Progress 09/01/08 to 08/31/11
Outputs
OUTPUTS: We accomplished the following outputs during a 3 year project. As we stated in our objectives; we determined the timeline for viral persistence in hemocytes vs. whole body levels; studied hemocyte subtypes associated with virus; followed the fate and persistence of viruses in hemocytes transferred from exposed to naive oysters; determined if low pH virus tolerance correlates with total oyster and hemocyte persistence; visualized virus localization patterns within phagocytic hemocytes with emphasis on the phagolysosomes; and conducted experiments on inhibiting phagocytic activities of hemocytes and effects of immunomodulation on virus persistence in hemocytes. A major intended benefit of this study was to develop an in-depth mechanistic understanding of virus retention in the oyster. The role of hemocytes in uptake and retention of NV and HAV, were determined and quantified. To a lesser extent Norovirus surrogates were employed, such as feline calcivirus (FCV), murine norovirus (MNV), and some less common human picornavirus such as parecho-, echo- and coxsackie viruses, for experiments comparing the acid tolerance and virus persistence. State-of-the-art methodology were employed (image analysis, RT-PCR, fluorescent probes, etc.) to allow us for the detailed analysis of the problem. Unfortunately, flow cytometry method did not provide sufficient sample volume during the sample sorting process and samples processed for virus detection was not possible in oyster hemocytes after the sorting. We employed molecular methods to monitor virus detection and persistence in oyster hemocytes. The overall goal was to determine and quantify mechanisms that control uptake and persistence of pathogenic viruses in shellfish, concentrating on the roles of blood cells (hemocytes) in these processes. The roles of hemocytes in virus persistence in the oyster included a timeline of raw tissue and hemocyte virus uptake, followed by the fate and persistence of viruses in hemocytes transferred from exposed to naive oysters. Hemocyte inactivation and sub hemocyte population studies added to the hypothesis of virion uptake and accumulation. Understanding how these human pathogenic viruses persist so efficiently in shellfish should facilitate development of intervention strategies designed to purge or eliminate the viruses from shellfish tissue. We strongly believe that the information obtained in this study will make a significant contribution toward the existing body of knowledge on shellfish physiology, immunology, and virology. This study does not only provide mechanistic understanding of virus retention, but collectively the information obtained from this basic research should aid in the development of practical measures useful in reducing the uptake and/or retention of human pathogenic viruses by edible shellfish. PARTICIPANTS: In summary, funding for this project provided stipends for students, travel support to attend national and regional conference to share research results, and supplies and equipment necessary to conduct the project. It has enhanced partnership between the project partners and advanced our understanding of virus retention mechanism in oyster hemocytes. One full and one part-time graduate students were involved in the project. Full-time graduate student, Ms. Keleigh Provost completed her research on this research topic in 2009 and entered the work force in the research institution in Florida. Active research partners for this project were UMD-CBL and USDA-ARS Lab in Delaware. Because of the successful partnership initiated with this program, the Project Director, Dr. Ozbay collaborated on another USDA-AFRI funded project as a Co-PI with the University Delaware PI, Dr. Haiqiang Cheng, for five million dollars food safety project with over 12 different partners and collaborators from various labs and institutions. Because this program increased her visibility in the seafood safety program, Dr. Ozbay was able to successfully secure funding from the USDA-FSIS to establish the Seafood Safety Lab in Delaware State University, College of Agriculture & Related Sciences to conduct the baseline monitoring of domestic and imported retail catfish for chemical and microbiological contaminants. TARGET AUDIENCES: Target audiences in this project were shellfish consumers, shellfish aquaculture farmers, food safety extension agents and policy makers. There has been an increasing concern both nationally and internationally on the shellfish seafood safety. Information gathered in this project will provide some new information and first time details on the hemocytes function for virus retention. PI, Dr. Ozbay and Co-PIs, Drs. Kingsley and Anderson observed some interest from audiences during her international conference and seminars she attended. We believe that this project targets all shellfish consumers, educators, extension agents, both state and federal government scientists and policy makers. We prepared a brief flyer during the first two years of this project that was given the visitors during their visits to Dr. Ozbay's and USDA-ARS Labs. Presentations given to the students during their class and seminar have increased some awareness among the students in the college. Dr. Ozbay also shared project information with the community volunteers involved in shellfish aquaculture for restoration and oyster revitalization program via Oyster Gardening Program Delaware State University has partnered with the Center for the Inland Bays and the University of Delaware Sea Grant Program. PROJECT MODIFICATIONS: Unfortunately, flow cytometry method did not provide sufficient sample volume during the sample sorting process and samples processed for virus detection was not possible in oyster hemocytes after the sorting. We employed molecular methods to monitor virus detection and persistence in oyster hemocytes.
