Infection Status of Zoonotic Trematode Metacercariae in Fishes from Vientiane Municipality and Champasak Province in Lao PDR

The infection status of fishborne zoonotic trematode (FZT) metacercariae was investigated in fishes from 2 localities of Lao PDR. Total 157 freshwater fishes (17 species) were collected in local markets of Vientiane Municipality and Champasak Province in December 2010 and July 2011, and each fish was examined by the artificial digestion method. Total 6 species of FZT metacercariae, i.e., Opisthorchis viverrini, Haplorchis taichui, Haplorchis yokogawai, Haplorchis pumilio, Centrocestus formosanus, and Procerovum varium, were detected in fishes from Vientiane Municipality. The metacercariae of O. viverrini were detected in 50 (49.5%) out of 101 fishes (6 species), and their average number was 154 per fish infected. The remaining 5 species of heterophyid metacercariae were detected in 36.8%, 65.8%, 9.4%, 23.9%, and 5.1% fishes examined, and their average densities were 12, 1,038, 4, 15, and 13 per infected fish, respectively. In fishes from Champasak Province, 3 species of FZT metacercariae, i.e., O. viverrini, H. taichui, and H. yokogawai, were detected. Only 2 O. viverrini metacercariae were found in only 1 Barbonymus schwanefeldi. The metacercariae of H. taichui and H. yokogawai were detected in 60.0% and 50.0% of fishes examined, and their average densities were 47 and 28 per fish infected. By the present study, it has been confirmed that several species of FZT metacercariae are prevalent in fishes from Vientiane Municipality, with P. varium being a new member of FZT in Lao PDR. In comparison, FZT metacercariae are less prevalent in fishes from Champasak Province.     

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.     

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.     

Zoonotic Trematode Metacercariae in Fish from Phnom Penh and Pursat,Cambodia

A survey was performed to investigate the infection status of freshwater fish with zoonotic trematode metacercariae in Phnom Penh and Pursat Province, Cambodia. All collected fish with ice were transferred to our laboratory and examined using the artificial digestion method. In fish from Phnom Penh, 2 kinds of metacercariae (Opisthorchis viverrini and Haplorchis yokogawai) were detected. O. viverrini metacercariae were positive in 37 (50.0%) of 74 fish in 11 species (average no. metacercariae/fish, 18.6). H. yokogawai metacercariae were detected in 23 (57.5%) of 40 fish in 5 species (average no. metacercariae/fish, 21.0). In fish from Pursat Province, 5 kinds of metacercariae (O. viverrini, H. yokogawai, Haplorchis pumilio, Centrocestus formosanus, and Procerovum sp.) were detected; O. viverrini metacercariae (n=3) in 2 fish species (Henicorhynchus lineatus and Puntioplites falcifer), H. yokogawai metacercariae (n=51) in 1 species (P. falcifer), H. pumilio metacercariae (n=476) in 2 species (H. lineatus and Pristolepis fasciata), C. formosanus metacercariae (n=1) in 1 species (H. lineatus), and Procerovum sp. metacercariae (n=63) in 1 species (Anabas testudineus). From the above results, it has been confirmed that various freshwater fish play the role of a second intermediate host for zoonotic trematodes (O. viverrini, H. yokogawai, H. pumilio, C. formosanus, and Procerovum sp.) in Cambodia.     

Foodborne Trematode Metacercariae in Fish from Northern Vietnam and Their Adults Recovered from Experimental Hamsters

The prevalence of foodborne trematode (FBT) metacercariae was investigated in fish from 2 localities of northern Vietnam in 2004-2005. Freshwater fish (9 species) were collected from local markets in Hanoi City (n=76) and Nam Dinh Province (n=79), and were examined for FBT metacercariae using the artificial digestion technique. Adult flukes were obtained from hamsters experimentally infected with the metacercariae at day 8 post-infection. Three (Haplorchis pumilio, Centrocestus formosanus, and Procerovum varium) and 6 (Haplorchis taichui, H. pumilio, C. formosanus, P. varium, Stellantchasmus falcatus, and Heterophyopsis continua) species of FBT metacercariae were detected in the 2 regions, respectively. Overall, among the positive fish species, H. pumilio metacercariae were detected in 104 (80.0%) of 130 fish examined (metacercarial density per infected fish; 64.2). C. formosanus metacercariae were found in 37 (40.2%) of 92 fish (metacercarial density; 14.7). P. varium metacercariae were detected in 19 (63.3%) of 30 fish (Anabas testudineus and Mugil cephalus) (metacercarial density; 247.7). S. falcatus metacercariae were found in all 10 M. cephalus examined (metacercarial density; 84.4). H. continua metacercariae (2 in number) were detected in 1 fish of Coilia lindmani. Morphologic characteristics of the FBT metacercariae and their experimentally obtained adults were described. The results have demonstrated that various FBT species are prevalent in northen parts of Vietnam.     

