Current assessment of the systematics and population genetics of Opisthorchis viverrini sensu lato (Trematoda: Opisthorchiidae) and its first intermediate host Bithynia siamensis sensu lato (Gastropoda: Bithyniidae) in Thailand and Southeast Asia.

The group 1 carcinogen, the liver fluke Opisthorchis viverrini is the causative agent of opisthorchiasis and subsequent bile duct cancer (cholangiocarcinoma; CCA), which is an important public health problem in Southeast Asia. Bithynia snails are known to be the sole intermediate host of O. viverrini, and distributed throughout endemic areas of opisthorchiasis. Since 2001, the genetic variation investigation of O. viverrini has progressively been investigated. Comprehensive genetic variation studies of O. viverrini and Bithynia snails were undertaken and consecutively published in 2007 by Saijuntha and colleagues. These studies provided genetic evidence that O. viverrini and Bithynia snails are both species complex with evidence of co-evolution. Later, several studies have provided data in support of this finding, and have continuously to date reinforced that both O. viverrini and Bithynia are species complexes. Moreover, studies have shown that genetic variation of O. viverrini is related to geographical, temporal, fish host species including geographical genetic variation of its snail host, Bithynia siamensis sensu lato. This is significant and important in our understanding of the evolution and phylogenetic relationships between species within the O. viverrini and Bithynia species complexes. A comprehensive knowledge of the systematics and population genetics of O. viverrini and Bithynia snails provides a sound basis to instigate and develop effective prevention and control programs targeting opisthorchiasis and CCA in the endemic areas of Southeast Asia. Thus, this review examines the historical series of investigations of the systematics and population genetics of O. viverrini including Bithynia spp. in Southeast Asia since molecular genetic investigations commenced some 20 years ago.

Liver Flukes: Clonorchis and Opisthorchis.

Clonorchis sinensis, Opisthorchis viverrini, and O. felineus are liver flukes of human and animal pathogens occurring across much of Europe and Asia. Nevertheless, they are often underestimated compared to other, better known neglected diseases in spite of the fact that many millions of people are infected and hundreds of millions are at risk. This is possibly because of the chronic nature of the infection and disease and that it takes several decades prior to a life-threatening pathology to develop. Several studies in the past decade have provided more information on the molecular biology of the liver flukes which clearly lead to better understanding of parasite biology, systematics, and population genetics. Clonorchiasis and opisthorchiasis are characterized by a chronic infection that induces hepatobiliary inflammation, especially periductal fibrosis, which can be detected by ultrasonography. These chronic inflammations eventually lead to cholangiocarcinoma (CCA), a usually fatal bile duct cancer that develops in some infected individuals. In Thailand alone, opisthorchiasis-associated CCA kills up to 20,000 people every year and is therefore of substantial public health importance. Its socioeconomic impacts on impoverished families and communities are considerable. To reduce hepatobiliary morbidity and CCA, the primary intervention measures focus on control and elimination of the liver fluke. Accurate diagnosis of liver fluke infections in both human and other mammalian, snail and fish intermediate hosts, are important for achieving these goals. While the short-term goal of liver fluke control can be achieved by praziquantel chemotherapy, a comprehensive health education package targeting school children is believed to be more beneficial for a long-term goal/solution. It is recommended that a transdisciplinary research or multisectoral control approach including one health and/or eco health intervention strategy should be applied to combat the liver flukes, and hence contribute to reduction of cholangiocarcinoma in endemic areas.

Low-Grade Endemicity of Opisthorchiasis, Yangon, Myanmar.

We performed an epidemiologic survey of opisthorchiasis in Yangon, Myanmar. The fecal egg-positive rate of residents was 0.7%, and we recovered an adult fluke after chemotherapy and purging of an egg-positive resident. We detected Opisthorchis viverrini metacercariae in freshwater fish. We found the Yangon area to have low-grade endemicity of opisthorchiasis.

