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CFSAN Regulatory Codes: V.B, XI.A
CFSAN Program Priority Codes: 1.6a
Start Date: 10/1/99    Completion Date: 9/30/02

Statement of Research Problem:
Some virulence determinants of Salmonella Enteritidis and Vibrio species are not well characterized or are, as yet, unknown. For example V. cholerae non-O1 strains cause sporadic food borne disease in the US, but the mechanisms of virulence are uncertain. Even well characterized vaccine strains of V. cholerae O1 cause mild illness in volunteers but the virulence mechanisms are unknown. Marine vibrios (such as V. tubiashii and V. fluvialis) found in various kinds of seafood (including commercial aquaculture operations) may be pathogenic for humans. To determine whether or not they are a food borne risk, it is necessary to study them in tissue culture and animal models and determine what, if any, virulence factors, they are producing. Salmonella Enteritidis is a pathogen found within some chicken eggs. Although invasive mechanisms have been identified in S. Enteritidis, others mechanisms, such as toxins (cytotonic and cytotoxic) remain elusive. In order to provide accurate microbial risk assessments for food borne pathogens, it is essential to know what virulence factors are involved in causing human disease so that these strains can be identified and eliminated from the food supply. Additionally, genes coding for virulence factors make an ideal target (s) for rapid molecular probes used in detecting pathogenic microorganisms in foods.

Statement of Project Objective(s):
This project addresses two major areas relevant to microbial pathogenicity, risk assessment and methods development. First, data will be provided on the virulence factors that have an impact on the risk associated with Salmonella or Vibrioinfection. Specifically, virulence factors will be identified, characterized and studied in tissue culture and animal models. Animal models will be used to determine dose response data. Second, rapid methods of detecting microbial pathogens in foods based on detection of virulence genes or proteins will be developed. Once biosensor/microarray technology is available, methods will be adapted to this system.

Anticipated Impact on FDA Regulatory Program:


1. Data on virulence factors and dose response will provide information for microbial risk assessments.
   
2. Development of rapid methods for detection of Vibrio species and Salmonella Enteritidis will result in quicker detection of these food borne pathogens.

Project Priority Changes During FY2000:
Component 1 - Emphasis on protein characterization has been redirected toward molecular biological methods. Vibrio cholerae work is nearly complete and exploratory research will be conducted on new (to this project) organisms. Component 3 - Since nearly 3 years have been spent on identification and characterization of the Vibrio parahaemolyticus toxin gene with little or no progress, emphasis has been shifted to studying the invasive mechanism(s) and corresponding genes.

Administrative Liaison(s): Barbara A. McCardell: 202/ 205-4262

Research Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
B. McCardell	OARSA/DVA	0.5, 0.5, 0.5	1
S. Lavu	OARSA/DVA/	1.0, 1.0, 1.0	1
M. Kothary	OARSA/DVA/VMB	0.5, 0.5, 0.5	2
V. Sathyamoorthy	OARSA/DVA/VMB	1.0, 1.0, 1.0	2
M. Miliotis	OARSA/DVA/VMB	0.5, 0.5, 0.5	3
L. Carter	OARSA/DVA/VMB	1.0, 1.0, 1.0	3
B. Tall	OPDFB/DMS/MEB	0.5, 0.5, 0.5	4
	Total FTE	5.0, 5.0, 5.0

Collaborators:James B. Kaper, Ph.D., UM School of Medicine, CVD, Baltimore; HACU Students; JIFSAN Students, UM, College Park; Mohamed Faisal, Ph.D., VA Institute Marine Science, College of William and Mary; Robert Bayer, Ph.D., Dept. of Bio-systems Science and Engineering, Lobster Institute, University of Maine; Dennis Kopecko, Ph.D., FDA/CBER

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 and other strains of V. cholerae O1, 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.

Component 1 Objectives:
Identify novel toxin gene in V. cholerae.


1. Clone and sequence gene.
   
2. Determine presence or absence in other Vibrio species.
   
3. Construct deletion mutants in V. cholerae.
   
4. Test mutants in virulence assays to determine role of toxin in virulence.
Component 1 FY 2000 Deliverables:

1. Clone gene for Vibrio toxin.
   
2. Initiate manuscript on cloned toxin.
   
3. Initiate construction of deletion mutants.
Component 1 FY 2000 Progress:

1. Cloned and sequenced toxin gene from 2 V. cholerae 01 vaccine strains (1 El Tor and 1 Classical).
   
2. Screened other vibrios by PCR for toxin gene.
   
3. Cloned, sequenced and compared toxin gene from V. cholerae non-O1, V. fluvialis, and V. parahaemolyticus.
   
4. Published peer-reviewed paper on an aberrant non-O1 hemolysin.
   
5. Published peer-reviewed on the purification and characterization of the V. cholerae toxin.
   
6. Initiated manuscript on cloning of toxin.
   
7. Began construction of deletion mutants.
Technical Barriers to Meeting Component 1 Objectives or Deliverables: none listed
Component 1 FY 2001 Deliverables:

1. Complete paper on the cloned genes.
   
2. Complete development of deletion mutants.
   
3. Introduce mutant gene into V. cholerae vaccine strain.
   
4. Complete in vitro and in vivo assays of strain with mutant gene.
Component 1 FY 2002 Deliverables:

1. Complete and publish manuscript on the deletion mutants.

Component 2 Objectives:


1. Newly identified virulence factors from V. cholerae will be purified and characterized.
   
2. Data from # 1 used to develop specific tests to identify food borne pathogenic strains.
   
3. Clinical, food and environmental isolates of S. Enteritidis (SE) will be examined for enterotoxin production.
   
4. Antiserum will be raised against the enterotoxin of SE, and its specificity will be evaluated.
   
5. Antiserum will be raised against the newly described enterotoxin of V. parahaemolyticus (VP) and will be evaluated for its ability to inhibit the accumulation of fluid caused by various strains of VP in the suckling mouse model.
   
6. Putative virulence factors of the newly identified pathogenic vibrio, Vibrio vulnificus biogroup 3 will be determined.
Component 2 FY 2000 Deliverables:

1. Complete dose response studies for the SE and VP toxins in the suckling mouse assay.
   
2. V. vulnificus toxin purified.
   
3. Complete purification and characterization of Vibrio toxin.
   
4. Initiate purification of cloned Vibrio toxin.
   
5. Initiate purification of cloned V. parahaemolyticus toxin.
Component 2 FY2000 Progress:

1. Completed purification and characterization of V. cholerae O1 toxin from vaccine strain.
   
2. Manuscript completed and submitted for publication.
   
3. Completed purification of the cloned V. cholerae O1 toxin.
   
4. Testing of cloned toxin in mouse model has been initiated.
   
5. Minimum amount of purified SE enterotoxin required for a positive response in a suckling mouse assay determined to be 0.2 µg (10,000 CHO cell units).
   
6. Determination of VP (toxin) dose response initiated.
   
7. Hemagglutinin/protease produced by V. tubiashii was purified and characterized.
Technical Barriers to Meeting Component 2 Objectives or Deliverables:

1. Determination of VP toxin dose response delayed by problems encountered in purification of large amounts of toxin.
   
2. Preparation of V. vulnificus toxin discontinued because crude prep of the toxin gave a negative response in suckling mice, therefore, purification of V. vulnificus toxin (deliverable #2) was discontinued.
   
3. Purification of cloned VP toxin omitted because of gene sequence similarity with that of V. cholerae.
FY 2001 Deliverables:

1. Purification and characterization of cloned V. cholerae non-O1 toxin.
   
2. Complete testing of purified non-O1 toxin in animal model.
   
3. Antiserums against purified VP and SE toxins prepared.
   
4. Putative virulence determinants of V. vulnificus biogroup 3 identified.
FY 2002 Deliverables:

1. Antisera against toxins evaluated for specificity in detection and for significance in preventing disease in an animal model.
   
2. Complete screening of clinical, food and environmental isolates of SE for the production of enterotoxin using an ELISA and/or a tissue culture assay.
   
3. Complete characterization of virulence determinants of V. vulnificus biogroup 3 (if different from biogroups 1 and 2).

Assays for virulence factors can be time-consuming and require specialized skills and facilities (tissue culture, etc.). Therefore, it is more efficient to identify gene sequences coding for virulence factors and use them as a means of detecting organisms harboring specific virulence determinants. Pathogenicity of VP is closely associated with the presence of thermostable direct hemolysin (TDH). However, TDH negative strains can cause gastroenteritis and fluid accumulation in animal models suggesting the presence of additional virulence factors. Other possible virulence factors include invasion of enterocytes and production of a cytotonic enterotoxin (VPE) that elongates Chinese hamster ovary (CHO) cells in vitro. SE produces at least two toxins that elongate CHO cells. One is neutralized by antibodies to cholera toxin (CT) and the other (SEE) is not neutralized by these antibodies.

Component 3 Objectives:


1. Isolation and characterization of the gene(s) involved with invasion are the main objectives of this work. Probes based on sequences from this gene can then be used to identify invasive strains that may or may not produce the hemolysin.
   
