Inflammatory responses to Opisthorchis viverrini infection in animal models: A comparison between susceptible and nonsusceptible hosts in different anatomical locations.

Background: Inflammation caused by Opisthorchis viverrini infection increases the risk of cholangitis, cholecystitis, and leads to bile duct cancer (cholangiocarcinoma or CCA). However, only certain infected individuals are susceptible to CCA, suggesting the involvement of host factors in cancer development. In addition, there are reports indicating differences in the locations of CCA. Aim: This study aims to investigate cellular inflammatory responses in the common bile duct (CB), intrahepatic bile duct (IHB), and gallbladder (GB) in susceptible and non-susceptible hosts following O. viverrini infection. Methods: Thirty Syrian golden hamsters (a susceptible host) and 30 BALB/c mice (a non-susceptible host) infected with O. viverrini were studied at six time points (five animals per group). Histopathological evaluations were conducted on samples from the IHB, CB, and GB. Inflammatory cell infiltration was quantitatively assessed and compared between groups and time points. Statistical analysis was performed using one-way ANOVA, with a significance level of p < 0.05. Results: Inflammation was significantly more pronounced in the IHB compared to the other two biliary locations. In comparison between susceptible and non-susceptible hosts, the intensity of inflammation was higher in the OV+H group than in the OV+M group (p < 0.05). Conclusion: This study highlights the association between host response to inflammation, tissue location, and host susceptibility, with the IHB showing particular susceptibility to inflammation and pathological changes. These findings contribute to our understanding of the increased risk of CCA in susceptible hosts.

Temperature dependence of Opisthorchis felineus infection in the first intermediate host snail, Bithynia troschelii.

Opisthorchiasis is one of the most serious trematodiases in Russia, where the world's largest focus is located in the Ob basin. Temperature is an important factor affecting the metabolism of cold-blooded animals. It determines the development of the causative agent of opisthorchiasis, Opisthorchis felineus, and the success of infection of an intermediate host, the snail Bithynia troschelii. In the present study, the effect of water temperature on the development of the liver fluke O. felineus in the host snail was assessed, as was the temperature threshold at which B. troschelii hibernation initiates. Adult uninfected B. troschelii individuals collected from natural bodies of water were infected with O. felineus and maintained at different temperatures of water (18-30 °C, intervals of 3 °C) in the laboratory. Each snail was fed with embryonated uterine eggs of O. felineus at 24 °C. O. felineus infection in snails was detected using polymerase chain reaction (PCR) using specific primers. The prevalence of O. felineus infection in B. troschelii depends on the water temperature in which the snails are maintained. The highest infection rate of 45.2 % ± 12.1 % was observed at 27 °C (p ≥ 0.1). The longest lifespan of infected and uninfected B. troschelii was recorded at water temperatures of 24 and 27 °C. The snails were more successfully infected at the beginning of the warm season. Among the infected individuals, the majority (up to 85 %) were large snails. Cercarial shedding was not detected in experimentally infected snails. Apparently, this is due to the natural physiological state of Bithynia snails during the autumn-winter diapause, when opisthorchiids development in snails stops. At 10 °C, complete hibernation of all B. troschelii snails was observed, and infection by the trematodes became impossible. The highest prevalence of infection was recorded at 27 °C, suggesting that during climate warming, an increase in opisthorchiid infection of snails may occur, which must be considered when epidemiological measures are planned.

Association between Opisthorchis viverrini infection in cats and humans: Non-spatial and spatial analyses.

