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United States Department of Agriculture

Agricultural Research Service

2010 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
Research was completed on project objectives related to the safety of aquaculture products, which falls under National Program 108 Food Safety. These objectives relate to the safety of shellfish, which contribute to bacterial and viral illnesses among shellfish consumers. Completed objectives included the development and evaluation of a novel enzyme-based assay for the rapid, simple, inexpensive, quantitative, and simultaneous detection of vibrio bacteria and E. coli in shellfish and seawater. This method allows for improved monitoring for naturally present vibrios and for potential sewage contamination. Another objective was to complete a human volunteer study in collaboration with Virginia Tech and Emory University to determine the effectiveness of high pressure processing to inactivate noroviruses in shellfish. Pressures required to inactivate noroviruses were higher than those typically used in commercial operations. Under another objective we identified mechanisms of virus persistence within shellfish and demonstrated the uptake and persistence of viruses within the blood cells (hemocytes) of oysters. Such persistence precludes the use of several processing interventions to inactivate viruses from shellfish and suggests the need for more aggressive treatments. Additional milestones were added to the original project because of early completion of existing milestones, successful grant submission, and requests for assistance by stakeholders. Stakeholders along the Pacific Northwest have experienced high mortalities in shellfish hatcheries, reaching an unprecedented 70% in recent years. Hatcheries have been plagued by a fish pathogen known as V. tubiashii, which is jeopardizing the availability of West Coast shellfish. This bacterium affects not only hatchery-produced shellfish, but natural stocks as well. A plan was developed to fight this bacterium using bacterial viruses (phages) and initial efforts to isolate and characterize the first phages against V. tubiashii were successful. To date, 15 phages have been isolated against V. tubiashii and characterized. Mixtures (cocktails) of these phages could serve as a useful intervention strategy for hatchery operators and will be evaluated in the last year of this project. Another new objective prompted by industry, regulatory, and consumer concerns was the potential threat of a pandemic strain of the human pathogenic bacterium, Vibrio parhaemolyticus, to U.S. shellfish. The pandemic strain originated in 1995 in India and has spread globally. Two shellfish-associated outbreaks have been reported in the U.S. Studies were performed in collaboration with the Univ. of Delaware to evaluate the uptake, growth and persistence of this strain in oysters. 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 were rapidly killed, thus there does not appear to be an immediate threat of broad scale outbreaks from this strain.

1. Improved tests to monitor contamination of seawater and shellfish. Escherichia coli and bacteria in the family Vibrionaceae are important causes of seafood-related illness. ARS researchers at Dover, DE, developed and published three assays to detect and quantify Escherichia coli and total Vibrionaceae in shellfish, seawater and other foods and environmental samples. Assays involve membrane overlays of overnight colonies on agar plates to detect enzymes for E. coli and Vibrionaceae, respectively. Cellulose membranes produced a bright blue fluorescence when overlaid onto colonies of E. coli, and green fluorescence when overlaid onto Vibrionaceae family members. Color development occurred within 10 min of applying the membranes and was based on the presence of specific enzymes in the bacteria. A multiplex assay was also developed for simultaneous detection of total E. coli and total Vibrionaceae in oysters and seawater. The overlays are rapid, simple, and cost effective for quantification purposes. This research provides practical alternatives for monitoring bacterial indicators and potential pathogens in complex samples, including oysters, clams, and mussels.

2. Characterized pandemic vibrio uptake and colonization in shellfish and effects of salt on vibrio survival. Vibrio parahaemolyticus serotype O3:K6 has been a major cause of shellfish-related illness in many parts of the world. Its pandemic spread to countries, including the U.S., raised concerns about the safety of U.S. shellfish and shellfish imported from other countries. ARS researchers at Dover, DE, in collaboration with the Univ. of Delaware staff evaluated the uptake and persistence of V. parahaemolyticus O3:K6 in oysters and concluded that the vibrios can proliferate in seawater, are efficiently cleared from the seawater by oysters, and are rapidly killed by digestion within oysters. Failure of this pandemic strain to persist in Eastern oysters, the principle oysters grown in the U.S., is good news and indicates that spread of this illness may be limited in the U.S. shellfish market. We also determined if environmental factors affected the growth of pandemic V. parahaemolyticus O3:K6. Different salt (NaCl) concentrations were evaluated and it was determined that salt can greatly affect vibrio response to pH and temperature stresses. In cultures containing 3% salt, there was a 65% survival of V. parahaemolyticus after freezing for 24 h at -20°C, but in cultures containing only 1% salt there was <1% survival. This suggests that oysters obtained from low salinity seawater may experience greater reductions of V. parahaemolyticus during frozen storage.

