Comparison of serological tests and faecal culture for the detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and analysis of the antigens involved

Three hundred and forty-one sera from cattle in Western Australia and 106 sera from Mycobacterium paratuberculosis faecal culture positive cattle were used to evaluate the performance of two absorbed enzyme-linked immunosorbent assays (ELISA) (one locally produced, the other a commercial test) and a complement fixation test (CFT) for the detection of Johne's disease in cattle. The diagnostic sensitivity (47.2%) of the local ELISA was significantly higher than that of the commercial ELISA (31.1%), and significantly higher than that for the complement fixation test (17.9%) and immunoblot (20.8%). Diagnostic specificity for the two ELISAs was 99.7% and 97.9% and similar for CFT and immunoblot (97.1% and 97.7%, respectively). The diagnostic sensitivity rose for both ELISAs and the CFT as the number of M. paratuberculosis isolated from the faeces increased. The ELISA antigen was characterised by polyacrylamide gel electrophoresis and electrophoretic immunoblotting and was found to consist mostly of a carbohydrate-type macromolecule of 32-42 kDa. This macromolecule was identified as lipoarabinomannan (LAM) by using a LAM-specific monoclonal antibody in immunoblots and purified LAM in absorption experiments. By applying more complex antigen preparations in immunoblots, serum antibodies against proteins of 47, 37, 30, 24 and 21 kDa, and against the 32-42 kDa carbohydrate component were frequently found in infected cattle, and of these the 47 kDa protein and the 32-42 kDa antigen were immuno-dominant. Pre-absorption of the sera with M. phlei sonicate indicated that the protein antigens contributed markedly to non-specific serological cross-reactions, while the 32-42 kDa non-protein macromolecule appeared to be specific.