<OL> <LI> Construct recombinant vaccine strains based on sefA, fimA and agfA fimbrial genes of S. Enteritidis. Analize expression of fimbrial proteins (SEF14, SEF17 and SEF21) by recombinant strains. <LI>Characterize immune response and protective effects of live recombinant vaccine strains against S. Enteritidis colonization in chickens. <LI>Compare recombinant vaccine strains (pCO5sefA, pCO5fimA or pCO5agfA) to 3 commercial vaccines against S. Enteritidis infection in chickens.
Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis I) is perceived as an expanding pandemic and a significant threat to the food safety and human health worldwide. In the majority of the human foodborne cases of S. Enteritidis, the consumption of contaminated eggs or egg products is implicated as the source of infection. Invasive strains of S. Enteritidis silently infect adult chickens and contaminate eggs before shells are formed, causing egg products to be considered a major source of infection to humans. Our long-term goal is to develop an effective control of S. Enteritidis in poultry in order to reduce the S. Enteritidis foodborne infections associated with contaminated eggs and to improve pre-harvest food safety and the United States agriculture sustainability.
We will conduct PCR amplification of the coding sequence of SEF17 and SEF21 major subunits (fimA and agfA genes, respectively). PCR products will be individually inserted into the unique Hind III cloning site of pCO5 temperature-inducible expression vector by ligation and, then, transformed into a non-pathogenic strain of E. coli, producing the pCO5fimA and pCO5agfA strains. This expression vector contains the temperature-inducible bacteriophage lambda promoter (pL), the cII ribosome-binding site, cII start codon and Hind III unique restriction site downstream from the initiator ATG for expression of unfused proteins. The recombinant pCO5-fimbrial gene will be transformed into a non-pathogenic strain of E. coli (strain 554B), which was originally isolated from a healthy chicken. Recombinant strains will be analyzed in vitro for the expression of SEF17 and SEF21 fimbrial proteins by SDS and western blotting. All strains (pCO5sefA, pCO5fimA and pCO5agfA) will be assayed in vivo for the production of secretory-IgA and humoral (IgG) antibodies to fimbriae in 1-day old chickens by ELISPOT and ELISA. Finally, comparison of the protective effects of the recombinant vaccine candidates against S. Enteritidis colonization in chickens will be performed by recovery of S. Enteritidis from challenged birds to evaluate the optimal protective response among all recombinant vaccine strains. We will examine persistence of recombinant strain in intestines; Number of cloacal swabs positive for S. Enteritidis; Number of antigen-specific IgA-secreting cells detected by ELISPOT in spleen and cecal tonsils; Number of colony forming units (CFU) of S. Enteritidis recovered from spleen, liver, intestines and cecum/cecal contents; ELISA titers for humoral IgG antibodies specific to fimbriae; ELISA titers for bile IgA antibodies specific to fimbriae. The recombinant vaccine strain (pCO5sefA, pCO5fimA or pCO5agfA), which demonstrated the best protective effect, will be compared to 3 commercial vaccines against S. Enteritidis infection in chickens.