Fishborne trematode (FBT) infections affect the health of more than 40 million people throughout the world and they are particularly prevalent in Asian countries. These parasites provoke a remarkable morbidity to humans and cause serious damages to aquaculture, which is a valuable source of food and employment in developing countries [1,2].

Lao People's Democratic Republic (Lao PDR) is located in the middle of Southeast Asia and bordered by 5 countries, i.e., China, Myanmar, Thailand, Cambodia, and Vietnam. This country is administratively divided into 18 regions, Vientiane Municipality and 17 Provinces. The Mekong River flows through the whole country from north to south. A quarter of its territory is located in the Mekong River basin. Like Thai people, Laotian people also have unique food habit. Some of them like to eat dishes containing raw freshwater fish, such as 'Koi pla', 'Pla som', and 'Som fak', and easily infect with FBT. Moreover, they have engaged in aquaculture in the fishponds with latrines which can play an important source of contamination [3,4].

It has been revealed that so many Laotians are infected with trematodes such as Opisthorchis viverrini, heterophyids, echinostomes, and lecithodendriids by the work of 'Korea-Lao PDR Collaborative Project of Parasite Eradication' [5-7]. Some investigators have previously reported that O. viverrini and some species of intestinal flukes are prevalent as FBT in Lao PDR [8-14]. However, the major source of infections has not been obvious yet. Therefore, we performed epidemiological surveys to know the second intermediate hosts of FBT and the infection status of metacercariae in freshwater fish from Vientiane Municipality and Savannakhet Province, Lao PDR.

We collected 156 freshwater fish of 17 species from fishermen and small-scale fish dealers in Savannakhet in July 2002 and 177 fish of 12 species in Vientiane in November 2002 (Fig. 1). All collected fish were transferred to the laboratories (local laboratories in Lao PDR and Department of Parasitology and Institute of Health Sciences, Gyeongsang National University College of Medicine). The fish species were identified with the aid of ichthyologists in Lao PDR and FishBase site in internet [15], and individually measured the length and weight according to the species (Tables 1, 2). They all were examined by the artificial digestion method except a few from Savannakhet which were examined by a muscle compression method.

The digenetic trematode metacercariae were segregated and collected from the sediment of digested materials using a stere-omicroscope. The metacercariae collected were categorized according to their size and morphological characters, and then the intensity of infection and the infection rate of each fish species were calculated. To obtain the adult flukes, each species of metacercariae was experimentally infected to a cat and hamsters. The adult flukes were recovered from the experimental animals at day 10 to day 30 after infection, and identified on the basis of their morphologies to confirm the definite species of metacercariae.

By the present study, it has been confirmed that FBT 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. Scholz et al. [16] described 5 species of trematode metacercariae, O. viverrini, H. taichui, H. pumilio, Stellantchasmus falcatus, and C. formosanus, from freshwater fish in rice fields around Vientiane Municipality and Nam Ngum water reservoir, Lao PDR. Ditrich et al. [9] and Giboda et al. [10] also detected 4 (O. viverrini, H. taichui, H. pumilio, and S. falcatus) and 3 (O. viverrini, H. taichui, and H. pumilio) species of trematode metacercariae in the freshwater fish from rice fields around Vientiane Municipality and Nam Ngum water reservoir. However, the data of Ditrich et al. [9] and Giboda et al. [10] are jointly owned by those of Scholz et al. [16]. Therefore, we would like to refer the findings of Scholz et al. [16].

Scholz et al. [16] examined 782 freshwater fish of 45 species collected from rice fields around Vientiane Municipality and Nam Ngum water reservoir for the presence of trematode metacercariae. We also examined 156 freshwater fish of 17 species from Savannakhet and 177 fish of 12 species from Vientiane Municipality. We mainly used the artificial digestion method to detect trematode metacercariae except for a few fish from Savannakhet which were examined by a muscle compression method. However, Scholz et al. [16] examined all fish by the muscle compressive method.

As the infection sources of O. viverrini, various species of freshfreshwater fish, including Cyclocheilichthys siaja (= C. armatus), H. dispar, Puntius orphoides, Puntius gonionotus (= Barbonymus gonionotus), Puntius proctozysron (= Puntioplites proctozystron), Puntius viehoeveri (= Barbonymus gonionotus), Labiobarbus lineatus, Esomus metallicus, and Osteochilus sp., have been reported in Thailand [17,18]. In Lao PDR, 7 fish species, H. dispar, Hampala macrolepidota, Barbodes gonionotus (= Barbonymus gonionotus), C. repasson, P. brevis and 2 Puntius spp., were verified to be the second intermediate hosts [16]. While we found O. viverrini metacercariae in 6 fish species, H. dispar, P. brevis, C. armatus, M. marginatus, P. falcifer, and E. metallicus, from Savannakhet Province, and in 7 fish species, H. dispar, P. brevis, C. armatus, C. repasson, O. elongatum, O. hasseltii and H. lagleri, from Vientiane Municipality. It has been confirmed by the present study that 5 fish species, M. marginatus, P. falcifer, O. elongatum, O. hasseltii, and H. lagleri, are the new second intermediate hosts for O. viverrini among the literature.

