Investigation and genetic polymorphism analysis of rodents infected with Echinococcus in Ili Prefecture, Xinjiang Uygur Autonomous Region, China

Abstract

Introduction: Alveolar echinococcosis (AE) is a life-threatening disease in humans caused by the larval stage of Echinococcus multilocularis. Domestic animals, dogs, foxes, and small mammals constitute the circular chain of AE. To evaluate the infection, distribution, and genetic polymorphism of AE in the Ili Prefecture (Nilka, Xinyuan and Zhaosu), we conducted this survey.

Methods: In June and July 2018, 267 small mammals were captured using water-infusion and mousetrap methods. Combined pathogenic and molecular biological methods were used to observe the histopathology of Echinococcus carried by rodents, amplify the mitochondrial nad1 gene of the pathogen, and investigate the genotype and haplotype diversity of Echinococcus in rodents in Ili Prefecture.

Results: Morphological identification revealed that these captured small mammals belonged to three species, with Microtus gregalis being the dominant species (183/267). Pathological and molecular biological results confirmed that E. multilocularis was the pathogen of echinococcosis in small mammals, with an infection rate of 15.73% (42/267). Among the three areas sampled, the highest infection rate of rodents was 25.45% (14/55) in Nilka County. However, there was no significant difference in the infection rates between regions (χ² = 5.119, p > 0.05). Of the three captured rodent species, M. gregalis had the highest infection rate of 17.49% (32/183), but there was no significant difference in infection rates between the rodent species (χ² = 1.364, p > 0.05). Phylogenetic analyses showed that the nad1 gene sequences obtained in this study clustered in the same clade as isolates from China. These isolates contained 21 haplotypes (Hap_1-21); Hap_2 was the most common haplotype (9/42). Furthermore, haplotype diversity (0.925 ± 0.027) and nucleotide diversity (0.01139 ± 0.00119) were higher in the Ili Prefecture than in other regions, indicating that population differentiation was high. Tajima's D and Fu's Fs tests were negative (p > 0.10), indicating that the population had expanded. The low fixation index (Fst) ranged from 0.00000 to 0.16945, indicating that the degree of genetic differentiation was different among different populations.

Discussion: In summary, Ili Prefecture is a high incidence area of AE, and Microtus spp. may play an important role in the transmission of AE in this area. The results of this study provide basic data for further study of the molecular epidemiology, genetic differences, and control of E. multilocularis in the Ili Prefecture, Xinjiang.

Keywords: Echinococcus multilocularis; Ili; genetic polymorphism; nad1 gene; rodent.

Figure 1

Small mammals were captured from parts of the high-altitude mountainous pasture areas in Ili Kazakh Autonomous Prefecture, Xinjiang, China. In the figure, “different shapes in different colors” indicate the sampling location.

Figure 2

Different appearances of rodent liver cysts. (A) Cysts in the liver of rodents, the reddish-brown is the liver, grayish-white translucent vesicles are cysts; (B) Cysts in the liver of rodents, the grayish-brown is the liver, grayish-white translucent vesicles are cysts; (C) Cysts attached to and protruding from the surface of rodent liver, the reddish-brown is the liver, the grayish-white spheres with blood vessels attached on the surface are cysts; (D) Cysts attached to and protruding from the surface of rodent liver, the reddish-brown is the liver, white tumor-like is the cyst.

Figure 3

Hematoxylin and eosin staining showing the characteristic pathological response in echinococcosis. (A) Liver cyst sections of rodents from herdsman’s original residence; (B) Liver cyst sections of rodents from artificial pasture; (C) Liver cyst sections of rodents from natural grassland. The original magnification of all tissue sections was 100×; black arrow indicates PSCs.

Figure 4

Phylogenetic analysis of Echinococcus nad1 gene sequences by the neighbor-joining method based on the Kimura 2-parameter model. The bootstrap method via 1000 pseudo replicates was used to assess the reliability of the tree. The pink area in the figure indicates sample sequences and sequences of different isolates of E. multilocularis; green area indicates other Echinococcus; purple area indicates outgroup sequences. The blue dots on the branches represent the bootstrap values; the smaller the bootstrap values, the smaller the blue dot, and vice versa. Phylogenetic tree landscaping was performed using an online website: https://itol.embl.de/.

Figure 5

The haplotype network for the nad1 gene of E. multilocularis. Geographical distribution of the haplotypes is indicated by different colors. Size of circles is proportional to the frequency of each haplotype. Each cross-sectional line represents a gene mutation site. Black circles: hypothetical intermediate haplotypes. Haplotype network was drawn with the PopART 1.7 software using the TCS network inference method.