Molecular phylogenetic and genetic variability of Fasciola gigantica in Kermanshah province, western Iran with an overview to understand haplotypes distribution in Asia and Africa

Abstract

Over the last decade, diagnostic tools to detect and differentiate Fasciola species have improved, but our understanding of the distribution of haplotypes and population structure of this parasite is less clear. This study was designed to survey this gap in the F. gigantica epidemiology in Kermanshah province, western Iran from 2015 to 2017. Sixty-eight Fasciola isolates were collected from slaughterhouses from this province. We evaluated the PCR-RFLP assay of the ITS1 genes for the identification of Fasciola species using the RsaI enzyme. After Fasciola species identification, the partial sequence of mitochondrial NADH dehydrogenase subunit 1 (ND1) gene of F. gigantica was used for subsequent construction of the phylogenetic tree and network analysis. Based on the PCR-PRFLP profile, one (6.25%) of sheep isolates and 19 (39.60%) of cattle isolates were detected as F. gigantica, whereas 93.75% of sheep isolates, 60.40% of cattle isolates and all of the goat isolates were F. hepatica. In the 20 analyzed flukes, five ND1 haplotypes were detected. Statistically significant genetic differentiation was demonstrated between the Iran population and all the other populations. Evidence is presented for the existence of two well-separated populations: African and West Asian gigantica flukes and East Asian gigantica flukes. Genetic relationships among haplotypes were associated with geographical divisions. Also, our results have heightened our knowledge about the genetic diversity of F. gigantic, providing the first evidence for the existence of two well-separated populations of this parasite.

Keywords: Fasciola; Genotyping; Iran; Kermanshah; NADH dehydrogenase subunit 1.

Fig. 1

The PCR-RFLP pattern of Fasciola after digestion with Rsa I restriction enzyme. Lane 1: Fasciola gigantica from cattle after digestion with Rsa I restriction enzyme; Lanes 2, 3 and 4: Fasciola hepatica from sheep, cattle, and sheep after digestion with Rsa I restriction enzyme, respectively; Lane 5: Fasciola gigantica from cattle; Lane 6: 50bp DNA ladder

Fig. 2

Alignment of NADH dehydrogenase subunit 1 sequence of Fasciola hepatica (NC_002546.1) and Fasciola gigantica (NC_024025.1) deposited in GenBank with Fasciola gigantica (MF428464.1 - MF428468.1) of Kermanshah province‎‎, Western Iran

Fig. 3

Phylogenetic relationship of the haplotypes of Fasciola gigantica identified in this study and known haplotypes previously published in GenBank as inferred by maximum-likelihood analysis of NADH dehydrogenase subunit 1 sequence calculated by Hasegawa-Kishino-Yano model. The numbers on the branches are percent bootstrap values from 1000 replicates. Haplotypes of Kermanshah Fasciola flukes detected in this study are highlighted with the red square

Fig. 4

Haplotype network inferred by used haplotypes of NADH dehydrogenase subunit 1 constructed by PopART software and median-joining algorithm. Each circle represents a unique haplotype, the circle size reflects frequency and colors indicate the region of origin. The mutational steps are indicated by the short marks crossing the connection lines

Fig. 5

Population structure of 42 haplotypes of Fasciola gigantica, according to ΔK, the most probable number of genetic clusters is two