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 pathogenicity AP-probe based on tdh gene.(A)
  2. Publish PCR technique for detecting tdh gene in enrichments of oyster samples.(A)
  3. Evaluate enrichment media to enhance recovery of tdh+ strains for use in combination with PCR.(A)
  4. Initiate characterization of tdh+ PCR bands from enrichments using PCR-RFLP to determine strain/ species responsible for band.(A)
  5. Continue development of a method for identifying V. parahaemolyticus (strains/clones) using filter bound DNA from colony lifts.(A)
  6. Complete data collection in collaboration with states.(B)
  7. Analyze data from all collaborators and publish.(B)
  8. Research for desired probe sequence, have probes synthesized and validate probe.(C)
  9. Evaluate the effect of various processing technologies on pathogenic strains of V. parahaemolyticus in oysters. (D)
  1. Complete research to determine if PCR-RFLP techniques can be used to determine the strain/species responsible for PCR bands that corresponding to tdh bands. (A)
  2. Complete development of a method for identification V.p. (strains/clones) using filter bound DNA from colony lifts.(A)
  3. Publish methodology and transfer technology to regulatory lab personnel.(A)
  4. Provide training on probe use to regulatory laboratories. Publish method. (C)
  5. Initiate studies on environmental levels of V. cholerae in seafoods and the effect of processing on these levels.(C)
Component 2
  1. Bio-Accumulation and Depuration of Enteric Organisms by Molluscan Shellfish.
  2. Inactivation of Caliciviruses in Bivalve Mollusks
Description Description
  1. Indicator organisms may not accurately predict the presence of enteric viruses in shellfish and their growing waters. This project will: 1) evaluate the effectiveness of alternative microorganisms, such as bacterial viruses, to better predict the hazards of human fecal pollution; and 2) determine the bio-accumulation/elimination kinetics of Caliciviruses and other enteric microorganisms from bivalve mollusks.
  2. 'Cooking' oysters prior to consumption may not provide protection from illnesses due to Caliciviruses . This study will determine if cooking and other remediation steps will inactivate and reduce the risk of illness to oyster consumers.
Deliverables

 FY1999

FY2000

FY2001
1. Initiate trials using Caliciviruses and bio-markers (fecal coliforms/male-specific coliphage).(A)

2. Publish data describing environmental factors that contribute to the occurrence of shellfish-related gastroenteritis outbreaks. (A)
  1. Complete studies using Calicivirus.(A)
  2. Determine if male-specific bacteriophage are suitable bio-markers for depicting the accumulation and elimination of Caliciviruses from Gulf coast oysters. (A)
  3. Determine a critical cooking matrix (time/temperature) to inactivate Caliciviruses in shellfish. (B)
  1. Initiate studies using hepatitis A.(A)
  2. Evaluate current indicator bacteria (fecal coliforms) or male-specific bacteriophage are suitable bio-markers for depicting the accumulation and elimination of hepatitis A from Gulf coast oysters.(A)
  3. Evaluation of the effect of various remediation technologies on Caliciviruses in shellfish (B)
Component 3 Molecular detection of human enteric viruses in shellfish focused on Norwalk-like virus (NLV) and hepatitis A virus (HAV).
Description Develop rapid processing procedures to concentrate viruses from shellfish tissue; optimize the overall virus recovery during processing; remove natural PCR inhibitors; and achieve sensitive and specific detection of NLV and HAV in contaminated shellfish.
Deliverables

 FY1999

FY2000

FY2001
  1. Complete optimization of HAV recovery.
  2. Define the detection sensitivity of HAV in contaminated shellfish.

1. Complete optimization of NLV recovery during various processing steps.

2. Define a detection sensitivity of NLV in contaminated shellfish.

1. Develop a quantitative PCR method for NLV in shellfish.

2. Construct, in vitro, a HLV vector for the quantitation assay.

NOTE: Some funding for FY1999 has been contributed by the Gulf of Mexico Program and funds are being sought for FY2000 through that same program. If those funds are not available, successful completion of this project will depend on additional technical staff (graduate student stipend or postdoctoral fellowship). Equipment need in FY2000: a biohazard hood ($10,000).

 

 

Legend | Abbreviations

FSI Project Number 9 RSVP Number: 38346     GPRA Goals: I.C., II.B.
Project Title Assessment of Technologies for Pathogen Reduction or Elimination
Personnel Name Office/Division FTE Component
S.E. Keller OPDFB/DFPP 1.0 1
G. Fleishman OPDFB/DFPP 0.5 4, 5
R. Reddy OPDFB/DFPP 1.0 4,6
V. Komolprasert OPDFB/DFPP 1.0 8
J.E. Schlesser OPDFB/DFPP 0.3 3
E.G. Murakami OPDFB/DFPP 1.0 7
M. Pascall OPDFB/DFPP 0.5 9
L. Jackson OPDFB/DFPP 0.5 7
C. Warner OPA/DPMU 0.8 2
L. Ali OPA/DPMU 1.0 3
D. Daniels OPA/DPMU 0.8 2
H. Solomon OSRS/DMS 1.0 6
T. Tran OSRS/DMS 0.5 2
R. Thunberg OSRS/DMS 1.0 2
T. Lilly OSRS/DMS 1.0 6
Additional Chemistry Support OPA/DPMU 0.5 8
Total FTE  

12.4
 

Non-FDA NCFST support Microbiologists and Technicians

 [5.0] 1,3,4,6,7,8
Administrative Liaisons David J. Armstrong, 708-728-4108

Richard E. McDonald, 708-728-4154.

D.B. Shah, 202-205-4981

Gregory Diachenko, 202-205-5320
Project Abstract This project will validate new approaches for controlling a variety of microbial contaminants in fruits and vegetables and processed food products and for reducing organisms or toxins that may cause foodborne illnesses from these products.
Project Description Several intervention strategies will be explored to improve the safety of fruits, vegetables, and processed food products. These strategies include improved sanitation techniques, alternative technologies (high pressure, pulsed electric field, ultrasound, and ultraviolet light processing),packaging, and irradiation.
Projected Impact Methods to improve the safety of raw agricultural products and processed commodities will be validated and published. Workshops and symposia will be organized to communicate the results of these studies. This project will provide the Agency with a substantial scientific basis for future FDA policies
Center Priorities Code 1.4a,b
Research Regulatory Needs Codes I.C; III.A; III.C
Component 1 Reduction of pathogens in fresh fruit juices.
Description Traditional and novel methods of disinfection will be investigated to determine the best approach to achieve a 5-log reduction in the appropriate target pathogen in fresh juices. To date, methods under examination include chlorine, ozone, UV, and surface heat treatments. Preliminary results have shown an approximately 1-log reduction in natural microflora on starting apples with chlorine (200 ppm, 5 min), and ozone (10ppm, 5 min). Data on surface heat treatment with inoculated apples (E. coli O157:H7) indicate a 2- to 3-log reduction at 95oC for under 60 seconds. Future plans include further testing and optimization of the above and additional testing of chlorine dioxide, detergents and various combinations of treatments.
Deliverables

FY1999

FY2000

FY2001

1. Finish evaluation of current ozone treatment system.

2. Complete surface heat treatment studies. Evaluate higher levels of chlorine.

1. Finish evaluation of higher Chlorine levels.

2. Begin evaluation of chlorine dioxide.

3. Examine use of detergents and/or surfactants in conjunction with other treatments.

1. Finish evaluation of chlorine dioxide and evaluation of combined treatments.

2. Recommend guidelines to improve safety of fresh fruit juice for use in HACCP plans.
Outside Funding $32,000 was received from NCFST for FY 99. Additional outside funding is needed to complete FY00 and FY01 deliverables.
Component 2 Effect of chemical disinfection on microflora in fresh produce
Description Recent studies indicate substantial regrowth of microflora after chemical intervention treatments of fresh produce. However, the nature of regrowth is unknown. The type of regrowth may effect the post processing infection, growth, and survival of foodborne pathogens. This study will examine the effect of the surviving population on the probability of foodborne illness in several ready-to-use fresh vegetables; namely, sprouts, broccoli, celery, lettuce and cauliflower. The results will be used to assess the potency and minimal doses of washing and ozone vs chlorine treatment. The chemistry of disinfectants used as interventions will also be studied to ensure that the compositions of the disinfectant solutions are exactly as specified for the experimental designs. A combination of commercial test kits and methods developed within CFSAN will be used to provide quality control for studies with sanitizing solutions.
Deliverables

FY1999

FY2000

FY2001
  1. Determine levels of natural microflora (pathogens & non-pathogens) on selected fresh produce.
  2. Study ozone stability as a function of concentration, pH, and total organic carbon (TOC) during treatment of fresh produce.
  3. Validate methods for iodate, bromate, and chlorite at ppb levels in produce.

1. Determine levels and regrowth of microflora on selected fresh produce after washing (ambient and hot), and/or different concentrations of chlorine or ozone.
  1. Study the effects of metal ions, short chain fatty acids, surfactants, and other chemicals on potency of ozone and chemical disinfectants.

1. Determine level and regrowth of microflora on selected fresh produce after inoculation with pathogens to determine effectiveness of sanitation treatments.

2. Fully characterize the physical and chemical parameters that must be controlled to maximize the effectiveness of ozone and other chemical sanitizers for fresh produce.
Component 3 Effectiveness of intervention strategies to control biofilms
Description It is well documented that current methods of testing the efficacy of antimicrobials/sanitizers using planktonic cells do not reflect the true efficacy of these agents against microbes that are present as biofilms in the environment including on foods. This discrepancy in methodology has adversely affected the regulatory agencies in assessing the real-life usefulness of antimicrobials. Also, methods are needed for the evaluation of treatments designed for the reduction of pathogens in processing environments and on produce. The Calgary Biofilm Device (CBD) has recently been introduced to determine the minimum inhibitory concentration (MIC) of antibiotics against biofilms on in-dwelling medical devices. The CBD will be used by us to develop a new biofilm-based antimicrobial efficacy test method. This method, after validation, can be incorporated in the Agency's guidelines for industry and will become a gold standard for evaluating intervention strategies to reduce or eliminate pathogens in food and food processing environments.
Deliverables

