The present work was to localize and isolate the anthelmintic substance from stem cortex of Machilis thunbergii which grows in all parts of the Korea peninsula. The dried M. thunbergii bark was extracted with a solvent mixture (Methanol: Ethyl acetate/1:1) by a Soxhlet extractor at 70C for 24 hours, and then the extract was separated into solid matter (negative wormicidal) and benzene extract (positive wormicidal) by benzene solvent. The anthelmintic action was observed on the cercaria, the liberated metacercaria and the adult of Clonorchis sinensis. On the silica gel thin layer chromatography with a spot of benzene extract in benzene solvent, nine spots were observed in iodine chamber and the spot of Rf. 0.16 value of them appeared the most strong wormicidal effect on the cercaria and the liberated metacercaria of C. sinensis. Afterward, a 0.3 g colourless solid substance which was equivalent to the Rf. 0.16 value spot was isolated from the benzene extract of 3 kg M. thunbergii stem cortex by means of silica gel column chromatography using benzene carrier, and the adult of C. sinensis was killed within 6 minutes by 0.05 percent solution of the substance.
parasitology-helminth-trematoda-Clonorchis sinensis ; chemotherpay-clonorchiasis ; rabbit ; liver ; Machilis thunbergii

A morphological study on the ultrastructures of the alimentary tract and the excretory system of Clonorchis sinensis was conducted. The liver flukes were collected from rabbit liver six months after the experimental infection The worms were washed with 0.85 percent saline solution and immediately moved to cold 2.5 percent glutaraldehyde in 0.1 M phosphate buffer pH 7.4. The materials were dissected and fixed for two hours. The blocks were post-fixed in 1 percent osmium tetroxide. The blocks were embedded in Epon 812. Ultra thin sections were cut with Sovall MT-2 ultramicrotome and stained with uranyl acetate and lead citrate. Sections were then observed with Hitachi HS-7S electron microscope. The following results were obtained in a series of observations. The walls of oral cavity and esophagus comprised tegumental syncytium, basement membrane, loose connective tissue, muscular layer and parenchymal cells. The apical surface and the base of the syncytium were covered with a protoplasmic membrane for each forming numerous invaginations. Granular endoplasmic reticulum was developed in the epithelium of the oesophagus. The gastrodermis of Clonorchis sinensis comprised two types of cells in general. The first cell type was numerous one forming a single continuous layer of epithelial cells. Each of the cells had outfolded cytoplasm into the caecal lumen and lamellae along the cell surface. Among the above epithelial cells, no considerable differences in structure reflecting their functional states were identified. The second cell type was less differentiated in nature and lay within the gastrodermis above the basement membrane but not in contact with the caecal lumen, being overlapped by neighboring gastrodermal cells of the type described above. At this portion the gastrodermis seemed to be a pseudostratified epithelium. There were well-developed lamellae along the surface of epithelia of all canals or duct concerning evacuation. The excretory pore was 7.5 micrometer in diameter and dorso-terminally opended. The epithelium of the excretory pore, a syncytial layer, contained many microtubules unlike the other part of tegumental layer of this worm. The epithelium thickness of the excretory pore was very irregular(1.3-5.5 micrometer).
parasitology-helminth-trematoda ; Clonorchis sinensis ; electron microscopy ; alimentary tract ; liver ; rabbit

The rabbits were infected with Clnorchis sinensis and were treated with praziquantel at the dose of 50 mg/kg x 2 x 2 days afer 1, 2, 4, 8 weeks and 7 months from the infection. Their livers were observed histopathologically 1, 4 and 12 weeks after treatment. The findings are summarized as below: The changes of the liver in control rabbits were relatively mild until 2 weeks after infection. However, widening and thickening of bile ducts, proliferation of biliary epithelium and periductal fibrosis were moderate after 4 weeks from infection and those changes were severe after 8 weeks and 7months. Goblet cell metaplasia was found after 8 weeks from infection. The mild changes of 2-week infection group were completely recovered by 4 weeks after the treatment. In the groups of 4 or more weeks after infection, the changes of bile ducts became milder in the degree after the treatment, but were still found 12 weeks after the treatment. As the infection duration was passed, more severe changes were observed after the treatment. In this context, it is concluded that the liver changes of acute clonorchiasis in the early two weeks are reversible by treatment while chronic biliary epithelial changes are irreversible. Therefore, early treatment should be recommended as possible to minimize the remaining histopathological changes of liver in clonorchiasis.
parasitolgy-helminth-trematoda ; Clonorchis sinensis ; liver ; clonorchiasis ; chemotherapy ; praziquantel ; pathology ; rabbit

