Elimination of an Infestation of Rat Fur (Radfordia ensifera) from a Colony of Long Evans Rats, Using the Micro-dot Technique for Topical Administration of 1% Ivermectin

SYLVIA Y. KONDO1, BS, DVM, ANDREW D. TAYLOR2, BA, MAS, PHD, AND STEWART S.C. CHUN3, BA, MLIS

Abstract Micro-dot delivery of 1% ivermectin was used in an effort to eliminate an infestation of rat fur mites (Radfordia ensifera) from a conventionally housed colony of Long Evans rats. The colony was used for breeding as well as for behavioral testing. A micropipette was used to apply a 1% solution of ivermectin (2 mg/kg of body weight) to the skin on the dorsal aspect of the shoulder. Three treatments were applied at approximately 2-week intervals. All rats in the colony were treated. However, to avoid toxicity to neonates, nursing females and their pups (control group) received mineral oil in lieu of ivermectin until after weaning, at which time they also were treated with ivermectin. During the treatment phase, skin scrapings were used to detect mites. Control rats remained positive for fur mites when treated with mineral oil. After 3 applications of ivermectin, all rats were found to be free of mites. During the posttreatment phase, skin scrapings, dorsal tape tests, and a washing method were performed on euthanatized rats to detect mites. Rats tested up to 129 days (18 weeks) after ivermectin treatment were still free of mites. In a breeding colony composed of rats of various sizes and ages, micropipette delivery of ivermectin allowed for accurate dosing to eliminate fur mites, while preventing inadvertent toxicosis. We did not detect obvious adverse effects on the breeding program or on the behavioral studies in which these rats were subsequently used, nor did we detect morbidity or mortality associated with ivermectin administration.

The rat fur (Radfordia ensifera) (Figure 1) is a common ectoparasite of wild rats as well as rats housed in conventional laboratory colonies (1–3). The life cycle has not been determined, although it may be similar to that of the mouse fur mite ( musculi), which varies from 10 to 21 days, depending on host and environmental conditions (4). The rat fur mite is a surface dweller and feeds on epithelial debris and tissue secretions of the host (4). Although some rats in affected colonies do not have clinical signs, those with clinical signs will scratch and have patchy alopecia with dermal ulcers. These signs may be caused by an allergic or pruritic stimulus induced by mites (4). Treatment protocols for fur mites have variable efficacy, and include the use of 2% AramiteTM, 2% malathion, and dichlor- vos-impregnated resin strips placed in cages (5). Use of 5% SevinTM dust and DursbanTM granules containing 0.5% chlorpyrifos have also been reported (6). Ivermectin has been administered at various concentrations and by several routes to FIG. 1. Photograph of a rat fur mite (Radfordia ensifera). (200x magnifi- treat with fur mite and pinworm infestations (7–11). cation) Micro-dot delivery of 1% ivermectin (12) was used in an effort to eliminate the rat fur mite from a conventionally housed colony included in the study reported here. This closed colony of Long of Long Evans rats. Ivermectin was chosen for its efficacy as a miti- Evans rats had been conventionally housed in the same facility cide (13) and delivered by use of the micro-dot technique to ensure for several years, and replacement rats are born in the colony. accurate dosing and selective treatment of rats in an effort to avoid The source of the mite infestation was not determined. Infesta- adverse affects on an ongoing breeding program and behavioral tion was diagnosed after clinical signs were observed in rats of studies conducted with these rats. In addition, the technique was the colony. In addition to infestation with fur mites, rats in this chosen because of ease of application, cost effectiveness, and mini- colony sporadically had clinical signs and positive serologic re- mal disruption of conventional husbandry procedures (12). sults for Mycoplasma pulmonis when tested by enzyme-linked immunosorbent assay and indirect fluorescent-antibody assay. Materials and Methods Rats in this colony also had pinworms (Syphacia muris), which : All Long Evans rats in the colony (n = 231), com- were diagnosed by perianal tape testing. During the last month posed of various ages (newborn to 7 months old) and sexes were of the study, an additional 46 rats not associated with this colony were housed in a separate room in the same facility. The 46 rats 1University of Hawaii, Laboratory Service, 2538 The Mall, Snyder Hall were not tested or treated for fur mites. 