Dr. Hassan has linked virulent S. typhimurium chi3761 to the development of Salmonella carrier status in infected chickens. Although we were able to document depletion of lymphocytes, immunosuppression, and persistent Salmonella excretion in chickens infected with chi3761, we do not have adequate understanding of what in Salmonella is responsible for inducing the host to manifest our observation. A year ago, Drs. Georg Plum and Josephine Clark-Curtiss in our laboratory developed a method to specifically isolate Mycobacterium avium genes that were expressed during growth of M. avium in human macrophages and not expressed when M. avium was grown in culture. This method employed isolation of mRNA from macrophages infected with M. avium, converting the mRNA to cDNA and then removing by subtractive hybridization host DNA sequences and M. avium cDNA's of genes expressed in vitro by using cDNA generated from mRNA obtained from in vitro grown M. avium. They thus succeeded in identifying genes principally or only expressed following uptake into macrophages.This same approach has been adopted by Dr. David MacLeod to investigate genes expressed by Salmonella in mice macrophages. He has shown that many of the genes expressed in vivo are encoded by genes that are unique to Salmonella and not present in E. coli as has been shown by Dr. Greg Mahairas and other postdocs in Dr. Curtiss group who have used genomic subtractive hybridization to identify genes of S. cholerauis, S. typhi and S. typhimurium that are not present in E. coli or not present in each other. The above methodology will be adapted and used to identify Salmonella specific cDNA sequences that are expressed in the bursa of Fabricius of chickens infected with chi3761. Dr. Curtiss laboratory is therefore the best environment for Dr. Hassan to carry out the experimental plan presented in this grant application. The long-term objectives are: 1) to characterize the molecular factors that enable S. typhimurium chi3761 to cause transient lymphocyte depletion, immunosuppression, and persistent Salmonella shedding in infected chickens and 2) to analyze specific responses that chi3761 induces in host tissues that lead to clonal depletion of lymphocytes thereby enhancing the development of tolerance to Salmonella by infected chickens.
