Validation of an in-house real-time PCR fecal assay and comparison with two commercial assays for the antemortem detection of Mycobacterium avium subsp. paratuberculosis infection in culled sheep

Abstract

Paratuberculosis is a chronic infectious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). In sheep, the antemortem detection of the infection is challenging given the slow progression of the disease and the lack of sensitive, specific, and cost-effective validated tests. We adapted an in-house real-time PCR (rtPCR) assay targeting the multi-copy IS 900 element of MAP. The sensitivity and specificity of this essay for the detection of MAP infection were estimated in a convenience sample of culled ewes from 7 infected flocks and compared to a commercial fecal rtPCR, a commercial ELISA, and fecal culture. An infected ewe was defined as a ewe with a positive culture of the ileum and/or mesenteric lymph node. A non-infected ewe was defined as a ewe negative in intestinal tissue culture, negative in fecal culture, and with no lesions consistent with paratuberculosis. The in-house rtPCR had a sensitivity estimate of 84% (95% confidence interval [CI]: 59%, 97%) among the 44 infected ewes, which was significantly higher ( p ⩽ 0.05) than the sensitivity of a commercial fecal rtPCR (52%, 95% CI: 27%, 76%; or 63%, 95% CI: 35%, 87% depending on the cutoff used), an ELISA (14%, 95% CI:2.0%, 41%), and fecal culture (21%, 95% CI: 2.7%, 59%). No statistical difference in assay specificities was observed for the 30 non-infected ewes. The in-house rtPCR is a promising tool that could be used advantageously for the antemortem detection of MAP infection in sheep.