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Development of a PCR assay for the detection of Spironucleus muris
Glenn A Jackson1, Robert S Livingston, Lela K Riley
1Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
Abstract:
Spironucleus muris is a protozoan that can colonize the intestinal tract of many rodent species. Although its effects on animal health and research are debated, S. muris is often included on exclusion lists for rodent facilities. Common diagnostic tests for S. muris are insensitive and typically are performed at postmortem examination. We sought to develop a PCR-based diagnostic test with sufficient sensitivity and specificity for use on fecal samples from live rodents. We designed and optimized a PCR assay that targeted the 16S-like rRNA gene of S. muris. The assay was highly specific, given that samples from mice contaminated with S. muris were PCR positive, whereas samples from mice contaminated with other protozoa were negative. The assay also was highly sensitive, detecting as few as 5 template copies per microliter diluent. All mice positive for S. muris on postmortem exams also were positive by fecal PCR. Moreover, S. muris was detected by PCR in mice negative by postmortem examination but from colonies known to be contaminated as well as in rats and hamsters. To assess protozoal loads in mice of differing ages, the PCR assay was adapted to a quantitative format. Fecal loads of S. muris were highest in 4-wk-old mice and declined with age. The PCR assay developed promises to be a highly specific antemortem diagnostic assay with higher sensitivity than that of existing postmortem tests.
Insights
A new PCR diagnostic test accurately detects Spironucleus muris in live rodents using fecal samples. This highly sensitive and specific antemortem method improves upon existing postmortem diagnostics for this common intestinal protozoan.
Area of Science:
- Veterinary Parasitology
- Molecular Diagnostics
- Rodent Health
Background:
- Spironucleus muris is an intestinal protozoan affecting rodents, often leading to exclusion from research facilities.
- Current diagnostic methods for S. muris lack sensitivity and are typically performed postmortem.
- Accurate detection is crucial for managing S. muris in research settings.
Purpose of the Study:
- To develop a sensitive and specific PCR-based diagnostic assay for antemortem detection of S. muris in rodents.
- To enable diagnosis using readily available fecal samples from live animals.
- To improve upon the limitations of existing diagnostic techniques.
Main Methods:
- Design and optimization of a PCR assay targeting the 16S-like rRNA gene of S. muris.
- Testing the assay's specificity using samples from rodents infected with S. muris and other protozoa.
- Evaluating the assay's sensitivity by detecting low template copy numbers.
- Validation against postmortem examination results and application to various rodent species and ages.
Main Results:
- The PCR assay demonstrated high specificity, correctly identifying S. muris and distinguishing it from other protozoa.
- The assay exhibited high sensitivity, detecting as few as 5 template copies/µL.
- All mice positive by postmortem examination were also positive by fecal PCR.
- S. muris was detected in mice negative by postmortem but known to be contaminated, as well as in rats and hamsters.
- Quantitative PCR revealed highest fecal S. muris loads in 4-week-old mice, decreasing with age.
Conclusions:
- A highly specific and sensitive PCR assay for antemortem diagnosis of S. muris in rodents has been developed.
- This fecal PCR test offers superior sensitivity compared to current postmortem diagnostic methods.
- The assay facilitates early detection and monitoring of S. muris infections in live rodent populations.
- This tool can aid in better management and control of S. muris in research facilities.
