Combination of cell culture and quantitative PCR (cc–qPCR) to assess disinfectants efficacy on Cryptosporidium oocysts under standardized conditions

Oocysts of Cryptosporidium parvum are resistant to environmental conditions and many disinfectants. A combination of cell culture and quantitative real time PCR (cc–qPCR) is established for evaluation of anticoccidial disinfectants against C. parvum. C. parvum oocysts were treated with disinfectants, washed and oocysts were incubated with HCT-8 cell monolayers in the presence of excystation medium for 3 h. Subsequently, unbound parasites were removed by washing with growing medium and the infected monolayers were further maintained in fresh growing medium for 48 h. Genomic DNA was extracted from each sample and qPCR performed targeting a specific sequence of the 70 kDa heat shock protein gene in order to quantify development. Treatment of oocysts with cresolic disinfectants demonstrated dose dependent reduction of viability of oocysts. More than 98% inactivations were recorded with at least 2% concentration of cresolic disinfectants after 2 h of treatment. Bleach (sodium hypochlorite) at 6% solution induced 92.7% inactivation of C. parvum oocysts after 2 h. Thermally treated oocysts (56 and 70 °C for 20 min) demonstrated complete inactivation, whereas at 38 °C no inactivation was observed. Application of Neopredisan® 135-1 and Aldecoc® TGE (4% for 2 h) as recommended according to the current guidelines stipulated by DVG (German Veterinary Society) consistently inactivated more than 99.5% of oocysts. The suggested cc–qPCR method appeared to be suited for standardized testing of inactivation measures, particularly for evaluation of chemical disinfectants and thus cc–qPCR is proposed as an alternative to the established chicken infectivity model for Eimeria tenella for testing anticoccidial disinfectants. A minimum inactivation of 99.5% in cc–qPCR model is claimed as a suitable threshold for certification of chemical products for disinfection of coccidia oocysts.