Progress 01/31/11 to 01/30/16
Outputs
Progress Report 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. 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. Bacterial and viral contamination of oysters, clams and mussels contribute to illnesses and deaths in the United States each year. Naturally-occurring Vibrio parahaemolyticus is the principle cause of shellfish-related illnesses and Vibrio vulnificus is the primary cause or shellfish-related mortalities in the U.S. Enteric viruses, principally norovirus, also contribute to a substantial number of illnesses from shellfish and other foods and is the primary cause of foodborne illness in the U.S. Together these pathogens have been problematic to the shellfish industry, regulatory agencies, and to consumers. Additionally, vibrios are important shellfish pathogens which contribute to mass mortalities in aquaculture and natural settings. Larval shellfish hatcheries have been particularly plagued by the presence of Vibrio coralliilyticus and Vibrio tubiashii, which have caused intermittent interruptions in hatchery production and shortages of seed oysters needed by the commercial oyster industry. Under Objective 1, we identified genetic and environmental factors responsible for V. parahaemolyticus and V. Vulnificus survival and persistence in the environment. We identified natural predatory bacteria against a pandemic strain of V. parahaemolyticus in seawater. Known as Halobacteriovorax, these predators were shown to attack and naturally modulate pathogenic V. parahaemolyticus and V. vulnificus in seawater and oysters. These predatory bacteria were the most significant cause of Vibrio reduction from among the environmental factors evaluated. A study on the seasonality and geographic distribution of these predators revealed their widespread presence in waters off the U.S. Atlantic and Gulf coasts and from Hawaiian seawater throughout the year. We evaluated the use of these Halobacteriovorax isolates as potential processing interventions to reduce pathogenic vibrios in market oysters. Oysters were subjected to the commercial process of depuration, where shellfish are placed in tanks of clean seawater and allowed to purge contaminants from their systems over several days. Conventional depuration is minimally effective in eliminating vibrios from shellfish, but by addition of Halobacteriovorax to the shellfish tanks before initiation of depuration, we expect to identify a modified depuration process effective against pathogenic vibrios. That work is continuing into the next project cycle. In addition to Halobacteriovorax against the vibrios, we identified strains against other important pathogens, namely E. coli O157:H7 and Salmonella Typhimurium DT-104. Host specificity studies showed that Halobacteriovorax isolated in E. coli and Salmonella infected a broader range of pathogens including the vibrios, whereas the earlier isolated Halobacteriovorax against V. parahaemolyticus had narrow host specificity, only infecting strains of V. parahaemolyticus. We have provided these strains to ARS collaborators who hope to use them to eliminate E. coli and Salmonella from the surface of produce. A publication on the host specificities of these Halobacteriovorax isolates and the newly identified isolates against E. coli and Salmonella was published earlier this year. The remediation of vibrios in commercial shellfish hatcheries and in market shellfish was the goal of Objective 2a. We isolated and identified 15 bacteriophages (bacterial viruses, also known as phages) which were effective in killing Vibrio coralliilyticus and Vibrio tubiashii in larval oysters. We also isolated phages against human Vibrio strains and completely DNA sequenced the genomes of a V. coralliilyticus and a V. tubiashii. Bacteriophages are increasingly being used as a green technology to combat pathogens in food products and can supplant the use of ineffectual antibiotic in aquaculture. Vibrio coralliilyticus and V. tubiashii have been associated with over 70% larval mortalities in West Coast hatcheries, nearly causing the hatcheries to go bankrupt. In laboratory studies, we showed that a mixture of these phages would reduce larval mortalities by up to 95%. With an industry collaborator (Intralytix, a phage based technology company), we obtained two USDA Small Business Innovative Research grants during this project period and are now working with an Oregon State University hatchery to further show commercial value to the use of phage technology. During this project period, we extracted and sequenced phage DNA from six isolates and travelled to Oregon State University in Corvallis, where we provided phage and vibrios and demonstrated procedures for determining phage and Vibrio concentrations. Further work to commercialize a mixture of phages will be a prime focus under the next project cycle. Several technologies to inactivate (kill) human enteric viruses were evaluated under objective 2b, �Developing intervention and control strategies for enteric viruses by E-beam, high pressure processing and