A novel nuclear marker, Pm-int9, for phylogenetic studies of Opisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis (Opisthorchiidae, Trematoda)

Abstract

Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, the trematodes of the family Opisthorchiidae, are important human parasites. Two previous studies (Kang et al. Parasitol Int 57:191-197, 2008; Katokhin et al. Dokl Biochem Biophys 421:214-217, 2008) have provided evidence using ribosomal and mitochondrial sequences that O. viverrini, O. felineus, and C. sinensis are closely related. We developed a novel nuclear marker, Pm-int9, which included the ninth intron of the paramyosin gene and flanking exon sequences. Samples of O. felineus from four localities of West Siberia, C. sinensis from the Russian Far East, and O. viverrini from Thailand were genotyped by Pm-int9. Little variation was detected in exon sequences, however, intron sequences turned out to be more variable than ribosomal internal transcribed spacers. We can conclude that Pm-int9 is valuable for interspecific variation studies. Phylogenetic analysis based on Pm-int9 revealed that O. viverrini and C. sinensis were closer to each other than either of them to O. felineus, supporting the opinion that C. sinensis should be considered the sister species of Opisthorchis spp.

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

“…Based on the ITS1 sequence, O. viverrini and O. felineus group together and are closely related to C. sinensis, whereas analysis of ITS2 and cox1 sequences suggests that O. felineus is more closely related to C. sinensis than to O. viverrini. In contrast, the ninth intron of the paramyosin gene (Pm-int9), which is more variable than nuclear markers, showed a closer relationship between O. viverrini and C. sinensis (375,376). Microsatellite DNA analysis of O. viverrini in Thailand using 12 microsatellite loci revealed the existence of genetic diversity and population substructuring in O. viverrini, which can be a useful tool to study transmission dynamics and control (377,378 used primarily for the detection and identification of Paragonimus species from metacercariae, crabs, and experimentally infected animals or for confirmation of eggs found in sputum or stool samples (Table 8).…”

Opisthorchiasis and Opisthorchis-associated cholangiocarcinoma in Thailand and Laos

“…O. felineus occurs in Russia, some republics of the former USSR, Kazakhstan and nearby countries, and has been recorded recently in Germany, Greece and Italy (Lun et al, 2005; Keiser and Utzinger, 2005; Armignacco et al, 2008; Shekhovtsov et al, 2009; Shin et al, 2010). In China, the liver fluke Clonorchis sinensis was recently estimated by the Chinese Center for Disease Control to infect 12.5 million people (Ministry of Health, National Institute of Parasitic Diseases, China CDC, 2005) (Table 1).…”

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

“…To overcome the pitfalls of the microscopic methods, various sensitive and specific molecular methods have been developed to discriminate C. sinensis and O viverrini eggs. Some of the examples are multiplex PCR approaches (Le et al 2006), PCR and sequencing of nuclear ribosomal and mitochondrial DNA (Park 2007) and nuclear marker sequences (PM-int9) (Shekhovtsov et al 2009), PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region (Kang et al 2008), and PCR targeting ribosomal DNA ITS regions (Sato et al 2009). A rapid, high-throughput, specific, and sensitive realtime PCR has been applied for the detection of C. sinensis (Kim et al 2009;Cai et al 2012;Rahman et al 2011) and O. viverrini (Intapan et al 2008a, 2008b, 2009aSuksumek et al 2008;Sri-Aroon et al 2011;Janwan et al 2011) DNA.…”