The efficacy of commercial (Strain 18) and field-isolate paratuberculosis vaccine preparations was investigated. The effect of prior exposure to Mycobacterium paratuberculosis and the adjuvant effect of rIL-12 on vaccine efficacy were also tested. Both Strain 18 and field-isolate vaccines induced strong local, systemic and enteric IFN-gamma responses. A significant reduction in mycobacterial colonization was observed when calves were vaccinated with the field-isolate prior to challenge, but not following vaccination with Strain 18 vaccine. Vaccination with rIL-12 prevented infection in some calves but its overall effect on IFN-gamma response and total mycobacterial load was not statistically significant. Efficacy of paratuberculosis vaccines may be enhanced if calves are vaccinated prior to M. paratuberculosis exposure with field-isolate vaccine instead of Strain 18 vaccine currently in use.