袋鼠源隐孢子虫分离株分子鉴定与种系发育关系分析

Molecular Identification and Phylogenetic Analysis of Cryptosporidium Isolates from a Kangaroo

【目的】对云南某动物园袋鼠粪样中分离到的1株隐孢子虫分离株进行种/基因型的分子鉴定,为云南动物园的袋鼠隐孢子虫病防控提供理论依据。【方法】针对隐孢子虫小亚基核糖体RNA基因序列设计引物,进行巢式PCR扩增,并对扩增产物进行测序和序列比对;使用DNAMAN和MEGA7.0等软件进行同源性分析并构建系统发育进化树,以确定该袋鼠源分离株与其他源隐孢子虫间的亲缘关系。【结果】成功扩增出隐孢子虫阳性片段。BLAST结果表明:获得的袋鼠源隐孢子虫分离株与鼠隐孢子虫(Cryptosporidium muris)一致,同源性可达100%。种系发育进化树表明:该分离株与鼠隐孢子虫处于同一分支,亲缘关系最近,属于胃寄生隐孢子虫的鼠隐孢子虫。【结论】该园袋鼠存在隐孢子虫感染,鉴定为具有人兽共患潜能的鼠隐孢子虫。本研究首次报道了中国袋鼠感染鼠隐孢子虫。

[Purpose]To identify the species/genotype of a Cryptosporidium isolated from faeces of kangaroos in a zoo in Yunnan,providing theoretical basis for the prevention and control of cryptosporidiosis in the zoo of Yunnan.[Methods]Primers were designed target to the conserved sequence of ribosomal RNA gene of Cryptosporidium for nested PCR amplification,and the PCR products was sequenced and compared.DNAMAN and MEGA 7.0 was used for homology analysis and phylogenetic tree was constructed to determine the genetic relationship between the kangaroo isolates and other Cryptosporidium.[Results]The positive fragment of Cryptosporidium was suc-cessfully amplified.BLAST results showed that the Cryptosporidium isolated from kangaroo was identical to Cryptosporidium muris,which was 100%homologous to C.muris.The phylogenetic tree showed that the isolated Cryptosporidium and C.muris were in the same genetic branch and most closely related,a gastric parasitic Cryptosporidium,and belonged to the gastro parasitic Cryptospor-idium category.[Conclusion]The kangaroo in this zoo shows the presence of Cryptosporidium in-fection and the pathogen is identified as C.muris,which has zoonosis potential.This is the first report of C.muris infection in kangaroo in China.