FY1999

FY2000

FY2001
  1. The CBD will be evaluated for usefulness as a model for biofilm research. Conditions for uniform growth and techniques for release and enumeration of cells will be developed.
  2. Initiate studies to evaluate the effects of chlorine and ozone on biofilms cultured on polished and brushed stainless steel, glass, and polyethylene film.
  1. Evaluate the performance of the CBD method and assess correlation to the current official methods and standards.
  2. Compare pure cultures with mixed microbial biofilms for efficacy of a selected antimicrobial agent.
  3. Continue to evaluate the effectiveness of ozone and different sanitizers to reduce/eliminate the microbial flora on food contact surface areas.
  4. Conduct studies using artificial inoculation of the food processing surface areas with E. coli O157:H7 and/or L. monocytogenes to determine the effectiveness of washing/sanitation.
  1. Validate the method refined earlier. Incorporate method in the Agency=s policy guidelines to industry and use in setting performance standards for putative antimicrobial agents.
  2. Continue inoculated biofilm studies to determine the effect of washing/sanitation using a variety of disinfectants and assess their effectiveness on biofilm elimination and reduction of potential microbial contaminants.
  3. Develop guidelines to improve sanitation practices for use in HACCP plans.
Outside funding $14,600 was received in FY 99 from NCFST and will be required for FY 00 and FY 01. JIFSAN application to complete CSLM analysis at Univ. of Maryland is pending.
Component 4 Safety Assessment of Pulsed Electric Field (PEF) Processing Technology
Description PEF Processing is a promising alternative method of preserving foods, which could be used to complement or replace traditional thermal processing methods. Previous studies have shown that PEF is capable of producing a pasteurization-type of effect on liquid foods to reduce the number of pathogenic microorganisms present. However, different implementations of PEF technology result in varying lethality, indicating a lack of sufficient understanding of the basic effect of PEF on pathogens. This understanding is vital if PEF is to be commercialized. The goal of this project is to quantify lethality as a function of PEF process variables. This approach allows for the prediction of lethality, which is necessary for process design and pathogen monitoring to assure that lethality is established and maintained.
Deliverables

 FY1999

FY2000

FY2001
  1. Modify static chamber using recommendations from the PurePulse Company.
  2. Establish an approach of gel casting to eliminate nonuniform field influence.
  3. Test chamber for consistency of treatment.
  1. Measure the effect of thermal energy and determine its effect on bacterial lethality to isolate the electric field contribution to lethality.
  2. Perform experiments to measure the influence of PEF parameters on the electric field lethality.
  3. Develop equation(s) to define the kinetics of PEF.
  1. Use kinetic equation(s) to predict lethality of microorganisms in a continuous flow PEF processor. Verify with a PEF continuous flow processor.
  2. Determine experimental protocol whereby the static chamber can be used to measure kinetic parameters of microorganisms.
Outside Funding $26,000 was received from the NCFST for FY 99 and will be needed for FY 2000 and FY 2001.
Component 5 Safety Assessment of High Frequency Ultrasound Technology.
Description Conventional ultrasound (20 kHz) has a partial bactericidal effect. A complete bactericidal effect is realized only in conjunction with other lethal agents, making this a challenge to use in food systems. HFUS (600 kHz) having higher energy may be capable of eliminating pathogenic bacteria purely by non-thermal ultrasonic action. This project will determine if significant lethality can be obtained through the use of HFUS.
Deliverables

FY1999

FY2000

FY2001
  1. Obtain or design / fabricate equipment for processing of surrogate bacteria.
  2. Establish experimental protocol with surrogate bacteria.
  3. Determine lethality of HFUS on surrogate bacteria.
  4. Evaluate containment issues for treatment of pathogenic bacteria. Implement equipment design based on this evaluation.
  1. Determine the effect of HFUS parameters (intensity, processing time) on lethality in surrogates.
  2. Investigate possible roles of gas sparging, temperature and other influences on the efficacy of HFUS in surrogates.
  1. Investigate the lethality of HFUS on E. coli O157:H7.
  2. Investigate the possible use of gas sparging, temperature and other influences on the efficacy of HFUS on E. coli O157:H7.
  3. Evaluate the potential for scale-up; develop proposal for a pilot scale project involving HFUS.
Component 6 Inactivation of Clostridium botulinum spores by high pressure processing (HPP).
Description HPP can be used to inactivate microorganisms in certain foods or food ingredients without a decrease in product quality. Since there are no published reports on the resistance of C. botulinum spores to HPP, there is a need for a kinetic-based process delivery calculation and validation protocols. Process establishment would then be a function of process conditions and the resistance of the appropriate organism of concern. We have initiated studies on the effect of pressure, temperature, and time on inactivation of C. botulinum types A and E and nonproteolytic type B spores in phosphate buffer (pH7.0). Up to a 3-log unit reduction of type A strains can be obtained at a maximum pressure (827 MPa) and temperature (75oC) combination for 15-20 min. Results of this study will benefit the FDA, industry, and NCFST in the application of HPP for inactivation of C. botulinum spores in various low-acid foods and food ingredients.
Deliverables

FY1999

FY2000

FY2001

1. Validate conditions that inactivate C. botulinum spores types A and E and nonproteolytic type B strains in a model food system.

1. Continue to study inactivation of C. botulinum spores of strains proteolytic type A and nonproteolytic type B in a model food system.

2. Determine D- and Zp-values of C. botulinum type E strains in a model buffer system.

1. Continue determination of D- and Zp-values of C. botulinum type E and other types.

2. Determine effect of pressure (up to 827 MPa) and temperature (up to 120oC) on inactivation of spores of C. botulinum type A and nonproteolytic type B strains in a model buffer system as well as a low-acid food system.

Outside Funding $35,000 was received in FY 99 from NCFST and will be needed to complete FY 00 and FY01 deliverables.
Component 7 Processing of fruit juices using ultraviolet (UV) light.
Description A big obstacle in the application of UV light processing in fruit juices is the low transmittance at the UV range. For UV processing to be effective, the required dosage at a specified wavelength must be delivered. In juices, dosage can be accurately calculated if transmittance data is available. A major focus of this project is to develop protocols to validate the UV dose for destruction of pathogens. Effectiveness of UV light against target pathogens and surrogates will be evaluated using a continuous type UV reactor. Although there are several reports of the effectiveness of UV light against several types of microorganisms, most of the studies were done in water and the UV dosage used were much higher than the recommended value. In water, the practice of overdosing is common for safety purposes. However, in juices, this practice is not recommended due to potential undesirable effects, (e.g., change in flavor and deterioration of nutrients). Chemical actinometry will be used to measure radiation dose in juices subjected to UV radiation. Natural components of juice will be monitored for suitability to use as radiation markers. One chemical marker to be studied in detail is vitamin A, which has been shown to degrade rapidly at wavelengths < 330 nm (Allwood and Plane, 1986). Finally, degradative photochemical reactions, such as browning and vitamin deterioration, will be studied in juices subjected to UV processing.
Deliverables

FY1999

FY2000

FY2001
  1. Install/design laboratory UV reactors, batch and continuous types.
  2. Develop protocols for microbiological studies and chemical actinometry for determining UV radiation dosage (in water and fruit juices).
  3. Conduct a trial study to assess the effectiveness of UV light in inactivating Cryptosporidium.
  1. Determine the germicidal effectiveness of UV on surrogates of E. coli 0157:H7 and other pathogens in water and several fruit juices.
  2. Evaluate various compounds that are present in commercially prepared juices for use in chemical actinometry.
  1. Develop computer model for evaluating intensity distribution in a reactor and predicting dosage delivery.
  2. Identify critical design parameters for UV reactors.
  3. Determine the effect of UV treatments on light sensitive nutrients of several fruit juices.
Outside Funding Funding has been requested from the NCFST for FY 00 and FY 01.
Component 8 Evaluation of packaging materials for irradiated pre-packaged foods
Description The selection and approval of polymeric packaging material for use in food irradiation (or other applications) depends on the resistance of the packaging to chemical or physical/ mechanical changes when subjected to applicable doses of radiation. This study will investigate the effects of ionizing radiation on several polymeric packing materials to determine the relationship of polymer structure to the extent to which irradiation either generates products in the polymer or alters the functionality of the polymer so that compounds migrate into food products. Effects of gamma and e-beam irradiation at various doses on chemical changes in the model test materials will be determined. Several test packaging materials of industry interest will be selected, irradiated at various conditions and subsequently analyzed to identify and quantify radiolytic products that may be generated. The results obtained will aid the petition review process of approving new packaging materials for food irradiation applications.
 

Deliverables

FY1999

FY2000

FY2001
  1. Develop and validate analytical methods for identification and quantification of volatile and nonvolatile compounds (ESR, HPLC, and GC/MS).
  2. Begin migration study using food simulants, and analyze the irradiated test materials in comparison with nonirradiated packaging materials.
  1. Continue analytical methods development and validation, continue e-beam and gamma irradiation experiments using the selected test materials, continue analysis of the irradiated test materials.
  2. Optimize use of mass spectroscopy (bench top, tandem, and time of flight) for the analysis of radiolysis products from irradiated polymeric packaging materials.
  1. Complete irradiation, migration study, data analysis and evaluation of the selected packaging materials.
  2. Determine whether the effects of e-beam and gamma irradiation on the test materials are equivalent or different.
  3. Develop test protocol for assessing packaging materials and assist the Office of Premarket Approval in developing guidelines or a points-to-consider document for industry.
Outside funding $28,000 funding was received from NCFST in FY 99 for work at Moffett Center. CFSAN received $20,000 from U.S. Army, Natick for high-dose radiation study in Washington. Additional outside funding is needed from NCFST, and the U.S. Army to complete FY 00 and FY 01 deliverables.
Component 9 Package integrity evaluation
Description The FDA regulations for all metal double seamed cans are clearly stated in 21 CFR 113.60(a), but the regulations for heat scaled flexible and semi-rigid plastic packages are not clearly specified. The inspection of semi-rigid plastic containers is mostly subjective and done primarily by visual inspection. This procedure is in contrast to the much more objective dms-down procedures for metal double-seamed cans. During the training of our FDA inspectors, the severe limitations of our current inspection techniques for plastic containers have become apparent. This reality, coupled with the increased use of plastic packaging for LACF foods makes it necessary to investigate the seal integrity of currently used plastic materials and packages. The results from the research work will suggest appropriate methods for inspection of heat-sealed (fusion and peelable) plastic packages. These methods will then be published and incorporated into the inspection protocols for plastic packages.
Deliverables

 FY1999

FY2000

FY2001
  1. Evaluate pressure differential and magnetic resonance imaging online, and pressure differential and ultrasonic imaging bench unit non-destructive inspection systems.
  2. Initiate distribution testing of package and correlate with non-destructive leak detection systems in #1.
  3. Participate in training exercises for FDA field inspectors and investigators involved in container inspection, post-processing contamination inquires and recalls.
  1. Complete distribution testing.
  2. Complete container integrity testing using different container types from those already tested.
  3. Write testing protocol and findings to help develop new FDA inspection guidelines.
  4. Participate in training exercises for field inspectors and investigators.
  1. Complete testing protocol.
  2. Publish results in packaging related journals.
  3. Conduct training exercises for field inspectors and investigators
Outside funding A proposal has been submitted to obtain funding from the NCFST for FY 00 and 01. Need additional outside funding to complete FY 00 and FY01 deliverables including $49,000 for an Instron peel tester.

