喀什地区某规模化引种场奶牛感染隐孢子虫的PCR检测与种系发育分析

doi:10.12160/j.issn.1672-5190.2024.03.015

摘要/Abstract

摘要： [目的]掌握新疆维吾尔自治区喀什地区某规模化引种场奶牛的隐孢子虫（Cryptosporidium）感染情况及其种类分布特征。[方法]采集该场不同年龄段奶牛粪便样本190份,基于小亚基核糖体RNA基因（SSU rDNA）,采用PCR方法检测隐孢子虫感染情况,经序列比对鉴定隐孢子虫种类。利用卡方（χ²）检验法,比较不同年龄段奶牛隐孢子虫感染率的差异。采用PCR方法扩增微小隐孢子虫（C. parvum）、牛隐孢子虫（C. bovis）和芮氏隐孢子虫（C. ryanae）的gp60基因,经序列比对鉴定3种隐孢子虫的基因亚型,构建种系发育树解析其遗传进化特征。[结果]32份奶牛粪便样本呈隐孢子虫阳性,总体感染率为16.84%（32/190）;鉴定出4种隐孢子虫,分别为微小隐孢子虫（n=8）、安氏隐孢子虫（C. andersoni,n=10）、牛隐孢子虫（n=10）和芮氏隐孢子虫（n=4）。以3月龄以下断奶前犊牛的隐孢子虫感染率较高,为28.00%（14/50）,不同年龄段奶牛隐孢子虫的感染率差异显著（χ²=8.679,P<0.05）。8份微小隐孢子虫阳性样本成功获得7条gp60基因亚型序列,鉴定出2种亚型,分别为ⅡdA15G1亚型（n=3）和ⅡdA19G1亚型（n=4）;10份牛隐孢子虫阳性样本成功获得6条gp60基因亚型序列,鉴定出3种亚型,分别为ⅩⅩⅥb（n=2）、ⅩⅩⅥc（n=3）和ⅩⅩⅥe（n=1）;4份芮氏隐孢子虫阳性样本均成功获得gp60基因亚型序列,均为ⅩⅩⅠb亚型。种系发育树分析结果显示,获得的微小隐孢子虫Ⅱd基因亚型序列与不同宿主源微小隐孢子虫Ⅱd亚型家族的序列同处于一个亚群,而与新西兰不同宿主源微小隐孢子虫Ⅱa家族亚型的序列形成不同亚群;获得的牛隐孢子虫和芮氏隐孢子虫的亚型序列均与我国不同地区牛源牛隐孢子虫和芮氏隐孢子虫的亚型序列同处一个大群。[结论]该引种场奶牛感染的隐孢子虫存在遗传多样分布特征,均为引进当地后感染,提示水源和草料污染可能是该场奶牛感染隐孢子虫的主要原因。

关键词: 隐孢子虫, 鉴定, 基因亚型, 遗传进化

Abstract: [Objective] This study was conducted to characterize the prevalence and species distribution of Cryptosporidium in dairy cattle on a large-scale introduction farm in Kashgar Prefecture, Xinjiang Uygur Autonomous Region. [Method] A total of 190 fecal samples were collected from dairy cattle of different age groups in this farm. Targeting on the small subunit ribosomal RNA gene （SSU rDNA）, PCR assay combined with sequencing were used to detect the Cryptosporidium infection. The species of Cryptosporidium were identified through SSU rDNA sequence alignment. Chi-square （χ²） test was performed to compare the differences in the Cryptosporidium infection rates among dairy cattle of different age groups. The gp60 genes of C. parvum, C. bovis and C. ryanae were amplified by PCR assay, and the subtypes of the 3 Cryptosporidium species were identified by sequence alignment. In addition, phylogenetic trees were constructed to analyze their genetic evolutionary characteristics. [Result] Thirty-two fecal samples of dairy cattle were positive for Cryptosporidium, with an overall infection rate of 16.84% （32/190）. Four Cryptosporidium species, including C. parvum （n=8）, C. andersoni （n=10）, C. bovis （n=10） and C. ryanae （n=4）, were identified. A higher infection rate of Cryptosporidium was observed in the pre-weaned calves under 3 months of age at 28.00% （14/50）. There were significant （χ²=8.679, P<0.05） differences in the infection rates of Cryptosporidium among dairy cattle of different age groups. Seven gp60 subtype sequences were obtained from the 8 C. parvum positive samples, with 2 subtypes identified as ⅡdA15G1 （n=3） and ⅡdA19G1 （n=4）. Six gp60 gene subtype sequences were obtained from the 10 C. bovis positive samples, with 3 subtypes identified as ⅩⅩⅥb （n=2）, ⅩⅩⅥc （n=3） and ⅩⅩⅥe （n=1）. The 4 C. ryanae positive samples were successfully subtyped with gp60 gene, all of which were identified as subtype ⅩⅪb. The phylogenetic tree analysis showed that the obtained Ⅱd subtype sequences of C. parvum in this study were in the same subgroup as the sequences of Ⅱd subtype family of C. parvum derived from different hosts, and were in different subgroups with the sequences ofⅡa subtype family of C. parvum derived from different hosts in New Zealand. Both the obtained subtype sequences of C. bovis and C. ryanae in this study were in the same group as those of cattle derived C. bovis and C. ryanae from different regions of China. [Conclusion] The Cryptosporidium prevalent in this introduction farm was characterized by diverse genetic distribution. The Cryptosporidium infection in dairy cattle occurred after being introduced locally, suggesting that water and forage pollution may be the primary causes of Cryptosporidium infection.

Key words: Cryptosporidium, identification, subtype, genetic evolution

中图分类号:

S858.232.723

朱亚楠, 张博文, 张振杰, 余复昌, 宋永生, 徐佳宁, 井波, 齐萌. 喀什地区某规模化引种场奶牛感染隐孢子虫的PCR检测与种系发育分析[J]. 畜牧与饲料科学, 2024, 45(3): 107-114.

ZHU Ya′nan, ZHANG Bowen, ZHANG Zhenjie, YU Fuchang, SONG Yongsheng, XU Jianing, JING Bo, QI Meng. PCR Detection and Phylogenetic Analysis of Cryptosporidium in Dairy Cattle on a Large-scale Introduction Farm in Kashgar Prefecture[J]. Animal Husbandry and Feed Science, 2024, 45(3): 107-114.

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