Use of Selamectin and Moxidectin in the Treatment of Mouse Fur Mites

Abstract

A breeding colony consisting of 250 different strains of mice was treated with the topical acaricide selamectin for the mouse fur mite Myocoptes musculinus, with no apparent ill effect, suggesting that this drug is safe for use in mice. To further evaluate their efficacy in treating Myocoptes spp., we compared selamectin with another acaricide, moxidectin, in a controlled manner. Infested mice were treated with selamectin or moxidectin at the time of cage change, and a subset of mice was retreated 10 d later. Mice underwent routine cellophane tape examination of the pelage for 1 y. Although no adult mites were found in any group at 1 mo after treatment, egg casings were found in the selamectin treatment group as late as 6 mo after treatment, prompting concern about its effectiveness. Moxidectin used in combination with cage changing was effective in eradicating mites, with mice negative for traces of mites on cellophane tape examination of the pelage from months 2 through 12 after treatment.

Fur mites are a common pathogen of laboratory mice. In 1 study, more than 33% of research institutions were reported to have had fur mites in at least 1 of their colonies.¹⁶ Myocoptes musculinus is the most common fur mite of mice, although coinfestations with Myobia musculi are possible. M. musculinus is a surface-dwelling mite, feeding on epidermal tissue. Its lifecycle ranges from 8 to 14 d, with eggs hatching in 5 d, and neonates becoming infested within 4 to 5 d of birth. Transmission is by close contact, and transmission via eggs or nymphs in bedding is the foundation of many sentinel programs. The mite M. musculi has a 23-d lifecycle, with eggs hatching in 7 to 8 d. Adults appear by day 15 and lay eggs within 24 h. These mites also dwell on the skin surface and are transmitted in the same way as M. musculinus. The potential adverse effects of fur mites include dermatitis, allergic-type responses, abnormal behavior and immune dysfunction.^17,18,25

Until recently, ivermectin was the drug of choice for treating fur mites in mice.⁵ However, this drug is labor-intensive to use because several treatments are required. Furthermore, ivermectin can cause neurotoxicity and confound behavioral research in some strains of mice, so it must be used with caution.^5,7 Recently the availability of antiparasitic medications for both companion and production animals has dramatically increased. These drugs may be underused by research institutions attempting to control parasite outbreaks. In 2004 our group used 1 relatively new drug, moxidectin, to treat fur mites discovered in 1 room of our facilities.²⁶ Moxidectin belongs to the milbemycin drug class, similar to avermectins, but is longer-acting in the prevention of heartworm in dogs¹⁹ and is ovicidal in the treatment of ticks in cattle.¹³ In our facility, a single dose of moxidectin appeared to eradicate the fur mites in 2 strains of knockout mice, with negative pelage tape tests at 2, 4, and 8 wk.²⁶ Unfortunately, in other mice treated with this regimen, fur mites were rediscovered approximately 1 y later. Whether the reappearance of fur mites constituted a new infestation or a treatment failure of the moxidectin is unclear. Because the mice were housed in microisolation caging and were not exposed to any other mice, a new infestation was unlikely. Treatment failure might indicate failure of the drug itself or its administration schedule to adequately eradicate mites; treatment failure might also result from human error leading to inadequate treatment. Moxidectin has been shown to be effective in treating mites in other species but may require more than a single dose.⁶

