Molecular Identification of the Human Pathogen Amphimerus sp. in the Freshwater Snail Aroapyrgus sp. in Ecuador.

Here, we report for the first time the snail intermediate host for the Amphimerus liver fluke, a foodborne trematodiasis. In Ecuador, Amphimerus of the Opisthorchiidae family, infects humans, cats, and dogs, in the tropical Pacific-coast region. Opisthorchiidae comprising also Clonorchis sinensis, Opisthorchis sp., and Metorchis sp., have complex life cycles involving a definitive and two intermediate hosts. We identified morphologically and investigated the presence and prevalence of Amphimerus cercaria and DNA in freshwater snails collected in a human-amphimeriasis endemic region in Ecuador, extracted DNA from snail tissue and emerged cercariae, performed real-time polymerase chain reaction (PCR) with the newly developed primers and probe amplifying the Amphimerus ribosomal internal transcribed spacer 2 (ITS2) region, and sequenced the amplified DNA fragment. We collected 2,800 snails, characterized four species Aroapyrgus sp., Melanoides tuberculata, Biomphalaria cousini, and Aplexa marmorata, isolated three cercariae morphotypes. Of the 640 snails analyzed by qPCR, only Aroapyrgus and one of the three cercariae resulted positive, at a 15% infection prevalence. Polymerase chain reaction revealed that the Aroapyrgus snail and cercaria-morphotype-3 corresponded to Amphimerus, but not to C. sinensis, Fasciola hepatica, or Paragonimus mexicanus. The sequence of amplified DNA product matched that of human-isolated Amphimerus. This finding constitutes the first documentation that Aroapyrgus sp. is the first intermediate host for the Amphimerus sp. that infect humans in Ecuador. The ITS2-gene PCR and sequencing analysis demonstrated a high prevalence of snail infection and proved useful for detecting the infection in snails, which findings can help the establishment of suitable control programs against transmission in any endemic region of interest.

A review of molecular identification tools for the opisthorchioidea.

The superfamily Opisthorchioidea encompasses the families Cryptogonimidae, Opisthorchiidae and Heterophyidae. These parasites depend on the aquatic environment and include marine and freshwater species. Some species, such as Clonorchis sinensis and Opisthorchis viverrini, have a high impact on public health with millions of infected people worldwide and have thus been the object of many studies and tool developments. However, for many species, tools for identification and detection are scarce. Although morphological descriptions have been used and are still important, they are often not efficient on the immature stages of these parasites. Thus, during the past few decades, molecular approaches for parasite identification have become commonplace. These approaches are efficient, quick and reliable. Nonetheless, for some parasites of the superfamily Opisthorchioidea, reference genomic data are limited. This study reviews available genetic data and molecular tools for the identification and/or the detection of this superfamily. Molecular data on this superfamily are mostly based on mitochondrial and ribosomal gene sequence analyses, especially on the cytochrome c oxidase subunit 1 gene and internal transcribed spacer regions respectively.

Morphological and molecular data for highly pathogenic avian parasite Erschoviorchis anuiensis sp. n. and phylogenetic relationships within the Opisthorchiidae (Trematoda).

Adult worms of Erschoviorchis anuiensis sp. n., parasites of the pancreas and liver of birds, were found by feeding the Muscovy ducks Cairina moschata dom. with freshwater fish (Phoxinus percnurus) from the Amur River basin (Russia). The trematodes obtained differ from the only previously known representative of the genus, E. lintoni by the large size of the ventral sucker, testes and ovary, the shape of the ovary (three-lobed vs irregular oval for E. lintoni), and the degree of vitellarium development (well-developed vitellarium with numerous follicles vs weakly developed vitelline fields for E. lintoni). In addition, genetic data were obtained for E. anuiensis sp. n., including nucleotide sequences of the ITS region and the 28S rRNA gene of nuclear DNA, and the mitochondrial сох1 gene. These data show that the genus Erschoviorchis is a sister group to the representatives of the genera Opisthorchis, Clonorchis, and Metorchis. At the same time, it did not cluster with species of Amphimerus, in which E. lintoni has sometimes been placed. The results of the study indicated that E. anuiensis sp. n., as well as E. lintoni, when it occurs in the pancreas, leads to significant associated pathological changes, manifested in an increase in size, changes of structure and tissue density.

