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United States Department of Agriculture

Agricultural Research Service

2009 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
A request was submitted and approved for changes in project direction and the addition of new milestones. The addition of new milestones was necessitated by early completion of existing milestones, new projects initiated in virus and vibrio research as a result of successful grant submission, and requests for assistance by the shellfish industry. The new milestones include: a) evaluating a pandemic strain of Vibrio parahaemolyticus, a particularly infectious bacteria found in oysters and other seafoods, b) developing methods to culture norovirus and hepatitis A virus in cell culture in order to facilitate their detection in shellfish and other foods, and c) isolating and charactering bacterial viruses (called phages) that might be used to disinfect larval shellfish and hatchery waters from bacteria (vibrios), which are causing high shellfish mortalities in commercial hatchery operations. To date, we have determined that the pandemic V. parahaemolyticus O3:K6 strain does not accumulate appreciably in oysters unless the bacteria are associated with algae or other constituents of the shellfish’s diet. Several strains of V. parahaemolyticus, including strains with and without genes believed to be responsible for their infectivity in humans, failed to accumulate in oysters in the absence of algae in the seawater. Methods to cultivate human norovirus and hepatitis A virus are underway using a variety of cell culture systems including a NASA bioreactor, which allows the growth of cell cultures under conditions of weightlessness. This allows cells to grow in a manner more consistent to those found in the human intestinal tract, which is believed to be the key in obtaining virus infection of these cells.

Over the past several years, the West Coast shellfish hatcheries have been plagued with high mortalities among larval shellfish due to the presence of a bacterium (Vibrio tubiashii) which has nearly driven the hatcheries to bankruptcy. A plan was developed to combat this bacterium, which threatens the welfare of the hatcheries and the entire West Coast shellfish industry. Acting on this plan, over 200 seawater samples from the U.S. were tested for a bacterial virus (phage), which kills Vibrio tubiashii. We discovered the first phage against this bacterium in seawater from Hawaii. This phage is currently under evaluation to determine if it might serve as an intervention to reduce or eliminated larval shellfish deaths caused by V. tubiashii.

A volunteer study to evaluate the effectiveness of high pressure processing on the inactivation of human norovirus in oysters is scheduled for completion in September 2009. Results indicate that a 5-min, 58,000-pounds per square inch (psi) treatment at room temperature is not sufficient to inactivate 1000 virus particles; however, a 5-min, 87,000-psi treatment at 7C completely inactivated 1000 virus particles. Results indicate conditions under which human norovirus may be inactivated in contaminated shellfish by high pressure processing.

1. Identified mechanisms of virus persistence in shellfish: Certain pathogens, such as human norovirus and hepatitis A virus, are able to persist within shellfish tissues for extended periods. Our research has implicated the oyster hemocytes, the blood cells of shellfish, as a site of virus uptake and persistence. We compared several different virus types (feline calicivirus, murine norovirus, hepatitis A virus, and poliovirus) for their abilities to persist in oyster tissues and within hemocytes. There is a correlation between the differing pH sensitivities of these viruses and persistence times within shellfish suggesting that persistence may relate to the virus’ resistance to acidic degradation within shellfish hemocytes. Transfer of hepatitis A virus-contaminated hemocytes to uninfected shellfish demonstrates that hepatitis A virus can persist within hemocytes for more than two weeks. This work has led to a greater understanding of the mechanisms for virus uptake and persistence within shellfish and contributes to a future milestone to investigate virus persistence mechanisms in shellfish.

2. Demonstrated practical application of ARS-developed test for total vibrios along Delaware Coast: Improved methods are needed to better monitor the quality and safety of shellfish and their harvesting areas. A Master’s student from Delaware State University participated in this project and successfully completed and defended her thesis entitled ‘An Investigation of the Relationship between Water Quality, Total Bacteria, and Vibrionaceae Concentrations in the Eastern Oyster (Crassostrea virginica) in Delaware’. The ARS colony overlay procedure for peptidases (COPP assay) was used to determine the levels of total bacteria and total vibrios at five oyster gardening sites in Delaware. Additional studies examined the relationship between various water quality parameters and bacterial levels. This study supports milestone 2 (to develop and evaluate colony overlay procedure for peptidase assays), provides baseline information to build a predictive index of conditions that are favorable for the proliferation of human and fish vibrios in the marine environment, and demonstrates the practical application of an ARS-developed assay for total vibrio detection.