other technologies�. Conditions have been defined showing that two types of human noroviruses (genogroup I.1 and II.4) are sensitive to high pressure processing (HPP). We showed in taste panel testing that HPP does generate a highly acceptable raw oyster for consumption by consumers when oysters are treated at pressure levels that would inactivate human norovirus and hepatitis A virus. We showed that E- beam irradiation can inactivate enteric viruses sequestered within shellfish tissues; however, the levels of E-beam radiation required to inactivate >90% of the virus present exceeded permissible irradiation limits. This technology may inactivate some viruses when applied as an intervention for other pathogens, by itself it will not likely be a viable intervention unless the FDA raises current permissible irradiation levels for food products. The concept of imparting harmonic vibrations, a potential new technology that might be sufficient to destroy viruses, was investigated. Research showed that ultra-short pulse visible lasers can inactivate murine norovirus (a surrogate for human norovirus) and hepatitis A virus while not substantially damaging blood proteins (suggesting that there would be little to no damage to oyster proteins to negatively affect their taste or appearance). Work on this is continuing into the next project cycle. Hepatitis E virus (HEV) work was performed using a newly described avian HEV strain in birds via a collaboration with the University of Delaware. Unfortunately the virus does not induce enough pathology in chickens to be useful as a surrogate for human HEV. Under objective 3, �Characterizing virus uptake and persistence in oysters�, the persistence of human norovirus within oysters as well as within hemocytes was characterized as a function of temperature. Results indicated that water temperature has a substantial effect on the ability of human norovirus to persist within oysters. Biogenic silver particles were generated on lactobacillus bacteria and fed to shellfish to determine if the silver could reduce viruses in oysters. Unfortunately, there was no indication that nanosilver had any effect on the persistence of viruses within live shellfish. Under objective 4, �Complete development of automated virus extraction and detection techniques for shellfish� a molecular biological method (RT-PCR) of assessing the inactivation of human norovirus was developed. It is able to differentiate infectious from inactivated noroviruses after application of chemical and physical treatments of the viruses. This has facilitated considerable new research regarding inactivation of human norovirus by high pressure processing and thermal processing. This assay has been confirmed as effective, using a pig model, and demonstrated that freezing has no effect on human norovirus. The assay was also used to assess the degree to which human norovirus is inactivated by irradiation. This work was further advanced to assess the effects of chemical sanitizers on human norovirus and was used to determine that traditional chlorine-based sewage treatment does not inactivate human norovirus. In addition, a simple, isothermal �LAMP� nucleic acid amplification method has been developed to test for the presence of human norovirus within oysters. Together, these studies have: identified novel predatory bacteria and bacteriophages against pathogens in shellfish, evaluated novel processing interventions to reduce bacterial and viral pathogens in oysters and other foods, developed improved technologies to better monitor for pathogenic viruses in shellfish, and helped steer research in important new directions. Work in some of these areas is continuing under project number 8072-42000-081-00D. Accomplishments 01 Characterized marine predatory bacteria against human pathogens. Pathogens in marine waters contaminate shellfish and cause shellfish- borne illness. ARS researchers in Dover, Delaware, isolated and characterized marine predatory bacteria (known as Halobacteriovorax species) against important food-borne pathogens, including E. coli, Salmonella Typhimurium, and pandemic strains of Vibrio parahaemolyticus. The ability of predatory bacteria to modulate pathogens in oysters was demonstrated and may lead to novel processing interventions for seafoods and other products. Application of naturally-occurring predatory bacteria (via rinse, spray or dip methods) to the surfaces of fruits, vegetables, meats, or fish may be a practical, green technology to eliminate or reduce E. coli, Salmonella, Vibrios, and other pathogens from foods to enhance food safety.
Impacts
(N/A)
Publications
- Dancho, B.A., Haiqiang, C., Kingsley, D.H. 2012. A method to discriminate between infectious and inactive human norovirus. International Journal of Food Microbiology. 155:222-226.
- Lou, F., Ye, M., Ma, Y., Li, X., Dicaprio, E., Chen, H., Krakowkal, S., Hughes, J., Kingsley, D.H., Li, J. 2015. A gnotobiotic pig model for determining human norovirus inactivation by high-pressure processing. Applied and Environmental Microbiology. 81(19):6679-6687.