501A, Honolulu, HI 96822; 2University of Hawaii at Manoa, Department of Rats in the colony were used for behavioral experiments as Zoology, 2538 The Mall, Edmondson Hall 152, Honolulu, HI 96822; 3Univer- well as being bred to supply subjects for ongoing research sity of Hawaii, Laboratory Animal Service, 2538 The Mall, Snyder Hall 514, projects. Behavioral studies included the Vogel test for anxiety, Honolulu, HI 96822 exposure to cats to test for fear, and colony aggression and de- 58 fense to test for stress. These uses constrained the design of our to detect mites on some of the euthanatized rats (16). study on elimination of fur mites. In particular, identification of Treatment phase: Treatment group—The experimental unit was rats, timing of treatments, and testing of live rats and samples a cage containing 1 or more rats. All cages were treated identi- obtained during necropsy were restricted. Although all adults cally. Rats in each cage were weighed and treated with 1% were identified, juveniles were only identified by cage (i.e., litter ivermectin at a dosage of 2 mg/kg of body weight applied topi- and gender). During the posttreatment period, only those rats cally to the skin on the dorsal aspect of the shoulder, using a that were euthanatized as part of behavioral testing were avail- micropipette (Eppendorf Digital Pipette 4710, Brinkman Instru- able for sample collection. In addition, the need to resume ments Co., Westbury, N.Y.). Rats in each cage received 3 ongoing behavioral studies and breeding necessitated movement ivermectin treatments at approximately 2-week intervals. of rats between rooms and cages as soon as the last ivermectin Skin scrapings were obtained on days 0 and 29 of treatment treatment was administered. and on day 44, 2 weeks after the third and final application of Facilities: Rats were housed in conventional polycarbonate ivermectin. Of the 48 treatment cages, 22 (11 adult and 11 juve- shoe-box cages (Allentown Caging Equipment Company, Inc., nile) were used for testing. For cages containing group-housed Allentown, N.J.) on corn-cob litter (Bed-O’Cobs, The rats, 1 rat (not necessarily the same rat each time) was used for Andersons, Maumee, Ohio) and hardwood bedding (Sanichips, testing on each date. P. J. Murphy Forest Products, Montville, N.J.). They were fed Two skin scrapings per rat were obtained from the dorsal as- chow formulated for rodents (Laboratory Diet 5001, pect of the neck and shoulder areas, using a No. 10 sterile surgical PMI Feeds, Inc., St. Louis, MO) and provided with tap water ad blade (Miltex Instrument Co., Inc., Lake Success, NY). The libitum. Environmental conditions included a 12-h light:12-h samples were mixed with mineral oil (White Mineral Oil USP, dark cycle, ambient temperature of 22 to 24 C, and 10 to 15 Chemtronics Inc, Leavenworth, KS) and examined by light mi- changes of fresh air per hour. croscopy at 4x magnification. Bedding in cages was changed 2 or 3 times per week, depend- Control Group—Cages in the other room, which contained ing on the amount of soiling in each cage. Treatments coincided nursing mothers with their litters, initially were designated as with days on which bedding was changed. To prevent spread of the control group to avoid ivermectin toxicosis of the neonatal fur mites between rooms, animal handlers wore caps, gowns, rats during the nursing period (14, 15). On day 0, each nursing gloves, shoe covers, and masks when working with the soiled female received 50 l of mineral oil applied topically to the skin bedding or cages. Soiled bedding and cages were dampened with on the dorsal aspect of the shoulder, using a micropipette. On a dilute (1:15) solution of hypochlorite acid (Aqua-Brite, ABC day 16, neonates were weaned and placed in 24 additional cages. Corp., Honolulu, HI) prior to removal from animal rooms. These At that time, all rats in the control group received mineral oil extra procedures were used during the treatment phase and for (adults, 50 l; juveniles, 25 l) applied topically to the skin on up to 20 days after the third and final administration of ivermectin the dorsal aspect of the shoulder. (Ivomec, MSD-Agvet, Rahway, N.J.). Husbandry procedures for All rats in the control group were subsequently