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Gymnophalloides seoi (Digenea: Gymnophallidae) is a human intestinal trematode contracted by eating raw oysters (Crassostrea gigas) in the Republic of Korea (=Korea). It has been known to be highly endemic in Aphae Island, Shinan-gun, Jeollanam-do (Province). However, recent epidemiological status of G. seoi has not been reported since the 1990s. In this study, we investigated the prevalence of G. seoi metacercariae in natural and cultured oysters collected from 3 islands and 2 coastal areas in western parts of Korea. The oysters were examined using the artificial digestion method followed by stereomicroscopy. The overall positive rate of G. seoi metacercariae in natural oysters was 66.0% (99/150), and the oysters collected from Yubu Island showed the highest infection rate (74.0%). However, the metacercarial density per oyster was relatively low (1.5-2.4 per oyster). By contrast, no metacercaria was found in cultured oysters purchased from 2 coastal areas in Chungcheongnam-do. Thus, we could confirm that natural oysters produced from 3 western coastal islands are infected with G. seoi metacercariae, whereas cultured oysters purchased from 2 coastal areas were free from infection.

Gymnophallid metacercariae found in the Manila clam Ruditapes philippinarum (‘Banjirak’ in Korean) from Gochang-gun, Jeollabuk-do, Korea were morphologically and molecularly confirmed to be Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974. The metacercariae were morphologically characterized by having a large oral sucker, small ventral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, which were all compatible with P. duboisi. Some of the metacercariae were experimentally fed to mice, and adult flukes were recovered at day 7 post-infection. The morphology of the adult flukes was basically the same as that of the metacercariae except for the presence of uterine eggs; the uterus was filled with up to 40 eggs. The nucleotide sequences (1,193 bp) from ITS regions (ITS1, 5.8S rDNA, and ITS2) of the metacercariae showed 99.7% identity with P. duboisi and 75.7% identity with Gymnophalloides seoi deposited in GenBank. These results confirmed the presence of P. duboisi metacercariae in the Manila clam R. philippinarum in an estuary region of Gochang-gun, Korea.

In intestinal helminth infections, Th2 immune respones are generally associated with mucin secretion for worm expulsion from the host intestine. In particular, IL-4 and IL-13 are the important cytokines related with intestinal mucus production via STAT6 signalling in nematode infections. However, this perspective has never been studied in Gymnophalloides seoi infection. The present study aimed to observe the STAT6 signalling and cytokine responses in C57BL/6 mice, a mouse strain resistant to infection with this trematode. The results showed that worm expulsion occurred actively during days 1-2 post-infection (PI), when goblet cells began to proliferate in the small intestine. The STAT6 gene expression in the mouse spleen became remarkable from day 2 PI. Moreover, G. seoi infection induced a significant increase of IL-13 from day 4 PI in the spleen of infected mice. Our results suggested that goblet cell hyperplasia and worm expulsion in G. seoi-infected mice should be induced by STAT6 signalling, in which IL-13 may be involved as a dominant triggering cytokine.

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.

The changing patterns of goblet cell hyperplasia, intestinal epithelial cell turnover, and intestinal motility were studied in ICR and C57BL/6 mice infected with Gymnophalloides seoi (Digenea: Gymnophallidae). Whereas ICR mice retained G. seoi worms until day 7 post-infection (PI), C57BL/6 mice showed a rapid worm expulsion within day 3 PI. Immunosuppression with Depo-Medrol significantly delayed the worm expulsion in C57BL/6 mice. Goblet cell counts were increased in both strains of mice, peaking at day 1 PI in C57BL/6 mice and slowly increasing until day 7 PI in ICR mice. In C57BL/6 mice infected with G. seoi, newly proliferating intestinal epithelial cells were remarkably increased in the crypt, and the increase was the highest at day 1 PI. However, in ICR mice, newly proliferating intestinal epithelial cells increased slowly from day 1 to day 7 PI. Intestinal motility was increased in G. seoi-infected mice, and its chronological pattern was highly correlated with the worm load in both strains of mice. Meanwhile, immunosuppression of C57BL/6 mice abrogated the goblet cell proliferation, reduced the epithelial cell proliferation, and suppressed the intestinal motility. Goblet cell hyperplasia, increased intestinal epithelial cell turnover, and increased intestinal motility should be important mucosal defense mechanisms in G. seoi-infected C57BL/6 mice.

