|Smith, Jim - MONTANA MICRO. SUPPLY|
|Veal, Duncan - MACQUARIE UNIV.|
|Dorsch, Matthias - MACQUARIE UNIV.|
Submitted to: American Waterworks Association Research Foundation
Publication Type: Book / Chapter
Publication Acceptance Date: August 8, 2002
Publication Date: December 1, 2002
Citation: JENKINS, M.C., TROUT, J.M., HIGGINS, J.A., SMITH, J., VEAL, D., DORSCH, M. ADVANCING CRYPTOSPORIDIUM PARVUM DETECTION METHODOLOGIES. American Waterworks Association Research Foundation. ISBN 1-58321-266-3. 2002. Interpretive Summary: A major problem facing water utilities is detecting Cryptosporidium parvum oocysts in source water that will be processed to drinking water. The difficulty is related to the high sensitivity required because the infectious dose of this parasite for humans is very low (~ 150 oocysts). In addition, C. parvum oocysts are very small (4-6 microns) and cannot be seen except with the aid of a microscope. This study showed that molecular methods could be used to detect low numbers of C. parvum oocysts in water and that these assays could also distinguish between live and dead oocysts. This study also showed that an inexpensive processing method, termed continuous flow centrifugation (CFC), could concentrate C. parvum oocysts such that the parasites would be detectable by the molecular assays. This study also showed that C. parvum oocysts were very stable in water at ambient temperatures. The oocysts remained viable and infectious up to 7 months in water. These results should not only provide tools to the water industry for detecting C. parvum oocysts in water, but also provide insight into the stability of the parasite in the environment.
Technical Abstract: Single real-time PCR using TaqMan probes based on a highly abundant gene for ribosomal protein showed sensitivity down to 50 C. parvum oocysts. This sensitivity was improved to 5 oocysts by nested PCR. Cell culture-PCR was capable of discriminating between live and dead C. parvum oocysts. Using these assays as well as fluorescent in-situ hybridization (FISH), C. parvum oocysts stored for 1-7 months at 15oC were infectious for mice and cell cultures and were viable as determined by FISH analysis. Mouse infection and cell culture PCR showed C. parvum oocysts stored beyond 7 months were non-viable. A two-fold decrease in FISH positivities was found with oocysts stored for 7 vs. 8 months. Oocysts stored for 9 months had a negligible FISH reaction. Oocyst amylopectin concentrations decreased two-fold from 1 to 2 months storage, and another two-fold between 2 and 3 months, remaining at this level until termination of the experiment. The CPAG RT-PCR signal decreased about 50% between 1 and 2 to 3 months storage at 15oC and another 50% at 4 months. No detectable RT-PCR signal was observed beyond 4 months storage. Continuous flow centrifugation (CFC) was evaluated as a method for concentrating oocysts in water. Source water oocyst recoveries always exceeded recoveries from finished water (83-106% vs. 25-92%). Oocysts recovered from source and finished water were detectable by PCR and were infectious for cell culture and viable as measured by FISH staining. The results showed that PCR, FISH, amylopectin, and RT-PCR assays, and cell culture coupled to PCR are useful for detecting and estimating viability of C. parvum oocysts in raw and finished water samples.