Impacts
This collaborative research program allowed comprehensive investigation of the oyster hemocytes for virus retention and increase defense and immune response for the virus with various forms of b-glucans compounds. This partnership also allowed the students involved to obtain extensive hands on training and be better familiar with the molecular methods and equipment. Project director had an opportunity to meet and collaborate with other peers because of her experience with this project and opportunities provided with the funding for this project. Our research results demonstrated an association of virus contamination with hemocytes but not with hemolymph. Live oysters contaminated overnight with hepatitis A virus (HAV) and murine norovirus (MNV) had 56% and 80% of extractable virus associated with hemocytes, respectively. Transfer of HAV-contaminated hemocytes to naive (virus-free) oysters resulted in naive oyster meat testing HAV positive for up to 3 weeks. Acid tolerance of HAV, MNV, poliovirus (PV), and feline calicivirus (FCV) correlated with the ability of each virus to persist within oysters. HAV persisted the longest (>21 days) and was most acid resistant, MNV and PV were less tolerant of acidic pH, persisting for up to 12 days and 1 day, respectively, and FCV did not persist (<1 day) within oysters and was not acid tolerant. This suggests that the ability of a virus to tolerate the acidic conditions typical of phagolysosomal vesicles within hemocytes plays a role in determining virus persistence in shellfish. We found that viruses were extracted more expediently from hemocytes than from whole shellfish tissues and gave similar RT-PCR detection sensitivities. In addition, to gain insight to the effects of these b-glucan compounds in molluscs to enhance disease resistance via hemocyte activation, changes in circulating oyster hemocyte numbers, population profiles, and adhesion were quantified after injection of b-1,3-glucans. These agents included zymosan A or MacroGard, an immune stimulant suggested for aquaculture applications. Results were compared with those from estuarine water-injected control oysters at daily intervals for 3 days. MacroGard produced a rapid (by 1 day) elevation of total circulating hemocytes, which remained significant for 3 days; zymosan A stimulation of this response was not statistically significant. MacroGard administration resulted in a sustained increase in percent granulocytes in circulation. Zymosan A also produced an increment in the numbers of granulocytes that became significant by 3 days. Granulocytes are considered to be the most immunologically active hemocyte subclass. Both b-glucan preparations promoted hemocyte aggregation at days 1-3 after injection. Analysis of avidity of hemocyte aggregation using an index of clump cellular density confirmed this observation. Hemocytic aggregation is thought to be an indicator of cell activation. These results suggest that b-glucans can act to increase the numbers of circulating hemocytes available for defense responses, to promote long term changes in the hemocyte profile favoring immunologically active cells, and to enhance hemocytic aggregation.
Publications
- Ozbay, G., Kingsley, D., Provost, K. and Anderson, R. (2010). The Retention of Enteric Viruses by the Hemocytes of the Eastern Oyster (Crassostrea virginica). International Food Technology (IFT) Meeting and EXPO. July 18-21, 2010. Chicago, Illinois, USA. Poster Presentation.
- Research results were published in two peer reviewed journal articles in year 2011.
- Provost, K., Dancho, B. A., Ozbay, G., Anderson, R.S., Richards, G. and Kingsley, G.H.(2011). Hemocytes are a Site of Persistence for Virus-Contaminated Oysters. Applied and Environmental Microbiology. (ARIS# 261982). Doi 10.1128/AEM.06887-11.
- Anderson, R.S., Ozbay, G., Kingsley, D.H. and Strauss, M.A. (2011). Oyster hemocyte mobilization and increased adhesion activity after -glucan administration (ARIS #271596). Journal of Shellfish Research 30(3):1-7.
- In addition to the journal publications, following publications were made on this project (2008-2011).
- Graduate Student Thesis, The Retention of Human Viruses by the Hemocytes of the Eastern Oyster, Crassostrea virginica.(2009). Masters Thesis, pp. 72, Natural Resources, Department of Agriculture and Natural Resources, Delaware State University, Dover, DE, USA.
- Outreach White Paper, Provost, K. and Ozbay, G. (2008). Human Virus Retention in Eastern Oyster (Crassostrea virginica) Endocytes. Department of Agriculture and Natural Resources, Delaware State University.