A small coastal village of Muan-gun, Chollanam-do, was surveyed for intestinal fluke infections, especially heterophyids such as Heterophyes nocens and Pygidiopsis summa by fecal examination on 108 inhabitants. The egg positive rate of heterophyids was very high, 75.0%, and that of other parasites was comparatively low, 0.9-3.7% by parasite species. After treatment of 20 patients showing high E.P.G. with praziquantel and purging with MgSO4, total 3,864 specimens of H. nocens were collected from the diarrheic stools of all the patients treated (3-1,338 individually) and total 703 P. summa were harvested from 18 patients (1-170 individually), together with several other species of flukes. Other flukes included Stictodora fuscata (164 specimens from 4 patients), Heterophyopsis continua (2 from 2 patients), and Gymnophalloides seoi (4 from 3 patients). From this study, the surveyed coastal area of Muan-gun, Chollanam-do was proven to be a new endemic focus of H. nocens and P. summa. The occurrence of a few infected cases suggests that this area should also be a low-grade endemic area of S. fuscata, H. continua, and G. seoi.

Gymnophalkides seoi has drawn medical attentions since the discovery of the first human case and a highly endemic area on a southwestern coastal island of Shinan- gun, Korea. Marine bivalves especially oysters were strongly suspected as the source of infection. In this study the oysters, Crassostrea girns, naturally produced (rom the endemic area were examined whether they contain gymnophallid metacercariae. All of 50 oysters examined were infected with the metacercaviae of a gymnophallid, with the metacercarial density per oyster of 610 on average (2-4,792 in range). Later they were identified as G. seoi by obtaining adult worms from experimental mice. The metacercariae were unencysted, and firmly attached on the mantle surface of the oysters with their oral sucker. In sectioned specimens they were equipped with the ventral pit, a peculiar organ of the genus Gymophalloides, and non-muscular genital pore which was connected dorsally to the seminal vesicle. The seminal vesicle was in a great majority mono-sac. By this study, it has been confirmed that the oyster is a 2nd intermediate host of G. seoi as well as the major source of human infection with this fluke. Key words: Gymnophalloines seoi, Gymnophallidae, human intestinal fluke, oysters, metacercariae , Shlnan-gun, Korea

