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Production of a New Vaccine for Poultry to Prevent Salmonella

Shabatura, J; Kremer, C J; Koo, O
Sea Star International LLC
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Our Phase I feasibility project will address four major objectives: 1) To generate a double deletion mutant by developing amino acid auxotroph and a transcriptional regulator deleted ?hilA mutant Salmonella Typhimurium; 2) To perform growth and survivability tests in minimal media, chicken feed and fecal broth; 3) To investigate the virulence of the mutants by cytotoxicity tests and adhesion and invasion studies in chicken cells; and 4) To assess the poultry vaccine market and needs of customers for our Phase II proposal.

The deliverable for this SBIR Phase I is a S. Typhimurium candidate vaccine strain that is not only completely effective but also safe for widespread commercial us in the poultry industry. As the technology and approaches become more optimized and proof of concept established we will be able to apply these strategies to other problematic pathogens in not only the poultry industry but other food animal production systems.

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Non-Technical Summary:
Despite concerted efforts on both the part of the food industry and Federal regulators, already in 2011 there have been Salmonella foodborne illnesses in 31 U.S. states. Salmonella is a leading cause of foodborne illness. It is responsible each year for an estimated 1 million illnesses, 19,336 hospitalizations and 378 deaths. Salmonella associated outbreaks are primarily related to poultry meat and eggs. Despite the effort to control pathogenic Salmonella during poultry processing, the number of illnesses has not declined. Therefore, the control of pathogenic Salmonella has been a top research priority of USDA, which in 2010 published even tighter performance standards for controlling Salmonella in poultry products. The U.S. poultry industry has been producing broiler meat and egg products valued at $45 billion and $6.52 billion respectively in 2010. Arkansas has been consistently one of the top two states in the U.S. for poultry and egg production, valued at $3.7 billion. To protect consumers' health, decrease bird morbidity, and prevent product recalls, there is a widespread interest by the poultry industry in the development and verification of an effective Salmonella vaccination. There are currently competing Salmonella vaccines in the market, but all have serious efficacy, risk and cost drawbacks that are preventing widespread industry adoption. To be a commercial success, our new vaccine must compete in the market place that already contains competitive Salmonella vaccines.

We will overcome the limitations of the current vaccines, because our new vaccine will provide:
Greater Efficacy: Using an avirulent, live vaccine is superior to many of the current vaccines that use dead Salmonella which provide only short-lived protection and are less effective in reducing horizontal (bird to bird) Salmonella transmission.
Improved Price Point: Our small company will have lower overhead than many commercial vaccines producers because the owners understand the limitations of the price-sensitive, commodity, poultry broiler and egg industries and embrace the marketing objectives as well as the science involved in the project.
Minimal Risk of Reversion: We will use and verify that our vaccine, a double deletion Salmonella strain, will not revert back to virulent, pathogenic Salmonella.
Controlled Retention: Our feasibility tests for Phase I will demonstrate that our avirulent Salmonella strain will be there when needed to protect the newly hatched chick when it is most vulnerable--then clear out of its system ahead of slaughter for broilers or egg production for layers. This minimizes the risk of the birds testing false-positive for pathogenic Salmonella.

Objective 1: In cooperation with University of Arkansas Center for Food Safety, we will generate a double deletion mutant S. Typhimurium. Growth conditions for bacterial strains - Salmonella Typhimurium LT2 is the parent strain for the construction of a live attenuated vaccine. Removal of the selection marker - For the second gene deletion, the cassette with selection marker (kanamycin) needs to be removed from hilA mutant and a FLP expression plasmid, 706-FLP (Gene Bridges) will be used to assist the deletion by site-specific FLP recombinase. Double deletion by homologous recombination - Using the expression plasmid pRedET (Gene Bridges) containing the second target gene, the essential amino acid related gene will be deleted in ?hilA S. Typhimurium mutant strain. DNA sequencing - All PCR products will be excised from the gel and eluted from the gel using the Qiagen Quick gel Extraction kit (Qiagen, Valencia, CA.).
Objective 2: Screen the mutants with the growth tests in feed and fecal samples. Minimal medium -The mutants will be grown in LB broth supplemented with kanamycin (15?g/ml) overnight at 37 C. Also survivability test will be evaluated without target amino acids for each mutant by measuring the rate of decrease of viable cell concentration and calculating the average time for 50% for the cells to become nonviable. Chicken feed/fecal broth - The mutants incubated overnight in LB broth supplemented with kanamycin (15 ?g/ml) will be washed and subcultured in the chicken feed/fecal broth and the survivability will be analyzed.
Objective 3: Virulence test the mutants by LDH, adhesion and invasion assay. HD11 cells and Caco-2 cells preparation - HD11 chicken macrophage cells and Caco-2 human colon adenocarcinoma cells will be incubated and supplemented with 5% (v/v) fetal bovine serum (FBS; Invitrogen) or 10% FBS, respectively in 95% air/5% CO2 at 37C. When the cells become confluent, the cells will be harvested by trypsinization and the responded cells will be seeded onto well plates at 10 5 - 10 6 cells/well and used for cytotoxicity tests and adhesion and invasion tests.
Objective 4: Gain an understanding of the technical and economic factors driving sales of animal vaccines. 1. From this Phase I - We will establish the feasibility of the double gene deletion mutant, control how the live vaccines persistent in the host animal (humans as well as environment), and investigate the survival and reduced sustainability of our candidate vaccine cultures. 2. SWOT Analysis - To successfully compete in the animal vaccine marketplace we must find niches that are not currently occupied by large animal pharmaceutical companies. 3. Economics - The best vaccines are those that do what they are designed to do and are affordable as repeat purchase by a number of customers. 4. USDA SBIR Phase II & III - In in vivo tests, applying the vaccine strains by oral administration of the vaccines to a limited number of one-day old chicks then applying wild type S. Typhimurium 24 hr later will be required to convince the market to accept our vaccine.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Viruses and Prions
Bacterial Pathogens
Meat, Poultry, Game