High prevalence of liver and intestinal fluke infections among residents of Savannakhet Province in Laos

The prevalence of liver and intestinal fluke infections was surveyed on residents of Savannakhet Province, Laos. Fecal specimens were collected from a total of 981 residents in 4 Mekong riverside villages and examined by the Kato-Katz thick smear technique. The results revealed that the overall helminth egg positive rate was 84.2%, and the positive rate for small trematode eggs, including Opisthorchis viverrini, heterophyids, or lecithodendriids, was 67.1%. To obtain adult flukes, 38 small trematode egg positive cases were treated with a 20-30 mg/kg single dose of praziquantel and purged. Diarrheic stools were then collected from 29 people and searched for helminth parasites using stereomicroscopes. Mixed infections with O. viverrini and 6 kinds of intestinal flukes were found, namely, Haplorchis taichui, Haplorchis pumilio, Haplorchis yokogawai, Prosthodendrium molenkampi, Phaneropsolus bonnei, and echinostomes. The total number of flukes collected was 7,693 specimens (av. no. per treated person; 265.3). The most common species was O. viverrini, followed by H. taichui, P. molenkampi, echinostomes, H. pumilio, P. bonnei, and H. yokogawai. The results indicate that foodborne liver and intestinal fluke infections are prevalent among residents of Savannakhet Province, Laos.     

Farm-Level Risk Factors for Fish-Borne Zoonotic Trematode Infection in Integrated Small-Scale Fish Farms in Northern Vietnam

Background

Northern Vietnam is an endemic region for fish-borne zoonotic trematodes (FZT), including liver and intestinal flukes. Humans acquire the FZT infection by eating raw or inadequately cooked fish. The production of FZT-free fish in aquaculture is a key component in establishing a sustainable program to prevent and control the FZT transmission to humans. Interventions in aquaculture should be based on knowledge of the main risk factors associated with FZT transmission.

Methodology/Principal Findings

A longitudinal study was carried out from June 2006 to May 2007 in Nam Dinh province, Red River Delta to investigate the development and risk factors of FZT infections in freshwater cultured fish. A total of 3820 fish were sampled six times at two-month intervals from 96 fish farms. Logistic analysis with repeated measurements was used to evaluate potential risk factors based on information collected through questionnaire interviews with 61 fish farm owners. The results showed that the FZT infections significantly increased from first sampling in June to July 2006 (65%) to sixth sampling in April to May, 2007 (76%). The liver fluke, Clonorchis sinensis and different zoonotic intestinal flukes including Haplochis pumilio, H. taichui, H. yokogawai, Centrocestus formosanus and Procerovum varium were found in sampled fish. Duration of fish cultured (sampling times), mebendazole drug self-medication of household members, presence of snails in the pond, and feeding fish with green vegetation collected outside fish farms all had a significant effect on the development of FZT prevalence in the fish.

Conclusions/Significance

The FZT prevalence in fish increased by 11 percentage points during a one-year culture period and the risk factors for the development of infection were identified. Results also highlight that the young fish are already highly infected when stocked into the grow-out systems. This knowledge should be incorporated into control programs of FZT transmission in integrated small-scale aquaculture nursery and grow-out systems in Vietnam.     