Cats are recognized as significant reservoir hosts for human opisthorchiasis, particularly in areas with a high prevalence of infection. Despite this, the precise role of cats in the transmission of Opisthorchis viverrini between humans and felines remains unclear. This study investigates the association between these two hosts through both spatial and non-spatial analyses in the endemic Thanya sub-district of Thailand. A total of 105 owned cats were randomly sampled from 15 villages within the sub-district for stool examination. A questionnaire was administered to 66 cat owners to explore the human-pet relationship. Household locations were collected using GPS devices. Non-spatial analyses revealed a positive association between the two hosts (P= 0.011; OR 7, 95% CI: 1.6-30.9), highlighting two independent significant risk factors: cat owners consuming raw fish (P = 0.028; OR = 4.52, 95% CI: 1.25-19.45) and feeding cats raw fish (P = 0.011; OR = 16.41, 95% CI: 2.78-317.04) according to multivariate analysis. Spatial analysis provided further support to the non-spatial findings (p = 0.0123; OR = 3.45, 95% CI = 0.88-13.61). Multiple autologistic regression confirmed two significant risk factors: cat owners consuming raw fish (p = 0.054; OR = 3.37, 95% CI: 0.98-11.59) and feeding cats raw fish (p = 0.014; OR = 7.43, 95% CI: 1.49-37.05). Risk mapping identified the western part of the study site as a hotspot for O. viverrini infection. Hyper-endemic focusing revealed a union of human and cat buffers at 0.46 km², with an overlapping area of 0.22 km² (47.83%). This study underscores the impact of owners' behaviors, specifically consuming and feeding raw fish to cats, on the increased probability of infection in cats. It emphasizes the need for effective opisthorchiasis control through health education targeting cat owners in endemic areas.

Inflammatory cell responses in biliary mucosa during Opisthorchis viverrini infection: Insights into susceptibility differences among hosts.

Background: Individual host susceptibility is believed to be a risk factor in the interaction between the host and the parasite. Since studying time series in humans is limited, animal models are replaced. Aim: This study aims to explore and compare the pattern of inflammatory cell types along the biliary tract and their association with proliferative lesions in the early development of cholangiocarcinoma from susceptible and nonsusceptible animal models. Methods: Thirty male Syrian golden hamsters and 30 BALB/c mice, serving as the susceptible and nonsusceptible animal models, were used in this comparative study. The animals were infected with 50 Opisthorchis viverrini metacercariae via gastric intubation. At days 1, 2, 7, 14, 28, and 56 postinfection (p.i.), five animals were randomly selected from each group and humanely sacrificed. The hepatobiliary tissues were collected and processed for histopathological study. Histochemical and immunohistochemical staining were applied to differentiate the inflammatory cell types. Kruskal-Wallis and Mann-Whitney tests were applied to assess all semi-quantitative and quantitative variables. The correlation between each variable was also analyzed using Spearman rank at a p-value < 0.05. Results: The results demonstrated that mice had different patterns of infiltrating cell types when compared to hamsters. This suggested that the cellular response to the infection in mice occurred earlier than that in hamsters. The response in mice reached its peak at D7 to D14 and then rapidly declined at D28. In contrast, although the inflammatory response in hamsters started slowly, the response reached the peak at D28 and maintained a high level until D56. Significant differences in the number of inflammatory cells between mice and hamsters were seen at D1 (p = 0.047), D7 (p = 0.049), D28 (p = 0.040), and D56 (p < 0.040). Conclusion: The inflammatory responses to O. viverrini infection in the nonsusceptible animal model occurred and declined earlier while the response in the susceptible animal model occurred later in a gradual manner. Both rodents are suitable animal models for the studies of opisthorchiasis susceptibility.

Microsatellite markers for Opisthorchis felineus to understand its genetic diversity and transmission patterns of opisthorchiosis.

Opisthorchis felineus is a food-borne trematode which causes opisthorchiosis and affects mainly the liver and bile ducts of the liver with a possible risk of bile duct carcinogenesis resulting in cholangiocarcinoma. In Russia, O. felineus is mainly endemic in Western Siberia (Ob and Irtysh river basins) and occurs throughout the Volga, Kama, Don, and Dnepr river basins. The prevalence, intensity, and clinical significance of human infections and the incidence of cholangiocarcinoma vary geographically in endemic regions. Currently, there is substantial evidence on genetic variation of O. felineus, but information on the population genetic structure is so far very scarce. Because microsatellite DNA of this parasite is not available, we for the first time isolated sufficient microsatellite loci to examine the genetic diversity and population structure of O. felineus, using multiple nuclear loci approach. A total of ten highly polymorphic microsatellite loci from a constructed enriched genomic DNA library were characterized, using 29 samples representing huge O. felineus metapopulation extended in latitude over 5000 km from Middle Europe to Western Siberia. At least three populations can be discerned as result of analysis of the microsatellite loci genetic diversity. Based on the results for the first time, a hypothesis was put forward about the formation of a modern habitat of O. felineus.

