U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition

Three Year Research Plan
National Food Safety Initiative
Produce and Imported Foods Safety Initiative
1999-2001 Update
August 1999

FSI Research Projects

Executive Summary | Table of Contents | Research Projects | Appendices


Legend | Abbreviations

FSI Project Number 01 RSVP Number 38017 GPRA Goal II.B.

Project Title

 Detection and quantitation of pathogens
Project Personnel  

Name

 Office/Division FTE [ 99,00,01] Component
W.A. Andrews OSRS/DMS 0.5 1
R.M. Amaguana OSRS/DMS 1.0 1
T. Hammack OSRS/DMS 1.0 1
N. Belay OSRS/DMS 1.0 2
A. Rasooly OSRS/DMS 1.0 5
R.H. Hall OPDFB/DVA 1.0 3
S. Lavu OCAC/DSAT 0.3, 1.0, 1.0 3
B. Goswami OPA/DMBRE 1.0 6
M.L. Tortorello OPDFB/DFPP 1.0 4
T. Fu OPDFB/DFPP 1.0 4
D. Stewart OPDFB/DFPP 1.0 4
TOTAL FTE   9.8, 10.5, 10.5

 

 

  

 
ORA Detailee (Hepatitis A detection in cilantro)   30 days  
OSN Personnel OSN/DSAT Management 2
Proposed but unfunded positions- support scientists OPA/DMBRE

OSRS/DMS

 [1.0 ] Support Scientist

[1.0 ] Support Scientist

  

6

5
Collaborators: Univ. Md.

Chinese Academy of Preventive Medicine

Bose Institute, India

Univ. of Washington

 3
CBER

 

 

 5
Administrative Liaison D.B. Shah, 202-205-4981

D. Danford, 202-205-5365
Project Abstract  Development of rapid detection and quantification methods for pathogens and their toxins, with special emphasis on certain imported and domestic perishable foods, that sporadically contain low levels of pathogen contamination.
Project Description  An essential component of a comprehensive strategy to enhance food safety is the development of an arsenal of rapid and sensitive methods for detecting pathogens or their toxic metabolites. Many perishable products, whether imported or domestic, do not undergo additional processing to inactivate harmful contaminants prior to consumption. Contamination may only occur sporadically and at low levels; but, in some products such as sprouts, low levels of pathogens in seeds also may be amplified during germination. High levels of background microflora often hamper detection of pathogens in produce. Food matrix interference is often encountered with all food testing methods; hence, gene based assays seem especially susceptible, i.e., RT-PCR for detection of Hepatitis A virus in produce.

The tasks listed in this project will provide new or refined methods for the detection and quantification of pathogens or their toxic metabolites. Real-time detection methods developed in this project may be useful for verification of critical control points, thus enhancing HACCP programs.
Projected Impact 1. Data on the presence of pathogens can enhance the development of agency policy on reducing the risk of illness from sprout/produce.

2. Methods developed will enhance the capability of the field labs in detecting pathogens in foods.

3. Methods developed will enhance microbiological safety of infant formulas

4. Promote international harmonization of microbiological methods used in food testing.

Center Priorities Code  1.3b, 1.4a, 1.4c
Research Regulatory Needs Codes 1A, 1C, 1D, IIA, IIC, IXB, IXE.
Component 1 A. Develop methods to detect low levels of Salmonella in fruits and fruit juices.

B. Collaborative study on the efficiency of selective media used for the recovery of Salmonella.
Description A. Preliminary evidence suggests that some fresh fruits may contain one or more substances that interfere with the detection of Salmonella. This work will assess the effectiveness of current methods and, if needed, implement modifications to detect low levels of Salmonella, including S. typhi and S. paratyphi in orange juice, apple juice and cider, mamey and selected high risk produce.

B. Complete the collaborative study on the effectiveness of selective media used for the recovery of Salmonella from foods with low microbial load and the study on the evaluation of the Universal Pre-enrichment medium for the detection of Salmonella in dairy foods. These data will contribute to the harmonization of reference methods to accelerate acceptance of imports and improve analytical capabilities of the field labs.
Deliverables 

FY1999

FY2000

FY2001
  1. Develop effectiveness of existing method for the recovery of Salmonella from mamay; improve method where possible.(A)
  2. Complete collaborative study, provide recommendation to the field, and prepare manuscript detailing results of the study.(B)
  1. Develop sensitive method for detecting Salmonella in juices.(A)
  1. Refine/develop methods for the isolation of S. typhi and paratyphi from selected fruits, juices, or produce.(A)
Component 2 B. cereus emetic toxin: development of a real-time detection method and evaluation of conditions for toxin production in infant formulas and medical foods.
Description  The standard method for detecting emetic toxin is monkey or kitten feeding assay, which is cumbersome and semi-quantitative. An in vitro quantitative emetic toxin assay has recently been developed in our lab; we are currently exploring the possibility of reformatting this assay into an electrode sensor method for real-time detection of toxin. These assays will be used to evaluate:

(1) the health significance of low levels of B. cereus in infant formulas and medical foods,

(2) the potential for low level contamination of emetic toxin in ingredients of formulas and medical foods, and

(3) the potential for maltodextrin to induce toxin formation when used as a formula ingredient.

Deliverables

FY1999

FY2000

FY2001
  1. Complete work on ionophore-based assay for emetic toxin and publish results.
  2. Validate use of this assay for testing infant formulas.
  1. Develop electrode sensor, real-time detection method for emetic toxin.
  2. Complete evaluation on the potential of maltodextrin to augment toxin production by B.cereus.
  1. Validate electrode sensor methodology in simulated processing environment
Component 3 Development of pathogen detection systems utilizing the ELISA cascade amplification system and a proprietary nucleic acid amplification system
Description These methods exhibit enhanced sensitivity over currently available rapid tests and overcome detection difficulties associated withfood samples. They are suitable both for low-level pathogen and toxin detection. This work will also identify likely candidates among virulence determinants of emerging or newly recognized pathogens for use as targets in these new assay systems.
Deliverables

 FY1999

FY2000

FY2001
  1. Evaluate ELISA cascade amplification assay for the detection of Staphylococcal enterotoxins.
  2. Evaluate proprietary nucleic acid amplification and detection assay for enterohemorrhagic E. coli serotypes including O157:H7.
  1. Identify novel virulence markers in emerging or newly recognized food borne pathogens such as Vibrios.
  1. Develop a set of detection methods adapted to a common platform instrumentation.
  2. Evaluate the system on contaminated produce and other food matrices.
Component 4 Development of tests for pathogen monitoring during processing of sprouts and other produce

Description

 Detection of pathogens present at low levels in seeds for sprouting or other produce presents unique challenges. High levels of competing microflora may intensify problems detecting pathogens. Methods to concentrate low level pathogens present in irrigation water prior to harvesting, in distribution or in produce wash water, followed by rapid detection, can be used to identify contaminated products and can be used to monitor Critical Control Points in production/processing. Year Deliverables
Deliverables

