Current assays used to detect Mycobacterium bovis infection lack accuracy, especially for recently infected animals, or are impractical for rapid field diagnostic applications. To overcome these limitations with serological assays, a synthetic peptide derived from early secretory antigenic target 6 (ESAT6-p) and a recombinant major secreted immunogenic protein (rMPB70) of M. bovis were used in an enzyme-linked immunosorbent assay (EIA), an immunochromatographic assay (ICGA), and a latex bead agglutination assay (LBAA). Sera from noninfected, M. bovis-infected, or M. avium subsp. Bovine tuberculosis (bTB) is caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex. bTB is one of the important bovine diseases causing great economic loss worldwide (21, 30). These are also diseases of concern for other ruminant species and humans (8, 21). The presence of M. bovis in wild-animal reservoirs (21,23,32,38,49) has also made control of the diseases more difficult.At present, the standard diagnostic assay for bTB is the single intradermal (SID) test (skin test) with purified protein derivative (PPD). There are many limitations to the SID test and other cell-mediated immunity (CMI)-based tests (e.g., gamma interferon [IFN-␥] and lymphocyte proliferation assays) for the diagnosis of bTB. The first limitation is that the delayed-type hypersensitivity response to the antigen has low sensitivity and specificity. It is not possible to determine if the response to PPD is attributable to exposure to M. bovis, M. avium subsp. paratuberculosis, or environmental mycobacteria, e.g., Mycobacterium avium subsp. avium. Second, there is a predominant CMI response during the early phases of infection with low bacillary loads which may be reduced or absent in animals at the advanced stages of disease with high bacterial loads. The animals become anergic to CMI-based tests (18,35,42,46). Third, animals need to be handled twice over a 72-h period to obtain results of the test. Fourth, other CMI-based tests are expensive and require trained staff to perform the tests and interpret the results. Fifth, false positives may result from exposure to environmental mycobacteria and/or vaccination with BCG. Therefore, there is a need to develop sensitive and easy-to-use M. bovis-specific serological tests that can be used to distinguish between animals exposed to M. bovis, M. avium subsp. paratuberculosis, and environmental mycobacteria.