Pa. Rochelle et al., AN ASSAY COMBINING CELL-CULTURE WITH REVERSE-TRANSCRIPTASE PCR TO DETECT AND DETERMINE THE INFECTIVITY OF WATERBORNE CRYPTOSPORIDIUM-PARVUM, Applied and environmental microbiology, 63(5), 1997, pp. 2029-2037
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.