It is known that mycobacterial heat shock proteins of pathogenic mycobacteria elicit cellular immune responses in both humans and animals. In the present study, recombinant M. bovis HSP 60 kD and 70 kD, and M. paratuberculosis PPD antigens were used in blastogenesis assays to test T cell reactivity of cows in various stages of infection with M. paratuberculosis. Subsequently overlapping 15-mer peptides from HSP 70 were used in epitope mapping studies. In both infected and vaccinated cows the stage of infection was determined by fecal culture results, clinical signs, and age. Cows from M. paratuberculosis free herds, as determined by fecal culture, served as negative control animals. Preliminary results suggest that cell mediated responses against HSP 70 kD and PPD correlate with the stage of infection while responses against HSP 60 kD, which were observed infrequently, did not. Average responses against PPD are higher in non-shedders vs. healthy cows, healthy shedders vs. non-shedders, and clinical cases vs. healthy shedders. Responses against HSP 70 kD parallel the PPD responses, except for clinical cases. In clinical cases the response against HSP 70 kD equals that of healthy negative control animals; while responses against PPD are still above the level observed in healthy shedders. Vaccinated animals have HSP 70 kD responses equal to healthy shedders. HSP 70 kD epitope mapping using PBMC's of a negative control animal, a healthy shedder, and a clinical cow revealed that all three animals recognized conserved aminoterminal epitopes. In addition, the subclinical shedder recognized carboxyterminal epitopes. In contrast, the clinical cow recognized the same epitopes as the negative control but non that were recognized by the healthy shedder. We are in the process of analyzing the nature of the regulatory events apparently causing differences in T cell reactivity in different stages of infection and their relevance for immunopathogenesis.