treated with the final 4 months of the study did not include use of caps, gowns, ivermectin on days 32, 39, and 50 of the treatment phase. Treat- shoe covers, or dilute hypochlorite acid. ment consisted of ivermectin (2 mg/kg) applied topically to the Procedures: On initial day of the treatment phase (day 0), a skin on the dorsal aspect of the shoulder. Each rat received 3 treatment group was designated, consisting of 24 separately caged treatments at 7- to 14-day intervals. adults and 99 six-week-old juveniles group housed (2 to 6 per Skin scrapings were obtained from the same 4 nursing females cage) with same-sex siblings in 24 cages, for a total of 48 cages in on days 0, 32, and 64 of the treatment phase. Skin scrapings 2 rooms. The control group was housed in another room. This were obtained from juveniles (not necessarily the same ones at group consisted initially of 8 nursing females and 100 neonates, each test date) representing 6 to 18 cages on days 32, 64, and 78 with each mother and litter housed in separate cages. Neonates of the treatment phase. were weaned at 21 days of age (day 16 of the treatment phase) Posttreatment phase: After the final ivermectin treatment was and were then grouped housed in same-sex groups of 2 to 6 administered to the control group on day 50, rats in the colony siblings per cage, resulting in 24 additional cages. On day 39 of were routinely moved between rooms and cages to facilitate the the treatment phase, 25 of the 100 juveniles from the control uses for which they were intended. After behavioral testing, 86 group were placed in 6 microisolator top cages in another room. rats from the treatment group and 9 rats from the control group This additional isolation was used in an attempt to eliminate a were euthanatized and samples were obtained. Rats were eutha- concurrent pinworm infestation. The number of pinworm eggs natized over a period of up to 129 days (18 weeks) after the final was substantially decreased, but pinworms were not eliminated. ivermectin treatment. At the time of euthanasia, detection of During the treatment phase, all rats were confined to their mites was performed by use of skin scrapings, dorsal tape tests, designated rooms, and new rats were not introduced to the and a washing technique (16). colony. In addition, behavioral studies were not conducted dur- During the posttreatment phase, the experimental unit was ing this time. During the final 3 months of the study (after the each rat. There were 95 skin scrapings, 84 dorsal tape tests, and third and final ivermectin treatment), rats were routinely moved 10 washes performed on the 95 euthanatized rats (Table 1). Rats between rooms according to the needs of investigators for be- were euthanatized by means of CO2 inhalation, then 2 skin havioral experiments and the breeding program for which they scrapings were performed on each rat. After the skin scrapings, were intended. Special measures were not used to decontami- the dorsal tape tests or the washing method was performed on nate the animal rooms or behavioral testing area during the each rat carcass. Dorsal tape tests were performed by placing a course of the study. 2-in wide piece of clear tape (3M Superior Performance Scotch Mites were detected by examination of skin scrapings obtained Mailing Tape, St. Paul, MN) on the dorsum of the rat. The tape from live rats, whereas skin scrapings and dorsal tape tests were was left on the rat’s body overnight (ambient temperature, 25 performed on rats that had been euthanatized. Choice of tests C), removed the following morning, and examined under a dis- was made on the basis of results of a previous study (12) in which secting microscope at 4x magnification. All skin scrapings were it was reported that the dorsal tape test was the most accurate examined by light microscopy at 4x magnification. method of detecting fur mites. In that study, examination of skin The washing technique (16) was performed by placing the scrapings was the second most reliable method, followed by gross carcass in a bottle containing a dilute (5%) solution of alconox examination of the coat. In addition, we used a washing method detergent in water (Alconox, Inc. NY, NY). The rat carcass was

