(Table of Contents)

CFSAN Regulatory Codes: 1A,1C,1D,IIA,IIC,IXB
CFSAN Program Priority Codes: 1.3b,1.4a,1.4c
Start Date: 5/1/99    Completion Date: 5/1/02

Statement of Research Problem:
An essential component of a comprehensive strategy to enhance food safety is the development of a variety of rapid and sensitive methods for detecting and quantifying pathogens or their toxic metabolites in foods. Many domestic and imported perishable products are not processed to inactivate harmful biological contaminants prior to consumption. Conditions that preserve the organoleptic qualities may lead to sublethal injury to pathogens that may not be detected by conventional cultural methods. Further complicating the detection of pathogens or their toxic metabolites is the sporadic or low levels of contamination, the presence of high level of background microflora, lack of suitable propagation systems for food borne viruses and the interference by components of the food matrix in currently available detection methods.

Statement of Project Objective(s):
Various components of this project will provide new or refined methods for the detection and quantification of pathogens and their toxic metabolites, and food sample preparation techniques such that functional real-time detection would be possible. Molecular typing or fingerprinting methods can contribute to our understanding of pathogen ecology and distribution. These tasks defined in this project will support and enhance Agency surveillance and enforcement activities.

Anticipated Impact on FDA Regulatory Program:


1. New, refined and real-time detection methods will enhance microbiological safety of foods, specifically high risk foods and infant formulas and reduce burden on industry dealing with perishable products.
   
2. Data on the presence of pathogens, their numbers and distribution will enhance development of risk assessment and verification of critical control points in HACCP programs.
   
3. New molecular real-time detection and typing methods will enhance timely response to bioterrorism involving foods.

Project Priority Changes During FY2000:
Validation of rapid methods specified in sprout guidance document resulted in diversion of work in component 4. Four new components have been added to this project for FY01-02.

Administrative Liaison(s): D.B. Shah: 202-205-4981

Research Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
W.H. Andrews	OPDFB/DMS	1.0,1.0,1.0	1 & 7
T.S. Hammack	OPDFB/DMS	1.0,1.0,1.0	1
M.L. Johnson	OPDFB/DMS	1.0,1.0,1.0	1
N. Belay	OPDFB/DMS	1.0,1.0,1.0	2
R.H. Hall	OARSA/DVA	1.0,1.0,1.0	3
T.J. Fu	OPDFB/DFPP	1.0,1.0,1.0	4
M.L. Tortorello	OPDFB/DFPP	1.0,1.0,1.0	4 & 8
D.S. Stewart	OPDFB/DFPP	1.0,1.0,1.0	4 & 8
A. Rasooly	OPDFB/DMS	1.0,1.0,1.0	5 & 10
A. Goswami	OARSA/DMB	1.0,1.0,1.0	6
M. Kulka	OARSA/DMB	1.0,1.0,1.0	6
A.P. Jacobson	OPDFB/DMS	0.0,1.0,1.0	7
S. Gendel	OPDFB/DFPP	1.0,1.0,1.0	9
S. Al-Khaldi	OPDFB/DMS	0.0,1.0,1.0	10
	Total FTE:	12, 14, 14

Collaborators: K. Reineke, NCFST; J. Ulaszek, NCFST; O. Vanpelt, NCFST; L. Restaino, R. & F. Labs. Chicago; S. Yee, Univ. of Washington, Seattle

The BAM Salmonella method will be evaluated for use with juices and produce that have been implicated in Salmonella outbreaks. In two outbreaks, involving orange juice and mamey pulp, the BAM culture method was unable to detect Salmonella. These results strongly suggest that the method should be evaluated for use with these foods, as well as with other juices and produce that might be involved in future outbreaks.

