Impact/Purpose:

Develop a method to detect microsporidians in water.

Description:

The health effects associated with various waterborne pathogenic microorganisms has been well documented. During the last quarter century protozoan parasites like Giardia and Cryptosporidium have been added to this group. To date, however, the causes of many cases of waterborne gastroenteritis have not been resolved. Microsporidians, which are on the Agency's Contaminant Candidate List, are obligate intracellular parasites of invertebrates, fish, laboratory rodents, rabbits, and primates. With the advent of the AIDS epidemic, microsporidians have become fully recognized as emerging agents of human disease. The transmission form is an environmentally resistant spore which range in size from 1 to 3 Fm in diameter. These protozoan parasites are transmitted in a fecal-oral mode by contaminated food, fomites, or water. Since several microsporidian genera from humans localize in either the gastrointestinal and urinary tracts, they can challenge wastewater treatment facilities and eventually get into surface drinking water sources. The purpose of this project is to develop a method to assess the levels of microsporidians in water sources and supplies. The approach will include the development of monoclonal antibodies, adaptation of standard immunofluorescent techniques used for Giardia and Cryptosporidium and the development of fluorescent in situ hybridization probes. To date, fluorescent in situ hybridization probes have been developed to the genus Encephalitozoon and to the species E. hellem.

Record Details:

Record Type:PROJECT( PUBLISHED REPORT/ REPORT)

Start Date:09/01/2003

Completion Date:09/01/2003

OMB Category:Other

Record ID: 15801

Show Additional Record Data

Project Information:

Progress :A fluorescent in situ hybridization (FISH) assay has been developed which targets a variable region of the rRNA of Encephalitozoon hellem with a fluorescent-labeled oligonucleotide probe. The specificity and reliability afforded by targeting unique genetic sequences within the rRNA far exceeds those requiring the biochemical expression of specific markers that may not be constant over time. In situ hybridization has the benefit of molecular information at the level of nucleic acid sequences, similar to PCR, but with the added advantage of morphological observation that PCR lacks. This assay appears to be highly sensitive and specific to an individual species, and has potential for being performed at relatively low cost. Furthermore, the FISH assay appears to work in conjunction with a fluorescently labeled antibody directed against the spore surface. Preliminary studies also have indicated that the FISH assay may be able to detect E. hellem spores seeded into source water.

Reports:

Hester, J.D., H.D.A. Lindquist, A.M. Bobst, and F.W. Schaefer, III. 2000. Fluorescent in situ detection of Encephalitozoon hellem spores with a 6-carboxyfluorescein-labeled ribosomal-RNA targeted oligonucleotide probe. Journal of Eukaryotic Microbiology 47:299-308.

Dufour, A.P., M. Feige, H.D.A. Lindquist, Editor, M. Messner, S. Regli, C. Rodgers, Frank W. Schaefer, III, S. Schaub, J. Sinclair, and L.J. Wymer. 1999. Criteria for evaluation of proposed protozoan detection methods. U.S. Environmental Protection Agency, Office of Research and Development, Cincinnati, OH. EPA 815-K-99-02.

Lindquist, H.D.A., A.P. Dufour, L.J. Wymer, F.W. Schaefer, III. 1999. Criteria for evaluation of proposed protozoan detection methods. J. Microbiol. Method. 37: 33-43.

Presentations:

Hester, J.D. H.D.A. Lindquist, A.M. Bobst, and F.W. Schaefer, III. 1999. In Situ Detetion of Encephalitozoon hellem Spores in Seeded Water Samples with a Fluorescent Labeled Ribosomal RNA-Targeted Oligonucleotide Probe. Ohio River Base Consortium for Research and Education; Cincinnati, OH.

Hester, J.D. 1999. Fluorescent In Situ Detection of Encephalitozoon hellem Spores in Environmental Water Samples. Ecology Division Seminar. Miami University, Oxford, Ohio.

Hester, J.D. H.D.A. Lindquist, A. M. Bobst and F.W. Schaefer III. 1999. Fluorescent In Situ Detection of Encephalitozoon hellem Spores in Environmental Water Samples. 2000 Joint Meeting of the American Society of Parasitologists and the Society of Protozologists, San Juan Puerto Rico. 24-28 June 2000.

Relevance :The Office of Research and Development and the Office of Water jointly published the Research Plan for Microbial Pathogens and Disinfection By-Products in Drinking Water in 1997. The support functions and methods being developed under this task relate to the plan's priority areas, "methods for detecting and enumerating Cryptosporidium and Giardia (including identifying the viability potential) in source and finished drinking water, and methods for other emerging protozoa." Specifically, research is being performed in exposure task EX.M.7, "new protozoan agents."