Description of a new species, Cryptocotyle lata sp. nov., and discussion of the phylogenetic relationships in Opisthorchioidea.

Adult Cryptocotyle lata sp. nov. worms were obtained from experimental studies. In the Russian southern Far East, the life cycle of this parasite is carried out using freshwater snails (Boreoelona ussuriensis), freshwater fish, and birds as the first intermediate, second intermediate, and definitive hosts, respectively. The morphological indices of C. lata sp. nov. are closest to Cryptocotyle concava; however, these two species differ in terms of their sizes of body, oral and ventral suckers, eggs, and the shape of their testes and ovaries. Analysis of the life cycles of the Cryptocotyle representatives suggested that C. concava were at least two cryptic species, one of which circulates using brackish water Hydrobia snails, and the other using freshwater Amnicola snails as the first intermediate hosts. Molecular data (i.e., the 28S gene and the second internal transcribed spacer (ITS2) of rDNA) were used to analyze the phylogenetic relationships of C. lata sp. nov. and other representatives of Opisthorchioidea. The long repeats and secondary structure of the ITS1 region were studied. Representatives of the Opisthorchiidae and several species from Heterophyidae (including the genus Cryptocotyle) were found to have molecular features that suggested that these species belonged to Opisthorchiidae. At the same time, the genetic relatedness of worms, which are united in common clusters on phylogenetic trees, is consistent with the use of the first intermediate hosts from different taxonomic groups in their life cycles; namely, snails of the Truncatelloidea are hosts of trematodes from a cluster with Opisthorchiidae and a number species of the family Heterophyidae, while snails of the Cerithioidea are hosts of worms from a cluster that includes only the Heterophyidae. In addition, the results of genetic studies indicate that Clonorchis sinensis, Metorchis ussuriensis, Metorchis bilis, Metorchis xanthosomus, and Metorchis orientalis should be included in the genus Opisthorchis.

High prevalence of zoonotic trematodes in roach (Rutilus rutilus) in the Gulf of Finland.

The intention to increase roach (Rutilus rutilus) consumption is in focus for ecological and economic reasons in Finland. However, its safety as food has not been considered comprehensively. We collected and artificially digested 85 roach halves originating from the south-eastern coast of Finland, and found trematode metacercariae in 98.8% of the samples. Based on polymerase chain reaction (PCR) and sequencing of amplicons generated from the ITS2 gene region, zoonotic parasites of the family Opistorchiidae were identified as Pseudamphistomum truncatum and Metorchis bilis, and also non-zoonotic Holostephanus dubinini (family Cyathocotylidae) and Posthodiplostomum spp. (family Diplostomidae) were identified. The species identity of other trematodes found is currently being investigated. Mixed infections of several trematode species were common. The prevalence of morphologically identified zoonotic P. truncatum was 46%, and zoonotic M. bilis was found in one sequence sample. The high prevalence of zoonotic trematode metacercariae in roach from the Gulf of Finland is alarming. Only thoroughly cooked roach products can be recommended for human or animal consumption from the area.

Integrative taxonomy of European parasitic flatworms of the genus Metorchis Looss, 1899 (Trematoda: Opisthorchiidae).

