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Detection of Cryptosporidium Spp. In the Wachusett Reservoir WatershedEPA Grant Number: U915773
Title: Detection of Cryptosporidium Spp. In the Wachusett Reservoir Watershed
Investigators: Jellison, Kristen L.
Institution: Massachusetts Institute of Technology
EPA Project Officer: Jones, Brandon
Project Period: August 1, 2000 through August 1, 2003
Project Amount: $102,000
RFA: STAR Graduate Fellowships (2000) RFA Text | Recipients Lists
Research Category: Academic Fellowships , Engineering and Environmental Chemistry , Fellowship - Civil/Environmental Engineering
The goal of this research project is to better characterize the behavior of Cryptosporidium oocysts in the Wachusett Reservoir watershed, the drinking water source for Boston, Massachusetts and surrounding municipalities. Specific aims of the work are to: (1) investigate seasonal trends in oocyst contamination of surface water; (2) identify Cryptosporidium species in surface water; (3) identify surrogate water quality parameters that can be used as markers for contamination risk; and (4) determine sources of oocyst contamination in the watershed.
Water samples are filtered through Gelman Envirochek Sampling Capsules from Pall Gelman Sciences Inc. (Ann Arbor, MI) according to manufacturer's recommendations. During filtration, water quality parameters including pH, specific conductivity, dissolved oxygen content, temperature, and turbidity are measured. Typically, ~40-80L of water are filtered. Filters are transported to the laboratory on ice and eluted according to manufacturer's recommendations within 36 hours of sample collection. Oocysts are purified from water pellets or fecal samples using immunomagnetic separation (IMS) with the Crypto-Scan IMS kit from ImmuCell (Portland, ME). DNA is extracted from purified oocysts with phenol-chloroform, precipitated with ethanol, and amplified using a nested PCR assay that targets a 434-bp variable region of the 18S rRNA gene. Nested PCR products are visualized on a 1.2 percent agarose gel stained with ethidium bromide, and the nested PCR products from positive samples are cloned into the pGEM-T Easy Vector System (Promega Corporation, WI). Selected clones of the nested PCR products are sequenced on an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems, CA).
The water sampling method will determine the contamination risk from oocysts in the environment that may pose a threat to public health, illustrating the need for specific oocyst detection methods that identify species type and thus health risk.