Food Safety Vaccine of Economic Benefit to Poultry Producers to Prevent Salmonella and APEC Infections and Foodborne Transmission to Humans

NON-TECHNICAL SUMMARY: Salmonella and Escherichia coli infections in poultry are a human health threat by transmission to humans via contaminated poultry products. Safe effective vaccines to prevent Salmonella infection of poultry exist but are not extensively used since there is no adverse economic consequence associated with Salmonella infection of chickens. On the other hand, avian pathogenic E. coli (APEC) infections cause morbidity, mortality and carcass condemnation and therefore have a negative economic impact. The difficulty of having an effective vaccine against APEC strains is related to the high level of diversity of these strains. Avirulent strains of Salmonella unable to cause disease will be engineered, these bacteria will possess many mutations to minimize induction of immune responses to serotype-specific antigens and maximize induction of cross-protective immunity to common related antigens of S. enterica strains of diverse serotypes; and these bacteria will be genetically modify to harbor plasmids from other enteric bacteria. Introduction of pAPEC-1 plasmid harboring common APEC antigens in these strains will enhance induction of cross-protective immunity to APEC serotypes. None of the bacterial vaccine cells are able to survive and thus exhibit complete biological containment. The objective is to develop and evaluate vaccines for chickens to prevent Salmonella and APEC infections and foodborne transmission to humans The purpose of this project is to develop safe and inexpensive vaccines for chickens to prevent Salmonella and APEC infections and foodborne transmission to humans. <P>APPROACH: A safe efficacious vaccine for the prevention of Salmonella and APEC infections in poultry will be engineered by using a regulated delayed attenuation phenotype such that vaccine strains at the time of immunization exhibit nearly wild-type attributes for survival and colonization of lymphoid tissues but after five to ten cell divisions in the host become avirulent. The first strategy is constructing S. Typhimurium vaccine strains with the mutations Äpmi-2426 and Ä(gmd-fcl)-26. These mutations will turn off ability to synthesize LPS-O-antigen in vivo. The second strategy is to replace promoters for virulence genes needed to display with regulatable promoters (ÄPfur81::TT araC PBAD fur and ÄPcrp527::TT araC PBAD) that will be on for vaccine strain growth and initial colonization of the gastrointestinal tract and be turned off in vivo. Growth of such strains in the presence of arabinose leads to transcription of the fur and/or crp genes but expression ceases in vivo since there is no free arabinose. Attenuation develops as the products of these genes are diluted at each cell division. We also delay onset of attenuation by including ÄaraBAD23, which prevents use of arabinose retained in the cell cytoplasm at the time of oral immunization, and/or ÄaraE25 which enhances retention of arabinose. Additionally, we will include ÄfliC180 and ÄfljB180 mutations which have deletions in the antigenically variable serotype-specific domains of FliC and FljB to enhance the likelihood for repeat use of the host-vector system for other vaccines. The portion of the flagellar protein thatinteracts with TLR5 to recruit/stimulate innate immune responses represents the conserved N- and C-terminal regions of the flagellar proteins. We designed our Salmonella vaccine strains capable of carrying either cloning vectors engineered to express a recombinant protective antigen or wild-type plasmids specifying synthesis of different antigens of targeted pathogens. We first cured S. Typhimurium of its pSTV plasmid and then cloned the spv region essential for virulence and internal tissue growth into either the endA or cysG gene thus deleting these genes in the process. The Salmonella cured strain containing spv in its chromosome will be attenuated as described above and then used to contain the virulence plasmid pAPEC-1 of an APEC strain which encodes three Fur-regulated operons important for iron and manganese uptake. pAPEC-1 will then be engineered to be DadB+ and Tra- to establish a balanced-lethal vector-host system. We will then fully evaluate our recombinant attenuated Salmonella Typhimurium strain with pAPEC-1 as an anti Salmonella and E. coli vaccine for poultry that should enhance food safety by lessening the likelihood of transmission of Salmonella and APEC to humans via contaminated poultry products.