 

 

Legend | Abbreviations

FSI Project Number 10 RSVP Number 38788   GRPA Goals II.C.
Project Title Mycotoxins
Personnel Name Office/Division

FTE

Component
 

R. E. Eppley

 OPDFB/DNP

1.0

 

1
M. E. Stack OPDFB/DNP

1.0

1, 2, 3
F. Thomas OPDFB/DNP

1.0

3
V. Tournas OPDFB/DNP

1.0

1,2
M. W. Trucksess OPDFB/DNP

0.5

3
K. Young OPDFB/DNP

1.0

1,3
L. S. Jackson OPDFB/DFPP

0.5

4
T. Black OSRS/DTR

0.5

5,6,7
T. Collins OSRS/DTR

0.5

5,6,7
L. Garthoff OSRS/DTR

1.0

5,6,7
D. Hinton OSRS/DTR

0.5

5,6,8
S. Long OSRS/DTR

0.4

5,6,7
N. Olejnik OSRS/DTR

0.6

5,6,7
D. Quander OSRS/DTR

0.4

5,6,7
R. Brown OSRS/DTR

0.3

5,6,7
J. Rorie OSRS/DTR

0.6

5,6,7
M. Scott OSRS/DTR

0.5

5,6,8
M. Smith OSRS/DTR

0.4

5,6,7
T. Sobotka OSRS/DTR

0.4

5,6,7
R. Sotomayor OSRS/DTR

1.0

6,8
R. Sprando OSRS/DTR

0.6

5,6,7
M. Washington OSRS/DTR

1.0

6,8
Total FTE

14.7
Administrative Liaison Mary W. Trucksess 202-205-4429,

Neil L. Sass 301-594-5800
Project Abstract To develop information needed for hazard identification (identification and production of mycotoxins), risk assessment (occurrence and toxicity) and risk management (analytical methods for monitoring and regulatory actions).
Project Description Mycotoxins produced by molds are common contaminants of many important food crops, including wheat, corn, and peanuts. Some mycotoxins are found in fruits and vegetables. These contaminants have a broad range of toxic effects, including carcinogenicity, neurotoxicity, and reproductive and developmental toxicity. In addition to their potential human toxicity, many mycotoxins have antibiotic activity and may influence the growth of pathogenic bacteria. Regulatory limits, including international standards, have tremendous economic impact and must be developed on science-based risk assessments to have validity. The purpose of this research is to generate the information, including exposure and toxicity data, needed for risk assessments and to develop analytical methods for control programs (regulatory and HACCP). The studies involved in the development of data also include microbial ecology, particularly the relationships of mycotoxins with pathogenic bacteria, and the effects of processing on mycotoxin levels in human foods, as well as assessment of various types of toxicity.
Projected Impact Project reduce exposure to mycotoxins in foods and feeds and will provide the Agency with necessary data for setting FDA guidelines on mycotoxins.
Center Priorities Code 2.16a, 2.16b
Research Regulatory Needs Codes IV. A.
Component 1 Production of Mycotoxins
Description Production of toxins in cultured corn and isolation and identification of suspect mycotoxins from Fusarium species, and cyclopiazonic acid from Aspergillus flavus, for use as analytical standards and for toxicological evaluation.
Deliverables

FY1999

FY2000

FY2001
  1. Isolate sufficient quantities of Fusarium toxins for preliminary toxicological evaluation and for use as standards in method development.
  1. Produce cyclopiazonic acid and isolate sufficient quantities for toxicological evaluation.
  1. Isolate sufficient quantities of Fusarium toxins for long term toxicological studies
Component 2 Profiling Mold Flora in Fresh Produce and Juice
Description Determination of mold flora in pretreated fresh produce, such as grapes, apples, tomatoes, berries, spouts and in Aready-to-eat@ salads; isolation and identification of potential toxin producing molds; and investigation of the occurrence of suspect toxins in fresh produce.
Deliverables

FY1999

FY2000

FY2001
  1. Identify produce that is frequently contaminated with molds.
  1. Isolate and identify the molds from the produce and investigate if they produce toxins.
  
Component 3 Development and Validation of Analytical Methods
Description Development and validation of improved methods for rapid testing; investigation of chromatographic methods, immunochemical methods, and emerging analytical techniques.
Deliverables

 FY1999

FY2000

FY2001
  1. Develop methods to detect and quantitate trichothecenes in grains.
  2. Develop general procedures to confirm identity of ochratoxin A in raisins and fumonisins in grains.
  3. Develop a solid phase extraction method for determining patulin in apple juice. Use the method for the patulin and E. coli interaction study.
  4. Determine the effect of patulin on E. coli growth in apple juice.
  1. Continue study of method for trichothecenes and prepare report for publication.
  2. Report results of patulin in fruit juices study.
  3. Develop method for ochratoxin A in grape juice and wine.
  4. Complete extensive study of interaction of patulin and E. coli O157:H7. Prepare manuscript for publication.
  5. Validate a rapid method for fumonisins in corn and sorghum by conducting an international collaborative study.
  6. Investigate the use of microwave extractor for the analysis of mycotoxin in foods.
  1. Develop analytical method for moniliformin in grains and prepare manuscript for publication.
  2. Develop chromatographic method for the major ergot alkaloids in wheat, rye and other grains and prepare a collaborative study to validate the method.
  3. Develop a quantitative method for cyclopiazonic acid in corn and peanuts.
 Component 4 Effect of Processing on Mycotoxins
Description Determination of the effects of processing/chemical treatments on mycotoxins in foods.
Deliverables

FY1999

FY2000

FY2001
  1. Determine the effects of added sugars on fumonisin levels in extruded corn grits.
  2. Report on the effects of electron beam irradiation on one mycotoxin in corn.
  1. Report on the effects of electron beam irradiation with and without chemical treatment(s) on a mycotoxin in corn.
  2. Report on the effects of thermal processing on one selected mycotoxin produced by Fusarium species.
  1. Report on the effects of processing/chemical treatments on a mycotoxin produced by a selected Fusarium species.
Component 5 In Vitro and in Vivo Studies to Determine the Immunotoxic, Neurotoxic, and Developmental Toxicity of Selected Mycotoxins
Description Toxicological investigation of the effects of sub-chronic exposure to dietary vomitoxin , deoxynivalenol [DON], in IL-6 Deficient Mice. Collaborative study with Michigan State Univ. Using the genetically modified IL-6 knockout mouse model.
Deliverables

FY1999

FY2000

FY2001
  1. Continue imaging analysis, pathology evaluation, and special (immunostaining) of lymphoid tissue from IL-6 study.
  2. Conduct planned analyses of brain tissue from mice exposed to dietary deoxynivalenol.
  3. Process tissues, write manuscript on the male testicular toxicity evaluation.
  1. Compare the pathology evaluation and imaging analysis of the lymphoid tissues, i.e., spleen, lymph nodes, including Peyer's patches and thymus, from the IL-6 study.
  2. Complete neurochemical and histological analyses of sections of brain tissue from DON exposed mice. These analyses include assessment of changes in neurocellular protein profiles and special histochemical stains to assess treatment-related neurodegeneration, microglial proliferation, and changes to brain barrier permeability.
  1. Collaborate with Michigan State University on completion of manuscript for the immunotoxicity study.
  2. Complete manuscript of work on neurotoxicity of DON in IL-6 study.
Component 6 Toxicological Study of Selected Fusarium toxins (Deoxynivalenol and zearalenone).
Description Toxin or combined toxins will be fed to pregnant rats from day 6-20 of gestation and the effects will be noted on the fetuses. A preliminary study will be run to assess the dose of the toxin to be fed to the pregnant rats. Then a final study will be run to assess the developmental toxicity and genotoxicity of the toxin.
Deliverables

FY1999

FY2000

FY2001
  1. Develop preliminary protocol for deoxynivalenol (DON) developmental toxicity study and male reproduction study and run studies. Develop final protocol. Run final study.
  2. Develop protocol for the male testicular toxicity study on DON.
  3. Develop preliminary protocol for zearalenone developmental toxicity study. This will be a range-finding study focusing on standard teratologic endpoints as well as anogenital (A-G) distance to evaluate the potential estrogenic effect.
  4. Initiate protocol development for the biochemical and immunotoxicity studies of DON in rats.
  1. Completion of DON developmental toxicity final study. Begin final report.
  2. Completion of DON male reproduction study.
  3. Run zearalenone developmental toxicity preliminary study. Develop zearalenone final study protocol.
  4. Develop protocol with OPDFB to investigate the neurologic effects of repeated exposure, reasonable comparable to human exposure levels, to selected Fusarium mycotoxins (e.g., DON or zearalenone) in rats. Measures of behavioral function (e.g., sensorimotor and cognitive), in conjunction with correlative morphochemical analyses, will focus on the detection of subtle treatment- related effects on the nervous system.
  5. Initiate biochemical and immunotoxicity studies of DON in rats, possible in vitro and in vivo studies after confirmation from OPDFB for risk relevance.
  1. Complete DON final report and manuscript.
  2. Run zearalenone final study and prepare final report.
  3. Initiate studies to investigate the neurotoxicity (behavioral and neurochemical) of select Fusarium mycotoxins.
  4. Compare biochemical and immunotoxicity studies. Initiate preparation of final report(s).
  5. Develop reproductive toxicity preliminary protocol for evaluation of combinations of DON and zearalenone.
Component 7 Toxicity Study of Fumonisins
Description Determine the toxic effect of fumonisins on male rats or female rats.
Deliverables

FY1999

FY2000

FY2001
  1. Initiate preparation of final report on the study of the neurobehavioral effects of fumonisin B1.
  1. Complete final report of totally hydrolyzed fumonisin B1developmental toxicity study.
  2. Complete report of neurobehavioral effects of fumonisin B1 in rats.
  1. Prepare manuscript for the developmental toxicity of totally hydrolyzed fumonisin B1.
  2. Publish report of neurobehavioral effects of fumonisin B1.
Component 8 Biochemical and Immunologic Endpoints in Toxicologic Studies with Rats Exposed to Aflatoxin B1 and Aflatoxin B1 in Combination with Selected Mycotoxins
Description Determine DNA/RNA adduct formation and detoxification enzymes in select tissues, as well as changes in lymphocyte cell subsets and function in lymphoid tissue as a function of time and cumulative dose in a study of continuous vs. intermittent dosing of aflatoxin B1 to assess the effects of interactions of AFB1 with fumonisin B1, and with cyclopiazonic acid (CPA) on biochemical endpoints, e.g., DNA and RNA adduct formation, and immunological endpoints.
Deliverables

FY1999

FY2000

FY2001
  1. Complete study on the effects of intermittent and continuous exposure to AFB1 on DNA/RNA adduct formation in liver, kidney and testis of rats.
  2. Complete manuscript on glutathione S-transferase and DNA adducts in liver and testes.
  3. Complete manuscript on the temporal patterns of DNAadduct formation by AFB1 in rat liver and testis.
  4. Complete first manuscript on the flow cytometric and cytokine measurements with rat spleen lymphocytes.
  5. Initiate manuscript on the effects of intermittent and continuous exposure, i.e., DNA and RNA adducts at all dose levels tested, for the rat liver.
  6. Continue image analysis of lymphoid tissues, pathology evaluation and special evaluation (immunostaining) of the lymphoid tissue.
  1. Complete manuscript on adduct formation in rat liver.
  2. Initiate manuscript preparation on comparison of adduct formation in the rat kidney and liver
  3. Complete a second manuscript comparing functional and ex-vivo analysis of immunologic endpoints with the histopathology results.
  4. Develop protocol(s) to evaluate biochemical and immunologic toxicity associated with combinations of AFB1 and fumonisin B1 (in vivo and in vitro studies) using the rat and/or rat hepatocytes and lymphocytes.
  5. Use human hepatocyes, lymphocytes, and/or explants for the above study.
  1. Initiate and/or continue studies on the interactions of AFB1 and fumonisin B1.
  2. Develop protocol(s) to evaluate biochemical and immunologic toxicity associated with combinations of AFB1 and cyclopiazonic acid (in vivo and in vitro) using the rat and/or rat hapatocytes and lymphocytes.
  3. Use human hepatocyes, lymphocytes, and/or explants for the above study.