- Abstracts & Presentations, Anderson, R.S., Ozbay, G., Kingsley, D.H. and Strauss, M.A. (2011). Activation of eastern oyster (Crassostea virginica) hemocytes following administration of β-glucans. Society for Invertebrate Pathology, 2011. International Congress on Invertebrate Pathology and Microbial Control & 44th Annual Meeting of the Society for Invertebrate Pathology, Halifax, Nova Scotia, CANADA, August 7-11, 2011. Oral Presentation.
- Kingsley, D.H., Provost, K., Dancho, B. and Ozbay, G. (2011). New perspectives on virus detection in shellfish: Hemocytes as a source of concentrated virus. The 8th International Molluscan Seafood Safety Workshop in Charlottetown, Prince Edward Island, CANADA, June 12-17, 2011. Oral Presentation.
- Ozbay, G., Lambert, M., Fay, J., Coyne, K., Reining, B. and Pappas, A. October (2010). Integrated Research Efforts in Environmental Monitoring, Food Safety and Genetics. October 13, 2010, Istanbul Technical University, Istanbul, Turkey. Invited Seminar.
- Ozbay, G., Kingsley, D., Provost, K. and Anderson, R. (2010). The Retention of Enteric Viruses by the Hemocytes of the Eastern Oyster (Crassostrea virginica). USDA-AFRI Project Directors Meeting. July 17-18, 2010. Chicago, Illinois, USA. Poster Presentation.
- Provost, K. and Ozbay, G. 2008. Retention Properties of the Oyster (Crassostrea virginica) Hemocytes for Human Enteric Viruses. April 6-13, 2008. 100th National Shellfisheries Association Biennial Meeting, Providence, RI. Oral Presentation.
- Provost, K. and Ozbay, G. (2008). Retention Properties of the Oyster (Crassostrea virginica) Hemocytes for Human Enteric Viruses. April 8, 2008. Northeast Shellfish Sanitation Association, Special Session, Providence, RI Oral Presentation.
- Provost, K. (2009). The Retention of Human Pathogens by the Hemocytes of the Eastern Oyster (Crassostrea virginica), Graduate Thesis Defense, Delaware State University, Natural Resources Program, Thursday, May 21, 2009, Agricultural Annex Building, Dover, Delaware. Thesis Defense.
- Provost, K., Kingsley, D., and Ozbay, G. (2008). Persistence of Viruses in Oyster (Crassostrea virginica) Hemocytes. AEA/ARD Land-Grant Conference, June 9-12, 2008, Memphis, TN. Oral Presentation.
- Provost, K. (2008). Delaware State University, 2008 Natural Resources Program Graduate Seminar, Dover, DE.
- Awards; Provost, K. (2008). Selected Abstract for Presentation Award. Persistence of Viruses in Oyster (Crassostrea virginica) Hemocytes, AEA/ARD Land-Grant Conference, June 9-12, 2008, Memphis, TN, USA.
- Ozbay, G. (2008). Selected Abstract for Presentation. Food Security and Agrisecurity: Tracking of Food Distribution by the Use of Geographic Information System and Global Information Systems, AEA/ARD Land-Grant Conference, June 9-12, 2008, Memphis, TN, USA.
- Newsletter Articles; Delaware State University Aquatic Sciences Quarterly Newsletter. (2008). Fall 2008: Graduate Student Keleigh Provost Studies Shellfish Disease.
- Delaware State University Aquatic Sciences Quarterly Newsletter. (2009). Summer 2009: Students Graduated in Aquatic Sciences Program: Keleigh Provost, Bellamy Reynolds, Alicia Revis-Mangum and Frank Marenghi. Outreach Events; Delaware Coast Day. (2008). University of Delaware, Lewes Campus, Delaware, Sunday, October 5, 2008.
- UDEL Faculty, Dr. Sharon Tucker and her Science Teacher Students Visit. (2008). Delaware State University, Aquatic Sciences Program, Dover, Delaware, September 26, 2008.
- East Fishermen Association Expo. (2008). Maryland Watermen Association, Annual East Coast Commercial Fishermen and Aquaculture Trade Show, Ocean City, Maryland, January 23-25, 2009.