FY1999

FY2000

FY2001
  1. Evaluate sprout irrigation water as a target for pathogen monitoring.
  2. Test the efficacy of selected rapid methods for the detection and quantitation of Salmonella and E. coli 0157:H7 in sprout irrigation water.
  1. Develop large-scale sampling protocols for concentrating and detecting pathogens in irrigation water without culture enrichment.
  2. Conduct baseline measurements to test quantitative methods for determining extent of pathogen injury in fresh produce.
  1. Test applicability of newly developed concentration methods for pathogen monitoring in other types of produce.
  2. Test efficacy of quantitative methods for testing pathogen injury in food processing systems.
Component 5 Real time detection of microbial toxins using biosensors.
Description Using the BIOCOR instrument at CBER, a sandwich biosensor method was developed and successfully evaluated for the detection of Staphylococcal enterotoxin. The assay sensitivity is 5-10 ng/ml of toxin and the results are obtained in 4 min. The current biosensor ligands are antibodies that are specific for one antigen. This project will attempt to develop multi-antigenic peptides that can detect several antigens simultaneously in the biosensor. Oligopeptides from a combinatorial random phage-display library of peptides on the surface of filamentous phages will be screened for binding capacity to specific toxins and tested as replacement to antibodies. Such a technology is adaptable for real-time analysis of toxins in foods and for HACCP monitoring. Year Deliverables
Deliverables

FY1999

FY2000

FY2001
  1. Produce new peptide ligands for use with the biosensor methodology.
  1. Select peptides and combine into multi-antigenic ligands.
  1. Develop multiple toxin detection system for 3 bacterial and 2 fungal toxins.
Component 6 Detection of Hepatitis A and other viruses from fruits and produce.
Description Food matrix interference is a major problem in the detection of viruses by gene based methods. This project will develop methods for concentrating low level contamination of Hepatitis A and other caliciviruses in fruits and produce followed by purification methods to produce viral RNA that are suitable for detection by RT-PCR.
Deliverables

FY1999

FY2000

FY2001
  1. Improve concentration and RNA purification procedures to increase the sensitivity of detection by 30-100 fold.
  1. Evaluate antibodies as capture supports for concentration of intact viral particles or nucleic acids for viral RNA binding after extraction
  1. Establish RNA fingerprint database to distinguish Hepatitis strains.

 

 

Legend | Abbreviations

FSI Project Number 2 RSVP Number 21076 GPRA Goals: II.B.
Project Title Molecular Characterization of Maverick Strains of Enterohemorrhagic E. coli
Personnel Name Office/Division FTE(FY99,FY00,FY01)
P. Feng OSRS/DMS 1.0
Funded position: Molecular Biologist OSRS/DMS 0.5,1.0,1.0
Total FTE 1.5,2.0,2.0
ORA 30 day detailee
Proposed but unfunded positions: Support Scientist OSRS/DMS [2.0]
Administrative Liaison P. Feng, 202-205-4518
Project Abstract Genetic analysis and characterization of atypical variants of E. coli O157:H7 to identify suitable target for detection method development.
Project Description New variant strains of enterohemorrhagic Escherichia coli, particularly O157:H7, are being isolated more frequently from foods, animals and humans worldwide. Initially, these variants elude detection due to changes in the markers targeted by current testing methods. Characterization of these variants will provide information to account for the emergence of these strains and also identify new genetic or phenotypic markers that can be used to test for these evolving pathogenic variants. This work relates directly to the objective of developing improved detection technology for emerging foodborne pathogens.

Strategy: O Rough strains of O157:H7 do not produce the O157 antigen even though they carry the genetic sequences to encode the O157 antigen. Hence, they are not detected by routine serological assays used to detect O157:H7. O rough strain will be examined to determine the absence of O antigen gene expression and develop suitable assays for detection.
Projected Impact The likely impact will be a more complete accounting of major hazards in the food supply, leading to a more thorough and rapid application of control measures.
Center Priorities Code 1.4b
Research Regulatory Needs Codes XI.A.
 

Deliverables

FY1999

FY2000

FY2001
  1. Finish characterization of the O rough mutant of O157:H7.
  2. Select appropriate virulence factors and use these markers for developing detection assays for the Arough strains@.
  3. Initiate characterization of other new strains that become known or suspected, i.e., non-motile and non-Shiga toxin-producing strains.
  1. Evaluate the effectiveness of the assay for detecting O Rough strains of E. coli O157:H7 in foods.
  2. Continue characterization of non-motile and non-Shiga toxin-producing strains of O157:H7.
  1. Develop assay that can be used to detect non-motile strains.
  2. Determine the health significance of non-Shiga toxin-producing strains that are being isolated from environmental samples worldwide.

1Proposed but unfunded need: travel to attend the VTEC2000 meeting in Kyoto, Japan.

 

 

Legend | Abbreviations

FSI Project Number 3 RSVP Number 38120 GRPA Goals: I.B.; II B.
Project Title Effects of Environmental Conditions, Phytochemicals, Modified Atmosphere Packaging and other Parameters on the Growth and Survival of Foodborne Pathogens on produce, Particularly Sprouted Seeds
Personnel Name

Office/Division

FTE

 Component
J. Betz OPDFB/DNP

0.4

2
B. Canas OPDFB/DNP

1.0

2
L. Miller OPDFB/DNP

1.0

2
R. Whiting OPDFB/DNP

0.5

5
S. Mammel OPDFB/DNP

1.0

5
G. Skinner OPDFB/DFPP

1.0

4
R. Bennett OSRS/DMS

0.5

1
A.D.Hitchins OSRS/DMS

0.5

3
R. Duvall OSRS/DMS

0.8

3
H. Wisneski OCAC/DSAT

0.5

2
Approved Position:

Support scientists

  

OPDFB/DFPP

  

[0.5-1.0]*

5
TOTAL FTE

8.2
Administrative Liaison R. C. Whiting, 202-260-05115110511
Project Abstract This project focuses on evaluating the effects of environmental and food formulations factors on pathogen growth in minimally processed foods and to manipulate these factors to reduce or eliminate pathogen growth.
Project Description Processing of commercial produce is a rapidly expanding industry that offers convenient products with fresh-like qualities. Preservation and extension of shelf life for produce is frequently achieved through refrigeration, bactericidal rinses, modified atmosphere packaging and other technologies. To assure the safety of minimally processed produce, it is essential to obtain detailed information on the effect of environmental and food formulation factors on the growth and survival of pathogenic bacteria that may be present. For instance, various plants contain phytochemicals that are capable of suppressing microbial growth. The resident microflora on produce, which can vary with product, can affect or alter the relative growth rates of pathogens on produce. In addition, the growth of pathogenic bacteria during germination of sprouted seeds may greatly increase the risk of foodborne diseases. These studies will evaluate the effects of phytochemicals, environmental conditions, modified atmospheres, microflora composition and other factors on the growth and survival of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Bacillus cereus, as well as appropriate surrogate microorganisms, on assorted fruits and vegetables. Also, sprouted seeds will be a target for investigating natural contamination by pathogens.
Projected Impact These data will serve as a basis for developing guidelines for produce handling and intervention technologies.
Center Priorities Code 1.4c
Research Regulatory Needs Codes I. A.; I.D.; II. C.
Component 1 Prevalence, growth and survival of toxigenic Bacillus and Staphylococcal spp.in sprouting seeds
Deliverables