 

 

Legend | Abbreviations

FSI Project Number 11 RSVP Number 38459 GPRA Goals I.D.
Project Title Virulence Assessment and Molecular Pathogenesis of Multi-Drug-Resistant Salmonella Typhimurium
Personnel  

Name

 Office/Division FTE

Component
D.Hanes OPDFB/DVA 0.5

1,2
K. Lampel OPDFB/DVA 1.0

3
L. Kornegay OPDFB/DVA 1.0

3
C. Giri OSRS/DMS 1.0

4
Don Burr OPDFB/DVA 0.5

1
Total FTE 4.0  
JIFSAN students  
Collaborators:

D. Kopecko

FDA/CBER

4
Administrative Liaison D. Hanes, 301- 827-8075

B. A. McCardell, 202-205-4262
Project Abstract This project will compare the virulence of multi-antibiotic resistant S. Typhimurium with non-resistant strains, use animal models to generate dose response data, determine means of transfer of resistance genes, and develop rapid detection methods for MDR ST and for Shigella.
Project Description Initial reports on multi-drug resistant S. Typhimurium (MDR ST) suggested that it was associated with a higher mortality rate than non-resistant S. Typhimurium. Using animal and tissue culture models, the virulence of MDR ST strains will be compared with non-resistant Typhimurium and other salmonellae isolates. An animal model that mimics diarrheal disease will be used to determine the dose response curve of MDR ST.

Chromosome- and plasmid-mediated antibiotic resistance will be examined. Mode of transfer will be studied by examining the flanking regions of the resistance genes. A rapid PCR method will be developed to detect any MDR ST.

Rapid methods for the detection of Shigella will be developed, adapted for produce and other relevant foods, and transferred to FDA field labs.
Projected Impact This project will supply information on MDR ST for science-based regulatory decisions. Dose response data will be collected from animal models for hazard assessment. New, rapid detection methods for MDR ST and Shigella will be designed, evaluated and transferred to FDA laboratories.
Center Priorities Code
Research Regulatory Needs Codes X.B, XI.A., I.A
Component 1 Development of an animal diarrhea model in healthy and chronically ill animals.
Description MDR ST will be tested in several animal models to find the best model of human disease. Another animal model utilizing animals with chronic illness also will be selected for further study. Dose response data will be colleted using these two animal models and compared with non MDR ST and other Salmonella spp.
Deliverables

FY1999

FY2000

FY2001

1. Develop animal models

2. Dose response data from diarrhea model.
  1. Publish animal diarrhea model.
  2. Develop chronic illness animal model..
 1. Publish results of chronic illness model.

2. Provide dose response data
Component 2 Study of a hemolysin and its role in virulence
Description Recently discovered a hemolysin will be studied by :
  1.   Cloning gene,
  2.   Using PCR use to determine distributions of gene is among Salmonella and other bacteria,
  3.   It's role in virulence will be tested in the animal diarrhea model using strains defective a hemolysin production.
Deliverables

FY1999

FY2000

FY2001
1. PCR developed based on sequences from related Aeromonas hydrophila a hemolysin. 1. Gene cloned. 1. Mutants produced and studied.
Component 3 Rapid Methods
  1.   Development of rapid method for detection of MDR ST
  2.   Development of templates for PCR assays by FDA field labs.
  3.   Development of rapid method for Shigella.
Description
  1.   Genes coding for antibiotic resistance to ampicillin, streptomycin, sulfa, tetracycline and chloramphenicol will be sequenced and primers selected for PCR.
  2.   Method will be transferred to FDA field labs. Antibiotic resistance transfer will be studied.
  3.   Current detection methods for Shigella are time-consuming and cumbersome. This aspect is focused on developing a rapid PCR detection. method. for In collaboration with FDA Field labs, this method will be used on all foods.
Deliverables

FY1999

FY2000

FY2001
  1. Develop PCR assay for MDR ST targeting at least 3 antibiotic resistance genes. (A)
  2. Determine which resistance genes are on plasmids.(A)
  3. Complete design of PCR template preparation for sprouts.(B)
  4. Complete study and further development of method with FDA field labs. (1999 Compliance Program- Imported Produce Sampling Assignment). (B)
  1. Develop rapid method for all 5 antibiotic genes.(A)
  2. Examine role of plasmids in rapid acquisition of resistance genes. Determine sequences of chromosomal flanking regions (integrons).(A)
  3. Complete design of PCR template preparation for other produce.(B)
  4. Publish Shigella methods. (C)
  1. Collaborate with FDA field on use of the method.(A)
  2. Publish results of plasmid study.(A)
  3. Determine role of flanking sequences in rapid acquisition of antibiotic resistance genes.(A)
  4. Publish results of study of chromosomal sequences in acquisition of resistance genes.(A)
  5. Transfer methods to FDA field labs.(B)
  6. Adapt method for any newly available technology such as biosensors. (C)
Component 4 Studies to identify new virulence genes functionally involved in MDR-ST pathogenesis.
Description Human intestinal epithelial and mouse macrophage tissue culture cell lines will be employed to identify MDRST genes that are differentially expressed during host cell invasion and excocytosis. Such genes will be identified by the techniques differential RT PCR display and by using of green fluorescent protein (GFP) gene insertions to monitor gene expression. The phenotypes resulting from expression of these genes may define unique pathogenesis traits of the MDR-ST strains.
Deliverables

FY1999

 FY2000

FY2001
  1. Develop/modify cell invasion/exocytosis assays for MDR-ST strains in CaCo-2 and INT-407 cells.
  2. Conduct kenetic analyses of MDR-ST into CaCo-2 or INT-407 cells.
  3. Develop differential RT-PCR display assay protocols for use in examining differential gene expression within infected host cells.
  4. Develop and optimize RNA purification procedures to obtain required quantities of bacterial RNAs from infected host cells.
  1. Develop/modify cell invasion/exocytosis assays for MDR-ST strains in mouse macrophage cell line, J774A.1.
  2. Construct a promotor-less procaryotic expression vector containing both the GFP and the CAT structural genes to clone MDR-ST inducible promoters upstream from these reporter genes.
  3. Optimize differential RT-PCR display protocols to examine MDR-ST induced genes in various host infected cell lines.
  4. With the use of differential display technique and the GFP/CAT expression plasmid vector, identify the isolated bacterial cDNA clones that are differentially regulated.
  5. Prepare the data on invasion kinetics and differential gene expression for presentation at meetings and publication.
  1. Molecularly characterize the isolated cDNA clones by partial DNA sequencing and subsequent gene bank analysis to identify functionally involved genes.
  2. Determine whether unique MDR-ST genes are induced in macrophage versus intestinal epithelial cell lines.
  3. Initiate studies to determine the pathogenic functions of differentially expressed genes.
  4. Prepare and analyze the data on characterization of the identified clones for presentation at meetings and for publications.

Propsed but unfunded needs:

Equipment: Sorval Centrifuge Rotors SS34 and GSA, Spectophotometer for MOD-1, DNA sequencing equipment

Travel: Each investigator plans to present at one major meeting such as ASM

International travel to a Salmonella meeting in France

Local US-Japan Cholera conference in Baltimore

Training: Chad Giri to a Differential Display Training Course

One meeting/course per investigator on the project

 

 

Legend | Abbreviations

FSI Project Number 12 RSVP Number 38652
Project Title Minimizing Biogenic Amine Formation in Seafoods and Other Commodities
Personnel Name Office/Division FTE Component
W. Staruszkiewicz OS/DSAT/WSL 0.5 1,2,3
P. Rogers OS/DSAT/WSL 0.9 1,2,3
G. Ziobro OPDFB/DNP 0.5 4
P. Valdes Biles OPDFB/DNP 0.5 4
TOTAL FTE   2.4  
 

Proposed but unfunded positions

 Support Scientists [2]
Administrative Liaison G. Hoskin, 202-418-3172

J. Gecan, 202- 260-2022
Project Abstract Effects of on-board and post-harvest handling parameters on the formation of decomposed and scombrotoxic fish.
Project Description Biogenic amines, which are formed by bacterial processes associated with spoilage, can have serious health consequences, including life-threatening responses in sensitive individuals. When held for excessive periods of time, the microflora of specific species of fish convert amino acids to biogenic amines (e.g., histamine), which produce characteristic symptoms in humans. Production of scombrotoxins in these fish is dependent on the time and temperature of storage prior to consumption. In addition to seafoods, biogenic amine concerns are also encountered with certain types of cheeses and have been suspected in several cases involving bean sprouts and certain fermented foods.
Projected Impact One goal is to develop improved methods for the detection of biogenic amines in a wide variety of foods. Another goal is to evaluate commercially available starter microbial cultures for their production of biogenic amines. A major thrust of the current project will be the use of microbial species known to rapidly produce biogenic amines, to establish the time and temperature limits for key seafood processing and distribution steps, and to ensure minimization of bioamine formation. The results will be used to develop guidelines for the seafood and fermented foods industries.
Center Priorities Code
Research Regulatory Needs Codes VI.C.2,3
Component 1 Effects of on-board and post-harvest handling parameters on the formation of decomposed and scombrotoxic fish.
Description This task involves the measurement of the effects of time/temperature abuse on fish during harvest and handling with respect to the development of scombrotoxic products. The study requires the capture of live fish and total control of holding conditions on-board the vessel and at dockside to measure the formation of the biogenic amines histamine, cadaverine and putrescine. Data have been collected for mahimahi (acquired by trolling) and for a few samples of skipjack and yellowfin tuna. Additional data are needed for tuna harvested by long liners and for unfrozen fish held under extended refrigeration. In our initial experiments on mahimahi, we have observed in certain types of samples, the production of amine decarboxylase, which later reacts after storage to produce large amounts of histamine. These phenomena may be most critical under the conditions on long liner vessels and in long term refrigerated storage.
Deliverables

