Contaminated liver fluke egg in the environment has led to the high prevalence of human opisthorchiasis associated with cholangiocarcinoma in Southeast Asia. To find the effective lessening methods of Opisthorchis viverrini eggs in the contaminated environment, we investigated the temperature conditions for killing of these trematode eggs in vitro. Numerous O. viverrini eggs were obtained in the proximal part of uteri of adult worms from experimental hamsters. Mature eggs with miracidium were allocated by experimental groups (2 control: positive and negative and 4 treatment: 50, 60, 70, and 80°C) with 0.85% saline, and treated by the experimental plan. Eggs in each experimental groups were observed under the confocal microscope after stain with Propidium Iodide (PI) to evaluate the effect of temperatures. Eggs in 70 and 80°C groups were all killed after over 10 min heated. Majority of eggs in 60°C (10, 15, and 30 min heated), 70 and 80°C (5 min heated) groups were inactivated. However in 50°C group, below half of eggs were to be killed in all time lapse (10, 15 and 30 min). In order to prevent O. viverrini infection and cholangiocarcinoma, direct treatment of sewage by heating at 70 or 80°C at least 10 min is essential. Therefore, treatment of O. viverrini eggs at a high temperature is a potential method for controlling egg contamination in sewage.
Adult ; Animals ; Asia, Southeastern ; Cholangiocarcinoma ; Cricetinae ; Eggs ; Fasciola hepatica ; Heating ; Homicide ; Hot Temperature ; Humans ; In Vitro Techniques ; Methods ; Opisthorchiasis ; Opisthorchis ; Ovum ; Prevalence ; Propidium ; Sewage ; Uterus

Adult specimens of Echinochasmus caninus n. comb. (Verma, 1935) (Trematoda: Echinostomatidae) (syn. Episthmium caninum Yamaguti, 1958) were recovered from 11 riparian people who resided along the Mekong River in Khammouane Province, Lao PDR. In fecal examinations done by the Kato-Katz technique, the cases revealed eggs of Opisthorchis viverrini/minute intestinal flukes, hookworms, and in 2 cases echinostome eggs. To recover the adult helminths, praziquantel 30–40 mg/kg and pyrantel pamoate 10–15 mg/kg in a single dose were given and purged with magnesium salts. Various species of trematodes (including O. viverrini and Haplorchis spp.), cestodes, and nematodes were recovered from their diarrheic stools. Among the trematodes, small echinostome flukes (n=42; av. 3.8 specimens per case) of 0.7–1.2 mm in length are subjected in this study. They are morphologically characterized by having 24 collar spines interrupted dorsally and anterior extension of vitellaria from the cirrus sac or genital pore level to the posterior end of the body. Particularly based on this extensive distribution of vitellaria, the specific diagnosis was made as Echinochasmus caninus. The cases were co-infected with various other helminth parasites; thus, clinical manifestations specific for this echinostome infection were difficult to determine. The present paper describes for the first time human E. caninus infections in Lao PDR. Our cases marked the 4–14th human infections with this echinostome around the world following the 3 previous cases reported from Thailand.
Adult ; Ancylostomatoidea ; Animals ; Cestoda ; Comb and Wattles ; Diagnosis ; Eggs ; Helminths ; Humans ; Magnesium ; Opisthorchis ; Ovum ; Parasites ; Praziquantel ; Pyrantel Pamoate ; Rivers ; Salts ; Spine ; Thailand ; Trematoda

An archaeoparasitological analysis of the soil samples from Nadym Gorodok site of Western Siberia has been carried out in this study. The archaeological site was dated as the 13 to 18th century, being characterized as permafrost region ensuring good preservation of ancient parasite eggs. Parasite eggs as Opisthorchis felineus, Alaria alata, and Diphyllobothrium sp. were found in the archaeological soil samples, which made clear about the detailed aspects of Nadym Gorodok people's life. We found the Diphyllobothrium sp. eggs throughout the 14 to 18th century specimens, allowing us to presume that raw or undercooked fish might have been commonly used for the foods of Nadym Gorodok inhabitants and their dogs for at least the past 400 years. Our study on Nadym Gorodok specimens also demonstrate that there might have been migratory interactions and strong economic ties between the people and society in Western Siberia, based on archaeoparasitological results of Opisthorchis felineus in Western Siberia.
Animals ; Archaeology ; Asia, Northern ; Diphyllobothrium ; Dogs ; Eggs ; Humans ; Middle Aged ; Opisthorchis ; Ovum ; Parasites ; Permafrost ; Siberia ; Soil