FY1999

FY2000

FY2001
  1. Generate data on the presence of S. aureus on three types of domestic sprouted seeds, including alfalfa, mung beans and mustard.
  2. Beginning mid-year, test imported sprouted seeds from the Import Compliance Program.
  1. Collect additional data on domestic and imported sprouts and mature produce that are marketed at the consumer level.
  2. Begin simulation studies of the effect of environmental conditions, including modified atmospheric packaging, to ascertain whether this approach will prevent the outgrowth of Salmonella and B. cereus.
  1. Continue data collection and simulation studies to determine growth and survival of these two pathogens in these products.
Component  2 Analysis and characterization of phytochemicals that will suppress growth of microbial pathogens. Analysis and characterization of phytochemicals that will suppress growth of microbial pathogens.
Deliverables

FY1999

FY2000

FY2001
  1. Complete model system for studying the effect of storage and ecological factors on the production of phytochemicals.
  2. Isolate and identify these phytochemicals from vegetables, seeds, and sprouted seeds or chemically synthesize them when necessary.
  1. Screen isolated and synthesized compounds for inhibitory activity against normal bacterial flora and pathogens.
  2. Characterize bioactive phytochemicals.
  1. Evaluate effects of various stressors on levels of potentially toxic phytoalexins in the product.
  2. Examine whether pytochemicals have potential to selectively inhibit normal flora or pathogens.
  3. Begin preliminary study to determine if phytochemicals from one plant may be used to treat other plants (i.e., compounds from cranberries may inhibit adhesion of pathogens to other plant surfaces).
Component  3 Development of a more sensitive method for quantitation of Listeria monocytogenes.
Deliverables

FY1999

FY2000

FY2001
  1. Develop a colony counting method to enumerate L. monocytogenes from time controlled selective enrichment of 24 hours or less.
  1. Test enumeration method using foods, especially fresh produce, and optimize method to account for problems due to normal microflora interference.
  1. Determine if the colony counting format for quantitation can be replaced by a more convenient DNA-based rapid method format.
Component 4 Effects of various processing parameters on the levels of foodborne pathogens in minimally processed foods
Deliverables

FY1999

FY2000

FY2001
  1. Determine growth or decline of E. coli O157:H7 population on a variety of produce, particularly fresh cut vegetables. Develop models that will predict the expected microbial behavior on these products.
  1. Determine the effect of various anti-microbials and inhibitors on the growth and survival of bacteria on produce.
  1. Complete modeling and compare to published data. Extend these studies to Listeria. Begin similar experimentation on modified atmosphere packaged produce.
Component 5 Modeling for thermal inactivation and growth of Listeria under modified atmospheresModeling for thermal inactivation and growth of Listeria under modified atmospheres
Deliverables

FY1999

FY2000

FY2001
  1. Develop basic protocol for thermal inactivation of L. monocytogenes, collect data and fit to models.
  2. Identify surrogates of E. coli O157:H7 for use in food plant validation studies.
  3. Investigate the potential for using competitive flora to prevent growth of pathogens in contaminated sprouts.
  1. Expand thermal inactivation modeling to include factors relating to the growth of the bacteria (ie: stage of growth, adaptation, etc) and injury/recovery from heating.
  2. Begin modeling growth under combinations of nitrogen and carbon dioxide atmospheres.
  1. Continue thermal inactivation modeling, fit data to inactivation equations, develop regression equations, incorporate models into Pathogen Modeling Program.
  2. Expand modified atmosphere modeling to include other environmental factors such as temperature, pH and salt content.3

*Effective as of date hired.

Legend | Abbreviations

FSI Project Number 4 RSVP Number 22660T GPRA Goals: I.D.
Project Title Molecular Mechanisms for Pathogen Emergence
Personnel Name Office/Division

FTE
T.A. Cebula OPA/DMBRE/MBB

0.5
J. E. LeClerc OPA/DMBRE/MBB

0.8
B. Li OPA/DMBRE/MBB

0.8
D. D. Levy OPA/DMBRE/MBB

0.8
M. J Kotewicz OPA/DMBRE/MBB

0.8
W. L. Payne OPA/DMBRE/MBB

0.8
S. Assar Student Career Exchange Program;

Univ. FL B Gainesville

0.5
Total FTE  

5
Administrative Liaison T.A. Cebula, 202-205-4217
Project Abstract The objective of this project is to understand the molecular genesis and emergence of antimicrobial resistance among bacterial pathogens. Research will focus on the role of mutators, specifically those deficient in methyl-directed mismatch repair, on establishing antimicrobial resistance by genetic change (mutation) and exchange (recombination).

Project Description

Recognizing that bacterial pathogen will continue to evolve, public health initiatives must include research to understand how these pathogens arise, propagate, and emerge. Bacteria have great ability to adapt rapidly and propagate to fill an existing niche. The role that antibiotics play in the emergence of antibiotic resistance bacteria has received ample attention, yet, the role of genetic diversity of a bacterial population or the effects of other selective pressures have on emergence of antibiotic resistance have not been adequately addressed. This project investigates the proposition that antibiotic resistant strains are emerging from specific "pools" that exist in bacterial populations at large. We have shown previously that high frequency (1-5%) of methyl-directed mismatch repair (MMR-) defective, pathogenic Escherichia coli and Salmonella enterica strains, exist and persist among natural populations. We will assess the role that MMR- mutators play in the emergence of antibiotic resistant strains by first determining the frequency of mutators among clinical and agricultural isolates of Salmonella. T he nature of these mutator phenotype will be characterized and phylogenetic analyses will be done to assess whether clonal theory adequately addresses lineages observed among antibiotic resistant strains of Salmonella. Finally, we will explore if an MMR- phenotype can be enriched under experimental conditions using an in vivo murine infection model.
Projected Impact The impact of this project includes the development of rapid methods to detect and identify antibiotic resistance pathogens in our food supply and to aid in our understanding of how antimicrobial resistance emerges. Moreover, in order for intervention strategies to be effective, it is essential to understand the process of emergence to be able to predict if a certain pathogens will be in a unprocessed food and to explore processing parameters that will eliminate them. Finally, by identifying critical bacterial subpopulations (like the mutators) that are more apt to resist antibiotics and antimicrobials, appropriate containment procedures can be implemented before these strains are disseminated globally.
Center Priorities Code  
Research Regulatory Needs Codes IX.
Deliverables