Another new drug, selamectin, is an avermectin like ivermectin, but has been modified to improve safety.¹ Selamectin is effective in the treatment of companion animals with both surface-feeding (Otodectes cynotis, Cheyletiella yasurgi) and burrowing (Sarcoptes scabei) mites.^6,8,29 Some studies in companion animals have achieved complete resolution of mites after a single topical application,² and others report the use of an additional application 1 mo later.^4,21 Controlled studies with selamectin administered to laboratory dogs, cats, and rabbits resulted in ectoparasite eradication with the use of a single dose.^1,21 The use of selamectin has been described in CD1 mice, which have been used as an animal model to determine the efficacy of flea-control compounds.²⁷ Safety testing, also in CD1 mice, showed no clinical signs at 3 times the recommended dose of 10 mg/kg, only mild and transient clinical signs at 10 times the recommended dose, and mild clinical signs at 30 times the recommended dose.¹ Like ivermectin, selamectin can be toxic in P-glycoprotein knockout mice, which are about 100 times more sensitive to the potential toxicity of avermectins than are wild-type mice.^1,20 The degree of residual action of selamectin in mice is unknown, but residual action occurs in other species.^9,10,28 In cats, for example, selamectin is effective against reinfestation of fleas for as long as 14 d after treatment and is partially effective for 30 d after treatment.⁹ Both avermectins and milbemycins act by potentiating the effect of γ-aminobutyric acid and interfering with glutamate-gated ion channels of parasites.^11,20

At our institution, 3 sentinel mice exposed to soiled bedding in the breeding colony showed Myocoptes musculinus fur mites on pelage tape examination; 1 sentinel had monitored mice previously infested with fur mites and treated with moxidectin. Pelage tape tests of the mice previously treated with moxidectin showed that they were again infested with fur mites. In addition, another infested mouse strain had passed through the inhouse quarantine program without detection of mite infestation. Although the mice underwent multiple pelage tape examinations, no mites were detected during the 6-wk quarantine period. In an effort to eradicate fur mites from our breeding colony, all mice within the colony (approximately 8400 mice) were treated. Selamectin was chosen because it had been shown to be safe in mice,^1,20 was ovicidal,²² and has been effective as a single treatment^1,21 and because we had lost confidence in moxidectin. In addition, to further evaluate the therapeutic efficacy of selamectin and moxidectin, we compared these 2 drugs in the treatment of infested CF1 mice.

Materials and Methods

Humane care and use of animals.

Animals were housed in an AAALAC-accredited facility and in compliance with the Guide for the Care and Use of Laboratory Animals.²³ All procedures involving animal use were approved by the Institutional Animal Care and Use Committee at Emory University.

Housing and husbandry.

Until June 2005, incoming mice that had not been purchased from approved vendors were quarantined at our facility. On arrival, mice were placed in polycarbonate shoebox cages with isolator tops and corncob bedding. The room was kept on a 12:12-h light:dark cycle, and mice were fed fenbendazole-containing rodent chow (150 ppm, Rodent Diet 5T40, Purina Mills Test Diet, Richmond, VA) and autoclaved tap water ad libitum. Each cage was assessed 6 times in 8 wk for ecto- and endoparasites by using cellophane tape tests of the pelage, fecal floats, and anal tape methods. At the 6th week after arrival, mice were anesthetized with isoflurane, and blood was collected from the retroorbital sinus. Serum was diluted 1:4 with normal saline, frozen, and sent to a commercial rodent diagnostic laboratory (Charles River Laboratories, Wilmington, MA) for serologic evaluation for Sendai virus, pneumonia virus of mice, mouse hepatitis virus, mouse minute virus, Theiler's murine encephalomyelitis virus, reovirus 3, Ectromelia, Mycoplasma pulmonis, rotavirus, mouse adenovirus, lymphocytic choriomeningitis virus, K virus, polyoma virus, and mouse parvovirus. Mice seronegative for the listed pathogens and negative on parasitology panels were released into the mouse maintenance and breeding colonies.

Beginning in June 2005, 2 changes were made to these quarantine practices. First, the quarantine program was contracted to an outside source, which placed each shipment of mice in its own isolation cage and followed the procedures described earlier. In addition, all incoming mice were treated once topically between the shoulder blades with moxidectin (0.5 mg/kg; Cydectin pour-on containing 0.5% moxidectin as active ingredient in coconut oil, Fort Dodge Animal Health, Fort Dodge, IA).