FY1999

FY2000

FY2001
  1. Report on effects of on-board and post-harvest handling for the development of decomposed and scombrotoxic mahimahi.
  2. Draft recommendation on regulatory limits for chemical and sensory indicators of decomposition in mahimahi.
  1. Determine the effects of on-board and post-harvest handling of tuna on long liner vessels.
  2. Measure the effects of various refrigeration temperatures on unfrozen tuna in extended storage.
Component 2 Compare techniques for detecting scombrotoxic fish and the consequences of gaseous treatments prior to storage.
Description This work will provide an objective comparison of traditional techniques, such as the AOAC GLC method & HPLC, to innovative approaches including the AElectronic Nose@ and new test kits in preparation by commercial firms. Due to the complex industrial processes used for various pelagic species, no single test protocol is expected to satisfy all applications. A balance of speed, reliability and cost can best be realized through evaluations of a range of techniques in an objective setting using standard samples. Such a comparison should include a measure of the limitations of sensory evaluations as well. The expanding applications of gaseous treatment of fish with Asmoking machines@ and carbon monoxide mixtures raises questions about alterations in spoilage pathways and rates and on techniques for identifying scombrotoxic products. The preparation of a standard pack to measure these effects is proposed in conjunction with the method evaluations.
Deliverables

FY1999

FY2000

FY2001
 
  1. Conduct analyses on a decomposition pack to determine the extent of changes produced by Asmoking machines@ and carbon monoxide on tuna.
  2. Evaluate the use of the AElectronic Nose@ in comparison to other approaches for the detection of scombrotoxic fish.
  
Component 3 Investigate the roles of specific decomposition metabolites in consumer illnesses from scombroid and non-scombroid fish
Description Description There appears to be more than one type of consumer illness due to the ingestion of decomposed fish. In addition to the traditional problem caused by fish containing high levels of histamine, there have been many samples collected and analyzed that exhibit increased levels of biogenic amines but histamine is absent. The need to better understand all of the causative properties of spoiled fish responsible for human illnesses will be explored by collecting samples incriminated in poisoning incidents for chemical analyses and for animal testing by the Division of Toxicological Research.
Deliverables

FY1999

FY2000

FY2001
 

1. Report on the analyses of samples of fishery products associated with consumer illnesses.
  1. Results from animal tests to better define the health risks of mixtures of biogenic amines in fisheries products.
  2. Survey of fresh and frozen fish at dockside and at retail to estimate the incidence of potentially scombrotoxic fish.
Component 4 Biogenic amines in products other than seafood.
Description Determine presence of and levels of risk for bioamines in products other than seafood.
Deliverables

FY1999

FY2000

FY2001
  1. Evaluate current methodologies for the detection and recovery of bioamines from a wide variety of cheeses, vegetables, and other non-seafood products.
  1. Survey food products (e.g. cheeses) for bioamines.
  2. Develop model system to study bioamine production using mung bean sprouts or other appropriate substrate.
  1. Based on survey results and literature review of health effects of bioamines, prepare official draft guideline that will support direct reference regulatory action based on potential hazard to health from a chemical health hazard.

Proposed but unfunded equipment:

A replacement HPLC solvent delivery system is needed. $30,000.

 

 

Legend | Abbreviations

FSI Project Number 13 RSVP Number 39667  
Project Title Survival of Food Pathogens during the 60-Day Aging Period of Hard Cheeses Made from Unpasteurized Milk
Personnel Name Office/Division FTE
J. E. Schlesser OPDFB/DFFP 0.5  
Microbiologist NCFST {1.0}  
Total FTE   0.5  
Proposed but unfunded position: Postdoctoral research associate    

[1.0]

  
$45, 000 per year    

 

  

 
Administrative Liaison D. Armstrong, 708-728-4108; J. Mowbray, 202-205-1731
Project Abstract An evaluation of the current practice of 60-day aging of hard cheeses made from unpasteurized milk will be conducted.
Project Description Research is needed to assess whether the 60-day aging process of cheese made from unpasteurized milk is adequate to eliminate foodborne pathogens. Cheddar cheese will be made from milk inoculated with food pathogens (Escherichia coli O157:H7 and Listeria monocytogenes). Food pathogen levels will be monitored during the cheese making and aging period. Heat treatment of 64.4 °C for 16s in combination with 60-day aging will also be evaluated. The data will be incorporated into microbial risk assessment models to assist in determining whether this procedure provides an adequate level of public health protection.
Projected Impact In the event that 60-day aging is found to be inadequate to provide the appropriate level of public health protection, an evaluation of alternative control measures would assist the agency in the development of policy in this area. Validation of the effectiveness of current or alternative process control measures used in the manufacture of aged hard cheese would result in a greater assurance of a safe food supply and enhanced public confidence in these products.
Center Priorities Code
Research Regulatory Needs Codes V.A.1.
 

Deliverables

 FY1999

FY2000

FY2001
  1. Produce cheese made from milk inoculated with E. coli O157:H7.
  2. Monitor the food pathogen levels during the aging period.
  3. Establish baseline for producing cheese made from milk inoculated with E. coli O157:H7 and L. monocytogenes.
  1. Set up of process to conduct thermalization studies.
  2. Continue monitoring studies on pathogen reduction during cheese making and aging.
  3. Establish effectiveness of thermalization and, other processing conditions on destruction of E. coli O157:H7 and L. monocytogenes during cheese making and aging.
  1. Incorporate data into risk assessment models to establish criteria for cheese processing.
Outside Funding $7000 for supplies in FY99 from NCFST. NCFST received $75,000 of FSI money in FY1998 and $45,000 of FSI money in FY1999 to help fund this project. Proposed outside funding: Additional NCFST funding for supplies in FY 2001.

 

 

Legend | Abbreviations

FSI Project Number 14   RSVP Number  
Project Title Pathway Analysis: Assessment of Pathogen Transmission Capacities of Disease-Carrying Insects
Personnel Name Office/Division FTE Component
Alan R. Olsen OPDFB/DNP 1.0 1,2
Thomas Hammack

(traceback microbiologist)

 OSRS/DMS 1
Alen Karamian University of Maryland/JIFSAN (student intern) 2
Administrative Liaison J. Gecan, 202- 260-2022
Project Abstract This project develops an FDA science base for insect vectors of Salmonella, Listeria and E. coli that is comparable to similar science bases under development for livestock. Flies are a recognized contributing epidemiological factor in the spread of foodborne pathogens, especially E. coli, Listeria, Salmonella and Shigella. House flies are a recognized risk factor as vectors of E. coli O157:H7 in dairy farms and cattle farms and as vectors of Salmonella in broiler houses and dog kennels. The house fly is the most competent fly vector of Salmonella. Recent studies show that wild populations of house flies and blow flies harbor pathogens over entire breeding seasons. Studies conducted on litter beetles in broiler houses found that litter beetles infected with Salmonella transmit the infection to previously uninfected flocks under laboratory and field conditions. No research has been conducted to determine if flies have a similar vector competence for infecting layer house flocks. No studies have been conducted to assess the reservoir competence or other risk factors associated with insects in or near layer houses and fresh produce facilities. Recent findings that fruit flies can transmit E. coli O157:H7 to apples and that litter beetles can also transmit E. coli indicate that this project should be expanded to include fresh produce and to explore the vector competencies of vectors other than the house fly.
Project Description Layer house phase: Although rural environments have many potential reservoirs of Salmonella, the physical realities of layer house construction and operation limit the likely vehicles of contamination of uninfected flocks to human workers, water, feed, rodents and insects. The layer house phase of the project is designed to determine if the wild flies are a risk factor as natural reservoirs of Salmonella in and around infected layer house flocks. Flies are aseptically collected during traceback inspections for salmonellosis outbreaks involving shell eggs and tested for Salmonella using the procedures that are used for the other traceback environmental samples. Analysis of samples from the first, and to date only, traceback inspection of the project isolated S. enteriditis and S. infantis from house flies in and around the infected layer house. Samples of water and feed were negative for Salmonella. The relative densities of fly populations are also measured during the traceback inspections using the WHO/CDC protocol for estimating fly densities.

Fly colony phase: This phase is designed to determine the mechanical vector competence of house flies for Salmonella and Listeria. Individual fly adults are tested to determine the mean pathogen carrying capacity of a single fly under laboratory conditions. Vector competence is an important factor for comparing the relative risks presented by different species of flies. There are 12 species of flies, including the house fly, that are reasonably competent mechanical vectors of pathogens but there is evidence that vector competence may vary among fly species. Tests will be conducted to compare the vector competence of the other species with the house fly baseline. This phase is in abeyance until such time as the FSI program provides microbiological analytical support.

Projected Impact The vector competence studies determine which vectors should be included in risk assessment models for shell eggs and produce. The studies also provide estimates of vector population densities, expressed in the standard WHO/CDC epidemiological formula, for use in risk assessment modeling. The fly colony phase, when funded, will provide a science base for developing CPGs for unacceptable numbers of flies in layer houses or other food production facilities. The results of the fly colony tests will also be used to develop HACCP guidance for fruit juice and seafood. The entire project provides a needed addition to the FSI science base for the Center=s enforcement strategy for filth and extraneous materials.

Suggested project expansion: In view of the newly-discovered capability of fruit flies to contaminate apples with E. coli O157:H7, it is recommended that the layer house phase be expanded to include FDA surveys and traceback inspections involving pathogen contamination of fresh produce. As is the case with layer house tracebacks, the produce study can be accomplished using existing survey and traceback resources. In view of the diversity of vectors of foodborne pathogens and the diversity of vector competencies, it is further recommended that the project be expanded to include other vector species in all phases of the project.
Center Priorities Code 1.4d
Research Regulatory Needs Codes X.
Component 1 Fly Colony Phase
Description Testing individual flies for transmission capacity in the laboratory
Deliverables

FY1999

FY2000

FY2001
  1. Establish fly colony ; begin preliminary laboratory testing.
  1. Laboratory testing.
  1. Complete laboratory testing; analyze data for publication and CPG development.
Component 2 Layer House Phase
Description Collecting wild flies during traceback inspections and testing pools of the flies for pathogens
Deliverables

FY1999

FY2000

FY2001
  1. Est. three to five layer house traceback inspections; complete laboratory testing, fly identifications and specimen preparation.
  1. Est. three to five layer house traceback inspections; complete laboratory testing, fly identifications and specimen preparation.
  1. Complete laboratory testing; analyze data; deposit voucher specimens at Smithsonian; prepare data for publication.

