**-  North Atlantic  
-  Eastern Regional Rsch. Ctr. 
-  Microbial Food Safety Research Unit 
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**Location: Microbial Food Safety Research Unit**
**2006 Annual Report**
**1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)?  How serious is the problem?  Why does it matter?**													The research is conducted under National Program 108, Food Safety.  Food Safety falls under goal 3 of the Agency Strategic Plan: to Enhance Protection and Safety of the Nation’s Agriculture and Food Supply.  Research under this CRIS falls within component 1.2 of the Agency Strategic Plan which involves pathogens, toxins and chemical contaminants in post-harvest foods.  Within component 1.2 are sub-components directly linked with this project, specifically 1.2.1 “Detection and Quantitation of Pathogens”, 1.2.4 “Processing Intervention Strategies” and 1.2.8 “Pathogenicity”.  
Enteric viruses and bacteria cause a variety of illnesses among shellfish consumers. The noroviruses (formerly called the Norwalk-like viruses) and hepatitis A virus are among the most significant viral pathogens affecting molluscan shellfish safety, while members of the genus Vibrio cause the majority of bacterial illnesses associated with shellfish.  To better protect the consumer, analytical methods and improved shellfish processing strategies are needed.  Research will focus on four main objectives designed to enhance the safety of aquaculture products, to: a)develop rapid, enzyme-based assays to detect bacterial pathogens in aquaculture products; b)identify RT-PCR inhibitors and develop real-time molecular methods to detect and quantify viral pathogens in shellfish tissues; c)investigate physical and chemical parameters influencing the efficiency of high hydrostatic pressure inactivation of hepatitis A virus, norovirus, and surrogate viruses; and d) investigate the mechanisms of enteric virus persistence within live shellfish.
Enteric viruses jeopardize the health and safety of consumers of virtually any raw or lightly cooked food or foods contaminated by the hands of food-handlers.  There are an estimated 23 million cases of norovirus illness and 80,000 cases of hepatitis A in the United States each year.  Due to the epidemic nature of these pathogens, outbreaks may spread rapidly.  Vibrios are major pathogens, particularly in underdeveloped countries where cholera can kill thousands annually.  In the United States, Vibrio vulnificus and V. parahaemolyticus are the major shellfish-borne bacterial pathogens and are naturally present in shellfish-harvesting waters and in oysters, clams, and mussels.  Vibrio vulnificus is particularly deadly with mortality rates approaching 60% among those who become ill.  Diabetics and those with liver disease seem most at risk of V. vulnificus infection and death.  Vibrio parahaemolyticus outbreaks have been responsible for massive closures of shellfish harvesting areas along the Atlantic, Gulf, and Pacific coasts.  In fact, portions of the Delaware Bay were closed in June, 2005, due to a high level of V. parahaemolyticus, while major closings of shellfish harvesting areas along the Washington coast were put into place as a consequence of 177 V. parahaemolyticus illnesses in New York, Oregon, and Washington during June and July, 2006.  						
**2.List by year the currently approved milestones (indicators of research progress)**													Year 1 (FY 2006)Identify enzyme-associated mechanisms that are likely to enhance virulence of V. vulnificus in the human host.  Identify RT-PCR inhibitors in shellfish extracts and design improved virus extraction procedures for oysters to remove the inhibitorsComplete the characterization of murine norovirus inactivation by high hydrostatic pressure processing 
Year 2 (FY 2007)Isolate potential virulence enzymes in non-vulnificus bacterial pathogensDevelop real-time RT-PCR technique to quantify noroviruses in oystersComplete a human volunteer study to assess the inactivation of human norovirus in oysters using high pressure processing 
Year 3 (FY 2008)Complete the development of new COPP assays for non-Vibrio pathogensComplete studies on the mechanisms of virus inactivation by high hydrostatic pressure.Complete structural and functional evaluation of at least one newly identified bacterial enzyme
Year 4 (FY 2009)Complete studies on the isolation and characterization of bacterial enzymes.Complete studies to determine the organoleptic quality of high pressure processed shellfish
Year 5 (FY 2010)Complete field testing of the COPP assayComplete studies on the persistence of viruses in oyster hemocytes

