Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe

Abstract

Aims: Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum. Methods and Results: FISH was performed using a 5¢-Texas red-labelled DNA oligonucleotide probe at 1 pmol ll )1 (G. Vesey, N. Ashbolt, E.J. Fricker, D. Deere, K.L. Williams, D.A. Veal and M. Dorsch (1998) Journal of Applied Microbiology 85, 429). Intact and heat-permeabilized oocysts were treated with 1-100 lg ml )1 RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h. Conclusions: Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mmol l )1 VRC before FISH permeabilization steps should neutralize RNase activity. Significance and Impact of the Study: Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.

A Real-Time PCR Method for Quantifying Viable Ascaris Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA

“…One option to degrade DNA in inactivated eggs would be to pretreat the eggs with proteinase and nucleases before PCR. This technique has been used effectively to decrease false-positive signals from inactivated viruses and Cryptosporidium oocysts (26,32).…”

Differential expression of the two distinct replication protein A subunits from Cryptosporidium parvum

“…Aliquots (10 ml) containing UV treated or mock treated samples were spotted onto poly-L-lysine coated coverslips and air-dried overnight at ambient temperature. Unexcysted samples were heated at 808C for 10 min to permeabilize oocysts [Smith et al, 2004]. Oocyst or sporozoite preparations were extracted with cold methanol, blocked in 10% BSA-PBS for 1 h, incubated individually with CpRPA1A and CpRPA1B polyclonal antibodies and TRITC-conjugated secondary antibodies as described previously [Millership and Zhu, 2002].…”

Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping of Viable Cryptosporidium Oocysts

“…Other more recent approaches utilize molecular biology-based techniques to assess oocyst viability. Fluorescence in situ hybridization (5,10,20,26,40,47), nucleic acid sequence-based amplification (4), reverse transcriptase PCR (RT-PCR) (17,22,50), and an integrated cell culture/ PCR assay (12,25) all show promise in providing a rapid and relatively inexpensive viability assay for Cryptosporidium. However, it still remains to be determined if these methods can detect all Cryptosporidium species and genotypes found in the environment.…”