Comparative Characterization of Four Calcium-Binding EF Hand Proteins from Opisthorchis viverrini

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.

Effects of physicochemical factors on development and survival of Opisthorchis viverrini uterine eggs

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

Effects of albendazole, artesunate, praziquantel and miltefosine, on Opisthorchis viverrini cercariae and mature metacercariae

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

Effects of albendazole, artesunate, praziquantel and miltefosine, on Opisthorchis viverrini cercariae and mature metacercariae

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 and LC) of these drugs were determined. Mature metacercariae previously exposed to various concentrations of the drugs were administered to hamsters. Worms were harvested 30 d post infection and worm recovery rates calculated. Al, Ar, Pzq and Mf produced morphological degeneration and induced shedding tails of cercariae after 24 h exposure.@*RESULTS@#The LC and LC of Al, Ar, Pzq and Mf on cercariae were 0.720 and 1.139, 0.350 and 0.861, 0.017 and 0.693, and 0.530 and 1.134 ppm, respectively. LC and LC of Ar on mature metacercariae were 303.643 and 446.237 ppm and of Mf were 289.711 and 631.781 ppm, respectively but no lethal effect in Pzq- and Al-treated groups (up to 1 ppt). No worms were found in hamsters administered Pzq-treated metacercariae. The adult worms from Al-treated metacercariae were significantly bigger in size compared to the control group (P < 0.05). Fecundity and body width were greater in adults from Mf-treated metacercariae compared to the control group (P < 0.05).@*CONCLUSIONS@#The larvicidal effects of these drugs were high efficacy to O. viverrini cercariae but lesser efficacy to metacercariae. It should be further studied with the eventual aim of developing a field-based control program.

Molecular and Biochemical Characterization of Opisthorchis viverrini Calreticulin

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.

Histopathological Changes in Tissues of Bithynia siamensis goniomphalos Incubated in Crude Extracts of Camellia Seed and Mangosteen Pericarp

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.

Distribution and Abundance of Opisthorchis viverrini Metacercariae in Cyprinid Fish in Northeastern Thailand

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.

Effects of the extract from Desmodium pulchellum in rats

 Desmodium pulchellum is a medicinal herb which has been reported to possess anthelminthic effects against the liver fluke (O. viverrini) in hamsters.  This report shows pharmacological effects of the herb extracts in concious rats.  Locomotion associated with muscle relaxation was obserbed.  Meanwhile, rats received the extracts had rapid respiration and became red at ears, nose and feet compared to control rats.  These results suggest that the herb has major effects on motor, respiratory and cardiovascular system.  Both gripping of mice on inclined plane and locomotion of rats were decreased by the herb extracts.  The extract from bark of the root exhibited the most potent effect compared to wooden part of the root, leaves and stem.  Intravenous injection of these extracts into anaesthetized rats increased respiratory volume (both expired and inspired air), blood pressure and amplitude of QRS-complex.  These results indicate that bark of the root exhibits all of these major pharmacological effects and is the most potent part of the herb.

Preliminary study : Effects of Desmodium pulchellum’s root on Opisthorchis viverrini in hamsters

 This report is the preliminary study on effects of the extract from Desmodium pulchellum’s root on the fluke “Opisthorchis viverrini” in hamsters. After 45 days of infection with metacercariae of this liver fluke, all six groups of 5 hamsters had similar number of eggs in feces (p\>0.7). The treatment effects of water extract from this herb (10 g root/1 kg b.w) given for 1,2,3,4 or 5 days on the number of eggs in feces and the number of adult fluke in the liver were compared with those of controls. The results show that the number of eggs in feces from hamsters treated with the herb-extract is significantly decreased from controls (p 0.1). These results suggest that the extract from this herb may have anthelminthic effect on this fluke, at least , on the egg production. 

Effects of Aflatoxin B1 and Poor-Protein Diet In Hamsters Infected with Opisthorchis viverrini

Forty-two  male Syrian Golden hamsters, maintained on a 5.3% protein diet, were experimentally infected with 50 metacercarine of Opisthorchis viverrini. An oral dose of 0.05 mg aflatoxin B1 was then administered to each animal once weekly for 12 weeks and the low protein diet further maintained for a subsequent period of 6 months. Histopathological examination of the livers revealed bile ductular proliferation, dilatation of larger bile ducts with granuloma formation and pseudopapillary epithelial proligeration associated with nuclear pleomorphism, hyperchromicity and pasudostratifica tion of the lining columnar cells. Atypia of hepatocytes  was pronounced within the hepatic lobules in 64% of the hamsters. Some livers also had focal hyperplasia of Kupffer cells and parenchymal architectural alteration due to fibrous tissue proliferation within the hepatic parenchyma and around portal areas. The epidemiology of primary hepatic epithelial tumours in Thailand is discussed with particular reference to the contributory roles of endemic liver fluke infestation , low-protein diet and widespread contamination of food by aflatoxin. 