Dear Editor, Multiple infections with small liver flukes and minute intestinal flukes are the serious public health concern in the lower Mekong basin [1,2]. Although the epidemiological survey for those trematode infections are primarily carried out based on copro-parasitological examination, detection/identification of fecal eggs/worms is a tedious job and often problematic because of the morphological similarities of eggs/worms. Along with the popularization of PCR-sequencing methods, copro-DNA diagnosis and molecular phylogenetic identification/speciation have been introduced in epidemiological studies. Among various genes and non-coding lesions of nuclear and mitochondrial DNAs, mitochondrial cytochrome c oxidase subunit I (COXI) is one of the most widely used inter- and intra-species marker. Using COXI and some other markers, Lee and his colleagues performed molecular phylogenetic analyses on small liver flukes (Lee SU, Huh S. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis. Korean J Parasitol 2004; 42: 145-148) and on minute intestinal flukes (Lee SU, Huh S, Sohn WM, Chai JY. Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai. Korean J Parasitol 2004; 42: 129-135). The COX1 gene sequences appeared in those articles are; Clonorchis sinensis ({"type":"entrez-nucleotide","attrs":{"text":"AF184619","term_id":"296940320","term_text":"AF184619"}}AF184619, {"type":"entrez-nucleotide","attrs":{"text":"AF181889","term_id":"285026682","term_text":"AF181889"}}AF181889, {"type":"entrez-nucleotide","attrs":{"text":"AF188122","term_id":"285809842","term_text":"AF188122"}}AF188122), Metagonimus yokogawai ({"type":"entrez-nucleotide","attrs":{"text":"AF096230","term_id":"297039733","term_text":"AF096230"}}AF096230), Metagonimus takahashii ({"type":"entrez-nucleotide","attrs":{"text":"AF096231","term_id":"297039734","term_text":"AF096231"}}AF096231), Metagonimus miyatai ({"type":"entrez-nucleotide","attrs":{"text":"AF096232","term_id":"297039735","term_text":"AF096232"}}AF096232), Pygidiopsis summa ({"type":"entrez-nucleotide","attrs":{"text":"AF181884","term_id":"288563287","term_text":"AF181884"}}AF181884), and Stellantchasmus falcatus ({"type":"entrez-nucleotide","attrs":{"text":"AF181887","term_id":"285804435","term_text":"AF181887"}}AF181887). In addition, Park [3] compared his COXI sequence of Opisthorchis viverrini Laotian isolate (AY055 382) to those of Gymnophalloides seoi ({"type":"entrez-nucleotide","attrs":{"text":"AF096234","term_id":"297039736","term_text":"AF096234"}}AF096234) and Neodiplostomum seoulense ({"type":"entrez-nucleotide","attrs":{"text":"AF096233","term_id":"285026845","term_text":"AF096233"}}AF096233) registered in the DNA database (Lee et al. unpublished). For the phylogenetic analyses of COXI gene of minute intestinal flukes of our own data, we have downloaded all those above mentioned COXI of Lee et al. and aligned them including our own COXI sequence of Haplorchis taichui ({"type":"entrez-nucleotide","attrs":{"text":"EF055885","term_id":"119855482","term_text":"EF055885"}}EF055885) [4] and Paragonimus bangkokensis ({"type":"entrez-nucleotide","attrs":{"text":"AB354227","term_id":"155369203","term_text":"AB354227"}}AB354227) [5]. Surprisingly, those sequence data were divided into 2 distinct groups without any similarities (Fig. 1). Eventually, we realized that this astonishing result is due to the reverse complementary sequences of COXI data deposited by Lee et al. (in the bottom half of the figure). We also noticed similar mixed-up deposition of the forward and reverse sequences of COXI gene of Fasciola spp., which were also included in Fig. 1 ({"type":"entrez-nucleotide","attrs":{"text":"AJ628024","term_id":"88319716","term_text":"AJ628024"}}AJ628024, {"type":"entrez-nucleotide","attrs":{"text":"AJ628039","term_id":"88319746","term_text":"AJ628039"}}AJ628039, {"type":"entrez-nucleotide","attrs":{"text":"FJ469984","term_id":"238631966","term_text":"FJ469984"}}FJ469984; Zhu XQ et al. unpublished). Fig. 1 The DNA sequence alignment of the partial COXI gene of some trematodes obtained from the GenBank. Seven sequences from the top are the forward strands with JB3 primer sequence, and 2 in the middle are the forward strands without primer. Eight sequences ... For the determination of partial COX1 sequences of Platyhelminthes, the primer set of JB3 (5'-TTT TTT GGG CAT CCT GAG GTT TAT-3') and JB4.5 (5'-TAA AGA AAG AAC ATA ATG AAA ATG-3') [6] was widely used for investigating the inter- and intra-species variations of trematodes and cestodes. We noticed the mixed-up of the forward and reverse COXI sequences by Lee et al. as well as Zhu et al. because of the presence of the characteristic feature of this primer set (boxed in Fig. 1) in the sequences. The primer sequence should be deleted from the sequence data because it is not always identical with the real DNA sequence of the gene and the inclusion of the primer sequences sometimes causes the misreading in phylogenetic analyses [7]. In 3 reverse sequences, {"type":"entrez-nucleotide","attrs":{"text":"AF181884","term_id":"288563287","term_text":"AF181884"}}AF181884, {"type":"entrez-nucleotide","attrs":{"text":"AY055380","term_id":"22203992","term_text":"AY055380"}}AY055380, and {"type":"entrez-nucleotide","attrs":{"text":"AF096233","term_id":"285026845","term_text":"AF096233"}}AF096233 seems to contain also the partial sequence of the cloning vector, which should be trimmed off before deposition. In general, raw data of forward and reverse sequences obtained from the sequencer should be aligned manually by cross-checking of the wave patterns because some 10-20 bases downstream from the forward primer and upstream from the reverse primer often contain erroneous base pairs [8]. Deposition of the reverse sequence means that those sequences were not aligned against forward sequence and not quite reliable. Since each sequence data in GenBank are opened for the public use, an accuracy of the sequence data is critically important for the mutual reliability of the scientists. The scientists should aware how to deposit accurate sequence data to the DNA data base. The reappraisal and correction of those sequences mentioned above is urgently necessary.