Mitochondrial and nuclear ribosomal DNA dataset suggests that Hepatiarius sudarikovi Feizullaev, 1961 is a member of the genus Opisthorchis Blanchard, 1895 (Digenea: Opisthorchiidae).

The taxonomy and classification of the family Opisthorchiidae have been revised by several authors with the exclusion or synonymization of some genera. The genus Hepatiarius Feizullaev, 1961 accommodated two species: Hepatiarius sudarikovi Feizullaev, 1961 and H. longissimus Linstow, 1883. Recently, some experts have suppressed Hepatiarius as a junior synonym of Opisthorchis Blanchard, 1895 based on morphological features alone. Prior to the present study, no molecular data either from nuclear or from mitochondrial DNA was available for any species of this genus. In the present study, four specimens of H. sudarikovi Feizullaev, 1961 were recovered from the bile ducts of the little egret, Egretta garzetta. The complete sequences of the internal transcribed spacers (ITS-1 and ITS-2) of ribosomal DNA (rDNA) and the nearly complete mitochondrial genome sequences were determined and the phylogenetic relationship of H. sudarikovi with related taxa was assessed based on the mitochondrial (mt) DNA sequences. The sequence similarity in the ITS rDNA between H. sudarikovi and Opisthorchis felineus was higher (97.62% in ITS-1 and 96.22% in ITS-2) than with other opisthorchiids. Phylogenetic analysis using Bayesian inference (BI) based on the concatenated amino acid sequences of 12 protein-coding genes (PCGs) clustered H. sudarikovi into the clade of opisthorchiids, with O. felineus being the closest related species, which supports the affinity of H. sudarikovi with trematodes in the genus Opisthorchis. This is the first avian liver fluke whose nearly complete mitochondrial genome was sequenced. The mtDNA sequences of H. sudarikovi, in combination with its rDNA sequences, provide novel resources of genetic markers for the identification, species differentiation, and systematic studies of H. sudarikovi with other avian opisthorchiid flukes.

Opisthorchis felineus and Metorchis bilis Metacercariae in Cyprinid Fish Leuciscus idus in Nura-Sarysu River, Kazakhstan.

Aim of the present study was to provide presence of opisthorchiid metacercariae in cyprinid fish Leuciscus idus in Nura-Sarysu river, Kazakhstan. Infection rate of the ides by the metacercariae was 42%. The metacercariae, similar morphologically to those of the liver flukes, were found: elliptical in shape, 0.19-0.25×0.15-0.22 mm, oral and ventral suckers nearly equal size, and excretory bladder O-shape with black content, occupying posterior part of the body. The metacercariae were divided into 2 groups with differences in size and thickness of cyst wall. Adult flukes were recovered from the Syrian hamsters infected with the opisthorch metacercariae and identified with morphological characters to Opisthorchis felineus and Metorchis bilis. DNA sequences of ITS1, ITS2, and cox1 supported the taxonomic assignment.

Taxonomy, Ecology and Population Genetics of Opisthorchis viverrini and Its Intermediate Hosts.

There have been considerable advances in our understanding of the systematics and ecology of Opisthorchis viverrini; however, this new knowledge has not only clarified but also complicated the situation. We now know that what was once considered to be a single species is, in fact, a species complex, with the individual species being confined to specific wetland areas. There is also a strong genetic association between the members of the O. viverrini species complex and their Bithynia snail intermediate hosts. Although this does not negate data collected before the recognition of this situation, it does lead to the caveat that regional and temporal variations in data collected may be related to the species examined. The advances in ecology have generally been spatially limited and have led, in part, to contradictory results that may well be related to nonrecognition of the species studied. It may also be related to natural temporal and spatial variation related, for example, to habitat characteristics. To understand the variation present, it will be necessary to conduct long-term (several years at least) sampling projects after defining the genetic characteristics of O. viverrini sensu lato and its Bithynia snail intermediate hosts.

Human liver fluke Opisthorchis viverrini (Trematoda, Opisthorchiidae) in Central Myanmar: New records of adults and metacercariae identified by morphology and molecular analysis.

