杭州市规模化养殖场湖羊芽囊原虫的PCR检测与基因亚型鉴定

PCR Detection and Subgenotype Identification of Blastocystis in Hu Sheep in Large-scale Farms in Hangzhou City,China

[目的]掌握杭州市规模化养殖场中湖羊芽囊原虫的感染情况和基因亚型分布。[方法]从杭州市4个规模化养殖场采集167份湖羊的新鲜粪便样本,提取粪便全基因组DNA;基于小亚基核糖体RNA(SSU rRNA)基因,对粪便DNA样本进行PCR扩增,对阳性产物进行测序;经序列比对鉴定芽囊原虫基因亚型,构建系统进化树解析其遗传进化关系。[结果]167份样本中,检测出芽囊原虫阳性样本18份,感染率为10.78%(18/167);3个养殖场呈芽囊原虫阳性,感染率依次为21.43%(3/14)、21.21%(14/66)和2.13%(1/47),1个养殖场未检测出芽囊原虫;不同养殖场之间湖羊芽囊原虫的感染率存在极显著(P<0.01)差异。鉴定出7个芽囊原虫基因亚型,分别为ST7(n=1)、ST10(n=9)、ST21(n=3)、ST23(n=1)、ST24(n=2)、ST26(n=1)、ST30(n=1),以ST10亚型为优势感染亚型(9/18,50.00%);基于芽囊原虫SSU rRNA基因的系统进化树分析发现,6种基因亚型序列聚类形成1个大群,其中ST10和ST23聚类形成亚群,ST21、ST26和ST30聚类形成亚群,ST24聚类形成亚群;而ST7聚类形成另外1个群。[结论]杭州市湖羊芽囊原虫感染较常见,其基因亚型存在遗传多样性。研究结果为我国绵羊芽囊原虫的感染情况调查提供了基础数据。

[Objective]This study aimed to understand the infection status and subgenotype distribution of Blastocystis in Hu sheep in large-scale farms in Hangzhou City,China.[Method]A total of 167 fresh fecal samples of Hu sheep were collected from four large-scale farms in Hangzhou City,and their whole genomic DNA were extracted.PCR assay targeting the small-subunit ribosomal RNA(SSU rRNA)gene was performed using the fecal DNA samples as templates,and the obtained positive amplification products were sequenced.The subgenotypes of Blastocystis were identified through sequence alignment.Phylogenetic tree was constructed to analyze their genetic evolutionary relationships.[Result]Among the 167 samples,18 were positive for Blastocystis,with an infection rate of 10.78%(18/167).Three farms had the prevalence of Blastocystis,with infection rates of 21.43%(3/14),21.21%(14/66)and 2.13%(1/47),respectively.One farm had no Blastocystis detection.There were extremely significant(P<0.01)differences in the infection rates of Blastocystis among different farms.Seven subgenotypes of Blastocystis were identified,including ST7(n=1),ST10(n=9),ST21(n=3),ST23(n=1),ST24(n=2),ST26(n=1)and ST30(n=1).ST10 was found as the dominant subgenotype(9/18,50.00%).Based on the phylogenetic tree analysis of the SSU rRNA gene of Blastocystis,it was found that six gene subgenotypes were clustered into one large group,with ST10 and ST23 clustering into one subgroup,ST21,ST26 and ST30 clustering into one subgroup,and ST24 clustering into one subgroup as well,while ST7 was clustered into another group.[Conclusion]Blastocystis infection in Hu sheep was common in Hangzhou City,and the subgenotypes of Blastocystis had genetic diversity.The results obtained in this study provided basic data for the investigation of sheep Blastocystis infection status in China.