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Simplified methods for obtaining purified oocysts from mice and for growing Cryptosporidium parvum in vitro
1WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, School of Veterinary Studies, Murdoch University, Australia.
Abstract:
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.
Insights
Researchers developed an improved in vitro cultivation method for Cryptosporidium parvum. This new protocol yields significantly higher parasite numbers in HCT-8 cells compared to previous methods, aiding in Cryptosporidium research.
Area of Science:
- Parasitology
- Cell Biology
- Infectious Diseases
Background:
- Cryptosporidium parvum is a significant cause of diarrheal disease.
- Effective in vitro cultivation is crucial for studying Cryptosporidium parvum pathogenesis and developing treatments.
- Previous in vitro culture methods have limitations in parasite yield and efficiency.
Purpose of the Study:
- To optimize in vitro cultivation conditions for Cryptosporidium parvum.
- To achieve significantly higher parasite yields in a human cell line.
- To establish a more robust and reproducible culture system for Cryptosporidium research.
Main Methods:
- Purification of Cryptosporidium parvum oocysts from infected Arc/Swiss mice.
- Optimization of oocyst pretreatment (low pH, trypsin) and RPMI-1640 media composition.
- Inoculation of confluent HCT-8 cell monolayers and incubation under specific atmospheric conditions (candle jar, 37°C).
Main Results:
- A refined protocol using specific RPMI-1640 supplements, pH, and atmospheric conditions was identified.
- Significantly superior growth of Cryptosporidium parvum was achieved in HCT-8 cells compared to previously reported methods.
- The optimized method allows for routine isolation of millions of purified oocysts per mouse.
Conclusions:
- The developed in vitro cultivation method provides a highly efficient system for growing Cryptosporidium parvum.
- This optimized protocol represents a significant advancement for Cryptosporidium parvum research, enabling more extensive studies.
- The improved culture conditions facilitate higher parasite yields, supporting drug screening and mechanistic studies.