The present commercial vaccines against Mycobacterium paratuberculosis do not prevent infection. One of the reasons for this may be the route of administration-parenteral route may favor strong systemic immunity but may not induce mucosal (enteric) immunity. We carried out studies aimed at targeting/enhancing the enteric immune response by oral vaccination. Two groups of calves were vaccinated on their first day of birth orally with heat-killed field strain M. paratuberculosis containing 8 x 10⁸ and 8 x 10⁷ CFU (high and low dose respectively). The calves were challenged with two dose of live organisms (8 x 10⁸ CFU) on days 21 and 22 post immunization. Calves were euthanized at day 42 and 40 tissues were collected for mycobacterium culture. Spleen, prescapular and ileocecal lymph node cells were isolated, cultured in vitro in the presence of heat-killed M. paratuberculosis antigen or concanavalin A (Con A) and the concentration of IFN-g in supernatant was measured by commercial ELISA kit. Although immunization enhanced the systemic IFN-g response (PBMCs and prescapular lymph node), it did not enhance enteric IFN-g response beyond that induced by infection alone. Neither vaccination nor infection caused any significant change in Con-A-induced production of IFN-g by cells from spleen, prescapular and ileocecal lymph nodes. Paradoxically, immunization with low, but not high dose, caused a significant reduction in both the number of bacterial colonies and total number of colony positive tissues. The data suggest that oral vaccination may be a means of targeting and enhancing local enteric immunity against M. paratuberculosis, and that protection may be vaccine dose-dependent.