Johne's disease is a chronic granulomatous infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis). Clinical disease is characterized by weight loss, diarrhea, decreased milk production, and ultimately death. To date, there is no effective treatment for paratuberculosis and current vaccines do not prevent infection, but rather delay the onset of clinical disease. Furthermore, currently available vaccines interfere with bovine tuberculosis skin testing and paratuberculosis diagnostic tests. In an effort to identify protective M. paratuberculosis genomic sequences, an expression library of M. paratuberculosis was generated and subdivided into 78 pools of clones. Each clone pool, which contained an average of 1500 clones, was evaluated by PCR with a set of seventeen known M. paratuberculosis sequences. C57BL/6J mice (6 week old, male, 20 grams) were immunized with 2 ìg of DNA in the abdominal area with each clone pool via gene gun delivery. The mice were boosted three weeks after initial immunization and inoculated in the tail vein with live, virulent M. paratuberculosis (strain 19698, 10⁸ bacteria per mouse) two weeks following the boost. Protective effects of each clone pool were evaluated based on culture of viable bacteria from liver, spleen, mesenteric lymph node, and ileum obtained at necropsy three months post-challenge. Five of twenty-six clone pools examined thus far have demonstrated at least a hundred-fold reduction in M. paratuberculosis colonization in mice when compared to other clone pools and nonvaccinated, infected control mice.