Universal Preenrichment (UP) broth, has been shown to be effective for use with orange juice and apple cider. UP broth has replaced lactose broth in the BAM Salmonella culture method for orange juice. Recent investigations, involving UP broth, have centered on its effectiveness with select dairy foods. The evaluation of UP broth for the recovery of Salmonella from dairy foods will be completed. In the future, UP broth should be evaluated with both low and high microbial load foods including produce.

There is no BAM method specifically designed for S. typhi and paratyphi. In 1998, there was a typhoid outbreak in which imported mamey pulp was implicated. S. typhi was not detected in mamey pulp with the BAM Salmonella method. This incident suggests that the BAM Salmonella method is ineffective for S. typhi. Moreover, other imported produce may be implicated in future typhoid outbreaks. Therefore, a method for S. typhi and paratyphi should be developed.

Component 1 Objectives:


1. To provide a more sensitive Salmonella culture method for use with orange juice, apple juice and cider, mamey and other selected high-risk produce.
   
2. Evaluate UP broth for the detection of Salmonella in dairy foods.
   
3. Develop and refine methods for the isolation of S. typhi and paratyphi for produce.
Component 1 FY 2000 Deliverables:

1. Provide a more sensitive culture method for orange juice.
   
2. Complete the evaluation of UP broth for the detection of Salmonella in dairy foods.
Component 1 FY 2000 Progress:

1. A method for orange juice has been developed.
   
2. A method for apple cider and apple juice is being developed.
   
3. The evaluation of UP broth for use with dairy foods, except casein and whole milk, has been completed.
Technical Barriers to Meeting Component 1 Objectives or Deliverables:

1. Delay. a second brand of unpasteurized apple cider was not available until Fall, 2000 apple crop harvested.
   
2. The evaluation of UP broth for use with selected dairy foods was not completed within FY2000, because it was necessary to extend the evaluation to include casein and whole milk.
Component 1 FY 2001 Deliverables:

1. Complete the development of a method for apple cider and apple juice.
   
2. Complete the development of a method for mamey.
   
3. Complete the evaluation of UP broth for use with dairy foods.
Component 1 FY 2002 Deliverables:

1. Evaluate and modify, if necessary, the BAM Salmonella method for tropical fruit, such as mangoes and papayas.
   
2. Develop a method for the isolation of S. typhi and paratyphi for produce.
   
3. Determine the relative effectiveness of UP broth for use with produce.

Conventional methods for detection of the emetic toxin include monkey-feeding tests and mammalian cell toxicity assays. We recently developed an in vitro microbial inhibition assay for the toxin with C. perfringens as the test organism and based on the ionophoric property of the toxin. The new test is as sensitive as the Hep-2 tissue cell assay. Further work is needed to improve sensitivity and to explore ways for automation of the test.


Component 2 Objectives:


1. Sensitivity Improvement: Identify factors that would enhance the emetic toxin's action against C. perfringens and thus improving assay sensitivity. Such factors include pH, growth phase of cells, temperature, and substances known to show synergistic effect with ionophores.
   
2. Automation of Test: The use of chromogenic substrates/indicators will be examined for the purpose of automation of the C. perfringens inhibition test. This is a particularly promising approach. With a colorimetric substrate in the test system, the emetic toxin-induced C. perfringens inhibition can be registered easily. Both spectrophotometric detection and accurate visual monitoring would be possible. Moreover, miniaturization of the test to the scale of the microtiter dish would be possible, allowing automated monitoring by a plate-reader. A microtiter dish methodology would also provide increased testing capacity and lower expenses.
Component 2 FY 2000 Deliverables:

1. Publish the C. perfringens growth inhibition assay for emetic toxin.
   
2. Develop potassium-ion electrode assay for emetic toxin.
   
3. Evaluate effect of maltodextrin on toxin production by B. cereus.
Component 2 FY 2000 Progress:

1. Manuscript describing the new emetic toxin assay submitted to Journal of Food Protection.
   
2. When using an anaerobe in the test system a potential drawback with the proposed electrode sensor method was realized. The method would not be suitable for continuous ionophore activity monitoring as open-air setup and continuous mixing are involved. Moreover, the need to emphasize automation goals was indicated in last year's review of the project. Thus, the work stated in objective #2 was initiated. Several chromogenic substrates including MTT, amaranth, and methyl orange were screened and amaranth and Direct-blue 15 were found most suitable.
   
