Behaviour and eating habits as determinants for human opisthorchiasis in the Bolsena Lake area, Italy.

In Italy, food-borne outbreaks of opisthorchiasis have occurred since 2003, all of them originating from some lakes in Central Italy where the only European liver fluke, Opisthorchis felineus (Rivolta, 1884) is endemic. The aim of the present study is to investigate the factors that may facilitate the life cycle of O. felineus in the area of Bolsena Lake, the knowledge of the local population about opisthorchiasis, and preventative healthcare measures. We performed a descriptive observational study through the submission of questionnaires to three target populations: fishermen who fish exclusively in Bolsena Lake, restaurateurs whose businesses are close to the lake and general population living in the province of Viterbo. Results showed hazardous behaviours and eating habits in the three target populations, as well as a poor knowledge of opisthorchiasis and preventative healthcare measures.

Modeling impacts of climate change on the potential distribution of the carcinogenic liver fluke, Opisthorchis viverrini, in Thailand.

Global climate change is now regarded as imposing a significant threat of enhancing transmission of parasitic diseases. Maximum entropy species distribution modeling (MaxEnt) was used to explore how projected climate change could affect the potential distribution of the carcinogenic liver fluke, Opisthorchis viverrini, in Thailand. A range of climate variables was used: the Hadley Global Environment Model 2-Earth System (HadGEM2-ES) climate change model and also the IPCC scenarios A2a for 2050 and 2070. Occurrence data from surveys conducted in 2009 and 2014 were obtained from the Department of Disease Control, Ministry of Public Health, Thailand. The MaxEnt model performed better than random for O. viverrini with training AUC values greater than 0.8 under current and future climatic conditions. The current distribution of O. viverrini is significantly affected by precipitation and minimum temperature. According to current conditions, parts of Thailand climatically suitable for O. viverrini are mostly in the northeast and north, but the parasite is largely absent from southern Thailand. Under future climate change scenarios, the distribution of O. viverrini in 2050 should be significantly affected by precipitation, maximum temperature, and mean temperature of the wettest quarter, whereas in 2070, significant factors are likely to be precipitation during the coldest quarter, maximum, and minimum temperatures. Maps of predicted future distribution revealed a drastic decrease in presence of O. viverrini in the northeast region. The information gained from this study should be a useful reference for implementing long-term prevention and control strategies for O. viverrini in Thailand.

Immunization and challenge shown by hamsters infected with Opisthorchis viverrini following exposure to gamma-irradiated metacercariae of this carcinogenic liver fluke.

Here we report findings to optimize and standardize conditions to attenuate metacercariae of Opisthorchis viverrini by ionizing radiation to elicit protective immune responses to challenge infection. Metacercariae were gamma-irradiated and the ability of irradiated metacercariae to prevent patent infection of challenge metacercariae in hamsters was determined, as well as their ability to induce a host antibody response. Metacercariae irradiated in a dose-dependent manner, with 3, 5, 10, 12, 20, 25 and 50 Gray, were used to infect Syrian golden hamsters by stomach gavage to ascertain the effect of irradiation on ability of the worms to establish infection. In addition, other hamsters were infected with metacercariae irradiated with 20-50 Gray, followed by challenge with intact/wild-type (non-irradiated) metacercariae to determine the protective effect as established by the numbers of adult flukes, eggs of O. viverrini in hamster faeces and anti-O. viverrini antibody titres. Significantly fewer worms were recovered from hamsters immunized with metacercariae irradiated at 20, 25 and 50 Gray than from control hamsters infected with intact metacercariae or 0 Gray, and the worms showed damaged reproductive organs. Faecal egg numbers were decreased significantly in hamsters immunized with 25 and 50 Gray metacercariae of O. viverrini. Moreover, hamsters administered metacercariae that were protected elicited a robust, specific anti-fluke immunoglobulin G response compared to control hamsters, suggesting a role for antibody in protection elicited by radiation-attenuated metacercariae.

Anthelmintic, anti-inflammatory and antioxidant effects of Garcinia mangostana extract in hamster opisthorchiasis.

