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On the basis of our 2011 microbiological monitoring tests, we report here the current microbiological status of mice and rats housed in experimental facilities in Japan. We tested more than 14,000 mice, 6,000 serum samples, 500 fecal or cecal samples, and 200 lung samples from 3,549 mouse facilities within Japanese universities and institutes (U/I), pharmaceutical companies and contract research organizations (P/C). We also tested more than 1,500 rats, 1,600 serum samples, and 20 fecal or cecal samples from 772 U/I and P/C rat facilities. Bacterial cultures, serology, microscopy, PCR, and DNA analysis using DNA chips were performed. Staphylococcus aureus (18.8% in mouse facilities, 58.6% in rat facilities) was the most prevalent agent in both the mouse and rat facilities. The next most prevalent agents in the mouse facilities were murine norovirus (11.97%), intestinal protozoa (0.05-8.49%, from various species), Pasteurella pneumotropica (5.32%), and Helicobacter hepaticus (3.17%), while intestinal protozoa (0.74-6.84% from various species), Syphacia muris (6.20%), Pseudomonas aeruginosa (3.61%), and Pasteurella pneumotropica (3.05%) were the subsequent most prevalent agents in the rat facilities. These results suggest that the currently prevalent microbes in laboratory mice and rats in Japan are mainly opportunistic pathogens, intestinal protozoa, and microbes with low pathogenicity.

We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.

The RNA polymerase gene of murine norovirus (MNV) was isolated from feces and organ samples by the reverse transcription (RT)-polymerase chain reaction (PCR). For experimental infection, homogenate of cecum obtained from an MNV-infected mouse was gavaged to 3 C.B-17-Prkdc(scid) (scid) mice and 3 ICR mice at 6 weeks of age. Sixty days after oral inoculation, MNV was isolated from the cecum (3/3 scid and 3/3 ICR), feces (3/3 scid and 3/3 ICR), duodenum (1/3 scid and 3/3 ICR), liver (1/3 scid and 1/3 ICR), and spleen (3/3 ICR) samples, but MNV was not detected in the brain, heart, kidney, lung, salivary gland, ovary, thymus, or uterus samples of any of the orally inoculated mice. Feces of males cohabiting with MNV infected dams were positive for viral RNA after 18 days of cohabitation, but 8 fetuses (embryonic day 18.0) derived from the dams were negative for the virus. The results suggest that the cecum and feces are the most suitable sample types for the detection of MNV in infected animals and that caesarean section is efficient for the elimination of the virus. In terms of spontaneous infection, the RNA polymerase gene of MNV was isolated from 33/245 (13.1%) cecum samples derived from 15/59 (25.4%) facilities, and the sequence analysis revealed that at least 5 types of the virus were prevalent. This is the first report on MNV infection in mouse colonies in Japan.

To reveal the current status of the prevalence of Bordetella hinzii in mice in experimental facilities in Japan, a survey of this agent was performed by culture of tracheal swabs from a total of 12,923 mice from 1699 facilities (12,192 mice from 1572 facilities in universities and research institutes and 731 mice from 127 facilities in pharmaceutical companies) in total. In the results, 195 out of 12,192 mice (1.6%) from 44 out of 1572 facilities (2.8%) in universities and research institutes were positive for B. hinzii. No B. hinzii-positive mice were found in 127 pharmaceutical companies surveyed. Gross lesions in the lungs with isolation of B. hinzii were observed in seven mice from four universities, and the lesions were identified as bronchopneumonia histopathologically. To our knowledge, this is the first report to reveal the prevalence of B. hinzii in laboratory mice.