AN ASSAY COMBINING CELL-CULTURE WITH REVERSE-TRANSCRIPTASE PCR TO DETECT AND DETERMINE THE INFECTIVITY OF WATERBORNE CRYPTOSPORIDIUM-PARVUM

The presence of Cryptosporidium in drinking water supplies is a signif icant problem faced by the water industry. Although a variety of metho ds exist for the detection of waterborne oocysts, water utilities curr ently have no way of assessing the infectivity of detected oocysts and consequently are unable to accurately determine the risks posed to pu blic health by waterborne Cryptosporidium. In this paper, the developm ent of an infectivity assay for waterborne Cryptosporidium parvum is d escribed, Oocysts were inoculated onto monolayers of Caco-2 cells and grown on microscope slides, and infections were detected by C. parvum specific reverse transcriptase PCR of extracted mRNA, targeting the he at shock protein 70 (hsp70) gene, A single infectious oocyst was detec ted by this experimental procedure. The use of concentrated samples ob tained from 250 liters of finished water had no observable effect on t he integrity of cell monolayers or on the infectivity of oocysts seede d into the concentrate, Intracellular developmental stages of the para site were also detected by using fluorescently labeled antibodies. One pair of PCR primers targeting the hsp70 gene was specific for C. parv um, while a second pair recognized all species of Cryptosporidium test ed. The C. parvum-specific primers amplified DNA from 1 to 10 oocysts used to seed 65 to 100 liters of concentrated environmental water samp les and were compatible with multiplex PCR for the simultaneous detect ion of C. parvum and Giardia lamblia. This paper confirms the utility of PCR for the detection of waterborne C. parvum and, most importantly , demonstrates the potential of an in vitro infectivity assay.