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Development of Rapid Methods for Detection and Enumeration of Vibrio Vulnificus, Vibrio Parahaemolyticus and Enteric Viruses in Seafood and Harvesting Areas


In the United States, seafood accounts for 26.5% of foodborne outbreaks. A majority of these cases are due to consumption of contaminated raw bivalve molluscan shellfish. Two of the major bacterial pathogens associated with raw oysters are Vibrio vulnificus and Vibrio parahaemolyticus. Because of the rapid progression and high mortality rates of V. vulnificus infections in humans, especially those victims with underlying chronic disease, gulf coast oysters are being scrutinized for the presence and level of this organism. Conventional methods for V. vulnificus, V. parahaemolyticus and enteric virus detection in raw oysters or other seafood involves lengthy selective cultural enrichment. Thus, the recovery, identification and enumeration of V. vulnificus, V. parahaemolyticus and enteric viruses in oysters or seawater can be overwhelming in logistics, materials and time, while the expeditious and specific identification of V. vulnificus, V. parahaemolyticus and enteric viruses in the laboratory is desirable. In addition, the need for user-friendly methods for generating quantitative data from raw oysters for HACCP and food and drug administration (FDA) regulatory purposes is another serious criterion for developing alternative methods for V. vulnificus, V. parahaemolyticus and enteric virus enumeration. <P>

The specific objectives for enumeration and identification of pathogenic Vibrios in oysters are as follows; produce species specific antibodies against pathogenic and/or total Vibrio parahaemolyticus and Vibrio vulnificus, produce monoclonal antibodies reactive with and specific for the thermostable direct (TDH) and TDH-related hemolysins and the H antigen(s) displayed on V. parahaemolyticus polar flagella, develop a direct colony immunoblot assay for detection of V. parahaemolyticus using anti-V. parahaemolyticus H and anti-hemolysin monoclonal antibodies, employ the colony immunoblot assay to detect known clinical and environmental V. parahaemolyticus isolates and examine any cross-reactions with other Vibrio species, employ the colony immunoblot assay to detect and enumerate environmental V. parahaemolyticus isolates in raw oysters harvested from the Gulf of Mexico over a 1 year period, and develop sensitive IMS (immunomagnetic separation) /DNA hybridization-based, IMS/colony Immunoblot and/or IMS/qPCR-based assays to detect Vibrio parahaemolyticus virulent and non-virulent strains and Vibrio vulnificus in oyster homogenates and seawater. The final goal of this research project will be to optimize qRT-PCR methods for detection and quantification of FRNA phages, Hepatitis A and Noroviruses in seawater samples and to demonstrate the integrated sampling and test methods to assess the reliability of FRNA phages as indicators for HAV and/or NV in shellfish harvests areas.

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NON-TECHNICAL SUMMARY: Economic Impact on Louisiana Oyster Industry: Vibrio vulnificus and V. parahaemolyticus continues to harass the Louisiana oyster industry because it is found in the marine environment and its ability to cause disease in certain consumers. Research efforts are in progress to develop technologies to eliminate or greatly reduce the V. vulnificus and V. parahaemolyticus burden in oysters. To complement these efforts, and permit investigators to assess their endeavors, methods are needed which can easily detect and enumerate V. vulnificus and V. parahaemolyticus cells by direct examination of shellfish. Our research project will develop rapid antibody methods that can be used by scientists, seafood processors and regulatory agencies. There is a continuing strong governmental and commercial demand for development of less costly, user-friendly antibody-based assays specific for pathogenic V. vulnificus and V. parahaemolyticus in raw oysters and other seafood products. Since Louisiana is one of the leading states in oyster harvesting, benefits should accrue to Louisiana and sister states on the Gulf of Mexico. Large multistate outbreaks of Norwalk virus in 1993 and Hepatitis A virus in 2005 were traced to consumption of oysters obtained from Louisiana harvesting sites. These outbreaks further highlighted the fact that there is very little data on virus occurrence in seawater. The lack of data is due to the absence of efficient methods that enable rapid detection of viruses. Most current methods are labor or time intensive, expensive, or impractical. Therefore, there is an acute need for the development of technologies that enable efficient detection of viruses in seawater samples. Our project will be to optimize methods for extraction of enteric viruses from large volumes of seawater, develop DNA methods for enumeration and detection of enteric viruses from seawater and assess the reliability of FRNA phages as indicators for Hepatitis A and/or Norovirus in shellfish harvests areas. FRNA phages are reliable indicators of the possible presence of human enteric viruses in seafood and seafood growing waters because they behave like water-borne viruses. Results from this project could lead to development of methods that can be used to monitor enteric viruses in seawater.


APPROACH: Monoclonal antibodies reactive with V. parahaemolyticus TDH/TRH and H antigens will be developed by previously described methods. Once the monoclonal antibodies for the species-specific hemolysins and H antigens are developed, then adaptation and optimization of the DCI (direct colony immunoblot) method for the detection of total and pathogenic V. parahaemolyticus isolates on selective and non-selective agar plates will be conducted. Next the specificity of the assay will be determined using other Vibrio species and pathogenic and non-pathogenic V. parahaemolyticus strains in mixed cultures on spread plates employing duplicate immunoblots. Finally, the assay will be used to detect and enumerate total and pathogenic V. parahaemolyticus isolates in seeded and unseeded oyster homogenates on selective agar plates in a 24-hour time frame. The V. parahaemolyticus DCI assay will be compared to the DNAH (DNA hybridization) method over a one year period following ISSC methods for a single laboratory validation of a new analytical method. The next stage of the investigation will be dedicated to the adaptation and optimization of existing IMS (Immunomagnetic separation) methods to isolate and concentrate V. vulnificus isolates in pure and then mixed cultures. Next, quantification of V. vulnificus in spiked oyster homogenates, with and without IMS, will be determined by established qPCR methods. The efficiency of the IMS/qPCR procedure will be compared to the Food and Drug Administration's DNA probe hybridization method. If good correlation between the two methods (IMS/qPCR and DNA probe hybridization) is observed, then the recovery and enumeration of environmental V. vulnificus isolates from freshly harvested oysters by IMS/qPCR will be determined every three months for one year. To assess the reliability of FRNA phages as indicators for HAV and/or NV in seawater collected from shellfish growing areas, seawater samples will be taken from 6 locations in the Gulf of Mexico over a two year period. Seawater samples will be collected in sterile 10 liter carboys. During each visit, a one liter sample of seawater will be obtained and labeled with the collector's initials, a unique sample identification number, time of sampling, date, and location. All sample containers will be capped, labeled, and stored in a sample cooler that had been previously chilled to 4degreeC. Samples will be immediately transported to the laboratory for analyses. All samples will be subjected to the filtration/anion exchange resin capture method and RNA will be extracted from enteric viruses attached to the resin beads and the presence of FRNA phages, HAV and NV will be determined by qRT-PCR assays. If positive HAV and NV are detected in the environmental samples they will be sent to a commercial lab for cloning and sequencing. Results from this research project will allow us to develop rapid enteric viral indicator methods that correlate to the presence of Hepatitis A and/or Noroviruses in shellfish growing areas.

Janes, Marlene
Louisiana State University
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