A Single Dose of Topical Moxidectin as an Effective Treatment for Murine Acariasis Due to Myocoptes musculinus

JENNIFER K. PULLIUM, MVB, DIPLOMATE, ACLAM,1,2* WILLIAM J. BROOKS, BA,1 APRIL D. LANGLEY, RVT,1 AND MICHAEL J. HUERKAMP, DVM, DIPLOMATE, ACLAM1,2

Murine fur mites are reported to exist in over one-third of research institutions and can be problematic to eliminate. Current treatment strategies can be labor-intensive, toxic, and may confound research studies. The ideal method would be technically simple, safe, effective, and relatively inexpensive. When we found that mice from a noncommercial vendor were infested with Myocoptes musculinus, the animals were treated topically with Cydectin pour-on (containing moxidectin 0.5%) at 5 mg/kg. After one treatment, mites were eradi- cated from all infested mice. No toxic effects or clinical signs of illness were observed in the mice. To the authors’ knowledge, this is the first report of topical moxidectin as a treatment for murine acariasis.

As the use and transfer of genetically engineered mutant mice in- limited, however, it has been shown to be safe when administered creases, the risk of introducing rodent pathogens increases as well. A to hamsters, rats, and gerbils (16-18). Because of the surface-feed- recent study states that over one third of research institutions had fur ing nature of M. musculinus, we decided to administer it topically mites in at least one of their mouse colonies (1). The potential ad- to affected mice, with the hope of needing no more than one treat- verse effects of Myocoptes musculinus infestation include dermatitis, ment to eradicate the mites. To the authors’ knowledge, this is the hypersensitivity, abnormal behavior, and immune dysfunction (2- first report on the use of topical moxidectin for the treatment of 6). The decision regarding how to treat murine acariasis can be murine acariasis. difficult, as current effective methods can be labor-intensive and, consequently, expensive. For example, caesarean rederivation can be Materials and Methods used to eliminate a mite infestation (2-4); however, it requires spe- Humane care and use of animals. Animals were housed in an cialized skills and may be difficult to perform reliably in fragile mouse Association for the Assessment and Accreditation of Laboratory Ani- strains (7). Subcutaneous administration of ivermectin and dichlor- mal Care, International-accredited facility and in compliance with vos in the bedding require multiple treatments, an inconvenience in the Guide for the Care and Use of Laboratory Animals. All procedures a large research institution (8). Other treatment methods may be involving animal use were approved by the Institutional Animal Care detrimental to the health of the mice or interfere with research stud- and Use Committee at Emory University. ies. Dichlorvos carries the risk of toxicity (9), as does ivermectin, Mice. Two strains of mice were infested with and treated for M. particularly in young mice and those with neurological aberrations musculinus. The first strain was B6.Cg-Spp1tm1Blh, obtained from a (10, 11). Ivermectin treatment can also confound some behavioral private institution that did not have a rodent health monitoring pro- tests (12) and, although effective when administered in drinking water gram. These mice have a null mutation for osteopontin (OPN–/–). (13), this method may not be practical for treating large numbers of The OPN–/– mice then were bred with B6;129-Apoetm1Unc/J (The Jack- mice or those maintained on an automatic watering system. son Laboratory, Bar Harbor, Maine), which have a null mutation for When a mouse with ulcerative dermatitis was found to have M. apolipoprotein E (Apoe–/–), to create mice that were double knock- musculinus on pelage tape examination at our institution, further outs for OPN and Apoe. These OPN–/–Apoe–/– mice were the second evaluation of the other mice of the same strain and their progeny mouse genotype treated for M. musculinus. revealed that nearly every mouse that could be traced to the origi- Housing and Husbandry. The OPN–/– mice (two breeder pairs) nally affected strain was infested with M. musculinus. Although were housed in rodent quarantine upon arrival and placed in poly- approximately 10% of the mice exhibited some degree of hair loss, carbonate shoe box cages with isolator tops and corncob bedding. no other mice