Metorchis spp. are flukes (Platyhelminthes: Digenea) that infect vertebrates, including humans, dogs, cats, poultry and wild game, with cyprinid freshwater fish serving as typical second intermediate hosts. In their definitive hosts, the Metorchis spp. are difficult to identify to species. We provide and analyze sequences of two nuclear (18S rDNA and ITS2) and two mitochondrial (CO1 and ND1) DNA loci of four morphologically identified European species of the Metorchis, namely Metorchis albidus, Metorchis bilis, Metorchis crassiusculus and Metorchis xanthosomus, and of another opisthorchiid, Euamphimerus pancreaticus. DNA analysis suggests that the Metorchis specimens identified morphologically as M. albidus (from Lutra lutra), M. bilis (from Phalacrocorax carbo) and M. crassiusculus (from Aquila heliaca and Buteo rufinus) represent a single species. Thus, M. albidus (Braun, 1893) Loos, 1899 and M. crassiusculus (Rudolphi, 1809) Looss, 1899 are recognized as junior subjective synonyms of M. bilis (Braun, 1790) Odening, 1962. We also provide comparative measurements of the Central European Metorchis spp., and address their tissue specificity and prevalence based on the examination of extensive bird cohort from 1962 to 2015. M. bilis and M. xanthosomus can be morphologically diagnosed by measuring the extent of genitalia relative to body length and by the size ratio of their suckers. They also differ in their core definitive hosts, with ducks (Anas, Aythya) and coots (Fulica) hosting M. xanthosomus, and cormorants (Phalacrocorax), the birds of prey (Buteo, Aquila, etc.), piscivorous mammals (Lutra, Vulpes, Ursus, etc.) and humans hosting M. bilis. Previous reports on the Metorchis spp. contain numerous suspected misidentifications.

Telomeric DNA in chromosomes of five opisthorchid species.

The analysis of telomere repeat distribution in chromosomes of five opisthorchid species (Opisthorchis felineus (Rivolta, 1884), Opisthorchis viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), Metorchis bilis (Braun, 1890), Clonorchis sinensis (Cobbold, 1875)) was performed with fluorescent in situ hybridization (FISH) of labeled (TTAGGG)n DNA-probe and PNA telomere probe on mitotic and meiotic chromosomes of these species. It was shown that chromosome telomeres of all studied species contain large clusters of (TTAGGG)n telomeric repeats. Interstitial clusters of the (TTAGGG)n repeats have not been revealed in the chromosomes of any studied species even when FISH of PNA telomere probe on pachytene chromosomes was performed. Furthermore interstitial clusters of the (TTAGGG)n repeats have not been detected in the chromosomes of O. viverrini, one of chromosomes of this species is the result of a fusion of two ancestral opisthorchid chromosomes.

Families Opisthorchiidae and Heterophyidae: are they distinct?

Superfamily Opisthorchioidea Looss, 1899 consists of three well-known families, Opisthorchiidae, Heterophyidae, and Cryptogonimidae, with basic similarities in morphology and life-cycles. Many species in the first two of these families are human pathogens, such as Opisthorchis viverrini, O. felineus, Clonorchis sinensis, Haplorchis spp. and Metagonimus spp. Recently, a molecular phylogenetic study on the classification of Digenea revealed a paraphyletic relationship between Opisthorchiidae and Heterophyidae. For our study, we gathered and analyzed all available data in GenBank, together with new data of nuclear 18S ribosomal DNA and ribosomal internal transcribed spacer 2 (ITS2) sequences of the families within the Opisthorchioidea. Maximum likelihood and Bayesian inference analyses suggested that families Opisthorchiidae and Heterophyidae are inseparable from each other, with the former nested within the latter. Groupings in molecular trees are generally consistent with morphological features used in taxonomy.

Confirmation of the paraphyletic relationship between families Opisthorchiidae and Heterophyidae using small and large subunit ribosomal DNA sequences.

Opisthorchiidae and Heterophyidae are classified into different families based on morphological identification. However, recent molecular phylogenetic studies suggested the possible paraphyletic relationship between these two families. In this study, the paraphyletic relationship between these two families was confirmed further by maximum likelihood and Bayesian inference analyses using the combined sequences of SSU and LSU rDNA.

Discrimination of Opisthorchis viverrini from Haplorchis taichui using COI sequence marker.

