Genotypes of Echinococcus granulosus in Animals from Yushu,Northeastern China

Abstract

Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance worldwide. The aim of this study was to determine the genotype distribution of E. granulosus in animals in Yushu, Qinghai Province, northeastern China. The mitochondrial nicotinamide adenine dinucleotide+hydrogen (NADH) dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes from 30 echinococcosis isolates were analyzed by sequence alignment, generating two unique sequence profiles at both nad1 and cox1 loci. Phylogenetic analysis demonstrated that 28 isolates (93.3%) belonged to the well-known G1–G3 complex (E. granulosus sensu stricto), and 2 (6.7%) were placed in G6–G10 complex (E. canadensis). The present study provides the genetic composition of E. granulosus from animals in Yushu, Qinghai Province, and confirms that, for the first time, the E. granulosus G6–G10 complex (E. canadensis) is not only limited to Xinjiang and might be of greater public health significance than previously believed in China.

Introduction

E chinococcosis is a serious zoonotic disease caused by the tapeworms Echinococcus granulosus, E. multilocularis, E. oligarthrus, and E. vogeli. The latter 2 species cause polycystic echinococcosis in humans, only limited to central and South America, whereas E. granulosus and E. multilocularis are pathogens of cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively. E. granulosus is distributed worldwide, whereas E. multilocularis is more limited to the Northern Hemisphere, including central and northern Europe, Asia, and North America. CE and AE are highly endemic in western and northwestern China, from Xinjiang, Tibet, Qinghai, Sichuan, Gansu to Ningxia (Chai 1995, Eckert and Deplazes 2004). A new species of Echinococcus, E. shiquicus, has recently found in the Qinghai–Tibet plateau (Xiao et al. 2006).

E. granulosus shows high genetic variability, with 10 genotypes (G1–G10) described thus far, presenting different host ranges, geographical distribution, and pathogenicity (Eckert and Deplazes 2004, Jenkins et al. 2005). Recently, E. granulosus has been reclassified into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10) (Sharbatkhori et al. 2009, Jabbar et al. 2011).

Previous studies from different regions have revealed the presence of two E. granulosus genotypes in China: Genotype G1 (sheep strain) is a major cause of infection in both humans and animals, whereas genotype G6 (camel strain) is limited to Xingjiang (Yang et al. 2005, Bart et al. 2006a, Nakao et al. 2010). However, the transmission of E. granulosus in some endemic regions of China is poorly understood. The aim of our study was to determine the genotypic distribution of E. granulosus in animals in Yushu, Qinghai Province, northeastern China.

Materials and Methods

The Yushu Tibetan Autonomous Prefecture includes Yushu, Zaduo, Chenduo, Nangqian, Zhiduo, and Qumalai counties (Fig. S1) (Supplementary Data are available at www.liebertonline/vbz/.) The average elevation is >4000 meters above sea level, and the annual mean temperature is −0.8°C. The population is about 288,000, of which 95% are Tibetan and transhumant herdsmen. There are 4,200,000 livestock, including yaks, sheep, goats, and horses. Most families keep at least 1 guard dog, and there are also large numbers of stray dogs and some wild carnivores, such as snow leopard (Panthera uncia), brown bear (Ursus arctos), and lynx (Felis lynx).

Thirty E. granulosus cysts (1 per animal) were obtained from slaughterhouses in Zaduo (2 from sheep and 5 from yaks), Nangqian (6 from yaks), Qumalai (2 each from sheep and goats), Yushu (11 from yaks), and Zhiduo (2 from sheep). DNA was extracted from the cysts using a genomic DNA extraction kit (TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.4.0), as previously described (Bart et al. 2006b). PCR was conducted to amplify the mitochondrial nicotinamide adenine dinucleotide+hydrogen (NADH) dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes. The primers used were egND1-for and egND1-rev, which amplified a 332-bp fragment of the nad1 gene, and JB3 and JB4.5, which amplified a 450-bp fragment of the cox1 gene (Bowles et al. 1992, Busi et al. 2007).

PCR products were cloned into the pMD18-T vector (TaKaRa) and sequenced on an ABI 3130 Genetic Analyzer. Nucleotide sequences were aligned using the program ClustalX with reference sequences of known E. granulosus genotypes (Bowles et al. 1992). Phylogenetic analysis was carried out using MEGA v5.05.

Results and Discussion

The mitochondrial nad1 and cox1 genes were amplified from the collected E. granulosus cysts and sequenced. The sequence alignments generated 2 unique sequence profiles at both cox1 and nad1 loci. Twenty-eight of the 30 E. granulosus cysts produced cox1 and nad1 sequences with the highest homology to the corresponding sequences of genotypes G1–G3. The remaining 2 cysts from goats of Qumalai produced 100% sequence identity to G6 genotype in cox1, and 99.7% to G6 and G7 genotypes in nad1 (Fig. S2).

