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A desirable test to diagnose infections withMycobacterium avium subsp.paratuberculosis facilitates identification of infected cattle prior to the state ofM. avium subsp.paratuberculosis shedding. This study aimed at adjusting a flow cytometry (FC)-based assay, using intactM. avium subsp.paratuberculosis bacteria as the antigen, for diagnosis ofM. avium subsp.paratuberculosis infections in calves. Serum samples were collected from experimentally infected (n = 12) and naturally exposed (n = 32) calves. Samples from five calves from positive dams were analyzed to determine the dynamics of maternal antibodies. Samples from adult cattle with defined infection status served as the standard (18M. avium subsp.paratuberculosis shedders, 22M. avium subsp.paratuberculosis free). After preadsorption withMycobacterium phlei , sera were incubated withM. avium subsp.paratuberculosis andM. avium subsp.avium bacterial suspensions, respectively, followed by the separate detection of bovine IgG, IgG1, IgG2, and IgM attached to the bacterial surface.M. avium subsp.paratuberculosis -specific sample/positive (S/P) ratios were compared to enzyme-linked immunosorbent assay (ELISA) S/P ratios. In adult cattle, the FC assay for IgG1 had a sensitivity of 78% at a specificity of 100%. Maternally acquired antibodies could be detected in calves up to 121 days of life. While all but two sera taken at day 100 ± 10 postnatum from naturally exposed calves tested negative, elevated S/P ratios (IgG and IgG1) became detectable from 44 and 46 weeks postinoculation onwards in two calves infected experimentally. Even with the optimized FC assay,M. avium subsp.paratuberculosis -specific antibodies can only occasionally be detected in infected calves less than 12 months of age. The failure to detect such antibodies apparently reflects the distinct immunobiology ofM. avium subsp.paratuberculosis infections rather than methodological constraints.

Flow Cytometric Detection of Mycobacterium avium subsp. paratuberculosis-Specific Antibodies in Experimentally Infected and Naturally Exposed Calves