• 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.

• Parasites, Hosts and Diseases
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• v.31 no.4
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• pp.347-352
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• 1993

Six early third-stage larvae of Gnnthostoma nipponicum were recovered from the muscle of 376 loaches, Misgurnw anguiILicnudatus, imported from China. They were 614 × 114㎛ in average size, almost colorless except brownish intestine, and encircled by about 229 transverse rows of minute cuticular spines. Their head bulbs provided with 3 rows of hooklets, of which average number were 34.5 on the first, 36.7 on the second and 39.7 on the third. According1y, it is revealed that the Chinese loach is a natural second intermediate host of G. nipponicum and G. nipponicum has been distributed somewhere in China. This parasite may infect human when the imported loaches are consumed raw.

• Journal of agricultural medicine and community health
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• v.8 no.1
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• pp.66-80
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• 1983

As a part of the epidemiological studies of clonorchiasis in Korea, this study was conducted to evaluate the current endemicity and the natural transition of the Clonorchis infection in Goyang Gun a low endemic area in recent years, prior to the introduction of praziquantel which will eventually influence to the status of the prevalence. The data obtained in this study in 1983 were evaluated for natural transition of the infection in comparison with those obtained 16 years ago in 1967 by the author (Kim, 1974). The areas of investigation, villages and schools surveyed, methods and techniques used in this study were the same as in 1967, except for the contents of the questionnaire for raw freshwater fish consumption by the local inhabitants. 1) The current prevalence rate of Clonorchis infection among the inhabitants was 7.5% on the average out of a total of 479 persons examined. The prevalence rate was 9.0% in the riverside area and 4.2% in the inland area. Among the schoolchildren, the prevalence rate was 1.1% out of a total of 1 319 examined. By area, it was 1.4% in the riverside area and 0.7% in the inland area. By sex, the prevalence rate was 13.3% in the male and 1.3% in the female in the inhabitants and no difference was seen in the schoolchildren. 2) In the natural transition of the infection, the prevalence rate in the inhabitants has decreased from 22.5% in 1967 to 7.5% in 1983, and in the schoolchildren, from 9.5% in 1967 to 1.1% in 1983. The reduction rate was higher in the riverside area than in the inland area. 3) In the prevalence rate by age, 1.2% was seen in the 10-14 age group and gradually increased to 8.1% in the 30-39 age group and reached peak 18.1% in the 40-49 age group. By sex, in the male, the prevalence rates have increased to 31.9% and 33.3% in the 40-49 and 50-59 age groups, respectively and decreased thereafter. In the female, the prevalence rate less than 5% was seen only in between the 10-14 and 30-39 age groups. 4) In the natural transition of the prevalence rate by age, sharp decrease was seen in the male from around 50% in 1967 between 15-19 and 30-39 age groups. The generation over 40s showed less decrease. In the female, the prevalence rate has decreased from 13% in 1967 to 5% in 1983 in the middle age groups and dropped to 0% in the rest of the age groups. 5) The intensity of the infection among clonorchiasis cases by mean EPmg (number of eggs per mg feces) value was 1.4. In the inhabitants, the value was 2.0 in the riverside area and 0.4 in the inland area. While in the schoolchildren, the value was 0.2 in both riverside and inland areas. 6) In the transition of the intensity of the infection, EPmg among the inhabitants has decreased from 3.9 in 1967 to 2.0 in 1983 in the riverside area, and from 2.9 to 0.4 in the inland area. In the schoolchildren, the reduction was similar in both riverside and inland areas resulting from 1.0-1.1 in 1967 to 0.2 in 1983. 7) In the intensity of the infection by age, EPmg 3.4 was peak at the 40-49 age group and 0.2-1.0 was seen in the rest of the age groups. The mean value was 1.5 in the male and 0.6 in the female. 8) In the natural transition of the intensity of the infection, the EPmg has decreased from 2.7 in 1967 to 1.4 in 1983. By age, reduction was seen in all of the age groups, particularly in the young and the old age groups of 50s and over, except in the 40-49 age group in which reverse phenomenon was seen. By sex, it has decreased from 3.5 in 1967 to 1.5 in 1983 in the male and from 1.0 to 0.6 in the female. 9) In the distribution of the clonorchiasis cases by the range of EPmg value, 70.3% of the cases were placed in the range of 0.1-0.9 as the most and 16.2% in 1.0-4.9 as the next. With such figures, those included in the range less than 0.9 as light infection were 78.4% and under 5.0-9.9 up to moderate infection 99.3% of the cases were covered. The cases were distributed up to 20.0-39.9 in the male and to 1.0-4.9 in the female. 10) In the transition of the distribution of the clonorchiasis cases by EPmg, the highest intensity reached up to 60.0-79.9 in 1967 and to 20.0-39.9 in 1983. In the range of light infection, under 0.1-0.9, the distribution in rate was 64.5% in 1967 and 78.4% in 1983. Up to the range of moderate infection, under 5.0-9.9, 91.7% in 1967 and 97.3% in 1983 were seen respectively. 11) In a survey for raw freshwater fish consumption among the local inhabitants,78.3 of the clonorchiasis cases interviewed admitted their experience of the raw consumption. However, those who practised in the past two years were 34.8% 55.6% of those who have such experience in the past professed that they did not practise raw freshwater fish consumption in the past two years. 12) The major cause of the reduction of the raw freshwater fish consumption among the inhabitants were the wide spread water pollution in the locality. The most common reason professed for stopping raw freshwater fish consumption among the inhabitants was the risk of the fluke infection. 13) In animal survey, 3.1% of dogs were found infected with Clonorchis, decreasing from 21.6% in 1967. 14) The distribution of the first intermediate host, Parafossarulus manchouricus has greatly diminished in this locality and found only in two localized ponds. No Clonorchis infection was found from the snails examined. 15) The second intermediate freshwater fish host has been further limited by extended water pollution. No susceptible fish host could be examined. 16) In conclusion, the endemicity of Clonorchis infection in Croyang Gun, low endemic area, has significantly decreased during the past 16 years. The major cause of the regressive transition of the infection was the water pollution of the freshwater system of this locality. This has upset the ecosystems of the intermediate host of Clonorchis sinensis in many areas of waterbodies and further discouraged to a significant extent the local inhabitants from raw freshwater fish consumption.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.34 no.6
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• pp.720-728
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• 2001