The infection rates and intensities of O. viverrini metacercariae in fish were much higher in the present study compared with those of Scholz et al. [16]. Especially, 2 fish species, C. armatus and P. brevis, from Savannakhet Province, and 3 species, H. dispar, C. armatus, and O. elongatum, from Vientiane Municipality were heavily infected with the metacercariae of O. viverrini. These fish are highly dangerous if eaten raw by people because of the high density of metacercariae. This liver fluke species can provoke severe pathological changes in the biliary passage, such as dilatation, wall thickening, inflammation and mucosal hyperplasia of the bile duct, and liver cirrhosis [17]. Moreover, this fluke species has been recognized as an important risk factor in the development of cholangiocarcinoma in humans [19,20].

H. taichui metacercariae have been recorded in fish from several Asian countries, i.e. India, China, Taiwan, the Philippines, and Laos [16,21-23]. They were detected in 4 fish species, Cirrhinus reba (= Labeo ariza), Amblypharyngodon mola, Labeo bata, and Puntius sophore, in India, 3 fish species, Puntius binotatus, Puntius palata (= Spratellicypris palata), and Ophicephalus striatus (= Channa striata), in the Philippines, 3 fish species, B. gonionotus, P. brevis, and Puntius orphoides, in Thailand, and 4 fish species, H. dispar, H. macrolepidota, B. gonionotus, and Mystacoleucus maginatus, in Lao PDR [16,21-24]. In the present study, H. taichui metacercariae were detected in 3 fish species, P. brevis, M. marginatus, and P. falcifer, from Savannakhet Province, and in 6 fish species, H. dispar, C. armatus, C. repasson, O. elongatum, L. leptocheila, and C. molitorella, from Vientiane Municipality. It has been confirmed by the present study that 6 fish species, P. falcifer, C. armatus, C. repasson, O. elongatum, L. leptocheila, and C. molitorella, are recorded as new second intermediate hosts of H. taichui in the literature.

H. yokogawai metacercariae have been reported in Asian countries such as Taiwan, India, and Thailand, and in Egypt [25]. They were found in 11 fish species, Mystus vittatus, Channa punctatus, C. reba (= Labeo ariza), A. mola, L. bata, Nandus nandus, Puntius chola, Puntius sarana, P. sophore, Glossogobius giurus (= G. giuris), and Ompok bimaculatus in india, 6 fish species, Puntius binotatus, P. orphoides, Puntius leiacanthus, Cyprinus carpio, Osteochilus lini, and Puntius gonionotus (= Barbonymus gonionotus), in Thailand, and 2 fish species, Tilapia nilotica (= Oreochromis niloticus niloticus) and Tilapia zillii, in Egypt [22,24,26-29]. In the present study, H. yokogawai metacercariae were detected in 9 fish species, H. dispar, C. armatus, C. repasson, O. elongatum, L. leptocheila, O. hasseltii, H. wetmorei, H. lagleri, and C. molitorella, from Vientiane Municipality. These 9 fish species have never been reported as the second intermediate hosts for H. yokogawai in the literature. Therefore, here we record aforementioned 9 fish species as new second intermediate hosts for H. yokogawai.

C. formosanus has been recorded in China, Taiwan, Japan, the Philippines, and India [30,31]. Scholz et al. [16] detected C. formosanus metacercariae in only 1 fish species, Esomus longimana (= E. longimanus), among 45 fish species from rice fields around Vientiane Municipality and Nam Ngum water reservoir, Lao PDR. Recently, Han et al. [32] found C. formosanus metacercariae in P. brevis fish from Vientiane Municipality, Lao PDR. In the present study, we found them in 4 fish species, C. repasson, P. brevis, O. hasseltii, and C. molitorella, from Vientiane Municipality. Three among the 4 fish species, C. repasson, O. hasseltii, and C. molitorella, are added as new second intermediate host for C. formosanus.

The eggs of opisthorchiid flukes including O. viverrini are indistinguishable from those of heterophyid flukes in Kato-Katz fecal smears. Chai et al. [6,7] tried to recover adult flukes from people to know the exact status of trematode infection in 3 administrative regions of Lao PDR, i.e., Vientiane Municipality, Saravane Province, and Savannakhet Province, after praziquantel treatment and purgation with MgSO₄. In these studies, it was confirmed that O. viverrini and H. taichui are the 2 most common fluke species, and various fluke species are mixedinfected in the inhabitants. However, the intensity of infection with each fluke species varied by locality surveyed. In the case of O. viverrini, the worm burdens were high in Vientiane Municipality and Savannakhet Province, whereas the burden was comparatively low in Saravane Province. The intensity of O. viverrini infection was the highest in Savannakhet Province, followed by Vientiane Municipality, and Saravane Province [6,7]. However, in the case of H. taichui, the average number of worms recovered per person was remarkably high in Saravane Province, whereas it was comparatively low in Savannakhet Province and Vientiane Municipality [6,7]. From these findings, it was proved that O. viverrini is more endemic in Vientiane Municipality and Savannakhet Province, and H. taichui is highly endemic in Saravane Province.

On the other hand, survey data on larval fluke infections in intermediate hosts can also be used as an epidemiological index. In the present study, the metacercariae of O. viverrini and H. taichui were detected in the fish from Savannakhet, and the metacercariae of O. viverrini, H. taichui, H. yokogawai and C. formosanus were detected in the fish from Vientiane. Among them, O. viverrini and H. taichui were the 2 predominant species in both localities. The metacercarial density was the highest for O. viverrini in the fish from the 2 localities. This finding is well corresponded to the report of Chai et al. [6,7]. Conclusively, it has been shown by the present study that various freshwater fish from Vientiane Municipality and Savannakhet Province, Lao PDR will play a role for infection sources of FBT, i.e. O. viverrini, H. taichui, H. yokogawai, and C. formosanus.