In these studies, an implant device was developed to detect vaccination-induced, cell-mediated immunity in the subcutaneous tissue of calves. The long-term, future aim of these combined studies is to use the implant device to efficiently screen novel Mycobacterium avium subsp. paratuberculosis (MAP) vaccine candidates. This in vivo screening test would rapidly and inexpensively provide knowledge of a vaccine’s

efficacy in the host species; vaccine candidates exhibiting evidence of efficacy could then be selected for more extensive vaccine challenge studies. A better understanding of in vivo cell-mediated immunity in response to MAP immunization is essential for the development of a novel vaccine screening test. In all of the studies presented, Mycoparà ® vaccinated bull calves were compared to unvaccinated, control calves. Our first specific aim was based on a proof-of-concept study that focused on the development of a platform for in vivo detection of cell-mediated immunity in cattle. The results of the study showed that MAP-specific in vivo measurements can be achieved with the use of our designed implant device. Histological analysis revealed a lack of cellular infiltrate in naà ¯ve calves as well as the vaccinated calf not challenged with the MAP antigen, purified protein derivative-johnin (PPD-J). This demonstrated antigen-dependent immune cell recruitment into the collagen of the implant and illustrated the collagen’s capacity for cellular migration. IFNwas only detected in the antigen-containing implants that were placed in vaccinated calves. IL-10 did not exhibit antigen or vaccine-dependent trends. In a longitudinal analysis, the implants were measured at 0, 2, 4, 6, and 8 weeks post- vaccination. IFNlevels in the MAP antigen-containing implants placed in vaccinated calves were significantly higher than the implants retrieved from naà ¯ve cattle at weeks 4

and 6 post-vaccination. These responses paralleled the responses observed in MAP- stimulated peripheral blood leukocytes (PBL) from vaccinated calves. Significant differences in IL-10 levels between the implants within vaccinated and naà ¯ve calves were not observed. The intradermal caudal fold test (CFT) demonstrated false-positive rates when one of our calves reacted to PPD-J prior to vaccination. This indicated that our implant device was capable of identifying each of the 3 vaccinated calves, suggesting an increased specificity over the intradermal CFT using PPD-J. Using live MAP bacteria, we explored the device’s potential as a bacterial killing assay; collagen and bacteria- containing implant devices were placed in the subcutaneous tissues of calves divided into two groups, naà ¯ve and vaccinated calves, at 14 and 20 days post-vaccination. After 5 days, implants were retrieved from the subcutaneous tissues and collagen was processed for evaluation of IFNcytokine levels and flow cytometric analysis of bacterial viability. Three weeks post-vaccination, bacteria was removed from the 5-day implanted collagen, and flow cytometric analysis of the bacteria demonstrated a significantly greater percentage of propidium iodide (PI) stained-MAP in the vaccinated calves compared to the naà ¯ve calves. This finding indicated an enhancement of bacterial killing within collagen implants placed in vaccinated calves. Collagen from vaccinated calves at the three week post-vaccination time point also demonstrated a significantly higher production of IFNthan collagen from naà ¯ve calves. The work of this dissertation provides supporting evidence that a subcutaneous bacterial challenge model could prove to be an efficient and cost-effective method for screening novel vaccine candidates in a natural host model.