2007 Annual Report 1a.Objectives (from AD-416) Research will focus on four main objectives designed to enhance the safety of aquaculture products, to: a) continue to 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. Develop more effective means for decontaminating fresh and minimally processed fruits and vegetables containing human pathogens to ensure food safety and security by assessing the efficacy of new and/or improved intervention technologies. This maintains the flexibility to expand research efforts on produced when and where necessary. 1b.Approach (from AD-416) We propose to use a wide variety of protein chemistry, biochemistry, microbiology, virology, molecular biology, and food technology principles and techniques to: a) develop molecular biological and enzyme-based assays to detect specific pathogens in shellfish tissues as well as processing interventions to inactivate enteric viruses that contaminate shellfish; b) screen for, identify and characterize novel enzyme activities associated with bacterial pathogens to develop rapid, enzyme-based assays for their decteion; c) develop improved virus extraction procedures for shellfish with the intent to characterize and eliminate potential inhibitors of real-time RT-PCR methods; d)explore the mechanism by which high pressure processing inactivates hepatitis A virus and noroviruses to determine the physical and chemical parameters that influence processing effectiveness, e) participate in a human volunteer study to determine the effectiveness of high pressure processing to inactivate noroviruses in oysters; and f) evaluate the mechanism by which enteric viruses persist within shellfish with the goal of developing improved shellfish disinfection and detection methods. We will accomplish these tasks in collaboration with Federal, State, and industry partners and distribute new methods and information to our stakeholders, especially the aquaculture industry and regulatory agencies. Together, these studies will enchance seafood safety and quality for all Americans. 3.Progress Report High pressure inactivation of human norovirus in oysters. Several phases of a human volunteer study to assess the effects of high pressure processing on norovirus inactivation in shellfish were completed under a three-laboratory collaboration, and a no-cost extension to the National Research Initiative Grant is being requested to provide additional time to complete the final stages of the study (see subordinate project 1935-42000-059-03N). National Program Component 108, Problem: 1.2.4, Processing Intevention Strategies. 1935-42000-059-02N (Evaluation of Virus Inactivation by Hydrostatic Pressure) with the University of Delaware (see separate annual report for further details). Recent work with HAV-contaminated shellfish has shown that this phenomenon does not appear to hold up for the virus within shellfish tissues, suggesting a specific media-dependent effect of pressure on HAV. Current experiments are seeking to ferret out this issue. For a complete report on the progress of this agreement, see the report for 1935-42000-059-02N. Progress is monitored through work visits and conference calls. 1935-42000-059-03N (Norwalk Virus Inactivation by High Hydrostatic Pressure Processing: A comprehensive and Integrated Program for Research and Outreach.) with Virginia Polytechnical Institute and State University. (see separate annual report for further details). Initial evaluation of human norovirus’ capacity to be inactivated in water samples is planned this summer as well as verification that freezing after high pressure does not negatively effect remaining virus counts in pressure-treated oysters (accomplished using MNV). Once this has been completed, pressure inactivation studies on human norovirus-contaminated oysters will begin. For a complete report on the progress of this agreement, see the report for 1935-42000-059-02N. Progress is monitored thorugh work visits and conference calls. 1935-42000-059-6N (Development of Methids and Strategies to Improve the Microbiological Safety of Aquaculture Products.) with Rutgers Agricultural Research and Extension Center. (see separate annual report for further details). Work has been initiated to evaluate the potential of inactivation of murine norovirus (MNV) within clams as part of a larger USDA National Research Initiative Grant with Rutgers University Department of Food Sciences to evaluate the application of high pressure processing for the New Jersey clam industry. For a complete report on the progress of this agreement, see the report for 1935-42000-059-02N. Progress is monitored through work visits and conference calls. 4.Accomplishments Isolation of a potential virulence-enhancing enzyme in Shewanella species. An enzyme was identified in members of the Shewanella family of bacteria. Shewanella include flesh-eating species that can be lethal to humans. A novel, enzyme-based test was developed to detect and quantify Shewanella species and the test was used in a two-year study of oysters and seawater in the Delaware Bay. DNA sequencing was performed on the isolates and several species of Shewanella, including the flesh-eating species, were identified for the first time in the Delaware Bay. Our assay provides a novel means to quickly test aquaculture products, especially oysters, for Shewanella bacteria. 2006-2010 Action Plan National Program Component 108, Problem Statements: 1.2.1, Detection [of pathogens] and 1.2.8, (Pathogenicity). Developed and applied real-time molecular techniques to quantify noroviruses in oysters. Noroviruses are the primary cause of food-related illness in the United States with an estimated 23 million cases occurring annually. Real-time molecular methods were developed and evaluated to quantify noroviruses in oysters. The methods involved development of oyster processing strategies to extract the viruses from shellfish tissues and to analyze the extracts by real-time testing method. In addition, several methods containing internal controls were developed to quantify the levels of norovirus in the shellfish. Benefits of this research include the development of improved methods to monitor virus levels in naturally-contaminated oysters and to determine the effectiveness of processing interventions on the elimination of viruses in shellfish. 2006-2010 Action Plan National Program Component 108, Problem Statement: 1.2.1, Detection [of pathogens]. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of active CRADAs and MTAs 2 Number of invention disclosures submitted 1 Number of non-peer reviewed presentations and proceedings 8 Review Publications Richards, G.P. 2006. Shellfish-associated viral disease outbreaks. Book Chapter. In Viruses in Foods, (S.M. Goyal, Editor), Springer, New York. pp. 223-238. Kingsley, D.H., Guan, D., Hoover, D.G., Chen, H. 2006. Inactivation of hepatitis a virus by high pressure processing: the role of temperature and pressure oscillation. Journal of Food Protection. 69:2454-2459 Kingsley, D.H., Holliman, D., Calci, K., Chen, H., Flich, G. 2007. Inactivation of a Norovirus by High Pressure Processing. Applied and Environmental Microbiology. 73:581-585. Kingsley, D.H. 2007. An rna extraction protocol for shellfish-borne viruses. Journal of Virological Methods. Vol.141:58-62.