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。

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.