关键词: 微小隐孢子虫, 卵囊, 改良饱和盐水漂浮法, Percoll密度梯度离心法, 不连续蔗糖密度梯度离心法, 氯化铯密度梯度离心法, 牛输卵, 管上皮细胞

Abstract: Objective To explore an applicable method for isolation and purification of Cryptosporidium parvum oocysts with high purity， recovery and vigor from mouse feces. Methods Four techniques were used for isolating and purifying C.parvum oocysts from mouse feces： modified saturated saline flotation， percoll gradient centrifugation， CsCl gradient centrifugation and the classical discontinuous sucrose gradient centrifugation. Oocysts received from the methods were used respectively to infect in vitro bovine fallopian tube epithelial cells （BFTE） and the development of the oocysts was examined under microscope after 48 h and 72 h cultivation. Results The number of oocysts received by the classical discontinuous sucrose gradient centrifugation [（2.86±0.08）×10⁷］ was significantly higher than that of percoll gradient centrifugation [（1.52±0.08）×10⁷］ （P<0.01） and CsCl gradient centrifugation [（2.46±0.13）×10⁷］ （P<0.05）， but similar to that of the modified saturated saline flotation [（2.88±0.15）×10⁷］. No significant difference was found on the number of oocysts by BFTE cultivation at 48 and 72 hours post-inoculation（P>0.05）. Oocysts received from CsCl gradient centrifugation showed higher purity than those by discontinuous sucrose gradient centrifugation. Conclusion In comparison to the classical discontinuous sucrose gradient centrifugation, operation of the modified saturated saline flotation is easier and faster， and the purity of oocysts isolated by CsCl gradient centrifugation is higher.

Key words: Cryptosporidium parvum, Oocyst, Modified saturated saline flotation, Percoll gradient centrifugation, Discontinuous sucrose gradient centrifugation, CsCl gradient centrifugation, BFTE