微小隐孢子虫感染绵羊模型的建立

为建立稳定、可靠的微小隐孢子虫(Cryptosporidium parvum)感染绵羊模型,将4只2日龄绵羊分成2组,分别经口接种1×106个(低剂量组)和1×107个(高剂量组)的C.parvum卵囊,观察绵羊每日精神状况,并采集粪便样品,提取DNA,用基于TaqMan探针的实时荧光定量PCR方法进行检测,计算每日排卵囊数量。结果表明,两组羔羊均从第4天开始出现精神沉郁、食欲下降及严重的腹泻症状,镜检发现有大量的C.parvum卵囊排出,其中高剂量组的2只羔羊在感染后的第10天死亡。TaqMan实时荧光定量PCR检测结果显示,两组羔羊均从第1天开始有少量的卵囊排出,从第5天开始增多。其中低剂量组的1#羔羊第9天达到高峰,2#羔羊第7天达到高峰,随后排卵囊量开始下降,2只羔羊直到实验结束的第36天仍有卵囊排出,并且首次发现羔羊感染C.parvum后出现3个排卵囊高峰期。高剂量组的2只羔羊均在第6天达到高峰,其中3#羔羊第7天仍然有大量卵囊排出,4#羔羊第7天和第8天仍有大量卵囊排出,随后开始下降;高剂量组羔羊高峰期排卵囊数量明显高于低剂量组羔羊高峰期排卵囊数量。对死亡羔羊进行的肠道组织学观察,发现虫体寄生部位肠黏膜层出现大面积实质细胞脱落,局部可见肠腺数量减少,被增生的结缔组织取代。提取死亡羔羊肠道内容物和粪便DNA,进行套式PCR扩增,经测序鉴定,证实为C.parvum感染,与羔羊接种的虫种一致。上述结果表明,接种1×106个C.parvum卵囊,绵羊出现明显的临床症状,并伴有大量的卵囊排出,可以作为C.parvum的感染模型;而接种1×107个C.parvum卵囊,绵羊会发生死亡,但是能够排出更多的卵囊,适合进行隐孢子虫的繁殖试验。为进一步利用该模型研究C.parvum的致病机理、疫苗和药物筛选奠定了良好的基础。

Establishment of Cryptosporidium parvum Sheep Infection Model

In order to establish a stable and reliable sheep infection model of Cryptosporidium parvum,four 2-day-old sheep were divided into two groups,and inoculated orally with 1×106(low dose group)and 1×107(high dose group)C.parvum oocysts,respectively.The daily mental status was observed,and fecal samples were collected every day.DNA was extracted from all fecal samples and detected by TaqMan real-time PCR method.The number of oocysts were calculated every day.The results showed that lambs inoculated with C.parvum oocysts were depressed and had a decreased appetite on day 4 post-inoculation(pi).Severe diarrhea symptoms occurred and a large number of oocysts were detected by microscopic examination,and the lambs in the high dose group were died on day 10 pi.The TaqMan real-time PCR results showed that both lambs excreted oocysts on day 1 pi,increased significantly on day 5 pi.All the lambs excreted oocysts increasing significantly on day 5 pi.In the low dose group,the number of oocysts excretion peaked on day 9 pi in lamb 1#,and on day 7 pi in lamb 2#,then began to decline,and continued to excrete up to day 36 pi.This is the first time to find that three peak periods of oocysts excretion were existed in lambs.In the high dose group,the number of oocysts excretion peaked on day 6 pi,and still had large number of oocysts excreted on day 7 pi in lamb 3#and on day 7 pi and 8 pi in lamb 4#,then began to decline.The number of oocysts excreted in high dose group was significantly higher than that in low dose group.Histological observation of intestinal in died lambs showed that large areas of parenchymal cells were exfoliated in intestinal mucosa,and partial intestinal glands were replaced with proliferated connective tissues.DNA from intestinal contents and feces of dead lambs were amplified by nest PCR,and C.parvum infection was verified in sheep by sequence analysis,which was consistent with the species of sheep inoculation.These results showed that sheep inoculated with 1×106 C.parvum oocysts can cause significant symptoms,and accompanied with a large number of oocysts excretion,suggest that sheep may be a good animal model for C.parvum infection.However,sheep inoculated with 1×107 C.parvum oocysts can cause sheep death,but more oocysts were obtained,and this model will be suitable for reproductive experiments.This study laid a good foundation for further study in pathogenesis,vaccines and drugs of C.parvum.