Efficacy of strain RB51 vaccine in protecting infection and vertical transmission against Brucella abortus in Sprague-Dawley rats

Immunizing animals in the wild against Brucella (B.) abortus is essential to control bovine brucellosis because cattle can get the disease through close contact with infected wildlife. The aim of this experiment was to evaluate the effectiveness of the B. abortus strain RB51 vaccine in protecting infection as well as vertical transmission in Sprague-Dawley (SD) rats against B. abortus biotype 1. Virgin female SD rats (n = 48) two months of age were divided into two groups: one group (n = 24) received RB51 vaccine intraperitoneally with 3 x 10(10) colony forming units (CFU) and the other group (n = 24) was used as non-vaccinated control. Non-vaccinated and RB51-vaccinated rats were challenged with 1.5 x 10(9) CFU of virulent B. abortus biotype 1 six weeks after vaccination. Three weeks after challenge, all rats were bred. Verification of RB51-vaccine induced protection in SD rats was determined by bacteriological, serological and molecular screening of maternal and fetal tissues at necropsy. The RB51 vaccine elicited 81.25% protection in SD rats against infection with B. abortus biotype 1. Offspring from rats vaccinated with RB51 had a decreased (p < 0.05) prevalence of vertical transmission of B. abortus biotype 1 compared to the offspring from non-vaccinated rats (20.23% and 87.50%, respectively). This is the first report of RB51 vaccination efficacy against the vertical transmission of B. abortus in the SD rat model.

A Three-year Follow-up Study of Human Brucellosis in Korea

BACKGROUND: The first probable case of human brucellosis in Korea was reported in 2002. Since then there has been a gradual increase in the incidence and prevalence of the disease. There has not been any long-term follow-up investigation of the clinical course and serologic profiles of the disease in Korea. The present investigation is a 3 year clinical and serologic follow-up of human brucellosis patients in Chonbuk province. MATERIALS AND METHODS: The investigation involved a total of 11 patients who were positively diagnosed with brucellosis in 2003. Clinical manifestations were evaluated either directly or via telephone interviews. The serologic follow-up was done by standard tube agglutination test (SAT). Negative sero- conversion was defined as an SAT titer less than 1:40. Early convalescence was defined as clinical manifestations of brucellosis observed within 12 months of diagnosis, and delayed convalescence was defined as clinical symptoms persisting longer than 12 months. RESULTS: A total of eleven patients (8 males and 3 females; with a mean age of 45 years) participated in the study. Ten patients were livestock workers and one was a practicing veterinarian. Three years after the initial diagnosis, chronic fatigue and arthralgia were persistently observed in more than 60% of the patients in delayed convalescence. There was no evidence of relapse or reinfection of the disease. The median duration of antibiotics treatment and serologic follow-up were 11 weeks and 22 months, respectively. Negative sero-conversion was noted between 5 and 16 months after the initiation of treatment (with a median 11 months). CONCLUSION: Although the human brucellosis patients developed chronic symptoms such as fatigue and arthralgia even after negative sero-conversion, there was no evidence of any relapse or reinfection. This may mean that antibiotic intervention is not a justifiable recommendation. We also suggest that serologic monitoring be performed for at least 16 months after the initiation of treatment.

A Three-year Follow-up Study of Human Brucellosis in Korea

BACKGROUND: The first probable case of human brucellosis in Korea was reported in 2002. Since then there has been a gradual increase in the incidence and prevalence of the disease. There has not been any long-term follow-up investigation of the clinical course and serologic profiles of the disease in Korea. The present investigation is a 3 year clinical and serologic follow-up of human brucellosis patients in Chonbuk province. MATERIALS AND METHODS: The investigation involved a total of 11 patients who were positively diagnosed with brucellosis in 2003. Clinical manifestations were evaluated either directly or via telephone interviews. The serologic follow-up was done by standard tube agglutination test (SAT). Negative sero- conversion was defined as an SAT titer less than 1:40. Early convalescence was defined as clinical manifestations of brucellosis observed within 12 months of diagnosis, and delayed convalescence was defined as clinical symptoms persisting longer than 12 months. RESULTS: A total of eleven patients (8 males and 3 females; with a mean age of 45 years) participated in the study. Ten patients were livestock workers and one was a practicing veterinarian. Three years after the initial diagnosis, chronic fatigue and arthralgia were persistently observed in more than 60% of the patients in delayed convalescence. There was no evidence of relapse or reinfection of the disease. The median duration of antibiotics treatment and serologic follow-up were 11 weeks and 22 months, respectively. Negative sero-conversion was noted between 5 and 16 months after the initiation of treatment (with a median 11 months). CONCLUSION: Although the human brucellosis patients developed chronic symptoms such as fatigue and arthralgia even after negative sero-conversion, there was no evidence of any relapse or reinfection. This may mean that antibiotic intervention is not a justifiable recommendation. We also suggest that serologic monitoring be performed for at least 16 months after the initiation of treatment.

