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Development of a Quantitative Cell Culture-Based Infectivity Assay for Cryptosporidium parvum in Source and Finished Water

EPA Grant Number: R825146
Title: Development of a Quantitative Cell Culture-Based Infectivity Assay for Cryptosporidium parvum in Source and Finished Water
Investigators: Leon, Ricardo De , Rochelle, Paul A.
Institution: Metropolitan Water District of Southern California
EPA Project Officer: Rosenthal, Sheila
Project Period: October 1, 1996 through September 30, 1998
Project Amount: $214,502
RFA: Drinking Water (1996)
Research Category: Drinking Water

Description:

Detection of Cryptosporidium oocysts in finished water is of concern because these protozoa have low infectious doses and high resistance to chlorination. Nonetheless, current detection methodology used for water samples, the immunofluorescent assay (IFA), does not determine whether the oocysts are viable or infectious. The purpose of this project is to develop a cell culture infectivity assay in combination with the rapid and specific methods of in-situ gene probe hybridization and polymerase chain reaction (PCR). The project objectives are: 1) optimize cell culture assay for infection with low levels of oocysts and develop a reverse transcriptase (RT)-PCR assay which targets Cryptosporidium parvum specific mRNA from a heat shock protein; 2) develop a quantitative in-situ (IS)-PCR cell culture-based infectivity assay for Cryptosporidium with confirmatory non-radioactive in-situ probing; 3) develop sample clean-up procedures which are compatible with cell culture and optimally isolate viable or infectious oocysts from environmental samples; and 4) evaluate the developed method with environmental concentrates from source and finished water. The expected outcome and benefits to public health and the water industry are the development of a quantitative method for determining the infectivity of C. parvum oocysts isolated from water. The assay will provide a better method to assess the risk to public health posed by C. parvum oocysts in finished water. It may also be used to determine the efficacy of oocyst inactivation by disinfectants and to monitor oocyst survival in water.

Supplemental Keywords:

pathogens, drinking water, genetic probes, California, CA, region 9, exposure, environmental testing, human health. , Water, Geographic Area, Scientific Discipline, RFA, Drinking Water, Analytical Chemistry, Health Risk Assessment, Environmental Chemistry, Genetics, State, drinking water system, drinking water contaminants, treatment, California (CA), finished water, exposure and effects, pathogenic protozoa, gene probe, cryptosporidium parvum oocysts, monitoring, emerging pathogens, human health effects, infectivity assays, immunofluorescent assay, source water, exposure

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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