Molecular characterization and phylogenetic analysis of Fasciola gigantica from western Java, Indonesia

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Highlights

  • Fasciola flukes from western Java were identified by using pepck and pold genes.

  • All the flukes from western Java were identified as Fasciola gigantica.

  • Phylogenetic analyses were employed for Asian F. gigantica by using nad1 gene.

  • The Indonesian F. gigantica population was closely related with that of Thailand.

Abstract

Fasciola gigantica and aspermic (hybrid) Fasciola flukes are thought to be distributed in Southeast Asian countries. The objectives of this study were to investigate the distribution of these flukes from unidentified ruminants in western Java, Indonesia, and to determine their distribution history into the area. Sixty Fasciola flukes from western Java were identified as F. gigantica based on the nucleotide sequences of the nuclear phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold) genes. The flukes were then analyzed phylogenetically based on the nucleotide sequence of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene, together with Fasciola flukes from other Asian countries. All but one F. gigantica fluke were classified in F. gigantica haplogroup C, which mainly contains nad1 haplotypes detected in flukes from Thailand, Vietnam, and China. A population genetic analysis suggested that haplogroup C spread from Thailand to the neighboring countries including Indonesia together with domestic ruminants, such as the swamp buffalo, Bubalus bubalis. The swamp buffalo is one of the important definitive hosts of Fasciola flukes in Indonesia, and is considered to have been domesticated in the north of Thailand. The remaining one fluke displayed a novel nad1 haplotype that has never been detected in the reference countries. Therefore, the origin of the fluke could not be established. No hybrid Fasciola flukes were detected in this study, in contrast to neighboring Asian countries.

Introduction

Fasciolosis is predominantly a parasitic disease of livestock, which reduces productivity by causing a liver disorder in the host. The economic losses associated with the disease are estimated to be over two billion US dollars every year [1]. The disease also has zoonotic potential, and 91.1 million people are at risk of infection worldwide [2]. The disease is caused by two well-known species, Fasciola hepatica, mainly distributed in temperate zones, and Fasciola gigantica, mainly distributed in tropical zones [3]. These two species have mature spermatozoa in their seminal vesicles, which undergo fertilization in the bile ducts of the host [4]. In addition to the two species, aspermic Fasciola flukes, which have few or no spermatozoa in their seminal vesicles, have been reported in Asian countries [4], [7]. Recently, novel nuclear markers, the phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold) genes, have been used for the precise discrimination of F. hepatica, F. gigantica, and aspermic Fasciola flukes [5], which can be clearly distinguished with a multiplex polymerase chain reaction (PCR) and/or a PCR–restriction fragment length polymorphism method. By using these methods, aspermic Fasciola flukes display a mixed fragment pattern combining those of the two species, and are therefore thought to be their descendants, generated by hybridization between F. hepatica and F. gigantica [5]. So, they are termed as “hybrid Fasciola flukes” in this study.

The phylogenetic relationships among these species can be analyzed according to the nucleotide sequences of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene [6]. Fasciola gigantica is thought to be the predominant species in Southeast Asia [10], [11], [12]. A previous study based on the nad1 gene showed that the artificial movement of domestic ruminants, including zebu cattle and water buffaloes, has been involved in the spread of F. gigantica in Southeast Asian countries [7]. However, molecular information on the Fasciola flukes in Indonesia is limited till date. Therefore, the objectives of this study were to investigate the Fasciola species that are distributed in western Java, Indonesia, using the pepck and pold genes, and to determine the distribution history of F. gigantica by analyzing its phylogenetic relationships with populations in neighboring countries based on the nad1 gene.

Section snippets

Collection of Fasciola flukes and analysis of their spermatogenetic status

Fasciola flukes were collected from the bile ducts of 10 livers from a slaughter house and two meat markets in Subang, western Java, Indonesia, in March and May 2015 (Fig. 1, Table 1). Unfortunately, the ruminant species from which the livers were collected could not be precisely identified in this study, although cattle are predominant at these collection sites, buffaloes are only occasionally present. One to nine flukes were collected from each liver, and in total, 60 flukes were examined.

Species identification

Many sperm were observed in the seminal vesicles of 44 Fasciola flukes (spermic). These flukes displayed the F. gigantica fragment patterns for both the pepck and pold genes [5]. The remaining 16 flukes contained no sperm in their seminal vesicles, but they also displayed the F. gigantica fragment patterns for the pepck and pold genes. Therefore, all the Fasciola flukes examined in this study were identified as F. gigantica. None of the western Java samples showed the mixed fragment pattern of

Discussion

All 60 Fasciola flukes collected from western Java in Indonesia were identified as F. gigantica in an analysis of the nuclear pepck and pold genes (Table 1) [5]. These results are consistent with the nad1 haplotypes insofar as all the flukes had haplotypes belonging to the F. gigantica haplogroup based on the nad1 gene (Table 1, Fig. 2). Therefore, the pepck and pold regions are confirmed as robust markers that can precisely distinguish F. gigantica from hybrid Fasciola flukes. Sixteen flukes

Acknowledgments

The authors would like to thank the staff of Balai Veteriner Subang, Dinas Peternakan of Districts/Cities, and the farmers of western Java, Indonesia, for their valuable help in collecting the samples.

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