INTERNATIONAL COLLABORATIVE CELL CULTURE AND U/V STUDIES
Report Ref. No. 08/DW/06/21
In 2001 an agreement was entered into by representatives of the Drinking Water Inspectorate (DWI), the United Kingdom Water Industry Research (UKWIR), the American Water Works Association Research Foundation (AWWARF), the United States Environmental Protection Agency (US EPA), Kiwa and the Water Services Association of Australia (WSAA) to collaborate to implement a research programme into “Cryptosporidium Cell Culture and UV Treatment”. The aims of the programme were set out as:
The first phase of the project was put out to tender on two separate occasions without eliciting sufficient response to undertake proficiency trials for assessing infectivity of cryptosporidial oocysts amongst a number of laboratories. Having been unable to let a contract to compare a number of available tissue culture infectivity technologies, an optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in ‘blind’ trials. Flow cytometry sorted suspensions consisting of between 0-100% viable oocysts were prepared, shipped to the testing laboratory and analyzed ‘blindly’ by cell culture-IFA. Excystation of oocysts was also assessed. Data indicated the control (100% live) oocyst suspensions yielded statistically similar results to a cell culture dose response curve data developed previously at the testing laboratory. For test samples containing oocyst suspensions of unknown infectivity, cell culture-IFA analyses indicated a high degree of correlation with the infectivity of the samples as prepared. Cell culture infectivity has been shown to correlate well with neonatal mouse infectivity assays and these ‘blind’ validation trials provide credibility for the cell culture-IFA procedure as a cost-effective and expedient alternative to mouse infectivity assays for determining in vitro infectivity of C. parvum oocysts.Attempts by the Moredun Research Institute, Edinburgh to implement the tissue culture/RT-PCR infectivity assay for Cryptosporidium parvum, developed by Metropolitan Water Company of Southern California at the time of the study, were unsuccessful. Moredun found the method insufficiently sensitive and reproducible to allow its routine use. At best, the assay proved significantly more sensitive than neonatal mouse infectivity, but such performance was relatively infrequent, while reproducibility could vary considerably day to day.
The preliminary testing of a novel low pressure UV device for disinfection of Cryptosporidium parvum oocysts in a lowland river water demonstrated that disinfection could occur in turbidities up to 5 ntu, using in vitro excystation as a demonstration of oocyst viability.
For further information please contact UK Water Industry Research Limited,
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