3. Examined the effect of maltodextrin on emetic toxin production: no effect was observed.
   
4. Two other collaborative manuscripts completed (in clearance stage) and two poster presentations done.
Component 2 FY 2001 Deliverables:

1. Complete assay sensitivity improvement as stated in Objective #1 and publish results
   
2. Complete studies on use of chromogenic substrates/indicators for emetic toxin test improvement as stated in objective #2 and publish.
   
3. Develop a microtiter plate method of the chromogenic test.
Component 2 FY 2002 Deliverables:

1. Test the colorimetric microtiter plate method for its use to monitor the emetic toxin in a variety of foods.
Technical Barriers to Meeting Objectives or Deliverables: None anticipated

Sensitive and specific detection methods for food-borne pathogens offer important tools in food safety programs when applied to prevention, surveillance and trace back studies. Specific molecular targets in bacterial pathogens will be identified, characterized, and evaluated for a role as targets in the development of pathogen-specific assays. Obtaining the required assay sensitivity requires powerful signal amplification methods that must demonstrate resistance to interferents present in sample matrices, or function with suitable separation technology. This project will develop targeting and amplification strategies for a range of food-borne bacterial pathogens.

Component 3 Objectives:


1. To develop appropriate detection solutions initially for use in FDA surveillance and compliance activities.
   
2. Identify specific pathogen targets; optimize and evaluate target-signal amplification techniques.
   
3. Identify promising signal read-out methods; and work to deploy methods into FDA Field Labs and beyond.
   
4. Objectives are to evaluate hlyA as a specific gene target for enterohemorrhagic E. coli (EHEC), O157:H7 and other EHEC serotypes, and evaluate other potential targets.
   
5. Evaluate SDA and other techniques in comparison to PCR as a common platform for target amplification; and evaluate fluorescence polarization as a common read-out method for a range of food-borne hazards. Recent funding of a post-doctoral research position will permit expansion to include additional pathogens into SDA, FP (Fluorescence Polarization), or other systems. Collaboration with FDA Field Labs is an objective.
Component 3 FY 2000 Deliverables:

1. Identification of potential pathogen targets for detection systems based on SDA and FP.
   
2. Evaluate performance and matrix compatibility of hlyA/SDA/FP detection system in comparison to PCR.
   
3. Develop sampling strategy for water-borne pathogens from a variety of sources.
   
4. Submit for publication collaborative research data on putative virulence determinant of Vibrio.
Component 3 FY 2000 Progress:

1. Several food-borne hazards have been identified which are compatible with SDA and FP. The rapid and quantitative nature of FP is particularly useful for detection of toxins and high infectious dose pathogens.
   
2. Detailed performance specifications for hlyA/SDA/FP were obtained and the results submitted to Applied & Environmental Microbiology. Comparison with PCR showed SDA currently to be much more sensitive and rapid, but similarly specific and matrix-tolerant.
   
3. A sampling method for water obtained from several drinking, environmental and recreational sources was developed and presented at the American Society for Microbiology. A manuscript reporting a novel toxin from Vibrio was written and submitted to Infection & Immunity. The reviewers' comments have been received, addressed, and the manuscript re-submitted.
Technical Barriers to Meeting Component 3 Objectives or Deliverables:
(Logistics Barrier) Lack of experienced technical assistance remains a problem, but the continued availability of excellent students (e.g. through JIFSAN and HACU programs) mitigates this problem to a significant extent. The pending post-doc hire will provide significant additional expertise and momentum.
Component 3 FY 2001 Deliverables:

1. Develop Fluorescence Polarization as a signal readout system for toxins and pathogens in foods under SDA, PCR and ELISA formats.
   
