed that, although safe, topical selamectin was 100% ineffective in eliminating Syphacia spp. infections in rats and mice. Treatment with fenbendazole-medicated chow resulted in negative anal tape impressions beginning at week 2 in rats and week 1 in mice. Negative anal tape impressions in fenbendazole-treated animals were confirmed by negative intestinal content evaluations. Of the 2 treatments evaluated, fenbendazole-medicated chow remains an effective and practical method to eliminate pinworm infections in mice and rats.

Abbreviation: GABA, gamma aminobutyric acid

The rat pinworm, Syphacia muris, and the mouse pinworm, barriers. Young mice have been reported to develop whole body S. obvelata, inhabit the cecum and colon of rodents and are seizures or tremors after ivermectin treatment. Central nervous common contaminants in contemporary laboratory animal systems signs were attributed to increased sensitivity of suckling facilities.13 Although infections are generally nonpathogenic in rodents to ivermectin due to an immature blood–brain barrier immunocompetent animals, pinworm infections may have del- and a high concentration of the drug in maternal milk.20 Other eterious effects on behavior, growth, intestinal physiology, and strains sensitive to ivermectin toxicosis include subpopulations immunology.10,12,16,21 These effects and the demand for defined of CF-1 mice and mdr1a (Abcb4) and mdr1b (Abcb1) knockout experimental animals, possible hindrances to interinstitutional mice.19 Ivermectin overdose in a group of BALB/c mice resulted collaborative research, and increased operating cost associated in microvesicular fatty changes in the liver and diffuse tubular with treatment and environmental decontamination make necrosis.6 Ivermectin can also affect some behaviors in mice effective pinworm eradication of paramount importance for and rats.19 In contrast, fenbendazole is usually administered many facilities. Rederivation of rodents via hysterectomy and in a commercially milled rodent chow. Therefore, the drug Caesarian section or embryo transfer remains the ‘gold stan- concentration in the diet and the delivery schedule may not dard’ of pathogen eradication.19 However, in many instances, deliver consistent therapeutic doses, thereby permitting some rederivation is not practical, and the selection of appropriate animals to harbor pinworms and serve as a nidus for continued anthelmintics should include evaluation of drug safety, efficacy, proliferation and dissemination.7 and mechanism of action, as well as assessment of potential Since the late 1990s, progress in the area of small animal para- compromise to current or future uses of the animals.4 site control has resulted in the creation of synthetic avermectin Avermectins and benzimidazoles are currently the most analogs. Selamectin, 1 of these new analogs, has a spectrum of widely used anthelmintics for pinworm eradication. In veteri- activity encompassing a broad range of external and internal nary literature, the avermectins, which paralyze parasites by parasites of dogs and cats.2 Topically administered selamectin increasing Cl– permeability in the cell membranes of peripheral has been shown to be safe and effective in cats,2,9 rabbits,11 nerve tissues and possibly potentiating gamma aminobutyric and dogs, including Collies, some of which are sensitive to acid (GABA)-gated sites,5 are usually represented by ivermectin. ivermectin toxicosis.2,15 In an uncontrolled study, Winchester The benzimidazoles, mainly represented by fenbendazole, bind and colleagues reported selamectin (6 mg/kg) to be effective in to nematode B-tubulin and inhibit microtubule formation.5 treating S. obvelata and Aspicularis tetraptera infections in mice.24 Despite their wide use, both ivermectin and fenbendazole Labeled for once-monthly use, selamectin could represent a safe, are fraught with problems. Toxicity can result from ivermectin cost-efficient method for treating pinworm infection in rats and treatment, especially in animals with compromised blood-brain mice, especially those refractory to ivermectin treatment.

