This thesis focuses on vaccination-based control of bovine paratuberculosis, a chronic mycobacterial infection of the small intestine. Bovine paratuberculosis is a highly prevalent disease affecting ruminants worldwide, leading to substantial economic losses. There are concerns that the causative agent is involved in the pathogenesis of human Crohn’s disease, a debilitating inflammatory bowel disease. Therefore, dairy industries have installed national programs to limit human exposure via consumer products, like milk and cheese. Control programs for paratuberculosis are in need of an effective vaccine, however currently there is no vaccine registered for use in cattle in the EU. This thesis evaluates a candidate subunit vaccine, based on recombinant mycobacterial Heat shock protein 70, against bovine paratuberculosis for efficacy, safety and ability to differentiate vaccinated animals from infected animals. Young calves, most susceptible to acquire infection with Mycobacterium avium subspecies paratuberculosis (MAP), often get infected shortly after birth, before a classical vaccine can offer a protective response. Therefore, a vaccine is needed that also has efficacy in exposed animals, so called therapeutic vaccination. It was shown that vaccination of chronic naturally MAP infected cows resulted in reduced fecal shedding and prolonged survival in a proportion of animals, which correlated with vaccination-induced Hsp70 antibodies. Analysis of the Hsp70 vaccination-induced immune response revealed that the B cell response was dominant above Th1 reactivity. Vaccination-induced antibodies can bind Hsp70 in the cell wall of MAP, and provide a possible explanation for the observed protection. Proteome analysis showed that Hsp70 is an abundant protein identified in tuberculin used for the diagnostic skin test for diagnosing bovine tuberculosis. Surprisingly however, vaccination with Hsp70 did not interfere with diagnosis of bovine tuberculosis by skin testing. Antibody-based assays for paratuberculosis can be adapted by inclusion of Hsp70 in the pre-absorption step to remove cross-reactive proteins and thereby differentiating infected animals from vaccinated animals. Furthermore, it was concluded that the use of the candidate vaccine is safe, based on the observation of limited adverse effects after immunization. These characteristics are important pre-requisites for registration of a new paratuberculosis vaccine. Overall, these results show that the candidate vaccine Hsp70/DDA is safe and does not interfere with diagnosis of paratuberculosis and tuberculosis infected cattle. The efficacy of Hsp70/DDA needs to be improved, for example by combining it with other vaccine components, in order to contribute to paratuberculosis disease control.