Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai

We compared the DNA sequences of the genus Metagonimus: M. yokogawai, M. takahashii, and M. miyatai. We obtained 28S D1 ribosomal DNA (rDNA) and mitochondrial cytochrome c oxidase subunit I (mtCOI) fragments from the adult worms by PCR, that were cloned and sequenced. Phylogenetic relationships inferred from the nucleotide sequences of the 28S D1 rDNA and mtCOI gene. M. takahashii and M. yokogawai are placed in the same clade supported by DNA sequence and phylogenic tree analysis in 28S D1 rDNA and mtCOI gene region. The above findings tell us that M. takahashii is closer to M. yokogawai than to M. miyatai genetically. This phylogenetic data also support the nomination of M. miyatai as a separate species.     

Susceptibility of inbred mouse strains to infection with three species of Metagonimus prevalent in the Republic of Korea

Susceptibility of inbred mouse strains to Metagonimus yokogawai, Metagonimus miyatai, and Metagonimus takahashii infections was studied using BALB/c, ddY, C57BL/6J, C3H/HeN, and A/J mice, with H-2 haplotypes d, s, b, k, and a, respectively. Two hundred metacercariae were orally fed to each mouse, and the worm recovery rates (WRR), worm dimensions, and intrauterine egg numbers were measured at days 3, 7, 14, 21, and 28 postinfection (PI). On day 14 PI, the WRR of M. yokogawai was highest in ddY mice (average, 62.2%); those of M. miyatai and M. takahashii were highest in ddY (19.5%) and BALB/c mice (10.4%), respectively; worm maturation was best in C3H/HeN (M. yokogawai), C57BL/6J (M. miyatai), and ddY mice (M. takahashii). All mouse strains showed higher susceptibility to infection with M. yokogawai than with M. miyatai or M. takahashii. The results show that susceptibility of mice to Metagonimus infection varies according to mouse strain and parasite species but is suggested to be independent of the mouse H-2 haplotype.     

Surface ultrastructure of Metagonimus takahashii metacercariae and adults

A scanning electron microscopic study was performed on the surface ultrastructure of metacercariae and adults of Metagonimus takahashii. Metacercariae were collected from the scale of crucian carp (Carassius auratus), and adult flukes were harvested 1-4 weeks after infection to rats. In excysted metacercariae, the oral sucker had type I (numerous) and type II (seven in total) sensory papillae. Tegumental spines were dense and digitated into 5-7 points on the surface anterior to the ventral sucker, but became sparse and less digitated posteriorly toward the end of the body. In adults, seven type II sensory papillae were characteristically arranged around the lip of the oral sucker, and on the inner side of the lip four small and two large type I sensory papillae were symmetrically seen on each side (12 in total). Tegumental spines on anterior two-thirds of the body, were digitated with 9-12 tips ventrally and 8-13 tips dorsally. Sperms entering into the Laurer's canal were observed. The results show that the surface ultrastructure of M. takahashii is generally similar to those of M. yokogawai and M. miyatai except for the digitation of tegumental spines.     

Infection status of sweetfish from Kwangjung-stream and Namdae-stream in Yangyang-gun, Kangwon-do with the metacercariae of Metagonimus yokogawai

This study was performed to observe the infection status of sweetfish, caught from Kwangjung-stream and Namdae-stream in Yangyang-gun, Kangwon-do in August 1989, with the metacercariae of Metagonimus yokogawai. All of 28 sweetfish from Kwangjung-stream were infected with the metacercariae of M. yokogawai. The number of metacercariae in a fish ranged 89-521(224 on average), and the number of larvae/g of flesh was 12 approximately 55 (22 on average). On the other hand, no metacercariae were detected in the fish from Namdae-stream. It has been confirmed that the Kwangjung-stream in Yangyang-gun, Kangwon-do is one of the endemic foci of metagonimiasis in Korea.     

High endemicity of Metagonimus yokogawai infection among residents of Samchok-shi, Kangwon-do

A small-scale epidemiological survey was undertaken during 1997-1998 on the residents along the Osib-chon (Stream), Samchok-shi (City), Kangwon-do (Province), to evaluate the status of Metagonimus yokogawai infection. A total of 165 fecal samples was collected and examined by cellophane thick smear and formalin-ether sedimentation techniques. The egg positive rate of M. yokogawai was 29.7%, showing a remarkable difference between males (46.6%) and females (16.3%). To obtain the adult flukes of M. yokogawai, 11 egg positive persons were treated with praziquantel and purged with magnesium sulfate. A total of 242,119 adult flukes (average 22,010 per person, 367-119,650 in range) was collected from diarrheic stools, all of which were identified as M. yokogawai. The results show that M. yokogawai is still highly endemic in this area.     