- Delaware State University 3rd Annual Fishing Derby. (2008). Smyrna, Delaware,June 18, 2008.
|
Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Shellfish are an important vector for transmission of enteric pathogens. Interventions, such as depuration, do not adequately clear oysters of virus, while fecal bacteria levels are significantly reduced. Why viruses are retained in the bivalve flesh is not well understood. We hypothesize that phagocytic cells of the oyster (hemocytes) play an important role for the retention of virus particles within bivalves. The project focus was to gain a better understanding of the oyster's hemocytes as they relate to the persistence issue. Specifically, are viruses associated with the hemocytes; is there a correlation between virus pH tolerance and the persistence time in the oyster tissue; can transferred hemocytes test positive for virus for an extended period of time; if we inhibit phagocytic function of hemocytes, will there be a reduced uptake of virus First, hemocytes were separated from the hemolymph and results indicate that hepatitis A virus (HAV) is hemocyte cell-associated and the persistence times of hemocytes in oyster correlates with the presence of HAV within hemocytes. Second, a transfer of HAV-contaminated hemocytes to virus-free oysters indicated that the contaminated hemocytes both survived the transfer and retained HAV for up to 2 weeks. Since the process of phagocytized virus would likely result in sequestration of viruses within the phagolysome of the hemocyte, acidic pH tolerance of several viruses was evaluated to see if this property relates to the ability of the virus to persist within oysters. We find viability within increasingly acidic buffer to have the same order as acid tolerance, with HAV persisting the longest in shellfish tissues and having limited titer reduction after exposure to pH 2, while FCV does not readily persist within bivalves or tolerate low pH exposure. MNV and PV both display intermediate persistence times and intermediate tolerances to low pH buffer. Antiviral activity of hemocytes was evaluated. No significant antiviral activity was seen after incubation of MNV with whole hemolymph, hemocytes or plasma from untreated oysters; however, these components all have strong antibacterial activity. Overall these studies suggest that hemocytes play an important role in the retention of human viruses in oyster tissues. Outcome of this research project has been presented during the regional, national, and international conferences and seminar provided in the publication section of this report. Studies are under way to see if treatment with a beta-glucan immunostimulant commonly used in aquaculture will affect viral retention in oysters. This study focuses on inhibiting and stimulating phagocytic activities of hemocytes and evaluation effects on virus persistence in hemocytes. We attempt experimentally block the uptake of virus by temporal injection of silica particles (before and after virus contamination) into the open circulatory system of the oyster. While this would probably not be directly useful as a practical intervention, this may prevent dissemination of the virus throughout the shellfish or prevent long-term persistence of the virus, supporting the hemocyte retention hypothesis. PARTICIPANTS: This project has been conducted in collaborations with the USDA-ARS Microbial Safety of Aquaculture Products Center of Excellence and University of Maryland, Chesapeake Biological Laboratory. Ms. Keleigh Provost was a Graduate Student Researcher to assist with the project. Ms. Provost completed her M.S. Degree in May 2009 and following is the citation of her thesis. Provost, K. 2009. The Retention of Human Viruses by the Hemocytes of the Eastern Oyster, Crassostrea virginica. Masters Thesis, pp. 72, Natural Resources, Department of Agriculture and Natural Resources, Delaware State University, Dover, DE, USA. Mr. Brian Reckenbeil is a current Graduate Student Researcher assisting Dr. Ozbay with flow cytometer testing of oyster hemocytes. Dr. David Kingsley is the collaborating USDA-ARS scientist specializing in Virology and also was Co-Advisor to Ms. Provost. Dr. Robert Anderson, Co-PI for the project from the University of Maryland, Chesapeake Biological Laboratory and expert in Immunology. He is assisting on the last outlined objective of this project to investigate the effects of oyster immunomodulation on viral retention. TARGET AUDIENCES: Target audiences in this project are shellfish consumers, shellfish aquaculture farmers, food safety extension agents and policy makers. There has been an increasing concern both nationally and internationally