Mechanical Washing Prevents Transmission of Bacterial, Viral, and Protozoal Murine Pathogens from Cages.

Infectious agents have varying susceptibilities to thermal inactivation and/or mechanical removal from cages by the use of heated, pressurized water. In this study, we tested whether 5 specific infectious organisms (Candidatus savagella [segmented filamentous bacterium (SFB)], Helicobacter sp., mouse norovirus (MNV), Tritrichomonas sp., and Entamoeba muris) could survive the cage wash process and still infect naïve mice. These 5 organisms were chosen due to their prevalence in rodent colonies, environmental stability, and/or potential to influence experimental outcomes. Cages that had housed mice shedding all 5 organisms were assigned to 1 of 3 treatment groups: 1) sanitization in a tunnel washer followed by autoclaving (121 °C [250 °F] for 20 min; n = 40 cages); 2) sanitization in a tunnel washer (82 °C [180 °F] for an average of 30 s; n = 40 cages); or 3) control (bedding change only; n = 40 cages). The presence of these agents in the cage was assessed by performing PCR on swabs of the empty soiled cage interior before and after the treatment. In addition, to determine if any residual nucleic acid was infectious, 2 Swiss outbred (J:ARC(S)) female mice were housed for 7 d in cages from each treatment group. The above procedures were then repeated so that every week each pair of J:ARC(S) mice ( n = 10 pairs of mice/treatment group) were housed in another cage that underwent the same treatment; this was done for a total of 4 consecutive, 1-wk-long periods. Swabs collected from soiled cages were PCR-positive for SFB, Helicobacter, MNV, Tritrichomonas, and Entamoeba in 99%, 97%, 39%, 63%, and 73% of the cages tested, respectively. Cages in the tunnel wash group that were PCR-positive for SFB, Helicobacter, Tritrichomonas, and Entamoeba before treatment remained PCR-positive in 8%, 15%, 43%, and 10% of positive cages, respectively. None of the cages from the autoclave group were PCR-positive for any of the agents after treatment. None of the mice housed in cages in either the autoclave or tunnel wash groups became infected with any of the agents. However, 80%, 60%, and 100% of the pairs of mice housed in untreated cages were PCR-positive for SFB, MNV, and Entamoeba, respectively. None of the mice housed in untreated cages were positive for Helicobacter or Tritrichomonas. Our results suggest that nucleic acids from these bacterial and protozoal organisms may remain in cages after mechanical cage washing, but these nucleic acids are not infectious, and autoclaving is not necessary to prevent transmission.

Shared and distinctive features of the gut microbiome of C57BL/6 mice from different vendors and production sites, and in response to a new vivarium.

Animal models play a critical role in establishing causal relationships between gut microbiota and disease. The laboratory mouse is widely used to study the role of microbes in various disorders; however, differences between mouse vendors, genetic lineages and husbandry protocols have been shown to contribute to variation in phenotypes and to non-reproducibility of experimental results. We sought to understand how gut microbiome profiles of mice vary by vendor, vendor production facility and health status upon receipt into an academic facility and how they change over 12 weeks in the new environment. C57BL/6 mice were sourced from two different production sites for each of three different vendors. Mice were shipped to an academic research vivarium, and fresh-catch stool samples were collected from mice immediately from the shipping box upon receipt, and again after 2, 6 and 12 weeks in the new facility. Substantial variation in bacterial proportional abundance was observed among mice from each vendor at the time of receipt, but shared microbes accounted for most sequence reads. Vendor-specific microbes were generally of low abundance. Microbial profiles of mice from all vendors exhibited shifts over time, highlighting the importance of environmental conditions on microbial dynamics. Our results emphasize the need for continued efforts to account for sources of variation in animal models and understand how they contribute to experimental reproducibility.

Antibiotic-associated Manipulation of the Gut Microbiota and Phenotypic Restoration in NOD Mice.

Segmented filamentous bacterium (SFB) a gram-positive, anaerobic, and intestinal commensal organism directly influences the development of Th17 helper cells in the small intestine of mice. In NOD mice, SFB colonization interferes with the development of type 1 diabetes (T1D), a T-cell-mediated autoimmune disease, suggesting that SFB may influence Th17 cells to inhibit Th1 populations associated with the anti-ß-cell immune response. This effect is a serious concern for investigators who use NOD mice for diabetes research because the expected incidence of disease decreases markedly when they are colonized by SFB. A room housing mice for T1D studies at The Jackson Laboratory was determined by fecal PCR testing to have widespread SFB colonization of multiple NOD strains after a steady decline in the incidence of T1D was noted. Rederivation of all NOD-related mouse strains was not feasible; therefore an alternative treatment using antibiotics to eliminate SFB from colonized mice was undertaken. After antibiotic treatment, soiled bedding from NOD mouse strains housed in SFB-free high-health-status production barrier rooms was used to reintroduce the gastrointestinal microbiota. Over the past 16 mo since treating the mice and disinfecting the mouse room, regular PCR testing has shown that no additional SFB colonization of mice has occurred, and the expected incidence of T1D has been reestablished in the offspring of treated mice.

