Development of a qPCR for the detection and quantification of spp. in sheep feces and tissues.

spp. are common causes of disease in intensive livestock production systems, and contamination of foodstuffs is of significant concern for public health. Therefore, the identification and quantification of spp. is important for monitoring the level of fecal shedding or tissue colonization in infected animals and animal products. We developed and evaluated a quantitative PCR (qPCR) method on spiked sheep tissue and fecal samples for the detection and quantification of spp. Without the use of a pre-enrichment step, the qPCR limit of detection (LOD) results for sheep fecal (4 × 10-6 × 10 cfu/g) and tissue (4 × 10-4 × 10 cfu/g) samples were not adequate for detection purposes. With the inclusion of a 6-h pre-enrichment step in buffered peptone water (BPW), the LOD was 9 cfu/g (2.57 × 10 copies/g) in sheep feces, and 5.4 cfu/g (3.22 copies/g) sheep tissue. Comparison of the 6-h BPW qPCR method with a 24-h mannitol-selenite-cystine broth enrichment culture method using spiked samples revealed a sensitivity of 91% and 92%, respectively, and a specificity of 100% for both methods. The correlation was significant between the quantity (copies/mL) of spp. in BPW at 6 h and at 0 h, allowing semiquantitative analysis. Our results demonstrate that, following inclusion of a 6-h pre-enrichment step in BPW, qPCR is semiquantitative with improved LODs of spp. in sheep fecal and tissue samples.