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CFSAN Regulatory Codes: IA, IC, IF
CFSAN Program Priority Codes: none
Start Date: 4/99    Completion Date: 9/02

Statement of Research Problem:
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 water-borne contamination. Since the spring and early summer of 1996, major outbreaks in North America attributed to C. cayetanensis have been epidemiologically linked to the consumption of fresh 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 fresh basil. Unpasteurized apple cider and possibly scallions have been sources for C. parvum infections. Contaminated water is also suspected as a major factor in the transmission of all three parasitic protozoa.

The difficulties in assessing and controlling possible food borne contamination and infections with these microorganisms are many. There is a general lack of knowledge concerning life cycles (C. cayetanensis, Microsporidia spp), animal vectors and/or reservoirs, biochemistry, and the inability to efficiently culture the parasite either in an animal- or a tissue culture-based model. An inadequate supply of C. 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 food borne contamination lack the necessary sensitivity and reliability.

Statement of Project Objective(s):
Parasitic protozoa such as C. cayetanensis, C. parvum and Microsporidium spp. have emerged as important human pathogens and are closely associated with food and waterborne illness. In this 3 year plan, a continuing effort to improve sampling and detection methods will be pursued. These efforts will include fast, reliable, and highly sensitive PCR methodologies that can be applied to a variety of food and water sources. The development of model systems to evaluate intervention strategies will include continuing studies for the in vitro cultivation of C. cayetanensis oocysts.

Development of animal models that mimic human illness caused by these protozoa will be attempted and dose-response studies in normal and immuno-compromised animals will 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 the above-listed parasites. This project will continue to pursue three (3) aspects of research as they relate to food safety:


1. Continued development of sensitive detection methods in food and water sources;
   
2. In vitro cultivation; animal modeling development; and risk evaluation.
   
3. Intervention strategies

Anticipated Impact on FDA Regulatory Program:


1. Detection methods with increased sensitivities to satisfy needs for regulatory and surveillance roles.
   
2. Identify a suitable in vitro model to pursue infectivity studies and develop risk assessment models.
   
3. Evaluation of current Cyclospora intervention strategies (particularly those in place for approved Guatemala raspberry farms) will provide efficacy data and their applicability to similar protozoan parasites and environments.

Project Priority Changes During FY2000: No major changes are anticipated during FY2001. Research emphasis on C. parvum will be expanded because of its potential as a surrogate model for research, its availability, and its growing presence as a food- and water-borne pathogen.

Administrative Liaison: Palmer A. Orlandi: 202/205-4460

Research Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
Palmer A. Orlandi	OARSA/DVA/VMB	1.0, 1.0, 1.0	1
Darcy E. Hanes	OARSA/DVA/VMB	0.5, 0.5, 0.5	2
Jeff Bier(retired 8/31/00)	OS/DSAT	1.0, 0.0, 0.0	1
George J. Jackson	OSci/Staff College	0.2, 0.0, 0.0	3
Dan-My Chu	OPDFB/DMS/MEB	0.0, 1.0, 1.0	1
	Total FTE:	2.7, 2.5, 2.5

Other Personnel:


Name	Office/Division	FTE [00,01,02]	Component
Carrie M. Schneider	Contract Employee	0.5, 0.0, 0.0	2
Teresa Shea	Contract Employee	0.0, 0.5, 0.5	2

Collaborators:
Rafael Pratdesaba, Central American and Panama Institute for Nutrition (INCAP); Mike J. Arrowood, CDC/NCID/DPD; Mark L. Eberhard, CDC/NCID/DPD; Alex J. da Silva, CDC/NCID/DPD; Norman Pieniazek, CDC/NCID/DPD; Randy Worobo, Cornell University; Leonid Margolis, Ph.D., NICHD; Joshua Zimmerberg, M.D., Ph.D., NICHD; Wendy Fitzgerald, NICHD; Saul Tzipori, Tufts University

External Contracts:
CDC/NCID/DPD - Winter C. cayetanensis oocyst supply and primer development for PCR detection.
Uniformed Services University of the Health Sciences - Summer C. cayetanensis oocyst supply.
Stanford University - Molecular differentiation of protozoan species and strains.

Component 1 Objectives:


1. Continued development of methods for the isolation, detection and differentiation of pathogenic protozoan parasites from complex matrices.
   
2. Use of improved detection methodology for periodic surveying of incoming produce for possible parasitic contaminants.
   
3. Analysis of foods and water sources implicated in outbreaks of cyclosporiasis and cryptosporidiosis.
Component 1 FY 2000 Deliverables:

1. Development of a multiplex PCR detection strategy for the identification of contaminating parasitic protozoa in produce and water sources.
   
2. Evaluation of UV irradiation on C. parvum infectivity from apple cider samples (Combined component 1 and 2 objective).
   