- Hickey, M.E., Richards, G.P., Lee, J. 2014. Development of a two-step, non- probed multiplex real-time PCR for surveilling Vibrio anguillarum in seawater. Journal of Fish Diseases. 38:551-559.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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. Molluscan shellfish (oysters, clams and mussels) cause seafood-associated illnesses and occasional deaths in the U.S. Illnesses are commonly from viruses, like norovirus, which is the principle cause of foodborne illness in the U.S., and from bacteria of the genus Vibrio. Norovirus is associated with fecal pollution of foods while vibrios 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. Together, norovirus and vibrios are the leading causes of seafood-related illnesses in the U.S. Other vibrios, like V. tubiashii and V. coralliilyticus, are species which cause high mortalities in larval shellfish in U.S. shellfish hatcheries and have impacted the availability of seed oysters and clams, which are needed by the commercial shellfish industry. Under Objective 1, �to 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�, ARS researchers at Dover, Delaware, completed several lines of research. Under this objective, we found that natural seawater contains some factor that inhibits the survival and growth of pathogenic vibrios in both seawater and shellfish. We identified this factor as a naturally-occurring predatory bacterium of the genus Bacteriovorax. In small-scale laboratory trials, these salt- water Bacteriovorax effectively regulated the levels of pathogenic vibrios in oysters and seawater and may have practical application as a post-harvest processing technology to reduce vibrios in market-sized shellfish. Over the summers of 2014 and 2015, we used a mobile, pilot- scale depuration unit on loan from a clam depuration company in New Jersey. Depuration is a commercial process where shellfish are allowed to purge contaminants in tanks of clean seawater. This depuration unit is capable of processing 1.5 bushels of oysters in 325 gallons of seawater. We are using this system to show proof-of-concept that Bacteriovorax may have commercial application to reduce pathogenic vibrios (V. parahaemolyticus and V. vulnificus) in market-sized oysters. Objective 2a was �to develop and evaluate intervention and control strategies for Vibrio species through identification, characterization and application of phages to remediate shellfish mortalities in hatchery settings, and for use in commercial shellfish processing.� ARS researchers at Dover, Delaware, focused efforts on V. tubiashii, a bacterial pathogen responsible for high mortalities in larval shellfish in U.S. hatcheries � mortalities that cause shortages of seed oysters needed by the commercial shellfish industry. At industry request, a plan was developed and implemented to control V. tubiashii in shellfish hatcheries using lytic bacteriophages (phages). These phages are bacterial viruses that kill bacteria but are harmless to humans. Under this objective, we isolated and characterized the first phages ever discovered against V. tubiashii, demonstrated their ability to reduce larval mortalities by 80%, wrote and received a phase I Small Business Innovative Research (SBIR) grant, and established a CRADA with Intralytix Inc., a phage-based technology company, to commercialize the phage treatment for use by shellfish hatcheries. A phase II SBIR grant has recently been submitted to promote commercialization of the product. Overall, the study demonstrated proof-of-principle that these phages effectively reduce mortalities in both Eastern and Pacific oyster larvae. This research directly addressed a critical need expressed by the West Coast shellfish industry and led to significant technology transfer of ARS-discovered phages under a CRADA and SBIR grant for future product licensing and potential commercialization. Research to date on V. tubiashii is encouraging and is a prelude to future research to develop phage-based methods to eliminate human pathogenic vibrios from commercially harvested shellfish. It became clear during these studies that Vibrio coralliilyticus was also associated with larval shellfish mortalities and that several vibrios thought to be V. tubiashii were actually V. coralliilyticus. Both V. coralliilyticus and V. tubiashii are closely related genetically. Characterization of several strains of V. tubiashii,,which were being used in our research and were previously reported in the literature as V. tubiashii, were found to be V. coralliilyticus. The ARS-Dover lab, in collaboration with ARS in Clay Center, Nebraska, and ARS in Wyndmoor, Pennsylvania, completely sequenced and published the genome of one of these �V. tubiashii� and confirmed that it was actually a strain of V. coralliilyticus which is particularly pathogenic toward both Eastern and Pacific oyster larvae. The same group published the first complete genome sequence for a V. tubiashii (American Type Culture Collection type strain 19109). Both complete genome sequences were entered into GenBank. Thus, it was determined that the phages used in our Phase I SBIR trials were active against not only V. tubiashii, but V. coralliilyticus as well, and that they were effective in significantly reducing both pathogens and associated mortalities in larval oysters. Studies were also conducted to determine which