Improving Oyster Quality by Enhancing Natural Immunity

Non-Technical Summary: <BR>The most important cause of seafood-borne illness in the U.S. is contamination of oysters with bacteria of the genus Vibrio, the most important of which are Vibrio vulnificus and Vibrio parahaemolyticus. Of greatest concern is V. vulnificus, the cause of 95% of all deaths resulting from seafood consumption in this country. Despite intensive research, no effective means of reducing vibrio contamination in live oysters has been developed. However, American oysters have potent natural defenses against these human pathogens. This natural immunity has yet to be exploited in reducing the number of vibrios within oysters. We have recently discovered that when American oysters are placed in dry storage, they dramatically increase the levels of a highly potent component of this natural immunity known as ?antimicrobial polypeptides (AMPPs)?. These small proteins are highly active against human-pathogenic vibrios, suggesting that they may play a key role in regulating vibrio levels in oyster tissues. In this project, we will develop methods to quantify these antimicrobial peptides. We will then optimize the dry storage conditions that result in maximum AMPP levels, determine the relationship between AMPP levels and bacterial contamination in the 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 less susceptible to these AMPP defenses than environmental isolates. 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 differences in resistance to these compounds exist among various individuals and populations of oysters. To accomplish these goals, we will I) Determine the environmental factors (e.g. temperature, oxygen levels) responsible for increases in these small proteins (antibiotics) by exposing groups of oysters to various stresses to determine which of these factor(s) and under which result in the maximum increase of the antibiotic response. 2) Determine the length of time the antibiotics remain elevated in the tissues after stress. After exposing the oysters to the stress conditions of Objective I, we will sample oysters for up to 3 weeks in order to determine the decay rate of the antibiotics under typical commercial storage conditions. 3) Determine if there is a relationship between tissue antibiotic levels and the concentrations of pathogenic bacteria in those tissues, using procedures standard in our laboratories. We will also measure the bacterial concentration in the oyster tissues. This task will also allow us to determine how increased antibiotic levels are related to oyster safety and shelf life. 4) Determine if there is a difference in by season. We will repeat Objective 4 spring, summer, fall and winter. This will be done because our data suggest that there may be seasonal variation in the antibiotic levels and this would be important to determine if the antibiotic response is to be optimized. <P> Approach: <BR> Objectives and Approach: I) Determine the environmental factors responsible for antibiotic up-regulation. We will expose replicate groups of oysters to various stressors (temperature, hypoxia, salinity change) to determine which factor(s) and under which specific set of conditions result in the maximum up-regulation of the antibiotic response. To this end, we will measure both gene and protein expression using tools that we have already developed. 2) Determine the length of time that the antibiotics remain elevated in the tissues after stress. After exposing the oysters to the optimized stress determined in Objective I, we will sequentially sample replicate groups of oysters at various time intervals for up to 3 weeks in order to determine the decay rate of the antibiotics under typical commercial storage conditions. We will do this for oysters that have been induced to express three different levels of antibiotics. 3) Determine if there is a relationship between tissue antibiotic levels and the concentrations of pathogenic bacteria in those tissues. After optimizing the antibiotic response we will sample simultaneously various oyster tissues for total bacterial and vibrio concentrations, using procedures standard in our laboratories Gill, mantle, muscle hemolymph (blood) and digestive gland from individual oysters will be examined We will also measure the bacterial concentration m the entire oyster (all tissues combined) We will then determine if there is a correlation between bacterial levels and antibiotic concentration. This task will also allow us to determine how the up-regulated antibiotic levels are related to the safety and shelf life of oysters. 4) Determine if there is a difference in antibiotic up-regulation between different oyster stocks. We will compare the ability of two diploid oyster stocks with that of a triploid strain to up-regulate their antibiotic response. We will use this triploid strain because they were hybridized to produce a more disease-resistant stock. Oysters will be held in pens in an estuary. We will sample 10 oysters of all three strains immediately after removal from the pen (time 0). We will then place 50 oysters of each strain under the standardized stress identified in Objective 1. Blood, mantle, gill, muscle and digestive gland will be sampled and antibiotic levels will be measured as in Objective 1. 5) Determine if antibiotic up-regulation varies with season. We will repeat Objective 4 at four times during the year, including spring, summer, fall and winter. This will be done because our data suggest that there may be seasonal variation in the antibiotic levels and this would be important to determine if the up-regulation response is to be optimized.