After successful completion of quarantine at the contract location, mice were transferred back to our facility. Those comprising the inhouse breeding colony, mice were housed in polycarbonate shoebox cages with isolator tops and corncob bedding. The room was kept on a 14:10-h light:dark cycle, and mice were fed rodent chow (Rodent Diet 5021, Purina Mills Test Diet, Richmond, VA) and autoclaved tap water ad libitum. Mice on the acaricide study were kept inhouse in similar cages in a cubicle suite on a different floor from the breeding colony. The study room was kept on a 12:12-h light:dark cycle, and mice were fed rodent chow (Rodent Diet 5001, Purina Mills Test Diet) and autoclaved tap water ad libitum. The study mice were treated in sequence after the breeding colony was treated, and rooms were entered and managed by entirely different personnel, eliminating the possibility of reinfestation from the breeding colony

Mice.

The breeding colony at our facility consisted of 4 rooms, containing 15 ventilated racks, 4 static racks, and more than 250 different strains of mice. Mouse strains included both transgenics and knockouts but not P-glycoprotein knockout mice. The total number of mice treated was estimated at 8400. Mice originated from approved commercial vendors and other biomedical research institutions. Some mice in the colony had undergone quarantine at our facility, and more recent arrivals had undergone quarantine at the contract institution. Outbred mice of the CF1 strain were used as sentinels; outbred female CF1 mice were used for the therapeutic evaluation of moxidectin and selamectin.

The pathogen status of mice in our colonies was monitored by means of a sentinel program. Two dedicated sentinel mice on each side of a ventilated rack were exposed to soiled bedding by being placed directly into a soiled cage that had just been vacated by its occupant(s). Sentinels were placed into new soiled cages in a rotating fashion 3 d per week. Quarterly, sentinels were evaluated for ecto- and endoparasites by using cellophane tape tests of the pelage, fecal floatation, and anal tape methods. Mice were anesthetized with isoflurane, and blood was collected from the retroorbital sinus or facial vein. Serum was diluted 1:4 with normal saline, frozen, and sent to a commercial rodent diagnostic laboratory (Charles River Laboratories) for serologic evaluation for Sendai virus, pneumonia virus of mice, mouse hepatitis virus, mouse minute virus, Theiler's murine encephalomyelitis virus, reovirus 3, Ectromelia, Mycoplasma pulmonis, rotavirus, mouse adenovirus, lymphocytic choriomeningitis virus, K virus, polyoma virus, and mouse parvovirus. Ectromelia serology was performed every 6 mo. The panel was expanded once yearly to include lymphocytic choriomeningitis virus, K virus, polyoma virus, and mouse adenovirus. Starting in September 2007, sentinel mice were euthanized quarterly and replaced with new sentinels.

Breeding colony treatment and testing.

Consistent with recommended practice, breeding colony mice were treated only after a representative sample of mutant mice had been treated to evaluate selamectin for safety (data not shown).³² Mice were treated topically with 10 mg/kg selamectin (Revolution, Pfizer Animal Health, Exton, PA) at the time of cage changing. Adults were treated with 5 μl selamectin between the scapulae by using an automatic pipettor (Pipetman, Gilson, Middleton, WI), and unweaned pups were treated with 3 μl. Hairless neonates were not treated, but the dams and sires with which they were caged were. Mice were placed into clean cages immediately after treatment and were observed daily for signs of illness or injury, including neurologic complications. Efficacy of treatment was evaluated by using the routine sentinel surveillance program.

Acaricide treatment and evaluation.