A new endemic focus of Gymnophalloides seoi infection has been discovered on Aphae Island (Shinangun, Jeollanam-do), Republic of Korea. This area, which is referred to as Bokyong-ri, is a small seashore village located in the northern portion of the island. Fecal samples were collected from a total of 57 residents and examined by the Kato-Katz and formalin-ether sedimentation techniques. Helminth eggs were detected in 37 samples (64.9%); 33 samples were positive for G. seoi eggs (57.9%), 4 for Pygidiopsis summa (7.0%), 13 for other heterophyids (22.8%), 1 for Clonorchis sinensis (1.7%), and 1 for Trichuris trichiura (1.7%). Women (70.4%) revealed higher rates of G. seoi infection than did men (46.7%), and individuals older than 50 years of age also evidenced higher rates of G. seoi infection than in other age groups (P < 0.05). In worm collection from 13 G. seoi egg positive cases, G. seoi (total 86,558 specimens), Heterophyes nocens (278), Stictodora sp. (10), Heterophyopsis continua (3), P. summa (3), and unidentified flukes (42) were collected. Oysters, the source of G. seoi infection, were collected from an area proximal to the village and 50 were examined for metacercariae; 47 (94%) were found infected and the observed metacercarial density was 9.5 +/- 8.9 per oyster. The results of this study indicate that Bokyong-ri is a new endemic area of G. seoi infection, with high rates of infection in humans and oysters.

A nationwide survey was performed to know the distribution and prevalence of human Gymnophalloides seoi infection on western and southern coastal islands in the Republic of Korea. A total of 4,178 fecal specimens were collected from residents on 45 (24 western and 21 southern) islands, and examined by Kato-Katz and formalin-ether sedimentation techniques. Eggs of G. seoi were detected from 160 (3.8%) people living on 22 (13 western and 9 southern) islands. The prevalence varied by the location of islands; higher on western islands than on southern islands. The highest prevalence was found on Amtaedo (25.3%), followed by Cheungdo (25.0%), and Anchwado (20.9%) (Shinan-gun). A little lower prevalence was observed on Munyodo (13.3%), Shinshido (12.9%), and Sonyudo (10.3%) (Kunsan-shi). Of the remaining islands, the regions showing the prevalence greater than 5% included Kohado, Dallido (Mokpo-shi), Pyeongildo, Kogumdo (Wando-gun), and Keogumdo (Kohung-gun). A strong age predilection was noted (p < 0.05); 95% of the infected people were over 40 years old. Females showed a little higher prevalence than males. The results indicate that human G. seoi infection is more widely distributed than previously considered. Nine of 11 islands (excluding the 2 known areas Munyodo and Sunyudo) that showed greater prevalence than 5% are regarded as new endemic foci of G. seoi.