FY1999

FY2000

FY2001
  1. Determine the frequency and nature of mutators among clinically and agriculturally relevant isolates of Salmonella.
  2. Examine if genes located near mutators are more susceptible to genetic exchange events, and determine if they are involved in stress responses in E. coli O157:H7.
  1. Compare genetic relatedness between mutator strains and representative antibiotic-resistant strains isolated from clinically and agriculturally relevant collections of Salmonella.
  2. Determine whether mutator strains of Salmonella have an adaptive advantage for survival during infection in a murine model system.
  1. Determine whether strains of Salmonella, adapted for successful infection, have modified genes within or near mutator loci that would signal recombinational events.
  2. Establish whether particular mutator populations of Salmonella can be enriched during serial infection using a murine model system.

 

 

Legend | Abbreviations

FSI Project Number 5   RSVP Number 38233
Project Title Identification and Characterization of Virulence Determinants in Salmonella enteritidis and Vibrio species
Personnel Name Office/Division FTE Component
B. McCardell OPDFB/DVA 0.5 1
Mahendra Kothary OPDFB/DVA 0.5 2
V. Sathyamoorthy OPDFB/DVA 1.0 2
M. Miliotis OPDFB/DVA 0.5 3
Approved position: Support Scientist OPDFB/DVA 1.0[2000,2001 only] 3
B. Tall OSRS/DMS 0.5 4
TOTAL FTE 3, 4[2000,2001]  
Proposed but unfunded position for support scientist OSRS/DMS (1.0)[2000] 4
Collaborators Center for Vaccine Development, University of Maryland School of Medicine 1
   

JIFSAN students

   

2,3,4

Administrative Liaison B. McCardell , (202) 205-4262
Project Abstract Assessing the risks associated with pathogenic microorganisms and developing effective methods for their detection and control are dependent on having detailed knowledge about the factors that contribute to their virulence. Some virulence determinants of Salmonella and Vibrio species are unknown or poorly characterized.

Work on this project includes :

  • Characterizing virulence determinants;
  • Developing detection systems based on sequences of genes encoding virulence factors;
  • Studying the effects of virulence factors in animal models;
  • Using animal models to collect dose response data.
Project Description Bacterial strains from foodborne outbreaks will be characterized using in vitro and in vivo virulence assays. Those strains that have novel virulence factors will be studied in depth. Virulence factors (primarily toxins, pili and proteases) will be purified by standard protein chemistry methods. When N-terminal sequences have been determined, the virulence gene will be cloned. Primers for virulence genes will be selected and assays developed for the gene. The virulence of strains with and without the specific virulence factor will be tested in animal models. Dose response data will be generated. When available, detection systems will be coupled with biosenor technology.
Projected Impact Detection methods will be developed. Dose response data for science-based risk assessments will be generated and evaluated. CFSAN will gain science-based information on which to base regulations and industry guidelines.
Center Priorities Code
Research Regulatory Needs Codes V.B.; XI.A.
Component 1 Identification and characterization of novel toxin gene from Vibrio cholerae vaccine strain.
Description In collaboration with the Center for Vaccine Development, University of Maryland School of Medicine, this toxin gene will be cloned, sequenced and characterized. Primers will be selected for PCR. Other strains of V. cholerae O1 and non-O1, and other Vibrio species will be screened for this toxin. Toxin will be purified from cloned toxin and tested in an animal model.
Deliverables

FY1999

FY2000

FY2001
  1. Characterize mode of action of toxin.
  2. Complete manuscript on purification of protein from native strain.
  1. Clone Gene for Vibrio toxin.
  2. Complete manuscript on cloning and characterization of toxin gene.
  1. Generate virulence mutants and complete animal testing.
Component 2 Purification and characterization of virulence factors, including toxins and proteases of Salmonella Enteritidis and pathogenic Vibrio spp.
Description A novel toxin from V. cholerae O1 will be purified in sufficient quantity to test in the sealed infant mouse model. Toxins produced by cloned toxin genes of Vibrio cholerae O1 and non-O1, Salmonella enteritidis and Vibrio parahaemolyticus will be purified.

Newly identified virulence factors produced by Salmonella Enteritidis, Vibrio parahaemolyticus and Vibrio vulnificus will be purified and characterized. Then, this information can be used to develop specific tests to distinguish pathogenic strains from harmless environmental strains.
Deliverables

FY1999

FY2000

FY2001
  1. Purify Vibrio toxin and collect animal data.
  2. S. Enteritidis and V. parahaemolyticus toxins purified and characterized.
  3. V. vulnificus toxin partially purified.
  4. Hemagglutination by V. vulnificus protease characterized.
  1. Complete purification and characterization of Vibrio toxin.Initiate purification of cloned Vibrio toxin.
  2. Initiate purification of cloned Vibrio parahaemolyticus toxin.
  3. Complete dose response studies for the S. Enteritidis and V. parahaemolyticus toxins in the suckling mouse model.
  4. V. vulnificus toxin purified.
  1. Complete purification of cloned S. enteritidis toxin.
  2. Complete dose response studies for V. vulnificus toxin in the suckling mouse model
Component 3 Cloning of the genes for Vibrio parahaemolyticus and Salmonella Enteritidis enterotoxins.
Description Assays for virulence factors can be tedious and time consuming. Gene sequences associated with these factors can be used as probes to identify organisms harboring specific virulence determinants.

Pathogenicity of V. parahaemolyticus (VP) is closely associated with the presence of thermostable direct hemolysin (TDH). A cytotonic enterotoxin (VPE) that elongates Chinese hamster ovary (CHO) cells in vitro has been isolated from strains that test positive for hemolysis as well as nonhemolytic strains. TDH-VPE+ strains can cause gastroenteritis; therefore, VPE could be a contributing factor to this illness. Isolation and characterization of the gene(s) involved with VPE are the main objectives of this work. Probes based on sequences from this gene can then be used to identify strains VPE+ strains that may or may not produce the hemolysin.