on the shellfish seafood safety. Information gathered in this project will provide some new information and first time details on the hemocytes function for virus retention. PI, Dr. Ozbay noticed interest from audiences during her international conference and seminars she attended. We believe that this project targets all shellfish consumers, educators, extension agents, both state and federal government scientists and policy makers. We prepared a brief flyer during the first year of this project that was given the visitors during their visits to Dr. Ozbay's and USDA-ARS Labs. Presentations given to the students during their class and seminar have increased some awareness among the students in the college. Dr. Ozbay also shared project information with the Community Volunteers involved in shellfish aquaculture for restoration and oyster revitalization program via Oyster Gardening Program initiated in 2003. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Major impacts include increase our level of understanding with roles of hemocytes, virus introduction and retention. In this project, we demonstrate that viruses are located within blood cells of oysters and can remain within these cells for several weeks. Comparing several viral species, it is noted that the more acid-resistant the virus, the more persistent it appears to be within oyster tissues. Overall these studies suggest that hemocytes play an important role in the retention of human viruses in oyster tissues. While this research publication is primarily of a basic nature, understanding how and why food-borne viruses can stay sequestered within oysters may potentially lead to alternate intervention strategies for shellfish. Following specific tasks are completed and the manuscript article is prepared for the journal submission. We determined the timeline for viral persistence in hemocytes versus whole body levels and results are discussed in the manuscript. Although we identified hemocytes subtypes using Flow Cytometry, their association with virus was very difficult to obtain due to low detectable concentration of the virus in oyster hemocytes and dilution effects of the FACS Buffer used during sorting. We also determined the correlation of low pH tolerance with total oyster and hemocyte persistence. We studied the fate and persistence of viruses in hemocytes transferred from exposed to naive oysters and visualized virus localization patterns within phagocytic hemocytes with emphasis on the phagolysosomes using the high performance microscope systems. Following results summarize our findings as stated in specific tasks above: Hepatitis A virus (HAV) was found to be associated with hemocytes harvested and separated from the hemolymph of HAV-exposed oysters. Transfer of hemocytes from HAV-contaminated oysters to naive virus-free oysters results in the meat of the naive oyster testing HAV-positive for up to three weeks. Evaluation of the temporal persistence of HAV, murine norovirus (MNV) and poliovirus (PV) within hemocytes correlates with the presence of these viruses in oyster meat. Since phagocytosis would likely result in sequestration of virions within the acidic phagolysosome of the hemocyte, acidic pH tolerance of several viruses (HAV, MNV, PV, and feline calcivirus (FCV)) was evaluated to see if acid tolerance correlates with the virus' ability to persist within oysters. The degree of viral of persistence within oysters and their ability to survive low pH were similar: HAV persisted the longest and was most acid resistant; MNV and PV were less tolerant of acidic pH and persisted to a lesser degree than HAV while FCV did not persist within oysters and is not acid tolerant.
Publications
- Provost, K., Ozbay, G., Anderson, R.S., Richards, G.P. and Kingsley, D.H. 2010. Hemocytes as Sites of Persistence for Virus-Contaminated Oysters. Applied and Environmental Microbiology Journal. In Review.
- Ozbay, G., Lambert, M., Fay, J., Coyne, K., Reining, B. and Pappas, A. October 2010. Integrated Research in Environmental Monitoring, Food Safety and Genetics. October 13, 2010, Istanbul Technical University, Istanbul, Turkey. Invited Seminar.
- Ozbay, G., Kingsley, D., Provost, K. and Anderson, R. 2010. Retention of Enteric Viruses by the Hemocytes of the Eastern Oyster (Crassostrea virginica). USDA-AFRI Project Directors Meeting. July 17-18, 2010. Chicago, Illinois, USA. Poster Presentation.
- Ozbay, G., Kingsley, D., Provost, K. and Anderson, R. 2010. Retention of Enteric Viruses by the Hemocytes of the Eastern Oyster (Crassostrea virginica). International Food Technology (IFT) Meeting and EXPO. July 18-21, 2010. Chicago, Illinois, USA. Poster Presentation.