 

 

Legend | Abbreviations

FSI Project Number 15 RSVP Number 39338
Project Title Effects of a Variety of Stress Factors on the Immune Systems of Poultry and Subsequent Infection of Shell Eggs by Salmonella Enteritidis
Personnel Name Office/Division FTE
U. Babu OSN/DSAT 0.5  
P. Wiesenfeld OSN/DSAT 0.3
Total FTE   0.8  
Collaborators: Institution    

 
W.Song,

S. Joseph

Univ. Maryland

Univ. Maryland

    

 
CVM Personnel:      

 
D. Wagner and M. Myers    

 

  

 
 

 

  

 
Administrative Liaison R.B. Raybourne, 301-594-5820
Project Abstract The role of the hen immune system in influencing the contamination of eggs by Salmonella Enteritidis will be studied. The goal will be to discover stress factors that can weaken the immune system and increase the chances of contamination of eggs. Potential strategies to control these stresses or to enhance immune resistance will result.
Project Description Salmonella enteritidis (S. E.) carried by chickens and transmitted via shell eggs has become a major source of human intestinal infections. Despite the tremendous efforts made by the poultry industry, no effective measurements for elimination of S. E. colonization have been generated. Since the rate of horizontal transmission among chickens and egg-laying hens is very rapid, general hygiene measurements are not as effective as desired. The purpose of this study is to examine variables affecting the immune response of hens against S. E., especially under stress conditions. Hens with a weak immune system are likely to be more susceptible to S. E. infections. Activating the hens= immune system, such as by immunization, can prevent or eliminate the infection. This study will help us identify the factors that can up-regulate or down-regulate the immune system of hens, leading to approaches for inhibiting the colonization of the reproductive tissues by S.E. leading to a decreased incidence of contaminated shell eggs and reduction of exposure to consumers.

Using the methods that we established in the first year of this project, we will examine the immune response of hens receiving different doses of S. E. in different phases of the hens= production cycle. We will also compare the immune response of hens at a time just after receiving the bacteria versus a designated time(s) after the inoculation.
Projected Impact Will aid in program priority to foster farm eggs quality assurance programs
Center Priorities Code
Research Regulatory Needs Codes V.B1,2, X
Deliverables

FY1999

FY2000

FY2001
  1. Use GFP- fluorescent S. E., to follow the colonization and invasion process of S. E . in hen=s intestine and ovarian tissues.
  2. Measure antibody response and cell mediated response (cytokines, blast transformation) in S.e. infected or immunized hens.
  1. Compare the immune response of hens that received a low dose of S. E. with those that received a high dose.
  2. Compare the immune response of hens that received the bacteria early in the production cycle (chicks) with those that received bacteria at the start of the egg production cycle (18 weeks) and with those infected during production (30 weeks).
  1. Determine the effects of immunosuppresive agents on S.e infection and egg contamination in hens.
  2. Determine the effects of the following stress factors on S. e infection: temperature, air quality, lighting and cage space.

 

 

Legend | Abbreviations

FSI Project Number 16 RSVP Number 39225 GPRA Goal: I.B., 2.A.
Project Title Levels of Vibrio vulnificus and Vibrio parahaemolyticus in Retail Seafood
Personnel Name Office/Division FTE

Component
David W. Cook OS/DSAT/GCSL 0.5

1
Scott R. Rippey OS/DPEP 0.5

1
Angelo DePaola OS/DSAT/GCSL 0.2

1,2
Jessica Jones OS/DSAT/GCSL 0.2

2
Total FTE   1.4
Bio. Tech. OS/DSAT/GCSL 0.1

2
[Technician personnel] provided by ISSC 0.75

1
[Lab Personnel] SERL and Denver Lab. NA

1
State personnel collecting samples NA

1
CFSAN Statisticians OSAS/DM 0.3

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

R. M. McPhearson, 334-694-4480
Project Abstract Project will develop data on consumer exposure to vibrios for use in developing risk assessment models.
Project Description This collaborative study involving CFSAN, ORA, ISSC and NMFS is determining the levels of Vibrio vulnificus and Vibrio parahaemolyticus to which consumers are being exposed when they consume raw oysters. This project involves collections of consumer samples in nine locations throughout U.S. A library of V. vulnificus strains is being assembled to represent isolates from different coasts and seasons. These isolates will be used to determine if strain differences exist.

A. Analysis of retail oyster for Vibrio vulnificus and Vibrio parahaemolyticus.Approximately 36 oyster samples per month are being analyzed by both MPN and direct plating techniques for Vibrio vulnificus and Vibrio parahaemolyticus. Sample collection and analysis will end in June 1999.

B. Determine if strains of Vibrio vulnificus differ depending on source (clinical/environmental), sample location (Gulf, East, West Coast) and season..The retail study will provide an opportunity to obtain V. vulnificus strains representating all coasts and seasons. These strains can be used to determine if strain differences may be related to only Gulf coast oysters being linked to illness caused by V. vulnificus. Related to citizen position 98P-0504.
Projected Impact These data will provide a more accurate exposure estimate and thereby enhance current estimates of the microbiological risks associated with oysters.
Center Priorities Code
Research Regulatory Needs Codes VI., X.
Deliverables

 FY1999

FY2000

FY2001
  1. Vibrio vulnificus and Vibrio parahaemolyticus counts of oyster samples. (A)
  2. Construct database containing all information collected during project. (A)
  3. Assemble collection of representative strains from retail samples.(B)
  1. Analyze data for incorporation in risk assessment models for vibrios. (A)
  2. Conduct in vitro characterization of V. vulnificus strains. (May require targeting of extramural contracts to investigate LPS, VivB, RAPD-PCR, capsule, plasmid.). (B)
  1. Conduct in vivo characterization of V. vulnificus based on findings of UMD and FDA contract for vibrio infectious dose.(B)

 

 

Legend | Abbreviations

FSI Project Number 17 RSVP Number 39441 GPRA Goal: 1..A., II.B.
Project Title Development of Gastroenteritis Animal Models and Biomarkers for Food borne Pathogens to Serve as Surrogate Models for Human Disease
Personnel Name Office/Division FTE Component
D.H. Burr OPDFB/DVA 0.5 1
M.H. Kothary OPDFB/DVA 0.5 1
R.B. Raybourne OPDFB/DVA 0.5 5
K.M. Williams OPDFB/DVA 1.0 4
M.A. Principato OPDFB/DVA 1.0 2
E. Bigley OPDFB/DVA 1.0 4
T. Gaither OPDFB/DVA 1.0 4
T. Flynn OSRS/DTR 0.7 3
D. Gaines OSRS/DTR 0.5 4
S. Sahu OSRS/DTR 0.5 3
U. Babu OSN/DSAT 0.5 4
P. Wiesenfeld OSN/DSAT 0.4 5
Total FTE 8.1
Collaborators:

C. Pontzer

 Univ. MD  

 

  

 

  

 
M.A. Smith Univ. GA  

 
       

 
Administrative Liaison R.B. Raybourne, 301-594-5820
Project Abstract This project uses animal models and develops biological measures for dose response studies with foodborne pathogens. The results are used to estimate the numbers of bacteria that are likely to produce illness in both healthy and at-risk persons.
Project Description This project is intended to provide risk assessors with data for use in dose response modeling using the following research approaches :
  • Use and develop in vitro (cell culture) systems to assess the virulence of food isolates of Listeria monocytogenes and other food borne pathogenic bacteria.
  • Determine the effects of the fat content of the food matrix on acid resistance and pathogenic potential of L. monocytogenes and other food borne pathogenic bacteria.
  • Use and develop animal models of gastrointestinal illness to assess the virulence characteristics of Salmonella and Campylobacter.
  • Identify biomarkers of exposure and susceptibility in a primate dose response model of L. monocytogenes.
  • Identify biomarkers of exposure and susceptibility in human dose response studies with Vibrio cholera non-01 as a surrogate organism for V. parahaemolyticus.
 
  • Use animal dose response models to develop biomarkers and surrogate endpoints which correlate with human gastrointestinal illness caused by pathogens and their toxins (e.g. staphlococcal enterotoxins).
  • Explore the applicability of noninvasive human biomarkers of exposure and susceptibility.
Projected Impact This project will provide data, which reduce the uncertainty of the Center's risk assessment efforts with Listeria monocytogenes and Vibrio parahaemolyticus with respect to dose response, pathogen virulence and host susceptibility. The principles developed should be broadly applicable to other pathogens as well.
Center Priorities Code
Research Regulatory Needs Codes X.B.
Component 1
  1.   Molecular pathogenesis and dose response to enteropathogenic Campylobacter, Eschercia coli and Vibrio parahaemolyticus using animal models of gastrointestinal disease.
  2.   Development of suckling mouse model for gastroenteritis caused by food-borne pathogens.
Description Description
  1.   Development of animal models and experimental genetic systems to study the pathogenesis of the above pathogens will enable a better understanding of virulence genes in the disease process. These models will be used to determine differences in virulence potential between genetically characterized strains and provide dose response data for risk assessment. Improved detection and sub-typing methods will also result from these studies.
  2.   The ability of a pathogen to cause disease will be determined by an endpoint that mimics human symptoms (i.e., diarrhea). Selected doeses of pathogens from an inventory of known clinical trials using foodborne pathogens will be fed to suckling mice and the amount of fluid (diarrhea) accumulating in the stomach and intestine will be measured. Dose response curves will be generated for isolates with different virulence characteristics. Oral dose responses for selected pathogens will be determined in this model.
Deliverables

FY1999

FY2000

FY2001
  1. Utilize animal data to develop dose-response models for Campylobacter and EHEC.(A)
  2. Use ferret model for evaluating efficacy of candidate Campylobacter vaccines. (A)
  3. Develop animal model for evaluating virulence of Vibrio parahaemolyticus.(A)
  4. Oral dose responses for selected clinical Salmonella typhimurium (DT104 and non-DT104) and Vibrio parahaemolyticus isolates determined. (B)
  1. Develop primate model for Campylobacter dose response and virulence studies. (A)
  2. Study effect of food matrix in dose response models. (A)
  3. Oral dose responses for Salmonella enteritidis, Vibrio vulnificus and Listeria monocytogenes determined. (B)
  1. Develop detection and subtyping system for Campylobacter, EHEC and V. parahaemolyticus.(A)
  2. Provide necessary animal data to support development of risk assessment models.(A)
  3. Validate models with animal studies and human clinical and epidemiological studies. (A)
  4. Oral dose responses for Escherichia coli (enterotoxigenic, enteroaggregative, enterohemorrhagic, enteropathogenic) determined. (B)
Component 2 Immunologic biomarkers of exposure to Staphylococcal enterotoxins.
Description Description A mouse model of oral exposure to Staphylococcal enterotoxin will be used to examine and compare peripheral and local immunological effects of dose dependant exposure to toxin on gut associated lymphoid tissue. These effects will be compared in adult and aged mice.
Deliverables

FY1999

FY2000

FY2001
  1. Use an established mouse model to describe the immunologic response elicited by ingestion of Staphylococcal enterotoxin food toxin.
  2. Evaluate dose-response effects upon circulatory and gastrointestinal lymphatics.
  1. Assess the immunologic response to Staphylococcal enterotoxin using a newly developed aged mouse model.
  2. Evaluate dose effects and identify biomarkers associated with exposure in the aged mouse model as compared to normal adult.
  1. Adaptation of a mouse model system to describe the immune responsiveness of aged, T cell deficient individuals against Staphylococcal enterotoxin.

