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Live Recombinant Salmonella Vaccination with Novel Universal Antigen Presentation and Immune Potentiation


OBJECTIVE 1: Construct several live attenuated Salmonella strains that express codon-optimized epitopes of fliC and evaluate these strains for their ability to invade, colonize, and persist in tissues and elicit specific immune responses against homologous and heterologous Salmonella serovars in infected chickens. <P>OBJECTIVE 2: Determine the ability of CD154 expression to enhance immune responses against multiple Salmonella serovars. <P>OBJECTIVE 3: Evaluate the most effective candidate vaccine strains, identified in Objectives 1 and 2, for protection of chickens against infection following challenge with wild-type homologous and heterologous Salmonella strains.

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NON-TECHNICAL SUMMARY: Food-borne illness is a significant worldwide public health problem. In the United States, an estimated 1.4 million cases of human Salmonella infections occur annually and are responsible for approximately 580 deaths and 15,000 hospitalizations, resulting in a cost of $3 billion each year (Anonymous, 2005). The most common sources of human Salmonella infection are poultry and other meat products, as well as eggs and egg products that are infected or become contaminated with intestinal contents during processing (Glynn et al., 2004; Kimura et al., 2004; CDC, 2005). To date, live Salmonella vaccines have not been widely adopted for production poultry by the industry (Hargis et al., 2001). Therefore, an inexpensive live vaccine that could rapidly, persistently, and effectively protect poultry from a broad range of S. enterica serovars, by oral administration, would be valuable tool for the commercial poultry industry, and ultimately the public. Control of Salmonella in commercial poultry has a potential two-fold benefit: reduction of the impact of low level disease on performance, and reduced potential of poultry products to cause food-borne illness.<P>APPROACH: Attenuation of wild-type Salmonella strains will be achieved by deletion mutation of one or more of the genes(Maestroni et al., 2000) or by the insertion of multiple copies of a foreign epitope. To accomplish this in wild-type Salmonella strains, the target gene sequence in the bacterial genome of S. enteritidis will be replaced with the kanamycin resistant (KmR) gene sequence. This will be performed using 3S-PCR and electroporation of the 3S-PCR products into electrocompetent Salmonella cells containing the pKD46 plasmid. The resulting cell mixture will be plated on LB agar plates supplemented with Km to select for positive clones now containing a KmR gene. The KmR gene will be inserted into the genomic region containing the genes of interest (dam, aroA or htrA) by flanking the KmR gene with sequences homologous to the genes of interest. Once KmR mutants are obtained, the deletion mutations will be confirmed by PCR and DNA sequencing. These attenuation mutants, with fliC epitope insertion, will be compared for invasiveness, antigen-presenting potential, and persistence within vaccinated poultry. Mutants inducing effective seroconversion yet persisting for only a brief period are obviously preferred candidates. Candidate recombinants and appropriate controls will be administered by oral gavage at 104-107 cfu/mL at 10-fold increments to day-of-hatch broiler chicks obtained from a commercial hatchery. Chicks will be housed in individual isolators at age-appropriate temperatures with ad libitum access to appropriate feed and water. Serum samples will be obtained at 10, 20, 30, and 40 days post-inoculation for primary immune response, and at 10-day intervals after booster vaccination (to be determined based upon persistence of antibody response). Serum collected from birds in the challenge studies will be used in an antigen capture ELISA to determine relative antibody responses. In brief, individual wells of a 96-well plate will be coated with Salmonella or the fliC peptide conjugated to BSA. Antigen adhesion will be allowed to proceed overnight at 4C. Plates will be rinsed and incubated for 2 hours with the previously colleted sera. The plates will be rinsed again followed by incubation with a secondary antibody for an additional hour. After subsequent rinsing, the plates will be developed using a peroxidase substrate kit and absorbances read on a spectrophotometer at 450nm and 405nm. Each plate will contain a positive control and negative control where the fliC antigen or Salmonella polyclonal antibody and chicken serum from an untreated bird respectively replace the serum from the treatment groups. The absorbance obtained for the positive control, negative control and experimental samples will be used to calculate Sample to Positive control ratios (S/P ratios; Brown et al., 1991; Davies et al., 2003) using the following calculation: S/P ratio calculation: (sample mean-negative control mean)/ (positive control mean-negative control mean).

Hargis, Billy
University of Arkansas
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