|Title:||Pathogen Detection and Intervention Methods for Shellfish|
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.
Approach: 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.
|Funding Source:||United States Department of Agriculture (USDA), Agricultural Research Service (ARS)|
|Institutions:||USDA/ARS - North Atlantic Area|
|Project Reports:||2013 Annual Report|
2012 Annual Report
2011 Annual Report
|Pending USDA ARS Publications (NP 108):|
ARS (NP 108):
Inactivation of human norovirus in contaminated oysters and clams by high-hydrostatic pressure
Ye M, Li X, Kingsley DH, Jiang X, Chen H.
Appl Environ Microbiol. 2014 Apr;80(7):2248-53.
Temperature Effects for High-Pressure Processing of Picornaviruses
Kingsley DH, Li X, Chen H.
Food Environ Virol. 2014 Mar;6(1):58-61.
Studies of inactivation mechanism of non-enveloped icosahedral virus by a visible ultrashort pulsed laser
Tsen SW, Kingsley DH, Poweleit C, Achilefu S, Soroka DS, Wu TC, Tsen KT.
Virol J. 2014 Feb 5;11:20.
Loss of sigma factor RpoN increases intestinal colonization of Vibrio parahaemolyticus in an adult mouse model
Whitaker W, Richards GP, Boyd E .
Infect Immun. 2014 Feb;82(2):544-56.
High pressure processing of bivalve shellfish and HPP’s use as a virus intervention
Seasonal levels of the Vibrio predator Bacteriovorax in Atlantic, Pacific and Gulf Coast Seawater
Richards GP, Watson MA, Boyd F, Burkhardt Iii W, Lau R, Uknalis J, Fay JP .
Int J Microbiol. 2013 Nov;2013:ID 375371.
The influence of temperature, pH, and water immersion on the high hydrostatic pressure inactivation of GI.1 and GII.4 human noroviruses
Li X, Chen H, Kingsley DH.
Int J Food Microbiol. 2013 Oct;167(2):138–43.
Sensitivity of hepatitis A and murine norovirus to electron beam irradiation in oyster homogenates and whole oysters - quantifying the reduction in potential infection risks
Nair C, Dancho BA, Kingsley DH, Calci K, Meade GK, Mena KD, Pillai S .
Appl Environ Microbiol. 2013 Jun;79(12):3796-801.
Susceptibility of murine norovirus and hepatitis A virus to electron beam irradiation in oysters and quantifying the reduction in potential infection risks
Praveen C, Dancho BA, Kingsley DH, Calci KR, Meade GK, Mena KD, Pillai SD.
Appl Environ Microbiol. 2013 Jun;79(12):3796-801.
Murine macrophage inflammatory cytokine production and immune activation in response to Vibrio parahaemolyticus infection
Waters S, Luther S, Joerger T, Richards GP, Boyd EF, Parent MA.
Microbiol Immunol. 2013 Apr;57(4):323-8.
Lack of Norovirus Replication and Histo-Blood Group Antigen Expression in 3-Dimensional Intestinal Epithelial Cells
Herbst-Kralovets MM, Radtke AL, Lay MK, Bolick AN, Sarker SS, Kingsley DH, Arntzen CJ, Estes MK, Nickerson C.
Emerg Infect Diseases. 2013 Mar;19(3):431-8.
High pressure processing and its application to the challenge of virus-contaminated foods
Food Environ Virol. 2013 Mar;5(1):1-12.
Resilience of norovirus GII.4 to freezing and thawing: implications for virus infectivity
Richards GP, Watson MA, Meade GK, Hovan GL, Kingsley DH.
Food Environ Virol. 2012 Dec;4(4):192-7.
Predatory bacteria as natural modulators of Vibrio parahaemolyticus and Vibrio vulnificus in seawater and oysters
Richards GP, Fay JP, Dickens KA, Parent MA, Soroka DS, Boyd EF.
Appl Environ Microbiol. 2012 Oct;78(20):7455-66.
