CFSAN Regulatory Codes: X, B
CFSAN Program Priority Codes:
Start Date: 5/1/99    Completion Date: 5/1/02

Statement of Research Problem:
The goal of the FDA microbial risk assessment effort is to provide a transparent, rational and scientifically sound basis for risk management policies that will ensure the safety of the food supply. In terms of risk assessment for microbial pathogens in the food supply, the intent is to develop risk assessment models which cover all aspects of the food supply from production or harvest of food, up to and including consumption by the public (from farm to fork). The predictive power of the model is dependent upon the quality and reliability of available data. It is generally accepted that data gaps exist for all the major food borne pathogens regarding the number of organisms in a food required to produce clinical illness. An accurate model would be complex, factoring in interactions between characteristics of the food vehicle, variability in the pathogen populations and variability in host susceptibility. Pathogens of interest include Salmonella spp.; pathogenic Eschericia coli, Listeria monocytogenes, and Vibrio vulnificus, and protozoa such as Cryptosporidium spp.and Cyclospora. This project seeks to provide data useful for risk assessment by conducting human clinical and animal model studies that are complementary and as closely linked as possible.

Statement of Project Objective(s):
This project is intended to provide risk assessors with data for use in dose response modeling using the following research approaches:

1. Use and develop in vitro (cell culture) systems to assess the virulence of food isolates of Listeria monocytogenes and other food borne pathogenic bacteria.
2. 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.
3. Use and develop animal models of gastrointestinal illness to assess the virulence characteristics of Salmonella and Campylobacter.
4. Identify biomarkers of exposure and susceptibility in a primate dose response model of L. monocytogenes.
5. Identify biomarkers of exposure and susceptibility in human dose response studies with Vibrio cholera non-01 as a surrogate organism for V. parahaemolyticus

Anticipated Impact on FDA Regulatory Program: (max 10 lines)

1. This project will provide data that reduces 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 risk assessments of other pathogens as well.

Project Priority Changes During FY2000:
Component 5 has been reduced to an information gathering function

Collaborators: Dr. Carol Pontzer, UMD (Component 4); Dr. Mary Alice Smith, UGA (Component 4); Dr. Patricia Guerry, NMRC (Component 1); Dr. David Rollins, NMRC (Component 1)

1. Develop animal models and experimental genetic systems to study the pathogenesis and immune response of the above pathogens.
2. Develop a better understanding of virulence genes in the disease process.
3. Use these models to determine differences in virulence potential between genetically characterized strains and provide dose response data for risk assessment.
4. Improve detection and sub-typing methods as a result of these studies.
5. The ability of a pathogen to cause disease will be determined by an endpoint that mimics human symptoms (i.e., diarrhea) using the suckling mouse oral feeding model
6. Dose response curves will be generated for isolates with different virulence characteristics. Oral dose responses for selected pathogens will be determined in this model.

1. Study the effect of food matrix in dose response models
2. Oral dose responses for Salmonella enteritidis, V. vulnificus and Listeria monocytogenes determined in the suckling mouse model

1. The oral dose required for causing fluid accumulation in suckling mice was determined for Salmonella enteritidis and V. parahaemolyticus.
2. Studies aimed at determining the dose for Listeria monocytogenes is in progress.
3. An oral-challenge diarrheal disease model for V. parahaemolyticus was developed in ferrets.
4. Demonstrated for the first time the involvement of a plasmid in the virulence of Campylobacter jejuni.

1. Exposure of suckling mice to V. vulnificus resulted in death consequently oral dose could not be determined.

1. Study the effect of food matrix in dose response models
2. Use animal models to assess immunologically-based exposure and susceptibility biomarkers
3. Continue characterization of virulence genes in C. jejuni and V. parahaemolyticus.

1. Develop detection and subtyping system for Campylobacter, EHEC and V. parahaemolyticus.
2. Provide necessary animal data to support development of risk assessment models
3. Validate models with animal studies and human clinical and epidemiological studies
4. Oral dose responses for E. coli (enterotoxigenic, enteroaggregative, enterohemorrhagic, enteropathogenic) determined.

1. 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.

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. The immune response of aging mice (>1.5 years) clearly demonstrates that T cells from aged animals respond to SEB superantigen.
2. Histologic observations confirm that the lymphocytic infiltrate/proliferative areas are composed of uniform mononuclear cells.
3. Ingestion of SEB results in elevations of IL-4, IL-10, and interferon over a course of a week follwing exposure. Taken together, these observations are suggestive of an adjuvant effect of SEB upon the immune response of the aged.

Component 3 Objectives:

1. To assess the possible protective effects of high lipid content foods against the stomach acid barrier to food borne pathogens using in vitro models.
2. To assess the possible inhibitory effects of food isoflavonoids (e.g., genistein) on virulence of food borne pathogens in vitro and in vivo.
3. To assess iron uptake kinetics as a possible distinguishing factor between virulent and non-virulent strains of food borne pathogens.

1. Complete in vitro studies on food matrix lipids.
2. Initiate in vivo studies

1. Completed studies on inhibitory effects of genistein on in vitro cytotoxicity of 11 strains of Vibrio vulnificus and began preparation of a manuscript on this work.
2. Prepared a protocol for mouse, in vivo studies with soy isoflavonoids and Vibrio vulnificus.

1. Continue in vivo studies with soy-based diets and food borne pathogens of interest
2. Complete iron uptake kinetic studies with multiple strains of one species of food borne pathogen.

1. Complete in vivo studies, analyze data and prepare manuscript.

1. Peripheral blood samples from subjects in CFSAN-funded dose response trails with L. monocytogenes , V. parahaemolyticus and C.parvum 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 and dose response based on these biomarkers.

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. Continue banking of specimens, evaluate microarray methodology for V. parahaemolyticus mouse model.

1. Samples from the Rhesus-Listeria range finding study have been used in a series of studies with immune endpoints. The results indicate that a functional immune suppression may be associated with Listeria infections that induce stillbirth in the Rhesus model. Significant changes also were noted in the types of lymphocytes present in peripheral blood during infection. Increased cytokine responses following infection were also observed. Increased antibody responses to L. monocytogenes were seen in animals that had a stillbirth following infection.
2. Peripheral blood samples from animals in the dose response study have been received and immune biomarker studies have been started using the same parameters as in the range finding study.

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.

1. Conduct animal model studies to determine effects of immune stimulation and suppression on dose response relationship in Listeria mouse model.

Williams, K.M., Bigley, Elmer C., and Raybourne, R.B. 2000. Identification of Murine B-Cell and T-Cell Epitopes of Escherichia coli Outer Membrane Protein F with Synthetic Polypeptides. Infect. Immun. 68: 2535.

Bacon D.J., R.A. Alm, D.H. Burr, L. Hu, D.J. Kopecko, C.P. Ewing, T.J. Trust and P. Guerry. 2000. Involvement of a plasmid in virulence of Campylobacter jejuni 81-176. Infect. Immun. 68: 4384-4390.

Lee, L.H., E.Burg, S. Baqar, A.L. Bourgeois, D.H. Burr, C.P. Ewing, T.J. Trust and P. Guerry. 1999. Evaluation of a truncated recombinant flagellin subunit vaccine against Campylobacter jejuni. Infect. Immun. 67: 5799-5805.