Evaluation of an Enzyme-Linked Immunosorbent Assay Licensed by the USDA for Use in Cattle for Diagnosis of Ovine Paratuberculosis

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Abstract
A commercially available Mycobacterium phlei-absorbed enzyme-linked immunosorbent assay (ELISA) approved to detect antibodies to Mycobacterium paratuberculosis in cattle was evaluated for its applicability in sheep. The potential for interference with ELISA results from cross-reacting antibodies to Corynebacterium pseudotuberculosis was also investigated. Serum samples were randomly selected from a collection of samples obtained in 1986-1991 from 6 infected and 5 noninfected sheep flocks varying in breed, age, and geographic origin. Tests were performed on sera from 27 paratuberculous sheep, confirmed by histopathology, bacteriologic culture, and/or acid-fast staining of ileal mucosal smears, and on sera from 246 noninfected sheep. The optical density of each sample was expressed as a percentage of the optical density of a known positive sheep serum sample tested on the same plate. These values were log-transformed to achieve normality of distribution, and sensitivity and specificity estimates were calculated based on 2 and 3 standard deviations above the mean of the percent positive value (PPV) of the noninfected sheep. A cutoff value of PPV ≥ 55.74 resulted in an estimated sensitivity of 0.48 and a specificity of 0.95. Sera from 10 noninfected sheep with PPV above the cutoff level of 55.74% were absorbed with heat-treated C. pseudotuberculosis organisms in addition to M. phlei antigens. Sera from 14 ELISA-positive paratuberculous sheep and 23 ELISA-negative noninfected sheep were similarly treated, and results were compared. Absorption with C. pseudotuberculosis resulted in a significant decrease in PPV in all 3 groups of sheep sera, but a greater decrease was observed in the noninfected sheep with PPV above the cutoff level when compared with noninfected sheep with PPV below that level. Results of this study suggest that ELISA may be of value in screening sheep flocks for paratuberculosis, but further experimentation is needed to optimize the sensitivity and specificity of the assay. Exposure to C. pseudotuberculosis may confound results obtained by M. phlei-absorbed ELISA for paratuberculosis.

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