液氮保存微小隐孢子虫卵囊对小鼠感染力的研究

Research of the infectivity of Cryptosporidium parvum oocysts preserved in liquid nitrogen

目的观察两种不同保存方法对微小隐孢子虫卵囊感染小鼠能力的影响。方法用PBS稀释纯化的卵囊，加入二甲基亚砜（DMSO），置-40℃3h后，放入液氮中保存；或加入2．5％重铬酸钾中，4℃保存。3个月后取出，观察它们的脱囊率；分别感染用地塞米松免疫抑制后的BALB／c小鼠，计数卵囊排出量，计算平均每克粪便中的卵囊数目，同时记录两组小鼠的死亡时间。分别采用t检验和成组设计的两样本秩和检验进行统计学分析。结果液氮中保存3个月的卵囊能够感染免疫抑制的BALB／c小鼠，其脱囊率为7．2％-49．6％、卵囊排出量为（4．7～7．3）×10^5／g，接种后实验小鼠死亡时间为124～351h，与4℃保存的卵囊差别无统计学意义[8．3％-51．2％，（4．6～6．9）×10^5／g，132～348h，P均〉0．05）]。结论液氮中保存3个月的卵囊与4℃保存的卵囊对免疫抑制的小鼠具有同样的感染力，突破了微小隐孢子虫卵囊只能在4℃保存的传统观念，为隐孢子虫卵囊的长期保存提供了理论和实践依据。

Objective To observe the infectivity of Cryptosporidium parvum oocysts preserved in two different ways. Methods Purified oocysts were stored at 4 ℃ in 2.5% potassium dichromate or preserved in liquid nitrogen in the presence of cryoprotectant dimethyl suffoxide （DMSO） for 3 months, then inoculated to BALB/c immuno-depressed mice respectively. The number of oocysts per gram feces and the survival time of infected mice after inoculation in two groups were analyzed by t test and rank sum test respectively. Results Both the purified oocysts kept in liquid nitrogen or stored at 4℃ in 2.5% potassium dichromate exhibited infectivity to mice. There were no significant differences in two groups in terms of excystation rate （7.2%-49.6% , 8.3% -51.2% ） ,number of oocysts shedding [ （4.7-7.3） ×10^5/g, （4.6-6.9） ×10^5/g） ] and survival time of mice（ 124-351 h, 132-348 h）after inoculation （P 〉 0.05 ）. Conclusions Both the oocysts kept in liquid nitrogen or stored at 4℃ in 2.5% potassium dichromate exhibited infectivity to mice. This provides the rationale for longer preservation period of oocysts.