Fishborne Trematode Metacercariae in Luang Prabang,Khammouane, and Saravane Province,Lao PDR

Fishborne trematode (FBT) metacercariae were investigated in fish from 3 Provinces of Lao PDR. Total 242 freshwater fish of 40 species were collected in local markets of Luang Prabang (59 fish of 16 species), Khammouane (81 fish of 19 species), and Saravane (97 fish of 14 species), and each of them was examined by artificial digestion method. Four species of metacercariae (Opisthorchis viverrini, Haplorchis taichui, Haplorchis yokogawai, and Centrocestus formosanus) were detected. O. viverrini was detected in 35 fish (14.5%), and their density was 252 per infected fish (Luang Prabang, 88 metacercariae in 5 fish; Khammouane, 187 in 6 fish; Saravane, 303 in 24 fish). H. taichui was found in 102 fish (42.1%), and their density was 485 per infected fish (Luang Prabang, 260 metacercariae in 38 fish; Khammouane, 1,084 in 23 fish; Saravane, 359 in 41 fish). H. yokogawai was detected in 92 fish (38.0%), and their density was 222 per infected fish (Luang Prabang, 362 metacercariae in 17 fish; Khammouane, 126 in 20 fish; Saravane, 214 in 55 fish). Metacercariae of C. formosanus were found in 8 fish (3.3%), and their density was 3 per infected fish. In the present study, it has been confirmed that FBT metacercariae, in particular, H. taichui, H. yokogawai, and O. viverrini, are highly prevalent in fish from Luang Prabang, Khammouane, and Saravane Province, Lao PDR.     

Prevalence of Haplorchis taichui and Haplorchoides sp. metacercariae in freshwater fish from water reservoirs, Chiang Mai, Thailand

A parasitological investigation on trematode metacercariae was made on 62 freshwater fishes of 13 species in northern Thailand; Cyclocheilichthys apogon, Puntioplites proctozysron, Labiobarbus siamensis, Barbodes gonionotus, Barbodes altus, Henicorhynchus siamensis, Osteochilus hasselti, Notopterus notopterus, Mystacoleucus marginatus, Anabas testudineus, Systomus orphoides, Morulius chrysophykadian, and Hampala macrolepidota. The fish were caught over the summer period (February-May 2007) from 2 Chiang Mai water reservoirs, i.e., the Mae Ngad (UTM 47Q E 503200, 47Q N 2119300) and the Mae Kuang Udomtara (UTM 47Q E 513000, 47Q N 2092600) Reservoirs in Chiang Mai province, Thailand. The prevalence of heterophyid (Haplorchis taichui and Haplorchoides sp.) metacercariae in these fish was 83.9% and 74.2% in the Mae Ngad and Mae Kuang Udomtara Reservoirs, respectively. The highest intensity of heterophyid metacercariae in H. siamensis in the Mae Ngad was 120.4 and that in P. proctozysron in the Mae Kuang Udomtara was 180.0. The fish, A. testudineus, C. apogon, and M. chrysophykadian, were not found to be infected with H. taichui metacercariae. The results show that the freshwater fish in Chiang Mai water reservoirs are heavily infected with H. taichui and Haplorchoides sp. metacercariae.     

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.     

Fishborne Trematode Metacercariae Detected in Freshwater Fish from Vientiane Municipality and Savannakhet Province, Lao PDR

Freshwater fish from Vientiane Municipality and Savannakhet Province, Lao PDR were examined by the muscle compression and artificial digestion methods to know the infection status with trematode metacercariae. In the fish from Savannakhet, 2 species of metacercariae, Opisthorchis viverrini and Haplorchis taichui, were detected. O. viverrini metacercariae were found in 6 species of fish, Puntius brevis, Hampala dispar, Esomus metallicus, Mystacoleucus marginatus, Puntioplites falcifer, and Cyclocheilichthys armatus. H. taichui metacercariae were detected in 3 species of fish, P. brevis, P. falcifer, and M. marginatus. In the fish from Vientiane, 4 species of metacercariae, O. viverrini, H. taichui, Haplorchis yokogawai, and Centrocestus formosanus, were detected. Among them, O. viverrini metacercariae were found in 7 species of fish, Onychostoma elongatum, C. armatus, H. dispar, P. brevis, Cyclocheilichthys repasson, Osteochilus hasseltii, and Hypsibarbus lagleri. The metacercariae of H. taichui were detected in 6 species of fish, C. repasson, O. elongatum, C. armatus, H. dispar, Labiobarbus leptocheila, and Cirrhinus molitorella. The metacercariae of H. yokogawai were found in 9 species of fish, C. repasson, O. elongatum, C. armatus, H. dispar, Labiobarbus leptocheila, O. hasseltii. C. molitorella, Hypsibarbus wetmorei, and H. lagleri. The metacercariae of C. formosanus were detected in 4 species of fish, C. repasson, P. brevis, O. hasseltii, and C. molitorella. From these results, it is confirmed that fishborne trematode metacercariae, i.e. O. viverrini, H. taichui, H. yokogawai and C. formosanus, are prevalent in various species of freshwater fish from Savannakhet Province and Vientiane Municipality, Lao PDR.     