Roaming behavior of the owned domestic cats (Felis catus) with possible roles in the transmission of Opisthorchis viverrini in the endemic area in Khon Kaen, Thailand.

In the endemic area of the liver fluke (Opisthorchis viverrini) infection, effective control of opisthorchiasis using One Health approaches requires transmission interruption in all hosts in the parasite life cycle. Owned domestic cats (Felis catus) are considered an important species among mammalian reservoirs. Similar to humans, household cats become infected by eating raw, contaminated fish and spread the O. viverrini eggs to the environment by defecation. In rural areas, cat owners usually feed their cats with human food or food waste, especially fish dishes. There is a high risk for cats to become infected and deposit eggs in the peri-household environment. The lack of research on domestic cats led us to study their roaming behavior, roaming range, habitat preference and risk of disease transmission. GPS loggers were harnessed to 28 domestic cats in 7 villages around Lawa Lake, the endemic area of human opisthorchiasis. All cats were examined for the O. viverrini infection status using modified formalin-ether concentration technique (MFECT) before application of the GPS device used to observe and record their roaming distance and range area. Then, composition analysis for habitat selection was performed using the location data. Additionally, cat owners were interviewed using participatory epidemiologic methods for their perceptions and knowledge about the reservoir hosts of this trematode. Our study suggested that the prevalence of O. viverrini in cats in this area has not declined appreciably in comparison with our previous study four years ago, although the treatment and control has been applied intensively in humans. The longest distance that individual cats roamed ranged from 51 to 363 meters (mean±SD = 154.48±90.71). The cats traveled tracks around their home. There was no significant difference in the average distance that cats ranged between O. viverrini infected group (154.13±72.80 meters) and the non-infected group (154.67±101.39 meters) was found. Compositional analysis suggested that the domestic cats preferred urban habitats where food and safety were probably more available. It appears that the main transmission route for infection of cats was contaminated fish fed to cats or fish that remained in their houses. Therefore, owners should refrain from feeding their cats potentially contaminated foods, food scraps and raw fish remains and prevent their cats from accessing them. Moreover, the participatory epidemiology results suggest that awareness of villagers is not enough for the effective and sustainable control of the disease.

Molecular genetic species definition of Opisthorchiidae-like cercariae from Bithyniidae snails of Chany Lake (South-Western Siberia, Russia).

The studies of opisthorchiids larval stages associated with Bithyniidae snails can provide important and the most reliable data for opisthorchiidoses foci characterization due to the low mobility of such opisthorchiid's host species as Bithyniidae snails. The foci of opisthorchiosis (caused by Opisthorchis felineus) and metorchiosis (caused by Metorchis bilis) are overlapping in the basins of the Ob and Irtysh Rivers. Thus, difficulties with determining the species of cercariae significantly reduce the accuracy of epidemiological conclusions regarding opisthorchiosis, which has a much higher medical significance. Moreover, M. bilis cercariae identification is complicated by the fact that the focus of metorchiosis caused by Metorchis xanthosomus (infecting fish-eating predatory birds) occurs on the same territory. In this study, we for the first time carried out the molecular genetic identification of West-Siberian opisthorchiid cercariae to verify morphological identification and confirmed O. felineus's main association with Bithynia troschelii snail and M. bilis'-with B. tentaculata snail. Thus, our study applied a two-stage approach: the morphological identification of any opisthorchiid-like cercariae in Bithynia snails and subsequent molecular genetic precise species determination of cercariae in the reduced samples.

Susceptibility patterns of Bithynia siamensis siamensis and Bithynia funiculata to Opisthorchis viverrini infection: an indication of the risk of opisthorchiasis transmission in non-endemic areas.