Persistent infection with Strongyloides venezuelensis in the Mongolian gerbil (Meriones unguiculatus)

To examine the fate of Strongyloides venezuelensis. Mongolian gerbils (Meriones unguicalatus) were orally infected with 1,000 L3 larvae per animal. Altogether, 50 gerbils divided into 5 groups of 10 each were monitored for a period of 570 days to document the kinetics of faecal egg output, adults worm population, morphological development, fecundity, and hematological changes including peripheral blood eosinophilia. This study chronicled a life long parasitism of S. venezuelensis in the gerbil host, and showed that S. venezuelensis infection was quite stable throughout the course of infection and the worms maintained their normal development as evidenced by their body dimension. A progressive loss of body condition of the infected gerbils was observed as the level of infection advanced. However, no detectable pathological changes were observed in the gastrointestinal tract. The present findings indicate that an immunocompetent host, such as the Mongolian gerbil, can serve as a life long carrier model of S. venezuelensis if the worms are not expelled within 570 days after infection.

Immunogenicity and protective efficacy of solubilized merozoite-enriched Theileria sergenti immmunogens III. Characterization of immunodominant peptides

Immunoblot analysis utilizing bovine sera from naturally or experimentally infected with Theileria sergenti were used to determine the immunodominant polypeptides of T. sergenti (Korea isolate).The previously recognized major bands, 18 kDa, 29 kDa, 34 kDa, and 45 kDa, were excised after electrophoresis and trasferred to PVDF membrane. The individual bands were sequenced. The 34 kDa polypeptide which was the most antigenic and immunogenic peptide was observed in the Western blot. However, Chou-Fasman prediction sites (antiginic site) for antigen determinants of the 45 kDa,34 kDa, 29 kDa and 18 kDa polypeptide were 6, 4, 2 and 0, respectively. However, the 45 kDa polypeptide showed no reaction with anti-T. sergenti hyperimmune serum.

Fractionation of antigen for ELISA of bovine fascioliasis

In order to obtain the most specific and sensitive antigen from crude antigens of Fasciola hepatica for the immunodiagnosis of bovine fascioliasis by the enzyme linked immunosorbent assay(ELISA), phosphate buffered saline extract of F. hepatica was prepared. The crude extract was fractionated into 7 antigens using Sephadex G-100 column chromatography. Seven fractionated antigens were applied to ELISA, precipitation test and intradermal test, respectively. Results obtained are as follows: The specificity (95 per cent confidence interval in negative sera of bovine fascioliasis ; Mean+2 x SD of absorbance ) of the first (MW>150,000) and the second antigens (MW 120,000) were 93.7 per cent, but those of others including crude antigen showed 100 per cen.t. The sensitivity (positive sera of bovine fascioliasis having higher values with compared to the criterion) of the first, the sixth (MW 16,000) and the seventh antigen (MW<5,000) were 91.6 per cent, 87.5 per cent and 0 per cent, respectively, but those of others showed all 100 per cent. The absorbance by ELISA using the fifth antigen (MW 26,000) was 8.43-folds higher in the positive sera than that in the negative sera. This could be used as one of the most specific antigens for the immunodiagnosis of bovine fascioliasis. In Ouchterlony test, precipitin lines were not found in the sera naturally infected with F. hepatica, but some were found in the sera of rabbits immunized with the crude antigens. The numbers of precipitin lines in the sera of rabbits were different in the different fractionated antigens. They were 6 in the crude, 2 in the second and the third antigens, 1 in the forth, the fifth and the sixth antigens and absent in the seventh antigen, respectively. The wheal size for the bovine infected with F. hepatica was 2.46+-0.15 cm in the intradermal test antigen (saline extract of F. hepatica) supplied by the Veterinary Research Institute, Rural Development Administration, Korea. The wheal size of the first, the second and the third antigens were larger than that of intradermal test antigen, whereas those of the forth, the fifth, the sixth and the seventh antigens showed smaller than that of the intradermal test antigen. The results suggest that the fifth antigen may be specific antigen for the immunodiagnosis of bovine fascioliasis.

