First report on the phylogenetic relationship, genetic variation of Echinococcus shiquicus isolates in Tibet Autonomous Region, China

Abstract

Background Cystic or alveolar echinococcosis caused by the larval stages of Echinococcus spp. is a very severe zoonotic helminth infection. Echinococcus shiquicus is a newly discovered species that has only been reported in the Qinghai and Sichuan provinces of the Qinghai-Tibet plateau, China where, to date, it has only been confirmed in Tibetan foxes and wild small mammal populations of the Tibetan plateau. Information on its genetic and evolutionary diversity is scanty. The aim of this study was to investigate the prevalence of E. shiquicus in plateau pikas (Ochotona curzoniae), a known intermediate host, and to determine the genetic variation and phylogenetic relationship of the E. shiquicus population in the Tibet region of China based on mitochondrial DNA. Methods Echinococcus shiquicus samples were collected from Damxung and Nyêmo counties (located in Tibet Autonomous Region, China). The mitochondrial cox1 and nad1 gene sequences were analyzed, and the genetic diversity and epidemiology of E. shiquicus in the region were discussed based on the results. Results The prevalence of E. shiquicus in pikas in Damxung and Nyêmo counties was 3.95% (6/152) and 6.98% (9/129), respectively. In combination with previous public sequence data, the haplotype analysis revealed 12 haplotypes (H) characterized by two distinct clusters (I and II), and a sequence distance of 99.1–99.9% from the reference haplotype (H1). The diversity and neutrality indices for the entire E. shiquicus populations were: haplotype diversity (Hd) ± standard deviation (SD) 0.862 ± 0.035; nucleotide diversity (Hd ± SD) 0.0056 ± 0.0003; Tajima's D 0.876 (P > 0.05); and Fu’s F 6.000 (P > 0.05). Conclusions This was the first analysis of the newly discovered E. shiquicus in plateau pikas in the Tibet Autonomous Region of China. The neutrality indices suggest a deficiency of alleles, indicative of a recent population bottleneck.

Echinococcus shiquicus in Qinghai–Tibet plateau: population structure and confirmation of additional endemic areas

“…In this report, analyses of the concatenated mitochondrial cox1 and nad1 gene sequences were used to infer the population structure of E. shiquicus collected from the plateau in contrast to most previous studies that utilized partial gene sequences. The E. shiquicus population, as seen in the neutrality indices demonstrated by the cox1 and concatenated cox1-nad1 genes, suggested population expansion with significant negative values of Fu's F. This observation is in contrast to the population indices observed in some previous studies in Qinghai and Sichuan provinces where the neutrality test demonstrated positive nonsignificant values based on partial cox1 gene as well as a recent investigation of E. shiquicus populations from the Tibet Autonomous Region and Qinghai Province although with a longer DNA fragment, 2505 bp (Nakao et al, 2010;Zhu et al, 2020). With the geographically isolated and harsh climatic conditions of the plateau, one would expect that E. shiquicus population in the plateau would have experienced a population bottleneck event considering the geological events that led to the formation of the plateau millions of years ago (Harrison et al, 1992), but with the contrasting available reports mainly based on short gene sequences, the population structure and demographic history of E. shiquicus across the plateau remain to be further explored and it would be interesting to understand the population structure vis-à-vis the timeline of events during the plateau formation.…”

“…It was also, detected around Qinghai Lake in Ganzihe town of Haiyan County and Gonggongma village of Gangcha County towards the northeastern edge of the plateau. Until now, the diversity and population structure of E. shiquicus has been largely investigated by partial mitochondrial gene sequences of cox1 and/or nad1 (Ma et al, 2012(Ma et al, , 2015Wang et al, 2018;Han et al, 2019), and recently by mtDNA sequences of up to 2505 bp (Fan et al, 2016;Zhu et al, 2020). Moreover, studies have shown that by analysing longer DNA sequences or certain genes with higher resolution power, a better understanding of the intraspecies variation and phylogenetic resolution of Echinococcus species is realised (Laurimäe et al, 2018(Laurimäe et al, , 2019Ohiolei et al, 2020).…”

“…Genomic DNA was extracted by Qiagen Blood and Tissue Kit (Qiagen, Hilden, Germany) from the germinal layer of each cyst (retrieved from trapped pikas) and stored at −80°C before use. Using the following primer pairs, universal primers for nad1 (nad1-F: 5 ′ -TAAGWTRAGTGTGTGTGTTGGT-3 ′ and nad1-R: et al, 2016) and cox1 (cox1-F: 5 ′ -AGTTACTGC TAATAATTTTGTGTCAT-3 ′ and cox1-R: 5 ′ -TGATGTAAAAG GCAAATAAACC-3 ′ ) (Zhu et al, 2020). PCR was carried out in a 25 μL reaction mixture containing 12.5 μL Premix Ex Taq™ version 2.0 (Takara Bio, Kusatsu, Japan), 0.5 μL (10 pmol) of each primer, 0.5 μL of genomic DNA extract, and RNAse free water was used to complete the final volume to 25 μL.…”

“…Since the discovery of E. shiquicus, progress has been made in understanding the genetic diversity and population structure, howbeit by short mitochondrial gene sequences of the cytochrome c oxidase subunit 1 (cox1) and the NADH dehydrogenase subunit 1 (nad1) (Ma et al, 2012(Ma et al, , 2015Wang et al, 2018;Han et al, 2019), with only a few studies employing longer mtDNA sequences (2505 bp) (Fan et al, 2016;Zhu et al, 2020). Meanwhile, a better insight into the population structure will require an extensive investigation across the plateau as conflicting population demographics/indices have so far been observed, which is either a consequence of short DNA sequences or sample size limitations.…”

Echinococcus spp. and genotypes infecting humans in Tibet Autonomous Region of China: a molecular investigation with near-complete/complete mitochondrial sequences

“…Near-complete/complete mtDNA could be considered in areas with multiple species and genotypes such as TAR to identify the accurate species and genotypes among patients who need surgery to remove the lesion when the differential diagnosis such as imaging or qPCR is not conclusive. No E. shiquicus infection was identified with either qPCR or mtDNA method, which confirmed that humans were probably insusceptible to E. shiquicus , a new species found in the Tibetan Plateau only, despite the concern of its potential, given dog infection of E. shiquicus was reported [ 65 ]. Besides, near-complete/complete mitochondrial sequences of patients could serve as baseline data to monitor the Echinococcus species and genotype prevalence changes, mtDNA mutations, and trends of Echinococcus spp.…”