• Korean Journal of Fisheries and Aquatic Sciences
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• v.20 no.4
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• pp.317-327
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• 1987

As a series of studies on the suitability as a second intermediate host of Clonorchis sinensis, artificial infection experiments were applied to Tilapia mossambica. And then, in order to elucidate the defence mechanism of the fish to Clonorchis, clonorchicidal substance in the epidermal mucus of the fish was isolated by silica gel column and thin layer chromatography and analyzed for its chemical structure by UV, IR and NMR-spectroscopy. The results obtained are summarized as follows: 1. The cercariae which attempted to contact with the fish in the water were observed under stereomicroscope. After contact, the cercariae began to separate their tail from the body after several minutes and then the number increased to $80\%$ more than 10 minutes after the encounter. But very few cercariae could actually invade the epidermis of the fish. 2. The fish were reared with Parafossarulus manchouricus which were shedding numerous cercariae of Clonorchis in the aquarium for 24 hours. Only a few cercariae could invade the epidermis but most of the invaded cercariae died out before forming their cysts. Very few number of the remaining encysted cercariae were also found to be in a state of suspended animation within 42 hours. 3. In the cases of the control fish, Pseudorasbora parva, numerous cercariae of Clonorchis were found to invade the fish through the epidermis under stereomicroscope. Then many metacercariae of Clonorchis were also found in the fish while they were kept in the aquarium. 4. A sample of the epidermal mucus of Tilapia mossambica was extracted with ethly ether 6 times repeatedly. In silica gel column chromatography, using petroleum ether: chloroform/30:70(v/v) as a first solvent and MeOH as a second solvent, the extract was fractionated into the yellow and brownish red solutions in the first solvent and the clonorchicidal brownish yellow solution in the second sovent. 5. The clonorchicidal brownish yellow solution was added to petroleum ether, and the mixture was stored for 5 days at $5^{\circ}C$ and was, then, separated into supernatant fraction and precipitate. Ten mg/ml of the supernatant fraction killed, in vitro, the excysted metacercariae in 45 minutes but the precipitate in 600 minutes. 6. In silica gel column chromatography, using acetone: benzene/10:90(v/v) as a solvent, the more clonorchicidal supernatant fraction was fractionated into the first fraction with Rf. 0.2966 and the second fraction with Rf. 0.072. In vitro, 10mg/ml of the first fraction killed the excysted metacercariae in 28 minutes, the second fraction in 80 minutes and the first fraction was, therefore, determined to be a final clonorchicidal substance. 7. By this purification procedures, the most clonorchicidal substance from the epidermal mucus of Tilapia mossambica was purified 71-folds with $0.2075\%$ yield. Infra red, nuclear magnetic resonance and ultraviolet spectrometric analysis of the purified substance revealed that the substance is linoleic acid. According to the results of the present studies it seemed that this species can not serve as a proper intermediate host of Clonorchis sinensis, and that defence mechanism to the fluke seems to be correlated with linoleic acid in the epidermal mucus of this species.

• Korean Journal of Veterinary Research
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• v.36 no.3
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• pp.681-692
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• 1996

