Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint Molecular analysis reveals expansion of Fasciola hepatica distribution from Afghanistan to China Tran Nhat Thang a,b , Hakimullah Hakim c , Raihana Royan Rahimi c , Madoka Ichikawa-Seki a, ⁎ a Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan b Department of Animal Diseases, Faculty of Animal Science and Veterinary Medicine, Thai Nguyen University of Agriculture and Forestry, Quyet Thang Commune, Thai Nguyen City, Viet Nam c Department of Paraclinic, Faculty of Veterinary Sciences, Kabul University, Kabul 1006, Afghanistan ARTICLE INFO Keywords: Fasciola hepatica Afghanistan Pepck Pold nad1 ABSTRACT Recently, phosphoenol pyruvate carboxykinase (pepck) and DNA polymerase delta (pold) were established as reliable nuclear markers for species identification of Fasciola spp. in multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism-based assays, respectively. Currently, little is known about Fasciola species distribution in Central Asia. Therefore, the objective of the present study was to perform precise molecular species identification of liver flukes from Afghanistan and to reveal their dispersal route(s) via phy- logenetic analysis based on mitochondrial nad1 haplotypes. Ninety-two Fasciolaflukes collected from sheep in Kabul, Afghanistan, were identified as F. hepatica based on pepck and pold screening. Although the pepck frag- ment pattern obtained via multiplex PCR analysis could not distinguish the species of the seven Fasciola flukes, the pepck nucleotide sequence data confirmed that they were F. hepatica.The 20 nad1 haplotypes detected among the Afghani liver flukes were closely related to those from China and Egypt, with the F ST value (-0.003, P = .41) between the F. hepatica populations from Afghanistan and China confirming a very close relationship. Nucleotide diversity was greater in the population from Afghanistan compared with that from China, indicating that the Afghani population was older, and that the dispersal direction of F. hepatica was from Afghanistan to China. The results of the present study contribute to our understanding of the dispersal of F. hepatica from its predicted origin, the Fertile Crescent. 1. Introduction Fasciolosis is a globally-distributed zoonotic disease caused by the trematode parasites Fasciola hepatica and F. gigantica. The former spe- cies mainly occurs in Europe, the Americas, Oceania, and northern parts of Asia, while the latter is distributed in Africa and southern Asia [1,2]. In addition to these two better-known species, hybrid Fasciola flukes originating from interspecific hybridization [3] can be found throughout East, South East, and South Asian countries [3–14]. How- ever, the distribution of hybrid Fasciola flukes in the region between Central Asia and the Middle East has never been investigated to our knowledge. Several previous reports [15–18] attempted to identify the species of Fasciola flukes from Iran using morphological and morpho- metric features as well as molecular markers (nuclear internal tran- scribed spacer 1 and 2); however, these results have not been validated using more recently-established and reliable nuclear gene markers. Recently, novel nuclear markers phosphoenol pyruvate carboxykinase (pepck) and DNA polymerase delta (pold) were found to be useful for the precise discrimination of F. hepatica, F. gigantica, and hybrid Fasciola flukes [3,19]. In addition, the nucleotide sequences of mitochondrial nicotinamide adenine dinucleotide dehydrogenase sub- unit 1(nad1) and cytochrome oxidase subunit 1 (cox1) genes have been used to analyze intraspecific phylogenic relationships among Fasciola spp. [4]. Phylogenetic relationships as well as nucleotide diversity within the nad1 gene were then used in the previous studies [4–14] to determine the dispersal routes of Fasciola spp. in many Asian countries. Previous reports from Iran [15–18] analyzed the nucleotide se- quences of nad1 and cox1; however, frequency data for each haplotype could not be obtained from the reports. As such, based on currently available data, it is impossible to infer the dispersal route(s) of Fasciola flukes in Central Asia. Therefore, the objectives of the current study were to precisely identify Fasciola species in sheep from Kabul, Afgha- nistan, based on the nuclear pepck and pold genes, and to analyze their phylogenetic relationship with Fasciola populations from different parts https://doi.org/10.1016/j.parint.2019.101930 Received 30 January 2019; Received in revised form 17 May 2019; Accepted 19 May 2019 ⁎ Corresponding author at: Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan. E-mail address: madoka@iwate-u.ac.jp (M. Ichikawa-Seki). Parasitology International 72 (2019) 101930 Available online 21 May 2019 1383-5769/ © 2019 Elsevier B.V. All rights reserved. T