3. C. cayetanensis outbreak analysis.
Component 1 FY 2000 Progress:

1. Completion and publication of new detection methodologies.
   
2. Successfully isolated and identified C. cayetanensis in two food sources implicated in outbreaks of cyclosporiasis: July, 1999; Missouri; Chicken-basil pasta salad and June, 2000; Philadelphia, PA; Raspberry puree-filled wedding cake
   
3. Studies nearing completion: methods to differentially identify C. cayetanensis from other Cyclospora and Eimeria spp. by mismatch PCR; and, multiplex PCR for speciation of human-related Microsporidia.
   
4. Completion of a study to evaluate the effects of UV irradiation on the infectivity of C. parvum in two mouse models.
   
5. Conducted a Cyclospora workshop of FDA and CDC investigators to discuss issues in molecular detection methods.
   
6. Development of a multi-year collaboration between CFSAN and INCAP to study the seasonal emergence of C. cayetanensis in Guatemala.
Technical Barriers to Meeting Component 1 Objectives or Deliverables:

1. Lack of C. cayetanensis oocysts and the seasonality of the organism in the environment have slowed investigations into alternate molecular targets for detection and differential analysis.
Component 1 FY 2001 Deliverables:

1. Completion and publication of mismatch PCR and Microsporidium speciation PCR methodologies.
   
2. Initiation of Guatemalan study.
   
3. Develop strategy for surveying for C. cayetanensis, C. parvum, and Microsporidium spp. To investigate routes of contamination-implications for risk/safety evaluation; to include surface waters, soil samples, and human factors.
   
4. Conduct methods validation experiments prior to implementation of new molecular methodologies.
Component 1 FY 2002 Deliverables:

1. Interim progress report on Guatemalan study.
   
2. Implementation of survey.

Component 2 Objectives:


1. Develop a tissue culture system for the production of oocysts ex vivo
   
2. Develop an animal model for Cyclospora cayetanensis
   
3. Evaluation of the effects of UV irradiation on Cryptosporidium parvum infectivity from juice samples (Combined component 1 and 2 objective)
Component 2 FY 2000 Deliverables:

1. Continue to evaluate tissue culture models
   
2. Test and evaluate gnotobiotic pig as a model for C. cayetanensis
Component 2 FY 2000 Progress:

1. A method for limited oocyst production in tissue culture has been developed. Negotiations have been initiated to license this methodology to the private sector for optimization.
   
2. Gnotobiotic pigs have been challenged with C. cayetanensis on 2 separate occasions. An infection was not established in either challenge study.
   
3. Renewal of an IAG with NICHD to provide funding for research on developing a tissue culture model for C. cayetanensis
Technical Barriers to Meeting Component 2 Objectives or Deliverables:
Oocyst availability has been a limiting factor on research with C. cayetanensis. The seasonality of the disease makes this an inherent problem in working with this parasite.
Component 2 FY 2001 Deliverables:

1. Final evaluation of alternate host and culture system for C. cayetanensis oocyst propagation
   
2. Re-evaluate the immunosuppressed beagle dog as an animal model for C. cayetanensis
   
3. Continue research on the sand rat as an animal model for C. cayetanensis
Component 2 FY 2002 Deliverables:

1. Publication and completion of research on propagation of C. cayetanensis outside of the human host

Component 3 Objectives:


1. Analysis of environmental factors and farming practices to develop strategies for the prevention of C. cayetanensis
Component 3 FY 2000 Deliverables:

1. Evaluate epidemiologically the effect of a) water filters, b) hygiene measures, c) security measures on keeping the spring-summer crop of Guatemalan berries free of Cyclospora.
   
2. Evaluate the efficiency of Guatemalan government monitoring (PIPAA)
Component 3 FY 2000 Progress (Regress):

1. Unlike 1999, at least 2 U.S. outbreaks of cyclosporiasis were caused by Guatemalan raspberries produced by MPE farms. Was this due to negligent application of MPE or due to uncontrolled factors (animals, dust, rain splatter)?
Technical Barriers to Meeting Component 3 Objectives or Deliverables:

1. No detailed epidemiology on farms.
Component 3 FY 2001 Deliverables:

1. Determine reasons for the Summer 2000 outbreaks.

Jackson, G.J., L.A. Solorzano, J.E. Kvenberg, L.G. Flores, et al. 1999. Routes by which Cyclospora cayetanensis may contaminate food. Food Microbiology and Food Safety in the next Millennium. A.C.T. Tuijtelaars, et al., eds.) ICMFH.

Orlandi, P.A. and K.A. Lampel. 2000. Extraction-free, filter-based template preparation for rapid and sensitive pcr detection of pathogenic parasitic protozoa. J. Clin. Microbiol. 38(6):2271-2277.

Berhard, M.L., Y.R. Ortega, D.E. Hanes, E.K. Nace, R.Q. Do, M.G. Robl, K. Y. Won, C. Gavidia, N.L Sass, K. Mansfield, A. Gonzalo, J. Griffiths, R. Gilman, C. Sterling and M. J. Arrowood. 2000. Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals. J. Parasitol. 86(3):577-582.

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Hypertext updated by dav 2001-OCT-02