Thirty 6- to 7-wk-old female CF1 mice were exposed by means of direct contact to sentinel mice infested with Myocoptes mites and tested weekly by using cellophane tapes of the pelage weekly until the female CF1 mice were confirmed to be infested with mites. Because Myocoptes mites have a predilection for colonizing the ventral abdominal and inguinal areas,¹⁵ tape tests were collected from the axillary and inguinal areas as well as the more traditional area of the dorsal fur between the shoulders and neck.³² Infested mice were allocated randomly into 5 groups of 6 mice each: 2 groups each receiving 1 dose of selamectin or moxidectin (groups Moxidectin 1 and Selamectin 1), 2 groups each receiving 2 doses of moxidectin or selamectin 10 d apart (groups Moxidectin 2 and Selamectin 2), and 1 group receiving no treatment. Drugs were applied to the dorsum of the mice, between the scapulae, by using an automatic pipettor in a volume of 5 μl for selamectin (10 mg/kg) and 3 μl for moxidectin. Cages were changed at the time of treatment. Because the pharmacokinetics of moxidectin and selamectin in mice are unknown, the 10-d timeframe was chosen so as to interrupt the lifecycle of mites should the first treatment not prove to be ovicidal. Pelage tape tests were done on days 0, 2, 4, 7, 14, and, to determine long-term efficacy, on months 1, 2, 3, 6, 9, and 12. The presence of adult mites (live or dead), eggs, or empty egg casings were recorded. No distinction was made between live or dead adult mites, because finding even an adult carcass is considered a positive result in our facility. Mice were observed daily for signs of illness or injury, including neurologic complications. The group receiving no treatment showed signs of alopecia and mild pruritis by 6 mo, was euthanized, and therefore no tape tests were done for this group in months 9 and 12. In addition, 2 mice in the Moxidectin 1 group developed unrelated illness that necessitated euthanasia at 6 mo, so these additional 2 animals also did not receive tape tests at 9 and 12 mo. Thus a total of 312 pelage tape tests were evaluated over the course of 1 y. Subsequent to the 12-mo pelage tape test, mice were euthanized and submitted for pathologic examination. They underwent gross necropsy, with histopathology of the skin and of any gross abnormalities.

Cost analysis.

Costs were determined by calculating the cost of drug used per mouse. This figure was added to the cost of an average of 1 automatic pipettor tip per cage, the use of the automatic pipettor itself (assuming 25,000 uses over its lifetime), and labor at an average of 1 mouse per minute, which includes setup and cleanup.

Results

Breeding colony treatment.

Veterinary staff and animal care staff worked together during cage changeout to accomplish the single selamectin treatment of the breeding colony. Organization and administration of the treatment took 1 mo, with an estimated total of 100 man-hours of labor. No clinical signs or fatalities were attributed to the treatment in any of the more than 250 strains treated. The total cost for the treatment was estimated at US$5000, including drug and labor. At the time of this publication (2 y after treatment), the standard sentinel program has tested 443 sentinels, all of which have been negative for evidence of mites on cellophane tape examination of the pelage, showing that the breeding colony remains free of fur mites.

Therapeutic evaluation of moxidectin and selamectin.

Transmission of infestation from sentinel to study mice took approximately 2 mo of contact exposure. Pelage tape test results are presented in Table 1. Adult mites, eggs, or egg casings were found on the pelage of all mice in all treatment groups through day 14 after acaricide treatment. By 1 mo, adults were no longer found in either selamectin group, although eggs or casings were found in both the Selamectin 1 group (6 of 6 mice) and Selamectin 2 group (2 of 6 mice). In the moxidectin treatment groups at 1 mo, adult mites were found on 2 mice, 1 that had been treated once and 1 that had been treated twice. Eggs and egg casings also were found in both the Moxidectin 1 group (4 of 6 mice) and Moxidectin 2 group (3 of 6 mice). From months 2 through 12, adult mites and eggs were no longer found in any treatment group. Egg casings were found in 3 mice, all of which had been treated with selamectin: at 2 mo in a mouse treated twice, and at 6 mo in 2 mice treated once. Pelage tapes on months 9 and 12 were negative for all evidence of mites in all groups. Tape tests of the untreated control group continued to reveal adults, eggs, and casings through the 6th month, at which point the mice were euthanized. Necropsy with skin histopathology at 12 mo did not reveal any evidence of mites in any treatment group.

