Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269
Performing Department
PATHOBIOLOGY
Non Technical Summary
Salmonella enteritidis is one of the major foodborne pathogens that contaminate poultry and poultry products, leading to foodborne infections in humans. Since attachment to intestinal mucosa is a critical first step in the pathogenesis of S. enteritidis infection, reducing or eliminating its attachment to intestinal mucosa could potentially decrease Salmonella-contaminated eggs and poultry meat consumed by humans. The proposed research will help us to protect chickens from harboring S. enteritis in their intestinal tract, thereby reducing contamination of eggs and carcasses by this pathogen.
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Goals / Objectives
1. Sequence the 2.7 kb Sma1 DNA fragment containing the gene encoding the 82.3 kDa OMP, and identify and characterize the gene encoding the 75.6 kDa OMP. 2. Express and purify the 75.6 kDa and 82.3 kDa OMPs by cloning the genes in a suitable expression vector. 3. Determine the suitable adjuvant and route for administering S. enteritidis OMPs in chickens for optimum immune response. 4. Evaluate the efficacy of the 75.6 kDa and 82.3 kDa proteins to inhibit S. enteritidis attachment to chicken intestinal mucosa in vivo using the optimum adjuvant inoculation route.
Project Methods
1.Sequencing the gene encoding the 82.3 kDa OMP: The nucleotide sequence of the 2.7 kb Sma1 DNA fragment will be performed at the University of Connecticut Biotechnology Center. The nucleotide sequence will be compiled and analyzed using the DNAMAN software (Lynnon Biosoft, Quebec, Canada). The fragment of S. entertidis DNA which hybridize with the cDNA probe will be cloned in pUC19 plasmid vector, and its nucleotide sequence will be determined at the University of Connecticut Biotechnology Center. 2. Express and purify the 75.6 kDa and 82.3 kDa OMPs. We will use the pMALTM-2 vector system (New England Biolab, Inc, Beverly, Mass) for expressing and purifying protein from cloned genes for the 75.6 kDa and 82.3 kDa proteins. The yield of fusion protein from affinity purification ranges up to 100 mg/litter culture, with typical yield in the range of 10-40 mg/litter. 3. Determine the suitable adjuvant and route for administering S. enteritidis OMPs (75.6 kDa and 82.3 kDa)
for producing optimum immune response, and maximal reduction in carriage of the pathogen: Immunization of chickens with Salmonella OMPs using different adjuvants, and routes of inoculation has been shown to induce various degrees of antibody response. Adjuvants such as liposomes, imunostimulating complexes (ISCOM), and mineral oil will be utilized through different routes (subcutaneous and oral. 4. Evaluate the efficacy of the 75.6 kDa and 82.3 kDa OMPs to inhibit S. enteritidis attachment to chicken intestinal mucosa in vivo in a large population of birds using the optimum adjuvant and route of inoculation selected in objective 3. A group of one hundred eight-week old specific-pathogen-free, white leghorn chickens will be used for the study. The birds will be divided into four groups, each containing 25 birds. Group 1, Group 2, and Group 3 will be inoculated with 20 mg of 82.3 kDa protein, 20 mg 75.6 kDa protein, and a mixture of 10 mg each 75.6 and 82.3 kDa proteins, respectively
using the optimum adjuvant and route selected in the experiment under objective 1. Group 4, not inoculated with any proteins will serve as the control. All the treatment groups will be boosted twice with the same amount of proteins with a time interval of 2 weeks. One week after the last boost, chickens in all the groups will be challenged with a three-strain mixture of S. enteritidis by oral inoculation of 1 ml of culture containing approximately 108 CFU, using a syringe and a 10-cm rubber catheter. Following the challenge, chickens were observed for typical symptoms of Salmonellosis. Feces and fecal swabs will be collected daily for enumeration of S. enteritidis. The study will be terminated at 28 days post inoculation, at which time the birds will be sacrificed, and samples of tissues and contents from the intestinal tract will be enumerated for S. enteritidis. Pre-immunization serum as well as weekly samples of post-immunization serum from all the birds will be tested for S.
enteritidis-specific antibodies by enzyme-linked immunosorbent assay.