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

Abstract

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 μM) and monensin (0.00225 to 0.144 μM) on in vitro parasite development were determined in further trials in cultures inoculated each with 105 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 μM monensin reached a maximum of 17%, and inhibition of 40% was observed at 0.0045 μM. Halofuginone bromide also efficiently inhibited parasite in vitro reproduction, albeit at higher concentrations. At 12.5 μM or more, inhibition was at least 90%; 0.05 μM still yielded 80% inhibition, whereas at concentrations below 0.00625 μM, 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.

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Correspondence to A. Daugschies.

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Najdrowski, M., Heckeroth, A.R., Wackwitz, C. et al. Development and validation of a cell culture based assay for in vitro assessment of anticryptosporidial compounds. Parasitol Res 101, 161–167 (2007). https://doi.org/10.1007/s00436-006-0437-z

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Keywords

  • Cryptosporidium parvum
  • Halofuginone
  • Monensin
  • In vitro cultivation
  • Growth inhibition