The present study was conducted to isolate Babesia gibsoni by culture method of the microaerophilous stationary phase(MASP) and analyse the antigenic properties of the parasite by SDS-PAGE and immunoblot. The results obtained were summarized as follows. The protozoan parasite Babesia gibsoni multiplied in canine erythrocytes in RPMI 1640 medium(pH7.0) containing 20 40% normal canine serum under the MASP condition of 5% CO2 and 95% air at $37^{\circ}C$ incubator. The levels of parasitaemia in the erythrocytes were shown more higher by exchanging the medium at 24 hours interval. Under the above condition of MASP, the percentage of parasitized erythrocytes(PPE) after incubation for 8 days increased about 14 times more than that in the initiation of the 1% infected canine erythrocyte culture. The parasites were purely isolated from the MASP culture of red blood cells collected from dogs infected with Babesia gibsoni naturally or artificially. Among the total of 36 canine(Pit-bullterier) blood samples the parasites were isolated from 17 cases(47.2%) in the MASP culture while the parasites were detected from 20 cases(56%) and 12 cases(33.3%), respectively, by indirect fluorescent antibody(IFA) test and direct light microscopy(DLM). On the other hand, Babesia gibsoni was isolated by MASP culture from 15 cases(75%) and 11 cases(92%) of positive cases of IFA and DLM, respectively. In the analysis of the erythrocytic merozoite(AEOM) antigen derived from infected dog approximately 11 antigenic bands in molecular weight of 130, 120, 97.4, 92, 80, 52, 50, 42, 36, 30 and 29 KDa were observed on SDS-PAGE. Antigenic bands in the endoerythrocytic merozoite(CEOM) antigen derived from infected erythrocyte (sediment) in MASP culture were much similar to those of AEOM bands. In the exoerythrocytic merozoite(CEEM) antigen derived from supernatant of the infected erythrocyte culture approximately 20 antigenic bands were observed and the molecular weight of the major bands among these were 140, 120, 114, 105, 96, 93, 92, 80, 60, 52, 50, 38, 36, 30, 24, 18.5 and 16 KDa. In the protein patterns of AEOM and CEOM antigen by immunoblot 15 bands were observed and these patterns were much similar between each other. The molecular weight of the major bands in the both antigens were 130, 120, 80, 60, 52, 50, 42, 30, 29, 18.5 and 16 KDa. Approximately 21 bands were observed in CEEM antigen and the molecular weight of the major bands were 140, 120, 96, 92, 85, 80, 76, 60, 52, 50, 37, 30, 24, 16 and 15 KDa. The specific antigenic bands in the artificially infected dogs were firstly observed at 3 weeks afrer inoculation of infected blood and these antigenic bands were maintained up to 18 months after inoculation. In the immunoblot of the sera of the splenectomized dogs the specific antigenic bands with the molecular weight of 93 KDa and 52 KDa, respectively, were observed weakly comparing to those of non-splenectomized dog. In immunoblot of the sera collected from the naturally infected dogs the antigenic bands were observed as same as those of artificially infected dogs while antigenic band of 29 KDa in some individual dog showed strongly. In comparison of immunoblot of the sera collected from dogs non-treated and treated with diminazene aceturate(7mg/kg, IM) after artificial infection no differences of antigenic bands were observed. In analysis of antigenic bands by digoxigenin glycan/protein double labeling, antigenic bands in the molecular weight of 106, 60 58, 36, 30 and 29 KDa were determined as glycoproteins.

• Journal of Chest Surgery
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• v.29 no.2
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• pp.177-184
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• 1996

Between 1987 and 1994, 21 patients were treated surgically for aortic aneurysm involving the thoracoabdominal aorta. There were 11 males and 10 females, and their age ranged from 20 to 67 years old and mean age was 41.5 years. Many complained of back pain, chest pain or discomfort, and flank pain, but three patients were asymptomatic. 15 patients had chronic dissection (71.4%) and 6 had nondissecting fusiform or saccular aneurysm(28.5%), and of those 15 patients with chronic dissection, 6(28.5%) had atherosclerosis assniated with hypertension, 5 (23.8%) were Martian syndrome, and 2 (9.5%) were associated with pregnancy. The diameter of an aneurysm ranged from 6cm to 12cm, and their extent was classified as type I in 7(33.3%), type II in 8(38.1%), type III in 3(14.3%), and type IV in 3(14.3%) patients based on Crawford classification for TAA . Diseased aorta was replaced with artificial vascular graft in all but one patient. In whom the aortic tear site due to pseudoaneurysm was closed by primary suture. For the spinal cord protection during the operation, we used partial cardiopulmonary bypass (FV-FA or PA-FA bypass) in 12 patients (57.1%), Biopump (LA-FA bypass) in 4(19.0%), total circulartory arrest and CPB in 2 (9.5%), Gott's heparinized shunt in 1(4.7%), and simple aortic cross clamping in 2 (9.5%). The most common complication after the operation was hoarseness due to unilateral vocal cord palsy which onured in 5 patients (23.8%), and the next common complication was wound infection in 4 patients(19.0%), paraplegia in 2 patients (9.5%), chylothorax in 1 patient(4.7%). The hospital mortality rate was 9.5% (2deaths), and there was no late death. Our experience shows that the graft replacement of TAAA had reasonable rate of mortality, low rate of serious complication, and provided good post operative state of the pati nts, and since the thoracoabdominal aortic operation is not a high risky procedure anymore, we recommend a radical operation for the indicated patients.