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CFSAN Regulatory Codes: X.B, XI.A, I.A
CFSAN Program Priority Codes: 1.6a
Start Date: 5/1/99    Completion Date: 5/1/02

Statement of Research Problem:
Some risk factors associated with developing salmonellosis are not well established. Parameters such as host susceptibility, bacterial virulence, and vehicle of transmission all influence the outcome of a Salmonella infection. Many foods have been associated with Salmonella transmission, but the role of the food matrix in infection is not well defined. The food matrix may protect the bacteria from host defenses thus permitting infection. Understanding these host defenses is also crucial in determining risk. The ability of the host to respond to infection and the factors involved in this response greatly influence the outcome of infection. Finally, the virulence of the organism plays a major role in the disease process. Interaction with the host that permits expression of virulence genes is crucial if the organism is to establish an infection. Understanding the parameters that influence risk will provide a basis for action when Salmonella are identified in foods.
Rapid methods for detection of foodborne pathogens will strengthen the food safety initiative. In many cases, the food matrix is a major obstacle in developing and utilizing rapid methods. Background microflora present in some food items also inhibits the use of many methods developed for detection. Techniques for sample preparation that address these problems will enhance all aspects of the food safety initiative.

Statement of Project Objective(s):
This project addresses two major areas of research, risk assessment and methods development. On the first objective, this project will provide data on several parameters that impact the risk associated with a Salmonella infection. The effects of food matrices on Salmonella pathogenicity will be assessed using a rabbit diarrhea model. This will provide information on foods that may pose a greater risk if contaminated with salmonellae. The host responses to infection will also be examined to provide information on sections of the population that may have increased risk of developing infection with salmonellae. Finally, bacterial genes essential for infection in the host will be identified.
Addressing the second objective, this project will provide new methods of sample preparation that will be used in conjunction with rapids methods developed for foodborne pathogens. This technology will be transferred to FDA field laboratories.

Anticipated Impact on FDA Regulatory Program:


1. Data on infectious dose and food matrix effects will enhance development of risk assessment for Salmonellae species.
   
2. Rapid methods for Shigella will provide better enforcement tools.
   
3. Data on the role of host factors and host immune status on infection will impact risk assessment for sub-groups of the population.
   
4. Identification of virulence genes will advance the characterization of pathogenic Salmonellae.
   
5. Methods for sample preparation will enhance use or rapid methods for detection of foodborne pathogens.

Project Priority Changes During FY2000:
Project objectives and priorities remain compatible with FSI program goals and priorities. The project has shifted emphasis from working only on multi-drug resistant Salmonella Typhimurium (MDRST) to working on the genus Salmonellae. Using this approach data and methods can be generated that are applicable to all Salmonellae species. This will also allow a more rapid response if a particular species of Salmonellae would emerge as new foodborne pathogen.

Administrative Liaison(s): Darcy E. Hanes, Ph.D. 301/827-8075

Research Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
Chad Giri	OPDFB/DMS	1.0, 1.0, 1.0	1
Keith Lampel	OARSA/DVA/VMB	1.0, 1.0, 1.0	2,4
Leroy Kornegay	OARSA/DVA/VMB	1.0, 1.0, 1.0	2
Don Burr	OARSA/DVA/VMB	0.5, 0.5, 0.5	3
Saura Sahu	OARSA/DTR	0.0, 0.25,0.25	3
Darcy Hanes	OARSA/DVA/VMB	0.5, 0.5, 0.5	3,4,5
	Total FTE:	4.0, 4.25, 4.25

Collaborators: Anthony Morelli, Ph.D., USUHS; Dennis Kopecko, Ph.D., FDA/CBER

Component 1 Objectives:


1. Assess expression of genes required for invasion of and escape from cultured intestinal epithelial cells.
   
2. Identify genes expressed during exocytosis.
   
3. Assess gene expression during infection of an animal model.
Component 1 FY 2000 Deliverables:

1. Develop cell exocytosis assay in mouse macrophage cell line, J774A.1.
   
2. Construct promoter-less prokaryotic vector containing GFP and CAT promoters.
   
3. Optimize differential RT-PCR to examine salmonellae induced genes in cell lines.
   
4. Identify and isolate bacterial cDNA clones that are differentially regulated.
   
5. Prepare data for presentation and publication.
Component 1 FY 2000 Progress:

1. Exocytosis assay developed in INT407 cell line in place of macrophage cell line.
   
2. Vector containing GFP and CAT promoters constructed.
   
3. Salmonella cDNA library constructed in vector.
   
4. Plans to submit abstract for ASM meeting.
   
Technical Barriers to Meeting Component 1 Objectives or Deliverables:
New information suggests that RT-PCR may not be the best method to identify the genes that are expressed during cellular invasion and exocytosis. A method utilizing flow cytometry is being assessed to identify salmonellae genes differentially expressed during invasion and exocytosis. Deliverable 3 has been removed and deliverable 4 has been re-directed.
Component 1 FY 2001 Deliverables:

1. Using the GFP/CAT vector cDNA library and flow cytometry, identify and isolate bacterial cDNA clones that are expressed during invasion of and escape from tissue culture cells.
   