Among the snail species acting as hosts for medically significant trematodes, only three taxa of Bithynia are responsible for transmitting the carcinogenic liver fluke Opisthorchis viverrini to humans in different geographical areas. Although B. siamensis goniomphalos is the primary species responsible for O. viverrini transmission in endemic areas, B. siamensis siamensis and B. funiculata remain potential hosts for transmission. This study objects to determine the susceptibility of B. siamensis siamensis and B. funiculata to O. viverrini to assess the risk of O. viverrini transmission in non-endemic areas. The snails of both species were first introduced to O. viverrini eggs, after which O. viverrini infection was investigated using specific PCR primers after a period of 1, 7, 14, 28, and 56 days post-infection (dpi). Opisthorchis viverrini infection in both B. siamensis siamensis and B. funiculata was high in the early period (1 and 7 dpi) while decreasing over time. It was also shown that the odds of susceptibility to O. viverrini infection in B. siamensis siamensis were 64.5% higher relative to the odds of susceptibility in B. funiculata (P < 0.05). Results of this study provide an early insight into the Bithynia-Opisthorchis relationship and thus have great potential to assess risk and raise awareness of opisthorchiasis in non-endemic regions, especially in regions endemic for B. siamensis siamensis.

Does Opisthorchis viverrini circulate between humans and domestic cats in an endemic area in Thailand?

The liver fluke Opisthorchis viverrini is a foodborne trematode that, in chronic infection, is a leading cause of bile-duct cancer ­ cholangiocarcinoma. Cats and dogs are acknowledged as reservoir hosts of this parasite. However, this assumption is based on morphological similarity of flukes recovered from these hosts, without any molecular genetic evidence. The aim of this study was to obtain molecular data from O. viverrini eggs present in feces of humans and cats in the same locality in Thanya sub-district, Kalasin, Thailand. The mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was used as the marker for a population-genetic study. A DNA fragment of the cox1 gene was amplified from stool samples and subjected to nucleotide sequencing. Phylogenetic and haplotype network analyses were performed. The cox1 sequences of O. viverrini eggs from humans and cats largely formed separate clades on the phylogenetic trees, with an Fst value of 0.64 (P < 0.05), indicating largely distinct populations in the 2 species. However, 5 samples from cats were placed in the human cluster and 1 sample from a human was placed in the cat cluster. This suggests that host specificity of 'human' and 'cat' clades is not absolute. These results indicate that there are 2 populations of O. viverrini, one circulates primarily in humans and the other in cats. However, cross-transmission can occur between these 2 hosts. Taken altogether, the population-genetic evidence from this study partially supports the assumption that the cat can act as a reservoir host of O. viverrini.

Similarities and differences among the Opisthorchiidae liver flukes: insights from Opisthorchis felineus.

The foodborne liver trematode Opisthorchis felineus (Rivolta, 1884) is a member of the triad of phylogenetically related epidemiologically important Opisthorchiidae trematodes, which also includes O. viverrini (Poirier, 1886) and Clonorchis sinensis (Loos, 1907). Despite similarity in the life cycle, Opisthorchiidae liver flukes also have marked differences. Two species (O. viverrini and C. sinensis) are recognized as Group 1A biological carcinogens, whereas O. felineus belongs to Group 3A. In this review, we focus on these questions: Are there actual differences in carcinogenicity among these 3 liver fluke species? Is there an explanation for these differences? We provide a recent update of our knowledge on the liver fluke O. felineus and highlight its differences from O. viverrini and C. sinensis. In particular, we concentrate on differences in the climate of endemic areas, characteristics of the life cycle, the range of intermediate hosts, genomic and transcriptomic features of the pathogens, and clinical symptoms and morbidity of the infections in humans. The discussion of these questions can stimulate new developments in comparative studies on the pathogenicity of liver flukes and should help to identify species-specific features of opisthorchiasis and clonorchiasis pathogenesis.

Infection rate of Opisthorchis viverrini metacercariae in cyprinoid fish from the markets and its association to human opisthorchiasis in the local community in the Northeast Thailand.

