Objective: To investigate the maturity development of miracidia in uterine eggs from four portions of the Opisthorchis viverrini uterus and environmental factors possibly affected in maturation and infectivity of distal part uterine eggs. Methods: Uteri of adult worms were divided into 4 equal parts. Development of eggs was determined under light microscope. Only embryonated eggs were used to evaluate the effects of physicochemical factors: temperature, salinity, acidity, ultraviolet A, B, C. Infection success was evaluated by feeding treated eggs to intermediate host snails and determining by using a PCR approach. Results: Eggs obtained from the uterus closest to the ovary (regions 1 and 2) failed to develop in vitro. Eggs from region 4 of the uterus (close to the genital pore) were used to study effects of physicochemical factors. The highest survival and infection success was in groups of eggs kept at 30 ℃ (95.20％). The calculated period of loss infection success (LI50 and LI95) on miracidia in distal uterine eggs by exposure to UV-A, UV-B and UV-C were 73 and 1523 d; 8 and 20 d; 1 and 2 d, respectively. Lethal concentrations (LC50 and LC95) of salinity, HCl and NaOH on miracidia in distal uterine eggs were 45.43 and 120.09 ppt, 0.01 and 0.25 M; 0.01 and 0.11 M, respectively, after 24 h exposure. Conclusions: Opisthorchis viverrini eggs display a high tolerance to environmental conditions, especially after snail host eating for infection.

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection.
Animals ; Antigens, Helminth/analysis/*chemistry/immunology ; Clonorchiasis/*diagnosis ; Clonorchis sinensis/*immunology ; Cross Reactions ; Electrophoresis, Polyacrylamide Gel/veterinary ; Humans ; Immunoblotting/veterinary ; Molecular Weight ; Opisthorchiasis/*diagnosis ; Opisthorchis/*immunology

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

The present study was performed to observe histopathological changes in tissues of Bithynia siamensis goniomphalos (Gastropoda, Bithyniidae) incubated in crude extract solutions of camellia (Camellia oleifera) seed and mangosteen (Garcinia mangostana) pericarp, and furthermore to estimate the molluscicidal effects of 2 plant substances. Substantial numbers of bithyniid snails were incubated in various concentrations of 2 plant solution for 24 hr. As the positive control, snails incubated in various concentrations of niclosamide, a chemical molluscicide, were used. The histopathological findings were observed in sectioned snail specimens of each experimental and control groups. The results showed that both camellia and mangosteen extracts had molluscicidal effects at 24 hr with 50% lethal concentration (LC50) at concentrations of 0.003 and 0.002 g/ml, respectively, while niclosamide had LC50 at concentrations 0.599 ppm. B. siamensis goniomphalos snail tissues (foot, gill, and digestive system) showed disruption of columnar muscle fibers of the foot, reduction of the length and number of gill cilia, numerous mucous vacuoles, and irregularly shaped of epithelial cells. Irregular apical and calciferous cells, dilatation of the digestive gland tubule, and large hemolymphatic spaces, and irregular apical surfaces, detachment of cilia, and enlargement of lysosomal vacuoles of epidermis were also shown in all groups. By the present study, it is confirmed that 2 plants, camellia and mangosteen, are keeping some substance having molluscicidal effects, and histopathological findings obtained in this study will provide some clues in further studies on their action mechanisms to use them as natural molluscicides.
Animals ; Camellia/*chemistry ; Disease Vectors ; Garcinia mangostana/*chemistry ; Gastropoda/*drug effects ; Host-Parasite Interactions ; Plant Extracts/chemistry/*pharmacology ; Seeds/chemistry

To increase public health awareness for prevention of opisthorchiasis caused by eating raw freshwater fish, the distribution and abundance of Opisthorchis viverrini metacercariae (OV MC) was investigated in freshwater fish obtained from 20 provinces in northeastern Thailand between April 2011 and February 2012. A cross-sectional survey was conducted on 12,890 fish consisting of 13 species randomly caught from 26 rivers, 10 dams, and 38 ponds/lakes. Fish, were collected in each of the rainy and winter seasons from each province. Fish were identified, counted, weighed, and digested using pepsin-HCl. Samples were examined for OV MC by a sedimentation method, and metacercariae were identified under a stereomicroscope. OV MC were found in 6 species of fish; i.e., Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti, and Puntioplites proctozysron from localities in 13 provinces. Among the sites where OV MC-infected fish were found, 70.0% were dams, 23.7% were ponds/lakes, and 7.7% were rivers. The mean intensity of OV MC ranged from 0.01 to 6.5 cysts per fish (or 1.3-287.5 cysts per kg of fish). A high mean intensity of OV MC per fish (>3 cysts) was found in 5 provinces: Amnat Charoen (6.5 cysts), Nakhon Phanom (4.3), Mukdahan (4.1), Khon Kaen, (3.5) and Si Sa Ket (3.4). In conclusion, OV MC are prevalent in natural cyprinid fish, with the infection rate varying according to fish species and habitats.
Animals ; Cross-Sectional Studies ; Cyprinidae/*parasitology ; Fish Diseases/*epidemiology/*parasitology ; Metacercariae/*isolation & purification ; Microscopy ; Opisthorchiasis/epidemiology/parasitology/*veterinary ; Opisthorchis/*isolation & purification ; Parasitology/methods ; Prevalence ; Thailand

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

Objective: To investigate the maturity development of miracidia in uterine eggs from four portions of the Opisthorchis viverrini uterus and environmental factors possibly affected in maturation and infectivity of distal part uterine eggs. Methods: Uteri of adult worms were divided into 4 equal parts. Development of eggs was determined under light microscope. Only embryonated eggs were used to evaluate the effects of physicochemical factors: Temperature, salinity, acidity, ultraviolet A, B, C. Infection success was evaluated by feeding treated eggs to intermediate host snails and determining by using a PCR approach. Results: Eggs obtained from the uterus closest to the ovary (regions 1 and 2) failed to develop in vitro. Eggs from region 4 of the uterus (close to the genital pore) were used to study effects of physicochemical factors. The highest survival and infection success was in groups of eggs kept at 30 °C(95.20%). The calculated period of loss infection success (LI

Objective To explore larvicidal effects of anthelmintic drugs on Opisthorchis viverrini (O. viverrini) for alternative approach to interrupting its cycle for developing a field-based control program. Methods The larvicidal activities of albendazole (Al), artesunate (Ar), praziquantel (Pzq) and miltefosine (Mf) on O. viverrini cercariae and mature metacercariae were investigated. Lethal concentrations (LC