1a. Objectives (from AD-416)
The safety of aquaculture products, particularly molluscan shellfish, is jeopardized by vibrio and enteric virus contamination and the lack of processing interventions. Among the foods of greatest concern are raw or lightly-cooked oysters and clams, which result in substantial health risks to consumers. The objectives of this project are designed to identify the mechanisms by which bivalve shellfish become contaminated with pathogenic viruses and vibrios and to identify processing interventions to reduce illnesses and losses to the shellfish and associated industries. Objective 1: Characterize the uptake and depletion of pandemic V. parahaemolyticus, other virulent and avirulent strains of V. parahaemolyticus and V. vulnificus in shellfish as affected by diet, environmental factors, and virulence genes. Objective 2: Develop and evaluate intervention and control strategies for: a) vibrio species through identification, characterization and application of phages to remediate shellfish mortalities in hatchery settings, and for use in commercial shellfish processing. b) enteric viruses, such as hepatitis A and E viruses, human norovirus, and surrogates, using methods such as high pressure processing, e-beam, or other technologies. Objective 3: Characterize the uptake and persistence of norovirus and hepatitis A virus in oysters. Objective 4: Develop technologies to automate, simplify, or improve current virus testing methods to include the evaluation of assays for infectious (live) versus inactivated (dead) viruses.
1b. Approach (from AD-416)
Under objective 1, we will determine if differences in seawater salinity and pH significantly affect the growth and persistence of the human pathogens Vibrio parahaemolyticus and V. vulnificus in seawater; whether algae (Tetraselmis chui) will affect vibrio blooms in seawater or the levels of uptake in shellfish; and if vibrio persistence in oysters (Crassostrea virginica) varies depending on vibrio species, strain, or the presence of virulence genes. Oysters will be obtained from the Univ. of Delaware Marine Lab in Lewes, DE. Bacteriological analyses and titering of vibrio inocula, oysters, and seawater will be performed according to our newly developed and quantitative pour plate method which detects streptomycin-resistant mutants of the virulent and avirulent strains of V. parahaemolyticus and V. vulnificus. Oysters, vibrios, and algae will be added to tanks of seawater containing shellfish, both of which will be collected daily, serially diluted, and each dilution will be tested to enumerate specific pathogens. Under objective 2a, we will identify bacteriophages against V. tubiashii; isolate and characterize them biochemically and morphologically; propagate and quantify the phages using methods developed in this lab; and apply phage cocktails (multiple phage strains) in shellfish hatcheries to determine if they can significantly reduce larval shellfish mortalities. In addition, lytic phages against V. parahaemolyticus and V. vulnificus will be evaluated as a potential processing intervention to reduce human pathogenic vibrios in commercially harvested oysters. Under objective 2b, we will determine under what conditions high pressure processing (HPP), electronic-beam irradiation, and other processing techniques can eliminate viruses from shellfish. Various concentrations of acidic flavorings and ethanol will be evaluated to add novel flavors, develop altered product forms, and to increase the efficiency (reducing required pressure) of HPP against norovirus and hepatitis A virus. Hepatitis E virus (HEV) studies will be performed to evaluate the ability of HPP to inactivate HEV using a chicken model. Under objective 3, we will evaluate the uptake and persistence of viruses by oyster blood cells (hemocytes) through fluorescent microscopy or strepavidin-labeling and histological techniques. Intervention methods that specifically target, destroy, or eliminate theses hemocytes, or the pathogens within the hemocytes, will be evaluated. Biogenic silver nanoparticles will be evaluated for possible use in targeting viruses within lysosomal compartments in hemocytes. Under objective 4, we will explore hemocytes as a concentrated source of viruses within shellfish; determine if hemocytes are a suitable target for improvement of virus assay, extraction from virus-contaminated shellfish, and automated testing on a microscale format; and evaluate whether virus receptor interactions may be used to discriminate between potentially infectious and non-infectious viruses. We will automate extraction and detection methods, exploiting magnetic beads and/or chip-type formats.
3. Progress Report
Research under National Program 108 Food Safety, was initiated on 2/1/11 on project objectives related to the safety of shellfish, which contribute to bacterial and viral illnesses among consumers. V. vulnificus and V. parahaemolyticus are naturally-occurring marine bacteria which accumulate in shellfish and cause illnesses and deaths among consumers. V. tubiashii is another naturally-occurring marine bacterium which kills juvenile shellfish and has been particularly problematic in shellfish hatcheries, leading to shortages of seed oysters and clams for commercial aquaculture operations. Norovirus and hepatitis A virus are the two most prevalent causes of seafood-related viral illnesses in the US for which processing interventions and detection methods are needed. To date, objective 1 research has demonstrated the inability of the pandemic strain of V. parahaemolyticus to persist in seawater and shellfish. Efforts are underway to determine if this pandemic strain is unique or if other strains of V. parahaemolyticus and V. vulnificus respond similarly. Under objective 2a, we are responding to West Coast stakeholders to develop methods to reduce high mortalities in shellfish hatcheries as a result of the shellfish pathogen, V. tubiashii. As an intervention, we are evaluating the effectiveness of bacterial phages (viruses that kill bacteria) to reduce oyster mortalities in shellfish hatcheries. To date we have isolated 16 phages against V. tubiashii from seawater from Hawaii. Pilot studies are underway, in collaboration with Oregon State University, to determine the effectiveness of these phages to combat hatchery outbreaks of V. tubiashii. Phages against the human pathogen V. vulnificus have been received from the University of Florida and will be characterized for possible use as a processing intervention to reduce shellfish-related illness and mortality among consumers. Under objective 2b, initial studies evaluating e-beam technology as a possible processing intervention to reduce or eliminate enteric virus contamination in shellfish have been conducted in collaboration with Texas A&M University. Also under objective 2b, results of a collaborative study with Virginia Tech and Emory University on the effectiveness of high pressure processing to eliminate human noroviruses in shellfish was published. Results showed that pressures higher than those currently applied commercially are needed to eliminate human norovirus in contaminated shellfish. Under objective 3, a paper was submitted and accepted for publication showing that oyster hemocytes (primitive blood cells) are the sites of virus accumulation and persistence in contaminated shellfish. This opens the way for the development of new, simplified and innovative methods to monitor for the presence of viruses in shellfish. In addition, a book chapter on Foodborne Viruses was written and accepted for publication in the American Public Health Association’s Compendium of Methods for the Microbiological Examination of Foods. Together, these efforts are enhancing shellfish safety and supporting the needs of consumers, regulators, and the shellfish industry.
1. Sites of virus persistence in shellfish identified. Virus contamination of shellfish has led to frequent outbreaks of hepatitis A and norovirus illness. Once contaminated, shellfish retain high levels of these viruses for extended periods. ARS researchers at Dover, DE, identified primitive blood cells of oysters, known as hemocytes, as the site of virus accumulation and persistence. They devloped simple procedures to extract and test for viruses within the hemocytes. This is a dramatic improvement over traditional testing methods for viruses in shellfish which rely on tedious dissection and testing of intestinal tissues that contain only a portion of the viral contaminants. Improved assay methods will enhance monitoring efforts and support regulatory agency and industry goals to reduce shellfish-associated enteric virus illness.
Leon, J., Kingsley, D.H., Montes, J., Richards, G.P., Lyon, G., Abdulhafid, G., Seitz, S., Fernandez, M., Teunis, P.F., Flick, G.J., Moe, C.L. 2011. Human norovirus inactivation in oysters by high hydrostatic pressure processing: A randomized double-blinded study. Journal of Infectious Diseases. 77(15):5476-82.