ENHANCED DEVELOPMENT OF CRYPTOSPORIDIUM-PARVUM IN-VITRO BY REMOVAL OF OOCYST TOXINS FROM INFECTED CELL MONOLAYERS

Previous studies have suggested that coccidian oocysts of the genus Eimeria contain toxic substances that can inhibit parasite development in cultured cells. To examine whether or not oocysts of Cryptosporidium parvum may also contain such toxic substances, Madin-Darby bovine kidney cells were plated into 24-well tissue culture plates in RPMI 1640 medium supplemented with 10% fetal bovine serum and inoculated with CsCl purified oocysts of C. parvum. Plates were sealed in candle jars at 37 degrees C so that sporozoites could excyst. After 3 hr, candle jars were opened, and the medium containing excysted and unexcysted oocysts was removed and replaced with fresh medium. H-3-uracil was then added to wells, and plates were reincubated for an additional 65 hr at 37 degrees C in candle jars. Control cultures consisted of infected cultures sealed and incubated in candle jars but not opened, as well as infected cultures exposed to atmospheric oxygen concentrations but not washed free of old medium. Parasite growth was measured as incorporation of[H-3]uracil by scintillation counting. Results revealed that washing inoculated monolayers and then adding fresh medium significantly enhanced uracil incorporation.