关键词: 安氏隐孢子虫, 小亚基核糖体RNA, 卵囊囊壁蛋白, 种系发生分析

Abstract: 【Abstract】 Objective To isolate and identify Cryptosporidium oocysts from feces of naturally infected cow. Methods Fecal samples were collected from Cryptosporidium infected cows confirmed by modified acid-fast staining method. Oocysts were isolated and purified with Sheather sucrose density gradient centrifugation technique. Genomic DNA was isolated with Chelex-100. Both primers were designed to amplify Cryptosporidium small subunit ribosome RNA gene （SSU rRNA） and Cryptosporidium oocyst wall protein gene （COWP）， respectively. The PCR products were cloned into pGEM-T and pGEM-T Easy vector and sequenced subsequently. Homology and phylogeny were analyzed with BLASTn and MEGA software. Results The results suggested that the size of oocysts was （7.4±0.32） μm by（5.4±0.21） μm and the ratio of length and width was 1.37±0.07（n=20）. BLASTn revealed that the identity of SSU rRNA and COWP gene of Cryptosporidium isolated from cow to the counterparts of C.andersoni was 100% and 99% respectively. Phylogenetic reconstruction placed the isolated Cryptosporidium within the C.andersoni clade based on the sequence of SSU rRNA and COWP gene. Conclusion What isolated from naturally infected cow feces has been identified as C. andersoni.

Key words: Cryptosporidium andersoni, Small subunit ribosome RNA（SSU rRNA）, Oocyst wall protein, Phylogenetic analysis