Salmonella Enteritidis (SE) produces at least two toxins that elongate CHO cells, one is neutralized by antibodies to classic cholera toxin, the other is not neutralized by these antibodies (SEE). Gene(s) associated with SEE will be identified, isolated and characterized. Probes based on sequences from this gene can then be used to identify other salmonellae that produce this toxin.
Deliverables

FY1999

FY2000

FY2001
  1. Initiate cloning of VPE and SEE genes by transposon mutagenesis and conventional cloning techniques.
  2. Test mutants in CHO cell and esterase assays.
  1. Initiate sequencing of the toxin genes.
  2. Initiate development of detection method based on gene sequences.
  3. Test clones that produce positive toxin and mutants that do not produce toxin production in different animal models.
  1. Complete DNA sequencing of toxin genes
  2. Synthesize oligonucleotides based on the sequencing data, and test for DNA homology with other strains by PCR or nonradioactive hybridization using digoxygenin-labeled probes.
Component 4 Adherence and invasion mechanisms of Vibrio species.
Description In seafood species, Vibrio species, such as V. vulnificus cause systemic infections; in humans, they cause gastroenteritis, wound infections, and septicemia. Diseases caused by marine vibrios greatly affect aquaculture, marine fish farming and public health. Mechanisms such as adherence and invasion influence persistence of these bacteria. These characteristerics may have an impact on the emergence of Vibrio species in seafood hosts and their subsequent transmission to humans. Studies of these traits may lead to procedures for removing these pathogens from seafoods, consequently eliminating them from human foods.
Deliverables

FY1999

FY2000

FY2001
  1. Complete kinetic adherence and invasion studies of several vibrios, including both biotypes of V. vulnificus, V. fluvialis, V. tubiashii, V. alginolyticus, V.damsela, and V. ordalii with atlantic menhaden liver cells (AML) and primary menhaden and oyster cells.
  2. Isolate, purify, and characterize adherence factors such as, hemagglutinins/ pili expressed by V. vulnificus, V. mimicus, V. tubiashii, V. fluvialis, and V. alginolyticus.
  3. Characterize important virulence factors expressed by V. fluvialis strains isolated from diseased lobsters harvested from Atlantic coastal waters. Determine if strains are pathogenic to vertebrates using animal dose studies.
  1. Determine thermal kill parameters/heat resistance in V. fluvialis lobster isolates.
  2. Complete signal transduction inhibitor studies to determine role of host activation of protein kinases, rearrangement of cytoskeletal elements (microfilaments and microtubules) and receptor-mediated endocytotic pathways involved in the early stages of V. vulnificus entry into AML cells.
  3. Determine if V. vulnificus -mediated-cytotoxicity and invasion are independent events. Isolate, purify, and characterize adherence factors such as hemagglutinins/ pili expressed by V. damsela, V. anguillarum, and V. ordiali.
  1. Determine AML host receptor (s) involved in colonization and invasion of shellfish and finfish cultured cells by V. vulnificus using polyclonal antibodies, lectins, and inhibitors.
  2. Compare signal transduction inhibitor studies of V. vulnificus entry into AML cells and role of host receptors in adherence and invasion with other marine vibrios.

 

 

Legend | Abbreviations

FSI Project Number 6 RSVP Number   39770T GPRA Goals II. A.,B.
Project Title Cyclospora and Related Parasitic Protozoa: Detection and Viability Assessment
Personnel Name Office/Division FTE

Component
P.A. Orlandi OPDFB/DVA 1.0

1
D.E. Hanes OPDFB/DVA 0.5

2
J. BierH OS/DSAT 1.0

1
G. J. Jackson

Total FTE

 OSRS/OSRS 0.2

2.7

2
Proposed but Unfunded Positions: Microbiologist/Parasitologist [2.0]  
  Technical Support Personnel [2.0]  
Collaborators: *Moffet Center, ORA    
External ContractsI     Cost
Winter Cyclospora cayetanensis oocyst supply and primers for PCR detection. CDC/NCID/DPD   $30,000
Molecular differentiation of protozoan species and strains Stanford University   $25,000
Central American survey of mammalian hosts Northern Virginia Community College   $1,500
Uniformed Services University

of the Health Sciences

 Summer supply of Cyclospora cayetanensis oocysts   $15,000
Administrative Liaison P.A. Orlandi , (202) 205-4460G.J. Jackson, (202) 205-4051
Project Abstract Parasitic protozoa such as Cyclospora cayetanensis, Cryptosporidium parvum and Microsporidium spp. have emerged as important human pathogens and are closely associated with food and waterborne illness. This project will pursue three (3) aspects of research as they relate to food safety:
  1. continued development of sensitive detection methods and better sampling of food and water sources;
  2. in vitro cultivation and animal modeling development and risk evaluation;
  3. intervention strategies.
Project Description Within the last several years, the protozoan parasites Cyclospora cayetanensis, Cryptosporidium parvum and Microsporidium spp. have become increasingly recognized as important, rapidly emerging human pathogens in immunocompromised and immunocompetent individuals alike. Outbreaks of enteric infections caused by these microorganisms have been associated with food- and waterborne contamination. Since the spring and early summer of 1996, major outbreaks in North America attributed to Cyclospora cayetanensis have been epidemiologically-linked to the consumption of spring crop raspberries from Guatemala. Smaller outbreaks of cyclosporiasis in the United States during the 1990s have been associated with the consumption of other fresh produce-mesclun lettuce and basil. Unpasturized apple cider has been a source for Cryptosporidum parvum infections; scallions have also been implicated. Contaminated water sources are also suspected as a major route in the transmission of all three parasitic protozoa.

The difficulties in assessing and controlling possible foodborne contamination and infections with these coccidia are many. There is a general lack of knowledge concerning life cycles (Cyclospora cayetanensis, Microsporidia spp), animal vectors and/or reservoirs, biochemistry, and the inability to efficiently culture the parasite either in an animal model (Cyclospora cayetanensis) or a tissue culture-based system (Cyclospora cayetanensis and Cryptosporidium parvum). An inadequate supply of Cyclospora cayetanensis also contributes to our general lack of understanding. Neither a means for assessing their pathogenicity and survival after exposure to potential intervention treatments nor sufficient infectious dose information is available. Current methods to detect foodborne contamination lack the necessary sensitivity and reliability. In this 3-yr plan, improved sampling and detection methods will be pursued to include fast, reliable, and highly sensitive PCR methodologies that can be applied to a variety of food and water sources. The development of systems for evaluating intervention strategies will include in vitro cultivation of oocysts and identification of model hosts. Development of animal models that mimic human illness caused by these protozoa will be attempted and dose-response studies in normal and immunocompromised animals will then be conducted to provide data for developing risk assessment models. Alternate protozoa such as Eimeria spp. will also be evaluated as research models in the absence of adequate supplies of Cyclospora cayetanensis.
Projected Impact The results of this project will provide for an improved ability to detect and reduce the risk of food and water-borne illness attributed to parasitic protozoa.
Center Priorities Code
Research Regulatory Needs Codes I.A., I. C., I. F.
Component 1 Methods for the Detection of Cyclospora cayetanensis and Related Protozoa on Fresh Produce
Deliverables

FY1999

FY2000

FY2001
  1. Survey incoming Guatemalan raspberries using existing methods of oocyst detection.
  2. Develop and evaluate alternate method of oocyst isolation and extraction from food matrices for PCR detection and microscopic identification.
  3. Evaluate Cryptosporidium and Eimeria as model oocysts for developing better isolation and extraction methods.
  1. Begin produce survey for Microsporidia spp.
  2. Evaluate the effect of UV irradiation on Cryptosporidium infectivity from juice samples.
  3. Develop multiplex PCR detection strategy for identification of contaminating parasitic protozoa in produce and water sources.
  
