目的 了解深圳市某奶牛场引进育种奶牛的隐孢子虫感染情况及隐孢子虫分离株的分子特性。 方法 于2018年5月收集深圳市某奶牛场奶牛新鲜粪样,粪样分装后抽提隐孢子虫基因组DNA,巢式PCR扩增隐孢子虫小亚单位核糖体核糖核酸基因（SSU rDNA）,阳性样品进行测序和BLAST比对分析,鉴定隐孢子虫基因型,并构建系统进化树。采用Graphad Prism7.0软件进行作图和卡方检验。 结果 共收集20头奶牛粪样,其中成年产乳牛粪样5份,断奶后犊牛粪样10份,断奶前犊牛粪样5份。检出隐孢子虫阳性8份,阳性率为40%;其中成年产乳牛隐孢子虫阳性1份（1/5）,断奶后犊牛隐孢子虫阳性5份（5/10）,断奶前犊牛隐孢子虫阳性2份（2/5）。测序分析鉴定出2个隐孢子虫基因型,芮氏隐孢子虫（n = 5）和牛隐孢子虫（n = 3）,各年龄组中均有芮氏隐孢子虫感染。经与GenBank中隐孢子虫SSU rDNA基因型比对,5个芮氏隐孢子虫分离株中4个与埃塞俄比亚牛源芮氏隐孢子虫SSU rDNA基因（GenBank登录号为KT922233）序列相似度达100%,1个与华北地区牛源芮氏隐孢子虫SSU rDNA基因（GenBank登录号为HQ179574）序列相似度达99.9%;3个牛隐孢子虫分离株与埃塞俄比亚牛源牛隐孢子虫SSU rDNA基因（GenBank登录号为KT922231）的序列相似度达100%。基于SSU rDNA基因序列构建的隐孢子虫种系发育进化树,本研究获得的芮氏隐孢子虫序列与牛隐孢子虫分别在两群分支中,与不同地区的同一隐孢子虫虫种一致。 结论 深圳市某奶牛场奶牛感染隐孢子虫较为普遍,以芮氏隐孢子虫为优势感染虫种。

Objective To investigate the status of Cryptosporidium infection in imported breeding dairy cows in a dairy farm in Shenzhen, and analyze the molecular characteristics of Cryptosporidium isolates. Methods Fresh cow fecal samples were collected from a dairy farm in Shenzhen. Genomic DNA was extracted from the samples, and the small subunit ribosomal ribonucleic acid gene (SSU rDNA) of Cryptosporidium was amplified by nested PCR. The PCR products were sequenced and blasted to determine the genotype, and construct phylogenetic tree. Data were analyzed with Chi-square test using Graphad Prism 7.0. Results A total of 20 cow fecal samples were collected, including 5 from adult cows, 10 from weaned calves and 5 from pre-weaning calves. The PCR results showed that 8 samples were positive of Cryptosporidium, with positive rate of 40%, including 1 adult cow (1/5), 5 weaned calves (5/10), and 2 pre-weaning calves (2/5). Sequencing results showed that 2 Cryptosporidium genotypes were detected, including C. ryanae (n = 5) and C. bovis (n = 3). The C. ryanae infection was found in all age groups. After blasting with the SSU rDNA gene of C. ryanae, four of the five isolates of C. ryanae were 100% similar to the SSU rDNA gene (KT922233) of bovine-borne C. ryanae from Ethiopia, and the other one was similar to that of the isolate of C. ryanae (HQ179574, 99.9%) from Northeast China. The three isolates of C. bovis were 100% similar with the SSU rDNA gene (KT922231) of bovine-borne C. bovis from Ethiopia. After constructing the phylogenetic tree of C. ryanae based on the sequence of SSU rDNA gene, the sequences of C. ryanae and C. bovis were in two branches, which were consistent with the same isolated Cryptosporidium species in different regions. Conclusion Cryptosporidium infection is common in this dairy farm in Shengzhen, with C. ryanae as a dominant species in infection.