Table 1.

Positive signs of mites in mice treated with moxidectin and selamectin

	Day 2			Day 4			Day 7			Day 14			Day 28			Month 2			Month 3			Month 6			Month 12
Treatment	A	E	C	A	E	C	A	E	C	A	E	C	A	E	C	A	E	C	A	E	C	A	E	C	A	E	C
None	2	6	1	1	6	4	3	4	4	4	5	5	4	5	6	4	4	0	6	2	1	4	6	5	–	–	–
Moxidectin 1	1	6	6	2	6	6	1	6	4	1	5	4	1	1	4	0	0	0	0	0	0	0	0	0	0	0	0
Moxidectin 2	5	5	2	1	6	5	4	1	1	5	2	1	1	3	3	0	0	0	0	0	0	0	0	0	0	0	0
Selamectin 1	3	6	3	2	4	6	0	2	6	3	3	4	0	3	4	0	0	1	0	0	0	0	0	0	0	0	0
Selamectin 2	1	5	3	0	6	4	2	3	6	1	6	6	0	1	1	0	0	0	0	0	0	0	0	2	0	0	0

A, adults; E, eggs; C, egg casings

At the 9-mo point, all animals were negative for adults, eggs, and egg casings.

Discussion

Treatment of ectoparasites can be a costly and labor-intensive undertaking. The topic is of obvious interest to laboratory animal clinicians, and a recent review of the literature using the keywords mouse, acariasis, and treatment revealed 48 citations discussing various permutations of ectoparasite infestations in mice. Studies used various medications, combinations of medications and husbandry practices in attempt to eradicate ectoparasites, including Myocoptes spp., Myobia spp., Radfordia spp., and Orthnythonyssus bacoti. One of the more recent articles advocated a particularly cumbersome multimodal drug approach, using selamectin and nestlets, changed weekly, soaked in amitraz and fipronil,³ presumably an expensive method of fur mite treatment. Another recent article eradicated fur mites by using cross-fostering in combination with ivermectin.¹⁴ Although this strategy was effective, cross-fostering is labor-intensive and therefore costly. The goal of the work done here was to evaluate the efficacy of 2 relatively simple, and therefore presumably relatively cost-effective, treatment schemes in combination with the routine cage change and to test whether administering each drug twice instead of once improved efficacy. In addition, the comparison of the 2 drugs and timing of treatment at cage changing was driven by a treatment failure in a prior use of a single treatment with moxidectin without a cage change.

In our experience, treating 1 group of rooms (the breeding colony, with a census of approximately 8400 mice) with a single dose of selamectin for fur mites cost approximately US$5000, the majority of which was associated with labor (approximately 100 man-hours). Our calculations show the drug cost of selamectin to be US$0.0459 per mouse (US$385.56 for 8400 mice) and moxidectin to be US$0.0007 per mouse (US$5.88 for 8400 mice). The difference in drug cost initially may seem large, but it pales in comparison with the overall $5000 price tag, which reflects the intensive labor involved in topically treating each individual mouse. Although this expense and amount of labor may appear burdensome, it is likely to be considerably less costly than other recently reported effective methods, including the cross-fostering and multimodal drug approaches described above.

Over the long term and on the surface, both drugs appeared to be effective in eradicating mites, with no adults found in either treatment group after 1 mo. Treating twice with selamectin may have been of some benefit at 1 mo, potentially resulting in more rapid depletion of the mite population, but later months showed no difference between treating once and twice. Although no adult mites were found after the first month, the egg casings found at 2 and 6 mo in the selamectin group are a source of concern, because the hairs to which the casings adhered could have been shed by that time or the casings could have been removed by grooming. Although much is known about mouse hair cyclicity, the duration of telogen in the adult mouse varies by strain,³¹ and when the hair shaft would have been shed is unknown, although in CD1 mice, an outbred strain similar to CF1, the majority of hair is shed by 8 wk.²⁴ Likewise, while grooming is an intensive part of the normal behavioral repertoire of mice, it cannot be said how long it would take for a mouse to groom one particular hair. Therefore whether the remaining egg casings were evidence of continuing, albeit attenuated, infestation or a remnant of the original one which was now resolved is unclear. Our suspicion is that an attenuated infestation may have still been present.