Life cycle and morphology of Metagonimus miyatai (Digenea: Heterophyidae) from Nagano,Japan

The life cycle of Metagonimus miyatai Saito, Chai, Kim, Lee and Rim, 1997 (Digenea: Heterophyidae) was studied in the Hiroi River at Kotobuki, Iiyama City, Nagano Prefecture, central Japan. Daughter rediae and cercariae were found in Semisulcospira libertina (Gould) and S. dolorosa (Gould) (Gastropoda: Pleuroceridae). Metacercariae were found encysted in Phoxinus lagowskii steindachneri Sauvage (Osteichthyes: Cyprinidae). Cercariae were experimentally exposed to P. lagowskii steindachneri, and metacercariae were obtained from the fish. Metacercariae of natural and experimental infections were experimentally fed to respective golden hamsters, and gravid adults identifiable as M. miyatai were recovered from the small intestine of the golden hamsters. The daughter redia, cercaria, metacercariae and adults are described. The life cycle of M. miyatai is discussed.     

A cytogenetic study on human intestinal trematodes of the genus Metagonimus (Digenea: Heterophyidae) in Korea

In order to analyze chromosome numbers and karyotypes of intestinal trematodes belonging to the genus, Metagonimus, the gonad tissues of M. takahashii, M. miyatai, and M. yokogawai were prepared and examined. The number of bivalents in the first meiotic division of M. takahashii was nine (n = 9). The diploid number of M. miyatai was observed to be eighteen (2n = 18) and their chromosomes consisted of one pair of metacentric, 7 pairs of submetacentric, and one pair of telocentric chromosomes. The diploid number of M. yokogawai was thirty-two (2n = 32) and the chromosome complements were composed of two pairs of metacentric, 11 pairs of submetacentric, and three pairs of subtelocentric chromosomes. These results could be a supporting evidence for the validity of the new species, M. miyatai, distinct from M. yokogawai.     

Three morphological types of the genus Metagonimus encysted in the dace, Tribolodon taczanowskii, caught from the Sumjin River.

Three morphological types of the genus Metagonimus were found encysted in the dace, Tribolodon taczanowskii, caught from the Sumjin River, Korea. They include Metagonimus Yokogawa type (M. yokogawai), Miyata type, and Koga type according to Saito's classification. The metacercariae were experimentally fed to rats and hamsters and a total of 1,624 Metagonimus worms were recovered from their small intestine. The number of each type was 820 for Yokogawa type, 688 for Miyata type, 46 for Koga type, and 70 undetermined. The major differential keys between Yokogawa and Miyata types were in the position of two testes, distribution of uterine tubules, and size of the eggs, and the keys between Miyata and Koga types were in the position of two testes, distribution of vitelline follicles, and size of the eggs. The validity of Miyata type as a specific level should be retained until more convincing evidences are obtained. Koga type is regarded as a synonym of M. takahashii. The present results reveal that there are 3 morphological types of Metagonimus whose larvae are encysted in the dace, T. taczanowskii, from the Sumjin River.     

Molecular differentiation of three species of Metagonimus by simple sequence repeat anchored polymerase chain reaction (SSR-PCR) amplification

Genomic DNAs from 3 species of Metagonimus in the Republic of Korea, M. yokogawai, M. miyatai, and M. takahashii, were amplified by simple sequence repeat anchored polymerase chain reaction. Primers of 3' or 5' termini of the (CA)n repeats were used; 3 kinds of 3' anchored primers (CA)8RG, (CA)4RG, and (CA)8RY, and 2 kinds of 5' anchored primers BDB(CA)7C and DBDA(CA)7. The results showed distinguishable banding patterns among the 3 species of Metagonimus, suggesting that they have different genotypes.     