3. Completed field test of ARS vibrio assay for shellfish in an area impacted by wild horses and agricultural runoff: Assateague Island National Seashore, Maryland, is a site where 150 feral horses reside and where a Delaware State University Master’s student performed a 2-year field test using the ARS-developed colony overlay procedure for peptidases (COPP assay) to determine if the horses contributed significantly to contamination of the local shellfish. This work led to a recently published paper entitled ‘Seawater and Shellfish (Geukensia demissa) Quality along the Western Coast of Assateague Island National Seashore, Maryland: An Area Impacted by Feral Horses and Agricultural Runoff”. This paper evaluated the use of the COPP assay for monitoring shellfish quality and safety in ribbed mussels. Water and shellfish quality were poor along Assateague Island and suggest poor stewardship of the coastal environment, partially from contamination of the water by horses, but more substantially from agricultural runoff. This work supports milestone 2 (to develop and evaluate colony overlay procedure for peptidase assays) and demonstrates the practical application of an ARS-developed assay for total vibrio detection.

4. Determined the effectiveness of high pressure processing to inactivate hepatitis A virus in in-shell oysters: Since commercial in-shell oysters are occasionally high pressure treated, and to date all previous scientific studies on high pressure effects on oysters were performed with shucked oyster meats, a study was performed to evaluate whether the presence of shell had an effect on high pressure inactivation of hepatitis A virus within contaminated oysters. Results confirm that HPP readily inactivates hepatitis A virus in both shucked oyster meat and in whole in-shell oysters, suggesting that commercial HPP of oysters may be acceptable for the disinfection of microbial contaminants. This research supports milestone 3 (to investigate physical and chemical parameters affecting HPP) and demonstrates that commercial HPP protocols and experimental laboratory protocols for inactivating harmful pathogens in shellfish provide comparable results.

5. Showed that murine norovirus, a surrogate for human norovirus, is inactivated by high pressure processing: A study with murine norovirus (MNV), a virus genetically related to human norovirus and commonly used as a surrogate for human norovirus, was completed and demonstrated that virus inactivation by high pressure processing (HPP) was effective in preventing infection in immunosuppressed mice, thus confirming earlier cell culture-based assays that MNV can be inactivated by HPP. Pathology and progression of the infection in infected mouse controls was microscopically characterized in the tissues. The successful inactivation of a virus that is closely related to human norovirus is further evidence that HPP may be an effective intervention to inactivate human noroviruses in uncooked foods such as shellfish. This work supports milestone 3 (to investigate physical and chemical parameters affecting HPP) and demonstrates that HPP can block the infection of MNV that is administered via a foodborne route.

6. Identified similar enzyme activities in vibrio and cancer cells: Vibrio vulnificus is a highly invasive bacterium associated with the consumption of raw shellfish. We discovered an enzyme activity in V. vulnificus and attempted to determine if this enzyme activity might be responsible for the rapid spread of vibrios and the correspondingly high mortality rate in humans. In preliminary studies, we screened for similar enzyme activity in normal human and malignant cells speculating that if this enzyme enhanced the spread of vibrio bacteria, that a similar enzyme in malignant cells might increase the rate of cancer spread and help to validate a functional relationship for this enzyme in bacterial and cancer invasiveness. We tentatively identified annexin A6 as the possible source of this enzyme activity and, using confocal microscopy, identified the cellular localization of annexin A6 in melanoma cells, human colorectal cancer cells, and in normal small intestine cells. This research supports milestone 1 (structurally and functionally characterize vibrio-related enzymes), and provides clues to uncovering the mysteries of vibrio and cancer cell invasiveness.

5.Significant Activities that Support Special Target Populations
At the request of stakeholders on the U.S. West Coast, ARS researchers assisted shellfish hatchery owners and operators and the shellfish industry at large in attempting to define intervention strategies to reduce larval shellfish mortalities, which have been occurring in the hatcheries for the past several years. Mortalities are suspected to be caused by a pathogen of larval shellfish known as Vibrio tubiashii. Interventions that could be employed in a hatchery setting are under evaluation to combat this pathogen.

6.Technology Transfer

Number of the New/Active MTAs (providing only)6

Review Publications
Kingsley, D.H., Chen, H. 2009. Influence of pH, Salt and Temperature on Pressure Inactivation of Hepatitis A virus. Food Microbiology. 130:61-64.

Kingsley, D.H., Chen, H. 2008. Aqueous Matrix Compositions Influence Virus Inactivation by High Pressure Processing. Journal of Food Protection. 71(1):598-603.

Lambert, M.S., Ozbay, G., Richards, G.P. 2009. The Influence of Feral Horse Activity on Water and Shellfish (Gukensia demissa) Quality Along the Western Coast of Assateague Island National Seashore, Maryland. Estuaries and Coasts - Journal of the Estuarine Research Federation. 57:405-415.

Kural, A.G., Shearer, A., Kingsley, D.H., Chen, H. 2008. Pressure Inactivation of Vibrio parahaemolyticus in Oysters- Influence of Pressure level and Treatment Temperature. International Journal of Food Microbiology. 127:1-5.

Last Modified: 8/4/2015
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