Effect of artemether, artesunate, OZ78, praziquantel, and tribendimidine alone or in combination chemotherapy on the tegument of Clonorchis sinensis.

We investigated the morphological effects of half-strength treatments with praziquantel, artemether, artesunate, OZ78 and tribendimidine as well as combinations of praziquantel with artemether, artesunate, OZ78 and tribendimidine and an artesunate-tribendimidine combination in rats harboring adult Clonorchis sinensis. Rats were infected with C. sinensis, dosed orally with single agents or combination treatments and flukes recovered at 3 or 5 days post-treatment. The number of flukes was counted, the viability recorded and surface changes monitored by scanning electron microscopy. Drug effects induced by the individual drugs at sub-curative doses 3 days post-treatment were minor with the exception of flukes recovered from rats treated with artemether and tribendimidine. Treatment with the praziquantel combinations of artesunate, OZ78 and tribendimidine did not produce a greater disruption of the tegument than the individual drugs 3 days post-treatment. On the other hand, at this time point many worms treated with artemether-praziquantel had died and eruptions, roughening or blebbing were observed on all worms examined. Five days post-treatment flukes exposed to any of the praziquantel combinations in rats had died. Rats treated with an artesunate-tribendimidine combination resulted in a rapid death of flukes, 3 days post-treatment all worms had been expelled. In conclusion, we have confirmed the promising clonorchicidal properties of different drug combinations in rats. Differences in the extent and time-scale of tegumental disruption have been observed. The effect of drug combinations against C. sinensis requires further scientific inquiry, e.g. in transmission electron microscopy studies and in the C. sinensis-rabbit model.

Effect of single-dose oral artemether and tribendimidine on the tegument of adult Clonorchis sinensis in rats.

The tegument of trematodes plays a key role in nutrient absorption, exerts secretory functions, protects the parasite against the immune system of the host, and is a target for anti-trematocidal drugs. We performed a temporal examination of tegumental changes following artemether and tribendimidine administration on adult Clonorchis sinensis in rats using scanning electron microscopy. Rats infected with C. sinensis for 6 weeks were treated orally with a single dose of artemether (150 mg/kg) or tribendimidine (300 mg/kg). Worms were collected between 8 h and 7 days (artemether) and between 4 h and 2 days post-treatment (tribendimidine). Worms recovered from untreated rats served as controls. Eight hours after artemether administration, the tegument of C. sinensis was extensively disrupted, including severe swelling, fusion and vacuolization, and the suckers were damaged. Four hours after administration of tribendimidine, C. sinensis worms showed extensive tegumental alterations, characterized by massive sloughing, and the suckers were damaged. Interestingly, the severity of tegumental changes did not progress further with time. Our results show that both artemether and tribendimidine rapidly disrupt the tegument and damage the suckers of adult C. sinensis. The subtle differences in tegumental changes induced by artemether and tribendimidine might indicate different mechanisms of action of these drugs against C. sinensis.

Clonorchicidal properties of the synthetic trioxolane OZ78.

Because the synthetic trioxolane OZ78 is active against the liver fluke Fasciola hepatica, we were motivated to investigate the in vivo and in vitro activity against another liver fluke, namely Clonorchis sinensis. Rats infected with C. sinensis for 2 and 5 wk were treated orally with single doses of OZ78 (75, 150, or 300 mg/kg). Worm burden reductions were assessed against untreated control rats. Scanning electron microscopy (SEM) was used to observe adult C. sinensis after recovery from rats 1-3 days posttreatment with a single 300 mg/kg oral dose of OZ78 and after in vitro exposure to concentrations of 1, 10, and 100 microg/ml of OZ78. A single 300 mg/kg oral dose of OZ78 resulted in worm burden reductions of 78.5% and 98.5% against juvenile and adult C. sinensis, respectively. SEM observations revealed tegumental surface alterations, including blebbing and sloughing. OZ78 emerges as a new compound with a broad spectrum of activity against major foodborne trematode infections.

Ultrastructural localization of phosphoglycerate kinase in adult Clonorchis sinensis.

Phosphoglycerate kinase (PGK) is an enzyme that produces one ATP molecule in the glycolytic pathway. Clonorchis sinensis is largely dependent on glycolysis for energy production. We performed immunoelectron microscopy on adult C. sinensis by using mouse immune serum raised against recombinant C. sinensis PGK. A high density of gold particles was found in the microvilli of the intestinal epithelium and in lamellae of the sperm duct. PGK was common in the somatic cells of intra-uterine eggs and in excreted products. It was localized with moderate intensity in muscular fibers of the subtegumental muscle layer, and in the myoepithelia of the intestine and excretory bladder. We suggest that PGK plays an essential role in C. sinensis energy production for movement via muscle contraction.

Purification and characterization of a 7-kDa protein from Clonorchis sinensis adult worms.