In this study, to understand the pathogenesis of new rabbit hemorrhagic disease virus (RHDVa) serotype, we carried out to administrate RHDVa to rabbits, and to examine sequential electron microscopic changes and relationship between pathogenesis and apoptosis. TUNEL-positive cells began to be observed from 24 hours after inoculation (HAI) and the number of positive cells was slightly increased with the course of time. Whereas marked increase of positive cells was seen in the liver from the rabbits died acutely. Typical viral particles with cup-like projections and a diameter of 30~40 nm were detected in homogenized liver samples and tissues at 36 and 48, and 48 HAI, respectively. Ultrastructurally, glycogen deposition was observed from the first stage of hepatocellular degeneration by RHDVa infection and then, swelling and disruption of cristae of mitochondria by viral particles, swelling of smooth endoplasmic reticulum, vacuoles and vesicles were detected. Condensation, margination and fragmentation of chromatin were observed in degenerative hepatocytes at 36 and 48 HAI, indicating apoptotic bodies. These data offer that hepatocytic apoptosis by RHDV infection could be closely related with mitochondrial impairment in the hepatocytes.
Apoptosis ; Chromatin ; Electrons ; Endoplasmic Reticulum, Smooth ; Glycogen ; Hemorrhagic Disease Virus, Rabbit ; Hepatocytes ; Liver ; Mitochondria ; Rabbits ; Sprains and Strains ; Vacuoles ; Virion

In order to confirm whether the migrating larvae of parasites could carry pathogenic organisms into liver and cause hepatitis, a series of experiments has been carried out. Clonorchis sinensis: Recovery rate of larvae in the abdominal cavity of rabbits: One to seven days after the administration adolescariae were recovered from the abdominal cavity in less than l percent of the total number of metacercariae given. Generally, 1-6 larvae were found from each animal which was given 900-1,000 metacercariae, though many larvae were already found in the common bile ducts or remained still in intestine. Fate of Clonorchis sinensis in abdominal cavity: The young or mature worms which were introduced directly into the abdominal cavity were examined l5, 32, 40 and 42 days after the inoculation. Several larvae were found on the surface of liver in four animals. All the worms on the surface of the liver were dead and the biopsied liver tissues on the area where the worms were attached showed no pathological changes. Two of them were between bile duct and liver tissue but pus cell infiltration surrounding them was observed. In every case, pus cell infiltration was found in the peripheral portion of the liver and pus nodules on the surface of intestine and mesentery. The nodule in the intestinal wall contained the eggs of Clonorchis sinensis. Two worms in the abdominal cavity were still alive. From the above results it is suggested that the larvae of Clonorchis sinensis were capable of penetrating the intestinal wall and reaching the organs in the abdominal cavity and surviving for l5-42 days, but they were unable to penetrate the organs. No bacterial flora appeared from the lesion by culture method. Fate of Clonorchis sinensis which was inoculated into the peripheral region of liver: Small abscess was observed at the same area. Microscopically, the area became edematous and the vessels in the peripheral region were dilated. The parasites became necrotic and amorphous. Pathologically the lesions appeared as eosinophilic masses and neutrophile leukocytes were infiltrated surrounding the masses. In some cases, the dead worms were found apart from the original place of inoculation but no leukocyte infiltration was found. There was linear infiltration between the original site and the portion where the dead worm was found. The distance from the capsule varied from 0 to 4 mm. Sometimes, the eggs of Clonorchis sinensis were also found. In all cases, there were no living worms in liver tissues and hepatic ducts. In all case,. the bacteriological examination was negative. Do clonorchis sinensis transfer the microorganism? Five adult worms of clonorchis sinensis were incubated in the saline solution containing Staphylococcus aureus. The intestinal contents of these worms were cultured in the Nutient-agar plate and examined by Methylene Blue and Gram's stain. The area of liver tissue where the Clonorchis sinensis were inoculated showed no inflammatory changes after the 3 days of inoculation but no living Staphylococcus aureus was found in the culture media with which the pieces of liver tissues were smeared. Hookworm: Cutaneous infection: Four to eight days after the cutaneous infection of Ancylostoma caninum, the mice were sacrificed. Grossly, there was no abnormal finding in liver. The pieces of liver tissues were smeared on the Nutrient-agar plate, and cocci were found in four out of six examined. The microorganism were confirmed as the same species of Diplococcus pneumoniae which were grown in the hookworm culture media. Oral infection: 1,000 filariform larvae of Ancylostoma caninum were given orally. 24 hours later, the mice were sacrificed and the pieces of liver tissue were smeared on the Nutrient-agar plate. After 50 hours at 36 C, the bacterial colonies were examined bacteriologically. Staphylococcus albus was found from two out of four samples. Grossly there was no abnormality on the surface of liver, but microscopically there were spots like microabscesses which were infiltrated by leukocytes. The larvae were also found from other portions of liver tissues and they were surrounded by yellow colored material. In another experiment, a combination of Ancylostoma duodenale and Staphylococcus aureus was fed to mice. The mice sacrificed five days after the oral administration of Ancylostoma duodenale cultivated in the media containing Staphylococcus aureus. The liver pieces were examined routinely. The larvae cultivated in normal tap water which contained no Staphylococcus aureus was used as control. In the experimental mouse, the cocci appeared in the liver. Pathologically, microabscesses infiltrated with neutrophile leukocytes were found, but there was no manifestation of inflammatory change due to Staphylococcus aureus. There was only mechanical trauma due to the larvae penetration. Haemorrhage appeared only where the larvae were found.
parasitology-helminth-trematoda-nematoda ; Clonorchis sinensis ; Ancylostoma caninum ; pathogenicity ; Staphyllococcus aureus ; Staphyllococcus albus ; Diplococcus pneumoniae ; rabbit-liver