had dermatitis. The infested mice originated from a The room was kept on a 12:12-h light:dark cycle and animals were private institution that did not have a rodent health monitoring fed rodent chow (Rodent Diet 5010, Purina Mills Test Diet, Rich- program. Although the mice underwent weekly pelage tape exami- mond, Va.) and autoclaved tap water ad libitum. Each mouse was nations upon arrival, no mites were detected during the 6-week tested weekly for 4 weeks (16 total samples) for ecto-and endopara- quarantine period. sites via pelage and anal tape tests and fecal float, respectively, and all In an effort to use a simple, safe, rapid, one-time treatment, we tests were negative. At week 6 after arrival, blood was collected from chose topical moxidectin, which is an avermectin, as is ivermectin. the orbital sinus of mice that were anesthetized with isoflurane. Se- Moxidectin has been shown to be longer-acting than ivermectin in rum samples were diluted 1:4 with normal saline, frozen, and sent to the prevention of heartworms in dogs (14) and ovicidal in the treat- Charles River Laboratories rodent diagnostic laboratory (Wilmington, ment of ticks in cattle (15), potentially eliminating the need for Mass.) for serological evaluation for Sendai virus, pneumonia virus multiple treatments for mites. The use of moxidectin in rodents is of mice, mouse hepatitis virus, mice minute virus, Theiler’s murine encephalomyelitis virus, reovirus 3, ectromelia, Mycoplasma pulmonis, Division of Animal Resources,1 Department of Pathology and Laboratory Medicine,2 Emory University School of Medicine, 615 Michael Street, Suite G02, Atlanta, Georgia 30322 rotavirus, mouse adenovirus, lymphocytic choriomeningitis virus, K *Corresponding author virus, polyoma virus, and mouse parvovirus. The mice were serone-

26 CONTEMPORARY TOPICS © 2005 by the American Association for Laboratory Animal Science Volume 44, No. 1 / January 2005 Figure 1. Pelage tape from moxidectin-treated mouse 1 week after treat- Figure 2. Pelage tape from moxidectin-treated mouse 1 week after treatment, ment, demonstrating desiccated egg with clumped material (closed arrow), demonstrating dead larva with open desiccated egg shell. Bar, 100 µm. dead larva (arrowhead), and empty egg shell remnant (open arrow). Inset, normal egg from vehicle-treated control mouse pelage tape. Bar, 100 µm. ing those that had been neonates (untreated) at the time of treat- ment (n = 151) were evaluated by using a pelage tape examination. gative for all of the listed pathogens and were released into the ro- Two weeks after treatment, one mouse had a dead adult on pelage dent colony, where they were bred with the Apoe–/– mice to create tape, two mice had one dead larva each, and nine mice had dead the OPN–/–Apoe–/– mice. eggs. At 4 weeks, two mice had one egg shell remnant each. At 8 Moxidectin treatment and testing. Mice were treated topically weeks, none of the mice exhibited any evidence of fur mites. None with moxidectin (5 mg/kg, Cydectin pour-on containing moxidectin of the moxidectin-treated mice demonstrated live eggs, larvae, or 0.5% as active ingredient in coconut oil, Fort Dodge Animal Health, adult mites, whereas all six vehicle-treated control mice had both live Fort Dodge, Iowa). The formulation was modified at the manufac- eggs and adults on pelage tape examination throughout the course of turer; the purple dye normally in Cydectin had been removed. the experiment. Mice of other strains that had been housed on the Moxidectin was applied to the dorsum of the mice (3 µl), between same rack as the affected mice and those in the same room on differ- the scapulae, by using a Hamilton syringe (Harvard Bioscience, Inc., ent racks were tested at one mouse per cage (n = 469 cages), but no Holliston, Mass.). All adult (OPN–/–, n = 68; OPN–/–Apoe–/–, n = 9) ectoparasites were found on any of these mice. and weanling (OPN–/–, n = 35) mice were treated once with moxidectin. Hairless neonates (OPN–/–, n = 39) were not treated, Discussion however, the sires and dams with which they were housed were treated. A single topical dose of moxidectin proved to be effective in eradi- –/– Six OPN served as vehicle controls, each receiving one application cating M. musculinus in both strains of mice, thereby keeping the µ of topical coconut oil (3 l/mouse). required labor and subsequent expenses to a minimum. The 8-week = One week after treatment, one mouse per cage was tested (n 32 follow-up period was sufficient to cover four to six life cycles of the cages) for the presence of M. musculinus by using a pelage tape test. mites (4). Had the treatment been unsuccessful, a viable egg or adult Hair samples were collected from between the scapulae and the ab- should have been detected during this time. A sample size of 151 was = dominal and inguinal regions. All mice (n 151) underwent a pelage sufficient to find one positive mouse with a 95% confidence level if tape test at weeks 2, 4, and 8 after treatment, including those mice 2% of the mice were infested (19). Although only one mouse per that had been neonates (untreated) at the time of moxidectin treat- cage was evaluated at 1 week post-moxidectin treatment, all adults, ment. Mice were observed daily for any signs of illness or toxicity, larvae, and eggs were deformed and nonviable. Therefore, it is pos- including neurological abnormalities. sible that the moxidectin had killed all forms of the mites at this point, including eggs, as seen with ticks (15). A rapid elimination of Results all live stages of the mites would explain how nude neonates remained None of the treated mice exhibited apparent clinical signs of ill- uninfested, even though only the sires and dams were treated. Nude ness or avermectin toxicity. At 1 week after moxidectin mice do not become infested with fur mites, and evidence indicates administration, all pelage tapes collected from moxidectin-treated that the same is true for hairless pups (7). The eggs appeared to des- mice (n = 32 mice) demonstrated dead M. musculinus eggs and lar- iccate, with an irregular outline and clumped internal contents, an vae, and tapes from two mice showed one dead adult on each tape. observed effect consistent with ivermectin treatment (8). Avermectins The eggs were desiccated with angular exteriors and clumps of dark are classically known to be GABA-agonists (20), however, ovicidal material within the egg or were merely broken shells (Fig. 1). All effects on tick eggs suggest that there may be other mechanisms of larvae also were dead and cylindrical in shape, matching the dimen- action that are currently unknown. sions of the eggs (Fig. 1). When larvae that appeared to be “hatching” Moxidectin had successfully eliminated M. musculinus infesta- from the egg were found, the larvae actually were completely immo- tion by 4 weeks post-treatment, despite the presence of portions of bile and appeared nonviable. The exterior of the egg split open egg shells in 2 of 151 mice. As mite eggs are adhered to the hair longitudinally (Fig. 2), as though the larvae died inside the eggs, shaft, grooming or natural loss of the hair may be required in order with the desiccated, deformed exterior of the egg eventually slough- to remove all remnants of eggs shells post-treatment. Although it ing off the larva. can vary depending on the location and type of hair, the hair cycle At weeks 2, 4, and 8 after moxidectin treatment, all mice, includ- length can be up to 60 days (21-23). Although one study indicated

Volume 44, No. 1 / January 2005 CONTEMPORARY TOPICS © 2005 by the American Association for Laboratory Animal Science 27 that pelage tape tests are less accurate in general than are skin 5. Jungmann, P., J. L. Guenet, P. A. Cazenave, et al. 1996. Murine ac- scrapings or microscopic pelt inspections, they are satisfactory for ariasis. I. Pathological and clinical evidence suggesting cutaneous allergy M. musculinus (8), therefore the likelihood of false negatives is mini- and wasting syndrome in BALB/c mouse. Res. Immunol. 147:27-38. mal. In addition, another report equates the tape sampling method 6. Jungmann, P., A. Freitas, A. Bandeira, et al. 1996. Murine acariasis. II. Immunological dysfunction and evidence for chronic activation of sensitivity with that of skin scrapings or postmortem examinations Th-2 lymphocytes. Scand. J. Immunol. 43:604-612. of the fur (13). 7. Huerkamp, M. J., L. A. Zitzow, S. Webb, et al. Submitted for publi- There was no mortality and no clinical signs of neurological or cation. other disease detected during the treatment follow-up period. High 8. Burdett, E. C., R. A. Heckmann, and R. Ochoa. 