3. Discovery and characterization of the first bacterial viruses (phages) against Vibrio tubiashii. Major mortalities in shellfish hatcheries have been associated with the bacterial pathogen Vibrio tubiashii, which has reduced hatchery production by over 70% during the past few years. ARS researchers at Dover, DE, discovered and characterized the first phages against Vibrio tubiashii. A mixture of 15 of these novel phages was applied to larval oysters followed by the addition of Vibrio tubiashii. Proof-of-principle testing demonstrated that the phage cocktail reduced larval oyster mortalities by 70%. A search for additional phages is underway with the goal of producing a cocktail containing multiple phages, which would be applied to hatchery shellfish larvae to reduce Vibrio tubiashii mortalities. This research provides the first evidence for a successful commercial intervention against the fish pathogen, Vibrio tubiashii.

4. Foodborne viruses shown to persist within oyster hemocytes. Enteric viruses, like hepatitis A virus and norovirus, can persist within shellfish for extended periods. ARS researchers at Dover, DE, have identified three lines of evidence which support the hypothesis that foodborne viruses are sequestered within oyster blood cells (hemocytes) where they remain infectious in live oysters:.
1)there is a correlation between the molecular detection of the virus within the whole oyster and detection within hemocytes,.
2)virus-contaminated hemocytes can be transferred to clean oysters where viruses persist within the hemocytes for two weeks, and,.
3)the ability of different viruses to resist acid pH exposure correlates with virus persistence times within acidic hemocytes of live oysters. This research provided a better understanding of oyster biology as related to virus uptake and retention, and the information derived is being exploited to develop a more expedient means of testing shellfish for foodborne virus contamination.

5. High pressure processing shown to inactivate hepatitis A virus within mussels. In parts of the world, mussels are consumed raw or lightly cooked, making them a significant source of hepatitis A virus transmission. ARS researchers at Dover, DE, collaborating with a visiting scientist from the University of Bari, Italy, identified high pressure processing as a viable intervention for eliminating hepatitis A virus in both commercial blue mussels from the U.S. and Mediterranean mussels from Italy. A manuscript was submitted and published. The University of Bari is now evaluating the potential to use high pressure processing as an alternative to commercial mussel depuration in Italy.

5.Significant Activities that Support Special Target Populations
Activities designed to remediate vibrio problems in shellfish hatcheries target both hatcheries and small shellfish farmers, two important target populations.

Review Publications
Kingsley, D.H., Calci, K., Holliman, S., Dancho, B.A., Flick, G. 2009. High pressure inactivation of HAV within oysters: comparison of shucked oysters with whole in shell meats. Food and Environmental Virology. 1:137-140.

Richards, G.P., Watson, M.A. 2010. Fluorogenic membrane overlays to enumerate total coliforms, Escherichia coli, and total Vibrionaceae in shellfish and seawater. Internationl Journal of Microbiology. Vol 2010, Article ID 910486,

Whitaker, B.W., Michelle, P.A., Naughton, L.M., Richards, G.P., Blumerman, S.L., Boyd, F.E. 2010. Growth of Vibrio parahaemolyticus O3:K6 at Different Salt Concentrations Modulates Responses to pH and Temperature Stresses. Applied and Environmental Microbiology. 76:720-729.

Terio, V., Tantillo, G., Martella, V., Dipinto, P., Buonavoglia, C., Kingsley, D.H. 2010. High Pressure Inactivation of HAV within Mussels. Food and Environmental Virology. 2:83-88.

Richards, G.P., Macleod, C., Le Guyader, F.S. 2010. Processing Strategies to Inactivate Enteric Viruses in Shellfish: Limitations of Surrogate Viruses and Molecular Methods. Food and Environmental Virology. Available: doi:10.107/s12560-010-9045-2.

Last Modified: 9/5/2015
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