Exploring the second intermediate hosts and morphology of human- and cat-specific Opisthorchis viverrini-like populations.

Infection by the zoonotic fish-borne trematode, Opisthorchis viverrini, remains a crucial health issue in Thailand and neighboring countries. Recently, molecular analysis revealed two populations of putative O. viverrini: one found primarily in human hosts ("human-specific" population) and the other primarily in cats ("cat-specific" population). It is unclear how the infective stages (metacercariae) of these different populations circulate among definitive and reservoir hosts in nature. To gain an insight into this, mitochondrial cox1 and nad1 gene sequences of metacercariae from fish intermediate hosts were examined. None of 192 metacercariae from cyprinid fish in Lao PDR and Thailand had sequences typical of "cat-specific" O. viverrini, suggesting that cyprinid fish are not the main second intermediate hosts of this population. Interestingly, all 20 O. viverrini-like metacercariae from snakehead fish (Channa striata) shared 99.51-100% sequence identity with eggs from cats naturally infected in a previous study. Hence, we propose a modification of the known transmission dynamics of O. viverrini: consumption of metacercariae within snakehead fish provides another pathway for cats and (occasionally) humans to acquire infection. We also performed morphological comparisons of eggs, metacercariae, and adult flukes (raised in hamsters) of both Opisthorchis populations. The "cat-specific" population has eggs that are narrower and adults that are shorter and wider than in the human-specific population. The metacercaria of the "cat-specific" population is elliptical, while that of the "human-specific" population is oval, occasionally rounded. Our results confirmed that O. viverrini-like metacercariae from snakehead fish are the infective stages of the "cat-specific" fluke. This provides a new insight into the dissemination and transmission of each population in the second intermediate host. The identity of the cat-specific population is discussed.

The New Data on the Serotonin and FMRFamide Localization in the Nervous System of Opisthorchis felineus Metacercaria.

BACKGROUND: Trematoda Opisthorchis felineus Rivolta, 1884 is the causative agent of dangerous parasite disease-opisthorchiasis, widespread in the Russian Federation. The details of the neuroanatomical localization of the serotoninergic and FMRFamidergic neurotransmitter elements as well as their functional roles remain not studied enough in both adult and larval forms of O. felineus. The studies in this area are important in term of the development of a new pharmacological strategy of the struggle with the causative agent of opisthorchiasis affecting the neuronal signal substances and the function of its nervous system. PURPOSE: The aim of this work was the immunocytochemical study of the neurotransmitters serotonin (5-HT, 5-Hydroxitryptamine) and neuropeptide FMRFamide localization in the nervous system of the opisthorchiasis causative agent-O. felineus metacercaria. To study the relationship between the detected neurotransmitters and the muscular elements of the parasite, the muscle staining was carried out simultaneously using fluorophore-conjugated phalloidin. METHODS: The localization of 5-HTergic and FMRFamidergic nerve structures was determined by immunocytochemical method. The staining samples were analyzed using a fluorescent and confocal laser scanning microscopies. RESULTS: The new data on the presence and distribution of the serotonin-immunopositive (IP)- and FMRFa-IP components in the central and peripheral departments of the nervous system of  O. felineus metacercaria has been obtained. Besides that a number of the new anatomical details of the nervous system organization and of the innervation of the organs and tissues in the investigated parasite have been revealed. CONCLUSION: The data obtained on the presence and localization of the 5-HTergic and peptidergic (FMRFamide) components in central and peripheral departments of the nervous system of O. felineus metacercaria elaborated and expanded the existing information about the nervous system as well as the innervations of the tissues and organs in the causative agent of opistchorchiasis.

Paropisthorchis caninus Stephens, 1912: Synopsis of identity and pathological findings due to spontaneous infection in the liver of a street dog in Assam, India.

