Lasiopodomys fuscus as an important intermediate host for Echinococcus multilocularis: isolation and phylogenetic identification of the parasite

Abstract

Background: Echinococcus multilocularis causes alveolar echinococcosis (AE) and is widely prevalent in Qinghai Province, China, where a number of different species have been identified as hosts. However, limited information is available on the Qinghai vole (Lasiopodomys fuscus), which is hyper endemic to Qinghai Province and may represent a potential intermediate host of E. multilocularis. Thus, L. fuscus could contribute to the endemicity of AE in the area.

Methods: Fifty Qinghai voles were captured from Jigzhi County in Qinghai Province for the clinical identification of E. multilocularis infection via anatomical examination. Hydatid fluid was collected from vesicles of the livers in suspected voles and subjected to a microscopic examination and PCR assay based on the barcoding gene of cox 1. PCR-amplified segments were sequenced for a phylogenetic analysis. E. multilocularis-infected Qinghai voles were morphologically identified and subjected to a phylogenetic analysis to confirm their identities.

Results: Seventeen of the 50 Qinghai voles had E. multilocularis-infection-like vesicles in their livers. Eleven out of the 17 Qinghai voles presented E. multilocularis infection, which was detected by PCR and sequencing. The phylogenetic analysis showed that all 11 positive samples belonged to the E. multilocularis Asian genotype. A morphological identification and phylogenetic analysis of the E. multilocularis-infected Qinghai voles confirmed that all captured animals were L. fuscus.

Conclusions: L. fuscus can be infected with E. multilocularis and plays a potential role in the life cycle and epidemiology of E. multilocularis in the Qinghai-Tibetan Plateau of China.

Keywords: Alveolar echinococcosis; Echinococcus multilocularis; Lasiopodomys fuscus; PCR; Phylogenetic analysis; Qinghai voles; Sequencing.

Fig. 1

Sampling site (marked by a black star) in southeast Qinghai Province, China

Fig. 2

Snap trapper (yellow arrow) used to capture Qinghai voles in grassland

Fig. 3

Qinghai voles captured in a grassland area

Fig. 4

Suspected AE characterized by vesicles in the liver

Fig. 5

H-E staining of the vesicles and the protoscoleces (40×). The black arrow shows the entire vesicle, and the yellow arrow shows the protoscoleces in the vesicle

Fig. 6

Protoscoleces examined under a stereoscopic microscope: a 40× and b 100×

Fig. 7

Agarose gel electrophoresis assay of the protoscoleces cox 1 gene of E. multilocularis. Lane M, DL 2000 molecular marker; Lane P, Positive control; Lane N, Negative control; and Lanes 1-11: Samples of E. multilocularis 1 to 11

Fig. 8

Phylogenetic comparison of the cox1 isolated sequences with those deposited in the GenBank database. A phylogenetic tree was constructed using the neighbor-joining method and the p-distance matrix for nucleotides with the pair-wise gap deletion option. The E. multilocularis strains isolated from Qinghai as well as AB688127.1 to AB461417.1 belonged to the Asian genotype (marked by ▲ and △), AB688134.1 to AB461413.1 belonged to the European genotype (marked by ●), AB353729.1 to AB461418.1 belonged to the North American genotype (marked by ○), and AB510023.1 to AB510024.1 belonged to the Mongolian genotype (marked by ■); in addition, sequence KP161209.1 belonged to E. equinus, GQ168811.1 belonged to E. granulosus, AB235846.1 belonged to E. ortleppi and AB893262.1 belonged to E. canadensis (marked by □)

Fig. 9

Morphological characteristics of Qinghai voles from different aspects: a Dorsal view; b Ventral view; and c Right lateral view; d Left lateral view

Fig. 10

Local features of Qinghai vole: a Right lateral view; b Left lateral view; c Head in full face view; and d Tail

Fig. 11

Agarose gel electrophoresis assay of PCR-amplified products of the Qinghai vole’s cox 1 gene. Lane M: DL 2000 molecular marker; Lane 1: Negative control; Lane 2: Vole’s tail DNA; and Lane 3: Vole’s ear DNA

Fig. 12

Phylogenetic comparison of the cox1 sequence from the Qinghai vole with those from the GenBank database. The phylogenetic tree was constructed using the neighbor-joining method and the p-distance matrix for nucleotides with the pair-wise gap deletion option. QHJZ represented the Qinghai vole captured in Jigzhi County; JX962254.1, JX962258.1 and JX962265.1 represented Lasiopodomys fuscus; and KP190276.1, KP190278.1 and KP190280.1 represented Neodon fuscus