2. Research improvements in SDA work-flow and optimize deployability of hlyA/SDA/FP into Field Labs.
   
3. Develop channel for Field Lab deployment of new rapid methods developed in this project.
Component 3 FY 2002 Deliverables:

1. Deploy EHEC hlyA/SDA/FP into a Field Lab setting (contingent on post-doc appointment).
   
2. Field test new pathogen and toxin detection assays through channel developed for hlyA/SDA/FP.
   
3. Continue to develop new assays for detection of food-borne hazards.

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.

Component 4 Objectives:


1. Examine effectiveness of water rinse as a means to separate pathogens from contaminated produce.
   
2. Develop filtration systems for the concentration of pathogens from large volumes of rinse water.
   
3. Develop/evaluate improved methods for detection and enumeration of pathogens in sprouts and produce.
   
4. Develop integrated concentration and detection systems for real-time pathogen monitoring during processing of fresh produce.
Component 4 FY 2000 Deliverables:

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.
Component 4 FY 2000 Progress:

1. Demonstrated that spent irrigation water is a good target for pathogen monitoring during sprouting of alfalfa seeds and showed effectiveness of water rinse for removing E. coli O157:H7 from lettuce.
   
2. Developed two membrane filtration processes for concentrating 100 ml - 10 L of sprout irrigation water, and used these processes in conjunction with rapid immunoassays to improve detection limit to as low as 1 CFU/L and allow the use of 1000-fold less enrichment media.
   
3. Studied growth kinetics of E. coli O157:H7 and Salmonella during sprouting of inoculated and naturally contaminated alfalfa seeds, respectively.
4. Evaluated use of rapid tests (Assurance Gold, VIP, Reveal, Gene-Trak, Bax for Screening, and antibody-direct epifluorescent technique or Ab-DEFT) for detection and/or enumeration of E. coli O157:H7 or Salmonella in inoculated or naturally contaminated sprouts and irrigation water.
Technical Barriers to Meeting Component 4 Objectives or Deliverables: None anticipated.
Component 4 FY 2001 Deliverables:

1. Evaluate the applicability of the two developed membrane filtration systems (standard membrane filtration and tangential flow filtration) for concentrating pathogens in rinse water used during processing of other types of fresh produce.
   
2. Evaluate the use of a fiber-optic biosensor and a solid state cytometer (Scan RDI) in conjunction with Ab-DEFT for real-time detection of pathogens in rinse water.
Component 4 FY 2002 Deliverables:

1. Integrate the tangential flow filtration system to a flow-through fiber optic biosensor for large volume, real-time pathogen monitoring during fresh produce production.
   
2. Integrate membrane filtration with the Scan RDI system to allow enumeration of pathogen in large volumes of rinse water.

Antibodies, the traditional ligand for proteins and bacterial detection, are inconvenient for detecting multiple toxins because each antibody is specific to a particular toxin and because it is complicated to produce biosensor chips with numerous antibodies. Furthermore, although they are specific, antibodies can cross-react nonspecifically with materials in food thus interfering with detection. The possibility of using synthetic peptides as ligands as an alternative to antibodies was studied. In addition, the effectiveness of mass spectrometry-SPR for multi-toxin identification was studied.

Component 5 Objectives:


1. Develop multitoxin methodology for SPR analysis of microbial toxins in food using various antibodies for toxin identification.
   
2. Develop general chromatography method for partial purification of staphylococcal enterotoxins from food.
   
3. Develop multitoxin methodology for SPR analysis of microbial toxins in food using various peptides for toxin identification.
   
