乳牛源隐孢子虫种类的PCR-RFLP和分子种系发育分析鉴定

Species identification of cryptosporidia isolated from dairy cattle by PCR-RFLP and phylogenetic analysis

应用饱和蔗糖溶液漂浮法和改良抗酸染色法对从浙江省杭州市整体搬迁至河南省郑州市1个月的某奶牛场隐孢子虫的感染情况进行了调查。共检查624份粪样,隐孢子虫总阳性率为2.24%(14/624)。根据其形态结构等特征初步鉴定其为安氏隐孢子虫(Cryptosporidium andersoni,感染率为2.08%)和牛隐孢子虫(C.bovis,感染率为0.16%)。对这些分离株进行的小鼠感染试验结果表明,分离的C.andersoni不感染SCID小鼠、正常的及免疫抑制的昆明系小鼠。利用套式PCR成功扩增出12个隐孢子虫分离株的18 SrRNA。SspⅠ和VspⅠ酶切分析显示,牛源隐孢子虫与骆驼源C.andersoni的酶切图谱相同;系统发育分析结果显示,12个乳牛源隐孢子虫分离株均为C.andersoni。

A epidemiological survey on cryptosporidiosis was carried out on one farm in Zhengzhou Prefecture of Henan Province, where the dairy cattle were moved from Hangzhou Prefecture of Zhejiang Province one month ago. 624 fecal samples were examined by the saturated sugar solution flotation method and the acid-fast stain technique. The overall infection rate was 2. 24% （14/624）. According to the morphology characteristics,two different oocysts were identified to be Cryptosporidium andersoni （2.08%） and C. bovis（0. 16%）,respectively. Cross-transmission experiments showed that the C. andersoni isolates did not infect severe combined immunodeficiency （SCID） mice and immunosuppressed or immunocompetent Kunming mice. 18 S rRNA gene was successfully amplified from 12 Cryptosporidium isolates by nested- PCR analysis. And then the gene was digested by Vsp I and Ssp I ,the maps of Cryptosporidium isolates from calf and C. andersoni isolate from camel were identical. Phylogenetic analysis showed that the 12 isolates were all belonged to C. andersoni.