Procedure Use Acknowledgements Discussion Developing a Cost-effective and Efficacious Method of Pinworm (Aspicularis tetraptera) Treatment for Large Colonies of Mice DL Hickman, *M Swan, P Hartman, VA Medical Center, Portland, OR Developing a Cost-effective and Efficacious Method of Pinworm (Aspicularis tetraptera) Treatment for Large Colonies of Mice DL Hickman, *M Swan, P Hartman, VA Medical Center, Portland, OR The authors wish to thank Paul Bui for his assistance with the treatment and monitoring of these animals. They also wish to thank Will Beckman for his assistance with the photography. Case History Mice: This case involved a large breeding colony of multiple strains of mice utilized for autoimmune disease model research. The room averaged over 500 cages of transgenic, knockout and immune compromised strains of mice. The investigator designated strains included RAGKO, PD1, GFP, DTG (B10PL), B6IL-13KO, ALPHA KO, HLA DR4, DR2, 1501N5, DR2 1502, MBP/1501, GFP/1501, 1501/0101, 0101, µMT, MHCII -/- and MBP/1501/GFP. Husbandry: All mice in this room were housed in autoclaved polycarbonate mouse shoebox cages with filter and wire top on a ventilated caging system. Mice were housed on eco-fresh bedding with nestlet provided to singly housed animals and breeder cages. Cages were changed once weekly in a laminar flow changing station with filter and wire tops changed out monthly. Dirty and soiled caging was sanitized in a mechanical cage washer with a final rinse temperature of 180 degrees F or 82 degrees Celsius respectively. All animals were maintained on a 12:12h light/dark cycle with lights on at 0600h. Rodents were given autoclavable rodent diet, 5010 ad libitum. This diet has a guaranteed analysis of 23% minimum crude protein, 4.5% minimum crude fat, 6.0% maximum crude fiber, and 8% maximum ash. Animals were housed either on an automatic watering system or on autoclaved polycarbonate water bottles. Room temperature and humidity were maintained at 72 degrees F and 30%. All technicians handling mice wore PPE including latex gloves sprayed with a 10% bleach solution between cages. Mice were checked daily by an animal care technician for food, water and general condition to ensure health of the colony. Any sick or dead animals were reported to the Veterinary Medical Unit for appropriate follow up Diagnosis: Pathogen identification of Aspicularis tetraptera was identified through our routine quarterly sentinel screening program. Sentinels were female CD-1 mice that were exposed to dirty bedding from colony animals for a minimum of 5 weeks. Quarterly sentinel testing consisted of screening for Sendai virus, mouse parvovirus, minute virus of mice, ectromelia virus, reovirus type 3, pneumonia virus of mice, murine adenovirus, Mycoplasma pulmonis, lymphocytic choriomenigitis virus, mouse rotavirus, mouse encephalomyelitis virus, polyoma virus, murine cytomegalovirus, mouse coronavirus and rodent pinworms and mites. This round of sentinel testing for this colony identified Aspicularis tetraptera, a species of rodent pinworm. Identification of pinworms and mites is performed through fecal floatation (fecalizers), ear examination, tape test, and direct examination of colon and cecal contents. Both direct examination of colon contents and fecal flotation were identified as positive in this case. Evaluation of Eradication Methods Options for treatment were evaluated based on past treatments used by the facility. These included Ivermectin administered orally through autoclaved polycarbonate water bottles, Ivermectin administered topically, and Fenbendazole administered orally. Considerations for the method of treatment included type of pharmaceutical, effect on research, cost, labor and rapidity to when the facility could begin treatment. Ivermectin acts as a neurotransmitter blocker that causes paralysis of the nematode and then death but subsequently is not ovicidal. As demonstrated in Table 1, Ivermectin is considered an inexpensive option that can be easily administered (being water soluble), but would require a large expense in labor to effectively treat the colony. Fembendazole-treated feed, though effective as a broad spectrum adulticidal, larvicidal and ovicidal drug, is expensive in material cost (requires a special feed, not water soluble) and labor. In addition, this would require a delay due to the special order required to obtain the feed. Nalgene carboys