Prevalence of Metagonimus metacercariae in sweetfish, Plecoglossus altivelis, from eastern and southern coastal areas in Korea

The present study was performed to determine the current infecion status of Metagonimus yokogawai metacercariae in sweetfish, Plecoglossus altivelis, collected from several streams in eastern and southern coastal areas of Korea. The sweetfish collected were artificially digested with pepsin-HCl solution and examined under a stereomicroscope in August and September, 2007. Out of 145 sweetfish collected from 10 streams in eastern coasts (Gangwon-do and Gye-ongsangbuk-do), 88 (60.7%) were infected with Metagonimus metacercariae. The average metacercarial density was 61 per infected fish. Among 141 sweetfish collected from 10 streams in southern coasts (Gyeongsangnam-do, Ulsan Metropolitan city, and Jeollanam-do), 140 (99.3%) were infected with Metagonimus metacercariae, and their average density was 949 per infected fish. The present study confirmed that M. yokogawai metacercariae are still prevalent in the sweetfish from several eastern and southern coastal localities. The prevalence and metacercarial density were much higher in the sweetfish from streams of southern coasts than in those of eastern coasts. Therefore, attention should be paid to this small fluke infection, and consumption of raw sweetfish naturally produced in these areas should be prohibited.     

Comparative morphology of minute intestinal fluke eggs that can occur in human stools in the republic of Korea

The egg morphology of minute intestinal flukes (MIF) that can occur as human infections in the Republic of Korea, i.e., Metagonimus yokogawai, M. miyatai, M. takahashii, Heterophyes nocens, Heterophyopsis continua, Stellantchasmus falcatus, Stictodora fuscata, Pygidiopsis summa, and Gymnophalloides seoi, was studied in comparison with Clonorchis sinensis. The adult worms were obtained from residents of endemic areas, and their intrauterine eggs were studied and measured using light microscopy; the length, width, length-width ratio (LWR), and Faust-Meleney index (FMI). Several specimens were processed for scanning electron microscopy (SEM), and before gold-coating, the uterine portion of each fluke was etched with a sharp pin in order to expose the eggs. The MIF eggs were ovoid, pyriform, or elliptical with a size range of 21-35×12-21 μm. S. fuscata eggs revealed the highest FMI (largest in the area) and lowest LWR, whereas P. summa eggs showed the lowest FMI and medium LWR. SEM revealed that G. seoi and S. fuscata had remarkably clean shell surface lacking the muskmelon-like structure which is prominent in C. sinensis eggs. In Metagonimus spp., H. continua, H. nocens, and S. falcatus eggs, minute surface ridges were recognizable though less prominent compared with C. sinensis. On the surface of P. summa eggs, thread-like curly structures were characteristically seen. The results revealed that important differential keys for MIF eggs include the length, width, area (FMI), shape of the eggs, and the extent of the muskmelon-like structure or ridges on their shell surface and operculum.     

Activities of brush border membrane bound enzymes of the small intestine in Metagonimus yokogawai infection in mice

The present study intended to evaluate the influences of Metagonimus yokogawai on the activities of brush border membrane bound enzymes of the small intestine. Mice were infected with 500 metacercariae respectively, and the worm recovery, morphological changes and enzyme activities were observed chronologically. A part of them were followed after the treatment. Recovered worms decreased in number continuously after the infection, and they were less than 10% after 2 weeks and almost zero after 28 weeks. Villous atrophy and stromal inflammation were found at two locations of the proximal jejunum from 2 weeks to 4 weeks after the infection. The enzymes, alkaline phosphatase, leucine aminopeptidase and disaccharidases (sucrase, lactase, maltase, and trehalase), showed lowered activities in the duodenum and proximal jejunum of the infected mice but they increased in the distal jejunum for the first two weeks. From three weeks after the infection, the activities were gradually recovered. In one week treated mice, they recovered the activities at 2 weeks from the treatment, but there found no differences of the activities between the 3 week treated group and infected controls. The present data reveal that M. yokogawai infection induces degenerative changes of the host's intestinal mucosa not only morphologically but functionally during the initial phase of infection. The lowered enzyme activities in acute metagonimiasis should be associated with malabsorption and diarrhea.     

