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.     

Infection Status of Estuarine Fish and Oysters with Intestinal Fluke Metacercariae in Muan-gun, Jeollanam-do, Korea

The source of human infection with intestinal flukes was surveyed in estuarine fishes, including the dotted gizzard shad, common sea bass, common blackish goby, redlip mullet, black sea bream, and oyster collected from Muan-gun, Jeollanam-do, Korea during August and September 2007. Collected fishes and oysters were artificially digested in pepsin-HCl solution and examined under a stereomicroscope. In 36 shads (Konosirus punctatus) and 20 basses (Lateolabrax japonicus) examined, Heterophyopsis continua metacercariae were found in 58.3% and 100%, and their average numbers were 12.0 and 6.3 per infected fish, respectively. In 34 gobies (Acanthogobius flavimanus) examined, metacercariae of H. continua were detected in 79.4%, Stictodora lari in 97.1%, and Acanthotrema felis in 92.1%, and their average numbers were 45.8, 189.3, and 235.3 per infected fish, respectively. In 37 redlip mullets (Chelon haematocheilus), Heterophyes nocens metacercariae were found in 56.8%, Pygidiopsis summa in 94.6%, and Stictodora fuscata in 45.9%, and the average metacercarial densities were 17.4, 31.3, and 35.1 per infected fish, respectively. In 30 black sea breams (Acanthopagrus schlegeli) and 45 oysters (Crassostrea gigas) examined, no metacercariae were detected. From the above results, it has been confirmed that the dotted gizzard shad, common sea bass, common blackish goby, and redlip mullet from Muan-gun, Jeollanam-do, Korea are infected with the metacercariae of heterophyid flukes.     

Four Human Cases of Acanthotrema felis (Digenea: Heterophyidae) Infection in Korea

Acanthotrema felis is an intestinal trematode of cats originally reported from the Republic of Korea. Only 1 human case infected with a single adult worm has been previously recorded. In the present study, we report 4 human cases infected with a total of 10 worms recovered after anthelmintic treatment and purging. All 4 patients reside in coastal areas of Jeollanam-do, Korea, and have consumed brackish water fish including the gobies, Acanthogobius flavimanus. The worms averaged 0.47 mm in length and 0.27 mm in width, and had 3 sclerites on the ventrogenital sac; 1 was short and thumb-like, another was long and blunt-ended, and the 3rd was long and broad-tipped. They were identified as A. felis Sohn, Han, & Chai, 2003. Surveys on coastal areas to detect further human cases infected with A. felis are required.     

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.     

Centrocestus formosanus (Digenea: Heterophyidae) encysted in the freshwater fish, Puntius brevis, from Lao PDR

The metacercariae of Centrocestus formosanus, a minute intestinal trematode of mammals and birds, were detected in the freshwater fish, Puntius brevis, from Vientiane Municipality, Lao PDR. The metacercariae were experimentally fed to mice, and adult flukes were recovered in their small intestines 7 days later. The adult flukes were morphologically characterized by having 32 (rarely 34) circumoral spines arranged in 2 alternative rows, a large bipartite seminal vesicle, an oval-shaped ovary, and an X-shaped excretory bladder. Based on these characters, the adults were identified as Centrocestus formosanus (Nishigori, 1924). The taxonomic significance of C. formosanus, in relation to a closely related species, C. caninus (Leiper, 1913), is briefly discussed. It has been first verified by adult worm recovery that C. formosanus is prevalent in Vientiane areas of Lao PDR, taking the freshwater fish, P. brevis, as a second intermediate host.     

Prevalence of zoonotic metacercariae in two species of grouper, Epinephelus coioides and Epinephelus bleekeri, and flathead mullet, Mugil cephalus, in Vietnam

