Simplified methods for obtaining purified oocysts from mice and for growing Cryptosporidium parvum in vitro.
Seven- to 8-day-old Arc/Swiss mice were infected with 100,000-120,000 Cryptosporidium parvum oocysts. At 8 days postinfection (PI) the jejunum, ileum, cecum, colon, and rectum were removed. Using a simple extraction procedure and purification by Ficoll gradient centrifugation, we rountinely obtained between 3-6 million and up to 15 million purified oocysts per mouse. For in vitro cultivation, purified oocysts were pretreated in a low pH (2.5-3) 0.5% trypsin solution for 20 min, resuspended in supplemented RPMI-1640 containing glucose 0.1 g (5.55 mM), sodium bicarbonate 0.3 g, bovine bile 0.02 g, folic acid 25 micrograms, 4-aminobenzoic acid 100 micrograms, calcium pantothenate 50 micrograms, ascorbic acid 875 micrograms, penicillin G 10,000 U and streptomycin 0.01 g per 100 ml, and 1% fetal bovine serum (pH 7.4 before filtration), and used to inoculate confluent monolayers of the human adenocarcinoma cell line HCT-8. Incubation was in a candle jar at 37 C. We tested numerous supplements to RPMI-1640, different pHs, and atmospheric conditions and found the parameters described above produced the greatest parasite numbers in vitro. We obtained significantly superior growth of C. parvum grown in HCT-8 cells using the conditions described above than in culture conditions described previously.
Meloni
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BP
WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, School of Veterinary Studies, Murdoch University, Australia.
Thompson
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RC
D000230:Adenocarcinoma; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003457:Cryptosporidiosis / Q000469:parasitology*; D016785:Cryptosporidium parvum / Q000254:growth & development* / Q000302:isolation & purification; D003470:Culture Media; D004195:Disease Models, Animal; D005243:Feces / Q000469:parasitology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007078:Ileal Neoplasms; D007080:Ileocecal Valve; D051379:Mice; D014407:Tumor Cells, Cultured
D003470:Culture Media