A 7-kDa protein was purified from extracts of adult Clonorchis sinensis by a combination of ammonium sulfate precipitation, anion exchange chromatography, cation exchange chromatography, gel-filtration chromatography, and reversed-phase FPLC. The 7-kDa protein exists in the excretory-secretory products of adult C. sinensis, but not in extracts of adult Paragonimus westermani. Also, the 7-kDa protein reacted with the sera of patients with clonorchiasis but not with paragonimiasis or normal human sera. To observe the localization of the 7-kDa protein in the tissue of adult C. sinensis, an immunogold labeling method was followed using anti-7-kDa antibody. The gold particles were observed in the basal layer below the tegumental syncytium, in the interstitial matrix of the parenchyma, and in the content of the uterus. The 7-kDa cDNA was obtained through reverse transcription-polymerase chain reaction using a primer designed from N-terminal sequence analysis. Rapid amplification of cDNA ends (5'-RACE) was used to obtain the complete protein coding sequence. The sequence encodes a 90-amino acid polypeptide. The deduced amino acid sequence of the 7-kDa protein revealed no homology with proteins of different organisms reported so far. These results suggest that the 7-kDa protein is a fluid antigen and may be valuable as a tool for the immunodiagnosis of clonorchiasis.

Clonorchis sinensis: immunolocalization of 26 kDa glutathione S-transferase in adult worms.

A mu-class glutathione S-transferase (Cs26GST) of molecular mass 26 kDa was characterized from Clonorchis sinensis. In adult C. sinensis, the distribution of the Cs26GST was investigated by immuno-histochemistry and electron microscopy. Cs26GST was localized to the tegument and parenchyma. Immunogold labeling was strong in the tegumental cell bodies and moderate in the tegument and ova in the oviduct. It is suggested that Cs26GST plays a role in the metabolism and fecundity of C. sinensis.

Image analytical observation on the growth and development of Clonorchis sinensis in rats.

For the two-dimensional observation on the growth and development of Clonorchis sinensis, an image analysis system (IBAS 2000, Kontron, Germany) was used in this study. On 3, 6, 9, 12, 15, 21, 30 and 90 days of infection, 474 worms were collected from rats infected with 50 metacercariae each. The overall recovery rate was 31.6%. The worms grew rapidly in their length and width up to 30 days of infection and then, did slowly to 90 days of infection. However, the growth pattern of body area was not similar to that of body length and width, because the body area increased continuously even after 30 days of infection. With the image analysis system, the sizes of irregular-shaped genital organs were measured easily, which showed sigmoidal growth patterns. The growth rate of genital organs increased rapidly until 21 days of infection when the uterus was filled with numerous eggs, and then gradually slowed down to 90 days. There was no difference in growth pattern between the anterior and posterior testis. The seminal receptacle, however, showed an abrupt increase in size between 15 to 21 days of infection when it was filled with condensed semen. Therefore, the growth pattern of seminal receptacle might be used as another criteria to estimate the extent of sexual maturation of C. sinensis. From this study, it is suggested that image analysis system is very useful to reveal the growth and development pattern of C. sinensis, especially of their internal organs.

[The functional characteristics of the ultrastructure of the outer membranes of opisthorchid eggs. 2. The mechanisms of egg retention on substrates]. / Funktsional'nye osobennosti ul'trastruktury vneshnikh obolochek iaits opistorkhid. Soobshchenie 2. Mekhanizmy uderzhivaniia iaits na substratakh.

The studies of the functional features of the ultrastructure of the outer membranes of Opisthorchis felineus and Clonorchis sinensis eggs have been continued. The variants of egg behavior on different substrates (artificial, natural, organic and inorganic) have been experimentally studied. It has been established that O. felineus and C. sinensis eggs retain on a number of substrates due to covering with microflora (e.g. fungus mycelium clinging to the rubbed surface of the egg outer membranes); sticking in the uneven surface of the substrate; catching in the uneven surface of the substrate; catching on microfibers of plant fragments. The features found are to a great extent caused by a complex comb-like structure of egg membranes and are interpreted in terms of the earlier unknown mechanisms of parasite adaptation (at the egg stage) facilitating its contact with molluscs--their intermediate hosts.

[The morphofunctional characteristics of the ultrastructure of the outer membrane of opisthorchid eggs. 1. The hatching of miracidia]. / Morfofunktsional'nye osobennosti ul'trastruktury vneshnei obolochki iaits opistorkhid. Soobshchenie 1. Vyluplenie miratsidiev.

Some functional peculiarities of miracidia hatching from O. felineus eggs have been studied, using scanning electron microscope. The results were compared to the earlier data obtained by the method elaborated for the determination of opisthorchis eggs viability by stimulation of miracidia release in the artificial media. It is concluded that in the course of miracidia hatching the two release-stimulating factors (mechanical and chemical) are mutually complementing each other. First, the egg membrane is deformed, in which the radula of the mollusk host plays an essential role, then the process of chemical activation of the hatching gland is triggered and miracidia motor activity is stimulated. A complex comb-shaped structure of opisthorchis eggs outer membranes is associated with the necessity of long-term eggs survival in the water reservoirs and forms a light delicate carcass preventing the egg from deformation.

[Scanning electron microscopic observations on Clonorchis sinensis cercariae].

