Genetic Variation of Echinococcus Spp. in Yaks and Sheep in the Tibet

Ohiolei et al. Parasites Vectors (2019) 12:608 https://doi.org/10.1186/s13071-019-3857-1 Parasites & Vectors RESEARCH Open Access Genetic variation of Echinococcus spp. in yaks and sheep in the Tibet Autonomous Region of China based on mitochondrial DNA John Asekhaen Ohiolei1†, Chen‑Yang Xia2†, Li Li1, Jian‑Zhi Liu2, Wen‑Qiang Tang2, Yan‑Tao Wu1, Danqulamu2, Guo‑Qiang Zhu1, Bin Shi2, Bao‑Quan Fu1, Hong Yin1, Hong‑Bin Yan1* and Wan‑Zhong Jia1* Abstract Background: Cystic echinococcosis (CE) in humans and livestock is caused by Echinococcus granulosus (sensu lato). In China where CE is endemic, a number of studies have shown that Echinococcus granulosus (sensu stricto) is majorly responsible for CE. However, E. canadensis (G6) which is the second leading cause of CE is now being detected in most parts of the country. In this study, the species diversity and genetic variation of Echinococcus granulosus (s.l.) in four counties in Tibet Autonomous Region of China were investigated. Methods: Infection with Echinococcus granulosus (s.s.) in yaks and sheep was identifed using NADH dehydrogenase subunit 1 and 5 (nad1 and nad5) mitochondrial genes while the genotype G6 of E. canadensis initially diagnosed with NADH dehydrogenase subunit 1 (nad1) was further confrmed by analysis of the complete mitochondrial genome and a phylogenetic network constructed based on the nad2 and nad5 genes. Results: Out of 85 hydatid cyst samples collected from slaughtered sheep (n 54) and yaks (n 31), 83 were identi‑ fed as E. granulosus (s.s.) G1 (n 77), G3 (n 6) and 2 were identifed as E. canadensis= G6. Analysis= of the nad1/nad5 genes revealed 16/17 mutations= with 9/14 =parsimony informative sites resulting in 15/14 haplotypes, respectively. Haplotype diversity (Hd) and nucleotide diversity (π) of E. granulosus (s.s.) population were 0.650 and 0.00127 for nad1 and 0.782 and 0.00306 for nad5, respectively, with an overall negative Tajima’s D and Fu’s Fs. A low F ST indicated no genetic diference between isolates from sheep and yaks. Conclusion: Pockets of infection with E. canadensis (G6, G7, G8 and G10) have been previously reported in sheep, goats, yaks and/or humans in diferent parts of China. While the G6 genotype has been previously reported in sheep in the Tibet Autonomous Region, the detection in a yak in the present study represents the frst to the best of our knowledge. Therefore, we recommend future surveys and control eforts to comprehensively investigate other poten‑ tial intermediate hosts for the prevalence and genetic diversity of the E. canadensis group (G6, G7, G8 and G10) across the country and their inclusion into the existing CE control programme. Keywords: Echinococcus canadensis, Haplotypes, Genetic variation, Tibet Autonomous Region *Correspondence: [email protected]; [email protected] †John Asekhaen Ohiolei and Chen‑Yang Xia contributed equally to this manuscript 1 State Key Laboratory of Veterinary Etiological Biology/National Professional Laboratory of Animal Hydatidosis, Key Laboratory of Veterinary Parasitology of Gansu Province/Lanzhou Veterinary Research Institute, CAAS, Lanzhou 730046, Gansu, People’s Republic of China Full list of author information is available at the end of the article © The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Parasites Vectors (2019) 12:608 Page 2 of 10 Background 4000 m and covers a landmass of about 1.23 million Cystic echinococcosis (CE) is a parasitic zoonosis caused km2 bordering Qinghai Province and Xinjiang Uygur by tapeworm species of Echinococcus granulosus (s.l.), Autonomous Region to the north, Sichuan and Yunnan which infect humans and livestock upon ingestion of the provinces to the east, and to the south, Myanmar, India, infective egg stage from the defnitive host resulting in Bhutan and Nepal. Tibetans live mostly as seminomadic the development of hydatid cyst of the metacestode larval pastoralists with goats, sheep, yaks, horses and dogs as stages in the liver, lungs and sometimes in other organs. their main domestic animals. Te Tibet region has a total Within the E. granulosus complex, high genetic variation of 74 counties in 7 prefectures that include 692 townships which afects species/genotype infectivity and preference and 5260 villages with a total population of 3 million [19]. to intermediate