|
Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: During this first year, graduate student, Ms. Keleigh Provost was hired to assist with few of the proposed objectives in this project. Keleigh as a second year graduate student involved in the following objectives/goals of this project and completed her thesis during the summer of 2009. Overall objective of this project was to gain a better understanding of the oyster's hemocytes as they relate to the persistence issue. Her thesis focused on the following questions: Are viruses associated with the hemocytes; Is there a correlation between virus pH tolerance and the persistence time in the oyster tissue; Can transferred hemocytes test positive for virus for an extended period of time; If we inhibit phagocytic function of hemocytes, will there be a reduced uptake of virus Keleigh presented her research findings at both national and regional conferences and disseminated a white paper as an extension publication to share during the outreach events and visitors attending on-campus events in DSU along with Dr. Ozbay as PD. Keleigh defended her thesis on May 21, 2009. She also prepared a brief article about her project in the quarterly newsletter article published in Aquatic Sciences Program in Delaware State University and prepared a white paper to share her research outcomes. She participated at a number of outreach events including Delaware Coast Day, DSU Cooperative Extension Fishing Derby, East Fisherman's Association Expo and many others. Keleigh also volunteered to maintain phytoplankton culture laboratory to help Dr. Ozbay with upcoming research project dealing with the bacteria levels in oyster tissues in relation to water quality. In addition, this grant program provided baseline information and support for other student projects and enhance Dr. Ozbay's research capability that helped her obtaining funding from Delaware EPSCOR Program and DSU Professional Development Funding. Students involved are Ms. Keleigh Provost (received Master of Sciences) and Mr. Brian Reckenbeil (new graduate student in Natural Resources Program). Funding has provided stipends for students, travel support to attend national and regional conference to share research results, and supplies and equipment necessary to conduct the project. It has enhanced partnership between the project partners and advanced our understanding of virus retention mechanism in oyster hemocytes. PARTICIPANTS: Graduate students involved are Ms. Keleigh Provost (received Master of Sciences) and Mr. Brian Reckenbeil (new graduate student in Natural Resources Program). Funding has provided stipends for students, travel support to attend national and regional conference to share research results, and supplies and equipment necessary to conduct the project. It has enhanced partnership between the project partners and advanced our understanding of virus retention mechanism in oyster hemocytes. Dr. Robert Anderson as a Co-PI has been working on the proposed objectives: Inhibiting phagocytic activities of hemocytes and effects of immunomodulation on virus persistence in hemocytes and visualize virus localization patterns within phagocytic hemocytes with emphasis on the phagolysosomes. Dr. David Kingsley had trained and co-advised Ms. Keleigh Provost to complete her thesis research. Her thesis focused on the following questions: Are viruses associated with the hemocytes; Is there a correlation between virus pH tolerance and the persistence time in the oyster tissue; Can transferred hemocytes test positive for virus for an extended period of time; If we inhibit phagocytic function of hemocytes, will there be a reduced uptake of virus Dr. Gulnihal Ozbay trained Keleigh with flow cytometry operation for identification of hemocyte subtypes associated with virus. Research team continue working on the proposed objectives and is currently working on an article for jornal submission. TARGET AUDIENCES: Common measures for intervention like depuration and relaying, as well as testing shellfish water quality using coliform standards, have not been completely successful in eradicating virus transmission after consumption. This research identified hemocytes as a likely primary source by which viruses enter and are retained within the oyster's tissues, thus making purification of oysters by the commercial process of depuration or relaying unacceptable as a remediation for virus-contaminated shellfish. This information should prove valuable to the shellfish industry and regulators as they continue to investigate options to enhance shellfish safety. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Virus accumulation and persistence in bivalve mollusks has long been documented in the United States and throughout the world. Shellfish consumption is a particularly significant mode of transmission for enteric pathogens. Harvesting of shellfish is approved and regulated by the use of fecal coliform standards to monitor the harvesting areas; however, this screening process does not adequately correlate with the presence of virus in oyster tissue. Pathways for virus retention in oyster flesh are unclear. The phagocytic cells of the oyster, the hemocytes, are proposed here to play an important role for the retention of virus particles within bivalves. Common measures for intervention like depuration and relaying, as well as testing shellfish water quality using coliform standards, have not been completely successful in eradicating virus transmission after consumption. Viruses are concentrated by the filtering process of oysters into the gut and digestive glands of the oysters and hemocytes appear to transport the viruses into other tissues. Digestive tissues have been studied in regards to virus binding; however, the involvement of hemocytes has not been directly demonstrated. Three experiments were undertaken in an effort to present a multifaceted approach to the question of hemocyte role in virus retention. First, a virus pH tolerance test using plaque assays determined the sensitivity of hepatitis A virus (HAV), poliovirus, murine norovirus, and feline calicivirus, to acidic pH. We expect viruses with low pH tolerance to have a shorter retention time. Depuration does not adequately clear oysters of virus, while bacterial levels are reduced in the same amount of time. It may be that viruses are tolerant of the low pH encountered when in contact with the hemocyte, while bacteria are not. Trends indicated that HAV was least sensitive to low pH, and was significantly different from the other viruses. A kinetics study preformed in tandem with the pH tolerance study indicated that HAV had the longest retention time in the oyster flesh. Hemocytes were separated from the hemolymph and results indicate that HAV is hemocyte cell-associated. Second, an adoptive transfer of HAV contaminated hemocytes indicated that the contaminated hemocytes both survived the transfer and retained HAV for up to 2 weeks, indicating that viruses are withheld in the hemocytes for at least 2 weeks. Third, a phagocytic blocking compound was utilized to describe both the dissemination of virus through the oyster tissue and the feasibility of using silica to disrupt the virus retention cycle by blocking phagocytosis. Results suggest that silica is a feasible blocking compound and hemocytes are a likely area of persistence for human pathogens. This research identified hemocytes as a likely primary source by which viruses enter and are retained within the oyster's tissues, thus making purification of oysters by the commercial process of depuration or relaying unacceptable as a remediation for virus-contaminated shellfish. This information should prove valuable to the shellfish industry and regulators as they continue to investigate options to enhance shellfish safety.
