Low-Temperature Post-Harvest Processing for Decontaminating Vibrio Parahaemolyticus in Oysters

Non-Technical Summary: Vibrio parahaemolyticus is human pathogen that is commonly found in molluscan shellfish, particularly oyster. Consumption of raw or undercooked oyster containing this pathogen may lead to development of acute gastroenteritis. Several outbreaks of V. parahaemolyticus infection associated with raw oyster consumption have been recorded in the United States since 1997, including recent ones in 2006 and 2007. Illness resulted from consuming raw oyster is a concern for public health and causes substantial economic loss to the shellfish industries. This project addresses one of the CSREES goals Enhance Protection and Safety of the Nation??s Agriculture and Food Supply by developing a cost-effective post-harvest process for eliminating V. parahaemolyticus in oyster and generating new knowledge in understanding mechanisms underlying Vibrio-oyster interaction to allow future development of intervention strategies for producing safe oyster for consumption. The objectives of this project are to develop a low-temperature post-harvest process for decontaminating V. parahaemolyticus in raw oyster to a safe level for consumption and to increase our understanding of Vibrio-oyster interactions at different temperatures. Studies will be conducted to identify an optimal low-temperature post-harvest process for effective decontamination of V. parahaemolyticus in oyster without significant adverse effects on oyster by determining the relative efficacies of UV-sterilized seawater depuration at refrigeration temperature (2-5?XC) in decontaminating V. parahaemolyticus in oyster. Genetic analysis will be conducted to identify differences in gene expression levels of oyster and V. parahaemolyticus exposed to different temperatures (5 and 20?XC) and search for key proteins expressed by oyster or V. parahaemolyticus required for persistence of V. parahaemolyticus in oyster. The successful completion of this project would provide the oyster industry with a simple and effective means to decontaminate V. parahaemolyticus in oyster intended for raw consumption as well as an understanding of fundamental biological concepts related to V. parahaemolyticus ecology in the lumen of the oyster host, which might be applicable to other Vibrio species associated with shellfish and therefore offers potential wide-ranging applications. <P> Approach: Raw Pacific oysters inoculated with clinical strains of V. parahaemolyticus will be depurated in low-temperature (2-5 C) seawater in a recirculating system equipped with a Gamma UV sterilizer. Reductions of V. parahaemolyticus in oysters will be analyzed at 12, 24, 48, 72, and 96 h with the 3-tube most probable method (MPN) method and a multiplex PCR assay targeting tlh, tdh, and trh genes. The most effective process (time-temperature combination) will be selected to study its efficacy in reducing V. parahaemolyticus in oysters of different breeds (triploid) and different ages (between one and three years old). The optimal low-temperature depuration process for decontaminating V. parahaemolyticus in oyster will be validated using Pacific oysters harvested between July and September when high densities of V. parahaemolyticus are expected in oysters. Differences in gene expression levels of oyster at different depuration temperatures will be investigated by using an oyster microarray to search for potentially specific proteins expressed by the Pacific oyster mediating clearance of bacteria. The microarray data collected will be analyzed to identify genes that are differentially regulated between different depuration conditions. The intensity of fluorescence for each gene at each time point will be measured and normalized for comparison with assigned relative ranks. A 95% confidence interval will be calculated for each gene prior to heat shock. Genes that fall outside of the confidence interval will be considered differentially expressed. Specific proteins expressed by V. parahaemolyticus that might be important for its survival and colonization in oyster will be identified with V. parahaemolyticus microchip. Bacterial gene expression levels during the association of the bacteria with a marine host will be determined through data analysis of the microchip assay. Once candidate genes that are significantly up or down regulated when the bacteria are associated with oysters during the effective or ineffective depuration conditions have been identified, gene expression changes will be validated by real-time reverse transcribed PCR.