Of the global 57 million deaths each year, about 1.8 (WHO) to 2.2 (UNESCO) million are due to infections with enteric pathogens including Salmonella, Escherichia coli and Shigella sp., and other entero-pathogens such as Yersinia sp., Vibrio cholerae, Campylobacter jejuni, Helicobacter pylori, Listeria monocytogenes, Clostridium perfringens and C. difficile. These pathogens are responsible for significant morbidity and are transmitted to humans by contaminated food and water. In developing countries, bacterial enteropathogens in food animals are major factors that decrease productivity of livestock creating food shortages and under-nourishment in humans. We believe that improving health, nutrition and economic well-being (the latter dependent on the first two) provide the best means to enhance the quality of life globally. We therefore spent much effort to devise new improved means to construct recombinant attenuated Salmonella vaccine vector systems for animals and humans that are capable of inducing cross-protective immunity to enteric pathogens and of delivering genus- or species-specific protective antigens to further enhance induction of immunity to enteric pathogens. We propose to use these technologies to develop vaccines to prevent/reduce diarrheal diseases caused by bacterial enteropathogens in agriculturally important animals and humans. Our objectives include to: (i) complete construction of S. Typhimurium and S. Paratyphi A strains with regulated delayed attenuation attributes to expose and display cross-protective surface antigens, that minimize induction of immune responses to serotype-specific antigens, that possess mutations to enhance safety and immunogenicity, that can delivery multiple genus- and species-specific protective antigens and that display biological containment to preclude persistence in vivo or survival if excreted, (ii) complete construction of multiple vector systems using balanced-lethal, balanced-attenuation and regulated-lysis vector systems to maximize induction of protective immune responses by delivery of conserved protective antigens of Shigella, Yersinia, E. coli, C. jejuni and C. perfringens, (iii) conduct studies to evaluate (a) how best to maximize induction of cross-protective immunity, (b) ability to induce protective immunity against specific enteric pathogens, (c) consequences of vaccination on normal flora, (d) efficacy of mixtures of vaccines to protect against Salmonella and one or two other species versus a vaccine to protect against multiple enteric bacterial pathogens, and (e) means to experimentally validate a vaccine for humans to protect against multiple pathogens, and (iv) conduct studies to provide preclinical safety and efficacy data to satisfy APHIS for veterinary vaccines and FDA for human vaccines. We will annually amend our Master File, prepare and fully characterize candidate vaccine Master Seeds for stability and safety, prepare and submit protocols for IRB approvals, and submit information to facilitate conduct of clinical animal and human trials. We plan to ultimately develop vaccines to confer protection against V. cholerae, C. difficile, L. monocytogenes and H. pylori. PUBLIC HEALTH RELEVANCE: Our objective is to use numerous new innovative technologies and new discoveries for design, construction and evaluation of live recombinant attenuated Salmonella vaccines to develop a low cost safe orally administered vaccine or mixture of vaccines to induce direct and cross-protective immunity to the majority of bacterial enteric pathogens that are responsible for more than 2 million global deaths each year and substantial morbidity and malnourishment with dire economic consequences. If successful, this vaccine(s) should have a societal public health benefit of a magnitude similar to the impact of the smallpox vaccine.