2011 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 detection; 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 enhance seafood safety and quality for all Americans.
Research under National Program 108 Food Safety was completed on 1/31/11 and centered on enteric viruses and vibrio bacteria, which are significant causes of illness and deaths to shellfish consumers each year. Findings over the course of this project have enhanced shellfish safety. Under objective a, more rapid, simple, cost effective tests for monitoring disease-causing vibrios and fecal E. coli contamination in shellfish and seawater were developed and published. The practical application of this new technology was demonstrated in field studies conducted in cooperation with Delaware State University scientists and students. Now, both vibrio and E. coli levels can be determined in a single, quick test which will enhance efforts to monitor shellfish safety. Under objective 2, we identified inhibitors to conventional molecular-based detection methods for human viral pathogens in shellfish and developed improved techniques to reduce these inhibitors. Our previously developed protocol to extract hepatitis A virus and norovirus in shellfish was evaluated for other types of viruses and was highly effective. Under objective c, studies were conducted and published on factors which affect the inactivation of hepatitis A virus and norovirus in oysters by means of high pressure processing (HPP). Factors included an evaluation of temperature on HPP effectiveness for inactivation of hepatitis A virus and two norovirus surrogates. Refrigeration temperatures greatly enhanced virus inactivation by HPP, along with pH, sugar, and salt concentrations. HPP was shown to inactivate hepatitis A virus within mussels, murine norovirus in oysters, and human norovirus in oysters, which led to several publications. Together, these studies identify HPP as a useful and practical processing technique to inactivate virus contaminants in shellfish. Under objective d, we identified oyster hemocytes (blood cells) as a site of virus persistence in shellfish and a potential target for the development of improved virus testing methods for oysters. Additional vibrio research was added to the original project because of early accomplishment of existing objectives, successful grant submission, and requests for assistance by stakeholders. Under these studies, we developed a potential intervention to reduce the devastation caused by the fish pathogen Vibrio tubiashii on US West Coast shellfish hatcheries. We isolated and characterized the first phages (bacterial viruses) against V. tubiashii from Hawaiian seawater and found that a mixture of these phages added to infected oyster larvae significantly reduced larval mortalities. Studies were performed in collaboration with the University of Delaware to evaluate the uptake, growth, and persistence of a pandemic strain of V.parahaemolyticus in oysters and seawater. Mutant strains lacking known virulence genes were also evaluated to determine if these genes enhanced vibrio uptake or colonization within oyster tissues. This pandemic strain and the mutants were readily concentrated within the oysters, but did not persist, thus there does not appear to be an immediate threat of broad scale outbreaks in the U.S. from this strain.
High pressure treatments inactivate viruses in seafoods. ARS researchers at Dover, DE, in collaboration with scientists at Virginia Tech, evaluated the inactivation of murine norovirus, a surrogate for human norovirus, by high pressure processing using a cell culture system and a novel live mouse model. Virus was detected in the liver, spleen, and brain of all mice that were fed non-pressurized oysters, but not in mice receiving pressure-treated oysters. Results confirmed that pressure inactivated the murine norovirus, as determined in both systems. This is further validation that high pressure processing is effective in reducing virus contamination of oysters and may have commercial application to reduce shellfish-related foodborne illness.
Gogal, R.M., Kerr, R., Kingsley, D.H., Granata, L.A., Leroith, T., Holliman, S.D., Dancho, B.A., Flick, G.J. 2011. High hydrostatic pressure processing of murine norovirus 1-contaminated oysters inhibits oral infection in STAT-1 -/- deficient female mice. Journal of Food Protection. 74:209-214.