Administration of praziquantel for treatment of liver fluke infection may affect the host, with mild and severe effects after treatment caused by host immune response. Therefore, we focused on the antioxidant property, inflammatory and anthelmintic effects of the traditional folk medicine, G. mangostana pericarp extract, in hamster opisthorchiasis. Syrian hamsters were divided into four groups: normal (control) (N); administered G. mangostana alone (GM); infected with Opisthorchis viverrini alone (OV); and infected with O. viverrini and administered G. mangostana extract for 1.5 months (OVGM). Hamster livers were collected 45 days after infection to determine histopathological changes, i.e. aggregation of inflammatory cells. The morphology of adult O. viverrini (body size and sizes of reproductive organs) was analyzed, as well as worm burden, eggs per worm and eggs per gram of feces. Toxicity was tested by kidney function (blood urea nitrogen and creatinine); the results demonstrated that G. mangostana had no renal toxic effect. ABTS radical-scavenging assay indicated that the extract had antioxidant property. Reduction in aggregation of inflammatory cells surrounding the hepatic bile duct, especially at the hilar region, was found in the OVGM group. Worm burden was similar in both infected groups (treated or untreated with G. mangostana), but the average size of adults in the OV group was larger than in the OVGM group; moreover, eggs per worm and eggs per gram of feces were also comparatively higher. The present study suggests that G. mangostana extract possesses anti-inflammatory and antioxidant properties and can interfere with parasite development by affecting adult size and egg production. This may be useful for controlling the spread of OV infection and other parasites in endemic areas.

[A man in his thirties with icterus and itching]. / En mann i 30-årene med icterus og kløe.

BACKGROUND: We present a patient from South-East Asia who has been living in Norway for five years. He was referred to our department with jaundice caused by opisthorchiasis/clonorchiasis. This reason for jaundice is highly unusual in Europe. Worldwide, however about 35,000,000 people are infected. CASE PRESENTATION: A male in his thirties, originally from South-East Asia, experienced diffuse itching for five weeks and painless jaundice for two weeks. Blood samples showed increasing cholestasis. Abdominal ultrasound, MRCP and computed tomography showed no sign of bile duct obstruction or liver tumour. Serological tests and liver biopsy revealed no infectious or autoimmune liver disease. ERCP showed normal bile ducts, but large quantities of typical hookworms in the duodenum. Stool samples showed at least one egg typical of Opisthorchis/Clonorchis and a large quantity of hookworm eggs. We interpreted these findings as parasite-induced obstruction of some of the small bile ducts. The patient recovered completely after treatment with Praziquantel. INTERPRETATION: Jaundice due to bile duct obstruction by opisthorchiasis/clonorchiasis is a well-known problem in South-East Asia. It may become more common in Europe as well as a result of increasing migration. Treatment with Praziquantel is simple and effective.

Alcohol and alkalosis enhance excystation of Opisthorchis viverrini metacercariae.

The northeastern region of Thailand has long been known as an endemic area of the human liver fluke infection which is caused by Opisthorchis viverrini. Humans are infected by ingestion of uncooked cyprinoid fish in traditional dishes such as "koi-pla," "pla-som," "pla-jom," and "pla-ra." To date, the prevalence of this parasite infection remains high because of cultural behavior and local beliefs. The popular misunderstanding among people in this area is that alcohol, lemon juice, and fish sauce can kill the parasites. Thus, they believe that they can eat raw fish without the risk of infection. This study attempts to clarify the effects of ethyl alcohol and acidosis-alkalosis on O. viverrini metacercariae excystation. Metacercariae of O. viverrini were obtained from infected cyprinoid fish in a natural reservoir. Most metacercariae were obtained from small cyprinoid fish. Metacercariae were divided into three experimental groups and were treated with solutions containing four different concentrations of ethyl alcohol, four different concentrations of salt, and a range of acidic/basic pH. Metacercariae excystation was observed at the assigned times, and the data were then analyzed. Salt had no effect on excystation. Interestingly, the optimal conditions for O. viverrini excystation were pH 9 and 25 % ethyl alcohol. The present study suggests that raw fish should not be eaten while drinking alcohol or when consuming other ingredients with pH 9, because both alcohol and pH 9 could induce O. viverrini metacercariae excystation, leading to the early development of parasites in the hepatobiliary system.

Curcumin prevents bile canalicular alterations in the liver of hamsters infected with Opisthorchis viverrini.