1997. Evaluation of doses of ivermectin have been shown to be toxic in pre-weanling five treatment regimens and five diagnositc methods for murine mites mice (11), however, it is unknown whether such toxicity would oc- (Myocoptes musculinus and Myobia musculi). Contemp. Top. Lab. Anim. cur with topical moxidectin. Sensitive behavioral tests were not Sci. 36(2):73-76. performed, therefore, additional experiments are required in order 9. Wagner, J. E. and D. R. Johnson. 1970. Toxicity of dichlorvos for laboratory mice—LD50 and effect on serum cholinesterase. Lab. Anim. to determine whether moxidectin can have similar adverse effects as Care 20:45-7. ivermectin (12). Mice of different strains may not respond in the 10. Diggs, H. E., D. J. Feller, J. C. Crabbe, et al. 1990. Effect of chronic same way to a particular drug; consequently further research is nec- ivermectin treatment on GABA receptor function in ethanol with- essary to affirm that topical moxidectin can eliminate M. musculinus drawal-seizure prone and resistant mice. Lab. Anim. Sci. 40:68-71. from other types of mice. Pharmacokinetic studies are needed to 11. Skopets, B., R. P. Wilson, J. W. Griffith, et al. 1996. Ivermectin tox- establish whether the reason moxidectin is effective with a single dose, icity in young mice. Lab. Anim. Sci. 46:111-112. versus the extended or multiple treatments required for ivermectin, 12. Davis, J. A., R. Paylor, M. P. McDonald, et al. 1999. Behavioral ef- is prolonged blood levels with moxidectin compared with the pre- fects of ivermectin in mice. Lab. Anim. Sci. 49:288-296. cipitous decrease seen with ivermectin (13). 13. Conole, J., M. J. Wilkinson, and Q. A. McKellar. 2003. Some obser- vations on the pharmacological properties of ivermectin during The use of the isolator caging appeared to prevent the spread of treatment of a mite infestation in mice. Contemp. Top. Lab. Anim. mites to 469 other cages on the same rack and in the same room as Sci. 42(4):42-45. infested mice, a finding that is consistent with previous reports of 14. Lok, J. B., D. H. Knight, G. T. Wang, et al. 2001. Activity of an wire mesh preventing mite transmission (4). A sample size of 469 is injectable, sustained-release formulation of moxidectin administered sufficient to detect one positive cage with a confidence level of 95% prophylactically to mixed-breed dogs to prevent infection with Dirofi- if 0.4% of the mice were infested (19). It is currently unknown why laria immitis. Am. J. Vet. Res. 62:1721-1726. the mite infestation was not identified while the mice were in quar- 15. Guglielmone, A. A., A. J. Mangold, M. E. Munoz Cobenas, et al. antine. The mice may have arrived at an age where the mite load had 2000. Moxidectin pour-on for control of natural populations of the cattle tick Boophilus microplus (Acarina: Ixodidae). Vet. Parasitol. previously peaked and were at a basal level upon arrival (19, 24). 87:237-41. Mice with a low level of mites may require an extended testing pe- 16. Beco, L., A. Petite, and T. Olivry. 2001. Comparison of subcutane- riod in order to find an infested mouse. Furthermore, the personnel ous ivermectin and oral moxidectin for the treatment of notoedric performing and evaluating the pelage tape tests at the time had lim- acariasis in hamsters. Vet. Rec. 149:324-327. ited experience in rodent parasitology, and they may have either taken 17. Oge, H., E. Ayaz, T. Ide, et al. 2000. The effect of doramectin, an insufficient sample of hair or failed to detect a mite egg. In order moxidectin and netobimin against natural infections of Syphacia muris to rectify this problem, mice from noncommercial vendors are now in rats. Vet. Parasitol. 88:299-303. required to have a health report stating they are free of ectoparasites, 18. Molento, M. B. and R. K. Prichard. 1999. Effects of the multidrug- the mice are quarantined for approximately 12 weeks, and all per- resistance-reversing agents verapamil and CL 347,099 on the efficacy of ivermectin or moxidectin against unselected and drug-selected strains sonnel involved in ectoparasite evaluation are properly trained. of Haemonchus contortus in jirds (Meriones unguiculatus). Parasitol. Res. 85:1007-1011. Acknowledgments 19. Small, J. D. 1984. Rodent and lagomorph health surveillance: quality The authors wish to thank Fort Dodge Animal Health for their generous assurance, p. 709-723. In J. G. Fox, B. J. Cohen, and F. M. Loew gift of Cydectin, Kimberly Benjamin for technical assistance, and Dirck L. 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