Cercaria yamagutii Ito, 1957 (C. yamagutii) was found in Lunatia fortuni (L. fortuni) and Neverita didyma (N. didyma) collected from the tideland of Sim-po located at the estuary of the Mankyong River, Chonbuk. It was finally confirmed that the parasite is Acanthoparyphium tyosenense Yamaguti, 1939 (A. tyosenense) and its life history was clarified in this study. Mactra veneriformis (M. veneriformis) was artificially infected with C. yamagutii isolated from L. fortuni and N. didyma. It began to intrude into M. veneriformis through the inhalent canal. Five hours after infection, the tails of the cercaria began to be separated from the main body and the cercaria started to form cysts. Mature cysts were formed 340 hours (14 days) after infection. The cysts were $300\sim360{\mu}m$ in diameter and the encysted metacercarias were $790\sim800\times300\sim310{\mu}m$ in size. The metacercarias were administered orally to Larus crassiostris (L. crassiostris), and adult worms of $84.5\sim112.5\times55\sim65{\mu}m$ were found full of eggs with $2.20\~3.70$ mm long and $0.40\~0.59$ mm wide after 10 days. In a field study, it was observed that the infection rate of A. tyosenense is $99.5\%$ in M. venerifomis, $76.3\%$ Solen strictus (S. strictus), and $37\%$ Ruditapes philippinarum (R. philippinarum), No difference was found among different host sizes, It was concluded that the first intermediate hosts of A. tyosenense Yamaguti were L. fortuni, N. didyma, Tympanotonus microptera, Cerithidea (Cerithidea) largillierti, Cerithidea (Cerideopsilloa) cingulata, the second intermediate hosts M. venerifomis, S. strictus and R. philippinamn, and the final hosts L. crassiostris and Melanitta fusca stejnegeri.

