Asexual development of Cryptosporidium parvum within a differentiated human enterocyte cell line

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Abstract
Unremitting diarrhea with malabsorption is associated with Cryptosporidium parvum infection of the small intestine in patients with AIDS. The lack of a well-defined in vitro model of C. parvum infection has severely hampered research into the biology of cryptosporidial invasion of the host epithelial cell and development of new pharmacologic and immunologic therapies. The adherent human intestinal epithelial cell line HT29 when grown in glucose-free medium develops morphologic and functional characteristics of the small intestine enterocyte and was used to develop an in vitro model of infection. Cryptosporidium oocysts obtained from AIDS patients were applied to a monolayer of cloned, differentiated HT29.74 cells. Cells were fixed and stained to estimate the degree of parasite infection. Schizonts were easily distinguished from the host cell by light microscopy. Twenty-four hours after 10(5) oocysts were added to approximately 10(6) HT29.74 cells, Cryptosporidium infection rates varied from 50 to 120 schizonts per 1,000 cells. Among 14 different experiments, the mean infection rate was 91 (+/- 18) schizonts per 1,000 cells. Electron microscopy at 6 and 24 h confirmed intracellular localization and development of schizonts. The morphologic features of the cryptosporidial schizonts within HT29.74 cells, which included the presence of a dense band and feeder layer, were identical to those described during cryptosporidial infection of human enterocytes in patients with AIDS. Fewer schizonts were observed at 5 days and beyond. Infection of differentiated HT29.74 cells (62 and 65 schizonts per 1,000 cells at 24 and 72 h, respectively) was over five times more efficient than infection of undifferentiated HT29.74 cells (9 and 5 schizonts per 1,000 cells at 24 and 72 h, respectively). In vitro infection of differentiated HT29.74 cells will allow a better understanding of the mechanisms by which C. parvum infects the small intestinal epithelium and will allow a systematic evaluation of new therapeutic agents.