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CFSAN Regulatory Codes: VI, VII
CFSAN Program Priority Codes: 1.4g
Start Date: 10/1/98     Completion Date: 9/30/02


Statement of Research Problem:
The control of viral and bacterial pathogens in seafood requires knowledge of their acquisition by seafood species and the effect of handling/processing procedures on their densities. Gathering this information requires methodology to detect and enumerate the pathogens. Methodology such as that in the BAM is often inadequate for acquiring large amounts of data needed for risk assessment because those procedures are resource intensive. Additionally, biotechnology has presented an array of new tools (PCR, gene-probes, real time analysis, etc.) that may be adapted into techniques to speed analysis, increase sensitivity and specificity, and lower analytical cost.

New processing technology is continually being developed for seafood and some old processing technologies (depuration of shellfish) remain invalidated. Research is needed to ensure that proper critical limits are identified and critical control points are set for those processes.

Statement of Project Objective(s):
To provide federal and state regulatory/research laboratories with tested methodology for measuring the risk of illness to consumers from pathogens in seafood and the environment, and, where possible, assist in transfer of technology.
To provide to the Office of Seafood with science based information that can be used to insure that industry is identifying critical limits and setting critical control points that afford an appropriate measure of consumer protection.

Anticipated Impact on FDA Regulatory Program:


1. New analytical tools for detecting/measuring viral and bacterial pathogens will be available for regulatory use.
   
2. Critical limits for some seafood processing procedures will be identified.
   
Project Priority Changes During FY2000:
Research was initiated with the Moffett Center to study the effect of hydrostatic pressure processing on vibrio bacteria in oysters. The purpose of the effort is to define critical limits for processing of oysters.

Administrative Liaison(s): George P. Hoskin, 202-418-3172

Research Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
D. W. Cook	OS/DSAT/GCSL	0.5, 0.75, 1.0	1,2
S. A. McCarthy	OS/DSAT/GCSL	1.0, 1.0, 1.0	1,2
DePaola	OS/DSAT/GCSL	0.5, 0.75, 0.75	1
Y. C. Shieh	OS/DSAT/GCSL	1.0, 0.8, 0.8	2
W. Burkhardt	OS/DSAT/GCSL	1.0, 0.8, 1.0	2
K. R. Calci	OS/DSAT/GCSL	1.0, 1.0, 1.0	2,
J. Jones	OS/DSAT/GCSL	1.0, 1.0, 1.0	1,2,
G. Blackstone	OS/DSAT/GCSL	1.0, 1.0, 1.0	1,2,
Technician	OS/DSAT/GCSL	0.0, 1.0, 1.0	1,2
Technician	OS/DSAT/GCSL	0.0, 1.0, 1.0	1
Postdoctoral position	OS/DSAT/GCSL	0.0, 1.0, 1.0	1
Postdoctoral position	OS/DSAT/GCSL	0.0, 1.0, 1.0	1
	Total FTE:	7.0, 11.1, 11.55

Collaborators:

Dr. R. Reddie Moffett Center Shellfish regulatory laboratories in the states of CT, NY, MA, VA, LA and TX Interstate Shellfish Sanitation Conference

This component contains several diverse projects that focus on developing methodology to detect/enumerate viral and bacterial pathogens in seafood and uses that methodology to gather data on occurrence and distribution of pathogens in seafood and their environment. Method development is aimed at improving the speed, accuracy and sensitivity of analysis while reducing labor and material cost. When possible, the GCSL will play an active role in transferring the developed methodology to state and federal regulatory labs.
As described below, 7 deliverables from previous plan have been accomplished and this component contains those not completed or listed as targeted for dates after FY 2000 and new deliverables resulting from the results of the previous plan. This component consolidates the 2 components (bacteria and viruses) from the previous plan into a single component. Deliverables 4, 5, 6, 8, & 9 are new.

Component 1 Objectives:

1. Develop PCR and non-radioactive probe methodology for detection and enumeration of pathogens in seafood.
   
2. Evaluate "real time" analysis approach for studying pathogen and indicator organisms in seafood and marine environment.
   
3. Develop efficient processing procedures to concentrate viruses from shellfish tissue, optimize the overall virus recovery during processing/concentration, effectively remove natural PCR inhibitors, and achieve sensitive and specific detection of NLV and HAV in contaminated shellfish.
   
Component 1 FY 2000 Deliverables:

1. Publish methodology for V. parahaemolyticus AP-probe based on tdh gene.
   
2. Publish PCR technique for detecting tdh gene in enrichments of oyster samples.
   
3. Evaluate enrichment media to enhance recovery of tdh+ V. parahaemolyticus strains for use in combination with PCR.
   
4. Initiate characterization of PCR generated tdh fragments from enrichments using PCR-RFLP to determine strain/species of vibrios responsible for band.
   