Opisthorchis-like metacercariae were found in cyprinoid fish, Puntius brevis, bought from markets in the Bago region, Central Myanmar. Adult worms recovered from experimentally-infected hamsters resembled Opisthorchis viverrini. DNA was extracted from adults and metacercariae. A portion of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and the internal transcribed spacer 2 (ITS2) regions were amplified using the polymerase chain reaction and then sequenced. The sequences confirmed that the flukes were O. viverrini. In phylogenetic analyses, sequences of O. viverrini, including our new sequences, clustered in a group with high bootstrap support for ITS2 (80%) and the cox1 gene (99%). Interestingly, ITS2 and cox1 sequences of O. viverrini and O. lobatus were very similar, raising a question about the identity of the latter. This is the first report of O. viverrini in cyprinoid fish in Central Myanmar, and only the second report of the species in Myanmar. It is an urgent warning against consuming raw or semi-cooked freshwater fish dishes. Development of an effective food-safety strategy should be provided for the prevention and control of opisthorchiasis and other foodborne diseases.

Updated molecular phylogenetic data for Opisthorchis spp. (Trematoda: Opisthorchioidea) from ducks in Vietnam.

BACKGROUND: An opisthorchiid liver fluke was recently reported from ducks (Anas platyrhynchos) in Binh Dinh Province of Central Vietnam, and referred to as "Opisthorchis viverrini-like". This species uses common cyprinoid fishes as second intermediate hosts as does Opisthorchis viverrini, with which it is sympatric in this province. In this study, we refer to the liver fluke from ducks as "Opisthorchis sp. BD2013", and provide new sequence data from the mitochondrial (mt) genome and the nuclear ribosomal transcription unit. A phylogenetic analysis was conducted to clarify the basal taxonomic position of this species from ducks within the genus Opisthorchis (Digenea: Opisthorchiidae). METHODS: Adults and eggs of liver flukes were collected from ducks, metacercariae from fishes (Puntius brevis, Rasbora aurotaenia, Esomus metallicus) and cercariae from snails (Bithynia funiculata) in different localities in Binh Dinh Province. From four developmental life stage samples (adults, eggs, metacercariae and cercariae), the complete cytochrome b (cob), nicotinamide dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and near-complete 18S and partial 28S ribosomal DNA (rDNA) sequences were obtained by PCR-coupled sequencing. The alignments of nucleotide sequences of concatenated cob + nad1 + cox1, and of concatenated 18S + 28S were separately subjected to phylogenetic analyses. Homologous sequences from other trematode species were included in each alignment. RESULTS: Phylogenetic trees were inferred from concatenated (cob + nad1 + cox1) nucleotide sequences and combined 18S + 28S nucleotide sequences of five Opisthorchis sp. BD2013 samples and additional reference taxa. Both trees demonstrated the anticipated clustering of taxa within the superfamily Opisthorchioidea, the paraphyly of the genus Opisthorchis and the sister-species relationship of Opisthorchis sp. BD2013 with O. viverrini. CONCLUSIONS: While it is likely that Opisthorchis sp. BD2013 is distinct from O. viverrini, it is clearly a sister taxon of O. viverrini within the limited number of Opisthorchis species for which appropriate sequence data are available. The new sequences provided here will assist the diagnosis and the taxonomic clarification of the opisthorchiid species.

Molecular discrimination of Opisthorchis-like eggs from residents in a rural community of central Thailand.