provided a reduction in both material and labor costs but required the use of Ivermectin as the pharmaceutical. Modified 5 gallon carboys were hooked up to the automatic watering system to administer 1% Ivermectin solution dosed at 3.2cc per gallon. Each carboy was fitted for an attachment that would hook directly into the watering system line. The attachment consisted of removing the quick action spigot and white collar that was purchased with the carboy and replacing it with the female portion of a quick disconnect and a 7/16 tap (Figure 1). Once the disconnect was in place, Loctite Thread Easy Teflon sealant was applied to quick disconnect and tap to create a water tight seal. The white collar was then replaced over the new attachment (Figure 2). Each carboy could be easily autoclaved without the white collar. Ivermectin administered through carboys proved a rapidly implemented and effective treatment for this colony of mice. Elimination was achieved after seven consecutive weeks of treatment, as evidenced by repeated sampling during the treatment period and continued sentinel screening for the facility. Treatment was completed 15 months ago and no additional outbreaks have been identified. Ivermectin has been reported to have toxicity issues in some strains and ages of mouse. For this reason, it is generally recommended that a small representative subset of animals be exposed to Ivermectin in advance of widespread administration to assess potential toxicity issues. In this case, a subset of this colony had been previously treated with Ivermectin without toxicity issues. It was believed that the pinworms returned because not all animals had been treated in the initial outbreak (at investigator request). Described methods of Ivermectin administration are labor intensive, requiring frequent topical application or changing of water bottles. Fenbendazole treated food or gel packs are considered a superior alternative to the use of Ivermectin because this drug has a wider margin of safety. However, administration of Fenbendazole treated food or gel packs also requires a significant labor cost, especially in large colony rooms. Also, because institutions must order Fenbendazole treated products specifically for the treatment of their colonies, there can be a significant time delay in the initiation of treatment. The method of Ivermectin administration described here was a cost effective method that allowed rapid implementation of treatment. Procedure Use Each rack had one five gallon carboy placed on its top shelf (Figure 4). The Thoren racks were then flushed with the Ivermectin treated water to ensure that all lixits were filled with the medicated water. Gravity was then used to pull water through the system. The colony was treated on a four day on three day off cycle for seven consecutive weeks. After four days of treatment the carboys were unhooked, the rack flushed with regular filtered water and the normal watering lines reconnected for the three day off cycle. One 5 gallon carboy lasted the full 4 day treatment for a 231 cage rack, but carboys were checked each day for adequate water levels. An autoclaved carboy was used at the beginning of each treatment cycle (Figure 3). Weanling mice who were naïve to the watering system were given water bottles dosed to the same amount as the carboys and maintained on the same cycle as each rack. Disease elimination was determined through tape testing and fecal flotation of every 10th cage in the colony. Tape testing and fecal flotation was performed every two weeks during and after treatment. Confirmation of elimination was determined at quarterly sentinel testing. Ivermectin Treatment (individual bottles) Ivermectin Treatment (automatic watering system) Fenbendazole Treatment Cost of Pharmaceuticals $20.00 $3,200 Labor Costs$3,000 (assumes 500 cages x 1 minute per cage x changing bottle twice weekly x 7 weeks x $25 per hour) $700 (assumes 4 hours per week to set up carboys and flush lines x 7 weeks at $25 per hour) $3,000 (assumes 500 cages x 1 minute x changing feed once per week x 14 weeks x $25 per hour) Additional CostsNone$800 (purchasing of carboys) None Total Costs$3,020$1,520$6,200 Time to ImplementRapid (pharmaceutical available through veterinary supplier) Slow (requires special order of feed) Figure 3 Cost Differences for the Treatment Options Considered Figure 1 Figure 2 Figure 4 Table 1: Procedure Use