A phylogenetic analysis of the cox1 and nad1 sequences E. granulosus detected herein, together with the representative sequences from all recognized species of Echinococcus in China, and reference sequence from different genotypes of E. granulosus was conducted (Table 1). As shown in the Figure 1A (cox1 analysis), a large clade consisted of isolates N3, Y4, and Y6 from Yushu, Qinghai, and other isolates from China. Reference genotypes G1–G3 were present, forming the well-known G1–G3 complex (E. granulosus sensu stricto). In contrast, the isolate Q1 was placed in the clade containing the G6–G10 complex (E. canadensis), together with G6 from Xinjinag (GenBank accession no. DQ356884). Figure 1B (nad1 analysis) also shows similar results. In both figures, the G1–G3 isolates from this study formed a subcluster distinct from the reference genotypes G1–G3, although still belonging to G1–G3 complex (E. granulosus sensu stricto). This was especially the case at the nad1 locus, where G1–G3 identified in this study had sequence similarity to G6–G10 at the 5′ and 3′ ends of the fragment under analysis (Fig. S2).

FIG. 1.

Phylogenetic tree showing the relationship between E. granulosus isolates from Yushu, Qinghai, China (C1, Y6, N3, and Q1) and previously described isolates based on the 366-bp fragment of the cox1 gene (A) and the 332-bp fragment of the nad1 gene (B). The sequences used in the phylogenetic analysis are listed in Table 1. The nucleotide sequences of Echinococcus shiquicus, E. multilocularis, and Taenia solium were used as outgroups. The figure was drawn with MEGA (www.megasoftware.net) using the neighbor-joining method. Bootstrap values are calculated from 1000 repetitions. Scale bars represent genetic distances between isolates. The triangles indicate the cox1 or nad1 sequences of E. granulosus from isolates obtained in the present study, and the dots indicate the cox1 or nad1 sequences of reference genotypes G1–G10 of E. granulosus. (The cox1 and nad1 sequences of G9 are not available.)

Table 1.

Echinococcus spp. in the Present Study and Other Isolates in China, Together with Reference Sequences Used for Phylogenetic Analysis

	GenBank accession no.
Parasite species/isolate	cox1	nad1	Host	Origin	Genotype	Reference
Echinococcus granulosus
N3	JQ317991	JQ317985	Sheep	Qinghai	G1–G3	This study
Q1	JQ317990	JQ317986	Goat	Qinghai	G6–G10	This study
Y6	JQ318001	JQ317988	Yak	Qinghai	G1–G3	This study
C1	NA	JQ317989	Sheep	Qinghai	G1–G3	This study
Y4	JQ318000	NA	Yak	Qinghai	G1–G3	This study
H2	DQ356877	NA	Human	Xinjiang	G1–G3	Unpublished
H41	DQ356884	NA	Human	Xinjiang	G6–G10	Unpublished
Haplotype G40	AB491453	NA	Human	Sichuan	G1–G3	Nakao et al. 2010
ST11	AY572550	AY572545	Sheep	Gansu	G1–G3	Unpublished
XJ-h	NA	EU704125	Human	Xinjinang	G1–G3	Unpublished
Species other than E. granulosus
E. shiquicus	AB208064	AB208064	Ochotona curzoniae	Sihuan		Nakao et al. 2007
E. multilocularis	JF906153	NA	Sheep	Qinghai		Unpublished
E. multilocularis	NA	AY389984	NA	Xinjinang		Unpublished
E. multilocularis	AB491458	NA	Human	Sichuan		Nakao et al. 2010
E. multilocularis	NA	EU704124	NA	Xinjiang		Unpublished
Taenia solium	JN084225	NA	Human	France		Year et al. 2011
Taenia solium	NA	AY395068	Human	Korea		Chung et al. 2005
Reference sequences
G1	M84661	AJ237632	Sheep	Australia	G1	Bowles et al. 1992, 1993
G2	M84662	AJ237633	Sheep	Australia	G2	Bowles et al. 1992, 1993
G3	M84663	AJ237634	Buffalo	India	G3	Bowles et al. 1992, 1993
G4	M84664	AJ237635	Horse	India	G4	Bowles et al. 1992, 1993
G5	M84665	AJ237636	Cattle	Netherlands	G5	Bowles et al. 1992, 1993
G6	M84666	AJ237637	Camel	Africa	G6	Bowles et al. 1992, 1993
G7	M84667	AJ237638	Pig	Poland	G7	Bowles et al. 1992, 1993
G8	AB235848	AB235848	Moose	USA	G8	Nakao et al. 2007
G10	AF525457	AF525297	Reindeer	Finland	G10	Lavikainen et al. 2003

NA, Not available.

Consistent with previous studies (Li et al. 2008) the E. granulosus G1–G3 complex (E. granulosus sensu stricto) was most commonly detected in slaughtered animals in Yushu, Qinghai Province (93.3% of the 30 cyst isolates). Although only 2 isolates from goats were identified as the G6–G10 complex (E. canadensis), our preliminary data, for the first time, suggest that the E. granulosus G6–G10 complex (E. canadensis) is not only limited to Xinjiang. Therefore, the E. granulosus G6–G10 complex (E. canadensis) might be of greater public health significance than previously believed. Further studies are necessary to determine host specificity, geographical distribution, transmission dynamics, and infectivity to humans of this genotype in China. As the G1–G3 isolates detected in this study had distinct nucleotide differences from reference sequences of G1–G3 from other countries at both cox1 and nad1 loci, more studies are also needed to determine the true identity of these isolates from China and whether they represent yak-adapted E. granulosus sensu stricto.

Footnotes

Acknowledgments

We thank the staff of the Veterinary Stations in Yushu, Qinghai Province, China for collection of echinococcosis cysts. This study was supported by the National Basic Research Program of China (“973” Program) (2012CB722501).

Author Disclosure Statement

No competing financial interests exist.

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