BACKGROUND: Opisthorchis viverrini infection is a major public health problem in northern and northeastern Thailand. The chronic infection of O. viverrini is related to cholangiocarcinoma which causes high mortality in endemic areas. Therefore, the diagnosis, treatment, control and prevention of O. viverrini infection are necessary. The morphology of the egg is very similar to that of other species of human liver flukes (Opisthorchis felineus and Clonorchis sinensis) as well as that of small intestinal flukes in the family Heterophyidae. Thus, molecular characterization is crucially required to discriminate species of Opisthorchis-like eggs in fecal examination. METHODOLOGY/PRINCIPAL FINDINGS: We aimed to determine the prevalence of O. viverrini infection among villagers living in Sanamchaikate District, Chachoengsao Province, in central Thailand, where O. viverrini infection has previously been reported. A total of 2,609 fecal samples were examined for Opisthorchis-like eggs using microscopic examination. PCR-RFLP analysis of the ITS2 region was used to discriminate Opisthorchis-like eggs. The genetic structure of O. viverrini infection was demonstrated using nucleotide sequencing of cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 1 (nad1). Testing of evolutionary neutrality of the cox1 and nad1 sequences of O. viverrini was performed using Tajima's D tests and Fu's Fs tests. Moreover, the haplotype networks and phylogenetic trees were constructed to study the relationships of O. viverrini isolated from different endemic areas. A high prevalence of O. viverrini infection is still observed in a rural community of Chachoengsao Province, central Thailand. The overall prevalence of Opisthorchis-like eggs using microscopic examination was 16.8%. PCR-RFLP profiles showed the predominant infection of O. viverrini (9.6%) including very low infections of other small intestinal flukes, Haplorchis taichui (0.08%) and Euparyphium albuferensis (0.08%). The genetic structure of O. viverrini populations in central Thailand was also described and revealed a non-significant difference in genetic diversity. In addition, the genetic background of the O. viverrini populations was closely related to the isolate from Lao PDR. CONCLUSIONS/SIGNIFICANCE: Our study highlighted the prevalence of O. viverrini infection in central Thailand indicating that control programs and health education regarding opisthorchiasis is still required in this endemic area. Additionally, the study demonstrated the genetic structure of O. viverrini, in central Thailand which could provide information on the molecular epidemiology of this parasite.

Ribosomal DNA analysis of isolates of the liver fluke Opisthorchis pedicellata (Verma, 1927) from two siluroid fish species in India.

The aim of this paper was to evaluate the genetic relationship between two isolates of Opisthorchis (O. pedicellata sp. 1, O. pedicellata sp. 2) from two siluroid fish hosts, based on three nuclear DNA (18S, internal transcribed spacer 1 (ITS1) and ITS2). Molecular analysis revealed that both isolates of 18S sequences showed low intraspecific variability (1.6%), while this was 1.9% for ITS1 and 2.6% for ITS2. The secondary structure of the ITS2 region exhibited a remarkable four-helix model, with helices 1 and 4 being relatively short, three U-U mismatches in helix 2 and the longest helix (3) having two UGGG motifs. The phylogenetic analyses, using neighbour-joining (NJ) and maximum parsimony (MP) methods of MEGA 6, demonstrate that both isolates form a sister clade with significant support (100%). Therefore, we concluded that both the isolates of O. pedicellata are of the same species but some variations may be due to the sympatric environment of variant hosts.

Development and validation of species-specific molecular diagnostic tool for Opisthorchis felineus (Digenea, Opisthorchiidae) metacercariae.

Opisthorchis felineus (family Opisthorchiidae) is a parasitic flatworm representing a serious threat to humans in some countries. Opisthorchiasis occurs after consumption of raw or undercooked cyprinid fish infected by the metacercarial stage of the parasite. Due to its small size, detection of the parasite in fish fillet is time-consuming and difficult. Furthermore, isolated metacercariae can be identified to genus but not to species level using morphological features and molecular techniques are necessary. In this work, we describe the development of primers for a diagnostic PCR amplification of a 254-bp fragment of the cytochrome c oxidase I in the mitochondrion of Opisthorchis felineus metacercariae isolated from fish fillet, together with a validation protocol for this method.

Characterization and differential expression analysis of Toxocara canis aquaporin-1 gene.

