INTRODUCTION
Mekong river basin in Lao People's Democratic Republic (Lao PDR) is known to be endemic areas of infections with fishborne zoonotic trematodes (FZT). Riparian Laotian peoples habitually consume raw or fermented freshwater fish, and thus they are easily infected with FZT [1,2]. Especially in stool examinations and worm recoveries, it has been reported that so many Laotian peoples are infected with FZT including Opisthorchis viverrini and heterophyid flukes [3-9].
Moreover, epidemiological studies on the second intermediate hosts (or the infection sources) of FZT were also performed in riverside areas of Lao PDR [9-13]. Schotz et al. [10] surveyed freshwater fishes from rice paddies and Nam Ngum water reservoir in Vientiane Province. Rim et al. [11,12] and Sohn et al. [9] investigated the infection status of FZT metacercariae in fishes from Vientiane Municipality, Savannakhet, Luang Prabang, Khammouane, and Saravane Provinces in Lao PDR. In the present study, to obtain further epidemiological information on FBT infections in Lao PDR, we additionally surveyed on the infection status of FBT metacercariae in freshwater fishes from Vientiane Municipality and Champasak Province (Fig. 1).
MATERIALS AND METHODS
We collected total 157 freshwater fishes in 17 species in local markets of Vientiane Municipality (117 fishes in 10 species) on December 2010 and July 2011, and Champasack Province (40 fishes in 10 species) on December 2010 (Fig. 1). All fishes were transferred to the laboratories (on-the-spot local laboratories in Lao PDR and in Department of Parasitology and Tropical Medicine, Gyeongsang National University School of Medicine), and the fish species were identified with a book [14] and FishBase website (http://www.fishbase.org/search.php) (Tables 1, 3). Individual fish was finely ground with a mortar with pestle or a grinder. The ground fish meat was mixed with artificial gastric juice, and the mixture was incubated at 36˚C for 2 hr. The digested material was filtered with a sieve (1×1 mm of mesh), and washed with 0.85% saline until the supernatant became clear. The sediment was carefully examined under a stereomicroscope, and metacercariae were collected based on general features. These collected metacercariae were then categorized according to the measurements and morphological characteristics, and the intensity of infection and the infection rate were calculated for each fish species.
Identified metacercariae were experimentally infected to cats to obtain adult flukes. At day 7 after the infection, cats were killed by peritoneal injection with animal anesthetics. The small intestines of cats were extracted and longitudinally opened with a scissors in a beaker with 0.85% saline. Adult flukes were recovered in the sediment of intestinal contents which were diluted with 0.85% saline. Recovered worms were fixed with 10% formalin under a cover glass pressure, stained with Semichon's acetocarmine, and observed under a light microscope with a micrometer.
RESULTS
Infection status of FZT metacercariae in fishes from Vientiane Municipality
The metacercariae of Haplorchis taichui were found in 43 (36.8%) out of 117 fishes (in 10 species) examined, and their average density was 12 per fish infected. Haplorchis yokogawai metacercariae were detected in 77 (65.8%) fishes, and their average density was 1,038 per fish infected. Haplorchis pumilio metacercariae were found in 11 (9.4%) fishes, and their average density was 4 per fish infected. Centrocestus formosanus metacercariae were detected in 28 (23.9%) fishes, and their average number was 14.6 per fish infected. The detailed infection status of heterophyid fluke metacercariae is shown in Table 1. The metacercariae of Procerovum varium were found in 6 (37.5%) out of 16 climbing perch, Anabas testudineus, and their average density was 12.7 per fish infected.
The metacercariae of Opisthorchis viverrini were detected in 50 (49.5%) out of 101 fishes in 6 species (60.0%), i.e., Cyclocheilichthys apogon, Cyclocheilichthys armatus, Puntius brevis, Cyclocheilichthys repasson, Anabas testudineus, and Henichorhynchus siamensis. Their average density was 154 per fish infected. The detailed infection status is designated in Table 2.
Infection status of FZT metacercariae in fishes from Champasak Province
The metacercariae of H. taichui were detected in 24 (60.0%) out of 40 fishes (all 10 species), and their average density was 47 per fish infected. Total 20 (50.0%) fishes were infected with 28 H. yokogawai metacercariae in average. The infection status of heterophyid fluke metacercariae is shown in Table 3. Only 2 O. viverrini metacercariae were found in 1 fish (50.0%) of Barbonymus schwanefeldi from Champasak Province.