4. Collaborative assessment of the effectiveness of mass spectrometry-SPR for multi-toxin identification.
   
5. Collaborative assessment of wavelength interrogation SPR biosensor.
Component 5 FY 2000 Deliverables:

1. Mass spectrometry-SPR method for analysis of multiple toxins in food.
   
2. Analytical chromatography for recovery of small amounts of staphylococcal enterotoxins from food.
   
3. Multi-antibody method for SPR analysis of multiple toxins in food.
   
4. Development of peptides for SPR analysis of multiple toxins in food.
Component 5 FY 2000 Progress:

1. Development of mass spectrometry-SPR method for analysis of multiple toxins in food.
   
2. Complete analytical chromatography for recovery of small amounts of staphylococcal enterotoxins from food.
   
3. Development of multi-antibody method for SPR analysis of multiple toxins in food
Technical Barriers to Meeting Component 5 Objectives or Deliverables:
The peptides identified seem to have low affinity to SE and are probably not useable for SPR analysis.
Component 5 FY 2001 Deliverables:

1. A protocol for a multi-antibody method for SPR analysis of multiple toxins in food.
   
2. Development of an antibody array for analysis of multiple toxins in food
   
3. Further study of the use of wavelength interrogation SPR biosensor in food analysis (depending on the progress of our collaborators at the University of Washington who are developing the SPR optical system).
Component 5 FY 2002 Deliverables:

1. A method for multi-antibody SPR analysis for multiple toxins in food
   
2. Antibody array for analysis of multiple toxins in food
   
3. Mobile wavelength interrogation SPR biosensor for food analysis (depending on the progress of our collaborators University of Washington who are develop the SPR optical system).

Currently, there are no simple, rapid, reproducible, reliable and/or universally applicable methods for the detection of hepatitis A virus (HAV) or caliciviruses in fruits or produce. Additionally, 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 virus and caliciviruses in fruits and produce followed by purification methods to produce viral RNA suitable for detection by RT-PCR.

Component 6 Objectives:


1. Establish the utility of viral and cellular gene expression for application in viral detection and identification methods.
   
2. Establish basic procedure(s) for isolation and purification of viral RNA and/or particles from diverse foods.
   
3. Develop methods for subsequent viral detection by reverse transcription-PCR (RT-PCR).
Component 6 FY2000 Deliverables:

1. Evaluate antibodies as capture supports for concentration of intact viral particles for the detection of viral RNA after extraction.
Component 6 FY2000 Progress:

1. Currently available commercial antibodies to HAV (and caliciviruses) are not optimally suited to the study of HAV capture and expression. Work is in progress to develop rabbit polyclonal antibodies directed against specific antigenic domains of selected HAV proteins. Their efficacy in viral particle/protein detection will be evaluated.
   
2. Using fresh cilantro as a fresh produce model, two extraction protocols were tested to compare the recovery and detection of HAV RNA from cilantro. Reverse-transcriptase-polymerase chain reaction was used in the successful detection of isolated HAV RNA from cilantro RNA preparations.
Technical Barriers to Meeting Component 6 Objectives or Deliverables: None
FY 2001 Deliverables:

