Paratuberculosis is a chronic wastage disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), a facultative intracellular bacterium. The disease is a significant financial burden with combined losses to the Victorian beef and dairy industries of close to $6 million per annum. Vaccine development for paratuberculosis has slowed due to the costs and time associated with assessing novel vaccines. Furthermore, the novel vaccines developed often don’t provide enhanced protection compared to currently available commercial vaccines. Therefore, new ways of assessing vaccines must be explored to expedite this process. For these reasons the main aim of this thesis was to develop a platform to assess in vivo vaccine efficacy in the natural host using an in vitro assay. To do this, a rapid system to assess the ability of ruminant cells to kill intracellular MAP was developed and was used to determine vaccine efficacy in both sheep and cattle. In addition, the immune profiles of animals that were successfully protected by vaccination compared to vaccine non-responders were assessed. Profiles of different immune factors that relate to a successful vaccine response against paratuberculosis in sheep and cattle were then compiled. The results generated from this thesis have led to the development of a model that details the important stages in the immune response following vaccination and exposure of ruminant to MAP. Specifically, the ability to mount a rapid and potent IgG1 response, followed by infiltration of large numbers of CD4+ T cells, CD8+ T cells, T cells and B cells into the ileum. Finally, a tight regulation of the cell mediated and humoral immune response following clearance of MAP is required. The insights gained through this model will aid in the development and screening of novel vaccine candidates for paratuberculosis in the hopes that a suitable candidate that prevents infection can be found.