Received: 10 Nov 2005. Revision requested: 15 Dec 2005. Accepted: 15 Dec 2005. Materials and Methods 1 Department of Comparative Medicine, College of Medicine, The University of Tennes- Animals. Rats. We obtained 50 male, 24-d-old, Crl: SD rats see Health Science Center, Memphis, Tennessee; 2St Jude Children’s Research Hospital, Animal Resources Center, Memphis, Tennessee. (Charles River Laboratories, Wilmington, MA). Although ini- *Corresponding author. Email: [email protected] tially specific pathogen-free, 40 rats acquired patent S. muris

23 Vol 45, No 3 Journal of the American Association for Laboratory Animal Science May 2006 infections as a result of 3-wk continuous exposure to pinworm- between the scapulae by using an automatic pipette. Fenben- contaminated bedding from an in-house colony of parasitized dazole-treated animals were offered fenbendazole-medicated rats. Colony animals were antibody-negative for sialodacryoad- chow (Rodent Diet 962018 milled with 150 ppm fenbendazole, enitis virus, Sendai virus, lymphocytic choriomeningitis virus, Harlan Teklad Global Diets, Wilmington, DE) every other week rat parvoviruses, reovirus 3, Theiler’s murine encephalomyelitis for a total of 9 wk (5 wk of treatment). virus, and Mycoplasma pulmonis. In addition, colony animals Mice were randomized into 4 groups containing 10 animals were free of all other parasites. Syphacia muris infection was each: negative control, positive control, selamectin (6 mg/kg)- confirmed in individual rats by cellophane tape impressions of treated, and fenbendazole-treated. Selamectin was diluted with the anus. For each evaluation, 3 impressions of the anus were ethanol (final concentration, 2.5 mg/ml) and applied ondays made. Anal tapes were mounted on glass slides and examined 0 and 30 as described for rats. Fenbendazole-medicated chow microscopically for ova under a 4t objective lens. A single was offered as previously described for rats. investigator (WAH) performed all microscopic examinations; Treatment efficacy and safety. All rodents were weighed all negative tapes were confirmed by a 2nd investigator (SJL). and tested for pinworm ova weekly by perianal cellophane Animals were included in the study only if they had a positive tape impression. On days 2 and 15, rats were anesthetized tape test on the day treatment was initiated (day 0). by inhalational isoflurane, and blood was collected from Mice. We obtained 40 male, 24-d-old, specific pathogen-free the retro-orbital sinus for hematology and serum chemis- Crl: CD1 mice (Charles River Laboratories, Wilmington, MA); 30 try profiles. Nine weeks after the start of treatment, allrats mice acquired patent S. obvelata infections as a result of a 6-wk were anesthetized as described, and blood was collected by continuous exposure to pinworm-contaminated bedding from intracardiac puncture. Animals were not allowed to recover an in-house colony of parasitized mice. Colony animals were but were euthanized by cervical dislocation, and necropsy antibody-negative for the following viral and bacterial patho- examination was performed. Skin, liver, spleen, kidney, testis, gens: mouse hepatitis virus, Sendai virus, pneumonia virus thymus, heart, lung, and gastrointestinal tract were harvested of mice, reovirus 3, Theiler’s murine encephalomyelitis virus, and fixed in 10% neutral buffered formalin. Tissues were ectromelia virus, M. pulmonis, mouse parvovirus, mouse minute processed routinely and embedded in paraffin, and 4 Nm sec- virus, mouse rotavirus, and lymphocytic choriomeningitis virus. tions were cut and stained with hematoxylin and eosin. Light Mice were also free of other parasites. Infection with S. obvelata microscopic examination of the tissues was performed by an was confirmed in individual mice by microscopic evaluation of experienced veterinary pathologist (KLB). cellophane anal impressions as described earlier. Mice were euthanized and necropsied as described earlier. Husbandry. All studies were conducted in 2 separate, identical According to a method similar to that described by Battle and cubicles in a single room at the University of Tennessee Health others,1 cecal content examinations were performed in all ani- Science Center (Memphis, TN). Animals were singly housed in mals by longitudinally opening the cecum and colon from the static, polysulfone, isolator cages (Alternative Design Manufac- ileocecal junction to the rectum and washing that portion of turing and Supply, Siloam Springs, AR) on autoclaved, contact, the gastrointestinal tract with 200 ml water. The fecal mixture hardwood bedding (Northeastern Products, Warrensburg, NY) was passed through a 5-in. culinary strainer, and the result- and maintained on a 12:12-h light:dark cycle at 22 q 0.5 pC. ing filtrate was passed through a 3-in. culinary strainer. The Caging, food, and water bottles were changed weekly under remaining filtrate was passed through a 100-Nm screen (Miami strict aseptic