Technical Abstract: Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis either by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multi-antigen print immunoassay for determination of antigen recognition patterns, immunoblot for sensitive kinetic studies, and VetTB STAT-PAK™ test – a lateral flow, rapid test. Responses to MPB83 were detected for all M. bovis-infected animals, regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 wks after inoculation, were boosted upon injection of PPD’s for skin testing, and persisted throughout the course of each of the 4 challenge studies. MPB83-specific IgM was detected prior to that of IgG; however, these early IgM responses rapidly waned demonstrating a benefit of tests that detect both IgM- and IgG-specific antibody. The VetTB STAT-PAK™ test detected responses in sera from 60% (15/25) of the animals by 7 wks after challenge and 96% (24/25) by 18 wks. These findings demonstrate the potential for antibody-based tests in the early detection of M. bovis infection in cattle.