Although currently available paratuberculosis vaccines may reduce the incidence of clinical disease, they may not prevent infection with M. paratuberculosis. The purpose of these experiments was to evaluate the immune response to commercially available vaccine (Strain 18) compared with vaccine prepared from a field isolate. The adjuvant effect of IL-12 administration was also evaluated, as was the timing of vaccination in relation to challenge exposure to M. paratuberculosis organisms. Calves were vaccinated with Strain 18 vaccine +/- hrIL-12 at 2-7 days of age and given an oral challenge with field strain M. paratuberculosis 3 weeks later. Other calves were given the oral challenge at 2-3 days of age, then vaccinated (Strain 18+/- hrIL-12) one week later. Finally, some calves were vaccinated with heat-killed field strain M. paratuberculosis +/- hrIL-12 at 2-7 days of age, then given an oral challenge 3 weeks later. Calves were euthanized at 49 days, and 40 tissues collected for mycobacterium culture on HEYM. Lymph node cells from ileocecal lymph node and prescapular lymph node (draining vaccination site) were cultured in-vitro, stimulated with M. paratuberculosis antigen, and IFN-g concentration in supernatant determined by ELISA. Vaccination in all 3 experiments resulted in high concentrations of IFN-g from prescapular and ileocecal lymph node cultures. Inclusion of hrIL-12 with the vaccine seemed to have no significant effect on IFN-g production by lymphocytes, although it did result in reduced IL-4 gene expression in draining lymph node. Severity of infection (n of colonies M. paratuberculosis recovered by culture) was reduced when vaccination was given before challenge exposure (both commercial and field-strain vaccines) compared with vaccination after challenge. Also, field strain vaccine showed better protection than commercial vaccine. Finally, some calves vaccinated with IL-12 before challenge were culture-negative in all tissues, while none of the calves vaccinated after challenge were completely protected.