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 . 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 .   surveyed freshwater fishes from rice paddies and nam ngum water reservoir in vientiane province . 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 . 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 . 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   and fishbase website ( http://www.fishbase.org/search.php ) ( tables 1 , 3 ) . the ground fish meat was mixed with artificial gastric juice , and the mixture was incubated at 36c for 2 hr . the digested material was filtered with a sieve ( 11 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 . 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 . 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 . only 2 o. viverrini metacercariae were found in 1 fish ( 50.0% ) of barbonymus schwanefeldi from champasak province . 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 . 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 ) . 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 . eggs small , yellow , and 25 - 28 ( 27 ) by 13 - 15 ( 14 ) . 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 . 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 . only 2 o. viverrini metacercariae were found in 1 fish ( 50.0% ) of barbonymus schwanefeldi from champasak province . 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 ) . 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 . eggs small , yellow , and 25 - 28 ( 27 ) by 13 - 15 ( 14 ) . 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 . .   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 .   detected 4 species ( o. viverrini , h. taichui , h. yokogawai and c. formosanus ) of fzt metacercariae in fishes from vientiane municipality . in rim , 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 .   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 ( 175136 m ) detected in the present study were slightly smaller than those ( 187147 ) of the previous study performed in vietnam . 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 . 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 ( 304186 m ) ( 434223 m ) , which were recovered from a hamster experimentally infected with the metacercariae from fish of vietnam . the length of expulsor ( 123 m ) is more or less longer than that in chai et al . 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 m long . by comparison , p. calderoni has a very long expulsor measuring more than 200 m , and p. cheni has a bipartite seminal vesicle with thinwalled chambers less than 100 m long . .   surveyed total 782 freshwater fishes ( 45 spp . ) from vientiane province . examined 156 fishes ( 17 spp . ) from savannakhet and 177 fishes ( 12 spp . ) from vientiane municipality . .   investigated total 242 freshwater fishes ( 40 spp . ) from 3 provinces , i.e. , luang prabang , khammuane , and saravane . 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 . 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 . 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 . 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 .   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 .
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 .