Four isoforms of calcium binding proteins containing 2 EF hand motifs and a dynein light chain-like domain in the human liver fluke Opisthorchis viverrini, namely OvCaBP1, 2, 3, and 4, were characterized. They had molecular weights of 22.7, 21.6, 23.7, and 22.5 kDa, respectively and showed 37.2–42.1% sequence identity to CaBP22.8 of O. viverrini. All were detected in 2- and 4-week-old immature and mature parasites. Additionally, OvCaBP4 was found in newly excysted juveniles. Polyclonal antibodies against each isoform were generated to detect the native proteins in parasite extracts by Western blot analysis. All OvCaBPs were detected in soluble and insoluble crude worm extracts and in the excretory-secretory product, at approximate sizes of 21–23 kDa. The ion-binding properties of the proteins were analyzed by mobility shift assays with the divalent cations Ca²⁺, Mg²⁺, Zn²⁺, and Cu²+. All OvCaBPs showed mobility shifts with Ca²⁺ and Zn²⁺. OvCaBP1 showed also positive results with Mg²⁺ and Cu²⁺. As tegumental proteins, OvCaBP1, 2, and 3 are interesting drug targets for the treatment of opisthorchiasis.
Antibodies ; Blotting, Western ; Calcium-Binding Proteins ; Cations, Divalent ; Dyneins ; EF Hand Motifs ; Electrophoretic Mobility Shift Assay ; Fasciola hepatica ; Humans ; Molecular Weight ; Opisthorchiasis ; Opisthorchis ; Parasites ; Protein Isoforms

Adult specimens of Echinostoma ilocanum (Garrison, 1908) Odhner, 1911 (Trematoda: Echinostomatidae) were recovered from 2 riparian people who resided along the Mekong River in Savannakhet Province, Lao PDR. In fecal examinations done by the Kato-Katz technique, they revealed echinostome eggs together with eggs of Opisthorchis viverrini (and minute intestinal fluke eggs) and hookworms. To recover the adult flukes, they were treated with praziquantel 30–40 mg/kg in a single dose and purged with magnesium salts. A total of 658 adult fluke specimens were recovered from the 2 people; 456 from case 1 and 202 from case 2. Specimens from case 1 consisted of 335 echinostomes (301 E. ilocanum and 34 species undetermined), 120 O. viverrini, and 1 Haplorchis taichui, and those from case 2 consisted of 36 E. ilocanum, 134 O. viverrini, and 32 H. taichui. Thus, the number of E. ilocanum specimens was 337 in total (average per person, 168.5). From this study, it is suggested that foodborne intestinal flukes and liver flukes are highly prevalent along the Mekong River in Savannakhet Province. The present report describes for the first time human infections with E. ilocanum in Lao PDR.
Adult ; Ancylostomatoidea ; Echinostoma ; Echinostomiasis ; Eggs ; Fasciola hepatica ; Humans ; Magnesium ; Opisthorchis ; Ovum ; Praziquantel ; Rivers ; Salts ; Trematoda

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.
Adult ; Base Sequence ; Fasciola hepatica ; Humans ; Kazakhstan ; Mesocricetus ; Metacercariae ; Opisthorchis ; Rivers ; Trematoda ; Urinary Bladder

Codon usage bias (CUB) is a unique property of genomes and has contributed to the better understanding of the molecular features and the evolution processes of particular gene. In this study, genetic indices associated with CUB, including relative synonymous codon usage and effective numbers of codons, as well as the nucleotide composition, were investigated in the Clonorchis sinensis tyrosinase genes and their platyhelminth orthologs, which play an important role in the eggshell formation. The relative synonymous codon usage patterns substantially differed among tyrosinase genes examined. In a neutrality analysis, the correlation between GC₁₂ and GC₃ was statistically significant, and the regression line had a relatively gradual slope (0.218). NC-plot, i.e., GC₃ vs effective number of codons (ENC), showed that most of the tyrosinase genes were below the expected curve. The codon adaptation index (CAI) values of the platyhelminth tyrosinases had a narrow distribution between 0.685/0.714 and 0.797/0.837, and were negatively correlated with their ENC. Taken together, these results suggested that CUB in the tyrosinase genes seemed to be basically governed by selection pressures rather than mutational bias, although the latter factor provided an additional force in shaping CUB of the C. sinensis and Opisthorchis viverrini genes. It was also apparent that the equilibrium point between selection pressure and mutational bias is much more inclined to selection pressure in highly expressed C. sinensis genes, than in poorly expressed genes.
Bias (Epidemiology) ; Clonorchis sinensis* ; Codon* ; Genome ; Monophenol Monooxygenase* ; Opisthorchis ; Platyhelminths

