Objective—To monitor by use of 5-color flow cytometry the antigen-specific responses of subsets of peripheral T cells in cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis (MAP) vaccine and to compare results with those for 2 established cell-mediated immunity assays.

Animals—45 female Holstein cattle with negative results for MAP in skin tests conducted at time of inoculation with MAP.

Procedures—Cattle were allocated to 4 groups. Cattle of group 1 (n = 12) were 0 to 3 months old and inoculated with a killed MAP vaccine. The 10 cattle of group 2 were the same age as those in group 1 but were not inoculated with MAP vaccine. The 11 cattle of group 3 were 9 to 12 months old and inoculated with killed MAP vaccine. The 12 cattle of group 4 were the same age as those in group 3 but were not inoculated with MAP vaccine.

Results—Flow cytometry identified T-cell subsets that responded specifically to the recall antigen. Results of assays for CD25 expression and wholeblood interferon-γ had the strongest correlation with results for skin tests as well as results with each other. Intracellular expression of interferon-γ was not correlated as well with results for the other tests.

Conclusions and Clinical Relevance—Flow cytometry can be useful for characterizing the immune response after administration of MAP vaccine and should be evaluated with regard to its sensitivity and specificity when used in detecting cattle naturally infected with MAP.