Fishborne zoonotic metacercariae have not been reported from brackish water and marine fish from Vietnam waters although these parasites are common in the country's freshwater fish. Both wild-caught and cultured grouper (Epinephelus coioides and Epinephelus bleekeri), and mullet (Mugil cephalus) from brackish and marine waters located in Khanh Hoa province in central coastal Vietnam were examined, and found positive for zoonotic trematode metacercariae. From grouper, Heterophyopsis continua and Procerovum varium were recovered. The prevalence of H. continua ranged from 2.0 to 6.0% and that for P. varium ranged from 11.6 to 15.8%. Mullet were infected with Pygidiopsis summa and H. continua both of these species are new records for Vietnam. The prevalence of P. summa in mullet was generally high, ranging from 17.6 to 75.5%, and was significantly higher than the prevalence of H. continua (2.5 to 32.4%). There were no significant differences in the prevalence of metacercariae between grouper from natural or cultured habitats, indicating that the highest risk of infection occurs in the wild-caught state prior to their placement in culture. Further, there was no difference in metacercarial prevalence between the 2 species of grouper. Infected wild-caught seed were only observed from January to October. Monthly variation in prevalence suggests seasonal variation in mullet infections occurs in this region with the highest transmission taking place from October to December. Basic investigations on the ecology and epidemiology of these intestinal flukes need to be carried out to determine their significance as a public health problem and the aspects of their biology that may be vulnerable to control interventions.     

Surface ultrastructure of the adult stage of Acanthotrema felis (Trematoda: Heterophyidae)

The surface ultrastructure of Acanthotrema felis (Trematoda: Heterophyidae) adults, recovered from a kitten experimentally infected with the metacercariae, was observed using a scanning electron microscope. The worm was leaf-like, ventrally concave and covered with scale-like multi-pointed tegumental spines. The spines on the anterior surface were short but broad, and had 10-12 pointed tips. The cytoplasmic processes protruded around the spines, like pockets for the spines. The ventrogenital opening was crescent, or kidney-shaped, and had protuberances with minute spines on its surrounding tegument. The spines on the posterior surface were long, but narrow, with 6-8 pointed tips. The cytoplasmic processes on this tegument were ridge-like, and elevated along the row of the spines. The surface ultrastructure of A. felis is generally similar to that of other heterophyid flukes, but some features are characteristic, and may be of taxonomic and bio-ecological significance.     

Acanthoparyphium tyosenense: the discovery of human infection and identification of its source

Acanthoparyphium tyosenense Yamaguti, 1939 (Digenea: Echinostomatidae), was originally reported as an avian intestinal parasite; here, its presence is reported in 10 humans in the Republic of Korea. The patients were 9 adults aged 35-66 yr (males and females) and a young girl aged 7 yr residing in 2 coastal villages in Puan-gun, Chollabuk-do. The worms were recovered after treatment with praziquantel and purgation with magnesium salts. A total of 158 specimens (1-107 specimens/individual) was collected, together with varying numbers of other intestinal flukes. The patients had eaten various kinds of brackish water mollusks caught in an estuary near their villages. Five bivalves and a gastropod species suspected as sources of human infection were collected and examined. Two bivalves (Mactra veneriformis and Solen grandis) and the gastropod (Neverita bicolor) were found to be infected with the metacercariae of A. tyosenense; adult flukes were confirmed after the experimental infection of chicks. The results show that A. tyosenense infects humans and that brackish water mollusks are the source of human infection.     

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.     

Two endemic foci of heterophyids and other intestinal fluke infections in southern and western coastal areas in Korea

Two endemic foci of heterophyid infections were discovered in coastal villages of Puan-gun, Chollabuk-do, and Sachon-gun. Kyongsangnam-do, Korea. Fecal examinations were performed on 153 inhabitants of Puan-gun and 138 of Sachon-gun, using cellophane thick smear and formalin-ether sedimentation technique. The helminth egg and/or protozoan cyst positive rate was 21.5% (33/153) in Puan-gun and 39.1% (54/138) in Sachon-gun. In Puan-gun, the egg positive rate of heterophyids was the highest, 17.6%, and that of other parasites was 0.7-2.6% by parasite species. In Sachon-gun, that of heterophyids was 18.8%, followed by Clonorchis sinensis (12.3%), and other parasites (0.7-5.0%). Twenth-two (Puan-gun) and six (Sachon-gun) heterophyid egg positive cases were treated with praziquantel, and adult flukes were collected from their diarrheic stools. A total of 3,284 adult flukes of Heterophyes nocens was collected from all of the 22 patients treated in Puan-gun (3-778 individually), and other trematodes were also collected from 2-15 patients: Pygidiopsis summa, Stellantchasmus falcalus, Metagonimus yokogawai, M. miyatai, Stictodora fuscata, Heterophyopsis continua, Acanthoparyphium kurogamo, and Gymnophalloides seoi. In Sachon-gun, M. yokogawai (3,007 specimens), H. nocens (120), and S. falcatus (46) were collected from 5 of 6 treated patients, and H. continua and S. lari each from one patient. The present study revealed that heterophyid flukes, especially H. nocens and M. yokogawai, are prevalent in the southern and western coastal areas of Korea where fresh and/or brackish water fishes are popularly eaten raw.     