strains of V. coralliilyticus and V. tubiashii were most infectious toward Eastern and Pacific oysters, and the LD50 values of these pathogens were published. This information will allow us to develop the most effective phage therapies against the most virulent strains of V. coralliilyticus and V. tubiashii in future studies. A Phase II SBIR proposal was submitted to support continued commercialization of phage therapy as an intervention against V. tubiashii and V. coralliilyticus in shellfish hatcheries. Additionally, phages against the human pathogens V. parahaemolyticus and V. vulnificus have been isolated and phage interventions to inactivate these pathogens in shellfish are underway. Under objective 2b, which was �to develop and evaluate intervention and control strategies for 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�, ARS researchers at Dover, Delaware, investigated the inactivation of an avian hepatitis E virus (HEV) stain. Zoonotic HEV (from swine) and types 1 and 2 human HEV are extremely difficult to propagate in vitro. An HEV was obtained from Virginia Tech and evaluated with the goal of using it as a surrogate to gain preliminary information about whether high pressure processing might inactive zoonotic HEV in uncooked meats. Attempts to propagate HEV in embryonated eggs, primary avian liver cells, and primary avian fibroblasts were unsuccessful. The feasibility of using day old chicks in an isolator unit was pursued collaboratively with the University of Delaware. While virus could be detected in stools one week after inoculation of specific pathogen free chicks, there was no obvious pathology noted in these birds. Due to the expense of live birds in isolator units, cumbersome logistics, and lack of good in vitro or in vivo infection assay, no practical method was available to assess the inactivation of avian HEV. Previously, we showed good inactivation of hepatitis A virus and norovirus surrogates and limited inactivation of human norovirus by high pressure processing . Under objective 3, �to characterize the uptake and persistence of norovirus and hepatitis A virus in oysters�, ARS researchers at Dover, Delaware, investigated the feasibility of inactivating human hepatitis A virus and murine norovirus as a surrogate for human norovirus using e- beam, which led to the publication of a paper in a previous year. Conclusions were that e-beam can inactivate these viruses with a 5 KiloGray dose being sufficient to inactive approximately 99% of the virus in buffer and 90% of the virus in oyster homogenates. Under objective 4, �to develop technologies to automate, simplify, or improve current virus testing methods to include the evaluation of assays for infectious (live) versus inactivated (dead) viruses�, ARS scientists at Dover, Delaware, found that porcine gastric mucin could be used to distinguish between infectious and non-infectious human norovirus. A method to distinguish infectious from non-infectious norovirus extracted from oysters has been successfully developed. Papers were published previously on what sanitizers can and cannot inactivate norovirus, and the effects of freezing and thawing on the viability of human norovirus as determined by our mucin binding assay. The mucin technology was recently approved for a U.S. patent, and a company is expressing interest in licensing this technology. The assay has also attracted the interest of the United States Food and Drug Administration (FDA) who would like to use the method as a potential tool for shellfish risk assessment. ARS has a research agreement with the FDA to evaluate the mucin binding assay to measure the effects of sewage treatment and environmental factors, such as sunlight, on norovirus inactivation and persistence. Accomplishments 01 Identification of vibrios pathogenic to larval shellfish and their eradication by phage therapy. Vibrio tubiashii has been linked with production crashes in shellfish hatcheries on the U.S. East and West Coasts, reducing the availability of seed oysters needed to sustain the commercial shellfish industry. ARS Researchers at Dover, Delaware, completely sequenced and published the genomes of two reputed V. tubiashii and found one to be a V. coralliilyticus. The lethality of these and other strains toward larval Eastern and Pacific oysters was determined and demonstrated that both V. coralliilyticus and V. tubiashii infected and killed Eastern oyster larvae, but only V. coralliilyticus infected and killed Pacific oyster larvae. Bacterial viruses (phages), which were discovered by the ARS Dover laboratory, effectively reduced oyster mortalities caused by both V. coralliilyticus and V. tubiashii and may serve as a useful therapeutic treatment to prevent the loss of hatchery products. 02 Resonance energy inactivates hepatitis A virus. ARS Researchers at Dover, Delaware, have shown that femtopulse lasers using light in the visible spectrum can impart sufficient resonance energy vibrations to inactivate non-enveloped viruses (i.e., food- and blood-borne viruses). Research indicates that the mechanism for virus destruction is non- thermal. An investigation of hepatitis A virus inactivation within blood plasma showed that a 1.5-log reduction can be obtained with limited damage to blood plasma proteins, demonstrating that this technology should have minimal effect on food proteins and organoleptic qualities.
