The effectiveness of Mycobacterium avium ssp paratuberculosis (MAP) killed-cell in oil vaccine at full, half, and quarter doses for inducing T cell mediated immune responses was evaluated using three groups of 10 age-matched calves (1-35 days of age). Another group of 10 calves were mock vaccinated and served as a control group. At 2 and 4 months after vaccination, blood samples were collected for cell-mediated immunity assays. MAP-PPD was used as recall antigen in the whole blood (WB) IFNγ assay to detect the extracellular interferon gamma (IFNγ) expression and in the multi-parameter flow cytometry assay to detect the expression of high affinity IL-2 receptor (CD25), intracellular IFNγ and intracellular interleukin-4 (IL-4) expression by CD4+, CD8+, and γδ TCR+ T cells. At 2 months after vaccination, the WB IFNγ assay and the multi-parameter flow cytometry assay showed significant (P<0.05) increases in CD25, IFNγ , and IL-4 expression in all T cell subsets of the full dose vaccinated group compared to the control group (except that the γδ TCR+ T cells did not express IL-4). Among the 3 vaccinated groups, the responses were not significantly different. At 4 months after vaccination, the WB IFNγ assay responses of the full and quarter dose vaccinated groups were still significantly higher than the control group (P<0.05). By 4 months after vaccination, at least 2 of the 3 vaccinated groups had significant increases in CD4+ cell expression of CD25, IFNγ , and IL-4. The CD8+ T cells did not have detectable increases in expression of either CD25, IFNγ, or IL-4, and the γδ TCR+ T cells had increased expression of CD25 and IFNγ but not IL-4. There were no significant differences between the vaccinated groups. The results demonstrate that the WB IFNγ and the multi-parameter flow cytometry assays could detect significant T cell specific responses to MAP-PPD at 2 months after vaccination but the responses decreased when detected at 4 months after vaccination. All three doses of vaccine were able to induce similar significant responses to MAP-PPD with both assays.