59 Table 1. Results of examination of skin scapings, dorsal tape tests and to the third ivermectin treatment. Another juvenile female from a washing technique conducted on rats during the post-treatment a litter of 11 rats died one day prior to the third ivermectin treat- phase for detection of fur mites. Fur mites were not detected by ment. Although the cause was not determined, death of the use of the tests for up to 129 days after treatment. female did not appear to be related to administration of Days After Treatment ivermectin. In a colony of this size, it would not be uncommon to have a few rats die, especially runts from large litters. Test 52 87 94 108 115 129 Total Post treatment phase: During the posttreatment phase, skin Skin 0/52a 0/2 0/5 0/11 0/17 0/8 0/95 scrapings were performed on 86 euthanatized rats on days 52 scraping (7.4 weeks; n = 52), 108 (15.4 weeks; n = 9), 115 (16.4 weeks; n = Dorsal 0/52 0/1b 0/4 0/11 0/8 0/8 0/84 17), and 129 (18.4 weeks; n = 8) after final administration of tape test ivermectin to rats in the treatment group (Table 1). In addition, Washing ND ND 0/1 ND 0/9 ND 0/10 dorsal tape tests were performed on euthanatized rats on days 52 ( n = 52), 108 (n = 9), 115 (n = 8), and 129 (n = 8) after final a = number positive/number tested b = body of one rat required for necropsy administration of ivermectin to rats in the treatment group. Skin ND = not done scrapings were obtained from 9 euthanatized rats of the control group on days 87 (12.4 weeks; n = 2), 94 (13.4 weeks; n = 5), and 108 (15.4 weeks; n = 2) after final application of ivermectin. placed in a sealed jar and covered with dilute alconox solution. Dorsal tape tests were performed on euthanatized rats in the Vigorous shaking of the jar was performed intermittently dur- control group on days 87 (n = 1), 94 (n = 4), and 108 (n = 2) ing a 30-min period. After the wash, the carcass was removed, after final application of ivermectin. The washing method was and excess solution squeezed from the coat and placed in the performed on 9 euthanatized rats in the treatment group on jar. Wash water was then poured into a large glass cylinder. Each day 115 (16.4 weeks) and on 1 euthanatized rat in the control rat carcass was then rinsed 1 or 2 more times by placing it in the group on day 94 (13.4 weeks) after final administration of sealed jar, adding distilled water, and shaking the jar vigorously. ivermectin. Fur mites were not detected by any of the test meth- The additional rinse water was added to the cylinder. A small ods used on rats from the treatment or control groups during amount of a solution of 95% alcohol (Hyakol, Breen Laborato- the posttreatment phase, and we did not observe additional clini- ries, Adelanto, CA) was added to the cylinder to reduce surface cal signs associated with fur-mite infestation. foaming. Solution in the cylinder was allowed to sit undisturbed for at least 15 min so that mites would settle to the bottom. The supernatant was then carefully decanted, and the residue from Discussion the bottom of the cylinder was poured into a petri dish and ex- Micropipette application of 1% ivermectin to rats for 3 treat- amined under a dissecting scope at 4x magnification. ments at approximately 2-week intervals was effective in eliminating rat fur mites. The mites were initially obtained from skin scrapings Results collected from rats that had alopecia and pruritus. A distinguish- ing feature of rat fur mites are the paired and equal claws on the Treatment phase: Skin scrapings obtained on day 0 of the treat- tarsi of the second pair of legs, which differentiates it from mouse ment phase revealed that 6 of 11 cages of adults and 7 of 11 fur mites, which have paired but unequal claws on the second pair cages of juveniles from the treatment group were positive for of legs for R. affinis, and only a single empodial claw on these seg- fur mites. On day 29, prior to the third application of ivermectin ments for M. musculi (17, 18). In addition, rat fur mites have a to the treatment group, all of the cages were negative for fur series of 6 foliate posterior idiosomal setae that distinguishes them mites. Additional skin scrapings obtained on day 44 from 6 cages from mouse fur mites (19). Humeral setae on rat fur mites are of adults and 12 cages of juveniles also were negative for fur much broader than those on mouse fur mites (19). mites. Analysis of our results indicated that 1% ivermectin delivered Three of 4 adults in the control group were positive for fur by micropipette (micro-dot) was a safe, easy and effective way of mites on day 0. On day 32, prior to the start of ivermectin treat- eliminating fur mites and clinical signs associated