The present study describes a small liver fluke recovered at post-mortem and the pathological alteration produced by the parasite in the liver of a street dog from Assam, India. The spatulate parasite measured 3-4 mm in length and 1.0-1.25 mm in width with spiny body surface and showed a grossly visible peduncle projecting from the ventral surface at the anterior portion. The eggs of the parasite contained well developed miracidia. The parasite was identified as Paropisthorchis caninus. Identity of the parasite is discussed in the light of available literature and found to be morphologically different from other reported species of dog Opisthorchis. Pathological lesions observed in the liver included degeneration of hepatic parenchyma, hyperplasia of bile duct epithelium, fibrous tissue proliferation and formation of pseudo lobule which were the characteristics of chronic proliferative inflammation.

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.

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.

Functional Analysis of the Unique Cytochrome P450 of the Liver Fluke Opisthorchis felineus.

The basic metabolic cytochrome P450 (CYP) system is essential for biotransformation of sterols and xenobiotics including drugs, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms have numerous paralogues of the CYP gene encoding heme monooxygenases with specific substrate range. Notably, by contrast, the parasitic flatworms have only one CYP gene. The role of this enzyme in the physiology and biochemistry of helminths is not known. The flukes and tapeworms are the etiologic agents of major neglected tropical diseases of humanity. Three helminth infections (Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium) are considered by the International Agency for Research on Cancer (IARC) as definite causes of cancer. We focused our research on the human liver fluke Opisthorchis felineus, an emerging source of biliary tract disease including bile duct cancer in Russia and central Europe. The aims of this study were (i) to determine the significance of the CYP activity for the morphology and survival of the liver fluke, (ii) to assess CYP ability to metabolize xenobiotics, and (iii) to localize the CYP activity in O. felineus tissues. We observed high constitutive expression of CYP mRNA (Real-time PCR) in O. felineus. This enzyme metabolized xenobiotics selective for mammalian CYP2E1, CYP2B, CYP3A, but not CYP1A, as determined by liquid chromatography and imaging analyses. Tissue localization studies revealed the CYP activity in excretory channels, while suppression of CYP mRNA by RNA interference was accompanied by morphological changes of the excretory system and increased mortality rates of the worms. These results suggest that the CYP function is linked to worm metabolism and detoxification. The findings also suggest that the CYP enzyme is involved in vitally important processes in the organism of parasites and is a potential drug target.

The first comprehensive study of praziquantel effects in vivo and in vitro on European liver fluke Opisthorchis felineus (Trematoda).

The European liver fluke Opisthorchis felineus (Rivolta, 1884) is an epidemiologically important parasite infecting mammals, including humans. Opisthorchis felineus is widespread in Russia, Kazakhstan and Eastern European countries. Praziquantel (PZQ) is the drug of choice for the treatment of opisthorchiasis, but the effects of this drug on O. felineus are poorly studied. The aims of this work were (i) to perform a study of PZQ effects in vitro, (ii) to identify morphological markers of PZQ action on O. felineus, (iii) to analyse damage to the worm surface and (iv) to assess the efficacy of PZQ in vivo in a hamster model. Light microscopy, optical sectioning and fluorescence microscopy were used to study morphological changes. In vivo, PZQ at a dose of 400mg/kg reduced the rate of infection in experimental acute and chronic opisthorchiasis in hamsters by 70% and 79%, respectively. In vitro, the drug caused destruction and vacuolisation of the tegument of O. felineus, contractions of the worm musculature, paralysis, and irreversible changes in morphology (IC50=0.14µg/mL). Differences in susceptibility to the drug between adult and newly excysted metacercariae were also observed. Qualitative effects of PZQ in vivo and in vitro were similar to the drug's effects on other trematodes, including epidemiologically important liver flukes. Nevertheless, high heterogeneity of O. felineus specimens in terms of susceptibility to the drug was observed. In addition, we describe for the first time the high rate of recovery of O. felineus following the destructive action of PZQ.

Opisthorchis viverrini-like liver fluke in birds from Vietnam: morphological variability and rDNA/mtDNA sequence confirmation.