Toxocara canis is an intestinal nematode of canids with a worldwide distribution, causing an important but neglected parasitic zoonosis in humans. Aquaporins (AQP) are a family of water channel proteins, which function as membrane channels to regulate water homeostasis. In this study, the coding sequence of aquaporin-1 gene of T. canis (Tc-aqp-1) was cloned and characterized. The obtained Tc-aqp-1 coding sequence was 933 bp in length, which predicted to encode 311 amino acids. Two conserved asparagine-proline-alanine (NPA) motifs were identified in the multiple sequence alignments. Phylogenetic analysis revealed the closest relationship between T. canis and Opisthorchis viverrini based on aquaporin-1 amino acid sequence. A structure was predicted with ligand binding sites predicted at H93, N95, N226, L94, I79, and I210 and with active sites predicted at I256 and G207. Gene Ontology (GO) annotations predicted its cellular component term of integral component of plasma membrane (GO: 0005887), molecular function term of channel activity (GO: 0015250), and biological process term of water transport (GO: 0006833). Tissue expression analysis revealed that the Tc-aqp-1 was highly expressed in the intestine of adult male. The findings of the present study provide the basis for further functional studies of T. canis aquaporin-1.

Cercarial emergence patterns for Opisthorchis viverrini sensu lato infecting Bithynia siamensis goniomphalos from Sakon Nakhon Province, Thailand.

UNLABELLED: Opisthorchis viverrini sensu lato is a food-borne trematode which is classified as a class 1 carcinogen, with infection potentially leading to cholangiocarcinoma. Snails of the genus Bithynia act as the first intermediate hosts and an amplifying point in the parasite life cycle. In order to investigate seasonal effect on transmission dynamics of O. viverrini in Bithynia siamensis goniomphalos, cercarial emergence and output profiles were monitored at different season. A total of 4533 snails originating from Sakon Nakhon Province, Thailand, collected during the three main seasons, were analyzed for O. viverrini s.l. INFECTION: Emergence of O. viverrini s.l. cercariae from snails was monitored daily from 06:00 to 18:00 h for seven consecutive days. The prevalence of infection in the snails was highest in the hot-dry season and declined in the rainy and cool-dry seasons. Peak cercarial emergence occurred between 08:00 and 10:00 h during the rainy and cool-dry seasons and between 10:00 and 12:00 h during the hot-dry season. The cercarial output was highest in the hot-dry season, similar to a previous study from Lao People's Democratic Republic (PDR). Average cercarial output/snail in Thailand was higher than in Lao PDR. The number of cercariae emerging from the snails was strongly related to snail size, but the relationship between prevalence of infection and snail size differed between seasons. Observed discrepancies in the emergence patterns and per capita cercarial release may reflect differences in environmental, snail, and/or parasite factors particularly biological characteristics between the cryptic species of O. viverrini s.l. and B. s. goniomphalos from Thailand and Lao PDR.

The complete mitochondrial genome of Metorchis orientalis (Trematoda: Opisthorchiidae): Comparison with other closely related species and phylogenetic implications.

Metorchis orientalis (Trematoda: Opisthorchiidae) is an important trematode infecting many animals and humans, causing metorchiasis. In the present study, the complete mitochondrial (mt) genome of M. orientalis was sequenced. The complete mt genome of M. orientalis is 13,834 bp circular DNA molecule and contains 12 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. The gene content and arrangement of M. orientalis is the same as those of Opisthorchiidae trematodes (Opisthorchis viverrini, Opisthorchis felineus and Clonorchis sinensis), but distinct from Schistosoma spp. Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes with three different computational algorithms (Bayesian inference, maximum likelihood and maximum parsimony) revealed that M. orientalis and O. viverrini represent sister taxa. The mt genome provides a novel genetic marker for further studies of the identification, classification and molecular epidemiology of Opisthorchiidae trematodes, and should have implications for the diagnosis, prevention and control of metorchiasis in animals and humans.

Molecular characterization of Opisthorchis noverca (Digenea: Opisthorchiidae) based on nuclear ribosomal ITS2 and mitochondrial COI genes.