with fur-mite ment for the control group, these 3 adults were still positive for infestation from rats in a conventionally housed colony for up fur mites, whereas none of the 13 cages of juveniles, which had to 129 days (18 weeks) after treatment. This technique can be by then been separated from the adults, were positive for fur used with minimal disruption to ongoing breeding programs mites. After the second treatment with ivermectin, 25 juveniles and behavioral studies. There was minimal stress, because the from the control group were moved to another room and placed rats were handled only momentarily to weigh and apply the in 6 microisolator cages. All cages of the control group tested on ivermectin, and the technique worked well within the constraints day 64 and 77 of the treatment phase were negative for fur mites. of conventional husbandry procedures. Use of the micropipette Fisher’s exact test was used to analyze results from the treat- ensured accurate dosing of the various sizes of rats in the breed- ment phase. Sixteen cages were initially infested with fur mites ing colony and prevented inadvertent toxicosis. The conservation (13 in the treatment group and 3 cages housing adults in the of labor and supplies made micro-dot administration of 1% control group). After 3 treatments with ivermectin, all 13 cages ivermectin extremely cost effective. in the treatment group were negative for infestation with fur In susceptible nematodes and , ivermectin stimu- mites. On the other hand, the 3 cages housing adults in the con- lates increased presynaptic release and postsynaptic receptor trol group that had been given 2 doses of mineral oil continued binding of gamma-amino-butyric acid (GABA). Arthropods use to be infested with fur mites. The proportion of cages that im- GABA as a neurotransmitter between nerve and muscle cells (20). proved (mites not detected) was significantly (P = 0.0018, n = Prolonged stimulation of GABA release and binding by 16) different between groups. After the 3 cages from the control ivermectin results in neuromuscular blockade, paralysis, and group had completed 3 treatments with ivermectin, all of them death in many parasites (20). In addition, prolonged stimula- were negative for infestation with fur mites. Additional clinical tion of GABA can result in suppression of reproductive processes signs attributable to fur-mite infestation were not observed once in parasites (13). ivermectin treatments were initiated. Mammals use GABA as a neurotransmitter in the central ner- During the treatment phase, 2 juveniles died. One juvenile vous system. In most instances, ivermectin does not readily male died as a result of fighting with litter mates one day prior

60 penetrate the blood brain-barrier of mammals to affect GABA 4. Harkness, J. E. and J. E. Wagner. 1995. Acariasis in the rat, concentrations within the central nervous system (20). This re- p. 179–180. The biology and medicine of rabbits and ro- sults in a markedly lower sensitivity of mammals to the toxic dents. Williams & Wilkins, Baltimore. effects of ivermectin, compared with that of invertebrates (14). 5. Hsu, C. K. 1979. Parasitic Diseases, p 327. In H. J. Baker, J. However, there are reports of specific breeds and species that R. Lindsey, S. H. Weisbroth (ed), The laboratory rat. Vol. are sensitive to ivermectin. In one study (21), pregnant rats re- 1., Biology and diseases. Academic Press, New York. ceiving between 2 and 4 mg of ivermectin/kg/day in peanut oil 6. Pence, B. C., D. S. Demick, B. C. Richard, et al. 1991. The via stomach tube during gestation and lactation had newborn efficacy and safety of Chlorpyrifos (DursbanTM)for con- pups that were highly susceptible to the neurotoxic effects of trol of Myobia musculi infestation in mice. Lab. Anim. Sci. ivermectin. Behavioral development was affected and pup mor- 41:139–142. tality was increased in those newborn rats. 7. Ostlind, D. A., M. A. Nartowicz, and W. G. Mickle. 1985. Ivermectin is toxic to neonatal rats born to mothers chroni- Efficacy of ivermectin against Syphacia obvelata (Nematoda) cally treated with ivermectin at dosages as low as 0.4 mg/kg/day in mice. J. Helminth. 59:257–261. during breeding, gestation, and lactation (14). This toxic effect 8. Battles, A. H., S. W. Adams, C. H. Courtney, et al. 1987. is manifested as an increase in pup mortality for up to approxi- Efficacy of ivermectin against natural infection of Syphacia mately 10 days after parturition, a decrease in mean body weight muris in rats. Lab. Anim. Sci. 37:791–792. gain, and delayed development (14). On the basis of results of 9. Baumans, V., R. Havenaar, and H. Van Herck. 