Flukes were found in the bile ducts of domestic ducks (Anas platyrhynchos), necropsied in the Binh Dinh province of Central Vietnam. Following staining, morphological characteristics of the bird flukes were compatible with Opisthorchis viverrini, although some characteristics differed from those described in specimens collected from mammal hosts. Computation of the phylogenetic trees on the partial sequences of the second internal ribosomal spacer (ITS2) of the ribosomal DNA and cytochrome c oxidase subunit I (COI) markers of the mitochondrial DNA showed close similarity of the 'bird' Opisthorchis sp. with O. viverrini. We speculate that these bird flukes are O. viverrini that show intraspecies morphological and molecular variability compared to isolates from mammals. This demonstrates the complex epidemiological situation of opisthorchiasis in Vietnam and urges investigations on the potential of birds as a reservoir host of this zoonotic fluke.

Molecular characterization of a tetraspanin from the human liver fluke, Opisthorchis viverrini.

BACKGROUND: The human liver fluke, Opisthorchis viverrini, is designated as a group 1 carcinogen, and is the major risk factor for cholangiocarcinoma in endemic countries throughout Southeast Asia. Proteins in the excretory-secretory products and tegumental surface membranes of the fluke have been proposed to play pivotal roles in parasite survival in the host, and subsequent pathogenesis. These macromolecules are therefore valid targets for the development of vaccines and new drugs to control the infection. Tetraspanins (TSP) are prominent components of the tegument of blood flukes where they are essential for tegument formation, are directly exposed to the immune system, and are major targets for a schistosomiasis vaccine. We propose that similar molecules in the surface membranes of O. viverrini are integral to tegument biogenesis and will be efficacious vaccine antigens. METHODOLOGY/PRINCIPAL FINDINGS: The cDNA sequence encoding O. viverrini tetraspanin-1 (Ov-TSP-1) was identified and cloned. The Ov-tsp-1gene was isolated from a cDNA library. Ov-tsp-1 mRNA was expressed most highly in metacercariae and eggs, and to a lesser extent in juvenile and adult worms. Immunolocalization with adult flukes confirmed that Ov-TSP-1 was expressed in the tegument and eggs in utero. Western blot analysis of rOv-TSP-1 probed with sera from O. viverrini-infected humans and hamsters indicated that both hosts raise antibody responses against the native TSP. Using RNA interference we silenced the expression level of Ov-tsp-1 mRNA in adult flukes by up to 72% by 10 days after delivery of dsRNA. Ultrastructural morphology of adult worms treated with Ov-tsp-1 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. CONCLUSIONS/SIGNIFICANCE: This is the first report of a tetraspanin from the tegument of a liver fluke. Our data imply that tetraspanins play important structural roles in the development of the tegument in the adult fluke. Potential uses of O. viverrini tetraspanins as novel interventions are discussed.

Comparative histopathology of Opisthorchis felineus and Opisthorchis viverrini in a hamster model: an implication of high pathogenicity of the European liver fluke.

European liver fluke (Opisthorchis felineus) and Asian liver fluke (Opisthorchis viverrini) are similar in morphology but comparative pathology of the infections has not been described. We therefore did comparative histopathology of both parasites in an experimental animal model. The study was conducted in 3 groups of 105 Syrian golden hamsters; the first and second groups fed with 50 metacercariae of O. felineus (OF) or O. viverrini (OV) and the last group was uninfected controls. Five hamsters in each group were euthanized on weeks 1, 2, 4, 8, 12 and 24 post-infection. The liver tissue was fixed and processed for routine histopathology and immunohistochemistry for proliferation markers (BrdU or PCNA). Overall, the liver histopathology of O. felineus and O. viverrini infection was generally similar. However, various histopathogical features including intense inflammation, fibrosis, biliary and goblet cell hyperplasia and dysplasia occurred earlier in the OF group. In addition, the existence of precancerous lesions such as cholangiofibrosis in a long-term infection was observed only in this group. O. felineus is larger in size than O. viverrini which, together with its excreted and secreted antigens, likely is crucial in the induction of liver fluke induced disease. The differences in nature and timing of the histopathological profile indicate that opisthorchiasis caused by the European liver fluke O. felineus is more pathogenic than its Asian relative O. viverrini.

Opisthorchis felineus and Metorchis bilis are the main agents of liver fluke infection of humans in Russia.

