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:
Oysters, clams and mussels are common causes of seafood-associated illnesses and occasional deaths in the US. Illnesses are commonly from viruses, like norovirus, which is the principle cause of foodborne illness in the US, and from bacteria of the genus Vibrio. Norovirus is associated with fecal pollution of foods while Vibrio bacteria are naturally-occurring in the marine environment. Two Vibrio species (V. vulnificus and V. parahaemolyticus) are particularly problematic because they cause disease and deaths in shellfish consumers. Another Vibrio, Vibrio tubiashii, is a species which causes high mortalities in larval shellfish in US shellfish hatcheries and has impacted the availability of seed oysters and clams which are needed by commercial shellfishermen. ARS researchers in Dover, DE, in collaboration with the Univ. of Delaware completed a project showing the presence of Vibrio predatory bacteria in natural seawater – bacteria that suppress Vibrio levels in seawater and shellfish. They also completed a study showing an association of these predatory bacteria in Atlantic, Gulf and Pacific seawater as related to temperature, salinity, and season. Established a CRADA with Intralytix, a company in Baltimore, to evaluate bacterial viruses, known as bacteriophages or phages, for possible commercialization and distribution to shellfish hatcheries to reduce larval shellfish mortalities. Recent studies demonstrated that a mixture of these phages reduced Vibrio-induced oyster mortalities by over 95%. ARS researchers at Dover, DE, in collaboration with Texas A&M demonstrated the effectiveness of an irradiation source, known as e-beam, in reducing norovirus and hepatitis A virus within intact shellfish by about 90%. Higher doses of e-beam irradiation inactivated viruses more effectively, but were beyond current irradiation levels permitted by the FDA. Another project by ARS researchers at Dover, DE, in collaboration with Arizona State University, evaluated the potential of ultrahigh acoustic waves to inactivate norovirus and showed proof-of-principle that norovirus can be inactivated by ultrahigh frequency waves. ARS researchers at Dover, DE completed two projects which utilize a newly developed ARS assay which separates viable from inactivated norovirus for subsequent quantitative testing. The first evaluates the effectiveness of sanitizers such as chlorine, chlorine dioxide, hydrogen peroxide, peroxyacetic acid, and trisodium phosphate on human norovirus. Another evaluated optimal temperature and pH conditions for two strains of norovirus by high pressure processing. Another study evaluated the reported propagation of human norovirus in a rotating vessel.
1. Vibrio predatory bacteria kill vibrios in seawater. Vibrio bacteria remain the number one cause of shellfish-associated illnesses and deaths in the US each year. Seawater temperature, salinity, and season can influence the levels of Vibrio predatory bacteria and their effect on disease-causing vibrios in seawater. ARS researchers at Dover, Delaware, in collaboration with the University of Delaware and the US FDA, concluded a one-year survey of shellfish waters from the Atlantic, Pacific (Hawaii), and Gulf Coasts and showed significantly higher counts of Vibrio predatory bacteria in the Atlantic during the summer and the Gulf during the winter, but no significant differences in temperate waters of Hawaii. Salinity had some effect on the levels of Vibrio predatory bacteria. This study showed a near constant presence of predatory bacteria in seawater and opens the door for the possible use of Vibrio predatory bacteria as a shellfish-processing intervention to eliminate disease-causing vibrios.
2. Bacterial viruses (bacteriophages) reduce mortality of larval shellfish. Shellfish hatcheries in the US have experienced high larval mortalities due to Vibrio tubiashii, a bacterium that is highly infectious to larvae. ARS researchers at Dover, Delaware, identified bacteriophages that kill V. tubiashii and established a new CRADA with Intralytix, an industry leader in developing bacteriophage-based treatments, to further characterize these bacteriophages for potential commercialization. For the first time, results on East Coast oyster larvae have shown that a mixture of several bacteriophages was 95% effective in eliminating larval mortalities. Application of these bacteriophages in shellfish hatcheries should reduce mortalities significantly and lead to less interruption in the supply of seed oysters and clams for commercial planting operations.
3. E-beam irradiation kills viruses. Improved processing methods are needed to reduce the incidence of norovirus, hepatitis A virus and other viral contaminants in foods. Irradiation is a current commercial intervention used in food processing. ARS researchers at Dover, Delaware, in collaboration with Texan A&M University evaluated one form of irradiation known as e-beam to inactivate viruses. It was demonstrated that e-beam could inactivate about 90% of a marine norovirus and hepatitis A virus within oysters with higher doses inactivating proportionately more of these viruses. This work demonstrated that E-beam irradiation can readily penetrate an oyster shell and may be useful as a processing intervention.
Richards, G.P., Watson, M.A., Meade, G.K., Hovan, G.L., Kingsley, D.H. 2012. Resilience of norovirus GII.4 to freezing and thawing:implications for virus infectivity. Food and Environmental Virology. 4:192-197.