1988. The that cross-fostering study, neonatal toxicity was not associated use of repeated treatment with ivomec and neguvon spray with in utero exposure, but was associated with exposure to in the control of murine fur mites and oxyurid worms. Lab. ivermectin through maternal milk. The period of enhanced Anim. 22:246–249. neonatal toxicity (up to 10 days after parturition) corresponds 10. Huerkamp, M. J. 1993. Ivermectin eradication of pinworms to the delayed completion of the blood-brain barrier in this spe- from rats kept in ventilated cages. Lab. Anim. Sci. 43:86– cies (14). Authors of another study (15) indicated that spraying 90. a 1:10 dilution of ivermectin (dose of 2.4 ml per adult mouse 11. LeBlanc, S. A., R. E. Faith, and C. A Montgomery. 1993. [2.4 mg per cage]) appeared to be safe for adult mice, but could Use of topical ivermectin treatment for Syphacia obvelata in cause signs of neurologic dysfunction or death of young mice mice. Lab. Anim. Sci. 43:526–528. when applied to lactating mothers with litters < 3 weeks of age. 12. West, W. L., J. C. Schofield, and B. T. Bennett. 1992. Effi- Finally, a study (22) involving a single subtoxic dose of cacy of the “micro-dot” technique for administering topical ivermectin at a rate as low as 1.6 mg/kg, administered subcuta- 1% Ivermectin for the control of pinworms and fur mites neously to 58-day-old rats, caused a subtle and transient (< 48 h in mice. Contemp. Top. Lab. Anim. Sci. 31:7–10. after ivermectin) increased number of errors in rats during le- 13. Campbell, W. C. and G. W. Benz. 1984. Ivermectin: a re- ver-pressing behavioral tests. view of efficacy and safety. J. Vet. Pharmacol. Therap. In light of studies demonstrating neonatal toxicity to 7:1–16. ivermectin (14,15), caution was taken to avoid treating nursing 14. Lankas, G. R., D. H. Minsker, and R. T. Robertson. 1989. mothers, and all rats were at least 21 days old before ivermectin Effects of Ivermectin on reproduction and neonatal toxic- was administered to them. Adverse effects were not detected by ity in rats. Food. Chem. Toxic. 27:523–529. investigators who used these rats for behavioral testing, and we 15. Skopets, B., R. P. Wilson, J. W. Griffith, et al. 1996. did not detect morbidity or mortality associated with micropi- Ivermectin toxicity in young mice. Lab. Anim. Sci. 46:111– pette administration of ivermectin in this colony. 112. 16. Lipovsky, L. J. 1951. A washing method of ectoparasite re- Acknowledgments covery with particular reference to chiggers (Acarina–Trombiculidae). J. Kansas Entomol. Soc. 24:151– The authors thank Dr. Lee Goff for assistance with mite iden- 156. tification and for providing a photograph of the rat fur mite; 17. Ewing, H. E. 1938. North American mites of the subfamily Drs. Robert and Caroline Blanchard for providing the colony of Myobiinae, new subfamily (Arachnida). Proc. Entomol. Soc. Long Evans rats; Mr. Henry Morrow for assistance with photog- Wash. D.C. 40:180–97. raphy; and Debbie Chinna, Linda Magee, Janice Nako-Piburn, 18. Peper, R. L. 1994. Diagnostic exercise: mite infestation in and Dr. Karim Jeraj for assistance with the study. a laboratory rat colony. Lab. Anim. Sci. 44:172–174. 19. Tenorio, J. M. and M. L. Goff. 1980. Ectoparasites of Ha- References waiian rodents. p. 9, 28. Allen Press, Lawrence, Kansas. 1. Baker, E. W., T. M. Evans, D. J. Gould, et al. 1956. A manual 20. Barragry, T. B. 1987. A review of the pharmacology and of parasitic mites of medical or economic importance. p. clinical uses of Ivermectin. Can. Vet. J. 28:512–517. 78–80. National Pest Control Association, New York. 21. Poul, J. M. 1988. Effects of perinatal Ivermectin exposure 2. Sasa, M., H. Tanaka, M. Fukui, et al. 1973. Internal para- on behavioral development of rats. Neurotoxicol. Teratol. sites of laboratory animals. p. 195–214. In R. J. C. Harris 10:267–272. (ed), The problems of laboratory animal disease. Academic 22. Nafstad, I., E. Sannes, A. Hem, et al. 1991. Behavioral Press, New York. method to detect marginal neurotoxic effects of Ivermectin 3. Skidmore, L. V. 1934. Acariasis of the white rat (Rattus in DA/Orl rats. J. Exp. Anim. Sci. 34:81–86. norvegicus form albinus). Can. Entomologist 66:110–15.

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