青海省部分地区藏羊梨形虫感染情况及遗传多样性分析

doi:10.12140/j.issn.1000-7423.2025.05.010

中国寄生虫学与寄生虫病杂志 ›› 2025, Vol. 43 ›› Issue (5): 663-669.doi: 10.12140/j.issn.1000-7423.2025.05.010

青海省部分地区藏羊梨形虫感染情况及遗传多样性分析

韩元¹^,²()(), 李志¹^,², 林伟山¹^,², 李春花¹^,², 王小红³, 才让周在⁴, 雷萌桐¹^,²^,^*()()

1 青海大学畜牧兽医科学院，青海西宁 810016
2 青海省动物疾病病原诊断与绿色防控技术研究重点实验室，青海西宁 810016
3 青海省海南藏族自治州贵南县畜牧兽医站，青海贵南 813100
4 青海省海北藏族自治州刚察县沙柳河镇畜牧兽医站，青海刚察 812300

收稿日期:2025-03-06 修回日期:2025-05-28 出版日期:2025-10-30 发布日期:2025-08-14
通讯作者: *雷萌桐（ORCID：0000-0003-4055-4561），男，博士，副研究员，从事动物寄生虫病流行病学与防控研究。E-mail：leimengtong@163.com
作者简介:韩元（ORCID：0009-0002-5810-6732），女，硕士，助理研究员，从事动物寄生虫病流行病学与防控研究。E-mail：hanyuan9401@163.com
基金资助:
青海省科技厅项目(2022-NK-169);青海大学青年科研基金(2023-QNY-9);青海省中央引导地方科技发展资金(2024ZY014)

Piroplasm infection and genetic diversity in Tibetan sheep in selected areas of Qinghai Province

HAN Yuan¹^,²()(), LI Zhi¹^,², LIN Weishan¹^,², LI Chunhua¹^,², WANG Xiaohong³, CAIRANG Zhouzai⁴, LEI Mengtong¹^,²^,^*()()

1 Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining 810016, Qinghai, China
2 Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Diseases and Green Technical Research for Prevention and Control, Xining 810016, Qinghai, China
3 Animal Husbandry and Veterinary Station of Guinan County, Hainan Tibetan Autonomous Prefecture, Qinghai Province, Guinan 813100, Qinghai, China
4 Animal Husbandry and Veterinary Station, Shaliuhe Township, Gangcha County, Haibei Tibetan Autonomous Prefecture, Qinghai Province, Gangcha 812300, Qinghai, China

Received:2025-03-06 Revised:2025-05-28 Online:2025-10-30 Published:2025-08-14
Contact: *E-mail: leimengtong@163.com
Supported by:
Science and Technology Department of Qinghai Province(2022-NK-169);Young Scientists Fund of Qinghai University(2023-QNY-9);Central Guidance on Local Science and Technology Development Fund of Qinghai Province(2024ZY014)