The growth and development of Gymnophalloides seoi were studied in C3H/HeN mice and effects of immunosuppression of the host on the worm development were observed. Two hundred metacercariae of G. seoi were orally administered to each mouse, and worms were recovered on days 1, 3, 5, 7, 14 and 21 post-infection (PI). The worm recovery rate was significantly higher in immunosuppressed (ImSP) mice than in immunocompetent (ImCT) mice except on days 1 and 3 PI. The worms attained sexual maturity by day 3 PI with eggs in the uterus, and worm dimensions and the number of uterine eggs continuously increased until day 14 PI in ImSP mice. Worms recovered from ImSP mice were significantly larger in size than those from ImCT mice on days 1 and 3 PI, and the number of uterine eggs was significantly larger in ImSP mice on days 5 and 7 PI. Genital organs such as the ovary, testes, and vitellaria, that were already developed in the metacercarial stage, grew a little in size until day 14 PI. The results show that the C3H/HeN mouse is, though not excellent, a suitable laboratory host for G. seoi.

An epidemiological survey was performed to know the infection status of oysters with Gymnophalloides seoi metacercariae in 7 islands of the West Sea known as the habitat of paleartic oystercatchers, Haematopus ostralegus osculans, in Korea. The surveyed areas were Aphaedo (Shinan-gun, Chollanam-do), Jangjado, Sonyudo and Munyodo (Okdo-myon, Kunsan-shi, Chollabuk-do), Yubudo (Changhang-up, Sochon-gun Chungchongnam-do), and Polumdo and Chumoondo (Sodo-myon, Kangwha-gun. Inchon-shi). The oysters collected from Aphaedo, the known endemic focus, were examined monthly from August 1995 to October 1996 for observation of any seasonal variation of the metacercarial density. The average metacercarial burden was 761-2,077 by month, but the seasonal variation of the metacercarial density was not obvious. A total of 54 metacercariae was detected in 63 oysters collected from Yubudo. Out of 30 oysters from Sonyudo, 25 (83.3%) were infected with 1-66 metacercariae (12.6 in average). All of 50 oysters (100%) from Munyodo were infected with 3-162 metacercariae (53.5 in average). Only 4 metacercariae were detected in 100 oysters from Chumoondo. However, no metacercariae were found in 55 oysters from Jangjado and 50 oysters from Polumdo. From the above results, it was confirmed that G. seoi is still highly prevalent in oysters from Aphaedo, and several islands of the West Sea known as the habitats of paleartic oystercatchers are new endemic areas of this fluke.

Gymnophalloides seoi is a human intestinal trematode prevalent on southwestern islands in Korea. In the present study, we investigated whether G. seoi metacercariae can grow and develop into adults by in vitro cultivation. The metacercariae were obtained from naturally infected oysters, and cultured in vitro for 5 days under three conditions; 37 degrees C/5% CO2, 41 degrees C/8% CO2, or 41 degrees C/5% CO2, in NCTC 109 complete media containing 20% FBS and 1% antibiotics-antimycotics. The degree of worm growth and development was compared with that grown in vivo of C3H mice. The length of the worms cultivated in vitro was 200-300 microns not significantly different from metacercariae, whereas the length of the worms recovered from C3H mice was significantly larger, 300-400 microns. The worms produced eggs when grown in C3H mice or cultured in vitro for 2 days under 41 degrees C/8% CO2 or 41 degrees C/5% CO2, but not when cultured under 37 degrees C/5% CO2. Among the in vitro conditions, 41 degrees C/5% CO2 was best for egg production, although the number of eggs was about half of worms obtained from C3H mice. In conclusion, in vitro cultivation of G. seoi metacercariae into egg-producing adults was partially successful under culture conditions of 41 degrees C/5% CO2 or 41 degrees C/8% CO2.