Publications
- Delaware State University 3rd Annual Fishing Derby, June 18, 2008. Smyrna, Delaware.
- Provost, K. 2009. The Retention of Human Pathogens by the Hemocytes of the Eastern Oyster (Crassostrea virginica), Graduate Thesis Defense, Delaware State University, Natural Resources Program, Thursday, May 21, 2009, Agricultural Annex Building, Dover, Delaware. Thesis Defense.
- Provost, K., Kingsley, D., and Ozbay, G. 2008. Persistence of Viruses in Oyster (Crassostrea virginica) Hemocytes. AEA/ARD Land-Grant Conference, June 9-12, 2008, Memphis, TN. Oral Presentation.
- Provost, K. and Ozbay, G. 2008. Retention Properties of the Oyster (Crassostrea virginica) Hemocytes for Human Enteric Viruses. April 6-13, 2008. 100th National Shellfisheries Association Biennial Meeting, Providence, RI. Oral Presentation.
- Reynolds, B., Provost, K. and Ozbay, G. 2008. Delaware Bay Stock Assessment of Eastern Oyster (Crassostrea virginica) and Juvenile Recruitment Using Dredge Calibration. American Fisheries Society, Mid-Atlantic Chapter, Fall Meeting, November 13, 2008, Adventure Aquarium, Camden, NJ. Oral Presentation.
- Reynolds, B., Provost, K., and Ozbay, G. 2008. Quantitative Stock Assessment of Oyster Biomass in Delaware Commercial Oyster Beds. April 6-13, 2008. 100th National Shellfisheries Association Biennial Meeting, Providence, RI. Poster Presentation.
- Provost, K. and Ozbay, April 2008. Retention Properties of the Oyster (Crassostrea virginica) Hemocytes for Human Enteric Viruses. April 8, 2008. Northeast Shellfish Sanitation Association, Special Session, Providence, RI Oral Presentation.
- Provost, K. 2008. Delaware State University, 2008 Natural Resources Program Graduate Seminars.
- Provost, K. 2008. Selected Abstract for Presentation Award. Persistence of Viruses in Oyster (Crassostrea virginica) Hemocytes, AEA/ARD Land-Grant Conference, June 9-12, 2008, Memphis, TN, USA.
- Ozbay, G. 2008. Selected Abstract for Presentation Award. Food Security and Agrisecurity: Tracking of Food Distribution by the Use of Geographic Information System and Global Information Systems, AEA/ARD Land-Grant Conference, June 9-12, 2008, Memphis, TN, USA.
- Delaware State University Aquatic Sciences Quarterly Newsletter. 2008. Fall 2008: Graduate Student Keleigh Provost Studies Shellfish Disease.
- Delaware State University Aquatic Sciences Quarterly Newsletter. 2009. Summer 2009: Students Graduated in Aquatic Sciences Program: Keleigh Provost, Bellamy Reynolds, Alicia Revis-Mangum and Frank Marenghi.
- Provost, K. and Ozbay, G. 2008. Human Virus Retention in Eastern Oyster (Crassostrea virginica) Endocytes. Department of Agriculture and Natural Resources, Delaware State University.
- Delaware Coast Day, Sunday, October 5, 2008. University of Delaware, Lewes Campus, Delaware.
- UDEL Faculty, Dr. Sharon Tucker and her Science Teacher Students Visit, September 26, 2008. Delaware State University, Aquatic Sciences Program, Dover, Delaware.
- East Fishermen Association Expo 2008. Maryland Watermen Association, Annual East Coast Commercial Fishermen and Aquaculture Trade Show, January 23-25, 2009, Ocean City, Maryland.
|
|