摘要/Abstract

摘要：

目的了解青海省部分地区藏羊梨形虫感染情况及其遗传进化特征。方法采集青海省8个州（市）12个县（区）的藏羊全血样品，硅基质柱法提取DNA，以梨形虫18S rRNA V4高变区基因片段为靶基因，采用巢式PCR法扩增，阳性样品测序后在NCBI中应用BLAST程序进行序列比对，鉴定梨形虫种类。应用DnaSPv6软件对阳性样品序列进行单倍型分析和核苷酸多态性分析，利用PopArt构建单倍型网络图。以田鼠巴贝虫（GenBank登录号：AB242176）序列作为外群，采用最大似然法构建基于梨形虫18S rRNA V4高度区序列的系统进化树。结果共采集475份藏羊血样，检出193份阳性血样，总感染率为40.6%（193/475）。12个县（区）均有梨形虫感染分布，感染率由高到低前3位依次为门源（94.1%，96/102）、海晏（72.7%，24/33）、乐都（47.8%，11/23）。感染的梨形虫虫种为吕氏泰勒虫、绵羊泰勒虫、尤氏泰勒虫和Theileria sp. OT3，感染率分别为20.6%（98/475）、17.7%（84/475）、1.3%（6/475）和1.1%（5/475）。感染的吕氏泰勒虫共有13个单倍型，以TlH1为主（占76.5%，75/98），主要分布在门源、乐都和民和；绵羊泰勒虫共有9个单倍型，以ToH1为主（占85.7%，72/84），分布在海晏、泽库、门源、刚察、湟源、达日、贵南、天峻和祁连；尤氏泰勒虫共有4个单倍型，以TuH2为主（3/6），主要分布在门源；Theileria sp. OT3共有4个单倍型，以TsH3为主（2/5），主要分布在民和。系统进化树结果显示，从青海藏羊全血样品获得的吕氏泰勒虫18S rRNA V4高变区序列与GenBank中吕氏泰勒虫四川羚牛株，兰州、天祝、青海和陕西绵羊株聚为一支；Theileria sp. OT3与Theileria sp. OT3青海牦牛株、陕西山羊株和西班牙绵羊株聚为一支；绵羊泰勒虫与已发表的青海牦牛株，青海、苏丹、土耳其、埃及和新疆绵羊株聚为一支；尤氏泰勒虫则与青海田鼠株、湖南山羊株、印度山羊株、伊拉克绵羊株和土耳其绵羊株聚为一支。结论青海部分地区藏羊梨形虫感染较严重，感染的梨形虫虫种和单倍型呈多样性，并发现了青海藏羊感染Theileria sp. OT3，为青海地区藏羊梨形虫病的综合防控提供了科学支撑。

关键词: 藏羊, 梨形虫, 18S rRNA, 感染率, 单倍型, 系统发育分析

Abstract:

Objective To investigate the prevalence of piroplasm infections and genetic evolutionary characteristics of piroplasm in Tibetan sheep from some areas in Qinghai Province. Methods Whole blood samples were collected from Tibetan sheep across 12 counties (districts) in 8 states (cities) of Qinghai Province, and genomic DNA was extracted using the silicon matrix column method. The 18S rRNA V4 hypervariable region sequences of piroplasm was amplified using nested PCR assay, and samples positive for nested PCR assay were sequenced, followed by sequence alignment using the BLAST program in NCBI to identify the piroplasm species. The sequences of samples positive for nested PCR assay were subjected to haplotype analysis and nucleotide polymorphism analysis using the software DnaSP version 6, and a haplotype network diagram was constructed using the PopArt software. A phylogenetic tree based on the 18S rRNA V4 hypervariable region sequence of piroplasm was built using the maximum likelihood method with the sequence of Babesia microti (GenBank accession number: AB242176) as the outgroup. Results A total of 475 Tibetan sheep blood samples were collected, and 193 blood samples were positive for piroplasm, with 40.6% (193/475) overall prevalence of infections. piroplasm infection was detected across 12 districts (counties), and the three highest prevalence seen in Menyuan (94.1%, 96/102), Haiyan (72.7%, 24/33), and Ledu (47.8%, 11/23). The species of piroplasm infecting Tibetan sheep included Theileria luwenshuni, T. ovis, T. uilenbergi and Theileria sp. OT3, with infection prevalence rates of 20.6% (98/475), 17.7% (84/475), 1.3% (6/475), and 1.1% (5/475), respectively. There were 13 haplotypes of T. luwenshuni, with TlH1 as the dominant haplotype (76.5%, 75/98), which were mainly distributed in Menyuan, Ledu, and Minhe, and there were 9 haplotypes of T. ovis, with ToH1 as the dominant haplotype (85.7%, 72/84), which were prevalent in Haiyan, Zeku, Menyuan, Gangcha, Huangyuan, Dari, Guinan, Tianjun, and Qilian, while there were 4 haplotypes of T. uilenbergi, with TuH2 as the dominant haplotype (3/6), which were mainly distributed in Menyuan, and there were 4 haplotypes of Theileria sp. OT3, with TsH3 as the dominant haplotype (2/5), which were mainly distributed in Minhe. Phylogenetic analysis revealed that the sequence of the T. luwenshuni 18S rRNA V4 hypervariable region from the whole blood of Tibetan sheep in Qinghai Province was clustered into a clade with the sequences of Budorcas taxicolor-derived T. luwenshuni isolate and sheep-derived T. luwenshuni isolates from Lanzhou, Tianzhu, Qinghai and Shaanxi in GenBank, and the sequence of Theileria sp. OT3 was clustered into a clade with those of Theileria sp. OT3 from yak-derived parasite isolates in Qinghai, goat-derived strains in Shaanxi and sheep-derived strains in Spanish. In addition, the sequence of T. ovis was clustered into a clade with published sequences of yak-derived strains from Qinghai, and sheep-derived strains from Qinghai, Sudan, Turkey, Egypt, and Xinjiang, while the sequence of T. uilenbergi was clustered into a clade with sequences of Qinghai vole-derived strains, goat-derived strains from Hunan and India, and sheep-derived strains from Iraq and Turkey. Conclusion The piroplasm infection in Tibetan Sheep were serious, with diverse species and haplotypes of the infected piroplasms in some areas from Qinghai Province, and the Theileria sp. OT3 was detected in Tibetan sheep, which provides a scientific support for the comprehensive prevention and control of piroplasmosis in Tibetan sheep in Qinghai.

