The characterization of protective antigens is essential for the development of an effective, sub-unit based vaccine against paratuberculosis. Surface-exposed and secreted antigens, present abundantly in mycobacterial culture filtrate (CF), are among the well known protective antigens for M. tuberculosis and M. bovis. Here we have analyzed the antigenicity of CF from M. avium subsp. paratuberculosis (M. ptb.) in C57BL/6bg/bg mice and in calves that were infected at 1-2 weeks of age by the intravenous or oral route respectively with M. ptb. ATCC 19698. Infected mice produced elevated levels of Th1 type cytokines in spleen cell cultures following in vitro stimulation with crude CF from M. ptb., grown as a surface pellicle on synthetic Sauton medium. Similarly, PBMC from infected calves showed significant proliferative and IFN-γ responses following stimulation with this CF. Separaration of M. ptb. CF by SDS-PAGE followed by electro-elution, indicated that the most immunodominant CF fractions (in the 30-32 kD mw. range) contained homologs of the mycolyl-transferase Ag85, a promising sub-unit vaccine protein family for tuberculosis. T cell responses of infected mice and cattle were highly cross-reactive against native Ag85 purified from M. bovis BCG CF and against synthetic peptides from the Ag85A and Ag85B component of BCG. Cross-reactive T cell responses were much weaker against peptides from the Ag85C protein. These results indicate that Ag85 homologs are also immunodominant T cell antigens in M.ptb infection and that they may have a vaccine potential for bovine paratuberculosis.

Sponsorship

This work was partially supported by grants from the FWO-Vlaanderen (G.0266.00) and from Brussels Capital region. Va.Ro. holds a grant from DG6 (Former Federal Ministry of Agriculture, now Federal Public Service for Health, Food Chain Security and Environment). Vi.Ro. is a FRIA bursary.