JOHNE'S DISEASE (JD), CAUSED BY MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS (MAP), IS ONE OF THE MOST SIGNIFICANT PROBLEMS IN ANIMAL HEALTH. WE PLAN TO DEVELOP LIVE-ATTENUATED VACCINES THAT CAN DIFFERENTIATE VACCINATED FROM INFECTED ANIMALS (DIVA). THESE VACCINES ARE EXPECTED TO ELICIT CELLULAR IMMUNITY BY ACTIVATING T-CELLS. WE PLAN TO GENERATE ATTENUATED MUTANTS WITH DIVA CAPABILITIES, BUT WITHOUT DRUG MARKERS. MUTANTS NOT EXPRESSING MAP_1152 AND MAP_1156 ANTIGENS WILL BE CONSTRUCTED AND TESTED FOR ATTENUATION IN BOVINE MACROPHAGES, A FIRST TEST FOR REDUCED VIRULENCE. TO TEST DIVA, THE CORRESPONDING RECOMBINANT ANTIGENS WILL BE EVALUATED FOR INDUCTION OF CELLULAR IMMUNITY AND ANTIBODY PRODUCTION IN NATURAL AND EXPERIMENTALLY INFECTED ANIMALS. TO ASSESS THE IMMUNOGENICITY AND PATHOGENICITY, CALVES WILL BE ORALLY INOCULATED WITH THE MUTANTS. WE WILL DETERMINE HISTOPATHOLOGY, INTESTINAL COLONIZATION, FECAL SHEDDING AND IMMUNE RESPONSES. THE OVERALL IMPACT IS THE EMBODIMENT OF A JDVACCINE WITH DIVA CAPABILITIES. THIS ACHIEVEMENT WOULD BE SIGNIFICANT IN AMELIORATING THE NEGATIVE IMPACT OF JD ON NATIONAL AND INTERNATIONAL TRADE.