5. Continue development of a method for identifying V. parahaemolyticus (strains/clones) using filter bound DNA from colony lifts.
   
6. Research for desired sequence for a V. cholerae probe, have probes synthesized and validate probe.
   
7. Complete optimization of NLV recovery during various processing steps.
   
8. Define a detection sensitivity of NLV in contaminated shellfish.
Component 1 FY 2000 Progress:

1. Completed. Manuscript accepted for publication in Journal of Food Protection. In cooperation with the EPA Gulf of Mexico program and the ISSC, a training video and accompanying manual on direct plating gene probe technique for enumerating V. parahaemolyticus in shellfish has been produced.
   
2. Research completed. A manuscript is in internal review for submission to a peer reviewed journal and manuscript submitted for inclusion in the FDA Laboratory Information Bulletin.
   
3. Literature review completed, media composition selected for testing and test underway. Will be completed this fiscal year.
   
4. PCR-RFLP of the tdh gene did not discriminate V. parahaemolyticus strains. A different approach using group specific PCR of the toxRS gene and a phage insert, which are both specific for the new O3:K6 clone, has been initiated and preliminary results indicate either can distinguish O3:K6 from other V. parahaemolyticus strains. Effort on this deliverable will continue in 2001.
   
5. Amplification of filter bound DNA was demonstrated for tdh and tlh genes. PCR of toxRS and/or phage insert will be used instead of PCR-RFLP to determine if colonies hybridizing with tdh are the O3:K6 clone. Effort on this deliverable will be continued in 2001.
   
6. V. cholerae species specific alkaline phosphatase probe was designed and tested. It appears to be universal for V. cholerae but cross-reacts with V. mimicus and V. metschnikovii. Current field testing suggest V. cholerae levels of <10/g in oysters and no evidence of cross-reaction with other bacteria in oysters. Deliverable on schedule.
   
7. A methodology for extracting viruses from oysters and detecting them by PCR was published. Efforts to improve the method continue using simple eluents (e.g. amino acids) for better elution of viruses during processing steps.
   
8. The NLV detection sensitivity of the method was carried out by surveying NLV in the oysters that were relocated in close proximity to a municipal sewage out-fall. NLV strains (predominant Lorsdale cluster) were detected in 60% of the oysters samples examined during a 9-month study. Current and alternative indicators in water were compared to NLV presence.
Technical Barriers to Meeting Component 1 Objectives or Deliverables:
The NLV recoveries by processing/concentration steps were not done because of a lack of suitable lab space. In an effort to prevent contamination it was decided not to bring NLV from clinical samples into the laboratory where environmental and outbreak samples will be analyzed. The NLV survey was designed as an alternate approach to study method sensitivity.
Component 1 FY 2001 Deliverables:

1. Methodology using PCR of tox RS and/or phage insert to determine presence of O3:K6 strains of V. parahaemolyticus in seafood enrichments that yield PCR bands developing with tdh primers.
   
2. Methodology to identify strains/clones of V. parahaemolyticus using DNA from filter bound colony lifts.
   
3. Methodology based on a species-specific alkaline phosphatase-labeled gene probe to detect Vibrio cholerae.
   
4. An evaluation of the Handheld Nucleic Acid Analyzer (HANAA) unit for the rapid detection of bacterial pathogens in seafood and environmental samples.
   
5. Methodology using ERIC PCR to distinguish V. parahaemolyticus strains.
   
6. Quantitative methodology for Listeria monocytogenes in seafood.
   
7. Completion of report on NLV in oysters relocated to sewage impacted waters.
   
8. A simplified virus detection procedure based on a fluorescent molecular beacon.
   
9. A quantitative PCR method for NLV detection in shellfish.
Component 1 FY 2002 Deliverables:

1. Validation of quantitative methodology for Listeria monocytogenes.
   
2. Develop quantitative PCR method for pathogens and indicator organisms in seawater.
   
3. Further deliverables depend upon needs discovered, if any, from the present studies.

Seafood borne microbial illness usually is caused by pathogens acquired prior to harvest; the agents commonly are pathogens associated with discharges of human waste and naturally occurring marine pathogens. After harvest, the methods by which the seafood is handled and processed can result in an increase or decrease in numbers of these pathogens. This component contains research projects that will lead to a better understanding of how seafood acquires pathogen contamination, the fate of pathogens during various stages of product handling, and the means to reduce or eliminate pathogens from the seafood. Information on the suitability of male-specific bacteriophages as indicators of viruses and virus elimination in shellfish is also being developed. This continues deliverables 1, 2, 3, & 6 from components 1 & 2 of last year's plan. Deliverables 5 & 6 are new.