Impacts
(N/A)
Publications
- Richards, G.P., Watson, M.A., Needleman, D.S., Church, K.M., Hase, C.C. 2014. Mortalities of eastern and pacific oyster larvae caused by the pathogens Vibrio coralliilyticus and Vibrio tubiashii. Applied and Environmental Microbiology. 81:292-297.
- Richards, G.P., Bono, J.L., Watson, M.A., Needleman, D.S. 2014. Complete genome sequence for the shellfish pathogen Vibrio coralliilyticus RE98 isolated from a shellfish hatchery. Genome Announcements. DOI:10.1128/ genomeA.01253-14.
- Richards, G.P., Needleman, D.S., Watson, M.A., Bono, J.L. 2014. Complete genome sequence of the larval shellfish pathogen Vibrio Tubiashii type strain ATCC 19109. Genome Announcements. 2(6); e01252-14. DOI: 10.1128/ genomeA01252-14.
- Kingsley, D.H., Kuhn, D.D., Flick, G.J., Oh, J., Lawson, L.S., Meade, G.K., Giesecke, C.C. 2014. Desirability of oysters treated by high pressure processing at different temperatures and elevated pressures. American Journal of Food Technology. 9:209-216.
- Shaw-Wei, T., Kingsley, D.H., Kibler, K., Jacobs, B., Sizemore, S., Vaiana, S., Anderson, J., Kong-Thon, T., Achilefu, S. 2014. Pathogen reduction in human plasma using an ultrashort pulsed laser. PLoS One. 9(11)e111673.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. Molluscan shellfish (oysters, clams and mussels) cause 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. At a recent meeting of the Interstate Shellfish Sanitation Conference (a group which promulgates shellfish regulations in the US), the FDA, industry and state regulatory agencies listed vibrios as the pathogens of greatest concern in the US. Other vibrios, like V. tubiashii, are species which cause high mortalities in larval shellfish in US shellfish hatcheries and have impacted the availability of seed oysters and clams, which are needed by commercial shellfishermen. ARS researchers in Dover, DE, in collaboration with the University of Delaware, the US FDA and Kona Coast Shellfish in Hawaii, completed and reported on the presence of vibrio predatory bacteria in seawater and found that they kill vibrios in shellfish. A plan was developed and implemented, in collaboration with the US FDA and industry, to evaluate these vibrio predators in a process that has great potential to be applied commercially to reduce or eliminate vibrios from shellfish. Under objective 2a, ARS researchers at Dover, DE, in collaboration with Intralytix, a biotechnology company in Baltimore, worked under a CRADA to complete a Phase I Small Business Innovative Research (SBIR) grant to evaluate bacterial viruses, known as bacteriophages, for possible commercialization and distribution to shellfish hatcheries to reduce larval shellfish mortalities. Phase I studies showed successful reduction of a variety of larval pathogens using a mixture of phages. Under objective 2b, ARS researchers at Dover, DE, in collaboration with Virginia Tech and Delaware State University demonstrated that high pressure processing (HPP) of oysters treated under conditions that have previously been shown to inactivate human norovirus, hepatitis A virus, and vibrio bacteria, are actually preferred by consumers as compared to untreated oysters. This established that HPP is a viable intervention for these infectious agents in bivalve shellfish. Another related project by ARS researchers at Dover, DE, and Virginia Tech has demonstrated that HPP can be used to flavor oysters since they absorb surrounding liquids under pressure. It is envisioned that this may create uniquely flavored oyster that would represent a new pathogen- free oyster product. In another project with Arizona State University, ARS researchers have demonstrated proof-of-principle that hepatitis A virus can be inactivated by ultrahigh frequency waves within blood plasma. Under objective 4, ARS researchers at Dover, published a paper demonstrating that human norovirus is highly resistant to sanitizers and a collaborative study with the University of Delaware was completed that demonstrated that the most common strain of human norovirus in circulation today is more sensitive to high pressure than the prototypical laboratory strain of human norovirus. Eleven papers were published this year including two under objective 1, four under objective 2, one under objective 3, one under objective 4, plus two book chapters and a methods paper on the development of a novel vibrio detection method. Significant Activities that Support Special Target Populations: Research on human vibrios targets populations of consumers who are particularly susceptible to infection due to underlying disease, like liver disease, diabetes, or compromised immune systems. It also targets