β-Tubulin mRNA as a marker of Cryptosporidium parvum oocyst viability

Abstract

Determining the viability of waterborne Cryptosporidium parvum oocysts remains a technical challenge, rRNA and mRNA were evaluated in a reverse transcription (RT)-PCR assay as potential markers of oocyst viability. The rationale for this approach is the rapid turnover and postmortem decay of cellular RNA. The β-tubulin mRNA and an anonymous mRNA transcript were chosen as potential markers because they are the only mRNA species in C. parvum known to possess introns. This feature facilitated the distinction between genuine RT-PCR products and PCR products originating from copurifying DNA. Prolonged incubation at room temperature of initially viable oocysts resulted in a gradual decrease in mRNA levels, which correlated with the loss of oocyst infectivity to neonatal mice. In contrast, oocysts stored at 4°C for over 39 weeks maintained their infectivity and displayed no decrease in the level of β-tubulin RT-PCR product. The postmortem decay of two mRNA species demonstrates that RT-PCR analysis can provide information on the viability of C. parvum oocysts. The methodological similarity between PCR detection and RT-PCR viability analysis could facilitate the development of a combined detection and viability assay.