Helicobacter spp. infections in mice can have broad-ranging effects on gastrointestinal, reproductive, and immune systems. This can introduce significant confounding variables for research and may reduce scientific rigor. Screening mouse colonies for Helicobacter species can be accomplished via noninvasive PCR testing on filter paper placed in animal-free dirty bedding sentinel cages. In our facility, one tablespoon of dirty bedding from each cage on a rack is added to a designated sentinel cage every 3 wk at cage change, and PCR testing is performed on in-cage filter paper quarterly. We hypothesized that cages that received Helicobacter spp.-positive bedding at later time points would have a lower detection rate of Helicobacter spp. with PCR testing compared with cages that received positive bedding at earlier time points due to the filter paper becoming saturated. To determine if screening would be able to detect one positive row of cages on a rack, 9 tablespoons of Helicobacter-positive bedding and 71 tablespoons of negative bedding were added at the 3-, 6-, or 9-wk time points to 14 empty sentinel cages per time point. Negative bedding was added every 3 wk to cages not scheduled to receive positive bedding. Negative controls received 80 tablespoons of negative bedding and positive controls received 80 tablespoons of positive bedding at each time point. Filter paper was tested via PCR for Helicobacter spp. at 12 wk. All positive controls tested positive, and all negative controls tested negative. Two 3-wk cages, two 6-wk cages, and three 9-wk cages were positive, indicating no difference between time points. This resulted in a 16.7% Helicobacter spp. detection rate. These results indicate that PCR on in-cage filter paper may not be reliable in detecting low levels of Helicobacter spp. nucleic acid in dirty bedding.Abstract
Experimental cage with dirty bedding and filter medium. According to the University of Oklahoma Health Sciences standard operating procedures, each cage contains one tablespoon of dirty bedding from each cage on the rack and filter medium for PCR detection of infectious agents. Dirty bedding is added to the sentinel cage every 3 wk at cage change out. Filter media are tested quarterly for a list of defined excluded pathogens.
Experimental design and timeline. Each box represents each experimental group (5 groups, n = 14 per group) at each time point. Group A cages were exposed to Helicobacter spp.-positive bedding at the 3-wk time point. Group B cages were exposed to Helicobacter spp.-positive bedding at the 6-wk time point. Group C cages were exposed to Helicobacter spp.-positive bedding at the 9-wk time point. Blue boxes indicate that the cages were exposed to Helicobacter spp.-positive bedding. Green boxes indicate that the cages were exposed to Helicobacter spp.-negative bedding only. Filter media were present in each cage starting on day 0. All filter media were removed and submitted for PCR at the 12-wk time point.
Number of Helicobacter spp.-positive cages per experimental group. Group A cages were exposed to Helicobacter spp.-positive bedding at the 3-wk time point, group B cages were exposed to Helicobacter spp.-positive bedding at the 6-wk time point, and group C cages were exposed to Helicobacter spp.-positive bedding at the 9-wk time point. In-cage filter paper from each cage was submitted for PCR at the 12-wk time point. Any result with a copy number greater than zero was considered a positive cage. All positive control cages were positive on PCR. Groups A, B, and C had few Helicobacter spp.-positive cages compared with the positive control group. ns, not significant (P > 0.05).
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