The prevalence of Opisthorchis viverrini, a fish-borne zoonotic trematode that can provoke cholangiocarcinoma, is high in the Northeast Thailand. The aim of this study was to determine the prevalence of O. viverrini metacercariae in the cyprinid fish and determine its association of O. viverrini infection among the consumers who regularly buy fish in the markets. A cross-sectional study was conducted in nine provinces covering 20 districts of Thailand, and we examined 778 cyprinoid fish specimens belonging to five species purchased from local markets. Pepsin-HCl digestion method was used to recover O. viverrini metacercariae from fish. In all districts surveyed, O. viverrini metacercariae-positive fish were found with the infection rates ranging from 3.9 to 21.1%. All five fish species studied were positive for O. viverrini metacercariae: Henicorhynchus siamensis (13.7%), Cyclocheilichtys spp. (12.7%), Hampala spp. (8.1%), Systomus spp. (6.9%) and Barbonymus goniatus (5.0%). An average prevalence of O. viverrini infection was 7.1% in the fish consumers surveyed in the markets. The source of fish was determined and our results showed that parasitized fish are sold in markets up to 100 km away from the point of capture, which contributes to the dispersion and maintenance of this helminthiasis. Our results point to the transmission of liver flukes via markets, in spite of many active programs of health education, elimination, prevention and control aimed to reduce O. viverrini infection and subsequent cholangiocarcinoma in the endemic areas of Thailand.

Opisthorchis viverrini metacercarial infection in cyprinid fish in Nakhon Phanom Province, Northeastern Thailand.

Opisthorchiasis is endemic in parts of Southeast Asia, including the northern and northeastern regions of Thailand. In these regions, the transmission by fish intermediate hosts has received little attention. We investigated the intensity of Opisthorchis viverrini metacercariae (OV MC) in wild cyprinid fishes from five districts within the Nakhon Phanom Province, Northeast Thailand. Fishes were procured from local markets in five districts throughout three different seasons (hot, rainy and cold) between February 2018 and January 2019. The samples were identified, counted and weighed before metacercariae detection was performed via the artificial digestion method. A total of 2,149 freshwater fishes, representing 20 species were collected. The fish most commonly contaminated with OV MC were Anematichthys repasson and Hampala dispar. The intensity of OV MC in Nakhon Phanom was 0.23 OV MC/fish and varied among districts, ranging from 0.07 to 0.52. A low intensity of OV MC/fish (defined as <=1 cyst) was found in all three different seasons in the Nakhon Phanom Province; hot season (0.55), cold season (0.22) and the rainy season (0.13). The intensity of OV MC/fish was moderate (defined as >1 cyst) in the Renu Nakhon district (2.5) in the hot season. By fish species, H. dispar yielded the highest, with a moderate intensity of 2.1. In natural freshwater cyprinid fish in Nakhon Phanom, OV MC infection is endemic with intensity rates varying according to district, season and fish species.

A MULTIPLEX PCR ASSAY FOR THE DIFFERENTIAL DETECTION OF OPISTHORCHIS FELINEUS AND METORCHIS BILIS.

Opisthorchis felineus and Metorchis bilis are two common small worms that parasitize in the gallbladder and bile ducts of the liver of humans and carnivores. These parasites have a severe impact on health and are considered pathogens of serious diseases worldwide, such as cholangiocarcinoma. However, there are still no commercially available molecular diagnostic kits capable of simultaneously detecting these parasites in humans. Therefore, the study aimed to develop a multiplex PCR analysis that will differentially determine these two opisthorchiasis infections in one reaction. Two specific primer pairs for a multiplex polymerase chain reaction (PCR) were designed based on corresponding mitochondrial genome sequences. The multiplex assay detection limit was assessed by serial dilutions of the genomic DNAs of trematode worms examined. Naturally, infected samples of human bile and feces were tested using the developed assay. A multiplex PCR assay was developed based on mitochondrial DNA that accurately and simultaneously identifies two trematode species in one reaction using specific fragment sizes of 307 and 252 bp for O. felineus and M. bilis, respectively. The optimal reaction conditions, specificity, and sensitivity of the multiplex PCR assay were investigated. The lowest DNA concentration detected was 100 pg for M. bilis and O. felineus in a 25µl reaction system. This study provides an efficient tool for the simultaneous detection of O. felineus and M. bilis. The proposed multiplex PCR assay will be potentially useful in epidemiological studies, diagnosis, and treatment of this mixed opisthorchiasis infection.

