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Simplified methods for obtaining purified oocysts from mice and for growing Cryptosporidium parvum in vitro

Meloni, B.P. and Thompson, R.C.A. (1996) Simplified methods for obtaining purified oocysts from mice and for growing Cryptosporidium parvum in vitro. The Journal of Parasitology, 82 (5). pp. 757-762.

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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 μg, 4-aminobenzoic acid 100 μg, calcium pantothenate 50 μg, ascorbic acid 875 μg, 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.

Item Type: Journal Article
Murdoch Affiliation(s): School of Veterinary Studies
Publisher: American Society of Parasitology
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