Morphology of metacercariae and adults of Procerovum varium (unit; µm)
Metacercariae (n=15; Fig. 2): Cyst elliptical, 150-193 (175)×118-148 (136) in size, having yellow brownish pigment granules scattering in body areas of intestinal bifurcation, a pair of eyespots lateral to pharynx, a ventral sucker deflectively located from median, a long thick-walled expulser, and a D-shaped (half-moon-shaped) excretory bladder with grouped granules.
Adults (n=10; Fig. 3): Body small, pear-shaped, 280-325 (304) long, and 165-215 (186) wide, with greatest width at posterior 1/3. Oral sucker subterminal, 25-38 (32) by 38-43 (41). Pharynx subglobular or elliptical, 20-28 (25) by 15-25 (19). Esophagus short, 35-50 (43) in length. Ventral sucker very small, 13-20 (17) by 18-25 (21), embedded in ventrogenital sac. Expulsor long and thick-walled, 88-150 (123) by 15-23 (20). Ovary spherical or subspherical, 30-45 (38) by 35-53 (43), slightly dextral to midline. Seminal receptacle long saccular, 70-105 (75) by 18-38 (28), lying between expulsor and the left-side of testis. One testis globular or subglobular, 75-103 (87) by 88-115 (99), situated in middle of hind-body. Uterus with eggs occufying from anterior 1/3 to posterior end, most of hind-body. Vitellaria follicular, distributing from posterior border of ovary to posterior extremity. Eggs small, yellow, and 25-28 (27) by 13-15 (14).
DISCUSSION
In the present study, 6 species of FZT metacercariae, i.e., O. viverrini, H. taichui, H. yokogawai, H. pumilio, C. formosanus, and P. varium, were detected in fishes from Vientiane Municipality. Schotz et al. [10] found 5 species (O. viverrini, H. taichui, H. pumilio, Stellantchasmus falcatus, and C. formosanus) of FZT metacercariae in the fishes from Vientiane Province. Rim et al. [11] detected 4 species (O. viverrini, H. taichui, H. yokogawai and C. formosanus) of FZT metacercariae in fishes from Vientiane Municipality. In Rim et al. [12], 4 species (O. viverrini, H. taichui, H. yokogawai, and C. formosanus) of FZT metacercariae were also detected in fishes from 3 Provinces, i.e., Luang Prabang, Khammouane, and Saravane. Sohn et al. [9] detected 3 Haplorchis species, H. taichui, H. yokogawai, and H. pumilio, metacercariae in fishes from Luang Prabang Province. However, P. varium metacercariae were detected for the first time in fishes from Lao PDR in the present study, although these metacercariae were detected in only 1 fish species, Anabas testudineus. Meanwhile, P. varium metacercariae were detected in more than 10 fish species including A. testudineus from an adjacent country, Vietnam [15-17].
The metacercariae of P. varium (175×136 μm) detected in the present study were slightly smaller than those (187×147) of the previous study performed in Vietnam [17]. However, their general morphologies, i.e., elliptical in shape, presence of brownish pigment granules in the worm body, a pair of eye-spots lateral to the pharynx, a thick-walled expulsor, and a Dshaped excretory bladder, were almost identical to those of the previous studies [17,18].
Trematodes in the genus Procerovum (Heterophyidae) are characterized by possessing a single testis and a long prominent seminal vesicle modified into an expulsor. Only 3 species, i.e., P. varium, P. calderoni, and P. cheni, have been certified for their validity based on the morphology of the seminal vesicle. Our specimens (304×186 μm) were somewhat smaller than those of Chai et al. [17] (434×223 μm), which were recovered from a hamster experimentally infected with the metacercariae from fish of Vietnam. The length of expulsor (123 μm) is more or less longer than that in Chai et al. [17] (115 μm). In spite that there were small differences between the 2 studies, we were able to identify our samples as P. varium by the presence of a saccular seminal vesicle with thick-walled chambers, an expulsor, less than 160 μm long. By comparison, P. calderoni has a very long expulsor measuring more than 200 μm, and P. cheni has a bipartite seminal vesicle with thinwalled chambers less than 100 μm long [17,18].