1. Establish cDNA fingerprint patterns to distinguish HAV strains following viral genome RT-PCR/PCR amplification.
   
2. Production and purification of HAV polymerase using a prokaryotic cell expression system.
FY 2002 Deliverables:

1. Development of a eukaryotic cell line that expresses one or more HAV genes.
   
2. Analysis of cellular and viral gene expression during HAV infection using DNA array technology.

Shigella spp. occur in a variety of foods including fresh produce. Shigella spp. are fastidious organisms, requiring relatively neutral pH levels and moderate temperatures for optimal growth. Their growth is inhibited by less sensitive microorganisms commonly present in foods. Isolation of Shigella spp. from foods is therefore difficult, particularly when Shigella is present at low levels. Since the infectious dose of Shigella is low, it is important to use methods that isolate Shigella from all foods when these organisms are present at low levels.
Pre-enrichment of food samples in broth media is used to isolate Shigella from foods. In the BAM procedure, this step is done in anaerobic environments. Anaerobiosis aids in the resuscitation of injured Shigella cells while inhibiting populations of aerobic organisms. To create anaerobic conditions, laboratories need to be equipped with anaerobic jars and water baths. The time and space required to maintain and operate this equipment reduces the overall efficiency of the method. To increase efficiency, alternatives to using anaerobic jars need to be considered. One such approach may be the use of commercially available reducing agents.
Evaluations of the BAM culture method have not been conducted on selected fresh produce and the lowest detection levels (LDL) remain unknown. Thus, it is necessary to identify areas in the method needing improvement so as to make the method appropriate for use in surveillance of a wide variety of fresh produce.

Component 7 Objectives:


1. Determine the LDL of the BAM culture method for the detection of Shigella spp. in selected types of fresh produce.
   
2. Make improvements in the current culture method, where needed, to optimize the method for use with selected fresh produce.
Component 7 FY 2000 Deliverables:
Component 7 FY 2000 Progress:
Technical Barriers to Meeting Component 7 Objectives or Deliverables: None anticipated
Component 7 FY 2001 Deliverables:

1. Determine LDLs of the BAM culture method for the detection of Shigella spp. in selected fresh produce.
   
2. Identify areas in the current culture method that need improvement.
Component 7 FY 2002 Deliverables:

1. Evaluate effect of reducing agents (thioglycollate, Oxyrase) as alternatives to use of anaerobic jars.

Sublethal injury of pathogens in food processing is of concern because it may allow surviving populations to cause food borne illness, or it may give rise to populations that exhibit greater resistance to control measures or increased virulence. Conventional methods for determination of sublethal injury and resuscitation are imprecise and only generally descriptive. Molecular methods may give a more accurate assessment of sublethal injury and resuscitation, provide a better understanding of their nature, and may lead to more rapid assays for determination of these states. Furthermore, because sublethally injured pathogens may be present in a food but not detectable by conventional methods, molecular methods may lead to improved pathogen detection in general, e.g. in outbreak-implicated foods.

Component 8 Objectives:


1. To compare methods for determination of sublethal injury and resuscitation, including conventional culture, direct cellular probes, expression of molecular markers.
   
2. To use these methods in the context of evaluating the effectiveness of new processing technologies.
Technical Barriers to Meeting Component 8 Objectives or Deliverables: None
Component 8 FY 2001 Deliverables:

1. Develop baseline data for determination of sublethal injury and resuscitation by conventional methods in pathogens, e.g., L. monocytogenes or E. coli O157:H7.
   
2. Investigate use of molecular methods, e.g. RT-PCR, NASBA, microarray expression analysis or direct cellular probes, for assessment of sublethal injury and resuscitation.
Component 8 FY 2002 Deliverables:

1. Investigate use of molecular methods for determination of microbial cell injury after novel processes, e.g. high hydrostatic pressure, pulsed electric field, or irradiation.

Automated ribotyping will be used to identify and track strains of major food borne pathogens. Ribotype pattern libraries will be constructed for several pathogens using environmental and food isolates obtained from FDA field labs, government agencies, state public health labs and universities.

Component 9 Objectives:


1. Track changes in strain distribution patterns for food borne pathogens.
   
2. Determine whether particular subtypes or strains are more resistant to processing or other control strategies.
   
3. Determine the optimal enzymes or combination of enzymes to use for characterizing genetically homogeneous pathogens such as E. coli 0157:H7 and Salmonella Enteritidis.
Component 9 FY 2000 Deliverables: NEW COMPONENT
Component 9 FY 2000 Progress:

1. Completed ribotyping of outbreak associated V. parahaemolyticus 03:K6
   
2. Completed ribotyping of V. parahaemolyticus isolates from environmental and retail foods.
   
3. Completed ribotyping of sprout outbreak associated Salmonella isolates.
   
4. Initiated studies on identifying enzymes for ribotyping of Salmonella Enteritidis.
Technical Barriers to Meeting Component 9 Objectives or Deliverables: None
Component 9 FY 2001 Deliverables:

