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Polymerase Chain Reaction on In-cage Filter Paper at Different Time Points to Detect Helicobacter spp
Abby C Bernardini1, Wendy R Williams2
11Oklahoma Comparative Medicine Training Program, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.
Abstract:
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.
Insights
Screening mouse colonies for Helicobacter spp. using PCR on filter paper in sentinel cages may not reliably detect low-level infections. This method showed a low detection rate, suggesting potential limitations for research accuracy.
Area of Science:
- Veterinary Microbiology
- Laboratory Animal Science
- Infectious Disease Research
Background:
- Helicobacter spp. infections in research mice can significantly impact study outcomes across multiple physiological systems.
- Maintaining pathogen-free mouse colonies is crucial for scientific rigor and reproducible research.
- Noninvasive PCR testing of dirty bedding in sentinel cages is a common method for Helicobacter spp. screening.
Purpose of the Study:
- To evaluate the reliability of PCR testing on filter paper in sentinel cages for detecting Helicobacter spp. in mouse colonies.
- To determine if the timing of introducing positive bedding affects Helicobacter spp. detection rates.
- To assess the sensitivity of this screening method for identifying low-level infections.
Main Methods:
- Sentinel cages with filter paper were inoculated with varying amounts of Helicobacter spp.-positive and negative mouse bedding at 3, 6, and 9-week intervals.
- Negative and positive control cages received exclusively negative or positive bedding, respectively.
- Filter paper from all sentinel cages was tested for Helicobacter spp. DNA using PCR at 12 weeks.
Main Results:
- All positive controls tested positive, and all negative controls tested negative, validating the PCR assay.
- A low overall detection rate of 16.7% for Helicobacter spp. was observed across all time points.
- The timing of positive bedding introduction (3, 6, or 9 weeks) did not significantly alter the detection rate.
Conclusions:
- PCR testing on in-cage filter paper may not be a sufficiently sensitive method for detecting low levels of Helicobacter spp. nucleic acid in dirty bedding.
- The saturation of filter paper or other factors may limit the effectiveness of this screening approach.
- Further refinement or alternative methods may be necessary for reliable Helicobacter spp. surveillance in research mouse colonies.