Opisthorchis viverrini infection causes inflammation and liver injury leading to periductal fibrosis. Little is known about the pathological alterations in bile canaliculi in opisthorchiasis. This study aimed to investigate bile canalicular alterations in O. viverrini-infected hamsters and to examine the chemopreventive effects of curcumin on such changes. Hamsters were infected with O. viverrini and one group of animals was fed with 1% dietary curcumin supplement. Animals were examined during the acute infection phase, days 21 and 30 post-infection (PI) and chronic infection phase (day 90 PI). Scanning electron microscopy revealed that in the infected group fed with a normal diet, bile canaliculi became slightly tortuous by 30 day PI and more tortuous at day 90 PI. Transmission electron microscopy showed a reduction in microvilli density of canaliculi starting at day 30 PI, with a marked loss of microvilli at day 90 PI. These ultrastructral changes were slightly seen at day 21 PI, which was similar to that found in infected animals fed with 1% curcumin-supplemented diet. Notably, curcumin treatment prevented the reduction of microvilli density, reduced the dilation of bile canaliculi, and decreased the tortuosity of the bile canaliculi relative to non-infected animals on a normal diet at days 30 and 90 PI. These results suggest that curcumin reduces alteration of bile canaliculi and may be a promising agent to prevent the onset of bile duct abnormalities induced by O. viverrini infection.

Light and electron microscopy observations of embryogenesis and egg development in the human liver fluke, Opisthorchis viverrini (Platyhelminthes, Digenea).

Eggs of most species digenean flukes hatch in the external environment to liberate larvae that seek and penetrate a snail intermediate host. Those of the human liver flukes, Opisthorchis viverrini, hatch within the gastrointestinal canal of their snail hosts. While adult parasites are primarily responsible for the pathology in cases of human opisthorchiasis, their eggs also contribute by inducing granulomata and in serving as nidi for gallstone formation. In view of the peculiar biology of O. viverrini eggs and their contribution to pathology, we investigated embryogenesis in this species by light and transmission electron microscopy. Egg development was traced from earliest stages of coalescence in the ootype until full embryonation in the distal region of the uterus. Fully mature eggs were generally impermeable to resin and could not be examined by conventional electron microscopy methods. However, the use of high-pressure freezing and freeze-substitution fixation of previously fixed eggs enabled the internal structure of mature eggs, particularly the subshell envelopes, to be elucidated. Fertilization occurs in the ootype, and the large zygote is seen therein with a single spermatozoon wrapped around its plasma membrane. As the zygote begins to divide, the spent vitellocytes are pushed to the periphery of the eggs, where they progressively degrade. The early eggshell is formed in the ootype by coalescing eggshell precursor material released by approximately six vitelline cells. The early eggs have a thinner eggshell and are larger than, but lack the characteristic shape of, mature eggs. Characteristic shell ornamentation, the "muskmelon" appearance of eggs, appears after eggshell polymerization in the ootype. Pores are not present in the shell of O. viverrini eggs. The inner and outer envelopes are poorly formed in this species, with the outer envelope evident beneath the eggshell at the opercular pole of the mature egg. The miracidium has a conical anterior end that lacks the distinctive lamellar appearance of the terebratorium of other digeneans, such as the schistosomes. The miracidium is richly glandular, containing an apical gland in the anterior end, large cephalic gland, and posterior secretory glands. Each gland contains a secretory product with different structure. The paucity of vitelline cells associating with eggs, the reduced size of eggs, and reduced complexity of the extraembryonic envelopes are interpreted as adaptations to the peculiar hatching biology of the miracidia.

Anti-inflammatory effect of prednisolone on the growth of human liver fluke in experimental opisthorchiasis.

Opisthorchis viverrini is one of the risk factors for cholangiocarcinoma (CCA) development and is endemic in Southeast Asia including Thailand. CCA is induced by chronic inflammation from a combination of mechanical damage, parasite secretions, and immunopathology. Chronic infection with O. viverrini has been associated with several hepatobiliary diseases which affect the development of hepatobiliary cancer and CCA. Therefore, reducing the pathogenesis from O. viverrini infection may be one of the choices to reduce the risk of cholangiocarcinoma development. Prednisolone is one of the steroidal anti-inflammatory drugs that suppress inflammation, and its use has risen continuously in recent years. We therefore investigated the effect of prednisolone on pathological changes in Syrian hamster opisthorchiasis, in terms of gross and histopathological changes, worm size, eggs per gram, eggs per worm, and immunohistochemical staining for COX2. Syrian hamsters were divided into three groups: uninfected control; O. viverrini-infected (OV); and O. viverrini-infected plus prednisolone administration (OVP). The results showed an anti-inflammatory effect in the OVP group by a reduction of the inflammatory cells surrounding the intrahepatic bile ducts. However, in addition, parasite sizes for all times of observation were larger than for other groups, which was also correlated with increased eggs per worm and eggs per gram of feces. This result suggests that prednisolone is useful in suppressing inflammation in Syrian hamster opisthorchiasis, whereas it was also beneficial for parasites by enhancing their reproductive development. To clarify the mechanism of this phenomenon, further studies are under investigation.