A nationwide survey was performed to know the geographical distribution of Gymnophalloides seoi (Digenea: Gymnophallidae) metacercariae in Korea, by examining the infection status of locally produced oysters, Crassostrea gigas. A total of 24 coastal areas (myons) of 14 guns ( = counties) in Kyonggi-do, Chollabuk-do, Chollanam-do, Kyongsangnam-do, Kyongsangbuk-do, or Kangwon-do, where natural oysters are produced but G. seoi has never been reported, and 13 areas (myons) of Shinan-gun, Chollanam-do, nearby the known endemic area, were surveyed. Oysters from nonendemic areas were free from G. seoi infection, except Byonsan-myon of Buan-gun, Chollabuk-do, where one of 50 oysters examined was infected with 15 metacercariae of G. seoi. In Shinan-gun, oysters from 10 areas including Aphae-myon ( = township) and Anjwa-myon were infected with the metacercariae, with the infection rate ranging from 1.7% to 100% by areas. The intensity of infection was the highest in Aphae-myon. 785.9 metacercariae per oyster. The results indicate that high prevalence of G. seoi is confined to Shinan-gun, but low grade prevalence is also present in adjacent areas such as Buan-gun, Chollabuk-do.

An experimental study was carried out to observe the effects of radiation on the infectivity of metacercariae of Gymnophalloides seoi to C3H mice. Oysters, the second intermediate host, were collected from an endemic area, and non-irradiated control, metacercaria-irradiation, and oyster-irradiation groups were prepared. One hundred metacercariae were infected orally to each mouse, and worm recovery rate was compared by groups at 7th day post-infection. In the metacercaria-irradiation group, the worm recovery rate was significantly reduced at radiation doses higher than 200 Gy, and the number of intrauterine eggs significantly reduced at doses over 50 Gy. In the oyster-irradiation group, 50 Gy significantly reduced both the worm recovery rate and number of uterine eggs. In the two groups, no worm was recovered at 1,000 Gy irradiation. Conclusively, irradiation of oysters with 200-1,000 Gy was effective to control infectivity of metacercariae to C3H mice, and could be adopted as a control measure for gymnophalloidiasis.

A transmission electron microscopic study was performed on the ultrastructure of the tegumental layer of Gymnophalloides seoi (Digenea: Gymnophallidae) metacercariae and adults. The metacercariae were obtained from naturally infected oysters, Crassostrea gigas, and the adults from experimentally infected C3H mice. The tegumental layer generally revealed a small number of foldings, numerous small vacuoles, sines, and muscle bundles. Beneath the muscle layer, nuclei of the tegumental cells were located. There was little difference in the structure of the tegument between the metacercariae and adults. The oral sucker, having well-developed muscle layers, showed a similar structure to the ventral sucker except numerous foldings in the ventral sucker. The ventral pit was surrounded by a thin syncytial layer, where a number of microtubules and mitochondria were seen. Around the ventral pit located well-developed circular and longitudinal muscles. The results showed that the ultrastructure of the tegumental layer of G. seoi metacercariae and adults revealed little difference from other trematodes in general. The ventral pit, a peculiar structure of this trematode, seems to function as a sphincter or an accessory adhesive organ.

A highly endemic focus of human infection with Heterophyes nocens (Heterophyidae) was discovered from a small coastal village of Shinan-gun, Chollanam-do, for the first time in Korea. Fecal examinations by cellophane thick smear and formalin-ether sedimentation techniques revealed 42.9% heterophyid egg positive rate out of 98 inhabitants examined. It was difficult to confirm the species of heterophyids only by eggs. In order to collect the adult flukes, the egg positive cases were treated with 10 mg/kg single dose of praziquantel and purged with magnesium sulfate, and the adult worms were collected from the diarrheic stools. From each of 18 cooperative patients 1 to 1,124 (total 4,730) H. nocens worms were recovered, together with a few to large numbers of heterophyids (Pygidiopsis summa, Stictodora fuscatum) and/or gymnophallids (Gymnophalloides seoi). It is speculated that H. nocens might be widely distributed along the southwestern coastal areas where the brackish water fish such as the mullets or gobies are popularly eaten raw.