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

doi:10.12140/j.issn.1000-7423.2025.05.010

Abstract

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

CLC Number:

R531

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.

Figures/Tables 5

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References 28

[1]	Yin H, Schnittger L, Luo JX, et al. Ovine theileriosis in China: A new look at an old story[J]. Parasitol Res, 2007, 101(2): 191-195.
[2]	Tian Z, Gao S, Ren Q, et al. Mitochondrial genome of Theileria uilenbergi endemic in sheep and goats in China[J]. Parasitol Res, 2021, 120(10): 3429-3436.
[3]	郝桂英. 羊泰勒虫病的研究进展[J]. 中国兽医学报, 2020, 40(2): 424-428, 434.
	Hao GY. Progress on caprine and ovine theileriosis[J]. Chin J Vet Sci, 2020, 40(2): 424-428, 434. (in Chinese)
[4]	Zaeemi M, Haddadzadeh H, Khazraiinia P, et al. Identification of different Theileria species (Theileria lestoquardi, Theileria ovis, and Theileria annulata) in naturally infected sheep using nested PCR-RFLP[J]. Parasitol Res, 2011, 108(4): 837-843.
[5]	Li YQ, Guan GQ, Ma ML, et al. Theileria ovis discovered in China[J]. Exp Parasitol, 2011, 127(1): 304-307.
[6]	Nangru A, Maharana BR, Vohra S, et al. Molecular identification of Theileria species in naturally infected sheep using nested PCR-RFLP[J]. Parasitol Res, 2022, 121(5): 1487-1497.
[7]	Tian ZC, Liu GY, Yin H, et al. First report on the occurrence of Theileria sp. OT3 in China[J]. Parasitol Int, 2014, 63(2): 403-407.
[8]	Abdullah SH, Ali SA. Molecular investigation and phylogeny of Theileria spp. from naturally infected sheep and the first report of Theileria sp. OT3 in Sulaymaniyah governorate/Iraq[J]. Pol J Vet Sci, 2021, 24(2): 201-209.
[9]	Kirman R, Guven E. Molecular detection of Babesia and Theileria species/genotypes in sheep and ixodid ticks in Erzurum, Northeastern Turkey: First report of Babesia canis in sheep[J]. Res Vet Sci, 2023, 157: 40-49.
[10]	Al-Obaidi QT, Arslan SH, Sulaiman EG, et al. Clinical, haematological and biochemical studies of babesiosis in native goats in Mosul[J]. Iraqi J Vet Sci, 2010, 24(1): 31-35.
[11]	Zhou JL, Li ZX, Zhou ZC, et al. Epidemiological and molecular characteristics of piroplasmids and Anaplasma spp. in Tan sheep, Ningxia, northwest China[J]. Transbound Emerg Dis, 2024, 2024: 2529855.
[12]	Niu QL, Liu ZJ, Yang JF, et al. Genetic characterization and molecular survey of Babesia sp. Xinjiang infection in small ruminants and ixodid ticks in China[J]. Infect Genet Evol, 2017, 49: 330-335.
[13]	顾月月, 薄震宇, 刘欣超, 等. 羊巴贝斯虫病流行病学的研究进展[J]. 中国动物传染病学报, 2024, 32(3): 221-226.
	Gu YY, Bo ZY, Liu XC, et al. Advanced on epidemiological research of babesiosis in sheep[J]. Chin J Anim Infect Dis, 2024, 32(3): 221-226. (in Chinese)
[14]	林青. 青海藏羊肌肉蛋白类型及功能特征分析[D]. 青海: 青海大学, 2024: 1-2.
	Lin Q. Characterization of Muscle Protein Type and Function in Tibetan Sheep in Qinghai Province[D]. Qinghai: Qinghai University, 2024: 1-2.
[15]	Zhao HX, Zan XQ, Tao JZ, et al. Molecular characterization of tick-borne pathogens in Bactrian camels and ticks from Gansu Province, China[J]. Acta Parasitol, 2024, 69(1): 343-350.
[16]	Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets[J]. Mol Biol Evol, 2017, 34(12): 3299-3302.
[17]	Luo J, Yin H. Theileriosis of sheep and goats in China[J]. Trop Anim Health Prod, 1997, 29(4 Suppl): 8S-10S.
[18]	Li YC, Galon EM, Guo QY, et al. Molecular detection and identification of Babesia spp., Theileria spp., and Anaplasma spp. in sheep from border regions, northwestern China[J]. Front Vet Sci, 2020, 7: 630.
[19]	郝桂英, 杨丽静, 李瑞琪, 等. 四川省攀西地区羊梨形虫的分子流行病学调查[J]. 中国预防兽医学报, 2021, 43(1): 92-97.
	Hao GY, Yang LJ, Li RQ, et al. Molecular epidemiological survey of ovine and caprine infection with piroplasms in Panxi region of Sichuan Province[J]. Chin J Prev Vet Med, 2021, 43(1): 92-97. (in Chinese)
[20]	钟维, 杜雪梅, 郝力力, 等. 四川省阿坝州牦牛和藏绵羊梨形虫分子流行病学调查[J]. 中国兽医杂志, 2019, 55(1): 20-22, 27.
	Zhong W, Du XM, Hao LL, et al. Investigation on molecular epidemiology of Piroplasma infection in yaks and Tibetan sheep in Aba Tibetan Autonomous Prefecture of Sichuan Province[J]. Chin J Vet Med, 2019, 55(1): 20-22, 27. (in Chinese)
[21]	齐萌, 赵爱云, 王海国, 等. 新疆南疆绵羊泰勒虫病分子流行病学调查[J]. 中国病原生物学杂志, 2017, 12(11): 1102-1104, 1111.
	Qi M, Zhao AY, Wang HG, et al. Molecular epidemiological survey of Theileria spp. in sheep from southern Xinjiang[J]. J Pathog Biol, 2017, 12(11): 1102-1104, 1111. (in Chinese)
[22]	He YC, Chen WK, Ma P, et al. Molecular detection of Anaplasma spp., Babesia spp. and Theileria spp. in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibetan Plateau, China[J]. Parasit Vectors, 2021, 14(1): 613.
[23]	Yu ZQ, Song JK, Zhang HJ, et al. Molecular characterization of Theileria spp. in goats from Shaanxi Province, northwestern China[J]. J Parasitol, 2018. DOI: 10.1645/18-38.
[24]	Li JX, Jian YN, Jia LJ, et al. Molecular characterization of tick-borne bacteria and protozoans in yaks (Bos grunniens), Tibetan sheep (Ovis aries) and Bactrian camels (Camelus bactrianus) in the Qinghai-Tibetan Plateau Area, China[J]. Ticks Tick Borne Dis, 2020, 11(5): 101466.
[25]	弓超, 温丽翠, 李建龙, 等. 新疆和田地区于田县牛源环形泰勒虫种群基因型及遗传多样性分析[J]. 黑龙江畜牧兽医, 2024(12): 74-82.
	Gong C, Wen LC, Li JL, et al. Analysis of genotypes and genetic diversity of bovine-derived Theileria annulata populations in Yutian County, Hotan Prefecture, Xinjiang Uygur Autonomous Region[J]. Heilongjiang Anim Sci Vet Med, 2024(12): 74-82. (in Chinese)
[26]	Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism[J]. Genetics, 1989, 123(3): 585-595.
[27]	Fu YX. Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection[J]. Genetics, 1997, 147(2): 915-925.
[28]	An B, Zhang LX, Wang YT, et al. Comparative phylogeography of two sister species of snowcock: Impacts of species-specific altitude preference and life history[J]. Avian Res, 2020, 11(1): 1.

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