Epidemiological Survey on the Infection of Intestinal Flukes in Residents of Muan-gun, Jeollanam-do, the Republic of Korea

Infection status of intestinal flukes was investigated in residents of Muan-gun, Jeollanam-do, the Republic of Korea. Total 1,257 fecal samples of residents were examined by formalin-ether sedimentation technique and Kato-Katz thick smear method. Helminth eggs were detected from 95 (7.6%) residents, and eggs of heterophyid flukes and Clonorchis sinensis were found from 62 (4.9%) and 40 (3.2%) cases, respectively. The larger heterophyid eggs, somewhat dark-brown in color and 37.7 × 21.5 µm in average size, and found in 32 (2.6%) out of 62 egg positive cases of heterophyid flukes. To confirm the adult flukes, we performed worm recovery from 12 cases after praziquantel treatment and purgation with MgSO₄. A total of 1,281 adult flukes, assigned to 7 species, were recovered from 9 cooperative cases. Heterophyes nocens (total 981 specimens) was collected from 9 cases, Stictodora fuscata (80) from 7, Gymnophalloides seoi (75) from 5, Pygidiopsis summa (140) from 3, Stellantchasmus falcatus (3) from 2, and Stictodora lari and Acanthotrema felis (each 1 worm) from 1 case each. The intrauterine eggs of S. fuscata collected from the recovered worm were identical with the larger heterophyid eggs detected in the stool examination. By the present study, it was confirmed that A. felis is a new intestinal fluke infecting humans, and residents in Muan-gun, Jeollanam-do are infected with variable species of intestinal trematodes.     

Decrease of Metagonimus yokogawai Endemicity along the Tamjin River Basin

The Tamjin River which flows from Jangheung-gun via Gangjin-gun to the South Sea was reported to be a highly endemic area of Metagonimus yokogawai infection in 1977 and 1985. However, there were no recent studies demonstrating how much change occurred in the endemicity, in terms of prevalence and worm burden, of metagonimiasis in this river basin. Thus, a small-scale epidemiological survey was carried out on some residents along the Tamjin River basin in order to determine the current status of M. yokogawai infection. A total of 48 fecal samples were collected and examined by the Kato-Katz thick smear and formalin-ether sedimentation techniques. The egg positive rate of all helminths was 50.0%, and that of M. yokogawai was 37.5%, followed by C. sinensis 22.9% and G. seoi 4.2%. To obtain the adult flukes of M. yokogawai, 6 egg positive cases were treated with praziquantel 10 mg/kg in a single dose and purged with magnesium sulfate. A total of 5,225 adult flukes (average 871 specimens per person) of M. yokogawai were collected from their diarrheic stools. Compared with the data reported in 1977 and 1985, the individual worm burdens appeared to have decreased remarkably, although the prevalence did not decrease at all. It is suggested that the endemicity of M. yokogawai infection along the Tamjin River has been reduced. To confirm this suggestion, the status of infection in snail and fish intermediate hosts should be investigated.     

Holostephanus metorchis (Digenea: Cyathocotylidae) from chicks experimentally infected with metacercariae from a fish, Pseudorasbora parva, in the Republic of Korea

Holostephanus metorchis (Digenea: Cyathocotylidae) is a parasite of birds, transmitted by freshwater fishes. H. metorchis adults were recovered from chicks experimentally infected with metacercariae collected from freshwater fishes, Pseudorasbora parva. The metacercariae were oval, surrounded with thick fibrous capsules. In adult flukes, the holdfast organ occupied the ventral concavity, and the anterior testis did not reach the level of the ventral sucker. Based on these morphological characteristics, these flukes were identified as H. metorchis.     

