The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidium parvum oocysts

Abstract

A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence in the 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resulted in fluorescence of sporozoites within oocysts that were capable of excystation, while oocysts that were dead prior to fixation did not fluoresce. Correlation of the FISH method with viability as measured by in vitro excystation was statistically highly significant, with a calculated correlation coefficient of 0.998. Examination of sequence data for Cryptosporidium spp. other than C. parvum suggests that the Cry1 probe is C. parvum-specific. In addition, 19 isolates of C. parvum 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 fluorescence of FISH-stained oocysts was not bright enough to enable detection of oocysts in environmental water concentrates containing autofluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species-specific detection and viability determination of C. parvum oocysts in water samples.

Evaluation of fluorochromes for flow cytometric detection of Cryptosporidium parvum oocysts labelled by fluorescent in situ hybridization

“…Cryptosporidium parvum oocysts were purified as described previously (Vesey et al 1997b(Vesey et al , 1998. Saccharomyces cerevisiae yeast cells were prepared as described by Deere et al (1998a).…”

“…Fluorescent in situ hybridization (FISH) to ribosomal RNA has proved effective for specific fluorescent labelling of microorganisms (Amann et al 1990), with flow cytometry proving useful for their subsequent sorting or detection (Wallner et al 1993(Wallner et al , 1995Vesey et al 1998). The ribosomal RNA approach to such phytogenetic discrimination is reviewed by Amann et al (1995), and flow cytometry in microbiology has been the subject of a number of reviews (Lloyd 1993 ;Vesey et al 1994 ;Porter et al 1996Porter et al , 1997Deere et al 1996).…”

“…1997). Gene probe methods targeted to RNA can have better species specificity and correlation with viability (Vesey et al 1998).…”

“…This has proved successful for labelling oocysts if a cell sorting cytometer is used to purify the oocysts for visual confirmation (Vesey et al 1998). However, the detection of low numbers of oocysts in environmental samples by an analysis-only cytometer was not possible because of the poor signal intensity from FISH (Vesey et al 1998).…”

“…Recently, Deere et al (1998b) found that optimization of oocyst pre-treatment was not able to increase signal intensity beyond the levels reached by Vesey et al (1998) as the number of target ribosomes appeared to be the limiting factor. Therefore, the signal per ribosome must be increased to boost signal intensities.…”