Component 2 Objectives:


1. Investigate the kinetics of bio-accumulation and depuration of Caliciviruses and indicator organisms in oysters on a seasonal basis, including differentiating microorganism inactivation from elimination.
   
2. Provide baseline data on V. parahaemolyticus densities in shellfish for use in risk assessment.
   
3. Develop data on handling practices and processing techniques that may affect the density of pathogens in shellfish and that can be used to set critical control points/limits.
   
Component 2 Deliverables:

1. Complete the V. parahaemolyticus data collection in collaboration with states.
   
2. Analyze V. parahaemolyticus data from all collaborators and publish.
   
3. Complete studies on the bioaccumulation and depuration of Calicivirus and indicator organisms by oysters.
   
4. Determine if male-specific bacteriophages are suitable bio-markers for depicting the accumulation and elimination of Caliciviruses from Gulf coast oysters
   
5. Determine a critical cooking matrix (time/temperature) to inactivate Caliciviruses in shellfish.
   
6. Evaluate the effect of various processing techniques on pathogenic strains of V. parahaemolyticus in oysters.
Component 2 FY2000 Progress:

1. Data on density total and pathogenic V. parahaemolyticus in shellfish was collected from ~550 shellfish samples. Six Gulf and East Coast states and GCSL contributed to data collection. Data collection will end within the fiscal year.
   
2. Decisions were made with the FDA Division of Mathematics and Statistics on the appropriate analyses of data collected in Deliverable 1. Data analysis and manuscript preparation will be initiated early next fiscal year.
   
3. Twelve bio-accumulation/ depuration trials with Caliciviruses and indicator organisms were completed. Preliminary findings demonstrate that MSB elimination by oysters is significantly and positively correlated to elimination of Calicivirus.
   
4. Have demonstrated that MSB are more refractory to elimination than are Caliciviruses from oysters.
   
5. Determined an acceptable method of heating and monitoring shellstock oysters of various weights to 100°C.
   
6. Data was collected on the survival of V. parahaemolyticus strains in oysters subjected to heat treatment and freezing. Research was initiated on the effect of pressure on the survival of V. vulnificus, V. parahaemolyticus and V. cholerae.
   
Technical Barriers to Meeting Component 2 Objectives or Deliverables:
Deliverable 2 was not completed because some collaborating states initiated data collection later than expected. Progress on deliverable 3 and 5 has been slowed because environmental conditions (high water temperatures) are preventing the oysters from rapidly bioaccumulating microorganisms from seawater.
FY 2001 Deliverables:

1. Determine the rates Calicivirus (SM17) and indicator organisms are eliminated from oysters during depuration on a seasonal basis.
   
2. Determine the rates Hepatitis A virus and indicator organisms are eliminated from oysters during depuration.
   
3. Determine if male-specific bacteriophage are suitable indicators to predict the rate of Caliciviruses from oysters.
   
4. Determine the impact high water temperatures have on the survival of Caliciviruses and male-specific bacteriophage in oysters and seawater, and the to determine the effects of seasonal changes on the ability of oysters to bioaccumulate these microorganisms.
   
5. An evaluation of the effect of hydrostatic pressure treatment on Calicviruses.
   
6. Establish critical limits (pressure/time) for the destruction of vibrio species in oysters undergoing hydrostatic pressure treatment.
   
7. Evaluate health risks arising from hydrostatic pressure treatment to remove vibrio bacteria from oysters.
   
FY 2002 Deliverables:

1. Determination if male-specific bacteriophage are suitable indicators microorganisms in predicting the presence of enteric viruses in naturally contaminated oysters from the Gulf coast (qualitatively and quantitatively).
   

Shieh, Y.-S. Carol, S. S. Monroe, R. L. Fankhauser, G. W. Langlois, W. Burkhardt III, and R. S. Baric. 2000. Detection of Norwalk-like virus in shellfish implicated in illness. Journal of Infectious Disease 181(suppl 2):S360-6.


Matsumoto, C., J. Okuda, M. Ishibashi, M. Iwanaga, P. Garg, T.V. Rammamurthy, H. Wong, A. DePaola, Y.B. Kim, M.J. Albert, and M. Nishibuchi. 2000. Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analysis. J. Clin Microbiol. 38:578-585.

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Hypertext updated by dav 2001-OCT-02