An Overview of Typical Infections of Research Mice: Health Monitoring and Prevention of Infection.

There are many reasons to keep research mice healthy and free from infections. The two most important of these are to protect the health and welfare of research mice and to prevent infections from negatively impacting research. Just as the genetic integrity of a mouse strain will influence the reproducibility and validity of research data, so too will the microbiologic integrity of the animals. This has been repeatedly demonstrated in the literature of laboratory animal sciences wherein the direct impact of infections on physiologic parameters under study have been described. Therefore, it is of great importance that scientists pay close attention to the health status of their research animal colonies and maintain good communications with the animal facility personnel at their institution about mouse health issues. This overview provides information about animal health monitoring (HM) in research mouse colonies including commonly monitored agents, diagnostic methods, HM program, risk assessment, and animal facility biosecurity. Lastly, matters of communication with laboratory animal professionals at research institutions are also addressed.

The case for genetic monitoring of mice and rats used in biomedical research.

Currently, there is the potential to generate over 200,000 mutant mouse strains between existing mouse strains (over 24,000) and genetically modified mouse embryonic stem cells (over 209,000) that have been entered into the International Mouse Strain Resource Center (IMSR) from laboratories and repositories all over the world. The number of rat strains is also increasing exponentially. These mouse and rat mutants are a tremendous genetic resource; however, the awareness of their genetic integrity such as genetic background and genotyping of these models is not always carefully monitored. In this review, we make a case for the International Council for Laboratory Animal Science (ICLAS), which is interested in promoting and helping academic institutions develop a genetic monitoring program to bring a level of genetic quality assurance into the scientific interchange and use of mouse and rat genetically mutant models.

Validation of multiplex microbead immunoassay for simultaneous serodetection of multiple infectious agents in laboratory mouse.

Multiplex methodologies enable simultaneous detection of antibodies against several infectious agents allowing sample conservation, cost effectiveness, and amenability to high-throughput/automation. We have previously described a multiplex microbead immunoassay for serodetection of ten, high-priority mouse infectious pathogens. Here, we present a validation of this multiplex diagnostic system using approximately four hundred serum samples from different groups of mice. Computer assisted multivariate analysis of the resulting high volume data (8000 data points) was performed. This computational approach enabled presentation of data in a variety of easily interpretable formats (e.g., correlation tables and heat maps). Importantly, this computer aided approach was instrumental for the evaluation of assay accuracy, sensitivity, specificity, and robustness during the study. Crucial pieces of information were obtained to make timely adjustments for assay refinement. This progressive approach to developing an implementation-ready clinical assay, facilitated by computational analysis, produced a highly efficient, accurate and dependable serodiagnostics system. This system has effectively replaced the current state-of-the-art methodology (ELISA) used in mouse colony health management at the University of California and the Jackson Laboratory. A pathway to develop multiplex serology tests for infectious disease diagnosis described here serves as a model for multiplex immunoassay design, clinical validation, refinement and implementation.

Kinetics of transmission, infectivity, and genome stability of two novel mouse norovirus isolates in breeding mice.

Murine noroviruses are a recently discovered group of viruses found within mouse research colonies in many animal facilities worldwide. In this study, we used 2 novel mouse norovirus (MNV) wildtype isolates to examine the kinetics of transmission and tissue distribution in breeding units of NOD.CB17-Prkdc(scid)/J and backcrossed NOD.CB17-Prkdc(scid)/J x NOD/ShiLtJ (N1) mice. Viral shedding in feces and dissemination to tissues of infected offspring mice were monitored by RT-PCR over a 6-wk period postpartum. Histologic sections of tissues from mice exposed to MNV were examined for lesions and their sera monitored for the presence of antibodies to MNV. Viruses shed in feces of parental and offspring mice were compared for sequence homology of the Orf2 gene. Studies showed that the wildtype viruses MNV5 and MNV6 behaved differently in terms of the kinetics of transmission and distribution to tissues of offspring mice. For MNV5, virus transmission from parents to offspring was not seen before 3 wk after birth, and neither isolate was transmitted between cages of infected and control mice. Susceptibility to infection was statistically different between the 2 mouse strains used in the study. Both immunodeficient NOD.CB17-Prkdc(scid)/J mice and NOD. CB17-Prkdc(scid)/J x NOD/ShiLtJ offspring capable of mounting an immune response shed virus in their feces throughout the 6-wk study period, but no gross or histologic lesions were present in infected tissues. Progeny viruses isolated from the feces of infected offspring showed numerous mutations in the Orf2 gene for MNV5 but not MNV6. These results confirm previous studies demonstrating that the biology of MNV in mice varies substantially with each virus isolate and mouse strain infected.