The surface structure of Clonorchis sinensis cercariae was observed under scanning electron microscope. The cercaria was of the parapleuro-lophocercous type. The measurements of the cercariae were as follows: body 137-240 x 62-90 microns, tail 320-470 x 21-34 microns, oral sucker 27-46 x 23-33 microns. The ventral sucker was incompletely developed and much smaller than the oral one. The whole body surface was covered with backward pointing spines. Four horizontal rows of oral spines were found around the mouth opening of the oral sucker. These spines diminished in size with the distance of the rows from the opening and their numbers were 12, 18, 19 and 18 respectively. Transverse and longitudinal plicae formed from tegument were found on the surface of the tail, being in connection with dorsal and ventral fins along posterior half of the tail. Many sensory structures or papillae were observed on the body. They were of three different types: 1) papillae with long or short cilia distributed widely on the body surface and around the mouth opening, the number of papillae with cilia on the body surface being greater than that described by Komiya et al (1940); 2) a few sensory fossa on the tail, measuring 1.0 x 0.6 microns; 3) a few ring-type papillae with knob on the body surface with pore of 0.5 micron in diameter.

Clonorchiasis in Korea.

Since the first report on the incidence of egg positiveness of Clonorchis sinensis by Matsumoto in 1915, clonorchiasis has been recognized as one of the most important endemic diseases in Korea. Most of the works on clonorchiasis after then had been studied by Japanese workers until 1945. After the Korean War in 1950-1953, health problems of Koreans were extremely aggravated. This gave us attentions for the prevention of contagious diseases as well as parasitic infections in public health point of view. It was greatly recognized that not only ascariasis, hookworm infections, and trichuriasis, but also clonorchiasis and paragonimiasis constitute the important helminthic diseases in Korea. In the late 1950s, several Korean workers conducted studies of C. sinensis on its biology, epidemiology, pathology, clinical symptoms as well as treatment. Thereafter overall epidemiological surveys were carried out actively throughout the country since 1960s. From these surveys, it became clear that endemic areas of clonorchiasis are scattered all over the country along the Nakdong, Kum, Mankyong, Yeongsan and Han rivers. The most extensive and intensive endemic regions were found mainly along the Nakdong river. Since then, clonorchiasis has been one of the most extensively studied subjects during the past 30 years. The present review mainly deals with current results obtained by Korean investigators on the prevalence of clonorchiasis, as well as its biology, epidemiology, pathogenesis, clinical manifestations, diagnosis, treatment, prevention and control.

[The differentiation of Opisthorchis eggs by the ultrastructure of their outer membranes]. / Differentsiatsiia iaits opistorkhid po ul'trastrukture vneshnikh obolochek.

Using a TESLA-BS-300 scanning electron microscope, the ultrastructure of the outer membranes of oocysts of 8 opisthorchis species (Opisthorchis felineus, O. viverrini, O. geminus, Clonorchis sinesis, Amphimerus anatis, Metorchis albidus, Pseudamphistomum truncatum, Erchoviorchis lintoni) belonging to 4 subfamilies, as well as that of O. felineus oocysts of various geographical isolates (from the Ukraine, the Volga region, West Siberia, Kazakhstan) have been investigated. The nature of the ultrastructure of the membranes differs from species to species and can be used for the differentiation of opisthorchis oocysts. Marked differences have been observed in different opisthorchis subfamilies (Opisthorchinae, Metorchinae, Pseudamphistominae, Pseudamphimerinae). The membrane ultrastructure was not significantly different in O. felineus oocysts from various geographical isolates, however, there are some distinctions in the size of oocysts and the form of apical thorns and opercular endings. The work has been done within the framework of the international scientific cooperation between the E. I. Martsinovskii Institute of Medical Parasitology and Tropical Medicine, USSR Ministry of Public Health and the Institute of Parasitology, the Czechoslovakian Academy of Sciences.

[Ultrastructural changes in the body wall and gut of Clonorchis sinensis in rats after albendazole treatment].

The effect of albendazole on the body wall and gut of Clornorchis sinensis was studied with transmission and scanning electron microscopes after albendazole administration to rats infected with Chonorchis sinensis at a single dose of 150 mg/kg. The results showed that swelling and adhesion of the projections of the tegument and gut microvilli occurred 1h after medication. Necrosis and disruption of the projections and the gut microvilli were seen at the 24th h. By the 72nd h, detachment of the partial projections were seen. The dynamic process of the damages observed on the tegument was identical with that of the gut microvilli (Figs. 1-10).

[The outer membranes of the eggs of Opisthorchis felineus and Clonorchis sinensis]. / Vneshnie obolochki iaits Opisthorchis felineus i Clonorchis sinensis.

The outer coats of Opisthorchis felineus and Clonorchis sinensis eggs have been examined by scanning electron microscopy. The outer coat turned to be extremely sculptured, presenting as interlaced crests of various height. The authors suggested the possibility of using this indicator in the taxonomy of Opisthorchiidae and studying their distribution in biocenoses as well as their ability of accumulating on the leaves of aquatic plants, thus facilitating the contact with their intermediate hosts the mollusks.