For infection of cultured epithelial cells in vitro, 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 excysted to release infective sporozoites (with an excystation efficiency greater than 90%), as previously reported [23] (link). Infection was performed in culture medium (DMEM-F12 with 100 U/ml penicillin and 100 µg/ml streptomycin) containing viable C. parvum sporozoites (from oocysts in a 5∶1 ratio with host cells). All experiments were performed in triplicate.
We adapted a mouse model of biliary and intestinal cryptosporidiosis via gallbladder injection of C. parvum originally developed by Verdon [30] (link). Briefly, 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 adjusted to 200,000 per 25 µl PBS and directly injected into the gallbladder of wild-type C57BL/6J or TLR4-deficient mice, as previously reported [13] (link), [30] (link). C. parvum infection in the intrahepatic bile ducts in the wild-type and TLR4-deficient mice was observed one week and two weeks post-injection. Five animals from each group at both time points were sacrificed and liver tissues obtained for immunohistochemistry or in situ hybridization, as previously reported [13] (link), [30] (link), [48] (link). The C57BL/6J wild-type and TLR4-deficient (C57BL/10ScNJ; Tlr4lps-del) genotypes were purchased from the Jackson laboratory.
Real-time PCR and immunofluorescent microscopy were used to assay C. parvum infection, as previously reported [22] (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. Data were expressed as copies of C. parvum 18s versus 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, College Station, TX), 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.
We adapted a mouse model of biliary and intestinal cryptosporidiosis via gallbladder injection of C. parvum originally developed by Verdon [30] (link). Briefly, 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 adjusted to 200,000 per 25 µl PBS and directly injected into the gallbladder of wild-type C57BL/6J or TLR4-deficient mice, as previously reported [13] (link), [30] (link). C. parvum infection in the intrahepatic bile ducts in the wild-type and TLR4-deficient mice was observed one week and two weeks post-injection. Five animals from each group at both time points were sacrificed and liver tissues obtained for immunohistochemistry or in situ hybridization, as previously reported [13] (link), [30] (link), [48] (link). The C57BL/6J wild-type and TLR4-deficient (C57BL/10ScNJ; Tlr4lps-del) genotypes were purchased from the Jackson laboratory.
Real-time PCR and immunofluorescent microscopy were used to assay C. parvum infection, as previously reported [22] (link). Briefly, primers specific for C. parvum 18s ribosomal RNA (forward:
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