CHARACTERIZATION OF TRANSMISSIBLE GASTROENTERITIS CORONAVIRUS S PROTEIN EXPRESSION PRODUCTS IN AVIRULENT SALMONELLA-TYPHIMURIUM DELTA-CYA DELTA-CRP - PERSISTENCE, STABILITY AND IMMUNE-RESPONSE IN SWINE

The spike protein from transmissible gastroenteritis virus (TGEV) was expressed in attenuated S. typhimurium Delta cya Delta crp Delta asd c hi 3987. Three partially overlapping fragments of TGEV S gene, encodin g the amino-terminal, intermediate, and carboxy-terminal end of the pr otein, as well as the full length gene were inserted into the asd(+) p lasmid pYA292 to generate recombinant plasmids pYATS-1, pYATS-2, pYATS -3, and pYATS-4, respectively, which were transformed into S. typhimur ium chi 3987. Recombinant S. ryphimurium chi 3987 (pYATS-1) and chi 39 87 (pYATS-4) expressing constitutively a 53 kDa amino-terminal fragmen t of the S protein and the full length protein (144 kDa), respectively , showed high stability. After 50 generations in vitro 60% and 20% of the bacteria transformed with pYATS-1 and pYATS-4, respectively, expre ssed the S-protein antigen. Since S. typhimurium chi 3987 (pYATS-1) sh owed a better level of expression and stability in vitro, this recombi nant strain was selected as a potential bivalent vector to induce both immunity to Salmonella and TGEV in swine. In order to study colonizat ion of swine tissues by S. typhimurium Delta cya Delta crp, a gene con ferring resistance to rifampicin was cloned into the chromosome of S. typhimurium chi 3987, generating chi 4509 strain. Both S. typhimurium chi 4509 (pYA292) and chi 4509 (pYATS-1) colonized the ileum of orally inoculated swine with clearance of bacteria between days 10-20 post-i nfection. The expression of the amino-terminal fragment of the S prote in diminished the ability of S. typhimurium chi 4509 (pYATS-1) to colo nize deep tissues. The recombinant strain S. typhimurium chi 3987 (pYA TS-1) induced TGEV specific antibodies in both serum and saliva of ora lly inoculated swine. This strain, as well as S. typhimurium chi 3987 (pYA292), also elicited both systemic and mucosal immunity to Salmonel la antigens.