The wormicidal substances of fresh water fishes on Clonorchis sinensis VII. The effect of linoleic acid and ethyl linoleate on parasite viability

In an attempt to analyze the clonorchicidal activity of linoleic acid and ethyl linoleate in vitro, the wormicidal effects on Clonorchis sinensis were chronologically monitored in dose titration experiments. Excysted metacercariae were killed within a period of 31.0+-4.0 min, 149.3+-4.l min and 207.0+-13.5 min with 100.0 mg, 0.1 mg and 0.001 mg linoleic acid, respectively. The time required for the linoleic acid to kill adult worms was 167.0+-0.8 min with 100.0 mg, 253.0+-0.8 min with 0.1 mg, and 277.0+-0.8 min at 0.001 mg titration. Clonorchicidal activity of ethyl linoleate was relatively delayed as death was observed within 263.3+-2.9 min, 286.0+-0.5 min, and 318.0+-0.8 min for 100.0 mg/ml, 0.1 mg/ml and 0.001 mg/ml concentrations, respectively. The wormicidal effects observed with these pure anticlonorchal substances were found to be similar to the biological activity of native products derived from the mucus of the fresh water fish.

Karyotype of Fasciola sp. obtained from Korean cattle

As a series of systematic classification for Korean common liver fluke, Fasciola sp., karyotype was investigated by means of the modified air-drying technique and of the regular Giemsa staining, Also, C-staining method was applied for detailed karyological analysis from the germ cells of the fluke. The following is a brief summary of the leading facts gained through the experiment. Korean Fasciola sp. was classified into three types based on their chromosomal complements; individuals with 20 or 30 chromosomes and with a 20/30 mosaic constitution. Worms having 30 chromosomes represent a triploid form with 3 sets of 10 basic chromosomes, while those with 20 chromosomes were diploid and mosaic individuals were 2n/3n mixoploid. The frequency of the individual type calculated is as follows; 67.45% of 212 flukes examined was of diploid, 10.85%, triploid, and the rest, 21.7%, mixoploid, respectively. In many cases, two or three types were found in the peculiar bovine host while single type inhabitant was about 20% out of 52 cases. The twenty chromosomes consisted of 1 parir of large metacentrics, 4 pairs of medium-sized subtelocentrics, and 5 pairs of small submetacentrics, while constitution of the thirty chromosomes was nearly interpreted as a triploid form with 3 sets of 10 basic chromosomes. The high centromeric indexes of both types are the first pairs among all the examined, and 37.93% was of diploid and 47.93%, triploid, respectively. In mixoploid individuals, constitution of the chromosomes of diploid or triploid cells was the same as that of diploid or triploid individuals. All the chromosomes of the germ cells in both types showed C-band around the centromeic region and especially the chromosomes no's 3, 7, and 8 showed a remarkable C-band distinguished from other chromosomes. The variance for the sizes of the worms and the eggs were not parallel with three different genotypes in Korean common liver fluke.

Application of micro-ELISA in serodiagnosis of fascioliasis in cattle

Fascioliasis in cattle is one of the most common and very serious trematode diseases in Korea. In the present study, the enzyme linked immunosorbent assay (ELISA) was applied in the diagnosis of fascioliasis using antigen of Fasciola hepatica, peroxidase of conjugate anti-cattle IgG and orthophenylenediamine as a substrate by micro-method technique of Voller et al. (1976b) and MacLaren (1978) with a slight modification. Results obtained from the present study are as follows: In assay for optimal dilution of stock antigen, the antigen (protein contents; 0.8 mg/ml) was diluted from 1/50 to 1/600 with carbonate buffer (pH 9.6), and then absorbance values were measured with 1/100 diluted sera. The regression equations between the OD values of ELISA and dilution of antigen were log Y=-0.181-0.00127X in infected sera, and log Y= -0.578- 0.000879X in normal sera. The significantly higher (p<0.05) OD value was observed in the former. In assay for optimal dilution of sera, the sera were diluted from 1/25 to 1/400 with in PBS/ Tween 20(pH 7.4), and absorbance values were measured with 1/200 diluted antigen. The regression equation between the OD values of ELISA and dilution of sera were log Y=-0.1540-0.0007238X in infected sera and log Y=-0.4834-0.00116X in normal sera. The former was higher than the latter (p<0.05). In the 27 cases of negative intradermal test, OD values of the ELISA are 0.447 +/- 0.144, the 95 percent confidence interval (Mean+2 x SD) of the values was 0.735, and there was no case over the values. Therefore, the sensitivity of the antigen to diagnose fascioliasis was 100 percent in the negative case. The OD value 0.7 which is designed as a criterion (detection level of positive one) is useful for the performance of the ELISA in fascioliasis. According to the OD value of criterion in the regression equations, the optimal dilutions of stock antigen and serum were 1/250 and 1/100, respectively. In the 58 cases of fascioliasis from which the adult could be found in the bile ducts, the OD value was 0.846+0.224(-0.224). The 75 percent (44 cattle) among them had higher value with compared to the criterion, and the 60 percent (20 cattle) of the cases of proliferative cholangitis of 33 cattle which had been infected previousely with Fasciola sp. is higher than the criterion. Prevalence of fascioliasis was 43.4 percent in the application of the ELISA to 272 cattle which were reared in Jeonbug district.