Experimental studies on the second intermediate hosts of Clonorchis sinensis IV. Observations on the fate of metacercariae of Clonorchis sinensis in the fish host, Cultriculus eigenmanni

In order to judge appropriately the suitability as the second intermediate host of Clonorchis sinensis, the present experiments were proposed to survey the metacercaria of C. sinensis in the fish host, Cultriculus eigenmanni, in the field, and to observe, by experimental infection of the fish with Clonorchis cercaria, the penetrating ability of the cercaria, maturity, the process of degeneration and extinction, and infectivity of the metacercaria. The following is a brief summary of the leading facts gained through the experiments. All the ten fish (mean body weight 13.4 +/- 3.43 g) which were caught in Ok-ku irrigation reservoir were infected with the metacercariae and the average number of the metacercariae found in the fish body/gram was 4.55. The fish was easily subject to invasion of the cercaria, and the examination of the cercariae after having the fish infected disclosed that 24 hours after the infection, all of the cercariae formed their cysts in muscle and the metacercariae kept growing, that 7 days later the metacercariae were found folding their bodies twice, and that in 15 days the characteristic structure of the metacercariae was complete and made a vigorous rotary movement intermittently. In the case of control fish, Pseudorasbora parva, both penetrating ability of the cercariae and the infective degree of the metacercariae to control host were stronger as compared with the fish host because that the control fish had higher suceptibility to C. sinensis. Afterwards, the metacercariae in the fish host came to a state of maturity and beyond this stage some metacercariae started the process of degeneration and extinction in as early as 30 days, and as more days elapsed, mortality of the metacercariae was not uniformly increased but difference of mortality appeared to be according to the individual of fish host. That is, in 390, 540 and 586 days, all of the metacercariae in the fish hosts became dead, meanwhile almost all of those found in the fish host of 596 days after the infection survived (mortality; 8.51 percent). Percentage of orally administered metacercariae developing to maturity in rabbit is 10.46 percent in the last case. As the days were progressed the shapes of the metacercariae in the fish host tended to change from ellipsoid to almost round shapes, the length became shorter while the width was wider. The results shown above revealed that C. eigenmanni was, though it could not be proper host than P. parva, suitable as the second intermediate host in addition to our previous observation results.

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.

Survey on encysted cercaria of trematodes from fresh-water fishes in Tongjin riverside areas in Korea

In an attempt to clarify the epidemiological feature of distomiasis in Tongjin riverside area, the prevalence of distomiasis in the residents and infection rates of the metacercariae in fresh-water fishes were investigated at the upper, middle and lower reaches of the river from January to April 1984. The results obtained were summarized as follows: Out of a total of 931 fresh-water fishes which composed of 33 different species, 611 fishes (65.6 percent) of 31 species were found positive with digenetic trematode metacercariae of 16 different species, and there were some differences in infection rates of the metacercariae among the fishes in the3 parts of the river; 53.8 percent in upper, 80.7 percent in middle, and 61.0 percent in lower reaches, respectively. Infection rates of the metacercariae of Exorchis oviformis, Metagonimus yokogawai, Echinochasmus japonicus, Metorchis orientalis and Clonorchis sinensis in the fishes were 48 percent, 29 percent, 11 percent, 7.9 percent and 6.3 percent, respectively. The average number of the encysted larvae of Clonorchis found in fish body/gram showed 4.44 in Pseudorasbora parva, Gnathopogon coreanus (1.2), Microphysogobio yaluensis(0.76), Abbottina springeri(0.4), Acanthorhodeus asmussi (0.21) and Cultriculus eigenmanni (0.17), respectively. The average number of the metacercariae of Metagonimus found in fish body/gram disclosed 34.01 in Zacco platypus, Zacco temmincki (16.46), Carassius carassius (5.35), Moroco oxycephalus (1.54), Aphyocypris chinensis (1.5) and etc., respectively. Detection rates of the eggs of Clonorchis and Metagonimus among residents were 1.1 percent and 0.8 percent, respectively, out of a total 923 persons.

