Persistent Corynebacterium bovis Infectious Hyperkeratotic Dermatitis in Immunocompetent Epidermal-Mutant dep/dep Mice.

During a previously reported program-wide Corynebacterium bovis outbreak, both immunocompetent depilated (dep/dep) mutant mice and transgenic mice that express the papillomavirus E6 oncoprotein became persistently infected with C. bovis. An orthokeratotic, hyperkeratotic, acanthotic dermatitis developed in the C. bovis-infected dep/dep mice, which remained C. bovis PCR-positive for >45 days prior to euthanasia as part of the program-wide C. bovis eradication effort. Since both affected strains of mice have altered skin homeostasis, immune status or the presence of hair may not alone be sufficient to explain strain susceptibility to C. bovis-related cutaneous disease. In order to avoid invalidation of preclinical studies due to C. bovis infection, it may be necessary to isolate immunodeficient mouse strains, implement facililty-wide surveillance for C. bovis, and sterilize equipment with vaporized hydrogen peroxide.

Housing and Husbandry Alternatives for Naked Mole Rat Colonies Used in Research Settings.

Naked mole rats (Heterocephalus glaber) are a unique rodent species originating in Africa and are increasingly being used in research. Their needs and characteristics differ from those of other rodents used in research. Unique housing systems are necessary to address the special macro- and microenvironmental requirements of NMRs. Naked mole rats are one of the 2 known eusocial mammalian species, are extremely long-living, are active burrowers, and are accustomed to a subterranean environment. Unlike typical rats and mice, naked mole rats need specific, unique housing systems that mimic their natural subterranean environment to support health and longevity. Here we provide an overview of naked mole rats and a housing method that can be used in research settings.

PCR Prevalence of Murine Opportunistic Microbes and their Mitigation by Using Vaporized Hydrogen Peroxide.

Exposing immunodeficient mice to opportunistic microbes introduces risks of data variability, morbidity, mortality, and the invalidation of studies involving unique human reagents, including the loss of primary human hematopoietic cells, patient-derived xenografts, and experimental therapeutics. The prevalence of 15 opportunistic microbes in a murine research facility was determined by yearlong PCR-based murine and IVC equipment surveillance comprising 1738 specimens. Of the 8 microbes detected, 3 organisms- Staphylococcus xylosus, Proteus mirabilis, and Pasteurella pneumotropica biotype Heyl-were most prevalent in both murine and IVC exhaust plenum specimens. Overall, the 8 detectable microbes were more readily PCR-detectable in IVC exhaust airways than in murine specimens, supporting the utility of PCR testing of IVC exhaust airways as a component of immunodeficient murine health surveillance. Vaporized hydrogen peroxide (VHP) exposure of IVC equipment left unassembled (that is, in a 'static-open' configuration) did not eliminate PCR detectable evidence of microbes. In contrast, VHP exposure of IVC equipment assembled 'active-closed' eliminated PCR-detectable evidence of all microbes. Ensuring data integrity and maintaining a topographically complex immunodeficient murine research environment is facilitated by knowing the prevalent opportunistic microbes to be monitored and by implementing a PCR-validated method of facility decontamination that mitigates opportunistic microbes and the risk of invalidation of studies involving immunodeficient mice.

Effects of Corynebacterium bovis on Engraftment of Patient-derived Chronic-Myelomonocytic Leukemia Cells in NSGS Mice.

Modeling chronic myelomonocytic leukemia (CMML) in immunodeficient NSGS mice relies on unique human CMML specimens and consistent murine engraftment. Only anecdotal comments have thus far supported the notion that research data may be altered by Corynebacterium bovis, an opportunistic cutaneous pathogen of immunodeficient mice. C. bovis disseminated by asymptomatic and clinically affected mice with hyperkeratotic dermatitis, resulting in resilient facility contamination and infectious recurrence. Herein we report that, compared with C. bovis PCR-negative counterparts, C. bovis PCR-positive NSGS mice developed periocular and facial hyperkeratosis and alopecia and had reduced metrics indicative of ineffective human CMML engraftment, including less thrombocytopenia, less splenomegaly, fewer CMML infiltrates in histopathologic sections of murine organs, and fewer human CD45+ cells in samples from murine spleen, bone marrow, and peripheral blood that were analyzed by flow cytometry. All CMML model metrics of engraftment were significantly reduced in the C. bovis PCR-positive cohort compared with the - negative cohort. In addition, a survey of comprehensive cancer center practices revealed that most murine facilities do not routinely test for C. bovis or broadly decontaminate the facility or its equipment after a C. bovis outbreak, thus increasing the likelihood of recurrence of invalidated studies. Our findings document that CMML engraftment of NSGS mice is diminished-and the integrity of murine research data jeopardized-by C. bovis infection of immunodeficient mice. In addition, our results indicate that C. bovis should be excluded from and not tolerated in murine facilities housing immunodeficient strains.

