ELISA assays are broadly used as a diagnostic tool for paratuberculosis, in order to early identify infected cattle. Nevertheless, specificity problems happen due to the great number of shared antigens between Mycobacterium avium subsp. paratuberculosis (Map) and other Mycobacteria, especially the closely related M. avium subsp. avium. Despite of an M. phlei pre-adsorption step, which intends to eliminate cross-reactions with environmental bacteria, other infections such as bovine tuberculosis, a frequently occurring disease in Brazil and in many other developing countries, may interfere with the specificity of the test. In this study, 97 sera from M. bovis infected cows, with skin-test reactive and confirmation by histopathology and tissue baciloscopy were used. In a first step, sera were tested by a PPA (paratuberculosis protoplasmatic antigen - Allied Monitor, USA) ELISA that is routinely used for the diagnosis of paratuberculosis. From the 97 sera 25 (26.59%) presented OD >0.60, when cut-off point for this test was standardized on 0.35. From those, twenty sera were randomly chosen for Immunoblot analysis. This analysis demonstrated that 18 (90%) sera strongly reacted with four PPA bands; one 32-36KDa, one 42KDa and two others between 77 and 100KDa. A standard paratuberculosis positive serum also recognized these same four bands, which indicates that those proteins are shared between M. bovis and Map. In conclusion, although ELISA tests may be used as valuable tool for the detection of paratuberculosis infected herds, interpretation of results in tuberculous cattle should be very criterious.