• Parasites, Hosts and Diseases
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• v.58 no.5
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• pp.499-511
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• 2020

Echinostome metacercariae were investigated in freshwater snails from 26 districts in 7 provinces of upper northern Thailand. The species identification was carried out based on the morphologies of the metacercariae and adult flukes harvested from experimental hamsters, and on nucleotide sequences of internal transcribed spacer 2 (ITS2) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) genes. Twenty-four out of 26 districts were found to be infected with echinostome metacercariae in freshwater snails with the prevalence of 40.4%. The metacercariae were found in all 6 species of snails, including Filopaludina martensi martensi (21.9%), Filopaludina doliaris (50.8%), F. sumatrensis polygramma (61.3%), Bithynia siamensis siamensis (14.5%), Bithynia pulchella (38.0%), and Anenthome helena (4.9%). The echinostome metacercariae found in these snails were identified as Echinostoma revolutum (37-collar-spined) and Echinostoma macrorchis (45-collar-spined) morphologically and molecularly. The 2-week-old adult flukes of E. revolutum revealed unique features of the cirrus sac extending to middle of the ventral sucker and smooth testes. E. macrorchis adults revealed the cirrus sac close to the right lateral margin of the ventral sucker and 2 large and elliptical testes with slight indentations and pointed posterior end of the posterior testis. The ITS2 and nad1 sequences confirmed the species identification of E. revolutum, and the sequences of E. macrorchis have been deposited for the first time in GenBank. The presence of the life cycle of E. macrorchis is a new record in Thailand and the snail F. doliaris as their second intermediate host seems to be new among the literature.

• Parasites, Hosts and Diseases
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• v.53 no.2
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• pp.201-207
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• 2015

This study aimed to investigate the infection status, worm development, and phylogenetic characteristics of the intestinal trematode, Stellantchasmus falcatus. The metacercariae of S. falcatus were detected only in the half-beak (Dermogenus pusillus) out of the 4 fish species examined. Their prevalence was 90.0%, and the intensity of infection was 919 metacercariae on average. Worms were recovered from 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each, and the average recovery rate was 43.0%. The body size and inner organs of S. falcatus quickly increased in the experimental chicks over days 1-2 post-infection (PI). In addition, ITS2 sequence data of this parasite were analyzed to examine the phylogenetic relationships with other trematodes using the UPGMA method. The results indicated that the ITS2 sequence data recorded from trematodes in the family Heterophyidae appeared to be monophyletic. This study concluded that D. pusillus serves as a compatible second intermediate host of S. falcatus in Thailand and that S. falcatus can develop rapidly in the experimental chicks. Data collected from this study can help to close the gap in knowledge regarding the epidemiology, biology, and phylogenetic characteristics of S. falcatus in Thailand.

• Parasites, Hosts and Diseases
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• v.53 no.5
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• pp.641-645
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• 2015

Fascioliasis, a food-borne trematode zoonosis, is a disease primarily in cattle and sheep and occasionally in humans. Water dropwort (Oenanthe javanica), an aquatic perennial herb, is a common second intermediate host of Fasciola, and the fresh stems and leaves are widely used as a seasoning in the Korean diet. However, no information regarding Fasciola species contamination in water dropwort is available. Here, we collected 500 samples of water dropwort in 3 areas in Korea during February and March 2015, and the water dropwort contamination of Fasciola species was monitored by DNA sequencing analysis of the Fasciola hepatica and Fasciola gigantica specific mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 500 samples assessed, the presence of F. hepatica cox1 and 1TS-2 markers were detected in 2 samples, and F. hepatica contamination was confirmed by sequencing analysis. The nucleotide sequences of cox1 PCR products from the 2 F. hepatica-contaminated samples were 96.5% identical to the F. hepatica cox1 sequences in GenBank, whereas F. gigantica cox1 sequences were 46.8% similar with the sequence detected from the cox1 positive samples. However, F. gigantica cox1 and ITS-2 markers were not detected by PCR in the 500 samples of water dropwort. Collectively, in this survey of the water dropwort contamination with Fasciola species, very low prevalence of F. hepatica contamination was detected in the samples.

