OBJECTIVE-To evaluate the effect of vaccination of calves with a killed Mycobacterium avium subsp paratuberculosis (MAP) vaccine on colonization of tissues following oral MAP exposure. ANIMALS-12 healthy Holstein calves. PROCEDURES-At 14 days after birth, calves received the MAP vaccine (1.0 mL, SC) or saline (0.9% NaCl) solution (1.0 mL, SC [control treatment]). Each calf received 1.2 x 10(9) CFUs of live MAP orally 21 and 22 days after vaccination. Prior to vaccination and at subsequent intervals, a blood sample was collected for ELISA detection of antibodies against MAP and for whole blood, antigen-specific, interferon (IFN)-gamma-release assay. Nine weeks after MAP challenge, calves were euthanized and various tissue samples were collected for mycobacterial culture. Interferon-gamma production in prescapular lymph node cells was measured following in vitro stimulation with MAP antigens. RESULTS-Calves were seronegative for anti-MAP antibodies at all times. Compared with the findings in control calves, antigen-specific IFN-gamma production in circulating lymphocytes and prescapular lymph node cells from vaccinated calves was significantly higher. Culture of tissues from vaccinated calves yielded significantly fewer CFUs of MAP (2,417 CFUs/g), compared with tissues from control calves (15,709 CFUs/g). Furthermore, significantly fewer tissue samples from vaccinated calves yielded MAP in culture (21.8 tissues/calf), compared with findings in control calves (27.6 tissues/calf). CONCLUSIONS AND CLINICAL RELEVANCE-Inoculation of calves with a killed MAP vaccine was associated with reduced colonization of intestinal tissues following experimental exposure to MAP. Use of the vaccine could potentially reduce transmission of MAP to calves in infected herds.