Evaluation of Sentinel-Free Soiled Bedding as a Replacement for Direct Sampling in PCR Detection of Rodent Pathogens during Quarantine
Establishing a screening method for rodent pathogens is an integral component of an institution’s rodent health surveillance program. Most rodent quarantine programs use direct colony sampling (DCS) from live animals for pathogen PCR testing. DCS may elicit undue stress to the animal and be time consuming, contingent on the number of quarantined animals. Sentinel-free soiled bedding (SFSB) sampling has previously been used to monitor the pathogen status of research rodents. A pilot study was conducted to determine the feasibility of an SFSB-based quarantine by comparing it to DCS for the evaluation of pet shop mice of different age ranges. Enhanced detection by SFSB for the 6- to 10-wk age range supported further investigation. The subsequent main study evaluated whether SFSB sampling is as effective as traditional DCS for detecting rodent pathogens. We hypothesized that SFSB contact media sampling is either equivalent to or more effective than DCS for detecting pathogens in quarantined mice. The study included mice imported from various institutions between October 2023 and August 2024. The DCS and exposed contact media were tested using PCR analyses. The total number of positive agent assay detections by DCS was 157, compared with 173 with the SFSB method. These results suggest that contact media sampling provides equivalent or superior detection of rodent pathogens compared with the DCS method. Although not statistically significant, it was observed that delaying sample submission decreased detection rates for 2 RNA viruses: 80% for murine astrovirus-1 (MuAstV1; 4 out of 5 samples) and 67% for genogroup V norovirus (MNV; 4 out of 6 samples). Immediate submission restored the detection rate to 100% for MuAstV1 and 91% for MNV. To conclude, SFSB rapid submission during quarantine provides a reliable and effective alternative to the traditional DCS method for the detection of rodent pathogens. This nonintrusive methodology mitigates stress during sampling while enhancing the effectiveness and sensitivity of pathogen detection.
Main study timeline of protocol events for contact media exposure.
Percent positive assays by method for all main study samples positive by at least one method (percent agreement). Data are presented based on quarantine groups; SFSB from QG 1 to 10 were held before testing, and SFSB from QG 11 to 30 were submitted for testing immediately after collection. Data from all groups were combined for overall effect (bottom). Percentage of positive detections was determined by totaling the number of positives for DCS or SFSB and dividing by the total expected positives for each QG (determined by a positive for either DCS or SFSB) multiplied by 100 to determine percentage.
Total number of positives for each assay by method from the main study. The total number of positives for each agent is presented based on quarantine groups; SFSB from QG 1 to 10 were held before testing, and SFSB from QG 11 to 30 were submitted for testing immediately after collection. Data from all groups were combined to understand overall effect (bottom). *, P < 0.05, **, P < 0.005, ***, P < 0.0005; ns, not statistically different.
Estimated (Est.) copy numbers per MNV and MuAstV1 PCR reaction for paired SFSB collection media held for less than 4 wk before testing and held for greater than 4 wk before testing. Lines indicated paired samples. Given the protocol change between QG 1 to 10 and 11 to 30 based on detection of RNA viruses, paired samples were evaluated to determine the estimated copy numbers at each testing time point relative to the corresponding DCS sample. Samples are labeled with the QG number.
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