2. 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.
Component 3 FY 2000 Deliverables:

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 non-radioactive hybridization using dioxygenin-labeled probes.
Component 3 FY 2000 Progress:

1. Completed screening of possible VP toxin mutants by CHO cell assay. No toxin mutants were detected.
   
2. One thousand clones were made from VP DNA. Testing for toxin by CHO cell assay is underway.
   
3. Initiated invasion testing of VP clones in tissue culture assays.
Technical Barriers to Meeting Component 3 Objectives or Deliverables: none listed
Component 3 FY 2001 Deliverables:

1. Complete testing of 1,000 VP clones for ability to invade tissue culture cells.
   
2. Complete testing of SE mutants for toxin production.
   
3. Initiate cloning of SEE genes by conventional cloning techniques and test in CHO cells for toxin production.
Component 3 FY 2002 Deliverables:

1. If clones are found that are positive for invasion, then:
   
2. Initiate sequencing of the invasion genes.
   
3. Initiate development of detection method based on gene sequences.
   
4. Test clones that are invasive in selected animal models.

Marine vibrios are undoubtedly the bacterial pathogen of greatest concern in seafood disease. This is because they can cause multiple diseases (septicemia, gastroenteritis, and wound infections) in both seafood hosts and in humans. Mechanisms such as the ecology of adherence and invasion greatly influence both the ecology and the persistence of these bacteria in the seafood host and thus subsequently affect the infectious dose for human illness. These characteristics have an impact on the emergence of Vibrio species among the seafood hosts and their subsequent transmission to humans. Studies of these traits will lead to procedures for removing these pathogens from the seafood supply and better detection methods for these pathogens.

Component 4 Objectives:


1. Characterize virulence determinants and mechanisms of several marine vibrios many of which are being found to be common among the species comprising this group.
2. Develop detection systems based on virulence gene sequences.
   
3. Study the effect of virulence factors in animal models and Provides dose response data from animal models
Component 4FY 2000 Deliverables:

1. Determine thermal kill parameters/heat resistance in V. fluvialis lobster isolates.
   
2. Complete signal transudation 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, V. ordiali.
Component 4 FY 2000 Progress:
Determined that both Vibrio tubiashii and the lobster pathogen, V. fluvialis can cause a diarrhea in suckling mice possibly by two different mechanisms involving colonization (V. tubiashii) and intoxication (V. fluvialis). It was shown that V. fluvialis does not survive in the mouse or at 37°C. An hemolysin expressed by V. tubiashii was also preliminarily characterized. Other results showed that an hemagglutinin expressed by V. tubiashii was a metalloprotease and that both the protease and hemolysin possessed similar properties to those expressed by V. cholerae, V. mimicus, and V. vulnificus. Tissue culture studies provided evidence that internalization of V. vulnificus occurred at a limited number of sites; that invasion was dependent on microfilaments, microtubules, protein kinase (C and tryosine) activities, and that bacterial mediated cytotoxicity and invasion were two independent events. Overall, these results emphasized the need to continue the Center's research efforts with these lesser-known marine vibrios.
Technical Barriers to Meeting Component 4 Objectives or Deliverables: none listed
Component 4 FY 2001 Deliverables:

1. Determine AML host receptor(s) involved in colonization and invasion of shellfish and finfish cultured cells by V. vulnificus entry using polyclonal antibodies, lectins, and inhibitors.
   
2. Compare signal transudation inhibitor studies of V. vulnificus entry into AML cells and role of host receptors in adherence and invasion with other marine vibrios.
Component 4 FY 2002 Deliverables:
Characterization of the adherence and invasion mechanisms associated with the recently identified V. vulnificus biotype 3 cells. Isolation and characterization of the hemolysin expressed by V. tubiashii.

McCardell, B.A., M.H. Kothary, and J.M. Madden. 1999. Two-step purification and partial characterization of a variant of the Vibrio cholerae non-O1 hemolysin. FEMS Microbiol. Lett. 180: 177-182.

McCardell, B.A., M.H. Kothary, R.H. Hall, and V. Sathyamoorthy. 2000. Identification of a CHO cell-elongating factor produced by Vibrio cholerae O1. Microbial Pathogenesis 29: 1-8.

Fernandez-Prada, C. F., D. L. Hoover B. D. Tall, J. Kopelowitz, A. B. Hartman, and M. M. Venkatesan. 2000. Shigella flexneri IpaH_7.8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages. Infect. Immun. 68:3608-3619.

McLaughlin, S.M., B. D. Tall, A. Shaheen, E. E. Elsayed, and M. Faisal. 2000. Zoosporulation of a new Perkinsus species isolated from the gills of the softshell Clam (Mya arenaria). Parasite 7:115-122.

B.D., J.F. La Peyre, J.W. Bier, M.D. Miliotis, D.E. Hanes, M.H. Kothary, D.B. Shah, and M Faisal. 1999. Perkinsus marinus extracelular protease modulates survival of Vibrio vulnificus in eastern oyster (Crassostrea virginica) hemocytes. Appl. Environ. Microbiol. 65:4261-4263.

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Hypertext updated by dav 2001-OCT-02