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In this project, we will develop assays to quantify a suite of natural innate defenses (antimicrobial polypeptides - AMPPs) that have potent activity against human-pathogenic vibrios. <P> We will optimize the dry storage conditions that result in maximum AMPP upregulation, determine the relationship between AMPP level and bacterial contamination in stored oysters, and determine if elevated AMPPs result in oysters with a longer shelf life. We will also examine whether human pathogenic vibrio isolates are more susceptible to these AMPP defenses than environmental isolates. <P> Finally, we will determine if V. vulnificus in the viable but nonculturable (VBNC) state is susceptible to these AMPPs. We expect our results will lead to effective means of consistently reducing vibrio contamination in oysters and also allow a much better understanding of why vibrio prevalence varies among various individuals and populations of oysters.
Non-Technical Summary: The most important cause of seafood-borne illness in the U.S. is contamination of oysters with Vibrio bacteria, the most important of which is Vibrio vulnificus. Despite intensive research, no effective means of reducing vibrio contamination in live oysters has been developed. However, American oysters have potent natural immunity to these human pathogens. This natural immunity has yet to be exploited in reducing vibrio concentrations. We have recently discovered that when American oysters are placed in dry storage, they dramatically increase their levels of a highly potent aspect of this natural immunity known as antimicrobial polypeptides (AMPPs). In this project, we will develop simple tests for measuring these AMPPs and then determine the storage conditions that results in the highest AMPP levels. We will also determine how AMPP levels affect the concentration of vibrios and other bacteria in oyster tissues. We will also determine the susceptibility of a number of nonpathogenic versus pathogenic vibrio isolates to these AMPPs. We expect that these experiments will result in a consistent method of increasing AMPP levels in oyster tissue and subsequently greatly reduce vibrio contamination in oyster tissues. <P> Approach: We will: 1. Develop sensitive and specific methods to quantify the major AMPPs in oyster tissue 2. Determine the time-temperature relationship that optimizes AMPP upregulation and determine if time of the season (ambient temperature) affects the response. 3. Determine if there is a relationship between AMPP levels and the concentrations of pathogenic bacteria in oysters. 4. Determine if AMPP levels in oyster tissue are related to the shelf life of live oysters under commercial storage conditions 5. Determine if there is a difference in AMPP susceptibility of clinical versus environmental Vibrio isolates 6. Determine if there is a difference in AMPP susceptibility of Vibrio isolates in different metabolic states.