Johne's disease in ruminants is caused by the pathogenic bacterium Mycobacterium avium subspecies paratuberculosis (Map). Currently available Map commercial vaccines protect against clinical disease but not infection. In this study, the proprietary Johne's vaccine Neoparasec and an aqueous formulation of Map 316F (AquaVax) were tested in sheep. Detailed immunological examination of blood and gut-associated lymphoid tissues was carried out on animals after vaccination and challenge with virulent Map to identify markers of protective immunity. Neoparasec vaccination provided significant protection against disease while AquaVax did not. Immune animals had stronger cell-mediated responses and altered proportions of CD4+, CD8+, CD25+ and B cells in blood, spleen and the gut lymphatics, than diseased animals.