Opisthorchis viverrini infection induces chronic inflammation, and a minor proportion of infected individuals develop advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA). Inflammatory cytokines and/or their gene polymorphisms may link to these biliary pathologies. We therefore investigated associations among cytokine gene polymorphisms and cytokine production in 510 Thai cases infected with O. viverrini who presented with APF+ or APF−, as established by abdominal ultrasonography as well as in patients diagnosed with CCA. Levels of pro-inflammatory and anti-inflammatory cytokines were determined in culture supernatants after stimulation of peripheral blood mononuclear cells (PBMCs) with O. viverrini excretory-secretory (ES) products. Pro-inflammatory cytokines, IL-1β, IL-6, IFN-γ, LT-α, and TNF-α were significantly increased in CCA patients compared with non-CCA (APF− and APF+) cases. Polymorphisms in genes encoding IL-1β-511C/T, IL-6-174G/C, IFN-γ +874T/A, LT-α +252A/G, and TNF-α−308G/A were then investigated by using PCR-RFLP or allele specific-PCR (AS-PCR) analyses. In the CCA cases, LT-α +252A/G and TNF-α−308G/A heterozygous and homozygous variants showed significantly higher levels of these cytokines than the wild type. By contrast, levels of cytokines in wild type of IFN-γ +874T/A were significantly higher than the variants in CCA cases. IFN-γ +874T/A polymorphisms were associated with advanced periductal fibrosis, whereas IL-6 −174G/C polymorphisms were associated with CCA. To our knowledge, these findings provide the first demonstration that O. viverrini infected individuals carrying several specific cytokine gene polymorphisms are susceptible to develop fibrosis and CCA.
Alleles ; Asian Continental Ancestry Group ; Cholangiocarcinoma* ; Cytokines ; Fasciola hepatica* ; Fibrosis* ; Humans ; Inflammation ; Interleukin-6 ; Liver* ; Opisthorchis* ; Pathology ; Ultrasonography

Calreticulin (CALR), a multifunctional protein thoroughly researched in mammals, comprises N-, P-, and C-domain and has roles in calcium homeostasis, chaperoning, clearance of apoptotic cells, cell adhesion, and also angiogenesis. In this study, the spatial and temporal expression patterns of the Opisthorchis viverrini CALR gene were analyzed, and calcium-binding and chaperoning properties of recombinant O. viverrini CALR (OvCALR) investigated. OvCALR mRNA was detected from the newly excysted juvenile to the mature parasite by RT-PCR while specific antibodies showed a wide distribution of the protein. OvCALR was localized in tegumental cell bodies, testes, ovary, eggs, Mehlis’ gland, prostate gland, and vitelline cells of the mature parasite. Recombinant OvCALR showed an in vitro suppressive effect on the thermal aggregation of citrate synthase. The recombinant OvCALR C-domain showed a mobility shift in native gel electrophoresis in the presence of calcium. The results imply that OvCALR has comparable function to the mammalian homolog as a calcium-binding molecular chaperone. Inferred from the observed strong immunostaining of the reproductive tissues, OvCALR should be important for reproduction and might be an interesting target to disrupt parasite fecundity. Transacetylase activity of OvCALR as reported for calreticulin of Haemonchus contortus could not be observed.
Antibodies ; Calcium ; Calreticulin* ; Cell Adhesion ; Cell Body ; Citrate (si)-Synthase ; Eggs ; Electrophoresis ; Female ; Fertility ; Haemonchus ; Homeostasis ; In Vitro Techniques ; Mammals ; Molecular Chaperones ; Opisthorchis* ; Ovary ; Ovum ; Parasites ; Prostate ; Reproduction ; RNA, Messenger ; Testis ; Vitellins

Following the first report of Opisthorchis viverrini infection in a domestic duck in Phu My District of Binh Dinh Province, Central Vietnam, many other cases were observed in the province. We determined the infection rate and intensity of O. viverrini infection in ducks in 4 districts of the province. A total of 178 ducks were randomly selected from 34 farms for examination of flukes in the liver and gall bladder. An infection rate of 34.3% (range 20.7-40.4% among districts) was found; the intensity of infection was 13.8 worms per infected duck (range 1-100). These findings show the role of ducks as a host for O. viverrini, duck genotype, which is sympatric with the human O. viverrini genotype in this province. It also stresses the need for investigations on the zoonotic potential and the life cycle of this parasite.
Agriculture ; Ducks* ; Genotype ; Humans ; Life Cycle Stages ; Liver ; Opisthorchis* ; Parasites ; Prevalence* ; Trematoda* ; Urinary Bladder ; Vietnam*