2012 Annual Report
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.
Oysters, clams, and mussels are commonly consumed in the United States, but occasionally cause illness or death from viral or bacterial contamination. The viruses and bacteria of primary concern are norovirus and hepatitis A virus, Vibrio vulnificus and Vibrio parahaemolyticus. According to the CDC, noroviruses are the principle cause of food-borne illness in the US with an estimated 5.5 million cases annually. Vibrio vulnificus is the leading cause of seafood-related bacterial deaths in the US, while V. parahaemolyticus contamination of shellfish and the associated illnesses they cause is responsible for yearly closures of shellfish harvesting operations and worldwide recalls of domestic shellfish products. Research under objective 1, originally designed to evaluate Vibrio persistence in seawater and shellfish under different salt concentrations, has shown that naturally-occurring predatory bacteria exert a major role in controlling disease-causing vibrios in seawater and oysters. Predatory bacteria against vibrios have been isolated and characterized from Atlantic, Pacific, and Gulf Coast seawater. This work could lead to fundamental changes in monitoring seafood safety. Another Vibrio of concern to the shellfish industry is Vibrio tubiashii, which is a bacterium that kills juvenile shellfish. It has been particularly problematic in commercial, West Coast shellfish hatcheries, where it has been responsible for major hatchery die-offs. Under objective 2a, ARS researchers in Wyndmoor, PA isolated, identified, and characterized the first bacterial viruses (known as phages) from seawater originating from deep water off the Hawaiian coast. These phages were shown to reduce mortalities in larval shellfish by up to 78%. These phages will be made available to industry for use in hatchery settings. Under objective 2b, an evaluation of the effects of single and repeated freeze-thaw cycles was completed to determine if freezing and thawing could inactivate norovirus. It was hoped that freezing and thawing could be used as a food processing intervention; however, ARS researchers in Dover, DE determined that human noroviruses persist through 14 rounds of freezing and thawing. Under objective 3, ARS researchers in Dover, DE identified the mechanism of persistence for shellfish-borne viruses. Hepatitis A virus, poliovirus, and two surrogates for human norovirus persisted in shellfish hemocytes (primitive blood cells). Viruses were extracted more expediently from hemocytes than from whole shellfish tissues. Under objective 4, the first method to differentiate potentially infectious versus inactivated noroviruses was developed using porcine gastric mucin, a secreted protein from the pig gut. Noroviruses inactivated by heat, ultraviolet light, and high pressure processing lost their ability to bind to mucin. This work could fundamentally change the way scientists detect noroviruses in shellfish and other foods. Research on other portions of this project is proceeding on or ahead of schedule. Manuscripts have been submitted to journals for objectives 1, 2a, and 2b, and have been recently published for objectives 3 and 4.
Method to differentiate infectious from non-infectious human norovirus. Simple tests to detect infective human noroviruses are needed to replace much more complex human volunteer studies, the only other alternative test for virus infectivity. ARS researchers at Dover, Delaware, attached swine mucin to magnetic beads to selectively bind infectious human noroviruses, but not inactivated noroviruses. Viruses inactivated by heat, ultraviolet light, and high pressure processing were unable to bind to mucin-coated magnetic beads. The binding of only infectious viruses to the beads will, for the first time, allow the detection of infectious viruses in food and environmental samples. This technology has been provisionally approved for patenting and a manuscript has been published.
Predatory bacteria naturally suppress vibrios in seawater and oysters. Vibrio bacteria are a significant threat to shellfish safety causing numerous illnesses, some deaths, and the closure of shellfish harvesting areas each year. ARS researchers at Dover, Delaware, isolated, identified, and characterized naturally-occurring bacterial predators against a variety of pathogenic Vibrio species. Electron microscopic analysis revealed a broad group of bacteria responsible for the decline in vibrios. These predators were shown to reduce vibrios in seawater and oysters and appear to be a natural control mechanism to enhance seafood safety. A manuscript was submitted and indicates that Vibrio predatory bacteria are important modulators of pathogenic vibrios in seawater and oysters.
Bacterial viruses (phages) save larval shellfish. Oysters, clams and mussels begin life as free- swimming larvae which are susceptible to infection by the shellfish pathogen Vibrio tubiashii. This bacterium has caused major die-offs at shellfish hatcheries, particularly on the US West Coast. Using Hawaiian seawater, ARS researchers at Dover, Delaware, discovered 15 phage viruses that kill various strains of V. tubiashii. A mixture of these phages protected larval oysters against high levels of V. tubiashii, suggesting applicability of these cocktails in commercial shellfish hatcheries. The phages have been approved for licensing for commercial application and a manuscript on this work has been submitted.
Anderson, R., Gulnihal, O., Kingsley, D.H., Maureen, S.A. 2011. Oyster hemocyte mobilization and increased adhesion activity after beta glucan administration. Journal of Shellfish Research. 30(3):635-641.
Richards, G.P. 2012. Critical review of norovirus surrogates in food safety research: rationale for considering volunteer studies. Food and Environmental Virology. 4:6-13.
Huff, K., Aroonnual, A., Bae, E., Banada, P., Rajwa, B., Rajwa, B., Hirleman, E.D., Robinson, J.P., Richards, G.P., Bhunia, A. 2012. Light scattering sensor for real-time identification of Vibrio parahaemolyticus, V. vulnificus and V. cholera colonies on solid agar plates. Microbial Biotechnology. DOI: 10.1111/j.1751-7915.2012.00349.x.
Fay, J., Richards, G.P., Ozbay, G. 2012. Water quality parameters and total aerobic bacterial and vibirionaceae loads in eastern oysters (crassostrea virginica) from oyster gardening sites. Archives of Environmental Contamination and Toxicology. 64:628-637.
Provost, K., Ozbay, G., Anderson, R., Richards, G.P., Kingsley, D.H. 2011. Hemocytes are sites of persistence for virus-contaminated oysters. Applied and Environmental Microbiology. 77:8360-8369.
Whitaker, B.W., Parent, M.A., Aoife, B., Richards, G.P., Boyd, F.E. 2012. Vibrio parahaemolyticus ToxRS regulator is required for stress tolerance and colonization in a novel orogastric streptomycin-induced adult murine model. Infection and Immunity. 80:1834-1845.