Date of Award
Doctor of Philosophy
Human rectal tumor (HRT) and human embryonic lung (HEL) cell lines were inoculated with oocysts and sporozoites of Cryptosporidium sp. from calves. Development of the parasites through the mature merozoite stage was observed in HRT cells and development of gametes was observed in HEL cells. The parasites were seen with both light and electron microscopy. The parasites failed to establish persistent or productive infections of the cultured cells;Use of in vitro excystation techniques to assess sporulation of oocysts revealed no evidence that sporulation occurred if oocysts were subjected to in vitro sporulation procedures. The data show that it is not worthwhile to attempt sporulation in an effort to increase yields of sporozoites and supports the hypothesis that sporulation of Cryptosporidium oocysts is completely endogenous;Sodium taurocholate, pH, and temperature were found to affect survival of sporozoites (as measured by maintenance of motility and morphology) after excystation. Loss of sporozoite viability may contribute to the ineffectiveness of cell culture systems;Factors influencing in vitro excystation were studied in an effort to maximize sporozoite yields. Best yields were obtained when oocysts were pretreated with bleach, then exposed to 1.0-0.1% sodium taurocholate solutions, pH 7.0 at 37(DEGREES)C for 60 min. Results of kinetic experiments to determine the effect of trypsin and protease inhibitors failed to implicate proteolytic enzymes as effectors of excystation. These data suggest that the excystation mechanisms used by Cryptosporidium are different from those used by Eimeria;New methods were developed for the isolation of Cryptosporidium oocysts from calf feces and for the isolation of excysted sporozoites from oocysts and cast oocyst shells. Oocysts were partially purified using sucrose step gradients and further purified by extraction with chloroform or sodium dodecyl sulfate followed by washing over Nuclepore filters. Filter purified oocysts could be treated with bleach and excysted by exposure to 0.15% taurocholic acid solutions. Sporozoites were recovered by drawing them through Nuclepore filters.
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Douglas Byron Woodmansee
Woodmansee, Douglas Byron, "Isolation, in vitro excystation, and in vitro development of Cryptosporidium sp from calves " (1986). Retrospective Theses and Dissertations. 8128.