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Recent outbreaks of vibrio disease in the Northwest US and Alaska have brought increased significance to this public health threat in north temperate areas like New England. Though studies have shown Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) to be present throughout New England waters, little is known about the distribution and incidence of the virulent strains of these pathogens. <P>The goal of the proposed research is to determine the environmental conditions that could be used to reduce or eliminate these pathogenic vibrio species from oysters and other bivalve molluscs. The potential to eliminate a public health threat associated with consumption of raw oysters and other bivalve shellfish is of paramount importance to the shellfishing industry. Preliminary results suggest that this is possible. Present-day assays for detection and enumeration of vibrios and their virulent strains, however, have been improved to the point that more accurate and conclusive assessment of environmental conditions can now be made. <P>The specific project objectives are to 1) develop and refine detection methods for pathogenic vibrios and their virulent strains in oysters, and to 2) conduct field tests of the detection methods under different environmental and water quality conditions that may inform managed reduction of pathogenic vibrio concentrations in oysters.<P> The expected outcomes will include detailed descriptions of the detection methods developed for pathogenic vibrios, experimental results on the application of these methods under a variety of environmental and water quality conditions, surveys of the incidence and concentrations of pathogenic species and their virulent strains in northern New England waters, and assessment of harvest water and post-harvest processing conditions that may serve to reduce vibrio concentrations.
Non-Technical Summary: Vibrio parahaemolyticus (Vp) and Vibrio vulnificus (Vv) are significant causes of shellfish-borne disease in the US and worldwide. Outbreaks of illness caused by Vp, and the incidence of disease and death associated with Vv infections are growing concerns for consumers of shellfish, especially from the Gulf of Mexico. Outbreaks are more frequent where water temperatures are warm and favor high Vibrio populations, however several large outbreaks have recently occurred in the cooler waters of Washington, Alaska and Long Island Sound. In the Gulf of Maine, these species are typically not detected during winter months, summertime disease incidence is low, and it is thought these strains are not virulent, though there have been infections from shellfish harvested from the Northeast. The shellfish industry has suffered increasingly more frequent vibrio-associated disease outbreaks linked to shellfish consumption that have had a widespread and cumulatively devastating impact on markets. Because of this, the top priority of the Interstate Shellfish Sanitation Commission and regional shellfish growers industry is to reduce shellfish-borne illnesses. The key public health and shellfish industry interests in this project are the development and refinement of more rapid detection methods for detecting and enumerating virulent strains of Vv and Vp. Traditional detection and enumeration methods have well-documented limitations, including a high incidence of false negatives and their time-consuming requirements. The recent development of new molecular-based detection methods (QPCR) shows promise for overcoming many limitations for enumerating vibrios. Currently, there is a need for method refinement and field-testing under different environmental conditions. In addition, PCR allows for differential detection of a range of different virulence factors, though not all virulence factors are well characterized. The incidence of pathogenic vibrios is affected by a range of environmental factors, but the potential for management of pathogenic vibrios based on environmental conditions requires further study. The proposed work will establish the capabilities to accurately determine the presence of total and pathogenic Vv and Vp in all regionally significant bivalve mollusks. The study will also begin to establish a better understanding of the effects of environmental conditions, the incidence and potential risk of Vv and Vp in the Northeast US, and the potential to reduce or eliminate these vibrios from shellfish prior to human consumption. It is critical that this and related studies are conducted now as the incidence of vibrio infections is increasing and the added burden to public health and shellfish resource managers for assessing its significance is anticipated. The shellfish industry is at a critical juncture, and the potential impact of this research has clear economic implications. The recent development of promising new tools to detect pathogenic vibrios provides for a timely opportunity to address these issues at this time, and the anticipated findings will be innovative and useful in advancing knowledge. <P> Approach: The most recent and refined published QPCR-MPN procedures for both Vv and Vp will be followed using equipment and facilities available at UNH that are different from what has been used to develop these methods. Once successfully modified, these methods will be used to quantify Vv and Vp, and the presence of pathogenic Vv and Vp strains verified using multiplex PCR. Following development with pure culture control strains, the control Vv and Vp strains will be induced to enter the viable but non-culturable state and the assay tested for sensitivity. Other testing will then be conducted with the test strains inoculated into natural microbial populations estuarine water and oysters. Water samples or oyster batches will be run in parallel assays to compare the traditional culture-based MPN assay to QPCR-MPN results and validate use of the QPCR-MPN method. Oysters will be collected at low tide from April through December at sites in NH and ME that differ in environmental and water quality conditions. Multipurpose meters will be used to measure water temperature, salinity, pH and dissolved oxygen in situ to determine environmental conditions within the oysters prior to harvest. Harvested oysters will be assayed using the developed methods to provide data on the incidence and concentrations of Vv and Vp in the study areas, and transported to Spinney Creek Shellfish Co. in Eliot, ME where an experimental facility will be used to test different controlled environmental conditions on the vibrios in shellfish. Oysters will be subject to different salinity, temperature and biological conditions in parallel tanks to test effects of single factors. Sub-sampling of the oysters will take place to determine changes that may occur with time (up to 8d) under the experimental conditions. It is anticipated that the main focus will be on salinity differences and the presence or absence (filtration and sterilization) of natural communities of microorganisms in the source waters. As a final experiment, we will transplant oysters between locations differing in conditions and look at reduction/increase of Vv and Vp concentrations. The adapted QPCR-MPN assay will be used to quantify the targeted vibrios in oysters from the different environmental treatments. Isolated pure colonies will be confirmed using standard biochemical tests and PCR for Vv and Vv species-specific genes. Vv and Vp concentrations will be based on averages of triplicate sub-samples for each treatment and sample time. The differences in average concentrations from different treatments will be tested using ANOVA. Vibrio concentration reductions will be determined by comparing concentrations at time zero to each collection time during experiments and field tests. The project success will be evaluated through controlled experiments designed to test the success of each step in methods development and the effects of experimental and field conditions on the targeted vibrios. The results should provide unique information on the incidence of virulent Vv and Vp strains, and whether oysters can be exposed to conditions that reduce the public health threat of these bacteria for human consumers.