Paratuberculosis is responsible for major economic losses -particularly in the dairy sector- because of reduced milk production in affected animals. It is estimated that in Belgium, 10 % of the dairy farms are infected with M. ptb. The main mode of transmission within herds is through ingestion of faecal contaminated milk. As the existing vaccine, composed of whole, killed mycobacteria, interferes with the PPD skin test used for the diagnosis of bovine tuberculosis, its use is limited and submitted to the approval of the veterinary services. A sub-unit vaccine, not interfering with bovine PPD skin test, could offer a solution to this problem. Mycobacterial culture filtrates (CF) are a rich source of secreted and surface exposed protein anti-gens and they have been reported to contain major protective antigens for tuberculosis (1). Using this information, we have analyzed CF from M. ptb ATCC 19698, grown as a surface pellicle on synthetic Sauton medium, for the presence of immunodominant T cell antigens. CF proteins were separated according to their molecular weight in 30 fractions using the "Whole Gel Eluter" (Biorad) and these fractions were tested for their capacity to elicit positive lymphoproliferative and IFN-gamma responses in PBMC cultures from naturally and experimentally infected cattle and in spleen cell cultures from experimentally infected mice. Several fractions in the 25-40 kD m.w. induced strong T cell responses. Highest IFN-gamma responses were found to fractions containing the M. ptb. homologues of the Ag85 complex, which ranks among the most promising TB vaccine candidates (2, 3). We therefore cloned the genes encoding the Ag85A and Ag85B proteins from M. ptb in eucaryotic expression vectors and tested the plasmids for immunogenicity in C57BL/6 and BALB/c mice. Strong Ag85 specific IFN-gamma and IL-2 responses could be induced by plasmid immunization. Protection studies in these and in mutant beige mice are in progress.