Key words: Tibetan sheep, Piroplasm, 18S rRNA, Prevalence, Haplotype, Phylogenetic analysis

中图分类号:

R531

韩元, 李志, 林伟山, 李春花, 王小红, 才让周在, 雷萌桐. 青海省部分地区藏羊梨形虫感染情况及遗传多样性分析[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(5): 663-669.

HAN Yuan, LI Zhi, LIN Weishan, LI Chunhua, WANG Xiaohong, CAIRANG Zhouzai, LEI Mengtong. Piroplasm infection and genetic diversity in Tibetan sheep in selected areas of Qinghai Province[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2025, 43(5): 663-669.

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采样点 Sample points	阳性率/% Positive rate/%	梨形虫种类 species
采样点 Sample points	阳性率/% Positive rate/%	吕氏泰勒虫 T. luwenshuni	尤氏泰勒虫 T. uilenbergi	绵羊泰勒虫 T. ovis	Theileria sp. OT3
曲麻莱 Qumalai	10.3（3/29）	0	0	3	0
达日 Dari	46.9（15/32）	0	0	15	0
泽库 Zeku	23.3（7/30）	0	0	7	0
贵南 Guinan	5.0（1/20）	0	0	1	0
天峻 Tianjun	2.4（1/42）	0	0	1	0
门源 Menyuan	94.1（96/102）	86	5	2	3
刚察 Gangcha	46.0（23/50）	0	0	23	0
海晏 Haiyan	72.7（24/33）	0	0	24	0
祁连 Qilian	6.3（2/32）	0	0	2	0
湟源 Huangyuan	10.0（6/60）	0	0	6	0
民和 Minhe	18.2（4/22）	1	1	0	2
乐都 Ledu	47.8（11/23）	11	0	0	0
总计 Total	40.6（193/475）	98	6	84	5

梨形虫种类 species	18S rRNA
梨形虫种类 species	序列数 No. sequences	多态位点数 No. polymorphic sites	单倍型数量 No. haplotype	单倍型多样性 Haplotype diversity	核苷酸多样性 Nucleotide diversity	田岛D值 Tajima’s D value	傅-李F值 Fu and Li’s F value
吕氏泰勒虫 T. luwenshuni	98	55	13	0.410	0.007 18	-2.447	-3.850
尤氏泰勒虫 T. uilenbergi	6	5	4	0.800	0.010 73	-0.826	-0.851
绵羊泰勒虫 T. ovis	84	23	9	0.265	0.002 41	-0.438	-5.624
Theileria sp. OT3	5	47	4	0.900	0.052 02	-1.224	-1.297

青海省部分地区藏羊梨形虫感染情况及遗传多样性分析

Piroplasm infection and genetic diversity in Tibetan sheep in selected areas of Qinghai Province

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