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The long-term goal of this work is to use basic research to eliminate E. coli O157 from cattle and therefore eliminate the pathogen from fresh beef products and fresh foods at risk for contamination from bovine waste. We will develop an effective cattle vaccine.<P> The proposed work addressed two specific objectives: <P>Objective 1: Characterize a fusion protein containing H7 flagellin and intimin and use as a topical or injectable vaccine/immunostimulant to eliminate E. coli O157 in cattle. <P>Objective 2: Use novel specific Toll-like receptor inducing adjuvants (CpG, flagellin and/or monophosphoryl Lipid A) for use as a topical vaccine to eliminate E. coli O157 in cattle.
Non-Technical Summary: Escherichia coli O157:H7 continues to be a major concern for the Idaho and nation-wide beef industry and public health. Despite increased awareness and better detection and surveillance methods, significant outbreaks and subsequent food recalls continue to occur because the organism uses ruminant animals, particularly cattle, as a silent reservoir. This lack of overt symptoms in the reservoir host makes it difficult to determine point source contamination. In addition, the infectious dose for the bacteria in humans is very low, <100 organisms, compounding the problem of detection and safety since extremely low contamination levels constitute serious potential hazard if food is undercooked or mishandled during preparation. Outbreaks of E. coli O157:H7 continue to negatively impact the Idaho and national cattle industry. Our research is aimed at understanding the basic biology of E. coli O157 colonization of cattle and to build an effective vaccine that will prevent colonization in this silent reservoir. Our vaccine strategy employs the use of new vaccine adjuvants that are directed at stimulating the mammalian innate immune response thereby promoting a better specific and long lasting specific immunity. <P> Approach: To address Objective 1, we will characterize the flagellin-intimin fusion protein to show that the molecule is folded properly to expose intimin and to allow appropriate flagellin activation of TLR-5. First, the construct will be expressed in a flagellin-minus mutant and the bacterial cells tested for motility. Detection of motile cells will be an easy assay indicating that flagella are assembled properly. Second, the activation of TLR-5 will be tested using a murine macrophage cell line and measuring the production of IL-8, the signature interleukin induced by flagellin. Then, the intimin-flagellin fusion will be tested in cattle for specific protective antibody production. To address Objective 2, we will over-express the gamma intimin protein in E. coli strain M15. This strain contains a cloned copy of the intimin gene fused with a histidine-tag that allows rapid purification of the protein using standard techniques. Concurrently, we will purify H7 flagellin to homogeneity using the standard techniques of ultracentrifugation and ion exchange chromatography. In addition, purified monophosphoryl-Lipid A (MPL), a TLR-4 inducer, will be purchased. Because MPL preparations vary, we will confirm that we have the proper stimulatory mix in tissue culture, before testing in cattle. Objective 2 will compare vaccine formulas using a combination of these TLR inducers as adjuvants to generate adaptive immunity directed against intimin and thus E. coli O157:H7. Purification of reagents will be complete in 6 months and preliminary animal vaccine trials will be completed within one year with confirmatory trials completed in years 2-3. Functional flagellin-intimin fusion protein will be used in a vaccine trial with 10 Holstein steers. Groups of five animals will be vaccinated or mock vaccinated. A booster application will be applied two weeks post initial vaccination. At forty days post-vaccination, an infectious dose of wild-type E. coli O157 will be applied by rectal application of the bacteria to the RAJ mucosa of each animal using our standard technique. If the fusion protein is protective, we expect to see decreased numbers of colonizing cells, and a shorter period of shedding for the immunized animals vs. those receiving mock immunization. We will monitor antibody titers towards the flagellin-intimin fusion protein in cattle by standard enzyme immunoassay from the secretions at the RAJ mucosa and in blood sera. A confirmatory trial with a second group of 10 steers will be done.