Food-borne intestinal trematode infections in the Republic of Korea

A total of 19 species of food-borne intestinal trematodes have been reported in humans in the Republic of Korea. They include 12 species of the Heterophyidae, Metagonimus yokogawai, M. takahashii, M. miyatai, Heterophyes nocens, H. heterophyes (imported), H. dispar (imported), Heterophyopsis continua, Pygidiopsis summa, Stellantchasmus falcatus, Centrocestus armatus, Stictodora fuscata, and S. lari; four species of the Echinostomatidae, Echinostoma hortense, E. cinetorchis, Echinochasmus japonicus, and Acanthoparyphium tyosenense; and one species each of the Neodiplostomidae, Neodiplostomum seoulense, Plagiorchiidae, Plagiorchis muris, and Gymnophallidae, Gymnophalloides seoi. Fresh water fish harbor the metacercarial stage of M. yokogawai, M. takahashii, M. miyatai, C. armatus, E. hortense, E. cinetorchis, E. japonicus, or P. muris. Brackish water fish serve as the second intermediate hosts for H. nocens, H. continua, P. summa, S. falcatus, S. fuscata, and S. lari. Brackish water bivalves are the source of infection with A. tyosenense. Tadpoles and frogs are the second intermediate hosts for N. seoulense, but the major source of human infection is the grass snake Rhabdophis tigrina, a paratenic host. The metacercariae of G. seoi are observed in oysters. The natural definitive hosts are, in most cases, mammals such as rats, cats and dogs. However, several species (C. armatus, S. lari, E. japonicus, A. tyosenense, and G. seoi) have birds as natural definitive hosts. Host-parasite relationships, pathogenesis and pathology, immunity, clinical aspects, differential diagnosis, treatment, prevention and control of these intestinal trematodes are briefly discussed.     

Clonorchis sinensis metacercarial infection in the pond smelt Hypomesus olidus and the minnow Zacco platypus collected from the Soyang and Daechung Lakes

The pond smelt Hypomesus olidus and minnow Zacco platypus were collected from the Soyang and Daechung Lakes in January 2003, and their metacercarial infections was examined by the muscle compression and artificial digestion techniques. In the Soyang Lake, 161 metacercariae of Clonorchis sinensis (0.35 per fish) were harvested from 459 pond smelts examined. Also, 13 metacercariae of C. sinensis (0.43 per fish), 1 of Metagonimus sp., 4 of Echinostoma sp., 148 of Centrocestus armatus and 44 unidentified species were collected from 30 minnows. In the Daechung Lake, 369 metacercariae of C. sinensis (3.69 per fish) and 51 unidentified species were recovered from 100 pond smelts. The metacercariae of C. sinensis were fed to experimental rats, in which the adult flukes were identified. The pond smelts and minnows collected from the Soyang and Daechung Lakes were verified to be the second intermediate hosts and the sources of human C. sinensis infection.     

Antigenic localities in the tissues of Metagonimus yokogawai observed by immunogoldlabeling method]

In order to determine the antigenic localization in the tissues of the adult Metagonimus yokogawai, immunogoldlabeling method was applied using serum immunoglobulins(IgG) of cats which were infected with isolated metacercariae from Plecoglossus altivelis. The sectioned worm tissue was embedded in Lowicryl HM 20 medium and stained with infected serum IgG and protein A gold complex(particle size: 12 nm). It was observed by electron microscopy at each tissue of the worm. The gold particles were observed on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum. The gold particles were not observed on the basal lamina of the tegument, interstitial matrix of the parenchyma, the muscle tissue and mitochondria of the tegument. The gold particles were specifically labeled in the secretory granules in the vitelline cells. They were also labeled on the lumen of bladder and egg shell. The above findings showed that antigenic materials in the tissue of adult worms were specifically concentrated on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum.     

Epidemiological studies of digenetic trematodes in Yongyang County, Kyungpook Province.

The present study was undertaken to determine the endemicity of trematode infections in Yongyang County, Kyungpook Province, Korea. One hundred and seventy-seven out of 955 residents examined were found to be infected with one or more kinds of helminths. The prevalence rate was 18.5%. Among them, 70 were found to be infected with Clonorchis sinensis, 74 with Metagonimus sp. and 24 with both flukes. The eggs of Fasciolidae were demonstrated from two specimens. The prevalence rate of clonorchiasis in males was 12.6%, while that in females was 3.6% (p less than 0.05). The prevalence of metagonimiasis in males was 12.0% and was also significantly higher than 6.1% in females (p less than 0.05). The intensity of both infections was significantly heavier in males than in females. The prevalence of both flukes was higher in residents of over 30 years of age than in those below that age. No correlation was found between the intensity and the age group. Among eight species of the fresh-water fish collected at the Panbyon River, Zacco temmincki was found to be the most frequently and heavily infested with metacercariae of Metagonimus sp., and Gnathopogon atromaculatus alone was found to be infested with metacercariae of Clonorchis sinensis. The present results suggest that Yongyang area still remains endemic with Clonorchis sinensis and Metagonimus sp. infection.     