This study aimed to discriminate infections of two common fish-borne trematodes in Thailand, Opisthorchis viverrini from Haplorchis taichui, based on mitochondrial cytochrome c oxidase subunit I (COI) gene. Designed primers (COI-OV-Hap F&R primers) amplified partial COI fragments of O. viverrini and H. taichui with high sensitivity in different developmental stages (adult, metacercaria, and egg). Polymerase chain reaction (PCR) amplicons were generated with low genomic DNA concentration ( approximately 10(-4)ng) of O. viverrini and H. taichui at 50 and 56 degrees C annealing temperatures, respectively. At 50 degrees C, COI fragments of Clonorchis sinensis and H. taichui were also obtained, but this was less sensitive than O. viverrini. At 56 degrees C, only H. taichui could be amplified and discriminated from H. pumilio, H. yogokawai, O. viverrini, and C. sinensis. Between 50 and 56 degrees C, the PCR amplicons of H. pumilio and H. yogokawai were amplified with low specificity and low sensitivity. The genetic characters among O. viverrini, C. sinensis, and H. taichui were distinguished by PCR-RFLP method. The PCR-RFLP profiles might be useful for diagnosing mixed O. viverrini and H. taichui infections in endemic areas, and for detecting metacercariae of O. viverrini, C. sinensis and H. taichui in epidemiological surveys of infections in fish hosts.

PCR diagnosis of infections with different species of Opisthorchiidae using a rapid clean-up procedure for stool samples and specific primers.

Infections with the opisthorchiid liver flukes Clonorchis sinensis, Opisthorchis viverrini, and Opisthorchis felineus cause serious health problems in endemic areas of Southeast Asia and countries of the former Soviet Union. Chronic infections--even with low worm burdens--may lead to the development of fatal cholangiocarcinoma and related symptoms. A more sensitive diagnosis is needed since the tiny eggs of the worms are often not seen in microscopic examinations of stool samples, especially in patients with low infections. This communication reports a rapid cleanup procedure for human stool samples, which enables reliable identification of DNA by polymerase chain reaction from few eggs of opisthorchiid flukes in fecal samples.

[Ecological bases of the combination of natural foci of Trematoda infections. 1. One- and two-host population-combined foci of Trematoda infections in the floodplain-river ecosystem of the Konda River].

In the context of the present-day teaching of parasitocenoses and the proposition that the pathogen's population is the only compulsory and specific component of a natural focus, the author brings to light the ecological bases of the combination of natural foci of metorchiases, bilharziasis, opisthorchiasis, and methorchiasis (M. xanthosomus). Two first foci are one-host population-combined and the latter focus is a two-host population-combined focus. While analyzing the combination of foci, it is expedient to consider in pairs since this provides a way of identifying the bases of the combination, which are unique to these foci, and determining the level, pattern, type, and degree of the combination of foci and, on their basis, the type of a combined focus. It is noted that in biohelminthiases, detection of even hemipopulations of parasites points to the presence of natural foci. The morphological structure of the floodplain-river landscape of the Konda River acts as the abiotic basis of the combination of foci. The parasitic systems of flukes, the parasitocenoses of co-acting hemipopulations of the pathogens and populations of hosts, the structure of foci, the species-specific composition of ecosystems, and the ecological relations of Trematoda hosts act as the biotic bases of the combination of foci of Trematoda infections. The host susceptibility to infection with Trematoda hemipopulations and the multihostality of B. polonica and M. xanthosomus act as the epizootic bases of the combination of foci. The abiotic, biotic, and epizootic bases of the combination are, in the aggregate, the ecological bases of the combination of natural foci of zoonoses.

Molecular characterization and differentiation of opisthorchiid trematodes of the species Opisthorchis felineus (Rivolta, 1884) and Metorchis bilis (Braun, 1790) using polymerase chain reaction.

Adult specimens of the opisthorchiid liver fluke species Opisthorchis felineus and Metorchis bilis could be identified for the first time by molecular biological methods using species specific primers (OF and MB primers) in the polymerase chain reaction (PCR). The OF or MB primers were based on a part of the mitochondrial cytochrome c oxidase I gene. A specific product of approximately 200 bp could be amplified for O. felineus by means of the specific O. felineus primers. By contrast, the amplification of M. bilis DNA with MB primers produced a fragment of approximately 110 bp. A specificity of 100% could be demonstrated for both primer pairs. The sensitivities of the PCRs were 10 pg for the O. felineus DNA and 100 fg for the M. bilis DNA.