**4a.List the single most significant research accomplishment during FY 2006.**													High Pressure Inactivation of Norovirus.  Norovirus is the number one cause of foodborne illness in the US.  For the oyster aquaculture industry, norovirus contamination is a major problem.  Human norovirus can not be propagated in the laboratory and there are no suitable animal models for this virus.  A closely related virus, murine (mouse) norovirus (MNV-1), has recently been discovered.  This virus is easily propagated in the laboratory and is currently the closest genetic relative to human norovirus.  In recent years, high pressure processing (HPP) has emerged as a viable commercial non-thermal strategy for pasteurization of foods.  The principle advantage of this technology is that foods retain much of their uncooked character, taste, and nutrient content.  Bivalve shellfish, such as oysters and clams, are frequently consumed raw or only lightly cooked, and since they efficiently bioconcentrate and retain enteric viruses from human waste, can represent a significant risk of food-borne illness.  High pressure processing is currently used by the oyster industry to inactivate Vibrio bacteria and facilitate the shucking process.  Work at the USDA, Microbial Food Safety Research Unit at Delaware State University in Dover, DE, has recently demonstrated that murine norovirus can be inactivated by HPP.  This work has been accomplished both with liquid virus stocks and with MNV-1 contaminated oysters.  This result demonstrates great promise for inactivating human norovirus within oysters with HPP technology.   National Program 108, Component: Microbial Pathogens, Action Plan 1.2.4.						
**4b.List other significant research accomplishment(s), if any.**													Effects of temperature, pH, salt, and sugar on pressure inactivation of viruses.   High pressure processing is a cold pasteurization process that can be influenced by a number of processing parameters to inactivate pathogens.  These parameters include the pressure applied, the time of pressure application, the temperature at which pressure is applied, and the composition of the food.  Recent research demonstrates that temperature can dramatically influence virus inactivation.  We found that the inactivation of the human norovirus surrogates, feline calcivirus (FCV) and murine norovirus, was enhanced at refrigeration temperatures by several orders of magnitude.  For hepatitis A virus, the converse was true, refrigeration temperatures resulted in substantially less inactivation when compared with studies performed at room temperature.  For higher temperatures (above 86oF), all viruses tested showed enhanced inactivation relative to room temperature.  Investigation of the effect of pH, salt content, and sugar content indicates that FCV inactivation is reduced as salt and sugar concentrations are increased.  Acidic buffers (under pH 5.2) resulted in substantially reduced inactivation as compared to buffers with pH’s between 6 and 8.  In total, this work defines critical parameters for HPP of foods and should lead to guidelines for treatment of products. National Program 108, Component: Microbial Pathogens, Action Plan 1.2.4.						
**4c.List significant activities that support special target populations.**													This Project recently completed the NP 108 OSQR Review Prcess and was certified.  See the report for 1935-42000-045-00D.						
**4d.Progress report.**													1935-42000-059-02N:  This report serves to document research conducted under a Non-funded Cooperative Agreement between ARS and the University of Delaware.  A project has been completed involving an evaluation of high pressure processing time and temperature for the inactivation of hepatitis A virus.  A second high pressure project was completed in which feline calicivirus was used to evaluate matrix composition effects for virus inactivation.  Results indicate that increasing salt (NaCl) and sucrose composition reduce the efficiency of virus inactivation under pressure.  The pH effects were also evaluated.  At acidic pH, there was minimal inactivation compared to controls, while at pH 6-8, inactivation was more efficient.  
1935-42000-059-03N:  This report serves to document research conducted under a Non-funded Cooperative Agreement between ARS and Virginia Polytechnic Institute and State University.  A project is nearing completion on an evaluation of the pressure sensitivity of murine norovirus (MNV-1).  This is the first norovirus ever shown to be inactivated by high pressure and is a close relative, genetically, to human norovirus.  It was noted that pressure application at 5 degrees C gave greatly enhanced inactivation and that infectious MNV-1 could be reduced by 99.99% in MNV-1-contaminated oysters with a 5 degrees C treatment of 400 megaPascals (58,000 pounds per square inch) for 5 min.
1935-42000-059-01S:  This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the Haskin Shellfish Research Laboratory, Rutgers University, Port Norris, NJ.  A method developed by the ARS Dover Laboratory for the analysis of members of the Vibrionaceae family of bacteria was field tested during monthly samplings of oysters and seawater from moderate and low salinity areas of the Delaware Bay over the past 2 years. The final monthly sampling occurred in June 2006 and a no-cost extension was approved through September 30, 2006, so that the tremendous volume of data could be tabulated and manuscripts prepared over the summer.  Oysters and seawater were screened for Vibrionaceae using the colony overlay procedure for peptidases (COPP assay) and isolates were identified biochemically.  The vast majority of isolates were Vibrionaceae species.  Levels of V. vulnificus and V. parahaemolyticus exceeded 100,000/gram of oyster during the summer months.  Other Vibrionaceae members readily identified including bacteria in the genera Aeromonas and Photobacterium.  High levels of potential opportunistic pathogens in the genera Shewanella and Chryseobacterium were frequently isolated.  Overall, the study demonstrated the ability to detect a broad range of Vibrionaceae using the COPP assay.

**5.Describe the major accomplishments to date and their predicted or actual impact.**													This CRIS just completed the OSQR process, please see report for 1935-42000-045-00D.

**6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)?  What are the constraints, if known, to the adoption and durability of the technology products?**													This CRIS just completed the OSQR process, please see report 1935-42000-045-00D.						
**7.List your most important publications in the popular press and presentations to organizations and articles written about your work.  (NOTE:  List your peer reviewed publications below).**													Richards, G.P. 2006. Is hepatitis E virus and emerging pathogen? Institute for Food Technologists, Orlando, FL. June 2006.						
**Review Publications***Richards, G.P., Watson, M.A. 2006. A simple fluorogenic method to assess vibrio cholerae and aeromonas hydrophila in well water. American Journal of Tropical Medicine and Hygiene. 65(3):516-521*    Project TeamRichards, Gary Kingsley, David  
Project Annual Reports FY 2009 FY 2008 FY 2007 FY 2006 Publications  Publications 
Related National Programs Food Safety, (animal and plant products)(108) Related Projects  Validation of the Effect of Interventions and Process on Persistence of Pathogens on Food  Unraveling Vibrio Parahaemolyticus Pathogenesis by Using a Functional Genomics Approach  Development of a Robust Assay for Infective Noroviruses, for Use in Food Safety Diagnostics  							Last Modified: 03/24/2010 		ARS Home |USDA.gov |Site Map |Policies and Links FOIA |Accessibility Statement |Privacy Policy |Nondiscrimination Statement |Information Quality |USA.gov |White House

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