Diagnosis of bovine paratuberculosis by a novel enzyme-linked immunosorbent assay based on early secreted antigens of Mycobacterium avium subsp. paratuberculosis.

We previously reported that protein antigens of serodiagnostic potential were more abundant in culture filtrates than cellular extracts from liquid cultures of Mycobacterium avium subsp. paratuberculosis (D. Cho and M. T. Collins, Clin. Vaccine Immunol. 13:1155-1161, 2006). Based on this observation, a novel enzyme-linked immunosorbent assay (ELISA) using antigens secreted by young (early- to mid-log-phase) cultures of M. avium subsp. paratuberculosis JTC303 (a low-passage isolate originating from the ileum of a Holstein bull) in mycobactin-supplemented Watson-Reid medium (pH 6.0) was developed and evaluated using a previously described panel of bovine sera (M. T. Collins et al., Clin. Diagn. Lab. Immunol. 12:685-692, 2005) that included 444 paratuberculosis cases and 412 controls. The new assay, called JTC-ELISA, had a significantly higher diagnostic sensitivity and an equivalent specificity compared to those of five commercial paratuberculosis ELISA kits. By receiver-operating characteristic analysis, the JTC-ELISA had the highest area under the curve of the six assays evaluated. The JTC-ELISA was particularly sensitive at detecting low-level fecal shedders of Mavium subsp. paratuberculosis (40%; the sensitivity of the commercial kits was 20%). The JTC-ELISA works effectively on both serum and milk samples for the detection of cattle with subclinical M. avium subsp. paratuberculosis infections, providing a cost-effective diagnostic tool to support paratuberculosis control programs in cattle herds.