Quantification of Cryptosporidium parvum in anaerobic digesters treating manure by (reverse-transcription) quantitative real-time PCR, infectivity and excystation tests.

The survival of Cryptosporidium parvum oocysts in anaerobic digesters treating manure was investigated for mesophilic, thermophilic, and a combined treatment (mesophilic-thermophilic-mesophilic) under different retention times of oocysts in the reactors. C. parvum DNA was extracted with an optimised protocol, and its amount determined by quantitative real-time PCR (qPCR). Results indicated noteworthy differences in DNA content after the different treatments. DNA was not degraded during the process. However, excystation and infectivity tests showed a reduction of viable oocyst numbers of > or = 2 and > or = 5 log units after the thermophilic treatment in two different experiments. Thus qPCR-targeting DNA can overestimate the number of oocysts that survive and remain viable after anaerobic digestion. However, targeting DNA is suitable to indicate the presence or absence of oocysts. Reverse transcription qPCR (RT-qPCR) targeting C. parvum hsp70 mRNA successfully indicated the presence of viable fraction of oocysts.

Development and validation of a cell culture based assay for in vitro assessment of anticryptosporidial compounds.

In vitro culture of Cryptosporidium parvum oocysts in HCT-8 cells was combined with immunofluorescent labelling and digital image analysis to quantify the development of the parasite by detecting and measuring the labelled area in the respective cell cultures. The number of inoculated oocysts and the labelled area correlated reliably and significantly (R (2), 0.98-0.99). The effects of various concentrations of halofuginone bromide (0.00039 to 50 microM) and monensin (0.00225 to 0.144 microM) on in vitro parasite development were determined in further trials in cultures inoculated each with 10(5) oocysts. Monensin reduced the detected area in a dose-dependant manner. In comparison to the untreated controls, the area positive for C. parvum in the cultures treated with 0.144 to 0.009 microM monensin reached a maximum of 17%, and inhibition of 40% was observed at 0.0045 microM. Halofuginone bromide also efficiently inhibited parasite in vitro reproduction, albeit at higher concentrations. At 12.5 microM or more, inhibition was at least 90%; 0.05 microM still yielded 80% inhibition, whereas at concentrations below 0.00625 microM, labelled areas abruptly increased. Both drugs appeared efficient under in vitro conditions; the applied system is suited to screen drugs for their anti-cryptosporidial capacity.