Background.

Early diagnosis of paratuberculosis in cattle is essential for controlling the spread of Johne's disease. Available tests lack sensitivity. We have shown that a recombinant p35K antigen was Map specific by immunoblotting.

Purpose.

To develop a purified p35-based ELISA and p35-immunoblots and to evaluate their efficacy compared to commercial ELISAs.

Methods.

Map-specific p35 protein was purified as a histidine-tagged fusion protein. A purified p35-based ELISA was developed and optimized and its efficacy evaluated on 44 reference sera. The results were compared to that of commercial ELISA assays and immunoblotting. The efficacy of the ELISAs was evaluated on sera from 100 cattle in a suspected infected herd.

Results.

Sera from 12 cattle with clinically advanced JD were positive by all assays. Sera from 10 Map-free cattle and 3 cows artificially inoculated with multiple doses of viable Map were negative by all assays. For sera from cows with subclinical infection, p35-ELISA was positive in 13/19 (68%) as compared to 42%, 47% and 53% for the most commonly used commercial ELISAs, respectively. One additional sample (14/19; 74%) was positive only by p35 immunoblotting. If the p35 assay was used in parallel with one commercial assay, the sensitivity increased to 79% (15/19). The specificity and the positive predictive values of the tests were 100% and the negative predictive values (NPV) of p35-ELISA and the better commercial-ELISA assays were 62.5% and 52%, respectively. When testing the 100 serum samples from cattle in the infected herd, the p35-ELISA was comparable but not significantly different from commercial ELISAs.

Conclusion.

Based on this limited initial evaluation, the p35-ELISA shows promise as a potentially more sensitive and specific test for paratuberculosis. The addition of p35-based immunoblot test further improved detection of subclinical cases. Evaluation of p35-ELISA on a larger set of well-characterized sera is warranted.