technique within a biological safety cabinet (model Aqua-culture, Miami, FL). The screen then was washed with NU-425-600, NuAire, Plymouth, MN). All cages and implements 10 ml water. A 2-ml aliquot of the wash solution was placed were washed in a mechanical washer with a final rinse at 82.2 pC in a RODAC plate (Becton Dickinson Labware, Lincoln Park, and were autoclaved at 121.1 pC for 15 min prior to entry into NJ) and stained with iodine to facilitate worm counting. Worm the room. A temperature recording label (Temp-Tape 180, Phar- counts were multiplied by the dilution factor of 5. macal Research Laboratories, Naugatuck, CT) was used daily Data analysis. With use of a statistical software program to ensure final rinse temperature. Steam chemical integrators (GraphPad Prism, GraphPad Software, 161, San Diego, CA), (SteriGage LR, 3M, St Paul, MN) were used with each autoclave mean worm counts were compared using unpaired 2-tailed cycle to ensure sterility; quality control was further assessed Student t tests. The threshold for statistical significance was monthly using a biological indicator (Verify,Steris, Mentor, OH). set at P  0.05. Animals were provided acidified water (pH 3.0) and irradiated, pelleted, rodent chow (Rodent Diet 7912, Harlan, Indianapolis, Results IN) ad libitum except during fenbendazole treatment periods. Rats. In the negative control group, 8 of 10 rats survived This study was conducted pursuant to an institutional animal the 9-wk treatment period. One rat was euthanized via CO2 care and use committee-approved protocol. All animals were overdose during week 1 due to traumatic eye injury. Another housed, cared for, and used in compliance with the Guide for rat died during week 2 as a result of unintentional anesthetic 14 the Care and Use of Laboratory Animals in a program accredited overdose. All remaining rats survived until the conclusion of by the Association for the Assessment and Accreditation of the study. Laboratory Animal Care, International. During the treatment period, 574 perianal tape impressions Treatment allocation. Rats were randomized into 5 groups; were evaluated. Rats treated with topical selamectin had S. muris- each group contained 10 animals. Animals not exposed to positive perianal tape impressions throughout the entire 9-wk pinworm-contaminated bedding were designated as negative period. At necropsy, the worm burden (mean q standard error controls. Pinworm-positive animals were assigned to the posi- of the mean) was 31 q 13.31 for rats treated with 0.6 mg/kg se- tive control (no treatment) or selamectin (0.6 mg/kg), selamectin lamectin and 32.5 q 15.94 for those given 6.0 mg/kg selamectin. (6.0 mg/kg), or fenbendazole treatment group. Animals treated The worm burden for positive control animals was 11.5 q 2.48. with selamectin (Revolution, Pfizer Animal Health, Exton, PA) Of the 10 rats treated with 150 ppm fenbendazole-medicated received topical application of the drug (diluted in 70% ethanol chow, 4 began to have S. muris-negative perianal tapes after 1 to yield 10 mg/ml) on days 0 and 30. Selamectin was applied wk of treatment. At the end of week 2, all fenbendazole-treated 24 Selamectin for treating pinworm infections in rats and mice

animals had negative anal tapes, which remained negative In contrast to fenbendazole, selamectin is believed to work through the duration of the study. Moreover, no worms were by enhancing chloride permeability or enhancing the release recovered from the cecum and colon of the negative control of GABA at presynaptic neurons. In nematodes, GABA acts as and fenbendazole-treated animals. No hematologic or serum neurotransmitter that blocks postsynaptic stimulation of the chemistry abnormalities were observed in any of the 48 animals adjacent neuron. The release of GABA subsequently causes (results not shown). In addition, there were no noteworthy paralysis and eventual death of the parasite.18 histologic lesions. After topical administration of 12 mg/kg selamectin to the Mice. Of the 40 mice, 38 survived the 9-wk treatment period. shaved skin of rats, the maximal plasma concentration was Anegative control animal was found dead during week 1 due to achieved within 24 h; the elimination half-life was 60 h.17 Con- traumatic injury.Afenbendazole-treated animal was euthanized versely, in rats, a single oral dose of ivermectin results in peak during week 3 because of urine scalding. During the study, 384 plasma levels at 4 to 8 h postdosing, with a half-life of 24 h.3 perianal tape impressions were evaluated. Two mice treated The inefficacy of selamectin in rats and mice may be attributed with 6 mg/kg topical selamectin had intermittently negative S. to the drug’s bioavailability and rapid elimination after topical obvelata perianal tape impressions. One mouse had a negative administration. Acomparison of the drug’s efficacy in dogs and perianal tape impression beginning in week 3 and continued cats may illustrate