Stress-Induced Hsp70 Gene Expression and Inactivation of Cryptosporidium parvum Oocysts by Chlorine-Based Oxidants

Abstract

Our research on the mechanisms of action of chlorine-based oxidants on Cryptosporidium parvum oocysts in water revealed a dual-phase effect: (i) response to oxidative stress, which was demonstrated by induced expression of the Hsp70 heat shock gene, and (ii) oocyst inactivation as a result of long-term exposure to oxidants. The relative biocidal effects of sodium hypochlorite (bleach) and electrolytically generated mixed oxidant solution (MOS) on C. parvum oocysts were compared at identical free chlorine concentrations. Oocyst inactivation was determined by quantitative reverse transcription-PCR (qRT-PCR) amplification of the heat-induced Hsp70 mRNA and compared with tissue culture infectivity. According to both assays, within the range between 25 and 250 mg/liter free chlorine and with 4 h contact time, MOS exhibits a higher efficacy in oocyst inactivation than hypochlorite. Other RNA-based viability assays, aimed at monitoring the levels of beta-tubulin mRNA and 18S rRNA, showed relatively slow decay rates of these molecules following disinfection by chlorine-based oxidants, rendering these molecular diagnostic viability markers inappropriate for disinfection efficacy assessment.