Component 2 Propagation of Cyclospora cayetanensis in Tissue Culture (A); Evaluation of Potential Animal Models (A); and, Risk Evaluation of Cyclospora cayetanensis Contamination Routes (B)
Deliverables

FY1999

FY2000

FY2001
1. Continue to evaluate tissue culture models (A).

2. Final evaluation of dog model for Cyclospora cayetanensis (A).

3. Complete and compile first raspberry survey for Cyclospora cayetanensis (A).

 1. Continue to evaluate tissue culture models (A).

2. Test and evaluate gnotobiotic pig model for Cyclospora cayetanensis (A).

3. Begin produce survey for Microsporidia spp. (B).

4. Compile results of Central American reservoir host survey for coccidia (B).

 1. Final evaluation of alternate host and culture systems for Cyclospora cayetanensis oocyst propagation (A).

2. Risk/safety evaluation of routes of Cyclospora cayetanensis contamination of Guatamalan raspberries. (B)
Component 3 Intervention Strategies for Cyclospora cayetanensis and Related Protozoan Parasite Contaminants
Year Deliverables

FY1999

FY2000

FY2001
 
  1. Evaluate intervention strategies for Cyclospora contamination of produce.
  1. Evaluate effectiveness of UV irradiation on Cryptosporidium infectivity from juice samples.

HAs of 31Aug00, Dr. J. Bier will be retiring. A search for a trained parasitologist to replace Dr Bier will be essential for the successful completion of Project 6 objectives.

*CFSAN/Moffet Center collaboration to evaluate the effectiveness of UV irradiation on Cryptosporidium infectivity from juice samples (Component 1/3).

Additional Funding Needs:

Equipment: DPDX System (Division of Parasitology Diagnostics System, CDC/DPD): Microscopic image transmission system, $75,000

 

 

Legend | Abbreviations

FSI Project Number 07 RSVP Number 38891 GPRA Goals: II.C.
Project Title Characterization of Pathogenic Aquatic Eucaryotes and their Toxins
Personnel Name Office/Division FTE

Component
R.Dickey OS/GCSL 1.0

1,2,3,4,5
S.Plakas OS/GCSL 1.0

2
R.Granade OS/GCSL 1.0

1,2,3
E.Jester OS/GCSL 1.0

1,2,3
D.Mowdy OS/GCSL 1.0

3
K.El Said OS/GCSL 1.0

2
N.Sass OSRS/DTR 0.4

2,4
M.Scott OSRS/DTR 0.2

1
R.Jackson OSRS/DTR 0.2

1
D.Hinton OSRS/DTR 0.2

1
S. Hall OS/DSAT/WSL 0.5

6,7,8,9
P. Eilers OS/DSAT/WSL 1.0

6,8
S. Conrad OS/DSAT/WSL 1.0

6,8,9
V. Brewer OS/DSAT/WSL 1.0

6,7,9
TOTAL FTE   10.5  
Administrative Liaison G. Hoskin 202-418-3172; M. McPhearson, 334-694-4480; S. Hall 202-205-4818; N. Sass 301-594-5800.
Project Abstract Evaluate aquatic biotoxin seafood hazards by determination of identity, toxicity, critical points/limits and detection methods.
Project Description

 

 While plankton are, in general, a vital component of the marine biosphere, some species produce potent toxins that accumulate in seafood and put human consumers at risk. Existing management programs have dealt moderately well with the problem in the past, but are challenged by novel toxins, and different temporal and spacial patterns of occurrence. Pfeisteria Organism Complex (POC)-associated fish kills have aroused concerns that POC toxins might accumulate in seafood. Despite the lack of any evidence of a public health risk, solid data are needed to properly address the situation. There is a general need for a better understanding of the various kinds of organisms and toxins known to cause human illness, better detection methods, including replacement of animal bioassays, for them, and development of novel management strategies that will provide better detection at lower cost

Specifically, goals will focus on identifying biotoxins, such as the known toxins of paralytic shellfish poison ( PSP), neurotoxic shellfish poison ( NSP toxins, primarily brevetoxin and metabolites=PbTx), diarrhetic shell fish poison (DSP), ciguatera fish poisoning toxin (CFP), and totally unknown toxins such as those recently evident in POC and buffalo fish that may be present in seafoods. Levels of contamination likely to pose human health hazards will be assessed and the means/tools needed to control such biotoxins developed. These are multidisciplinary studies involving chemistry (including elucidation of structure as a basis for developing quantitative tests), toxicology and molecular biology. Toxin chemical standards for FDA and external regulatory and research laboratories are produced under this project.
Projected Impact
  • Provide tools for establishing control procedures, critical control points and critical limits (i.e. action levels) for all biotoxins affecting consumers of seafoods.
  • Develop management tools and strategies to address the problem of seafood contamination by natural toxins from plankton.
  • Provide improved detection methods, and, where possible,provide alternatives to animal bioassay methods.
  • To develop more cost-effective, reliable programs for scientifically based marine biotoxin management and to ensure that existing programs deal effectively with new threats.
  • Establishment of control procedures, critical control points and critical limits (i.e. action levels) for all biotoxins affecting consumers of seafoods.
Center Priorities Code
Research Regulatory Needs Codes VI. C.
Component 1 Alternative Methods to Mouse Bioassay for NSP Regulatory Screening and Confirmation of Violative Shellfish.
Description Develop and evaluate in vitro methods as alternatives to official mouse bioassay. Convert existing NSP radioimmunoassay to enzyme-linked immunosorbant assay (ELISA) format and evaluate as alternative to mouse bioassay
Deliverables

FY1999

FY2000

FY2001
  1. Assess potential of in vitro cytotoxicity assay as replacement for FDA approved NSP mouse bioassay. Recommend use, limitations for use, or rejection.
  2. Determine sensitivity and specificity of anti-brevetoxin antibodies against standard brevetoxins. Recommend continuation or discontinuation of NSP ELISA development.
  1. Adapt anti-brevetoxin antibodies to ELISA format. Test performance fidelity against brevetoxins and NSP shellfish matrices. Recommend continuation or discontinuation of NSP ELISA development.
  1. Reduce complexity of NSP-ELISA to suit field application of immunoassay (e.g. develope dip-stick test) and test against NSP shellfish matrices. Recommend use, limitations for use, or rejection.
  2. Explore feasibility of developing molecular imprintable polymer (MIP: a rugged "artificial" antibody) assay for NSP as a less expensive and durable alternative to biological antibody-based immunoassay. Recommend development or rejection of NSP-MIP assay concept.
Component 2 Identification of Molluscan Metabolites of NSP Brevetoxins, critical levels/limits for NSP biotoxins and metabolites in molluscan shellfish, and characterize brevetoxin absorbtion, metabolism, and elimination from shellfish.
Description Isolate molluscan metabolites of Brevetoxins and elucidate chemical structures. Investigate NSP biotoxin/metabolite in vivo dose response by intraperitoneal and peroral routes of administration. Identify relationship to naturally incurred NSP biotoxin/metabolite residues, and assess validity of current NSP guidance level. Investigate brevetoxin absorbtion, metabolism, and elimination by shellfish exposure to radiolabelled toxin under controlled laboratory conditions.
Deliverables

