关键词: 安氏隐孢子虫, 奶牛, 种类鉴定, 种系发育

Abstract: Objective   To isolate cow-origin Cryptosporidium in Hefei，and identify its species.  Methods   285 dairy cattle fecal samples collected from a farm in Hefei were examined by using floating saturated solution of sucrose and modified acid-fast staining. Cryptosporidium oocysts were isolated and purified from positive fecal samples. Genetic DNA was extracted to be the template. According to the sequence of 18S rRNA gene and HSP70 gene from Cryptosporidium sp.，the primers were designed and synthesized. The PCR products were amplified by PCR and nested-PCR. The nested PCR products were cloned and sequenced. Homology searches and phylogenic tree construction were done by DNAStar software.  Results   Five fecal samples were positive by morphological methods with an infection rate of 1.8%（5/285）. Oocysts from the 5 positive fecal samples were elliptical or ovoid detected by using floating saturated solution of sucrose and modified acid-fast staining with the size of 7.37 μm×6.13 μm and 7.58 μm×6.20 μm，and a shape index of 1.20 and 1.22，respectively. Nested-PCR resulted in a 18S rRNA and HSP70 gene fragments with approximately 250 bp and 325 bp，respectively. The five isolates showed a high level of nucleic acid identity with sequence data of the 18S rRNA gene of Cryptosporidium andersoni （DQ989573），and they were clustered in the same clade. The highest HSP70 gene sequence identity was found among the five isolates and other reported C. andersoni isolates （AY954892 and DQ989576），and they were placed into the same clade.   Conclusion   The cow-origin Cryptosporidium isolates derived from Hefei is Cryptosporidium andersoni.

Key words: Cryptosporidium andersoni, Cow, Species identification, Phylogeny