• Parasites, Hosts and Diseases
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• v.51 no.2
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• pp.191-196
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• 2013

The echinostome metacercariae encysted in Cipangopaludina sp. snails that were purchased from a market in Vientiane Municipality, Lao PDR, were identified as Echinostoma macrorchis (Digenea: Echinostomatidae) through recovery of adult flukes after experimental infection to rats and a cat. The metacercariae were round, $113-128(121){\times}113-125(120){\mu}m$, having a thick cyst wall, a head collar armed with collar spines, and excretory granules. The adult flukes recovered from the rats and cat at day 14 and 30 post-infection, respectively, were elongated, ventrally curved, and $3.9-6.3{\times}0.7-1.1mm$ in size. The head collar was distinct, bearing 43-45 collar spines with 5 angle spines on each side. Two testes were large (as the name implies), tandem, and slightly constricted at the middle, with irregular margins. Eggs were operculated, ovoid to elliptical, and $88-95{\times}56-60{\mu}m$. In scanning electron microscopy, the head collar was prominent, with 43-45 collar spines. Scale-like tegumental spines were densely distributed on the ventral surface between the oral and ventral suckers. Sensory papillae were distributed mainly on the tegument around the 2 suckers. It is confirmed that E. macrorchis is distributed in Lao PDR using Cipangopaludina sp. snails as the second intermediate host.

• Parasites, Hosts and Diseases
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• v.28 no.1
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• pp.39-44
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• 1990

The life history of Echinostoma cinetorchis was completed in the laboratory using Hippeutis cantors as the first and second intermediate host. The incubation for maturation of eggs and hatching of miracidia took 24 days at 28℃. On the 66th day after miracidial challenge, 16 snails were crushed and examined for the presence of E. cinetorchis larvae. The metacercariae were detected in all of the snails, and from three of them were found the rediae and cercariae. The morphological characteristics of the larvae and the experimentally obtained adults were compatible with that of E. cinetorchis.

• Parasites, Hosts and Diseases
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• v.59 no.5
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• pp.519-522
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• 2021

The Chinese edible frogs, Hoplobatrachus rugulosus (n=20), and the striped snakehead fish, Channa striata (n=34), were purchased from local markets in 3 administrative regions of Cambodia (Phnom Penh, Pursat, and Takeo Provinces) from May 2017 to April 2019, and their infection status with Gnathostoma sp. larvae was investigated. The frogs and fish were transported to the laboratory with ice and examined using the artificial digestion method. Advanced 3rd-stage larvae (AdL₃) of Gnathostoma spinigerum, 24 in total number (1-6 larvae/frog), were detected from 6 (60.0%) out of 10 frogs purchased from Phnom Penh. No gnathostome larvae were detected in 10 frogs purchased from Takeo Province and 34 snakeheads from Phnom Penh, Pursat, and Takeo Provinces. AdL₃ isolated from the frogs were 2.55-3.90 mm long and 0.31-0.36 mm wide. They had a characteristic head bulb (0.081×0.191 mm in average size) with 4 rows of hooklets, a muscular long esophagus (0.950-1.230 mm long), and 2 pairs of cervical sacs (0.530-0.890 mm long). The average number of hooklets in the 1st, 2nd, 3rd, and 4th rows was 41, 45, 48, and 51, respectively. These features were consistent with G. spinigerum AdL₃. By the present study, it has been first confirmed that the Chinese edible frog, H. rugulosus, from Phnom Penh serves as a second intermediate host for G. spinigerum, although their intensity of infection was not so high compared to other previously reported localities.