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.     

Relationship between Snail Population Density and Infection Status of Snails and Fish with Zoonotic Trematodes in Vietnamese Carp Nurseries

Background

Fish-borne zoonotic trematodes (FZT) are a food safety and health concern in Vietnam. Humans and other final hosts acquire these parasites from eating raw or under-cooked fish with FZT metacercariae. Fish raised in ponds are exposed to cercariae shed by snail hosts that are common in fish farm ponds. Previous risk assessment on FZT transmission in the Red River Delta of Vietnam identified carp nursery ponds as major sites of transmission. In this study, we analyzed the association between snail population density and heterophyid trematode infection in snails with the rate of FZT transmission to juvenile fish raised in carp nurseries.

Methodology/Principal Findings

Snail population density and prevalence of trematode (Heterophyidae) infections were determined in 48 carp nurseries producing Rohu juveniles, (Labeo rohita) in the Red River Delta area. Fish samples were examined at 3, 6 and 9 weeks after the juvenile fish were introduced into the ponds. There was a significant positive correlation between prevalence of FZT metacercariae in juvenile fish and density of infected snails. Thus, the odds of infection in juvenile fish were 4.36 and 11.32 times higher for ponds with medium and high density of snails, respectively, compared to ponds where no infected snails were found. Further, the intensity of fish FZT infections increased with the density of infected snails. Interestingly, however, some ponds with no or few infected snails were collected also had high prevalence and intensity of FZT in juvenile fish. This may be due to immigration of cercariae into the pond from external water sources.

Conclusions/Significance

The total number and density of potential host snails and density of host snails infected with heterophyid trematodes in the aquaculture pond is a useful predictor for infections in juvenile fish, although infection levels in juvenile fish can occur despite low density or absence infected snails. This suggests that intervention programs to control FZT infection of fish should include not only intra-pond snail control, but also include water sources of allochthonous cercariae, i.e. canals supplying water to ponds as well as snail habitats outside the pond such as rice fields and surrounding ponds.     

Prevalence of Zoonotic Trematode Metacercariae in Freshwater Fish from Gangwon-do,Korea

The infection status of zoonotic trematode metacercariae was investigated in a total of 2,293 freshwater fish collected from 11 rivers or streams in 9 administrative regions of Gangwon-do, Korea for 5 years (2009-2013). All fish were collected by netting methods and examined using the artificial digestion methods. Clonorchis sinensis metacercariae were detected in 4 fish species, i.e., Pungtungia herzi, Squalidus japonicus coreanus, Acheilognathus rhombeus, and Ladislabia taczanowskii, from only Hantangang in Cheorwon-gun. Metagonimus spp. metacercariae were found in 1,154 (50.3%) fish and their average number per infected fish was 55.8. Among the positive fish species, especially Tribolodon hakonensis from Namdaecheon in Yangyang-gun and Plecoglossus altivelis from Osipcheon in Samcheok-si were most heavily infected. Centrocestus armatus metacercariae were detected in 611 (26.7%) fish and the average metacercarial burden per infected fish was 1,032. Two chub species, Zacco platypus and Zacco temminckii were highly and heavily infected with C. armatus metacercariae in almost all regions surveyed. Echinostoma spp. metacercariae were also found in 24 fish from a few localities, but their numbers per fish infected were very low. From the above results, it is confirmed that the metacercariae of intestinal flukes, especially Metagonimus spp. and C. armatus, were heavily infected, while C. sinensis metacercariae were rarely found in fish from Gangwon-do, Korea.     