FY1999

FY2000

FY2001
  1. Isolate and elucidate chemical structures of brevetoxin metabolites from shellfish for use in breve detection methods development and toxicological risk assessment.
  2. Determine mouse bioassay dose-response for purified brevetoxins as basis for toxicological assessment of naturally incurred brevetoxin residues in NSP shellfish.
  3. Design study protocol and perform range-finding experiments for radiolabelled brevetoxin uptake, metabolism and elimination in the eastern oyster (Crassostrea virginica).
  1. Describe chemical identification of brevetoxin metabolites from eastern oyster.
  2. Determine toxicological significance of parent brevetoxins and brevetoxin metabolites from NSP shellfish.
  3. Complete study of radiolabelled brevetoxin absorption, metabolism and elimination in the eastern oyster.
  1. Report on NSP-mouse bioassay toxicological assessment.
  2. Prepare report in-house addressing relevance of study to current NSP guidance level.
  3. Report on brevetoxin absorption, metabolism and elimination in Crassostrea virginica. Propose criteria for regulatory applications.
Component 3 Preparation of ciguatoxin (CFP) standards and development of methods for determination in finfish.
Description Isolate/purify of ciguatoxins from toxic finfish collected from ciguatera endemic regions. Refine in vitro and instrumental methods for the determination of CFP in finfish.
Deliverables

FY1999

FY2000

FY2001
1. Isolate and characterize ciguatoxin standards for use in methods development and toxicological assessment.

2. Compare cytotoxicity and LC/MS methods for determination of ciguatoxins in finfish.

 1. Isolate and characterize ciguatoxin standards for use in methods development and toxicological assessment.

2. Describe comparison of cytotoxicity with LC/MS methods for determination of ciguatoxins in finfish . Recommend use, limitations for use, or rejection.

3. Compare other in vitro methods against cytotoxicity and LC/MS for the determination of ciguatoxins in finfish.

 1. Isolate and characterize ciguatoxins toxin standards for use in methods development and toxicological assessment.

2. Describe comparison of in vitro with LC/MS methods for determination of ciguatoxins in finfish. Recommend use, limitations for use, or rejection.

3. Explore feasibility of developing immunochemical assay for CFP. Recommend development or rejection of CFP immunoassay concept.

4. Explore feasibility of developing molecular imprintable polymer assay for CFP. Recommend development or rejection of CFP-MIP assay concept.

Component 4 Development of guidance level for CFP.
Description  Develop in vivo dose-response model for incurred ciguatoxin residues in finfish; determine correlations with in vitro and instrumental methods of analysis (incl. case/outbreak analyses where possible); and propose critical levels/limits for control of CFP seafood hazard.
Deliverables

FY1999

FY2000

FY2001
 

1. Design study protocol and perform range-finding experiments for mouse bioassay dose-response for purified and naturally incurred ciguatoxins.

1. Complete study of CFP mouse bioassay dose-response. Propose regulatory guidance level for CFP.
Component 5 Characterization of okadaic acid (DSP) absorption, metabolism, and elimination from shellfish and identification of molluscan metabolites of okadaic acid.
Description Prepare large-scale cultures of okadaic acid producing algae, isolate and purify okadaic acid standards. Characterize okadaic acid absorption, metabolism and elimination in shellfish under controlled laboratory conditions. Isolate molluscan metabolites of DSP toxins and elucidate their chemical structures to better evaluate toxicity based on chemical analyses and to better design chemical test methods.
Deliverables

 

 FY1999

FY2000

FY2001
  1. Prepare large-scale cultures; isolate and prepare okadaic acid standards for use in shellfish exposure studies.
  2. Design study protocol and perform range-finding experiments for okadaic acid absorption, metabolism and elimination in selected shellfish species.
 1. Complete study of okadaic acid absorption, metabolism and elimination in selected shellfish species

2. Begin isolation and chemical identification of molluscan metabolites of okadaic acid.
Component 6 Are there seafood safety risks associated with Pfiesteria (POC)?
Description First recognized about ten years ago, Aattack dinoflagellates@ of the genus Pfiesteria and related genera recently have received a great deal of publicity. It is necessary to establish whether these organisms present a threat to food safety. A novel incubation system, developed in our lab, is being used to search for active material and produce levels of toxicity sufficient for investigation
Deliverables

FY1999

FY2000

FY2001
1. Cultures of pfiesterioid (POC species) organisms ongoing.
2. Complete evaluation of tissue culture assay running and evaluated.
3. Clear study protocol (QA, IACUC) for mammalian oral toxicity studies using cultured POC material.
  1. Initiate feeding experiments of bivalves with cultures pfiesterioid material to evaluate accumulation of toxicity.
  2. Report findings from toxicity screening and determine whether evidence supports further studies on POC toxicity.
  1. Complete initial study of accumulation of toxicity from pfiesterioid cultures.
  2. Evaluate utility of detection methods for POC toxins and produce standard testing protocol for regulatory use and produce standard testing protocol for regulatory use.
  3. Begin characterizing pfiesterioid toxins.
Component 7 Toxigenic plankton; morphology and toxin composition
Description Strategies for the management of marine biotoxins require an understanding of which organisms are making what toxins. If possible, morphological characteristics that allow recognition of toxigenic species under the light microscope should be defined for transfer to field observer programs in the states.
Deliverables

FY1999

FY2000

FY2001
  1. Two additional strains of Alexandrium spp. Will be isolated, cultured, and characterized for toxin composition. At least one of the isolates will be from a region with detailed coverage by field plankton observers.
  2. One strain of Pseudonitzchia will be in culture and demonstrated to be toxic.
  1. Twenty randomly-chosen phytoplankton species will be cultured and evaluated for domoic acid production.
  2. Two additional strains of Alexandrum spp. Will be isolated, cultured and characterized for toxin composition. At least one of the isolates will be from a region with detailed coverage by field plankton observers.
  1. Two additional strains of Alexandrum spp. Will be isolated, cultured and characterized for toxin composition. At least one of the isolates will be from a region with detailed coverage by field plankton observers.
  2. Summary of known sources of domoic acid and tools available for their recognition.
Component 8 Development of marine biotoxin detection methods and reference standards Development of marine biotoxin detection methods and reference standards
Description Management of and research on marine biotoxins requires sensitive, efficient detection methods. Work will focus on receptor assays for the saxitoxins and other families of toxins, and on improvement of HPLC for domoic acid. Reliable standards are necessary for the implementation of detection methods. Work will focus on the production of a stable domoic acid standard and the production of other toxins and derivatives.
Deliverables