2. Initiate studies to examine the cDNA library in a mouse model for salmonellae pathogenesis.
   
3. Develop methods to identify genes differentially regulated during infection of the mouse model.
Component 1 FY 2002 Deliverables:

1. Characterize differentially expressed cDNA clones using DNA sequencing and gene bank analysis.
   
2. Initiate studies to determine the pathogenic functions of differentially expressed genes.
   
3. Determine whether unique genes are induced in tissue culture cells versus the whole animal.
   
4. Prepare data for presentation and publication.

Component 2 Objectives:


1. Focus on a rapid PCR method for the detection of Shigella in food.
   
2. Transfer methodology for Shigella to the field laboratories.
   
3. Improve PCR template preparation for the detection of pathogens in foods using specialized filters.
Component 2 Deliverables:

1. Expand PCR template preparation protocol to be applied to all foods.
   
2. Establish CRADA with company producing filters for PCR preparation protocol.
   
3. Continue work on rapid methods for the detection of Shigella in food.
   
4. Continue transferring new methodology to field laboratories.
   
5. Initiate studies to adapt methods to new technologies.
Component 2 FY2000 Progress:

1. A multiplex PCR for MDRST based on sequence from 3 antibiotic resistance gene sequence has been completed.
   
2. A small plasmid from MDRST that encodes kanamycin resistance has been identified. The plasmid is being characterized using DNA analysis.
   
3. A method for detection of Shigella in food has been transferred to the FDA field laboratories. Manuscript IN PRESS
Technical Barriers to Meeting Component 2 Objectives or Deliverables:
Note programatic priority shift. Research on antibiotic resistance is not being conducted on this project.
FY 2001 Deliverables:

1. Collaborate with FDA field labs on use of PCR method for Shigella.
   
2. Transfer methods for Shigella to FDA field labs.
   
3. Adapt methods for new technology such as biosensors.
FY 2002 Deliverables:

1. Develop PCR template preparation protocol.
   
2. Establish CRADA PCR preparation protocol.

Component 3 Objectives:


1. Develop an animal model that mimics diarrheal symptoms seen in the human host.
   
2. Develop an animal model for salmonellae in chronically ill individuals.
   
3. Examine the effects of the food matrix on infection with Salmonella.
   
4. Compare the virulence of different species of Salmonella in the animal model.
   
5. Examine host response to Salmonella.
Component 3 FY 2000 Deliverables:

1. Publish animal diarrhea model.
   
2. Develop chronic illness animal model.
Component 3 FY 2000 Progress:

1. The manuscript for the diarrhea model is in preparation. The manuscript will be completed and submitted when commercial vendor is finished with the pathology portion.
   
2. The sandrat is being assessed as a chronic disease model for salmonellae.
Technical Barriers to Meeting Component 3 Objectives or Deliverables:

1. The study is heavily dependent upon the animal use of other investigators since many of the animals for this study were excess animals from other researcher's protocols.
Component 3 FY 2001 Deliverables:

1. Publish results of chronic illness model.
   
2. Provide dose response data.
   
3. Develop methods to assess changes in liver enzyme levels during infection with salmonellae using in vivo and in vitro models.
Component 3 FY 2002 Deliverables:

1. Examine the role of food matrices in infection with Salmonella.
   
2. Compare the virulence characteristics of Salmonellae species in the diarrhea model.
   
3. Initiate protection studies with rabbits that recovered from an initial Salmonella infection.
   
4. Identify specific changes in liver enzymes that indicate infection with Salmonella.

Component 4 Objectives:


1. Clone the α-hemolysin gene.
   
2. Use PCR to determine the distribution of this gene among Salmonella spp.
   
3. Determine the role of this gene in virulence using the animal diarrhea model.
Component 4 Deliverables:

1. Clone gene.
Component 4 FY2000 Progress:

1. New data indicates that the small plasmid containing the α-hemolysin gene may represent a mobile transmissible element involved in transfer of virulence genes among Salmonellae species. Work from this project has identified highly conserved versions of this plasmid containing various virulence genes in several species of Salmonellae. This plasmid may play a vital role in transfer of genetic material among Salmonella and possibly other bacterial species. Work on this component has been re-directed to examine the role of small virulence plasmids in the transfer of virulence genes. Emphasis has been placed on the entire plasmid and not the single gene present in the plasmid.
   
Technical Barriers to Meeting Component 4 Objectives or Deliverables: none listed
FY 2001 Deliverables:

1. Determine mobility characteristics of small plasmid.
   
2. Determine role of plasmid in pathogenicity.
FY 2002 Deliverables:
Publish and complete project.

Component 5 Objectives:


1. Assess the role of selected human cytokines in Salmonella invasion of cultured intestinal epithelial cells and macrophage cell.
   
2. Assess the role of antibody in Salmonella invasion of cultured intestinal epithelial cells.
   
3. Examine the role of cytokines and antibody in diarrheal disease in the animal model.
Component 5 FY 2000 Deliverables: New component
Component 5 FY 2000 Progress: New component
Technical Barriers to Meeting Component 5 Objectives or Deliverables: none listed
Component 5 FY 2001 Deliverables:

1. Examine the role of the tumor necrosis factor in Salmonella invasion of cultured cells.
   
2. Initiate studies to determine the antibody component of rabbits that recover from diarrheal illness
Component 5 FY 2002 Deliverables:

1. Develop methods to examine cytokine expression in rabbits infected with Salmonella.

Hanes, D.E. 1999. FDA-CFSAN research activities: virulence assessment and molecular pathogenesis of multi-drug resistant Salmonella typhimurium. Proceedings of the Food Safety Symposium on Antimicrobial Resistance, pp.201-204.

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Hypertext updated by dav 2001-OCT-02