Prevalence of Opisthorchis viverrini-Like Fluke Infection in Ducks in Binh Dinh Province,Central Vietnam

Following the first report of Opisthorchis viverrini infection in a domestic duck in Phu My District of Binh Dinh Province, Central Vietnam, many other cases were observed in the province. We determined the infection rate and intensity of O. viverrini infection in ducks in 4 districts of the province. A total of 178 ducks were randomly selected from 34 farms for examination of flukes in the liver and gall bladder. An infection rate of 34.3% (range 20.7-40.4% among districts) was found; the intensity of infection was 13.8 worms per infected duck (range 1-100). These findings show the role of ducks as a host for O. viverrini, duck genotype, which is sympatric with the human O. viverrini genotype in this province. It also stresses the need for investigations on the zoonotic potential and the life cycle of this parasite.     

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.     

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.     

Clonorchis sinensis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination

We report a single, one-step PCR approach for detection and discrimination of Clonorchis sinensis and Opisthorchis viverrini in different life-stage forms (adults, metacercariae, and eggs) from fish intermediate hosts and from infected patients. Primers designed for species-specific PCR, amplifying portions of the mitochondrial (mt) genome, were also suitable for a multiplex PCR. The latter was a single, one-step reaction under high stringency conditions, using simultaneously 2 pairs of primers (1 pair for C. sinensis--product size 612 bp, and 1 pair for O. viverrini--product size 1357 bp). Assays using serially diluted templates demonstrated that as little as 0.78 ng of genomic DNA of either species could yield amplicons. Genomic DNA extracted from different life-stage forms including adult worms (of both species), eggs (of O. viverrini), eggs possibly of several trematode species (collected from patients infected with C. sinensis in Vietnam) and mixed metacercariae of common trematodes (collected from fishes in the C. sinensis endemic areas), yielded specific bands of the correct size and their identity was confirmed by sequence analysis. The multiplex PCR approach described here proved to be a species-specific, sensitive and fast tool for accurate diagnosis of clonorchiasis and/or opisthorchiasis, permitting the detection of their metacercariae in infected fishes or adult/eggs from patients in endemic areas.     

Rapid and simple identification of human pathogenic heterophyid intestinal fluke metacercariae by PCR-RFLP

Six species of heterophyid intestinal flukes (HIFs) constitute the major endemic zoonotic fish-borne pathogens in Asia: Haplorchis taichui, H. pumilio, H. yokogawai, Procerovum varium, Stellantchasmus falcatus, and Centrocestus formosanus. Several different species of these parasites are often found co-infecting the same second intermediate fish host. Because of their morphological similarities, differentiating between species of HIF metacercariae is difficult, time-consuming, and frequently results in misidentification. In this study, we aimed to develop an efficient and accurate method of identifying metacercariae of these 6 HIFs. Metacercariae were roughly grouped together, based on morphological characteristics seen under a stereomicroscope. Then, PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the exact species of each metacercaria, using the 28S ribosomal RNA gene as the genetic marker and MboII as the restriction enzyme. Using a combination of morphological and molecular methods eliminates the need for DNA sequencing and infecting animal subjects to obtain adult worms, increases accuracy, and decreases the need for laborious morphological identification. Because the method is simple, rapid, and relatively cheap compared with PCR-sequencing, it may be an effective tool for epidemiological studies of HIFs in endemic areas.     

Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a mountainous area of Phongsaly Province, Lao PDR

Phongsaly Province, located in the northernmost area of Lao PDR, was previously suggested to be endemic for the liver fluke Opisthorchis viverrini infection. To confirm, or rule out, this suggestion, the Phonxay village in the Khoua District, Phongsaly Province, was selected for a survey. Ten volunteers (8 men and 2 women aged 31-57 years) who consumed raw freshwater fish and had gastrointestinal troubles were treated with a single dose of praziquantel (40 mg/kg) and pyrantel pamoate (10 mg/kg) and purged with magnesium sulfate to recover any worm parasites. Eight of the 10 volunteers expelled 1 or more species of trematodes, nematodes, or cestodes (worm positive rate; 80%). The worms were morphologically identified as H. taichui (861 worms from 8 people), H. yokogawai (59 from 6 people), Phaneropsolus bonnei (1 from 1 person), Trichostrongylus sp. (2 from 2 people), Ascaris lumbricoides (2 from 1 person), Enterobius vermicularis (11 from 3 people), and Taenia saginata (1 strobila with scolex from 1 person). The results indicate that the mountainous area of Phongsaly Province, Lao PDR, is not endemic for the liver fluke but endemic for intestinal flukes, in particular, Haplorchis taichui and H. yokogawai.