MEDICINE UPDATE: DIAGNOSTICS AND TREATMENTS 2. Materials and Methods
For the purpose of this study, 677 equine fecal and 686 environmental samples were collected. Each sample was inoculated into selenite or tetrathionate broth and incubated for 18 to 24 hours. After incu- bation, the enrichment broth samples were subcul- tured onto xylose-lysine-tergitol-4 or hektoen agar plates. Suspected Salmonella spp. colonies were subcultured and further identified with the use of biochemical assays. Concurrently to the microbio- logical analysis, 1 mL of the enrichment broth was processed for DNA purification. The samples were analyzed individually (1363 samples) and in pools of up to 10 samples (139 pools) with the use of a Sal- monella spp. real-time PCR assay targeting the in- vasion A gene.
3. Results and Discussion
The pooling strategy was able to detect all fecal and environmental samples dually positive by PCR and
culture. Three environmental sample pools tested PCR-positive; each of these pools contained two to five individual culture and PCR-positive samples. Two additional PCR-positive and culture-negative environmental samples tested negative by PCR when pooled together. Eleven fecal samples cul- tured positive for Salmonella spp. All these fecal samples were also PCR-positive at the individual and pooled levels. Eight additional PCR-positive and culture-negative fecal samples gave rise to five positive and three negative pools.
Acknowledgment This study was supported by a grant from The
Center for Equine Health, University of Califor- nia, School of Veterinary Medicine at Davis and Zoetis.
