The goal of this proposal is to aid the East Coast shellfish industry by performing research on two key issues identified by shellfish farmers: a) contamination of shellfish by the human pathogen Vibrio parahaemolyticus; and b) tissue concentrations of trace metals, primarily cadmium and mercury, bioconcentrated in shellfish of the genera Crassostrea and Mercenaria. <P>The specific aims of this research are to: 1) Optimize and validate a protocol for the detection of pathogenic and non-pathogenic strains of Vibrio parahaemolyticus using multiplex quantitative polymerase chain reaction (Q-PCR); 2) Evaluate and optimize a method for the elimination of polymerase chain reaction (PCR) inhibitors from oyster tissue samples; 3) Survey the temporal and spatial distribution of pathogenic strains of V. parahaemolyticus in shellfish and environmental samples in Rhode Island; 4) Survey cadmium (Cd), mercury (Hg) and other trace metal levels in shellfish soft tissue from bivalve shellfish collected from farmed and wild populations throughout the Atlantic coastal region of the U.S., from Maine to Florida; and 5) Provide the results from this study to appropriate agencies to aid in the national and international discussions of V. parahaemolyticus and heavy metals (Cd and Hg) as human health risks associated with consumption of seafood,to growers, if necessary, to recommend strategies to minimize levels of V. parahaemolyticus, Cd and Hg in farmed shellfish,and to the general public to allay fears that shellfish consumption is a human health risk based on V. parahaemolyticus outbreaks in other states and current literature suggesting seafood has high trace metal contamination.
Non-Technical Summary: The American oyster and northern hard clam have been premier seafood products in the U.S. since pre-colonial times. In more recent times, clam and oyster landings represent a substantial fishery, with farming increasingly contributing to landings. The goal of this proposal is to aid the bivalve shellfish industry in the East Coast of the US by performing research on two key issues identified by shellfish farmers: a) contamination of shellfish by the human pathogen Vibrio parahaemolyticus; and b) tissue concentrations of trace metals, primarily cadmium and mercury, bioconcentrated in shellfish of the genera Crassostrea and Mercenaria. Real and perceived problems from Vibrio parahaemolyticus or trace metal accumulations in bivalve shellfish could have a significant impact on both the wild harvest fishery and the aquaculture industry by reducing markets in a time when farmed shellfish production is increasing. Outbreaks of V. parahaemolyticus in the Pacific Northwest and Texas during 1997 and 1998, resulted in the adoption by regulatory agencies of a control plan to control sporadic illnesses due to the consumption of raw shellfish with V. parahaemolyticus, based on temperatures and total levels of V. parahaemolyticus in shellfish. However, this plan is based on information gathered in the Gulf of Mexico and Pacific Northwest. More data about the presence of pathogenic strains and relationship with environmental conditions and illnesses in the Atlantic region is necessary in order to develop a rational management plan for the control of Vibrio parahaemolyticus illnesses. We will gather this information by surveying for the presence of pathogenic strains of Vibrio parahaemolyticus in local waters and shellfish in the Northeast US using a sensitive method developed by the Food and Drug Administration, and will relate levels of pathogenic strains to environmental conditions and reports of human illnesses. Cadmium (Cd) and mercury (Hg) are trace metals can pose significant health risks to living organisms, including humans, when concentrated to a degree that impacts normal physiological processes. Shellfish, and especially oysters, are able to accumulate trace metals from the marine environment, and international transport and marketing of oysters for human consumption is banned when levels of cadmium exceed 1 or 2 ppm. Our second goal is to investigate the accumulation of trace metals in bivalve shellfish from the Atlantic Coast of US using methods approved by the US Environmental and Protection Agency. This proposed research will provide data on the levels of Vibrio parahaemolyticus and the bioconcentration and dynamics of heavy metals that will help avoid too stringent regulations of the bivalve industry that could impact the marketability of our farmed seafood products. <P> Approach: Vibrio parahaemolyticus. We will first optimize and validate in our laboratory the multiplex real time PCR method developed by researchers at the Food and Drug Administration using known levels of pathogenic and non-pathogenic V. parahaemolyticus strains. In parallel, we will evaluate if an oyster sample preparation protocol consisting of differential centrifugation, combined with treatment of oyster homogenates seeded with known amounts of V. parahaemolyticus with activated charcoal coated with P. fluorescens allows for the full recovery of V. parahaemolyticus cells. If unacceptable levels of recovery are observed (less than 90%), the coating method for the charcoal will be optimized by evaluating the use of: a) alternative coating materials, like different gram negative bacterial strains; or b) the use of beads coated with antibodies known to bind to all serotypes of V. parahaemolyticus (immunomagnetic separation or IMS). We will then evaluate the effectiveness of the coated charcoal method in removing PCR inhibitors in oyster samples by side-by-side quantification using real-time PCR of pathogenic and total levels of V. parahaemolyticus in oyster homogenates seeded with known amounts of bacteria and either: a) processed using the FDA protocol; or b) processed using the differential centrifugation-activated coated charcoal. The efficacy and cost of both methods of sample preparation will be compared. The best method will be then used to quantify levels of pathogenic and non-pathogenic V. parahaemolyticus in environmental and oyster samples from Rhode Island, collected every two weeks from May to December at selected locations. Environmental conditions (i.e. temperature, salinity, dissolved oxygen, turbidity, and organic matter content) at the time of sampling will be recorded, so the relationship between environmental parameters and presence of pathogenic V. parahaemolyticus can be determined. Heavy metals. We will develop an understanding of the mass balance of trace heavy metals, specifically cadmium and mercury, bioconcentrated in shellfish of the genus Crassostrea and Mercenaria, by determining metal concentrations using nitric acid sample digestion following US EPA methods 3051a and 3052. Northern hard clams and American oysters will be collected from major shellfish culture areas throughout the Eastern Seaboard and will include samples harvested from farm sites as well as limited samples from wild areas. At each site, the farmed population will be sampled based on season (summer and winter) and age class (Years 0, 1, & 2 in states from Maine to Delaware and Years 0 & 1 in states from Virginia to Florida) Each sample will include ten individuals for metals analysis and ten individuals for measuring dry tissue weight and condition index to provide adequate replication of the specific site measurement. In addition, a C. ariakensis population will be sampled from an experimental grow-out site in MD, VA, or NC (as available), and lastly, C. gigas will be collected from oyster growers on the Olympia Peninsula in Washington State. A total of 1,300 individual samples will be analyzed for Cd, Hg and other trace metals.