this phenomenon. After topical selamectin until week 6. The other mouse had a negative perianal tape in administration in dogs, about 5% of the drug is bioavailable, week 9. Worms were recovered from both mice on postmortem and peak plasma levels occur about 3 d later; the elimination examination, confirming S. obvelata infection. At necropsy, the half-life is about 11 d. In contrast, after topical administration worm burden recovered from mice treated with selamectin was to cats, about 75% of the drug is bioavailable and peak plasma 50.5 q 14.42. The worm burden of positive control animals was levels occur about 15 h later; the elimination half-life is about 14.5 q 3.98. All mice treated with 150 ppm fenbendazole-medi- 8d.18 In cats, topically applied selamectin has been shown to cated chow began to have S. obvelata-negative perianal tapes at provide greater than 95.0% inhibition of nematode ova output week 1, which remained negative through the duration of the as determined by fecal sugar flotation. study. No worms were recovered from the cecum and colon of At necropsy, topical treatment was more than 98.0% effec- the negative control and fenbendazole-treated animals. tive against Ancylostoma tubaeforme and 100% effective against Toxocara cati. In dogs, selamectin was 100% effective against A. canium, Uncinaria stenocephala, T. canis, and T. leonia only when Discussion 2 Topical selamectin dosed at 0.6 and 6 mg/kg was 100% administered orally. As a result, topical selamectin is indicated ineffective at eliminating S. muris in rats. In addition, topical only for use against A. tubaeforme and T. cati in cats. Selamectin selamectin dosed at 6 mg/kg was 100% ineffective at eliminat- has no known ovicidal effects; as a result, reinfection from en- ing S. obvelata in mice. The 0.6 mg/kg selamectin dose trial was vironmental sources may be a cause of treatment failure. not conducted in mice after 6 mg/kg selamectin was shown to Another reason for treatment failure may be inadequate drug be ineffective in eliminating S. muris in rats. All rats and mice dosage or improper administration frequency. The recommend- treated with selamectin had confirmed pinworm infections at ed topical selamectin dose for dogs and cats is 6 mg/kg applied 18 necropsy. once monthly. In addition, Winchester and others reported us- Winchester and others had shown topical selamectin (6 mg/kg) ing a one-time dose of 6 mg/kg to eliminate pinworm infections 24 to be effective in treating S. obvelata and Aspicularis tetraptera in- in mice. In light of the manufacturer’s dosing recommendations fections in mice. In the Winchester study, 6 mice were randomly in dogs and cats and the work of Winchester and others, the selected for 1 treatment with selamectin. After 6 wk, all treated maximal dose we evaluated was 6.0 mg/kg given every 30 d. animals were pinworm-negative via tape tests. Treated animals We conclude that doses of 6.0 mg/kg or less are ineffective at remained tape test-negative after cohabitating with pinworm- eliminating pinworm infections, and further study using higher positive animals for an additional 6 wk. Tape test evaluations doses, increasing the frequency of administration, or decreasing were confirmed by negative microscopic evaluations of cecal the time interval between administrations may be warranted. A contents.24 Our report does not corroborate the findings of similar effect was observed with topical ivermectin treatment. Winchester and others. In our study, 2 mice in the selamectin- A single micro-dot administration of 2 mg/kg of ivermectin treated group did have intermittently negative perianal tape applied between the scapulae was ineffective at eliminating impressions. Intermittently negative S. obvelata perianal tape pinworms in mice. However, when ivermectin was applied 22 impressions also were observed in the positive control group. twice at a 10-d interval, eradication was achieved. Patent S. obvelata infections were confirmed on these animals at At the tested doses (0.6 and 6.0 mg/kg), selamectin did not necropsy. From these data, we conclude that S. obvelata ova are have any significant effect on hematologic or serum chemistry shed intermittently. Therefore, use of perianal tape impressions profiles of rats. In addition, there were no noteworthy histo- alone for S. obvelata screening should be performed and inter- logic lesions. According to a report by Bishop and others, mice preted with caution. We did not observe intermittent shedding given 100 mg/kg selamectin orally exhibited only mild ptosis 2 of S. muris ova in rats. and piloerection. Because selamectin was found to be ineffec- As previously reported, fenbendazole is effective for elimi- tive in mice, we did not evaluate their hematologic and serum nating rodent pinworm infections.4,8,23 Fenbendazole binds to chemistry parameters nor was histopathologic interpretation nematode tubulin, thus preventing polymerization of tubulin