Proposed but unfunded equipment: PCR thermocycler and associated equipment $10,000.

Component 3 The role of food matrix in dose response and susceptibility to food-borne pathogens.
Description The possible protective effects of high lipid content foods against the stomach acid barrier to food borne pathogens (e.g. Listeria monocytogenes , V. parahaemolyticus, and Salmonella Enteritidis) will be examined. The macromolecular interaction of pathogens with lipids and its possible effects on virulence will also be investigated.

Deliverables

FY1999

FY2000

FY2001
  1. Develop protocol and methods for in vitro models using cultured human intestinal epithelial cells and simulated human stomach environment.
  2. Assess non-cultural methods to evaluate bacterial viability after exposure to artificial stomach.
  3. Develop protocol and standardize methods for in vitro invasion/replication of L. monocytogenes in phagocytic and nonphagocytic cells.
  1. Complete studies and prepare manuscript on the effects of food matrix lipids on acid resistance and in vitro pathogenicity.
  2. Initiate studies on the effects of food matrix lipids on the dose response to Listeria monocytogenes in a mouse (or other) oral dosing model.
  3. Test in vitro invasion and replication of food isolates of L. monocytogenes in phagocytic and nonphagocytic cells.
  1. Complete in vivo studies and prepare manuscript.

Proposed but unfunded equipment: Fluorescence polarization detector, $17,000.

Component 4 Biomarkers and surrogate endpoints for dose response to L. moncytogenes and V. parahaemolyticus
Description Peripheral blood samples from subjects in CFSAN-funded dose response trails with L. monocytogenes and V. parahaemolyticus will be used to assess immunologically-based exposure and susceptibility biomarkers Coordinated animal studies will be used to develop mechanistically-linked surrogate endpoints for human illness based on these biomarkers.
Deliverables

FY1999

FY2000

FY2001
  1. Analyze peripheral blood lymphocyte phenotypes in pregnant rhesus monkeys infected with L. monocytogenes and normal controls (range finding study).
  2. Measure the lymphocyte proliferative response to mitogens and Listeria antigens in infected rhesus monkeys and controls.
  3. Measure cytokine responses in plasma and culture supernatants from Rhesus-Listeria range-finding study.
  4. Determine data gaps regarding oral infectious dose in immunocompromised mouse models systems.
  5. Bank peripheral blood cells from Rhesus studies and spleen cells from V. parahaemolyticus mouse studies for future microarray analysis.
  1. Complete biomarker studies in range finding study.
  2. Evaluate biomarkers based on outcomes in Listeria-Rhesus range finding study.
  3. Begin analysis of most useful biomarkers in large scale Rhesus dose response study.
  4. Conduct oral dose response studies in mouse-Listeria models of immune compromised states based on identified data gaps.
  5. Continue banking of specimens, evaluate microarray methodology for V. parahaemolyticus mouse model.
  1. Complete biomarker analysis in Rhesus dose response study.
  2. Conduct peripheral blood biomarker analysis on subjects in V. parahaemolyticus clinical dose response study in coordination with U.MD.
  3. Perform microarray analysis on peripheral blood lymphocytes from V. parahaemolyticus clinical study.
  4. Evaluate data from dose response studies to update Listeria and Vibrio risk assessment models.

Proposed but unfunded position: technical assistant

Component 5 Noninvasive approaches to estimate exposure and susceptibility to food borne pathogens
Description Methods and approaches are needed to identify both the susceptible portion of the population and the level of exposure to food borne pathogens. Initial efforts utilize sera collected during the pretest for the National Health and Nutrition Examination Survey (NHANES) to develop biomarkers of exposure and susceptibility
Deliverables

FY1999

FY2000

FY2001
  1. Convene expert group to study the feasibility of using antibody screening to determine exposure to L. monocytogenes.
  2. Conduct preliminary ELISA screening of NHANES dress rehearsal samples for antibodies to LLO.
  1. Evaluate results of ELISA screening compared to questionnaire data.
  2. Conduct additional testing on NHANES III banked samples.
  3. Draft proposal and questions for inclusion in NHANES IV.
  1. Continue collection and testing of serum samples possibly to include other measures.
  2. Correlate measurements with other data parameters from NHANES.

 

 

Legend | Abbreviations

FSI Project Number 18 RSVP Number 37241 GRPA Goals: I.B., II.A.
Project Title Developing alternative modeling tools for assessing dose response and severity
Personnel Name Office/Division FTE
Clark Carrington OPDFB/DPEP 0.3  
Chung Kim OSRS/DTR 0.3
Clark Nardinelli OSAS/DMS 0.3  
Total FTE   0.9  
Collaborators ERS and CDC economists, members of CFSAN economics team
Administrative Liaison Clark Nardinelli 202-205-8702
Project Abstract The project will develop new methods for performing microbial risk assessments and interpreting the results.
Project Description We know that certain bacteria contaminating our food can cause sickness and even death. Although science has found out a lot about some of these pathogenic (disease causing) bacteria, there are still gaps in our knowledge, such as:

- How many of the bacteria (dose) have to be in the food to cause a range of effects or responses from mild upset to death?

- What are the monetary costs associated with illnesses from different microbial hazards and doses.

- What are the effects on humans of microbial endotoxins (the poisons released during infection).

- Since direct testing of pathogens on human test subjects can be dangerous and ethically questionable, how can we relate animal or in vitro (human or animal cells grown in a laboratory) studies to humans?

Do different groups of people (such as the very young or elderly, immuno-compromised or ethnic groups) react differently to different bacteria, or even to the same level of contamination, and how can any differences in response be measured?

Risk assessments are scientifically based evaluations of the data we know, filling in the gaps in a variety of ways, so that an estimate or probability is derived of the likelihood of an illness or other adverse event. A common limitation of risk assessments is that a single endpoint, such as hospitalization, is identified as the adverse event. One of the ways that the gaps in our knowledge can be accounted for is by using statistical models to simulate the different scenarios we do not know. Statistical models can be created that would take into account the range of endpoints, from mild discomfort to acute pain, permanent disability and death, and incorporate these into a single risk assessment.
Projected Impact This research will provide well tested models of what happens when we are exposed to different levels of microbial hazards from our food supply. For many hazards, this is information that we currently do not have. The models would be created from data including disease outbreak investigations, and animal and in-vitro studies and then adjusted to reflect the effects on humans in general or on sub-populations, for instance the young, elderly or immuno-compromised individuals. The models would then make up a vital part of risk assessments which would give policy makers, regulators, educators, the food industry and others a scientifically based prediction of the risk associated with the microbial hazards and the probable effects of various mitigation, control, strategies.
Center Priorities Code
Research Regulatory Needs Codes X.A.
Component 1 Developing Formal Modeling Techniques for Drawing Inferences from Data
Description A search of statistical models or equations already developed will be carried out to determine if they are suitable for testing microbial pathogens as part of a risk assessment. Models found will be tested to see if they accurately measure what we presently know already, are simple enough to do the job without introducing new assumptions, and are consistent with other accepted theories. If no suitable models are found, one will be developed that does fit the above criteria. Finally, since the model will be simulating the situations where we do not have data, we will test the level of uncertainty so that we have an understanding of how close real life we are.
Deliverables

FY1999

FY2000

FY2001
  1. Completed two-dimensional Dose-Frequency function and companion curve-fitting program.
  1. Adaptation of code developed in FY1999 for use as a simple two-dimensional Frequency Distribution function, with a companion curve-fitting program.
  1. Development or enhancement of functions to incorporate additional data sets and respond to feedback from earlier products.
Component 2 Monetary Values of Illnesses from Different Hazards
Description This segment will attempt to place monetary costs on the health effects of various microbial hazards, and estimate the relationship between doses and monetary costs. It will be broken down into four steps. The first step will estimate the monetary value of a day of perfect health. The second step will combine the monetary values for a day of perfect health with the distribution of potential health outcomes (ex. mild upset, to requiring doctor visit or hospitalization, to death) for various hazards, including Salmonella, E. coli O157: H7, C. parvum, B. cereus, Listeria, Vibrio, and methylmercury. The third step will seek to find information linking dose and qualitative severity (doctor or hospitalization required) from these hazards. The qualitative outcomes will be linked to the monetary outcomes generated in step 2. The fourth step of the project will be to estimate dose-severity for as much of the data as possible. The form of estimation will likely be Tobit regressions, a technique that allows the integration of severity and confounding factors into the standard dose-response framework.
Deliverables

FY1999

FY2000

FY2001

1. Limited scope hazard ranking for food-borne pathogens.

1. Proposal for new general approach to valuing food-borne illness.

 		 
Component 3 Development of a Pharmacokinetic (PBPK) Model for Microbial Endotoxin, Lipopolysaccharide (LPS)
Description Lipopolysaccharides (LPS) are endotoxins released by most Gram-negative bacteria (a description of many disease causing bacteria). Inability to rid the body of LPS leads to hepatic (liver) disease and has been implicated in the development of neuronal and developmental changes in the body. Identifying and characterizing potential health risks require a multifaceted approach that includes the use of laboratory animals and in vitro tests to establish a scientific basis for evaluating human health risks. This research will link PBPK, exposure, data to the dose of contaminants and their metabolites that actually reaches target tissues and elicits a response, the PBPD information. These, then, will be used to construct models which will provide information directed toward reducing the uncertainty in assessing the potential risk of toxic and inheritable genetic effects in humans exposed to these toxic contaminants.
Deliverables

FY1999

FY2000

FY2001
  1. Physiological, biochemical, and chemical parameters for microbial endotoxin, lipopolysaccharide (LPS). Based on literature and in vitro experiments.
  1. A biological-based mathematical model with specific mechanistic step governing the tissue disposition and toxic action of LPS as a biomarker for gram-negative pathogens. The model will be quantitative and consist of a set of equations.
  1. Validation of mathematical model with experimental data obtained from animal experiments in vivo.

 

 

Legend | Abbreviations

FSI Project Number 19 RSVP Number 37241 GRPA Goals: I. B.
Project Title Risk Assessment Clearinghouse
Personnel Name FTE Office/Division
M. Walderhaug OSRS/DMS 0.5
D. Lowther OCAC/DSAT 0.5
Total FTE  

1.0
Proposed but unfunded position : Postdoctoral Student  

0.5
Administrative Liaison W. Long, 202-205-4064
Project Abstract The Risk Assessment Clearinghouse was described in the May 1997 Report to the President as a technical resource for risk assessors from industry, academe, and government.
Project Description The Risk Assessment Clearinghouse was described in the May 1997 Report to the President as a technical resource for risk assessors from industry, academe, and government. The clearinghouse staff will work with Risk Assessment Consortium (RAC) member agencies to assist in creating access to data from governments, industry, and other sources. A framework to address clearinghouse issues such as data quality, cataloguing, and confidentiality will be developed in conjunction with the RAC. The clearinghouse output will be used to assist government agencies, the food industry, and anyone who seeks to utilize risk assessment, to improve the scientific basis for decision making.
Projected Impact The clearinghouse output will be used to assist government agencies, the food industry, and anyone who seeks to utilize risk assessment, to improve the scientific basis for decision making.
Center Priorities Code 1.14B
Research Regulatory Needs Codes X.
 

