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

摘要

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 ml~(-1) (G. Vesey, N. Ashbolt, E.J. Fricker, D. Deere, K.L. Williams, D.A. Veal and M. Dorsch (1998) Journal of Applied Microbiology85, 429). Intact and heat-permeabilized oocysts were treated with 1–100 mg 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.