Liver fluke infections are gradually transforming from a local problem of individual geographic regions to a widespread problem. The observed expansion is likely to be connected with the ever-increasing intensity of traffic flow and migration of the infected carriers between cities, regions, and countries. Opisthorchis felineus, the trematode belonging to the family Opisthorchiidae, is a well known causative agent of the infection called opisthorchiasis. Metorchis bilis, also a member of the family Opisthorchiidae, causes metorchiasis, a disease very close to opisthorchiasis in symptomatology. Genetic markers can be used to develop methods for differential diagnostics of these diseases. However, the questions connected with epidemiology of these trematode infections, their clinical characteristics, prognosis and therapy remain open. This review briefs the general biological characteristics of O. felineus and M. bilis persisting in various countries of Eurasia, their geographical range, epidemiology and molecular diagnostics of these liver flukes.

Discovery of Opisthorchis lobatus (Trematoda: Opisthorchiidae): a new record of small liver flukes in the Greater Mekong Sub-region.

Metacercariae, morphologically similar to those of small liver flukes, were found to parasitize red-tailed snakehead fish, Channa limbata, collected from the city of Vientiane, Lao People's Democratic Republic. Adult worms that were recovered from experimentally-infected hamsters showed characteristics distinctly different from Opisthorchis viverrini, but closely similar to Opisthorchis lobatus, which was first reported in poultry (Anas sp.) from Pakistan. The present study aimed to redescribe O. lobatus based on the adult worms recovered from experimentally-infected hamsters. Additionally, it aimed to document the genetic relationships among O. lobatus and other opisthorchiid liver flukes using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and the internal transcribed spacer 2 (ITS2) region. DNA alignment of the O. lobatus and O. viverrini COI partial sequences (330 bp) showed 3.03% fixed differences (2.72% of amino acids changed) while the ITS2 region (350 bp) indicated a 0.86% difference for nucleotides. Species boundaries between the 2 parasites were determined by neighbor-joining analysis using the molecular sequence data. The phenogram confirmed that O. lobatus was distinctly different from O. viverrini, representing the first reported instance of O. lobatus in the Greater Mekong Sub-region (GMS) and the first record of C. limbata as the second intermediate host of a small liver fluke. Questions regarding human infection and the extent of the geographic distribution of these species should be investigated further.

Biological variation within Opisthorchis viverrini sensu lato in Thailand and Lao PDR.

Previous molecular genetic analyses indicate that Opisthorchis viverrini is a complex of at least 2 cryptic species in Thailand and the Lao People's Democratic Republic (PDR). Opisthorchis viverrini sensu lato (s.l.) populations can be divided into 6 genetic groups, which correlate with 5 different river wetlands systems. The present study examines the infectivity, growth, fecundity, and body size of O. viverrini s.l. isolates from these systems. Worm recovery was lowest in the Songkhram River, which differed significantly from all other wetland systems. In addition, the Chi River and Nam Ngum River populations also differed significantly. A similar pattern was found for fecundity. These results support the genetic/molecular data indicating a subdivision of O. viverrini populations on the basis of watershed and that propose the existence of cryptic species in Thailand and the Lao PDR.

Discovery of Opisthorchis viverrini metacercariae in freshwater fish in southern Cambodia.

Small liver flukes, Clonorchis sinensis and Opisthorchis viverini, are fish-borne trematodes (FBTs) causing significant public health problems in Asia. While C. sinensis is distributing mainly in far east Asia, O. viverini is distributing in Indochina peninsula. Recently, however, the geographical distributions of those small liver flukes were proven to be far wider than expected. Nevertheless, little is known about the epidemiology of small liver flukes in Cambodia. The present study is, therefore, aimed at clarifying the status of small liver fluke infections in various species of freshwater fish in southern Cambodia. A total of 1479 freshwater fish, 1316 (89%) comprised of 20 different species of the cyprinoid family and 163 (11%) belonging to 8 families of non-cyprinoids, were collected during May 2007 and February 2008, and the presence of small liver fluke metacercariae was examined by the compression method. Small liver fluke metacercariae were found in 10 species of cyprinoids with the infection rate ranging 2.1-66.7% and the mean intensity of infection of 1.0-15.0 (range: 1-65). For the speciation, adult worms were obtained by experimental infection in hamsters. The small liver fluke found in this study were identified as Opisthorchis viverrini by the morphological features of adult worms, and this identification was confirmed by partial COI sequencing of the metacercariae.