Opisthorchiasis is a public health problem in South-East Asian countries and Eastern Europe. The infection implicates mainly two species of Opisthorchis, namely O. viverrini and O. felineus, that occur mostly in fish-eating mammals and humans, although there are rare reports of human cases involving two other species, O. noverca and O. guayaquilensis. Opisthorchis noverca has been reported frequently in dogs and pigs from the Indian subcontinent, with rare reports from cattle and human subjects. With a view to supplementing morphology-based identification of this species, the present study aimed to provide molecular characterization of O. noverca, using rDNA internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase I (mt COI) markers so as to determine its genetic correlation with other species of Opisthorchiidae, and also to generate a taxon-specific molecular marker based on the ITS2 region. The phylogenetic relationship between O. noverca and other species of the genus was determined using molecular sequence data. To strengthen the result, secondary structure sequence analyses of ITS2 with hemi-compensatory base changes (hCBCs), and amino acid sequence analyses, were also evaluated. Our results confirm that O. noverca is a distinct and valid species.

Is Opisthorchis viverrini an avian liver fluke?

Recently, in the Journal of Helminthology (May 2013), Dao et al. reported that Opisthorchis viverrini-like flukes were found in the bile duct of domestic ducks in Vietnam. They stated that this is the first record of Opisthorchis sp. in birds in Vietnam. However, three Opisthorchis species--O. cheelis, O. longissimus and O. parageminus--in birds in Vietnam were described by Le in 2000. Amongst these, O. parageminus was first reported, by Oshmarin in 1970, as a new Opisthorchis species found in domestic ducks (Anas platyrhynchos) in Vietnam. Morphologially O. viverrini-like flukes described by Dao et al. are much more similar to O. parageminus than to O. viverrini. The phylogenetic trees of internal transcribed spacer 2 (ITS2) and cytochrome oxidase 1 (CO1) gene sequences also showed that the O. viverrini-like liver flukes from domestic ducks were closer to O. lobatus than to O. viverrini. Therefore, O. viverrini-like liver flukes reported by Dao et al. (2013) are most likely to be O. parageminus.

Retrotransposon OV-RTE-1 from the carcinogenic liver fluke Opisthorchis viverrini: potential target for DNA-based diagnosis.

Infections by the fish-borne liver flukes Opisthorchis viverrini and Clonorchis sinensis can lead to bile duct cancer. These neglected tropical disease pathogens occur in East Asia, with O. viverrini primarily in Thailand and Laos and C. sinensis in Cambodia, Vietnam, and China. Genomic information about these pathogens holds the potential to improve disease treatment and control. Transcriptome analysis indicates that mobile genetic elements are active in O. viverrini, including a novel non-Long Terminal Repeat (LTR) retrotransposon. A consensus sequence of this element, termed OV-RTE-1, was assembled from expressed sequence tags and PCR amplified genomic DNA. OV-RTE-1 was 3330 bp in length, encoded 1101 amino acid residues and exhibited hallmark structures and sequences of non-LTR retrotransposons including a single open reading frame encoding apurinic-apyrimidinic endonuclease (EN) and reverse transcriptase (RT). Phylogenetic analyses confirmed that OV-RTE-1 was member of the RTE clade of non-LTR retrotransposons. OV-RTE-1 is the first non-LTR retrotransposon characterized from the trematode family Opisthorchiidae. Sequences of OV-RTE-1 were targeted to develop a diagnostic tool for detection of infection by O. viverrini. PCR specific primers for detection of O. viverrini DNA showed 100% specificity and sensitivity for detection of as little as 5 fg of O. viverrini DNA whereas the PCR based approach showed 62% sensitivity and 100% specificity with clinical stool samples. The OV-RTE-1 specific PCR could be developed as a molecular diagnostic for Opisthorchis infection targeting parasite eggs in stool samples, especially in regions of mixed infection of O. viverrini and/or C. sinensis and minute intestinal flukes.