Along with limitations in knowledge about hair cycles and grooming are the inherent limitations of the pelage tape test. This test appears to be robust when an infestation is accompanied by a heavy burden, as we found in untreated mice and as has been shown previously.³³ However, there is a risk of false negatives in situations where the mite burden is low, either because the mice are early in the stage of infestation or because mature mice have stable and controlled burdens.¹⁴ In the case of Myocoptes mites, the sensitivity of the test likely was increased by sampling the axillary and inguinal areas as well as behind the neck,¹⁵ but false negatives are still possible, particularly in cases of light mite burdens. Mathematical calculations suggest that the evaluation of 312 samples would be sufficient to detect at least 1 positive sample with a 95% level of confidence if 1% of the samples were positive for parasites.³⁰ However, these calculations do not consider assay sensitivity, and previous work has estimated that the sensitivity of the pelage tape test for detecting Myocoptes is approximately 84%;¹⁴ therefore it remains an assay with some limitations. Because direct comparisons were not done, whether the sensitivity of the test was increased by sampling from the axillary and inguinal regions in addition to the more traditional site of between the shoulder blades is difficult to say.

In light of the possible limited sensitivity of the pelage tape test, the findings of egg casings several months after treatment with selamectin make selamectin a drug of dubious value for the treatment of fur mites in mice. This conclusion apparently contradicts another recent study indicating success in clearing mite infestation with selamectin.¹² However, the cited study evaluated mice only twice after they were treated, tested only until 3 wk after treatment, and did not consider eggs or egg casings in their assessment. The length of time of our study (1 y after treatment), evaluation of multiple tests, and consideration of any evidence of mite presence adds to the robustness of our findings. Even though subsequent sentinel results indicate that the breeding colony has remained free of mites, the finding of the egg casings several months after treatment in the selamectin group has led us to rule out selamectin as a treatment, and we have discontinued its use. Moxidectin, a less-expensive alternative, fared better therapeutically in our study, with nothing found on the pelage of mice from months 2 to 12. With that said, however, this study was inspired initially by perceptions of previous treatment failure involving moxidectin. A notable difference between this study and our previous experience was the cage change at the time of treatment. This intervention would have removed any eggs or nymphs from the environment, markedly ameliorating the chance for reinfestation from the environment should moxidectin prove to have minimal residual activity. The previous single treatment was not associated with a cage change, and perhaps, if there was little residual action from the moxidectin, mice could have been reinfested from eggs or larvae remaining in the bedding. Moxidectin is highly lipid-soluble (100 times more so than ivermectin), and when administered orally or by injection, redistributes to fat, where it acts as a reservoir to be released into the blood for redistribution to the site of parasite burden.²⁰ Similar residual activity after topical treatment in mice remains to be determined and is an important piece missing from the fur mite treatment puzzle.

The possibility also remains, as it always does, that human error was the source of the previous treatment failure and that a cage of mice was either not treated or was treated insufficiently. The initial moxidectin treatments were done by using a Hamilton syringe, which, although highly accurate, is tedious to use. A single person treating hundreds of mice this way might well miss a mouse. In addition, because mice were not placed into a fresh cage, a mouse might have been assumed to have been treated when it was not. Use of the automatic pipettor made the process less tedious and more rapid, and the addition of the cage change to the regimen allowed treated mice to be placed in a fresh cage, ameliorating the chance of personnel losing track of which mice had been treated. Moxidectin in combination with a cage change proved effective, and we currently use it as part of our routine program for mice entering the breeder colony and in quarantine.