performed on mice. during microtubule assembly. In addition to affecting micro- An interesting finding of unknown importance was the worm tubule assembly, binding of fenbendazole to tubulin prevents counts of selamectin-treated animals as compared with positive formation of spindles necessary for cellular reproduction.5,23 Fen- controls. Neither of the selamectin-treated rat groups differed bendazole also is reported to have ovacidal activity.5 The tubulin significantly from the positive control rats. However, when binding and ovacidal characteristics of fenbendazole make the the worm counts of selamectin-treated mice were compared drug effective at eliminating pinworms from both rats and mice with those of positive control mice, statistical significance was and preventing reinfection from environmental sources. observed (P  0.0271). This finding could not be explained and 25 Vol 45, No 3 Journal of the American Association for Laboratory Animal Science May 2006 may be due to small sample size or serendipity. 9. Krautmann MJ, Novotny MJ, Keulenaer KD, Godin CS, Evans In summary, our study is the 1st to evaluate the efficacy and EI, McCall JW, Wang C, Rowan TG, Jernigan AD. 2000. Safety safety of topical selamectin to eradicate pinworm (Syphacia of selamectin in cats. Vet Parasitol 91:393–403. spp.) infections in mice and rats. Although safe, selamectin 10. Lubcke R, Hutcheson FAR, Barbezat GO. 1992. Impaired intestinal electrolyte transport in rats infested with the common parasite was ineffective at eliminating pinworm infections at the tested Syphacia muris. Dig Dis Sci 37:60–64. doses (0.6 and 6.0 mg/kg). Fenbendazole treatment remains an 11. McTier TL, Hair JA, Walstrom DJ, Thompson L. 2003. Efficacy effective and practical method to eliminate pinworm infections and safety of topical administration of selamectin for treatment of in mice and rats. ear mite infestation in rabbits. J Am Vet Med Assoc 223:322–324. 12. Mohn G, Phillip EM. 1981. Effects of Syphacia muris and the anthelmintic fenbendazole on the microsomal monooxygenase Acknowledgments system in the mouse liver. Lab Anim 15:89–95. Lillian Zalduondo, RLAT, and Jacqueline Tubbs (Tuskegee University, 13. National Research Council. 1991. Digestive system. In: Infectious College of Veterinary Medicine, Nursing, and Allied Health, Class of diseases of mice and rats. Washington (DC): National Academy 2006) are acknowledged for providing technical assistance. The authors Press. p 156–158. also wish to thank David Armbruster, PhD, David Hamilton, DVM, 14. National Research Council. 1996. Guide for the care and use of and J Scott Jackson, DVM, for review of preliminary versions of this laboratory animals. Washington (DC): National Academy Press. manuscript. 15. Novotny MJ, Krautmann MJ, Ehrhart JC, Godin CS, Evans EI, McCall JW, Fun F, Rowan TG, Jernigan AD. 2000. Safety of sela- References mectin in dogs. Vet Parasitol 91:377–391. 1. Battles AH, Adams SW, Courtney CH, Mladinich CRT. 1987. 16. Pearson DJ, Taylor G. 1975. The influence of the nematode Syphacia Efficacy of ivermectin against natural infection of Syphacia muris obvelata on adjuvant arthritis in the rat. Immunology 29:391–396. in rats. Lab Anim Sci 37:791–792. 17. Pfizer. 2002. 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The effect of infection with the pinworm (Syphacia effective eradication of pinworms (Syphacia muris) in rats by use muris) on rat growth. Lab Anim Sci 38:476–478. of fenbendazole. Lab Anim Sci 43:481–487. 22. West WL, Schofield JC, Bennett BT.1992. Efficacy of the “micro- 5. Courtney CH, Roberson EL. 1995. Chemotherapy of parasitic dot” technique for administering topical 1% ivermectin for the diseases. In: Adams HR, editor. Veterinary pharmacology and control of pinworms and fur mites in mice. Contemp Top Lab therapeutics. Ames (IA): Iowa State Press. p 889–898. Anim Sci 31(6):7–10. 6. Hamlen HJ, Kargas SA, Blum JR. 1994. Ivermectin overdose in 23. Wilkerson JD, Brooks DL, Derby M, Griffey SM. 2001. Com- a group of laboratory mice. Contemp Top Lab Anim Sci 33(2):49– parison of practical treatment methods to eradicate pinworm 51. (Dentostomella translucida) infections from Mongolian gerbils 7. Huerkamp MJ, Benjamin KA, Webb SK, Pullium JK. 2004. Long- (Meroines unguiculatus). Contemp Top Lab Anim Sci 40(5):31–36. term results of dietary fenbendazole to eradicate Syphacia muris 24. Winchester MF, Farrell B, Hayes Y, Bellinger D. 2004. The use of from rat colonies. Contemp Top Lab Anim Sci 43(2):35–36. fipronil and selamectin for the treatment and control of parasites 8. Huerkamp MJ, Benjamin KA, Zitzow LA, Pullium JK, Lloyd JA, on mice. Contemp Top Lab Anim Sci 43(4):65. Thompson WD, Webb SK, Lehner NDM. 2000. Fenbendazole treatment without environmental decontamination eradicates Syphacia muris from all rats in a large, complex research institution. Contemp Top Lab Anim Sci 39(3):9–12.

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