Schotz et al. [10] surveyed total 782 freshwater fishes (45 spp.) from Vientiane Province. Rim et al. [11] examined 156 fishes (17 spp.) from Savannakhet and 177 fishes (12 spp.) from Vientiane Municipality. Rim et al. [12] investigated total 242 freshwater fishes (40 spp.) from 3 Provinces, i.e., Luang Prabang, Khammuane, and Saravane. Sohn et al. [9] examined 207 freshwater fish (17 spp.) purchased in a market in Luang Prabang Province. In the present study, we also examined total 157 fishes (17 spp.) from Vientiane Municipality and Champasack Province. However, the number of fish (40 fishes) from Champasack Province was not enough to evaluate the endemicity of trematode infections.
As the second intermediate hosts for O. viverrini, about 35 species of freshwater fish have been reported in Thailand, Cambodia, and Lao PDR [10-12,19-21]. In the present study, O. viverrini metacercariae were detected in 6 fish species, A. testudineus, C. apogon, C. armatus, C. repasson, H. siamensis, and P. brevis, from Vientiane Municipality, and in only 1 species, B. schwanefeldi, from Champasack Province. Among these fish hosts, A. testudineus is newly reported as the second intermediate host for O. viverrini by the present study.
The infection rates and intensities of O. viverrini metacercariae in fishes from Vientiane Municipality were much higher than those in fishes from Champasack Province. However, they were relatively low in the level when we compared these with those of the previous studies performed in Vientiane Municipality, Savannakhet, Khammuane, and Saravane Provinces [11,12]. On the other hand, it has been known that 5 fish species, i.e., Onychostoma elongatum, C. armatus, C. apogon, P. brevis, and Hampala dispar, are highly susceptible fish hosts for O. viverrini metacercariae in Lao PDR [11,12]. In the present study, 3 fish species, i.e., C. apogon, C. armatus, and P. brevis, from Vientiane Municipality were highly and heavily infected with O. viverrini metacercariae.
About 48 fish species have been recorded as the second intermediate hosts for H. taichui in endemic Asian countries, i.e., India, China, the Philippines, Tailand, Vietnam, and Lao PDR [9-12,22-26]. In the present study, H. taichui metacercariae were detected in total 15 fish species. Among them, 6 species, Osteochilus hasseltii, Osteochilus lini, A. testudineus, B. schwanefeldi, Puntioplites proctozysron, and Probarbus labeaminor, have not been reported as the fish hosts of H. taichui yet. However, the infection rates and densities were relatively low when they were compared with those of previous studies, especially in fishes from 3 Provinces, Luang Prabang, Khammuane, and Saravane, in Lao PDR [12]. In addition, it is known that the worm burden of H. taichui is remarkably high among the residents of aforementioned 3 Provinces by the results of worm recovery after praziquantel treatment and MgSO4 purgation [5-7,9].
The metacercariae of H. yokogawai have been detected in 47 fish species from some Asian and Middle East countries such as India, Egypt, Thailand, Cambodia, and Lao PDR [11,23,25,27-31]. In the present study, they were found in 9 and 7 fish species from Vientiane Municipality and Champasack Province, respectively. Among these fish hosts, 3 species, A. testudineus, B. schwanefeldi, and Mystacoleucus marginatus, are to be recorded as the new second intermediate hosts of H. yokogawai.
As the second intermediate hosts of C. formosanus, 12 species of freshwater fishes, i.e., Acheilognathus tonkinensis, Cirrhinus molitorella, C. armatus, C. repasson, Esomus longimanus, Hypsibarbus pierrei, Mystacoleucus greenwayi, O. hasseltii, Opsariichthys bidens, P. proctozysron, and P. brevis, have been reported in Lao PDR [10-13,32]. In the present study, the metacercariae of C. formosanus were found in 4 fish species, A. testudineus, C. armatus, H. siamensis, and P. brevis, from Vientiane Municipality. Therefore, the 2 species, A. testudineus and H. siamensis, are added among the list of fish intermediate hosts for C. formosanus in Lao PDR.
It was collectively proved that O. viverrini is more endemic in fish intermediate hosts in Vientiane Municipality, but it is less endemic in Champasack Province although Sayasone et al. [33] reported a high prevalence (64.3%) of opisthorchiasis among 669 residents of Champasack Province. Four heterophyid species, i.e., H. taichui, H. pumilio, C. formosanus, and P. varium, except for H. yokogawai, are not prevalent in fishes from Vientiane Municipality, and 2 species, i.e., H. taichui and H. yokogawai, are less prevalent in fishes from Champasack Province. However, to reveal the exact infection status of FZT in humans and fishes from Champasack Province, the more profound studies should be performed at near future.
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