THE USE OF A RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBE FOR FLUORESCENT LABELING OF VIABLE CRYPTOSPORIDIUM-PARVUM OOCYSTS

A fluorescence in situ hybridization (FISH) technique has been develop ed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oli gonucleotide probe (Cry1 probe) targeting a specific sequence in the 1 8S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 prob e resulted in fluorescence of sporozoites within oocysts that were cap able of excystation, while oocysts that were dead prior to fixation di d not fluoresce, Correlation of the FISH method with viability as meas ured by in vitro excystation was statistically highly significant, wit h a calculated correlation coefficient of 0.998. Examination of sequen ce data for Cryptosporidium spp, other than C, parvum suggests that th e Cry1 probe is C, parvum-specific. In addition, 19 isolates of C, par vum were tested, and all fluoresced after hybridization with the Cry1 probe. Conversely, isolates of C. baileyi and C, muris were tested and found not to fluoresce after hybridization with the Cry1 probe. The f luorescence of FISH-stained oocysts was not bright enough to enable de tection of oocysts in environmental water concentrates containing auto fluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species-specific detection a nd viability determination of C, parvum oocysts in water samples.