The wormicidal substance of fresh water fishes on Clonorchis sinensis V. Purification and chemical characterization of clonorchicidal substance from epidermal mucus of Cyprinus carpio

As a series of studies to clarify clonorchicidal substances in body surface mucus of some fresh-water fishes, the substance in the epidermal mucus of Cyprinus carpio was isolated by silica gel column and thin layer chromatography and analyzed for its chemical nature. Wormicidal trial was done in vitro, and the results obtained are summarized as follows: The mucus was extracted by ethyl ether and separated into 4 fractions by column chromatography using benzene as solvent. The second fraction with yellowish red colour among them showed the strongest clonorchicidal effect. The yellowish red fraction obtained by column chromatography was then fractionated into 6 spots by thin layer chromatography with petrol. ether/chloroform(30/70, v/v), and the Rf. 0.714 spot among the 6 spots showed the strongest effect. The Rf. 0.714 spot was further fractionated into 6 spots by thin layer chromatography with benzene/acetone (90/10, v/v), and the Rf. 0.800 spot among the later 6 spots revealed the strongest effect. The Rf. 0.800 spot was chromatographed on column with benzene and 2 fractions were obtained. The second fraction of light brown colour represented the final purified fraction. By these purification procedures, clonorchicidal substance was purified 15-fold with 0.03 percent yield from the mucus of C. carpio, and 10mg of the final fraction killed the cercaria in 26 min, the metacercaria in 115 min, and the adult in 443 min. Infra red and nuclear magnetic resonance spectrometric analysis of the purified substance revealed that the substance belongs to an ethyl ester of unsaturated fatty acid with 2 double bonds, 15 methylene groups and l methyl group.

Epidemiological studies of Clonorchis sinensis in Mangyeong riverside areas in Korea

In an attempt to clarify the epidemiological feature of C. sinensis in Mangyeong riverside area, the prevalence of clonorchiasis, infestation rate of the cercariae in Parafossarulus manchouricus, and detection rate of the metacercariae in fresh-water fishes were investigated from March 1 to September 30, 1983 at the upper, middle and lower reaches of the river. The results obtained were summarized as follows:Detection rate of C. sinensis egg among inhabitants was 8.2 percent out of a total of 1,266 persons. but the differences in detection rates were not statistically signifcant among upper, middle and 1ower reaches. According to sex, the detection rates were 10.3 percent in male and 6.1 percent in female (p<0.05), but by age groups, increases of the rates were observed as increase in age (p<0.05). Out of a total of 380 fresh-water fishes of 32 different species, 93 fishes (25 percent) of 12 species were found positive with Clonorchis metacercariae, and there were differences in infection rates of the metacercariae among the fishes in 3 parts of the river; 11 percent in upper, 35 percent in middle, and 34 percent in lower reaches respectively. The metacercarial detection rates from various fishes were 97 percent in Pseudorasbora parva, Cultriculus eigenmanni (85 percent), Gnathopogon strigatus (67 percent), Microphysogobio yaluensis (50 percent), Gnathopogon coreanus (47 percent), Pungtungia herzi(44 percent), Abbottina rivularis (40 percent), Moroco oxycephalus (33 percent), Coreoleuciscus splendidus (32 percent), Gnathopogon majimae (26 percent), Rhodeus ocellatus (7 percent), and Aphyocypris chinensis (3 percent) respectively. Although very few P. manchouricus were collected at upper reach, 12 snails (0.7 percent) among a total of 1,713 were found infected with Clonorchis cercariae. Also the cercariae of Echinochasmus japonicus (7.99 percent), Lexogenes liberum (0.99 percent), Cyathocotyle orientalis (0.75 percent), Exorchis oviformis (0.23 percent) and Asymphylodora japonica (0.05 percent) were detected from the snails.