Opisthorchis felineus in cat: case report

Opisthorchis felineus foi encontrado em um gato admitido no Hospital Veterinário da Universidade de Trás-os-Montes e Alto Douro. No momento de admissão, o animal apresentava-se em estado de choque, com hipotermia, desidratação e mucosas pálidas. As análises hematológicas revelaram linfopenia, eosinofilia e anemia moderada. A necrópsia observou-se distensão da vesícula biliar e intensa infestação parasitária no fígado. O estudo morfológico do parasita revelou tratar-se de Opisthorchis felineus.

Taxonomy and biology of liver flukes.

Opisthorchis viverrini is a human liver fluke endemic in Thailand, Lao PDR and Cambodia. Three subspecies of Bithynia siamensis and 18 species of cyprinoid fish are susceptible first and second intermediate hosts, respectively. The differentiation between the adult worm of O. viverrini and that of the closely related species, Opisthorchis felineus and Clonorchis sinensis, is mainly based on the shape and position of the testes and the arrangement of the vitellaria. Eggs of these flukes are morphologically similar and problematic in identification. However, the differentiation between opisthorchid and lecithodendrid eggs is possible. Morphology and biology of each stage are comparatively reviewed. Biological aspects of snail and fish hosts are also included.

Study on the surface morphology of the developmental stages of the liver fluke, Opisthorchis viverrini (Trematoda: Opisthorchiidae).

The external morphology of some developmental stages of the fluke Opisthorchis viverrini (Trematoda: Opisthorchiidae), parasitizing humans in Southeast Asia was studied for the first time using electron microscopy. The surface structure of the egg, as well as the rediae, cercaria, metacercaria, and adult found in naturally infected hosts from Laos are described herein and their morphological characteristics discussed.

Opisthorchis viverrini: the relationship between egg production, worm size and intensity of infection in the hamster.

Worm recoveries, egg production and worm lengths were determined in golden Syrian hamsters given between five and 200 Opisthorchis viverrini metacercariae. Animals given five, 10 or 20 metacercariae showed similar worm recovery rates of 38%, 36% and 37%, respectively, whereas animals given 50, 100 or 200 metacercariae gave recoveries of 49%, 65% and 54%, respectively. The distribution of worms between right, left and middle liver lobes was similar for animals with between one and 43 worms but different for animals with between 64 and 154 worms. A curvilinear relationship between eggs found per gram of faeces per worm (epg/worm) and eggs/worm/24 hours and worm burden was observed at 60 days post-infection, with worm egg output decreasing as worm burden increased. Regression analysis of the logarithmically transformed data gave regression coefficients of r = 0.62 for log 10 epg/worm v. worm burden and r = 0.71 for log 10 eggs/worm/24 hours v. worm burden. When the same analyses were performed substituting worm density (worms/gram liver tissue) for worm burden, little or no difference was seen in the correlations obtained. A similar relationship was also observed between worm length and worm burden with mean worm length appearing to decrease with increase in worm burden. These results clearly show that worm crowding in the hamster host can have a profound effect on worm egg output and growth. The possible mechanisms underlying these phenomena are discussed and their potential relevance in human opisthorchiasis considered.

[Experimental research on the life cycle of Opisthorchis chabaudi n. sp. Description of the adult (author's transl)]. / Recherches expérimentales sur le cycle biologique d'Opisthorchis chabaudi n. sp. Description de l'adulte.

O. chabaudi n. sp. is experimentally obtained from cercariae, born into rediae and naturally produced by the snail Gabbia neumanni (Martens, 1898). Cercariae encyst into some tadpoles. The adulte grows into the biliary ducts of the Cat. O. chabaudi is mainly characterized by its outline, therefore by the shape and position of its reproductive systems. Accidental human contamination possibility is not denied.