FY1999

FY2000

FY2001
  1. Complete protocol for preparation and storage of domoic acid reference standards.
  2. Complete preparation of test materials for comparison of mouse oral potency, mouse intraperitoneal potency, and response factor by receptor assay for two seafood toxins.
  1. Complete preparation of test materials for comparison of mouse oral potency, mouse intraperitoneal potency, and response factor by receptor assay for two seafood toxins.
  2. Begin preparations of reference standards for two other toxin derivatives.
  1. Complete preparation of test materials for comparison of mouse oral potency, mouse intraperitoneal potency, and response factor by receptor assay for two other toxin derivatives.
Component 9 Improvement of marine biotoxin management strategies through state field observer programs.
Description  

From experience with outbreaks and ongoing management programs, it becomes evident that novel strategies are required to ensure adequate public health protection in the face of the biological challenge and the resources available for management. We are developing a strategy that uses neglected resources to better focus the existing programs on the times, locations, and toxins of greatest concern.
Deliverables

FY1999

FY2000

FY2001
  1. Provide evaluation of performance of programs in California, Maine, and Massachusetts.
  2. Complete revised syllabus, hand outs and other related materials for use in field observer training.
  1. Establish observer groups in Washington State and Alaska.
  2. Develop field observation method for Gymnodinium and other delicate species not effectively sampled by net.
  1. Establish observer groups along the Gulf coast, using methods suitable for Gymnodinium.
  2. Set up system for routine regional communication of field observations.

 

 

Legend | Abbreviations

FSI Project Number 08 RSVP Number 39112 GPRA Goal: I.B.
Project Title Control of Viral and Bacteriological Pathogens in Seafood
Personnel Name Office/Division

FTE (FY99,FY00,FY01)

Component
D. W. Cook OS/DSAT/GCSL

0.5

1
S. A. McCarthy OS/DSAT/GCSL

1

1
A. DePaola OS/DSAT/GCSL

0.5

1
Y. C. Sheih OS/DSAT/GCSL

1

3
W. Burkhardt OS/DSAT/GCSL

1

2
K. R.Calci OS/DSAT/GCSL

1

2
J. Jones OS/DSAT/GCSL

0.7, 0.9, 0.9

1,2
Support Scientist, approved hire OS/DSAT/GCSL

0.2, 0.8, 0.8

1
Total FTE

5.9,6.7,6.7

  
ORA 30 day detailee

1
Administrative Liaison George P. Hoskin, 202-418-3172

R. M. McPhearson, 334-694-4480
Project Abstract This project will develop methodology to assess the presence and fate of natural and pollution-borne pathogens in seafood and provides information for risk assessment.
Project Description Indicator bacteria may not accurately reflect the presence of enteric viruses (Hepatitis A or Norwalk-like viruses) in shellfish or its growing waters. This project will evaluate whether alternative microorganisms (e.g. bacteriophages) better predict the hazards from human fecal pollution.

Bacteria of the genus Vibrio occur naturally in estuarine waters, and frequently are found in seafoods harvested from those waters. Some species of Vibrio are pathogenic for humans; one species V. vulnificus is the leading cause of seafood-related deaths.
Projected Impact Development of better methods to detect and enumerate seafood pathogens and determination of critical temperature limits to be used in intervention strategies.
Center Priorities Code 1.7b
Research Regulatory Needs Codes VI A., B.
Component 1
  1. Develop PCR and non-radioactive probe methodology for detection and enumeration of V. parahaemolyticus (V.p.) in shellfish.
  2. Provide baseline data on V. parahaemolyticus in shellfish.
  3. Development a species-specific alkaline phosphatase-labeled gene probe for V. cholerae
  4. Develop data on handling practices and processing techniques that may affect levels of V. parahaemolyticus. in shellfish.
Description
  1. Recent outbreaks of V. parahaemolyticus food illness related to shellfish consumption has highlighted of the needs of regulatory agencies and researchers for better methods to enumerate this organism and to detect the pathogenic strains. This research will provide needed methodology and transfer it to ORA laboratories. This task is related to Risk Assessment of V. parahaemolyticus.
  2. State shellfish laboratory personnel will be trained in new DNA gene probe techniques for enumerating in shellfish. Using these techniques, state laboratories will gather and share with FDA data on levels of. V. parahaemolyticus normally found in shellfish during different seasons and under a variety of environmental conditions. FDA will analyze the data for use in risk assessment. This task is related to Risk Assessment of V. parahaemolyticus
  3. Seafood related illnesses caused by naturally occurring V. cholerae non-O1 remain a chronic problem. Efforts to investigate this organism in the environment and to define how seafood handling and processing steps affects its numbers have not progressed because of inadequate enumeration procedures. This project will develop and validate an alkaline phosphatase labeled gene probe for enumerating V. cholerae, then study this organism in seafood products.
  4. This work is designed to find out if pathogenic strains of V. parahaemolyticus can multiply in post harvest oysters as does the total V. parahaemolyticus and, if so, the growth rate and temperature range. Information on the effect of mild heat treatment and freezing will be developed to determine the effectiveness of these processes on reducing the risk posed by pathogenic strains of V. parahaemolyticus in oysters. This task is related to Risk Assessment of V. parahaemolyticus.
Deliverables

 FY1999

FY2000

FY2001
  1. Publish methodology for species-specific alkaline phosphatase (AP) labeled probe based on tlh gene of V. parahaemolyticus. (A)
  2. Validate AP-probe methodology for tdh (pathogenicity) gene in V. parahaemolyticus. (A)
  3. Refine PCR technique for detecting tdh gene in enrichments of oyster samples. (A)
  4. Initiate development of a method for identification of V. parahaemolyticus (strains/clones) using filter bound DNA from colony lifts.(A)
  5. Assist with transfer of non-radioactive gene probe technology to regulatory laboratories through ORA Quality Assurance Program. (A)
  6. Train states shellfish regulatory lab personnel in gene probe techniques for enumeration of V. parahaemolyticus (B)
  7. Initiate collaborative data collection effort with states and ISSC. (B)
  8. Determine the post harvest multiplication of O3:K6 strains of V. parahaemolyticus in shellstock oysters.(D)
  9. Determine the growth rates and thermal death points of pathogenic strains of V. parahaemolyticus.(D).
  1. Publish methodology for V. parahaemolyticus<