Prevalence of zoonotic trematodes in fish from a Vietnamese fish-farming community

The prevalence of fish-borne zoonotic trematode (FZT) metacercariae was investigated in fish farmed by rural households in Nghe An Province, located in northern Vietnam. In total, 716 fish, including tilapia (Oreochromis niloticus) and 6 carp species, i.e., grass carp (Ctenopharyngodon idellus), bighead carp (Aristichthys nobilis), mrigal (Cirrhinus mrigala), common carp (Cyprinus carpio), silver carp (Hypophthalmychthys molitrix), and roha (Labeo rohita), collected from 53 fish farms were examined. The overall prevalence of FZT metacercariae was 44.6%, ranging from 12.5% to 61.0% in fish species collected from grow-out ponds, which are the production system for growing fish from fingerling size to market size. The overall prevalence was 43.6% in fingerlings cultured in nurseries, ranging from 7.4% to 62.8% for different fish species. The FZT species recovered were heterophyids and echinostomatids and included Haplorchis pumilio, H. taichui, H. yokogawai, Centrocestus formosanus, Stellantchas musfalcatus, and Echinochasmus japonicus, all of which are intestinal flukes in humans, other mammals, and birds. This is the first report of H. yokogawai and E. japonicus in fish in Vietnam, and the first record for S. falcatus in northern Vietnam. Although a previous cross-sectional survey of the people living in these fish farm households revealed a very low prevalence of FZTs (< 1%), our results demonstrate that intestinal flukes are common in farmed fish in this area, suggesting that reservoir hosts such as dogs, cats, and pigs are more important in sustaining the life cycles of these flukes in fish farms than human hosts. This has implications for the effectiveness of control programs focused mainly on treatment of humans.     

Heterophyid metacercarial infections in brackish water fishes from Jinju-man (Bay), Kyongsangnam-do, Korea

Heterophyid metacercarial infections in brackish water fishes, i.e., perch, shad, mullet, redlip mullet, and goby, of Jinju-man (Bay), Kyongsangnam-do, Korea, were investigated using a digestion technique. Among 45 perch (Lateolabrax japonicus), the metacercariae of Heterophyopsis continua were found in 55.6% (18.5 metacercariae per fish), Stictodora spp. in 28.9% (3.6), and Metagonimus takahashii in 6.7% (17.0). The metacercariae of H. continua were detected in 23 (65.7%) of 35 shad (Konosirus punctatus). Among 15 mullet (Mugil cephalus), the metacercariae of Pygidiopsis summa were found in 100% (105.9 metacercariae per fish), Heterophyes nocens in 40.0% (8.5), H. continua in 13.3%, and Stictodora spp. in 6.7%. Among 12 redlip mullet (Chelon haematocheilus), the metacercariae of P. summa were detected in 91.7% (1,299 metacercariae per fish), H. nocens in 16.7%, and Stictodora spp. in 16.7%. Among 35 gobies (Acanthogobius flavimanus), the metacercariae of Stictodora spp. were found in 82.9% (44.5 metacercariae per fish), and H. continua in 25.7% (2.8). The adult flukes recovered from a cat experimentally infected with metacercariae from perches were compatible with M. takahashii. The results confirm that the estuarine fish in Jinju-man (Bay) are heavily infected with heterophyid metacercariae. Furthermore, it was found for the first time that the perch acts as a second intermediate host for M. takahashii.     

Studies on the in vitro effects of bithionol and menichlopholan on flukes of Clonorchis sinensis, Metagonimus takahashii and Paragonimus miyazakii.

The present study was carried out in order to obtain information on the action of bithionol and menichlopholan on Clonorchis sinensis, Metagonimus takahashii and Paragonimus miyazakii. The drugs inhibited the motility of the newly excysted juveniles and adult worms in vitro. Moreover, the in vitro treatment slightly decreased lactate dehydrogenase activity in homogenates of adult worms. The drug treatments of adult worms in vitro also inhibited the activity of fumarate reductase, malic enzyme, phosphogluconate dehydrogenase and succinate dehydrogenase in the homogenate. In addition to this, these drugs caused morphological alterations in the ultrastructure of the tegument of the flukes.