First report of human infection with Gynaecotyla squatarolae and first Korean record of Haplorchis pumilio in a patient

Gynaecotyla squatarolae (Digenea: Microphallidae) is a minute intestinal trematode whose natural hosts are aves. We conducted a feces screening survey in a coastal village of Muan-gun, where the residents routinely consume brackish water crabs as a food. Through this survey, a 50-year-old female was found to shed gymnophallid and heterophyid eggs in her stool, and 845 adult flukes were collected from her purged stool. The adult worms were morphologically grouped into three species. A total of 841 worms were Gymnophalloides seoi. Three worms were identified as G. squatarolae, and the last one proven to be Haplorchis pumilio. This is the first worldwide report of G. squatarolae infection in humans, and the first H. pumilio infection in Korean people.     

Fishborne Trematode Metacercariae in Freshwater Fish from Guangxi Zhuang Autonomous Region, China

A survey was performed to investigate the infection status of fishborne trematode (FBT) metacercariae in freshwater fish from Guangxi Zhuang Autonomous Region, China. A total of 307 freshwater fish of 31 species were collected from 5 administrative regions of Guangxi Zhuang Autonomous Region. They were examined by artificial digestion method from July 2003 to August 2004. No metacercariae were detected in fish from Fusui-xian. In fish from Mashan-xian and a market in Nanning, 3 species of metacercariae, Haplorchis taichui, Haplorchis pumilio, and Centrocestus formosanus, were mainly detected. Metacercariae (8 in number) of Clonorchis sinensis were found in 1 Chanodichthys dabryi purchased from a market in Nanning. In fish from Yangshuo, Metagonimus yokogawai metacercariae were detected from all 18 fish species examined. Total 13 C. sinensis metacercariae were found in 3 out of 10 Hemibarbus maculatus from Yangshuo. All 7 Zacco platypus from Yangshuo were infected with 8-112 Echinochasmus perfoliatus metacercariae. In fish from Binyang-xian, H. pumilo metacercariae were mainly detected in all 5 fish species examined, and only 1 metacercaria of C. sinensis was found in a Hemiculter leucisculus. From the above results, it was confirmed that some species of freshwater fish play a role of second intermediate hosts for FBT in Guangxi Zhuang Autonomous Region, China. In particular, 4 species of intestinal flukes, M. yokogawai, H. taichui, H. pumilio, and C. formosanus, were prevalent in fish hosts, whereas C. sinensis metacercariae were detected only in 3 fish species.     

Ovarian germline stem cells in the teleost fish, medaka (Oryzias latipes)

In the mammalian testis germline stem cells keep producing many sperms, while there is no direct evidence for the presence of germline stem cells in the ovary. It is widely accepted in mammals that the mature oocytes are supplied from a pool of primordial follicles in the adult ovary. In other vertebrates, such as fish, however, there has been no investigation on the mechanism underlying the high egg-producing ability. In this review, we introduce the recently identified ovarian germline stem cells and the surrounding unique structure in teleost fish, medaka (Oryzias latipes) [Nakamura S et al. Science. 2010; 328: 1561-1563]. We also discuss about the expression and function of sox9 that characterizes this unique structure.     

Prevalence of Clonorchis sinensis metacercariae in freshwater fish from three latitudinal regions of the Korean Peninsula

A large-scale survey was conducted to investigate the infection status of fresh water fishes with Clonorchis sinensis metacercariae (CsMc) in 3 wide regions, which were tentatively divided by latitudinal levels of the Korean peninsula. A total of 4,071 freshwater fishes were collected from 3 regions, i.e., northern (Gangwon-do: 1,543 fish), middle (Chungcheongbuk-do and Gyeongsangbuk-do: 1,167 fish), and southern areas (Jeollanam-do, Ulsan-si, and Gyeongsangnam-do: 1,361 fish). Each fish was examined by the artificial digestion method from 2003 to 2010. In northern areas, only 11 (0.7%) fish of 2 species, Pungtungia herzi and Squalidus japonicus coreanus from Hantan-gang, Cheolwon-gun, Gangwon-do were infected with av. 2.6 CsMc. In middle areas, 149 (12.8%) fish were infected with av. 164 CsMc. In southern areas, 538 (39.5%) fish were infected with av. 159 CsMc. In the analysis of endemicity in 3 regions with an index fish, P. herzi, 9 (6.2%) of 146 P. herzi from northern areas were infected with av. 2.8 CsMc. In middle areas, 34 (31.8%) of 107 P. herzi were infected with av. 215 CsMc, and in southern areas, 158 (92.9%) of 170 P. herzi were infected with av. 409 CsMc. From these results, it has been confirmed that the infection status of fish with CsMc is obviously different among the 3 latitudinal regions of the Korean peninsula with higher prevalence and burden in southern regions.     