[The strategy of a parasite is to preserve and continue its species; some trends for achieving the goal].

The authors show that the strategy of parasites, which is to preserve and continue its species is accomplished mainly by providing of a trophic substrate. Opisthorichiasis is used as an example to show that the nutriceutic biomass of cholangiocytes may be increased due to gene mutations, induced proliferation of liver stem cells, their differentiation to committed ones and cholangiocellular differon elements; moreover, the proliferative processes of mesenchymal components become active in other organs of a host. During their ontogenesis in the intermediate and final hosts, the parasites work out the mechanisms for prolonging their life span to complete a full development cycle (Margaritifera margaritifera glochidia); however, predominantly the parasite-host symbiosis is attended by the latter's lameness. Predation is one of the types of symbiotic relations.

Expression, refolding and purification of Ov-GRN-1, a granulin-like growth factor from the carcinogenic liver fluke, that causes proliferation of mammalian host cells.

Granulins (GRNs) are potent growth factors that are upregulated in many aggressive cancers from a wide range of organs. GRNs form tight, disulphide bonded, beta hairpin stacks, making them difficult to express in recombinant form. We recently described Ov-GRN-1, a GRN family member secreted by the carcinogenic liver fluke of humans, Opisthorchis viverrini, and showed that recombinant Ov-GRN-1 expressed and refolded from Escherichia coli caused proliferation of mammalian cell lines at nanomolar concentrations. We now report on an optimized method to express and purify monomeric Ov-GRN-1 in E. coli using a straightforward and scalable purification and refolding process. Purified monomeric protein caused proliferation at nanomolar concentrations of cancerous and non-cancerous cell lines derived from human bile duct tissue. The expression and purification method we describe herein will serve as a backbone upon which to develop expression and purification processes for recombinant GRNs from other organisms, accelerating research on this intriguing family of proteins.

[Continuous blood coagulation in chronic opisthorchiasis].

Animal experiments have shown that chronic Opisthorchis invasion results in accelerated continuous blood coagulation, by inducing hypercoagulation and platelet activation. The state of hemostasis depends on the degree of infection and undergoes the largest changes during maximum invasion. Irrespective of invasion rates, chronic opisthorchiasis is accompanied by hypofibrinogenemia.

[The efficiency of the enzyme immunoassay test system opisthorchiasis-CIC-EIA-best to detect circulating immune complexes containing opisthorchis antigens in the serum of patients with opisthorchiasis].

The efficacy of a kit of Opisthorchiasis-CIC-EIA-Best reagents was evaluated using 270 sera from patients in the study and control groups. The kit showed a sufficient sensitivity (not less than 87.2%) and a high specificity (not less than 97.9%). The use of the above kit of the reagents for enzyme immunoassay in practical healthcare enables one to increase detection rates among the infested subjects on comprehensive examination of those with suspected opisthorchiasis.

[Complicated forms of superinvasion opisthorchiasis: pathogenetic aspects].

Superinvasion opisthorchiasis is characterized by host growth retardation and weight reduction, which are due to the cleptoeffect of Opisthorchis felineus; metabolic disturbances, proliferative processes, accelerated collagen biosynthesis and biodegradation in the organs reflect parasite-host interactions as components of the system. The productive reactions of visceral pathology are initiated by the parasite's metabolites located in the liver, lung, heart, and testes.

The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini.

Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products and, utilizing selective labeling and sequential solubilization, from the host-exposed tegument. The excretory/secretory included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti-apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno-modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin-2. Novel roles in pathogenesis were suggested for the tegument-host interface since more than ten surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke.

Turmeric reduces inflammatory cells in hamster opisthorchiasis.

The curcumin compound from turmeric is effective in the treatment of many inflammatory diseases. The aim of our present study was to evaluate the efficacy of turmeric on reducing the histopathological changes of hamster opisthorchiasis. Hamsters were infected with Opisthorchis viverrini and then administered turmeric. Using light microscopic observation, liver function tests for alanine transaminase (ALT), alkaline phosphatase, and direct bilirubin were investigated. The resulting histopathological changes show that turmeric has anti-inflammatory properties--during both N-nitrosodimethylamine administration and O. viverrini infection--by reducing the aggregation of inflammatory cells surrounding the hepatic bile ducts, which correlates with a decreased serum ALT level. The decrease in direct bilirubin levels in the hamsters treated with turmeric suggests that turmeric may enhance biliary contraction. The present study found that turmeric clearly reduces the inflammatory cells in hamster opisthorchiasis at an early stage. This finding may be connected with a reduction in the risk factors of cholangiocarcinoma development.