Ultrastructure of tegumentary papillae of the excysted Opisthorchis viverrini metacercaria

Background: Opisthorchiasis, caused by liver fluke  Opisthorchis viverrini  is  endemic  in many countries of the Southeast Asia such as Thailand, Laos PDR, Cambodia and South Vietnam. It is a medically important fluke which causes several hepatobiliary diseases and cholangiocarcinoma.  The tegument of O. viverrini is served as the site for sensory input.  When the metacercaria infects to human, it excysts and migrates into bile ducts by creeping to pass the hepatopancreatic ampulla. The metacercaria might be equipped with some sensory perceptions. The tegumentary papillae may have a role for sensation. No detail of the ultrastructural of the tegumentary papillae of the O. viverrini has been studied.Objective:  To study the ultrastructure of the tegumentary papillae of newly excysted O. viverrini metacercariae by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).Study design: Descriptive study based on morphological observation at the ultrastructureral level.Setting: Department of Anatomy and Department of Parasitology, Faculty of Medicine, Khon Kaen  University, Thailand.Materials and Methods:  O.  viverrini metacercariae obtained from naturally infected cyprinoid fish in endemic area in Khon Kaen were excysted by suspending in 0.1% trypsin solution. The newly excysted metacercariae were processed and observed with a scanning and                  a transmission electron microscopes. Results: SEM studies of tegumentary papillae identified four types of papillae. Type I was a small conical papilla with long cilium (Fig. 1). Type II possessed short cilium and a larger conical papilla than type I (Fig. 2). Type III was characterized by grouping of 2-3 conical papillae on a dome-shaped base with variable length of the cilia (Fig. 3).  Type IV was non-ciliated conical papilla and found solely on inner lip of the oral and ventral suckers (Fig. 4). TEM observation revealed two types of tegumantary papillae, ciliated (type A) and non-ciliated (type B) papillae. Only one cilium was found in all ciliated tegumantary papillae. Based on morphology of tegumentary papillae and ultrastructures of the bulbous ending, the ciliated papillae were further divided into three subtypes (A1, A2 and A3).   In type B, there was an elevation of the tegument and the bulbous ending was lack communication with the external surface.Conclusions:  As seen by SEM, there were four types of tegumentary papillae of the excysted  O.  viverrini  metacercaria. By TEM observation, only two types of tegumentary papillae, uniciliated and non-ciliated were found. Based on internal structures of the bulbous ending, the uniciliated tegumentary  papillae  were further divided into  three subtypes . The evidence of a nerve process leading to the bulbous ending in the TEM study supported that these tegumentary papillae may consider to serve as  a structure for sensory perception. Only the type II papilla in SEM study and uniciliated papilla type A1 in TEM were both located in the outer lip of the oral sucker. Therefore, they might be the same type of papillae.Key words:  Opisthorchis viverrini, metaceraria, papillae, SEM, TEM 

Opisthorchis viverrini infection in minute intestinal fluke endemic areas of Chiang Mai Province, Thailand

Background: Food-born trematode infections such as opisthochiasis are major causes of morbidity in Asia. Fish-borne trematode infections are commonly found in the northern and northeastern regions of Thailand. Several species of cyprinoid freshwater fish have been reported as secondary intermediate hosts. Objectives: To determine the prevalence of infections with Opisthorchis viverrini and other intestinal parasites in Chiang Mai Province, Thailand, which is an endemic area for minute intestinal fluke infections in Northern Thailand due to environmental characteristics and the traditional consumption of raw fish. Results: Stool samples were collected from 327 subjects from Cho Lae sub-district, Ban Pao sub-district, Mae Taeng district, Luang Nuea sub-district, Doi Saket district, and Nong Yaeng sub-district San Sai district, Chiang Mai. The subject ages were between 10 and 60 years. Of these, 144 were males and 183 were females (male to female ratio 1:1.27). Conclusion: Parasites were found in 36 stool samples (11.0%). The majority of detected parasites (17 cases, 5.2 percent) were Opisthorchis viverrini. Other intestinal parasites included hook-worms, Trichuris trichiura, and minute intestinal flukes. The highest prevalence (31.2%) was found in Cho Lae sub-district, Mae Taeng district. The prevalence of infection was significantly associated with the health behaviour of the subjects. There was no significant association with sex-distribution.

Morphological Features of the Testes of the Adult Liver Fluke, Opisthorchis viverrini

Background: Infection of Opisthorchis viverrini is widely endemic mainly in Northeast of Thailand including Laos, Cambodia and South Vietnam. Its prevalence and pathogenic effects are associated with several hepatobiliary diseases in human. The liver fluke is monoecious, seft-fertilization can be occurred individually. The male reproductive organ usually consists of two deeply lobed testes, located in the posterior region of the body next to the ovary. No description is available on the ultrastructure and development of the testes of the Opisthorchis viverrini.Objectives: This study aims to examine the morphology and ultrastructure of the testes of the adult flukes O. viverrini.Study design: Descriptive study based on morphological observation by using light microscope (LM), transmission electron microscope (TEM) and scanning electron microscope (SEM).Setting: Department of Anatomy and Department of Parasitology, Faculty of Medicine, Khon Kaen University, Thailand.Materials and Methods: The encyst metacercaria of Opisthorchis viverrini were identified and collected. Ten adult Golden Syrian hamsters were used. Each animal was infected with 50 encyst metacercaria by gastric intubation. The animals were then sacrificed by deep ether anaesthesia, at 28 days post infection. The recovered flukes were routinely processed for investigation of the testes by LM, TEM and SEM.Results: In the whole mount specimens, the pair testes of the adult O. viverrini were clearly defined at the posterior 1/3 of the worm body posterior to the ovary. They were multilobate, 4-5 lobes each and located one behind the other. The testes showed numerous germ cells at various stages of development. The spermatogonia were closest to the testicular wall. In the deeper regions, cluster of numerous spermatocytes and spermatids were observed. The early spermatid exhibited an elongated nucleus with exhibiting lamellar chromatin alongside the two sets of striated rootlets. The scanning electron microscopic studied revealed that the spermatozoa were long filiformshaped.Conclusions: This is the first report of the morphology of the testes of the adult flukes, Opisthorchis viverrini studied by using light and electron microscopes. The testes contained different stages of developing spermatogenic cells. Their three dimentional morphology were thread-like with no easily discernible heads and tails.Keywords: Opisthorchis viverrini, spermatogenic cells, testes, ultrastructure

Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini (Opisthorchidae, Trematoda)

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.