Whole transcriptome profiling of adult and infective stages of the trematode Opisthorchis felineus
Graphical abstract
Introduction
Opisthorchiidae is a large family of parasitic flatworms (Class Trematoda) comprising more than 30 genera [1], [2], including the liver flukes, Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis, which collectively have a significant socio-economic impact [3]. These flukes inhabit the human biliary tract where they cause the chronic diseases, opisthorchiasis (O. felineus, O. viverrini) and clonorchiasis (C. sinensis), which are predominantly characterized by damage to the hepatobiliary system [4]. While a substantial amount of genomic and transcriptomic data is available for oriental liver flukes (O. viverrini and C. sinensis), little is known about O. felineus, a parasite which is endemic to regions of northern Asia and Europe [5].
Incidence of O. felineus-induced opisthorchiasis has been mostly reported in Russia, Belarus, Kazakhstan and other countries of the former USSR, especially in the basin of Siberian river Ob where prevalence of the disease in humans can reach 30% of the population [6]. Less frequently, cases of O. felineus infection of domestic animals and humans have also been reported in Western Europe [7], [8].
Like the other opisthorchiids, O. felineus has a complex lifecycle including two intermediate hosts (a snail and a fish) and a definitive host (piscivorous mammals including humans) [9], [10]. The snail (from the genus Bithynia [6]) becomes infected via the consumption of eggs released with the feces of the definitive host. After asexual development within the snail, a cercarial stage is released, which penetrates the skin of a second intermediate host (most commonly a cyprinoid fish) and encysts as a metacercaria. The definitive host is infected via the consumption of raw or undercooked infected fish.
The level of O. felineus worm burden in humans varies depending on the number of viable metacercariae ingested. Accordingly, the severity of disease can vary from asymptomatic to severe infection with serious damage to the liver and pancreas, including cholangitis and/or hepatolithiasis [11]. Chronic inflammation and injury of the biliary epithelium are thought to be the major contributing factors to opisthorchiasis [4]. C. sinensis and O. viverrini are classified as Class I carcinogens, and are a major cause of cholangiocarcinoma in Thailand [12], [13]. As of yet, it is unclear whether O. felineus can induce cancer, although this species has been reported to be more pathogenic than the other opisthorchiids [14].
Treatment of opisthorchiasis and clonorchiasis relies solely on praziquantel [15]. However, excessive use of this drug can reduce treatment efficacy [16], induce inflammation of the biliary system [12] and potentially lead to praziquantel resistance. Thus, alternative strategies to intervene with O. felineus infection are urgently required. Such interventions may be derived from a deeper understanding of the molecular biology of O. felineus but little is known about the biology of O. felineus or their interactions with the hosts at the molecular level. In addition, parasitic flatworms lack homology to model organisms for which extensive molecular data is available. Together, these factors impede the development of new intervention strategies for O. felineus-induced opisthorchiasis.
O. viverrini and C. sinensis have recently been characterized at the genomic and transcriptomic levels [17], [18], [19]. Complementary studies of O. felineus have not been undertaken. Furthermore, full transcriptome of the infective stage of the opisthorchiids was not yet elucidated although some expressed sequence tag data is available for the C. sinensis metacercariae [20], [21]. To address this knowledge gap, we sequenced the O. felineus total RNA using Illumina technologies and used de novo assembled transcripts to uncover evolutionary aspect of the adaptations on the transcriptomic level. These data provides the first comprehensive insights into the molecular biology of O. felineus on genome-wide scale.
Section snippets
Preparation of parasite samples
Metacercariae were collected from infected cyprinoid fishes from the Ob river in Novosibirsk region and were used to orally infect inbred Syrian (golden) hamsters Mesocricetus auratus (50 metacercariae per hamster). Hamsters were euthanized at least 3 weeks following inoculation and adult stages of O. felineus were collected from the livers. The adult flukes were thoroughly washed in sterile saline solution and stored in RNAlater (Ambion, USA) at 4 °C until RNA preparation.
The animals were kept
Pre-processing of the data and initial characterization
In total, 40 and 110 million Illumina paired-end reads were generated from the metacercarial and adult stage cDNA libraries, respectively. Prior to assembling, the transcriptome, adapters, low quality sequence data and additional 1.3% of the reads homologous to the laboratory host (M. auratus) were removed. The remaining pooled, high-quality reads assembled into 89,727 transcripts (mean length and corresponding standard deviation are 773 nt ± 1099 nt), of which, 12,665 (1952 nt ± 1713 nt) transcripts
Conclusions
Opisthorchiidae infect millions of people worldwide and constitute a major health problem for some regions. O. felineus is still a serious pathogen on the territory of Russia, Kazakhstan and in some European countries. Despite the significance of this parasite, we lack it genomic data and elucidating its transcriptome can be a good starting point for further research.
In this paper, using the Illumina sequencing of the two lifecycle stages of O. felineus, we identified 12,665 transcripts which
Authors' contributions
AK carried out experiment design and sample collection. ML and AP carried out the sequencing. MP, NY and NE carried out the bioinformatics analysis and drafted the manuscript. VM organized the experiment and drafted the manuscript.
Acknowledgments
Financial support for this study was provided in part by the State Project of ICG SB RAS VI.60.1.1, by project no. 14.B25.31.0033 (resolution no. 220 of the Government of the Russian Federation of April 9, 2010), by SB RAS Research Partnership grant no. 19, and by the Russian Foundation for Basic Research (RFBR # 15-04-03551а). MYP was additionally supported by the fund of Mikhail Prokhorov. We acknowledge support and collaboration of the TOPIC (Tomsk OPIsthorchiasis Consortium), //www.topic-global.org
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