An improved in vitro infection model for viability testing of Cryptosporidium parvum oocysts

An in vitro infection model for the protozoan parasite Cryptosporidium parvum was evaluated for its suitability to determine the viability status of oocysts. Adherent HCT-8 cells were used as host cells and confluent monolayers were inoculated with oocyst suspensions in the presence of 0.4% sodium taurocholate which proved not to be cytotoxic. For a semi-quantitative detection of the infection a PCR-based assay was developed. The influence of physical (elevated temperature) and chemical (chlorocresole) inactivation methods on oocyst viability were evaluated. A minimum of 1000 untreated oocysts was necessary to establish a reproducibly detectable infection of the cells. With 10 and 100 oocysts, 30 and 78% of cell cultures, respectively, could be diagnosed as infected. For thermal inactivation two different temperature levels were used (38 and 55 °C). 55 °C, irrespective of incubation time, was sufficient to inactivate the oocysts to a degree below the detection limit. An elevation of temperature to 38 °C, in contrast, had no appreciable effect on oocyst infectivity in cell culture. Neopredisan® efficacy against the parasite was tested at 0.25, 1 and 4% concentration. 0.25 and 1% had no discernible inhibiting effect on the developmental potential of the oocysts, while 4% Neopredisan® resulted in a significant inhibition of Cryptosporidium development which was, however, not as prominent as heating to 55 °C, and not all oocysts could be inactivated.