The Safe Drinking Water Act (SDWA), as amended in 1996, requires the Agency to establish criteria for a monitoring program for unregulated contaminants and to publish a list of contaminants to be monitored. The Office of Water has established a Contaminant Candidate List (CCL) that identifies agents being considered for monitoring under this rule. This list includes microsporidia.

Clients :Office of Ground Water and Drinking Water (Dr. Paul Berger)

Research Component :M/DBP (MICROBIAL)

Risk Paradigm :EXPOSURE

Progress :A fluorescent in situ hybridization (FISH) assay has been developed which targets a variable region of the rRNA of Encephalitozoon hellem with a fluorescent-labeled oligonucleotide probe. The specificity and reliability afforded by targeting unique genetic sequences within the rRNA far exceeds those requiring the biochemical expression of specific markers that may not be constant over time. In situ hybridization has the benefit of molecular information at the level of nucleic acid sequences, similar to PCR, but with the added advantage of morphological observation that PCR lacks. This assay appears to be highly sensitive and specific to an individual species, and has potential for being performed at relatively low cost. Furthermore, the FISH assay appears to work in conjunction with a fluorescently labeled antibody directed against the spore surface. Preliminary studies also have indicated that the FISH assay may be able to detect E. hellem spores seeded into source water.

Reports:

Hester, J.D., H.D.A. Lindquist, A.M. Bobst, and F.W. Schaefer, III. 2000. Fluorescent in situ detection of Encephalitozoon hellem spores with a 6-carboxyfluorescein-labeled ribosomal-RNA targeted oligonucleotide probe. Journal of Eukaryotic Microbiology 47:299-308.

Dufour, A.P., M. Feige, H.D.A. Lindquist, Editor, M. Messner, S. Regli, C. Rodgers, Frank W. Schaefer, III, S. Schaub, J. Sinclair, and L.J. Wymer. 1999. Criteria for evaluation of proposed protozoan detection methods. U.S. Environmental Protection Agency, Office of Research and Development, Cincinnati, OH. EPA 815-K-99-02.

Lindquist, H.D.A., A.P. Dufour, L.J. Wymer, F.W. Schaefer, III. 1999. Criteria for evaluation of proposed protozoan detection methods. J. Microbiol. Method. 37: 33-43.

Presentations:

Hester, J.D. H.D.A. Lindquist, A.M. Bobst, and F.W. Schaefer, III. 1999. In Situ Detetion of Encephalitozoon hellem Spores in Seeded Water Samples with a Fluorescent Labeled Ribosomal RNA-Targeted Oligonucleotide Probe. Ohio River Base Consortium for Research and Education; Cincinnati, OH.

Hester, J.D. 1999. Fluorescent In Situ Detection of Encephalitozoon hellem Spores in Environmental Water Samples. Ecology Division Seminar. Miami University, Oxford, Ohio.

Hester, J.D. H.D.A. Lindquist, A. M. Bobst and F.W. Schaefer III. 1999. Fluorescent In Situ Detection of Encephalitozoon hellem Spores in Environmental Water Samples. 2000 Joint Meeting of the American Society of Parasitologists and the Society of Protozologists, San Juan Puerto Rico. 24-28 June 2000.

Relevance :The Office of Research and Development and the Office of Water jointly published the Research Plan for Microbial Pathogens and Disinfection By-Products in Drinking Water in 1997. The support functions and methods being developed under this task relate to the plan's priority areas, "methods for detecting and enumerating Cryptosporidium and Giardia (including identifying the viability potential) in source and finished drinking water, and methods for other emerging protozoa." Specifically, research is being performed in exposure task EX.M.7, "new protozoan agents."

The Safe Drinking Water Act (SDWA), as amended in 1996, requires the Agency to establish criteria for a monitoring program for unregulated contaminants and to publish a list of contaminants to be monitored. The Office of Water has established a Contaminant Candidate List (CCL) that identifies agents being considered for monitoring under this rule. This list includes microsporidia.

Clients :Office of Ground Water and Drinking Water (Dr. Paul Berger)

Research Component :CCL (MICROBIAL)

Risk Paradigm :EXPOSURE

Project IDs:

ID Code :EX.M.7

Project type :ORD-DW Plan

ID Code :1974

Project type :OMIS