Opisthorchis viverrini: an underestimated parasite in world health.

Infection with Opisthorchis viverrini and its associated cholangiocarcinoma (CCA) is an underestimated problem in the Mekong region of Southeast Asia, despite the widespread use of praziquantel and health education measures for parasite control. Although data from Cambodia, Laos and Vietnam are rare, data from Thailand often show wide-ranging variability in epidemiological parameters, including human morbidity and the prevalence and incidence of CCA. The recent discovery of high levels of population genetic variability in O. viverrini in different wetlands in Thailand and Laos, which indicates the presence of sibling species, suggests that we have underestimated the complexity of this epidemiological situation. Future research should determine the relationship between the genetic variability of O. viverrini and patterns of opisthorchiasis-related disease.

Opisthorchis viverrini: the carcinogenic human liver fluke.

Opisthorchiasis caused by Opisthorchis viverrini remains a major public health problem in many parts of Southeast Asia, including Thailand, Lao PDR, Vietnam and Cambodia. The infection is associated with a number of hepatobiliary diseases, including cholangitis, obstructive jaundice, hepatomegaly, cholecystitis and cholelithiasis. Multi-factorial etiology of cholangiocarcinoma, mechanical damage, parasite secretions, and immunopathology may enhance cholangiocarcinogenesis. Moreover, both experimental and epidemiological evidences strongly implicate liver fluke infection as the major risk factor in cholangiocarcinoma, cancer of the bile ducts. The liver fluke infection is induced by eating raw or uncooked fish products that is the tradition and popular in the northeastern and northern region, particularly in rural areas, of Thailand. The health education programs to prevent and control opisthorchiasis are still required in the high-risk areas.

[Rivers of the Orenburg Region: maintenance of the life cycle of Opisthorchis felineus].

Specific hydrological and climatic features contribute to the functioning of the parasitic system egg--mollusk--fish--man parasitic in the Orenburg Region. Four types of areas, such as floodplain, suprafloodplain-terrace, valley-ravine, and near-lake ones, are adequate for the formation of a natural opisthorchiasis focus. The periods of active functioning of a natural opisthorchiasis focus have been established in the Orenburg Region. These include the emergence of the first cercariae and, therefore, the first (in this epidemic season) infection of the supplementary host (fish) occur in mid-June to early July and then 6 weeks later metacercariae achieve invasiveness in the fish muscles and subcutaneous fat, as a result the first infection of the final host with Opisthorchis occurs in early August.

ABC transporters in the liver fluke Opisthorchis felineus.

ATP-binding cassette transporters (ABC transporters) are essential components of normal cellular physiological machinery in all eukaryotic and prokaryotic species, including parasites. Some ABC transporters, e.g., P-glycoproteins, are involved in the efflux of toxins and xenobiotics from the cell. At present, nothing is known about ABC transporter genes in epidemiologically important liver flukes from the Opisthorchiidae family, including European liver fluke Opisthorchis felineus. Opisthorchiasis caused by O. felineus is a serious public health problem on the territory of Russia and other Eastern European countries. ABC drug transporters are attractive objects of research on molecular markers of resistance and on ways to potentiate sensitivity to anthelmintics through suppression of the transporters themselves with specific inhibitors. Here we aimed at the identification of ABC transporters in the O. felineus transcriptome and identification of P-glycoproteins. In addition, our aim was to assess ABC transcript abundance in the RNA-seq data, to study the mRNA expression of P-glycoprotein genes by Droplet Digital PCR throughout the life cycle of O. felineus, and to test the gene induction in response to xenobiotics or anthelminthic agents. We found 23 nucleotide sequences encoding ABC transporters belonging to different subfamilies, including four sequences of P-glycoproteins. According to the transcript abundance in the RNA-seq data, one of P-glycoproteins (P4) has the highest expression among all ABC genes in the adult worm. P-glycoproteins showed substantially differential mRNA expression throughout the fluke life cycle, with high expression in the adult worms. Putative activity of P-glycoproteins as xenobiotic efflux pumps was found to be linked to the excretory system of O. felineus and to be inhibited by verapamil or tariquidar. Thus, ABC drug transporters in the liver fluke O. felineus are functionally active, indicating that ABC drug transporters are likely to be molecular targets for a combination therapy aimed at prevention of a xenobiotic removal from helminth tissues and at increasing the drug concentration in the tissues.