The Vibrio parahaemolyticus ToxRS regulator is required for stress tolerance and colonization in a novel orogastric streptomycin-induced adult murine model
Whitaker WB, Parent MA, Boyd A, Richards GP, Boyd EF.
Infect Immun. 2012 May;80(5):1834-45.
Critical review of norovirus surrogates in food safety research: rationale for considering volunteer studies
Food Environ Virol. 2012 Mar;4(1):6-13.
Shellfish-associated enteric virus illness: virus localization, disease outbreaks and prevention - (Book / Chapter)
Accepted Publication (28-Aug-13)
Enteric virus and vibrio contamination of shellfish: intervention strategies - (Proceedings)
Richards, G.P. 2013. Enteric virus and vibrio contamination of shellfish: intervention strategies. Symposium Proceedings. In: Proceedings of the Korean Society of Food Science and Nutrition.Food Safety for the Food Industry, Gwangju, South Kore, November 13-15,2013.pp.57-58
Detection of enteric viruses in shellfish - (Proceedings)
Richards, G.P., Cliver, D.O., Greening, G.E. 2013. Detection of enteric viruses in shellfish. Meeting Proceedings. G. Sauve, editor In Molluscan Shellfish Safety, Proceedings of the 8th International Conference on Molluscan Shellfish Safety, Charlottetown, Prince Edward Island, Canada, June 12-17, 2011.Springer, New York, pp. 177-183.
An introduction to food and waterborne viruses: diseases, transmission, outbreaks, detection and control - (Book / Chapter)
Cook, N., Richards, G.P. 2013. An introduction to food and waterborne viruses: diseases, transmission, outbreaks, detection and control. Book Chapter. Food and Waterborne Viruses, Woodhead Publishing Co, Cambridge, United Kingdom, pp.3-18.
Shellfish contamination and spoilage - (Book / Chapter)
Accepted Publication (01-Feb-13)
Natural modulators of Vibrios in seawater and shellfish - (Abstract Only)
Novel methods for detection of foodborne viruses - (Abstract Only)
Kingsley, D.H. 2012. Novel methods for detection of foodborne viruses. Meeting Abstract., American Chemical Society (ACS) meeting., Philadelphia, PA., August 19-23, 2012., Volume 1, Page 1.
Recent intervention and detection advances for shellfish-borne norovirus - (Abstract Only)
Kingsley, D.H. 2012. Recent intervention and detection advances for shellfish-borne norovirus. Meeting Abstract. National Committee for the Microbiologist Criteria Subcommittee., Washington, DC., May 8-10,2012.Volume 1, Page 1.
Food safety research at Delaware State University: keeping the runs from slowing you down - (Abstract Only)
Kingsley, D.H. 2012. Food safety research at Delaware State University: keeping the runs from slowing you down. Meeting Abstract.Delaware State University, College of Agriculture and Related Sciences, Dover, DE, April 13, 2012., Volume 1, Page 1.
An extraction method for discrimination between infectious and inactive norovirus using RT-PCR - (Abstract Only)
Kingsley, D.H., Dancho, B. 2012. An extraction method for discrimination between infectious and inactive norovirus using RT-PCR. Meeting Abstract. American Society for Virology., Madison, Wisconsin., July 21-25, 2012., Volume 1, Page 1.
Foodborne viruses - (Book / Chapter)
Accepted Publication (23-Aug-11)
In memoriam Dean Otis Cliver 1935-2011 - (Other)
Richards, G.P. 2011. In memoriam Dean Otis Cliver 1935-2011. Food and Environmental Virology. 3(3):99-108. DOI: 10.1007/s12560-011-9064-7.
Sensitive detection of multiple hepatitis A virus genotypes with a single polony-based assay - (Abstract Only)
Accepted Publication (04-Mar-11)
|Food Safety Categories:||Food and Feed Handling and Processing|
Government Policy and Regulations
|Farm-to-Table Categories:||On-farm food production|
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