Effects of Three-time Administration of a Supramolecular Complex of Praziquantel with Disodium Glycyrrhizinate on Trematode Opisthorchis felineus in Hamsters.

BACKGROUND: Praziquantel (PZQ) is the most commonly used anthelmintic drug for treating trematodiases. It was shown here that PZQ in complex with disodium glycyrrhizinate (PZQ-Na2GA, in the 1:10 ratio) has higher bioavailability than PZQ alone. Our aim was to determine the effects of three-time administration of PZQ-Na2GA in an experimental opisthorchiasis felinea model. METHODS: The PZQ-Na2GA complex (1:10) at a 400 mg/kg dose (meaning 36.4 mg/kg PZQ) was administered to Opisthorchis felineus-infected hamsters three times under a "9:00 am-6:00 pm-9:00 am" regimen (PZQ-Na2GA × 3). Effects of treatment were assessed as a reduction of helminth load in the hamsters and as changes in physiological, hematological, and blood biochemical parameters. The helminths extracted from the liver of the hamsters that received PZQ-Na2GA thrice were assayed for sensitivity to PZQ in vitro. RESULTS: PZQ-Na2GA × 3 reduced the number of O. felineus helminths in the liver by 87%, which is 30% better than a previously reported effect of one-time administration of the complex. Meanwhile, relative weights of the liver and thymus diminished, and some hematological parameters improved. The helminths extracted from the hamsters 1 month after the PZQ-Na2GA × 3 treatment showed elevated sensitivity to PZQ, as determined in vitro. CONCLUSION: Compared with previously published data on the effectiveness of various drugs in experimental opisthorchiasis felinea, PZQ-Na2GA × 3 exerts the most potent anthelmintic effect. In addition, PZQ-Na2GA × 3 improves physiological status of O. felineus-infected hamsters and sensitizes the surviving parasites to subsequent PZQ treatment.

Infection Dynamics of Opisthorchis viverrini Metacercariae in Cyprinid Fishes from Two Endemic Areas in Thailand and Lao PDR.

The infection dynamics of Opisthorchis viverrini metacercariae was analyzed in cyprinid fish from endemic areas in Mukdahan Province, Thailand, and Khammouane Province, Lao PDR. The fish were collected during the cool-dry (November-February), hot-dry (March-May), and rainy (June-October) seasons in 2017. They were examined by the digestion method, and the infection status was statistically analyzed by study area, season, and fish size. The prevalence (no. of fish positive/no. of fish examined × 100) and metacercarial intensities (no. of metacercariae detected/no. of fish positive) of O. viverrini in both study areas depended on season, being high in the cool-dry season and varying in the hot-dry and rainy seasons. In Mukdahan Province, the average prevalence was 18.3% (range 11.0-46.7%, n = 420) and the intensity was 4.07 ± 5.86 cysts/fish (mean ± SD), whereas in Khammouane Province, the prevalence was 51.9% (range 9.1-70.6%, n = 673) and the intensity was 6.67 ± 12.88 cysts/fish. Among the cyprinid fish species examined, the infection was associated with fish body size and predominantly found in Hampala dispar (86.5%), Cyclocheilichthys armatus (73.2%), and Puntius brevis (42.7%). The distribution of O. viverrini metacercariae in fish was skewed, with most of the fish having a low worm burden with an average of four to six cysts/fish. The findings that seasonality, sampling locality, fish size, and species of fish play roles in the risk of O. viverrini infection imply that these host and environmental factors are important for the transmission dynamics and control of O. viverrini.

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.

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.

Effects of aestivation on survival of Bithynia siamensis goniomphalos snails and the infection of Opisthorchis viverrini in the irrigation area of wet- and dry-season rice paddy.