Facility-wide Eradication of Corynebacterium bovis by using PCR-validated Vaporized Hydrogen Peroxide.

Facility-wide Corynebacterium bovis eradication was established using vaporized hydrogen peroxide (VHP) decontamination guided by C. bovis PCR surveillance. Prior attempts limited to culling PCR-positive mice and decontaminating affected rooms were ineffective in preventing recurrence. Because research aims often require trafficking to and use of procedural cores, a 12-mo facility-wide C. bovis PCR surveillance of 2064 specimens was performed and documented that, despite the presence of few clinically hyperkeratotic mice, 35% of the murine housing and use space was contaminated by C. bovis. The airways of IVC racks and air-handling units (AHU) provided a substantive niche for C. bovis survival, comparable to the primary enclosure, with 26% of murine and 22% of airway specimens PCR-positive for C. bovis. Equipment airway VHP sterilization in a 'flex room' required an 'active-closed' setting with the IVC rack connected to the AHU set to the VHP cycle, because 12% of specimens from 'static-open' VHP-exposed airways remained PCR-positive for C. bovis, whereas 0% of specimens from active-closed VHP exposures were positive. VHP decontamination of the 29,931-ft2 facility was completed in 2 mo. C. bovis PCR testing of IVC exhaust plenums for 200 d in previously C. bovis-affected rooms confirmed that none of the 259 specimens tested were PCR-positive for the organism. Monthly surveillance identified a single recurrence during June 2017 (month 9), ensuring rapid culling of C. bovis PCR-positive mice and acute VHP decontamination of equipment and rooms. Molecular persistence of C. bovis was resolved in procedural and personnel areas, and no murine or housing specimens tested C. bovis PCR-positive during study months 11 and 12. Furthermore, since the conclusion of the 12-mo study, none of the 452 additional murine, cell biologic, environmental, and monthly equipment surveillance specimens tested were C. bovis PCR-positive, documenting an 11-mo period of facility-wide C. bovis eradication to date. Study invalidation due to C. bovis can be avoided through PCR surveillance for the organism, immediate culling of PCR-positive mice, and acute VHP decontamination of affected areas.

Staphylococcus xylosus PCR-validated Decontamination of Murine Individually Ventilated Cage Racks and Air Handling Units by Using 'Active-Closed' Exposure to Vaporized Hydrogen Peroxide.

Vaporized hydrogen peroxide (VHP) is used to decontaminate clinical, biocontainment, and research animal rooms and equipment. To assist with its implementation in a murine facility, we developed a safe and effective method of VHP sterilization of IVC racks and air handling units (AHU). Safety of VHP decontamination was assessed by ensuring VHP levels dissipated to less than 1 ppm in the room prior to personnel reentry and inside the primary enclosure prior to the return of mice; this condition occurred at least 18 h after the VHP cycle. Efficacy of VHP sterilization was assessed by using chemical indicators, biologic indicators, and PCR testing for Staphylococcus xylosus, a commensal organism of murine skin and an opportunistic pathogen, which was present in 160 of 172 (93%) of specimens from occupied IVC racks and the interior surfaces of in-use AHU. Neither mechanized washing nor hand-sanitizing eradicated S. xylosus from equipment airway interiors, with 17% to 24% of specimens remaining PCR-positive for S. xylosus. 'Static-open' VHP exposure of sanitized equipment did not ensure its sterilization. In contrast, 'active-closed' VHP exposure, in which IVC racks were assembled, sealed, and connected to AHU set to the VHP cycle, increased the proportion of chemical indicators that detected sterilizing levels of VHP inside the assembled equipment, and significantly decreased PCR-detectable S. xylosus inside the equipment. Supplementing bulk steam sterilization of the primary enclosure with VHP sterilization of the secondary housing equipment during room change-outs may help to mitigate opportunistic agents that jeopardize studies involving immunodeficient strains.