1. Ribotype Listeria monocytogenes isolates from labs.
   
2. Ribotype V. parahaemolyticus isolates from environmental and retail samples
   
3. Complete studies on enzymes for strain differentiation of L. monocytogenes
Component 9 FY 2002 Deliverables:

1. Continue ribotyping of L. monocytogenes
   
2. Identify enzymes for strain differentiation of E. coli 0157:H7

DNA chips are a miniature array of gene-specific DNA probes immobilized on a chip surface. In combination with amplification of genetic material by the polymerase chain reaction (PCR), this technique might be a powerful tool for detection and simultaneous discrimination of food borne human pathogens. The aim of the program is to develop diagnostic DNA microarray chips for analysis of various microbial food pathogens and their virulence genes.


Component 10 Objectives:


1. Initiate the DNA microarray analysis of rDNA of various microbial food pathogens.
   
2. Initiate the DNA microarray analysis of various microbial virulence genes of food pathogens.
Component 10 Deliverables:

1. Develop diagnostic DNA microarray chips for analysis of food pathogens.
   
2. Develop diagnostic DNA microarray chips for analysis of various microbial virulence genes.
Component 10 FY2000 Progress:

1. Development of method for detection of the presence of six genes encoding bacterial antigenic determinants and virulence factors (eaeA, slt-I, slt-II, fliC, rJbE, and ipaH) by gene-specific oligonucleotides on a chip. This assay tested for Salmonella, Shigella, and several pathogenic Escherichia coli.
Technical Barriers to Meeting Component 10 Objectives or Deliverables: None
Component 10 FY 2001 Deliverables:

1. DNA microarray chip for the analysis of eaeA, slt-I, slt-II, fliC, rJbE, and ipaH genes
Component 10 FY 2002 Deliverables:

1. Sequence analysis of rDNA useable for microarray design.
   
2. Sequence analysis of various microbial virulence factors usable for microarray design.
   
3. Produce microarray chips for analysis of food borne pathogens.

McCardell, B.A., M.H. Kothary, R.H. Hall, & V. Sathyamoorthy. (2000). Identification of a CHO cell-elongating factor produced by Vibrio cholerae O1. Microb. Pathog. 29:1-8.

Sathyamoorthy, V., R.H. Hall, B.A. McCardell, M.H. Kothary, S.J. Ahn, and S. Ratnayake.(2000) Purification and characterization of a cytotonic protein expressed in vitro by the live cholera vaccine candidate CVD 103-HgR. Infect. Immun. 68:6062-6065.

Reineke, K. F. and M. L. Tortorello. 2000. Direct enumeration of Escherichia coli and enteric bacteria in water, beverages and sprouts by 16S rRNA in situ hybridization. Food Microbiology 17:305-313.

Tortorello, M. L., D. S. Stewart, K. F. Reineke and J. M. Ulaszek. 2000. Detection of Salmonella in alfalfa sprout spent irrigation water by rapid immunoassays. FDA Laboratory Information Bulletin No. 4214.

Tortorello, M. L. 2000. Escherichia coli O157:H7. In: Encyclopedia of Food Microbiology. R. K. Robinson, C. A. Batt and P. D. Patel, eds. pp. 646 - 652.

Primera Reunion Sobre Investigacion en Materia de Inociudad Alimentaria, Mexico City, Mexico, May 4 and 5, 2000.

Hartman,G., D. Stewart and S. Gendel.2000. Identification of Listeria monocytogenes from ice cream using the BAX System. Laboratory Information Bulletin. 4217, 1-7.

Gendel, S. and J. Ulaszek. 2000. Ribotype analysis of strain distribution in Listeria monocytogenes. J. Food Protection. 63:179-185.

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Hypertext updated by dav 2001-OCT-02