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.

Survey on encysted cercariae of trematodes from fresh-water fishes in Mangyeong riverside area

In order to clarify infestation pattern for the encysted larvae of digenetic trematodes from fresh-water fishes, this survey was carried out from March to September, 1983. A total of 380 fishes of 32 species were collected with netting at the three reaches, upper, middle and lower in Mangyeong riverside area. After the fishes were dissected into small scraps, they were pressed under cover glass and examined for the presence of those of digenetic trematodes with a microscope. The results obtained were as follows: Out of a total of 380 fishes inspected, 320 fishes (84 percent) from 31 species were found positive with digenetic trematode metacercariae; more than 10 species of the metacercariae were detected in Pseudorasbora parva; Gnathopogon majimae, Microphysogobio yaluensis, Cultriculus eigenmanni and Gnathopogon coreanus (more than 8 species); Aphyocypris chinensis(8 species) and etc. respectively. Clonorchis sinensis metacercariae were found positive from 93 fishes (25 percent) from 12 species detection rates in other species of digenetic trematode metacercariae from various fishes were; Exorchis oviformis, 261 fishes (57 percent) from 28 species; Cyathocotyle orientalis, 47 fishes (12 percent) from 12 species; Metorchis orientalis, 21 fishes (6 percent) from 12 species; Metagonimus yokogawai, 164 fishes (43 percent) from 26 species; Pseudexorchis major, 71 fishes (19 percent) from 18 species; Metacercaria hasegawai, 77 fishes (20 percent) from 25 species; Centrocestus armatus, 24 fishes (6 percent) from 7 species; Echinochasmus japonicus, 2 fishes (0.5 percent) from 2 species, and unidentified species, 34 fishes (9 percent) from 15 species respectively. The sums of average number of the encysted larvae of all species found in fish body/gram showed 83 in P. parva, Cobitis taenia (74.2), A. chinensis (28.5), Pseudoperilampus uyekii (26.6), G. majimae (19.6) and etc. respectively and the average peak number of each metacercaria in fish body/gram showed 21.7 C. sinensis, 24 E. oviformis , 15.3 M. orientalis and 6.1 E. japonicus in P. parva ; 42.7 C. orientalis and 25.1 M. yokogawai in C. taenia; 8.3 C. armatus and 8.3 M. hasegawai in P. uyekii; 6.3 P. major in Carassius carassius, and 2.9 unidentified species in G. majimae respectively.

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.

Localization and isolation of anthelmintic activity on Clonorchis sinensis from stem cortex of Machilis thunbergii

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.

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.

The wormicidal substances of fresh water fishes on Clonorchis sinensis II. Preliminary research on the wormicidal substances from mucous substances of various fresh water fishes

The present work which was investigated in July 1979, was to observe the wormicidal effects of the external mucous substances of 9 species of fresh water fishes (Cyprinus carpio, Parasilurus asotus, Anguilla japonica, Ophicephalus argus, Carassius carassius; golden crusian carp, Misgurnus anguillicaudatus, Zacco platypus, Pseudorasbora parva and Carassius carassius; crusian carp) on cercaria, liberated metacercaria and adult of Clonorchis sinensis. The mucous substances extracted by ether from the above 9 species of fishes were separated into many spots on the silica gel thin layer chromatography, and divided into many fractions in the silica gel column chromatography which used petroleum ether (30 percent) and chloroform (70 percent) as a solvent. On the silica gel thin layer chromatography of ether extracts from 9 species of fresh water fishes, each of the Rf. values which had wormicidal effect on the cercaria of C. sinensis were different from others; Rf. value of C. carassius (crusian carp) was 0.937, 0.709 in O. argus, 0.612 in A. japonica, 0.576 in P. asotus, 0.451 in C. carpio, 0.701 in Z. platypus, 0.385 in C. carassius(golden crusian carp) and 0.15 in P. parva. Time for wormicide was different from each other, too. It took 14 min. in a case of C. carassius(crusian carp), 25 min. in Z. platypus, 26 min. in C. carassius(golden crusian carp), 28 min. in C. carpio, 30 min. in P. asotus, 35 min. in O. argus, 40 min. P. parva and 180 min. in A. japonica. But any of the spots of M. anguillicaudatus did not show wormicidal effect on the cercaria within 10 hours. In the silica gel column chromatography of ether extracts from 9 species of fresh water fishes, the fractions which had wormicidal effect on the metacercaria of C. sienesis were different from each other; in a case of C. carassius(crusian carp), the first fraction only had wormicidal effect, the 2nd in P. asotus, the 3rd in O. argus, the 4th in C. carassius(golden crusian carp), the 2nd in C. carpio and the 4th in P. parva. Time for wormicide was 40 min. in C. carassius(crusian carp), 52 min. in P. asotus, 74 min. in C. carassius(golden crusian carp), 92 min. in O. argus, 95 min. in C. carpio and 140 min. in P. parva, but any of the fractions of A. japonicas, M. anguillicaudatus and Z. platypus did not show wormicidal effect within 10 hours. The fractions which had wormicidal effect on the metacercaria of C. sinensis had also the same effect on the adult, but it took longer time to kill them. It took 3 hours in a case of C. carassius(crusian carp), 4 hrs. in C. carassius(golden crusian carp), 5 hrs. in P. asotus, 6 hrs. and 6 min. in O. argus, 6 hrs. and 10 min. in C. carpio and half and 6 hrs. in P. parva. But any of the fractions of A. japonica, M. anguillicaudatus and Z. platypus did not show wormicidal effect within 24 hrs.