FY1999

FY2000

FY2001
  1. Develop and implement 1 yr., 3 yr. and 10 yr. Goals for clearinghouse.
  2. Focus resources on development of both the systems component and the data modeling components (development of data sources, structuring information for ease of use).
  1. Further development of data, methods, and models, and adaptation of these tools for the internet format of the clearinghouse
  2. Promotion of prototype knowledge base amongst government agencies for assisting risk assessors in making inferences, visualizing processes, and manipulating data.
  3. Promotion of clearinghouse usefulness among industry, academia, and governments.
  1. Application of data quality measures to existing data sources to further guide risk assessors.
  2. Continued focus on the development of knowledge-based tools.

 

 

Legend | Abbreviations

FSI Project Number 20 RSVP Number 47472
Project Title Scientific support group
Personnel Name Office/Division FTE Expertise
F. Hines OSAS/DSSOSAS/DSS 0.8 Pathologist
D.Gaines OSRS/DTR 1.0 Flow Cytometry
S.Gendel OPDFB/DFPP 0.5 Ribotyping
E. Mazzola OSAS/DSS 0.5 NMR
S.Musser OSAS/DSS 0.5 Mass Spectrometry
J.Roach OSAS/DSS 0.5 Mass Spectrometry
M.Robl OSAS/DSS 0.4 Veterinarian
B.Tall OSRS/DMS 0.5 Electron Microscopy
S.Curtis OSRS/DMS 1.0 Electron Microscopy
J.-J. Yin OSAS/DSS 0.5 Electron Spin Resonance
Total FTE 6.2
Administrative Liaison S. M Musser, 202-205-4644
Project Abstract Provide specialized instrumentation, support services and personnel to other FSI projects.
Project Description The purpose of this project is to provide all FSI research projects with a cost-effective means of accessing high cost, specialized instrumentation and skilled researchers. This group includes personnel with specialized knowledge in the preparation of biological specimens, handling of animals and pathology services, as well as, key personnel for the operation of highly technical instruments. Access to this type of state-of-the-art technology permits development and refinement of detection, identification and quantification methods.
Projected Impact This project allows the timely completion projects and validation of data and methods.
Center Priorities Code N/A
Research Regulatory Needs Codes N/A
Deliverables

FY1999

FY2000

FY2001
  1. Procurement, installation, testing and use of new analytical instruments. These instruments include a scanning electron microscope, laser scanning flow cytometer, laser desorption ionization/ TOF/ mass spectrometer and a LC/MS/MS mass spectrometer. Also, continued support to those FSI projects that require specialized assistance.
  2. Further development of data, methods, and models, and adaptation of these tools for the Internet format of the clearinghouse.
  3. Promotion of prototype knowledge base amongst government agencies for assisting risk assessors in making inferences, visualizing processes, and manipulating data.
  4. Promotion of clearinghouse usefulness among industry, academia, and governments.
  1. Continued support to those FSI projects that require specialized assistance.
  1. Continued support to those FSI projects that require specialized assistance.

 

 

Risk Assessments

Legend | Abbreviations

FSI Project Number 21 RSVP Number 37241   GRPA Goals: I.B.
Project Title Assessment of the Public Health Impact of Foodborne Listeria monocytogenes.
  Name Office/Division FTE
 

Project Resources

 Whiting, R.C. OPDFB 0.5
Long, W. OPDFB 0.35
Hitchins, T. OSRS/DMS 0.35
Bender, M. OFL/DTE 0.35
Raybourne, R. OPDBF/DVA 0.35
McCarthy, P. OSAS/ 0.35
Bryce, J. OPDFB 0.35
Carrington, C. OPDFB/ 0.35
Lerner, P. OSAS/ 0.35
Rouse, T OPDFB 0.35
Hanson, E. OFL/DTE 0.35
McCabe, N. OFL/DTE 0.35
LeGault, L. OFL/DTE 0.2
 

Total FTE

  

4.55
 

Collaborators:

 Agency  

 
Datoc, M.L. FDA/ORA  

 
Smith, K. FDA/CDER  

 
Ebel, E. USDA/FSIS  

 
Schlosser, W. USDA/FSIS  

 
Liaison  

 

  

 
Ranson, G. USDA/FSIS  

 
Swaminathan, B. CDC/NCID/BMD  

 
Administrative Liaison R.C. Whiting, 202-260-0511
Project Abstract This risk assessment will determine the prevalence and extent of exposure of consumers to foodborne Listeria monocytogenes and will assess the resulting public health impact of such exposure.
Project Description The microbial pathogen, Listeria monocytogenes, is widespread in the agricultural and food processing plant environment and frequently is present in many foods. The organism can also be found in the gastrointestinal tract of some healthy adults. Despite the pervasive prevalence of the bacteria, the rate of listeriosis is not high. The disease, however, is serious when it occurs, causing meningitis in immunocompromised individuals and stillbirths and abortions in pregnant women.

Our risk assessment will determine whether certain foods contribute a significant portion of the total ingestion of this pathogen, what the consumption levels are, and what numbers of L. monocygtogenes cause illness in different people. Exposure assessment will determine the frequencies of occurrences and numbers in different classes of foods, particularly the ready-to-eat foods. When combined with food consumption data bases, the major sources of dietary L. monocytogenes will be identified. Epidemiological evidence of the foods causing both documented outbreaks and sporadic cases, the pathogen numbers consumed, the populations which become ill, and the severity of illness will be collected. This evidence will be supported by in vivo and in vitro experimental information. Evaluation of the dose-response relationship will include the health effects from consuming specific numbers of L. monocytogenes by individuals with different immune states and factors from the food matrix and characteristics of specific strains that affect illness.
Projected Impact This risk assessment will provide the scientific information for the development of policies to create effective agency programs and minimize the public health impact of this pathogen. This risk assessment is the initial activity necessary for FDA and FSIS to review their regulatory programs.
Center Priorities Code 1.7a
Research Regulatory Needs Codes X.
 

Deliverables

FY1999

FY2000
  1. Finalize charge to the task force.
  2. Make initial presentation to NACMCF - call for scientific data.
  3. Present plans and compilation of scientific data to ISSC. Also to NACMCF (public meeting).
  4. Make compilation of scientific data available on Internet.
  5. Complete risk assessment, draft document and present to FDA, USDA, CDC, NMFS, EPA and ISSC.
  6. Present complete risk assessment to NACMCF (public meeting).
  7. Make complete risk assessment document available for public comment. Finalize charge to the task force.
  1. Modify risk assessment, taking into account NACMCF review and public comments.
  2. Make final version available to public health agencies and public.

Legend | Abbreviations

FSI Project Number 21 RSVP Number 37241   GRPA Goals I.B.
Project Title Assessment of the Public Health Impact of Vibrio parahaemolyticus in raw molluscan shellfish.
Personnel Name Office/Division

FTE
Miliotis, M. OPDFB/DVA

0.5
Watkins, W. OS/DPEP

0.35
Ross, M. OSAS

0.35
Bowers, J. OSAS/DM

0.35
Dinovi, M. OPA/DPMU

0.35
DePaola, A. OS/DSAT/GCSL

0.2
Cook, D. OS/DSAT/GCSL

0.2
Hoskin, G. OS/DSAT

0.2
McCarthy, S. OS/DSAT/GCSL

0.2
Elliot, E. OFP/DHP

0.2
Podoski, B. OFP/DHP

0.2
Kothary, M. OPDFB/DVA

0.2
Burr, D. OPDFB/DVA

0.2
Tall, B. OSRS/DMS

0.2
Walderhaug, M. OSRS/DMS

0.2
Khambaty, F. OSRS/DMS

0.2
Street, D. OSAS/DMS

0.2
Klontz, K. OSAS/DMS

0.2
Timbo, B. OSAS/DMS

0.2
Whiting, R.

Long, W.

 OPDFB

OPDFB

  

 
Total FTE

 

4.7

  

 
Collaborators:

Wekell, N.

 FDA/ORA  

 
Kaysner, C.

Hill, W.

 FDA/ORA

FDA/ORA

  

 

  

 
 

 
Administrative Liaison Marianne Miliotis, 202-205-4824
Project Abstract This risk assessment will determine the prevalence and extent of exposure of consumers to Vibrio parahaemolyticus in raw molluscan shellfish and will assess the resulting public health impact of such exposure.
Project Description V. parahaemolyticus occurs naturally in the estuarine environment, and thus is present in many seafoods, including raw molluscan shellfish and other seafood. Investigations of outbreaks in 1997 and 1998 in the Pacific Northwest, Gulf Coast, and Northeast regions implicated this microorganism in over 700 cases of foodborne disease. The 1998 outbreak data indicate the emergence of new pathogenic strains.
Project Description The risk assessment will be divided into three modules: harvest, post harvest, and public health; the public health module will be further divided into epidemiology, consumption, and dose-response. The harvest module will address regional differences and seasonal fluctuations, evaluate how environmental parameters such as temperature or salinity, influence levels in seafood in the water at time of harvest. The post harvest module will evaluate the effect of processing and handling after harvest on the levels of the microorganism in the seafood. The public health module will address the number of V. parahaemolyticus infections, the number of V. parahaemolyticus cells in the oyster at time of consumption, probability of illness with different levels of the bacteria, and the severity of illness among consumers with different immune conditions.
Projected Impact This risk assessment will provide the scientific framework for the development of food safety guidance and policies to reduce the risk of disease from this seafoodborne pathogen. The policy will provide guidance to the industry, State and Federal laboratories on the type of testing to be performed and what risk reduction measures need to be taken. The resulting guidance will be developed in cooperation with the Interstate Shellfish Sanitation Conference (ISSC).
Center Priorities Code 1.7b
Research Regulatory Needs Codes X
 

Deliverables

FY1999

FY2000
  1. Finalize charge to the task force.
  2. Make initial presentation to NACMCF - call for scientific data.
  3. Present plans and compilation of scientific data to NACMCF (public meeting).
  4. Make compilation of scientific data available on Internet.
  5. Complete preliminary risk assessment, draft document, and present to FDA and FSIS.
  6. Present preliminary risk assessment to NACMCF (public meeting).
  7. Make preliminary risk assessment document available for public comment.
  1. Modify risk assessment, taking into account NACMCF review and public comments.
  2. Make final version available to public health agencies and public.

 

 

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