Rabbit hemorrhagic disease is a highly acute and fatal viral disease caused by rabbit hemorrhagic disease virus (RHDV). Since first outbreak in Korea 1987, RHDV has been continually affected in the country, but the pattern of outbreak seem to be changed. In this study, to understand the pathogenesis of the new RHDVa serotype, we therefore carried out to inoculate RHDVa to rabbits, and to examine the sequential histopathologic changes and viral distribution. Macroscopically, various sized dark red or white spots or appearance were observed in the liver, lung, kidney uterus and ureter. In euhanized rabbits, significant pathologic findings such as infiltration of heterophils and mononuclear cells were observed at 24 hours after inoculation (HAI), and these were sequentially extended periportal to centrilobular area. However, in dead rabbits, severe hepatic degeneration and/or necrosis with relatively weak inflammatory responses were observed. RHDV antigens began to detect in liver, spleen, and lung from 12 HAI by PCR. Immunohistochemically, RHDV positive cells were seen in only liver from 24 HAI, and the degree of immunogen reactivity was stronger in dead rabbits than in euthanized ones. In conclusion, RHDVa caused the subacute or chronic infection accompanying low mortality and moderate to severe inflammatory reaction in rabbits, suggesting the possibility that RHD could become endemic.
Dental Caries ; Hemorrhagic Disease Virus, Rabbit ; Kidney ; Korea ; Liver ; Lung ; Necrosis ; Polymerase Chain Reaction ; Rabbits ; Spleen ; Sprains and Strains ; Ureter ; Uterus ; Virus Diseases

Precipitin reaction between host organ extracts and antisera for Paragonimus and Clonorchis were studied to know whether the antigens from two species of trematodes have common characters with those of host organs. The brain, lung, heart, liver and muscle of rat and rabbit were extracted in 0.1 percent saline solution by freezing-thawing technique. For immunization of rabbit, the extracts of Paragonimus and Clonorchis was emulsified with equal amount of Freund's adjuvant and 1.0 ml of the mixture was injected subcutaneously once every 10 days for 5 times, and 3 days after the last immunization, antisera were prepared. Precipitin bands by agar-gel double diffusion methods were examined between organ extracts of rabbit or rat and Paragonimus or Clonorchis antisera. The results could be summarized as follows: Precipitin bands were appeared between extracts of liver, heart and muscle of rat and anti-Clonorchis rabbit sera but not appeared against brain and lung. Precipitin bands were appeared between extracts of lung, muscle and liver of rat and anti-Paragonimus rabbit sera but not appeared against brain and heart. Precipitin bands were appeared between extracts of liver and muscle of rabbit and anti-Clonorchis rabbit sera but not appeared against brain, heart and lung. Precipitin bands were appeared between extracts of lung and muscle of rabbit and anti-Paragonimus rabbit sera but not appeared against liver. The extracts of Paragonimus westermani showed precipitin bands against liver and brain of rat and liver of rabbit in agar-gel double diffusion.
parasitology-helminth-trematoda ; Paragonimus westermani ; Clonorchis sinensis ; immunolgy ; brain-lung-heart-liver-muscle ; rabbit ; rat ; agar-gel double diffusion ; Freund's adjuvant