Experimental Infection of Clonorchis sinensis to Cyprinus carpio nudus

As a part of observation on the suitability as a second intermediate host of Clonorchis sinensis, various infection experiments were tried to Cyprinus carpio nudus. The results obtained were summarized as follows: The cercariae which attempted to contact with Cyprinus carpio nudus in the water were observed under the stereomicroscope. After contact, cercariae began to separate its tails from the bodies at 4 minutes increasingly, and then the number ranged to 80 percent at 13 minutes after the encounter. But very few cercariae could actually invade into the epidermis of the fish. When Cyprinus carpio nudus were exposed to a number of cercariae in the beaker, only a few cercariae could invade through the epidermis. Most of the invaded cercariae were killed before forming the cyst. Also rare encysted cercariae were found to be dead within 48 hours. Cyprinus carpio nudus were reared with Parafossarulus manchouricus shedding numerous cercariae in the indoor aquarium. When observed after 12 days, to determine the susceptibility, no metacercaria was found infected. Cyprinus carpio nudus were reared with Parafossarulus manchouricus shedding numerous cercariae in Kimhae lake for 2 months in summer of 1982, in order to check the natural infection. No metacercaria of Clonorchis sinensis was found from the fish. In the cases of control fish, Pseudorasbora parva, numerous cercariae were inspected on the course of invasion through epidermis of the fish under the stereomicroscope. And many metacercariae were also found from the fish whenever they were kept in beaker, indoor aquarium, or the lake for natural infection. The results suggested that Cyprinus carpio nudus might not be proper intermediate host of Clonorchis sinensis.

The wormicidal substance of fresh water fishes on Clonorchis sinensis IV. Preliminary research on the wormicidal substance from mucus of Cyprinus carpio nudus

As a series of studies on the suitability as a second intermediate host of Clonorchis sinensis, the wormicidal effects of mucus from epidermis of Cyprinus carpio nudus were observed. The results obtained were summarized as follows: On the silica gel thin layer chromatography with the greenish yellow supernatant in acetone:benzene/10:90 as carrier, seven spots were observed in iodine chamber. The spot of Rf. 0.225 value among them disclosed the strongest wormicidal effect on the cercaria, the excysted metacercaria, and the adult of C. sinensis. Meanwhile, no seasonal variations were found in Rf. values and effect of the wormicidal fractions. The wormicidal materials in epidermal mucus of C. carpio nudus and Carassius carassius were compared in their effect, and the former was more effective killing substance than the latter. It seemed that C. carpio nudus could not be proper intermediate host of C. sinensis according to results of the present study, in addition to our previous observation results.

Clavate Cells Of Epidermis In Cyprinus Carpio Nudus With Reference To Its Defence Activity To Clonorchis Sinensis

No abstract available.

Isolation of anthelmintic substance on Clonorchis sinensis from tuber of Bletilla striata

The present work was done to isolate the anthelmintic substance from the tuber of Bletilla striata which grows Gangweon-do of the Korea peninsula. The dried B. striata tuber was extracted with ethyl ether by a Soxhlet extractor at 40C for 24 hours, and then the extract was separated into insoluble material (negative wormicidal) and soluble material (positive wormicidal) by petroleum ether solvent. The wormicidal substance was separated in the silica gel column chromatography from the petroleum ether extract, using benzene (90 percent) and acetone (10 percent) as a solvent. The anthelmintic activity was observed in vitro on the cercaria, the excysted metacercaria and the adult of Clonorchis sinensis. On the silica gel thin layer chromatography with the wormicidal substance in benzene:acetone/90:10, five spots were observed in iodine chamber and the spot of Rf. 0.18 value of them appeared the most strong wormicidal effect on the cercaria of C. sinensis. Afterward, a substance which was equivalent to the Rf. 0.18 value spot was isolated from the wormicidal substance by means of silica gel column chromatography using 1 part of ethyl acetate and 6 parts of chloroform carrier, and the excysted matacercaria and the adult of C. sinensis were killed within 14 minutes and 128 minutes in the substance.