In vitro Model of Cryptosporidiosis

An in vitro model of human biliary cryptosporidiosis using H69 cells was employed in these studies. Before infecting cells, C. parvum oocysts were treated with 1% sodium hypochlorite on ice for 20 min followed by extensive washing with DMEM-F12 medium. Oocysts were then added to the cell culture to release sporozoites to infect cells [54] . Infection was performed in culture medium (DMEM-F12 with 100 U/ml penicillin and 100 µg/ml streptomycin) containing viable C. parvum oocysts (oocysts with host cells in a 5∶1 ratio). Inactivated organisms (treated at 65°C for 30 min) were used for sham infection controls. All experiments were performed in triplicate. For the inhibition experiments, SC-514 (Calbiochem) was added to the medium. Cells were pre-treated with SC-514 for 1 h prior to C. parvum infection. SC-514 was used at a concentration of 100 µM, which showed no cytotoxic effects on H69 cells or on C. parvum sporozoites, in these studies.
Real-time PCR and immunofluorescent microscopy were used to assay C. parvum infection as previously reported [24] (link). Briefly, primers specific for C. parvum 18s ribosomal RNA (forward: 5′-TAGAGATTGGAGGTTGTTCCT-3′ and reverse: 5′-CTCCACCAACTAAGAACGGCC-3′) were used to amplify the cDNA specific to the parasite. Primers specific for human plus C. parvum 18s were used to determine total 18s cDNA [24] (link). Data were expressed as copies of C. parvum 18s vs total 18s. For immunofluorescent microscopy, cells were fixed with 2% paraformaldehyde and incubated with a polyclonal antibody against C. parvum (a gift from Dr. Guan Zhu, Texas A&M University) followed by anti-rabbit FITC-conjugated secondary antibody (Molecular Probes) and co-staining with 4′, 6-diamidino-2-phenylindole (DAPI, 5 µM) to stain cell nuclei. Labeled cells were assessed by confocal laser scanning microscopy.