hosts has been recognised based on stud- Te study was carried out in four counties in the region ies of parasite mitochondrial DNA [1–3]. So far, known namely: Angren, Saga and Zhongba (Shigatse Prefecture), species in this complex include E. granulosus (sensu and Dangxiong (Lhasa City) (Fig. 1). stricto) (G1/G3), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6, G7, G8 and G10) and E. felidis [4]. Mean- Sample collection and DNA extraction while, the species status of the E. canadensis group is still Between October and December 2017, a total of 85 under debate as suggestions have been made to reclassify hydatid cysts were collected from the lungs and liver of the group into E. intermedius (G6/7), E. borealis (G8) and slaughtered sheep (age 1–11 years) and yaks (age 5–18 E. canadensis (G10) [5, 6]. Nonetheless, for the purpose years) during routine meat inspection in slaughterhouses of convenience, we refer to the group as E. canadensis all and slabs in the counties. Te slaughtered animals were through the report. raised in an extensive management system (free grazing) CE poses a serious public health concern in China as in the same counties where the study was conducted. Te a result of livestock infection, increasing morbidity in collected hydatid cyst samples (Additional fle 1: Figure humans and the consequent economic impact due to S1, Additional fle 2: Figure S2) were then cleaned with the cost of diagnosis, control and treatment. Te preva- 75% alcohol before the germinal layers were removed and lence of CE in China varies with location with the Qing- washed repeatedly with phosphate bufer solution (PBS) hai-Tibet Plateau being the most endemic region. In and then transported in an ice box to the laboratory China, a number of molecular epidemiological studies where they were stored at − 80 °C prior to DNA extrac- have identifed E. granulosus (s.s.) as the leading cause tion. Further, a piece of the germinal layer excised from of cystic echinococcosis [7–12]. However, pockets of each hydatid cyst was crushed in liquid nitrogen before infection due to E. canadensis (G6, G7, G8 and G10) are genomic DNA extraction was carried out using Qiagen now being reported [12–15]. Previously, infection with Blood and Tissue DNA Extraction Kit (Qiagen, Hilden, E. canadensis G6 genotype was frequently reported in Germany) according to the manufacturer’s instructions. the Xinjiang Uygur Autonomous Region [16, 17]. How- ever, recent reports of the G6 genotype and other mem- DNA amplifcation and sequencing bers of E. canadensis group in areas other than Xinjiang PCR was performed in a 25 µl fnal reaction mixture con- Uygur Autonomous Region, are raising concerns on its taining 12.5 μl Premix Taq ™ (Takara Bio, Kusatsu, Japan), host range and public health signifcance [12, 14, 15, 18]. 0.5 μl of each primer (10 pmol), 0.5 μl of the genomic Terefore, it becomes imperative to update the status DNA (c. 10–180 ng), and 11 µl RNAse-free water. Te of CE and the genetic diversity of species especially in PCR conditions were as follows: initial denaturation at counties or communities in endemic regions, which will 95 °C for 5 min; 35 cycles of denaturation at 95 °C for 30 assist in monitoring the progress of ongoing CE man- s, annealing at 55 °C for 40 s and elongation at 72 °C for agement and control programmes across the country. In 60–90 s; and a fnal extension step at 72 °C for 10 min. this study, the species diversity and genetic variation of Te complete mitochondrial NADH dehydrogenase Echinococcus species in four counties in the Tibet Auton- subunit 1 gene (nad1, 894 bp) was initially amplifed omous Region of China were investigated and for the for all isolates using the forward (5ʹ-ATT ATA GAA frst time, the presence of E. canadensis (G6) in yak was AAT TTT CGT TTT ACA CGC-3ʹ) and reverse (5ʹ- confrmed using mitochondrial DNA markers (nad2 and ATT CAC AAT TTA CTA TAT CAA AGT AAC C-3ʹ) nad5) and the complete mitochondrial genome analysis. primers [15]. In order to distinguish between geno- types of E. granulosus (s.s.) a primer pair amplifying a Methods fragment of the nad5 gene (about 680 bp) reported to Study area have conserved regions capable of discriminating G1 Te Tibet Autonomous Region is a plateau with grass- from G3 [20] was used, while the complete mitochon- land vegetation.

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