shellfish hatchery operators whose livelihoods are jeopardized by massive die-offs of larval shellfish and shellfishermen who rely on shellfish aquaculture to satisfy commercial demands for oysters, clams, and other products. Virus research targets the development of improved methods and intervention strategies to enhance the safety of shellfish that are marketed to the consumer. Accomplishments 01 Commercial application for vibrio predatory bacteria. Vibrios are the primary cause of shellfish-associated bacterial illnesses and deaths in the US each year. In evaluating the effects of pH, temperature and algae diet on the levels of vibrios in shellfish, ARS researchers at Dover, Delaware and collaborators at the University of Delaware discovered a group of naturally occurring predatory bacteria that have a substantial effect on reducing vibrio levels in both seawater and shellfish. ARS researchers, in collaboration with the US FDA and industry, are evaluating the use of these vibrio predators to eliminate or significantly reduce vibrio levels in market shellfish. A pilot- scale, portable processing facility is under evaluation to determine the effectiveness of vibrio predatory bacteria for eliminating vibrios in oysters. Success of this process will result in direct application of an ARS-developed technology to remediate the most significant pathogens in oysters and clams and will enhance shellfish safety worldwide. 02 Commercialization of bacterial viruses (bacteriophages) to reduce shellfish mortalities. Shellfish hatcheries in the US have experienced high larval mortalities due to Vibrio tubiashii and related bacteria, causing shortages of seed oysters and clams for commercial planting. ARS researchers at Dover, Delaware, identified and characterized bacteriophages that kill V. tubiashii and other shellfish pathogens. In collaboration with Intralytix, a phage-based biotechnology company, a Phase I SBIR grant to commercialize these phages was completed and a tentative treatment was developed. 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 and aquaculture operations. 03 High pressure inactivation of viruses in oysters. Sewage-borne viruses, like human norovirus and hepatitis A virus, can contaminate shellfish leading to outbreaks of norovirus illness and hepatitis A in unsuspecting consumers. ARS researchers at Dover, Delaware showed that high pressure processing of oysters eliminates these viruses and that processed oysters are acceptable and often preferred by consumers over untreated oysters. Collaborative work with Virginia Tech demonstrated that it is possible to flavor oysters by placing them in flavored solutions instead of water when pressurized. An invention disclosure has been filed for this novel application. This research shows a practical means to enhance shellfish safety and to produce alternatively flavored oysters for the marketplace. 04 Effectiveness of norovirus disinfectants determined. There is little information available on the effectiveness of disinfectants to reduce human noroviruses. An ARS-developed porcine mucin binding assay has been used by ARS researchers at Dover, Delaware, to evaluate the potential of common sanitizers to inactivate human norovirus. Results indicate that chlorine is a reasonably effective sanitizer, but chlorine dioxide, peroxyacetic acid, and hydrogen peroxide do not appear to be effective. Trisodium phosphate was found to be moderately effective against human norovirus. These findings are expected to lead to improved norovirus disinfection techniques for food processing plants.
Impacts
(N/A)
Publications
- Tsen, S.D., Kingsley, D.H., Poweleit, C., Achilefu, S., Soroka, D.S., Wu, T., Tsen, K. 2014. Studies of Inactivation Mechanism of non-enveloped icosahedral viruses by a visible ultrashort pulsed laser. Virology Journal. 11:20.
- Richards, G.P., Watson, M.A., Boyd, F., Burkhardt Iii, W., Lau, R., Uknalis, J., Fay, J.P. 2013. Seasonal levels of the Vibrio predator Bacteriovorax in Atlantic, Pacific and Gulf Coast Seawater. International Journal of Microbiology. doi:10.1155/2013/375371.
- Kingsley, D.H., Li, X., Chen, H. 2014. Temperature effects for high pressure processing of Picornaviruses. Food and Environmental Virology. 6:58-61.
- Ye, M., Li, X., Kingsley, D.H., Jiang, X., Chen, H. 2014. Inactivation of human norovirus in contaminated oysters and clams by high-hydrostatic pressure. Applied and Environmental Microbiology. 80:2248-2253.
- Li, X., Chen, H., Kingsley, D.H. 2013. The influence of temperature pH and water immersion on the high hydrostatic pressure inactivation of GI.1 and GII.4 human noroviruses. International Journal of Food Microbiology. 167:138-143.
