DEFINING THE ROLE OF SALMONELLA TYPHIMURIUM ARTAB TOXIN IN DIARRHEAL DISEASE TO PROMOTE BOVINE VACCINE DEVELOPMENT

Based upon previous studies of toxicity and homology to well characterized AB5-type bacterial toxins, we hypothesize that the S.Typhimurium ArtAB enterotoxin is expressed during infection and promotes Salmonella pathogenicity in bovines. The long-term goal of this research is to develop a mucosal Salmonella vaccine for bovines. The objectives of this proposal will deliver preliminary evidence for the contribution of this toxin to disease, and will provide a foundation for vaccine development. To test this hypothesis we propose the following objectives:Objective 1: Confirm the prevalence and expression of artAB from bovine Salmonella. Clinical and subclinical Salmonella from fecal bovine samples will be genotyped for the presence of artAB. Bovine anti-ArtAB antibody expression will be determined by ELISA to identify in vivo toxin expression, and confirmed by RT-PCR.Objective 2: Assay the contribution of purified ArtAB to Salmonella pathogenicity. Electric cell-substrate impedance and cellular cytotoxicity assays by microscopy will be used with purified ArtAB to quantify effects on cell viability and epithelial barrier function. The mouse ligated loop model will be used to determine contributions to intestinal fluid secretion.Objective 3: Construct a S.Typhimurium artAB targeted deletion mutant. The modified lambda red recombinase will be used to disrupt artAB on the chromosome of bovine S.Typhimurium DT104. The contribution of artAB to Salmonella pathogenicity will be assessed using the mouse ligated loop model.