Microphallus koreana n. sp. (Trematoda: Microphallidae) transmitted by a marine crab, Macrophthalmus dilatatus

Microphallus species occur primarily as intestinal parasites of birds and mammals, and metacercariae of a new species belonging to this genus have been discovered from the crab, Macrophthalmus dilatatus, in the Republic of Korea. The metacercaria of this fluke was round with 2 thick walls, and the excysted one had mature genital organs. The adult flukes recovered from experimentally infected chicks had numerous intrauterine eggs, well-developed pars prostatica, widely bifurcating ceca, and prominent uterine bulge. After observing internal structures, it was concluded that this species is different from any other known Microphallus spp. Based on the morphology of metacercariae and adult flukes, we describe this specimen as a new species, Microphallus koreana n. sp.     

Experimental Life History and Biological Characteristics of Fasciola gigantica (Digenea: Fasciolidae)

This study was conducted to investigate the life history, morphology, and maturation of larval stages and adult worms of Fasciola gigantica in experimental mice. Lymnaea auricularia rubiginosa was used as the intermediate host, and Oryza sativa was used for encystment of the metacercariae, while Mus musculus was used as the definitive host for maturation study. Fresh eggs from the gall bladder of water buffaloes fully developed into embryonated ones and hatched out at days 11-12 after incubation at about 29ºC. Free-swimming miracidia rapidly penetrated into the snail host, and gradually developed into the next larval stages; sporocyst, redia, and daughter redia with cercariae. Fully-developed cercariae were separated from the redia and shed from the snails on day 39 post-infection (PI). Free-swimming cercariae were immediately allowed to adhere to rice plants, and capsules were constructed to protect metacercariae on rice plants. Juvenile worms were detected in intestines of mice at days 3 and 6 PI, but they were found in the bile duct from day 9 PI. Juvenile and adult flukes were recovered from 16 mice experimentally infected with metacercariae, with the average recovery rate of 35.8%. Sexually mature adult flukes were recovered from day 42 PI. It could be confirmed that experimentally encysted metacercariae could infect and develop to maturity in the experimental host. The present study reports for the first time the complete life history of F. gigantica by an experimental study in Thailand. The obtained information can be used as a guide for prevention, elimination, and treatment of F. gigantica at environment and in other hosts.     

Development of a novel beta-cell specific promoter system for the identification of insulin-producing cells in in vitro cell cultures

Recently, it has been reported that islet transplantation into patients with Type 1 diabetes may achieve insulin independence for a year or longer [Shapiro et al., Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen, N Engl J Med. 343 (2000) 230-238]. However, the amount of donor islet tissue is limited, therefore, multiple approaches are being explored to generate insulin-producing cells in vitro. Some promising results have been obtained using mouse and human stem cells and progenitor cells [Soria et al., From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus, Diabetologia. 4 (2001) 407-415; Lechner et al., Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus, Am J Physiol Endocrinol Metab. 284 (2003) 259-266; Bonner-Weir et al., In vitro cultivation of human islets from expanded ductal tissue, Proc Natl Acad Sci U S A, 97 (2000) 7999-8004; Assady et al., Insulin production by human embryonic stem cells, 50 (2001) Diabetes 1691-1697]. However, the efficiency of obtaining populations with high numbers of differentiated cells has been poor. In order to improve the efficiency of producing and selecting insulin-producing cells from undifferentiated cells, we have designed a novel beta-cell specific and glucose responsive promoter system designated pGL3.hINS-363 3x. This artificial promoter system exhibits significant luciferase activity not only in insulin-producing MIN6 m9 cells but also in isolated human islets. The pGL3.hINS-363 3x construct shows no activity in non-insulin-producing cells in low glucose conditions (2 mM glucose) but demonstrates significant activity and beta-cell specificity in high glucose conditions (16 mM glucose). Furthermore, pGL3.hINS-363 3x shows significant promoter activity in differentiated AR42J cells that can produce insulin after activin A and betacellulin treatment. Here, we describe a novel beta-cell specific and glucose responsive artificial promoter system designed for analyzing and sorting beta-like insulin-producing cells that have differentiated from stem cells or other progenitor cells.