- Kingsley, D.H. 2014. High pressure processing of bivalve shellfish and HPP's potential use as a virus intervention. Review Article. DOI:10.3390/ foods3020336.
- Kingsley, D.H., Vincent, E., Meade, G.K., Watson, C., Fan, X. 2014. Inactivation of human norovirus using chemical sanitizers. International Journal of Food Microbiology. 171:94-99.
- Whitaker, W., Richards, G.P., Boyd, E. 2014. Loss of sigma factor RpoN increases intestinal colonization of vibrio parahaemolyticus in an adult mouse model". Infection and Immunity. 82:544-556.
- Kingsley, D.H. 2014. Shellfish contamination and spoilage.In: Batt, C.A., Totorello, M.L., Elsevier Ltd., Encyclopedia of Food Microbiology. Volume 3, pp.389-396.
- Richards, G.P., Cliver, D.O., Greening, G. 2013. Foodborne viruses.In: S. Doores, Y. Salfinger and M.L. Tortorello. Compendium of Methods for the Microbiological Examination of Foods, 5th Edition, American Public Health Association, Washington, DC. Book Chapter. Doi:10.2105/MBEF.0222.049.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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 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. Accomplishments 01 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. 02 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. 03 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.
Impacts
(N/A)
Publications
- Nair, C., Dancho, B.A., Kingsley, D.H., Calci, K., Meade, G.K., Mena, K.D., Pillai, S. 2013. Sensitivity of hepatitis A and murine norovirus to electron beam irradiation in oyster homogenates and whole oysters - quantifying the reduction in potential infection risks. Applied and Environmental Microbiology. 79:3796-3801.
- 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.
- Herbst-Kralovets, M.M., Radtke, A.L., Lay, M.K., Bolick, A.N., Sarker, S.S. , Kingsley, D.H., Arntzen, C.J., Estes, M.K., Nickerson, C. 2013. Correlation between lack of norovirus replication and histo-blood group antigen expression in 3D-intestinal epithelial cultures. Emerging Infectious Diseases. Volume 19(3):431-438.
- Waters, S., Luther, S., Joerger, T., Richards, G.P., Boyd, E., Parent, M.A. 2013. Murine macrophage inflammatory cytokine production and immune activation in response to Vibrio parahaemolyticus infection. Microbiology and Immunology. 57(4):323-328.
- Richards, G.P., Fay, J.P., Dickens, K.A., Parent, M.A., Soroka, D.S., Boyd, E. 2012. Predatory bacteria as natural modulators of Vibrio parahaemolyticus and Vibrio vulnificus in seawater and oysters. Applied and Environmental Microbiology. 78:7455-7466.
- 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..
- Kingsley, D.H. 2013. High pressure processing and its application to the challenge of virus-contaminated foods. Food and Environmental Virology. 5:1-12.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations: Research on the human vibrios (Vibrio parahaemolyticus and Vibrio vulnificus) targets needs of the shellfish industry and populations of people who are at increased risk of infection, such as those who have underlying illnesses, including liver disease, diabetes, and weakened immune systems. Activities designed to remediate Vibrio problems in shellfish hatcheries target both the hatcheries and small shellfish farmers, two important target populations who have been negatively impacted by high shellfish mortalities. Research on infectious noroviruses binding to porcine mucin will be of direct benefit to the shellfish industry and State and Federal regulatory agencies. Accomplishments 01 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 ha caused major die-offs at shellfish hatcheries, particularly on the US We Coast. Using Hawaiian seawater, ARS researchers at Dover, Delaware, discovered 15 phage viruses that kill various strains of V. tubiashii. mixture of these phages protected larval oysters against high levels of 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. 02 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 te for virus infectivity. ARS researchers at Dover, Delaware, attached swi mucin to magnetic beads to selectively bind infectious human noroviruses but not inactivated noroviruses. Viruses inactivated by heat, ultraviol light, and high pressure processing were unable to bind to mucin-coated magnetic beads. The binding of only infectious viruses to the beads wil for the first time, allow the detection of infectious viruses in food an environmental samples. This technology has been provisionally approved for patenting and a manuscript has been published. 03 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, identifi 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 vibrio 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 ar important modulators of pathogenic vibrios in seawater and oysters.