The snail Bithynia siamensis goniomphalos acts as the first intermediate host of the human liver fluke, Opisthorchis viverrini, a major cause of cholangiocarcinoma in Northeast Thailand. In this study, we investigated host-parasite interactions in wet- and dry-season rice paddy plantation. The snails that aestivated through 4-month-dried period after rice harvest had average mortality rate of 36.45% and monthly increasing from 17.76% to 54.57%. Surprisingly, at depths greater than 5 cm experienced a higher mortality rate than upper level. Average mortality rate at the depth more than 5 cm, was 42.97%. In the initial month of aestivation, mortality rate of 0-5 and 5-10 cm depth was 9.13% and 26.39% then increase to 57.58% and 51.97%, respectively in the last 4th month. The average prevalence O. viverrini infection in snails during the study period was 0.44%. The highest prevalence of O. viverrini infection was found in the cool dry and wet season. The odds of O. viverrini infection in female snails and large snails were higher relative to male snails and medium-sized snails. The physicochemical factors involved in increased prevalence of O. viverrini infection in snail hosts were mean daily air temperature and average monthly rainfall. Short aestivation period made lower mortality in irrigation area than the previous report of non-irrigation area, that is, the aestivation is one of snail population control.

Biliary cystadenoma associated with Opisthorchis viverrini infection in a domestic cat (Felis catus).

A 12-year-old, female, domestic cat (Felis catus) presented with dehydration, emaciation, anorexia, and lethargy. The cat was unresponsive to medical treatment and euthanized; the carcass was submitted for pathological diagnosis. Necropsy revealed icteric mucous membranes. The liver was enlarged, with multinodular, cystic, white masses, 0.5-4.0 cm in diameter, scattered throughout. Microscopically, the biliary epithelium presented with a proliferation of multifocal cystic masses, occasionally with periodic acid-Schiff-positive fluid within the cysts. Simple cuboidal epithelial cells showed small, round to oval, vesicular nuclei and rare mitotic figures. There were also multifocal trematode-like parasites situated within the biliary tracts. Immunohistochemistry of the cystic masses was positive for pan-cytokeratin and proliferating cell nuclear antigen, while negative for vimentin. Molecular analysis and gene sequencing of the parasite indicated that it was Opisthorchis viverrini. Based on the pathological findings and molecular analysis, the cat was diagnosed with biliary cystadenoma related to O. viverrini infection. This report described an unusual case of O. viverrini infection associated with biliary tumor in a cat, and raises the possibility of domestic cats as a reservoir host of the human liver fluke.

Effects of miconazole/clotrimazole and praziquantel combinations against the liver fluke Opisthorchis felineus in vivo and in vitro.

The liver fluke Opisthorchis felineus (Rivolta, 1884) is the causative agent of opisthorchiasis felinea in Eurasia. Opisthorchiasis is a serious human and fish-eating animal's disease affecting bile ducts and the gall bladder. Currently, the main drug for specific therapy of opisthorchiasis is praziquantel. We have previously shown that azole inhibitors of O. felineus cytochrome P450 significantly reduced survival of the worms in vitro. Here, we studied in vitro anthelmintic effects of drug combinations involving azole substances approved by the US Food and Drug Administration together with praziquantel against adult or juvenile O. felineus liver flukes. A synergistic interaction was shown for praziquantel-clotrimazole (CI = 0.68) combination and for praziquantel-miconazole (CI = 0.68) combination against adult helminths in vitro. Praziquantel-miconazole (CI = 0.30) had a strongly synergistic effect against newly excysted metacercariae. We also tested anthelmintic effects of azole substances and their combinations with praziquantel in vivo in an animal model of chemotherapy. The treatment of juvenile worms (1 day postinfection) with 100 mg/kg miconazole resulted in a worm burden reduction (WBR) of 37.5% (P = 0.049), with 100 mg/kg clotrimazole causing a WBR of 31.25% (P = 0.025). The treatment of adult worms (5-6 weeks postinfection) with 100 mg/kg or 200 mg/kg miconazole yielded a WBR of 23.8% (P = 0.01) and 21.4% (P = 0.006), respectively. When praziquantel was administered together with clotrimazole or with miconazole, a WBR slightly greater than the effect of ED50 praziquantel was observed (WBR of 59.5 and 54.7%, respectively).In conclusion, the synergistic effect of the praziquantel-clotrimazole and praziquantel-miconazole combinations observed in vitro was not confirmed in vivo. Thus, this combination chemotherapy revealed no benefits over praziquantel monotherapy in the treatment of opisthorchiasis felinea.