The wormicidal substances of fresh water fishes on Clonorchis sinensis III. Seasonal variation on the wormicidal substances of mucous substances of Carassius carassius

The epidermal mucous substances of C. carassius which were extracted by ether were separated into various spots on the silica gel thin layer chromatography and separated into various fractions in the silica gel column chromatography, using petroleum ether (30 percent) and chloroform(70 percent) as a solvent. The wormicidal effects of the spots and the fractions on the cercaria, the liberating metacercaria and the adult of C. sinensis, were observed by month giving attention to ambient water temperature. Among the four spots of the thin 1ayer chromatography of the ether extracts from C. carassius, the uppermost first spots (Rf. 0.924-0965) had a strong wormicidal effect on the cercaria in May, June, July, August and September, and the cercaria which came in contact with the materials was killed in 29-49 min., but in other months (Rf. 0.612-0.752) it was killed only after 50-115 min. In column chromatography of ether extracts from C. carassius, the first fractions which had a wormicidal effect killed the metacercariae within 38-163 min. in May, June, July, August, and September, but it took about 120-458 min. to kill them in other months, meanwhile the wormicidal time of the fractions on the adults was 445-835 min. in May, June, July, August and September, but was 878-2,130 min. in other months. The Rf. value of the uppermost first spot of the thin layer chromatography of ether extrart from the mucous substance of C. carassius which was reared in the aquarium controlled at 23 C was 0.79, and its first fraction of column chromatography killed the metacercaria and the adult of C. sinensis within 315, and 1,850 min. respectively. As summarized above, the wormicidal materials of the epidermal mucous substances of C. carassius had no relation to ambient water temperature, but the secretion period of this wormicidal materials was the same as the liberating period of the cercariae from Parafossarulus manchouricus.

The wormicidal substances of fresh water fishes on Clonorchis sinensis I Preliminary research on the wormicidal substance from mucous substance of Carassius carassius

The present work was to observe the wormicidal effects of the external mucous substance of Carassius carassius and Pseudorasbora parva on cercaria, metacercaria (liberating) and adult of Clonorchis sinensis. The mucous substance which was extracted by ether was separated into various spots on the silica gel thin layer chromatography and separated into various fractions in the silica gel column chromatography, using petroleum ether(30 percent) and chloroform(70 percent) as solvent. R(f) 0.952 value of the silica gel thin layer chromatography and the first fraction (reddish yellow) frorn the silica gel column chromatography of the ether extracts from C. carassius had the wormicidal effect on all of the developing stages of C. sinensis. Cercaria was killed within 14 minutes, metacercaria within 27 minutes and adult 2.5 hours by the first fraction. But in the normal saline solution (control) and in the other fraction, cercaria and metacercaria were alive more than 24 hours and adult more than 72 hours. The extracts from the mucous substance of P. parva that is well penetrated by cercaria had no wormicidal effect on them.