Trematodes of the family Opisthorchiidae: a minireview.

Examination of the original descriptions of genera placed within the family Opisthorchiidae has revealed that only 33 of the original 43 genera are valid members of this family. Further study of these descriptions should also reveal that many of the subfamilies are also invalid. With reference to the original descriptions of these genera, and subsequent literature, a brief survey of the family has been compiled. Information on the spectrum of definitive hosts that these helminths parasitize is provided, as well as information on the life cycles, geographic distribution, and socioeconomic impacts of the family. More in-depth information is given on those species that are of particular medical importance; namely, Clonorchis sinensis, Opisthorchis viverrini, and O. felineus. The final aims of this review are to provide information on the entire genera of the family Opisthorchiidae, which will aid understanding of the phylogenetic relationships not only within the family, but also within the Class Trematoda.

Amphimerus bragai n. sp. (Digenea: Opisthorchiidae), a parasite of the rodent Nectomys squamipes (Cricetidae) from Minas Gerais, Brazil.

Amphimerus bragai n.sp. (Digenea, Opisthorchiidae) from the bile ducts of a rodent from the State of Minas Gerais, Brazil, Nectomys squamipes (Cricetidae), is described. The new species as studied by both light and scanning electron microscopy. A table is presented comparing the measurements of the new species with those of A. lancea (Diesing, 1850) and A. vallecaucensis Thatcher; 1970, parasites of dolphins and marsupials, respectively. The new species is similar in size and body form to A. vallecaucensis from which it differs in having a vitellarium that extends to the acetabulum while that of the former species are limited to the posterior one-third of the body. Additionally, the new species is from a rodent.

Amphimerus bragai n.sp. (Digenea: Opisthorchiidae), a parasite of the rodent Nectomys squamipes (Cricetidae) from Minas Gerais, Brazil

Amphimerus bragai n.sp. (Digenea, Opisthorchiidae) from the bile ducts of a rodent from the State of Minas Gerais, Brazil, Nectomys squamipes (Cricetidae), is described. The new species was studied by both light and scanning electron microscopy. A table is presented comparing the measurements of the new species with those of A. lancea (Diesing, 1850) and A. vallecaucensis Thatcher, 1970, parasites of dolphins and marsupials, respectively. The new species is similar in size and body form to a A. vallecaucensis from which it differs in having a vitellarium that extends to the acetabulum while that of the former species are limited to the posterior one-third of the body. Additionally, the new species is from a rodent.

Species determination of eggs of opisthorchiid and heterophyid flukes using scanning electron microscopy.

Eggs of opisthorchiid flukes have very characteristic muskmelon-like surface structure of their shells and can be differentiated using this mark from eggs of heterophyid flukes. Surface structure and morphological details in operculum and abopercular end can be observed using scanning electron microscopy. Eggs recorded very often in stool of Laotians belong mostly to Opisthorchis viverrini and sporadically to Metagonimus yokogawai.

[Sensillae of cercariae of Haplorchis taichui (Digenea, Heterophyidae). Discussion of the systematics of the Opisthorchioidea]. / Les sensilles de la cercaire d'Haplorchis taichui (Digene, Heterophyidae). Discussion sur la systématique des Opisthorchioidea.

Cercarial chaetotaxy of Haplorchis taichui (Haplorchiinae, Heterophyidae) is described and compared to that of ten other Opisthorchioidea: three belonging to the Acanthostomidae (Acanthostominae), one to the Opisthorchiidae (Opisthorchiinae) and six to the Heterophyidae (among these, two Centrocestinae, two Apophallinae, one Euryhelminthinae and one Haplorchiinae). Cercarial chaetotaxy allow us to distinguish the considered species and genera, but does not support division of the Opisthorchioidea into families as these have been defined based on morphology of adults; it suggests that the Heterophyidae is heterogenous; it allows us to recognize the Opisthorchioidea characterized by a loss of hexaradial symmetry on the first cephalic cycle of sensillae and by the presence of two to five pairs of sensillae on a tail with proximal excretory apertures.