Genetic variation of mitochondrial genes among Echinococcus multilocularis isolates collected in western China

Abstract

Background: Alveolar echinococcosis (AE) is a life-threatening human disease caused by Echinococcus multilocularis transmitted between rodents and dogs/foxes in the Northern Hemisphere. The study aims to identify the genetic variation of the parasite in AE patients from China.

Methods: E. multilocularis isolates were collected from wild small mammals (n = 6) and AE patients (n = 56) from western China. Genomic DNA was extracted from different tissue samples including paraffin tissue blocks, ethanol fixed tissues and frozen tissues surgically removed. Two mitochondrial gene fragments (526 bp for cob and 474 bp for nad2) of E. multilocularis were amplified and sequenced.

Results: The parasite fragment sequences of cob fragments from AE patients showed two haplotypes, and nad2 gene fragment sequences had four haplotypes. The gene sequences from Microtus sp. were 100% identical to the sequences of some isolates from AE patients. These haplotypes were distributed in both Qinghai and Xinjiang provinces. Alignment analysis with the sequences from the GenBank databases showed five genotypes including three Asian genotypes, one from Europe and one from North America.

Conclusions: Most AE patients harbored the Asian genotype 1 which may be an indication of its relative frequency in the definitive hosts and the environment or of its pathogenicity to humans, which calls for further research.

Keywords: Alveolar echinococcosis; Echinococcus multilocularis; Genetic variation; Mitochondrial genes; cob; nad2.

Fig. 1

Alignment of single nucleotide polymorphisms among the three isolates of E. multilocularis maintained in laboratory animals (jirds) and three isolates from wild Microtus spp. (Mictus3) rodents. The top of each panel shows the position of the substitute mutations. Abbreviations: Em-XJ, E. multilocularis isolates were collected from Xinjiang; Em-NX, E. multilocularis isolates were collected from Ningxia; Mictus1-3, the three isolates collected recently from Microtus; Em-A, an E. multilocularis isolate was originally collected from Alaska, USA; cob, cytochrome b; nad2, NADH dehydrogenase subunit 2; C, Canada; F, France; J, Japan; S, Slovakia; U, USA. Only positions with mutations are listed in the figure, the identical nucleotides are represented by dots

Fig. 2

Neighbor-joining haplotype tree and parsimony network of E. multilocularis nad2. a Variation of nad2 sequences isolated from AE patients and rodents from China. b Phylogenetic tree from NJ analysis. Values on the nodes are bootstrap proportions (%). The scale-bar represents a divergence of 0.0005. c Parsimony network for nad2 combined with the sequences of E. multilocularis from the GenBank database. The circular icon represents a clade, and the small triangle icon represents a single base difference. NA1-3 represent 3 nad2 haplotypes. Liver samples were collected from AE patients from China including provinces of Qinghai (QH), Gansu (GS), Sichuan (SC), Xinjiang (XJ) with counties Wuqia County (WQ), Nileke County (NLK), Zhaosu County (ZS), Xinyuan County (XY), and Huocheng County (HC). Three isolates were collected from Microtus (Mictus1-3) in Yili valley in Xinjiang

Fig. 3

Neighbor-joining haplotype tree and parsimony network of E. multilocularis cob. a Variation of cob sequences isolated from AE patients and rodents from China. b Phylogenetic tree from NJ analysis. Values on the nodes are bootstrap proportions (%). The scale-bar represent a divergence of 0.0002. c Parsimony network for cob combined with the sequences of E. multilocularis from the GenBank database. The circular icon represents a clade containing identical sequences, and the small triangle icon represents a single base difference CA1 and CA2 represent 2 cob haplotypes. Liver samples were collected from AE patients from China including Beijing (BJ) and provinces of Qinghai (QH), Gansu (GS), Sichuan (SC), Xinjiang (XJ) with counties Wuqia County (WQ), Nileke County (NLK), Zhaosu County (ZS), Xinyuan County (XY), and Hejing County (HJ). Three isolates were collected from Microtus (Mictus1-3) in Yili valley in Xinjiang

Fig. 4

Worldwide distribution of E. multilocularis cob and nad2 haplotypes