Impacts
(N/A)
Publications
- 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.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. Research under National Program 108 Food Safety, was initiated on 2/1/11 on project objectives related to the safety of shellfish, which contribute to bacterial and viral illnesses among consumers. V. vulnificus and V. parahaemolyticus are naturally-occurring marine bacteria which accumulate in shellfish and cause illnesses and deaths among consumers. V. tubiashii is another naturally-occurring marine bacterium which kills juvenile shellfish and has been particularly problematic in shellfish hatcheries, leading to shortages of seed oysters and clams for commercial aquaculture operations. Norovirus and hepatitis A virus are the two most prevalent causes of seafood-related viral illnesses in the US for which processing interventions and detection methods are needed. To date, objective 1 research has demonstrated the inability of the pandemic strain of V. parahaemolyticus to persist in seawater and shellfish. Efforts are underway to determine if this pandemic strain is unique or if other strains of V. parahaemolyticus and V. vulnificus respond similarly. Under objective 2a, we are responding to West Coast stakeholders to develop methods to reduce high mortalities in shellfish hatcheries as a result of the shellfish pathogen, V. tubiashii. As an intervention, we are evaluating the effectiveness of bacterial phages (viruses that kill bacteria) to reduce oyster mortalities in shellfish hatcheries. To date we have isolated 16 phages against V. tubiashii from seawater from Hawaii. Pilot studies are underway, in collaboration with Oregon State University, to determine the effectiveness of these phages to combat hatchery outbreaks of V. tubiashii. Phages against the human pathogen V. vulnificus have been received from the University of Florida and will be characterized for possible use as a processing intervention to reduce shellfish-related illness and mortality among consumers. Under objective 2b, initial studies evaluating e-beam technology as a possible processing intervention to reduce or eliminate enteric virus contamination in shellfish have been conducted in collaboration with Texas A&M University. Also under objective 2b, results of a collaborative study with Virginia Tech and Emory University on the effectiveness of high pressure processing to eliminate human noroviruses in shellfish was published. Results showed that pressures higher than those currently applied commercially are needed to eliminate human norovirus in contaminated shellfish. Under objective 3, a paper was submitted and accepted for publication showing that oyster hemocytes (primitive blood cells) are the sites of virus accumulation and persistence in contaminated shellfish. This opens the way for the development of new, simplified and innovative methods to monitor for the presence of viruses in shellfish. In addition, a book chapter on Foodborne Viruses was written and accepted for publication in the American Public Health Association�s Compendium of Methods for the Microbiological Examination of Foods. Together, these efforts are enhancing shellfish safety and supporting the needs of consumers, regulators, and the shellfish industry. Significant Activities that Support Special Target Populations Activities designed to remediate vibrio problems in shellfish hatcheries target both the hatcheries and small shellfish farmers (under $250,000 gross receipts), two important target populations that have been negatively impacted by high shellfish mortalities. Research on the human vibrios (Vibrio parahaemolyticus and vulnificus) targets populations of people who are at increased risk of infection, such as those who have underlying illnesses, including liver disease, diabetes, and weakened immune systems. Research on hepatitis A virus and noroviruses in shellfish target interventions to benefit the consumer, the shellfish industry, and State and Federal regulatory agencies. Accomplishments 01 Sites of virus persistence in shellfish identified. Virus contamination of shellfish has led to frequent outbreaks of hepatitis A and norovirus illness. Once contaminated, shellfish retain high levels of these virus for extended periods. ARS researchers at Dover, DE, identified primitiv blood cells of oysters, known as hemocytes, as the site of virus accumulation and persistence. They devloped simple procedures to extrac and test for viruses within the hemocytes. This is a dramatic improveme over traditional testing methods for viruses in shellfish which rely on tedious dissection and testing of intestinal tissues that contain only a portion of the viral contaminants. Improved assay methods will enhance monitoring efforts and support regulatory agency and industry goals to reduce shellfish-associated enteric virus illness.
Impacts
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Publications
- Leon, J., Kingsley, D.H., Montes, J., Richards, G.P., Lyon, G., Abdulhafid, G., Seitz, S., Fernandez, M., Teunis, P.F., Flick, G.J., Moe, C.L. 2011. Human norovirus inactivation in oysters by high hydrostatic pressure processing: A randomized double-blinded study. Journal of Infectious Diseases. 77(15):5476-82.
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