zoonotic trematode infections are public health problems in asian countries , including lao people 's democratic republic ( lao pdr ) , vietnam , thailand , china , and korea .
especially , fishborne trematodes ( fbt ) provoke a remarkable morbidity among local people as well as a serious damage in aquaculture industry [ 1 - 3 ] .
fbt infections in humans are almost entirely caused by habitual consumption of raw fish containing infective larvae ( = metacercariae ) .
these infections are highly localized in riverside areas , especially where riparian populations have the raw fish eating habit .
it has been known that riverside areas in southeast asia , especially the mekong river basin in vietnam , lao pdr , and thailand , are highly endemic with fbt infections [ 4 - 9 ] .
cambodia is located in the southern part of the indochina peninsula in southeast asia , and bordered by thailand to the northwest , lao pdr to the northeast , vietnam to the east , and the gulf of thailand to the southwest .
administratively , it is divided into 24 provinces including phnom penh municipality which is located in the central south region , and on the banks of the tonle sap lake , mekong , and bassac rivers .
pursat province is located in the western part of the country and between the tonle sap lake and the northern end of the cardamom mountains .
it has been known that many cambodian people are infected with helminth parasites , such as soil - transmitted nematodes and fbt including opisthorchis viverrini [ 11 - 13 ] .
this was reconfirmed by fecal examinations during the korea - cambodia international collaboration project on intestinal parasite control in cambodia ( 2006 - 2011 ) . with regard to fbt infections ,
several investigators have previously reported that o. viverrini are prevalent in some limited areas of cambodia [ 14 - 17 ] .
reported infection of freshwater fish with o. viverrini metacercariae in areas adjacent to the lake 500 near the border of kandal and takeo provinces , southern cambodia .
subsequently , adult o. viverrini specimens were recovered from humans and experimental animals , and metacercariae were detected in fish from takeo and kratie provinces . however , in other localities of cambodia , no studies have been available on fbt metacercarial infections in freshwater fish .
therefore , in this study , we investigated on the infection status of freshwater fish with fbt metacercariae in phnom penh and pursat province , cambodia .
we purchased a total of 184 freshwater fish ( 22 species ) from local markets of phnom penh municipality through 3 different times ( in june 2006 , december 2007 , and november 2010 ) and also purchased 36 fish ( 5 species ) from a local market in pursat province ( in june 2007 ) ( fig .
the fish were transported to the department of parasitology and institute of health sciences , gyeongsang national university school of medicine , jinju , korea under refrigeration .
the fish species were identified with the aid of the fishbase website ( http://www.fishbase.org/search.php ) ( tables 1 - 3 ) .
individual fish was finely ground with a mortar or a grinder , the ground fish meat was mixed with artificial gastric juice , and the mixture was incubated at 36 for 2 - 3 hr .
the digested material was filtered with 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 separately collected by their general morphological features .
the collected metacercariae were categorized according to the size and morphological characteristics , and then the intensity of infection and the infection rate were calculated according to the species of fish positive for metacercariae .
in fish from phnom penh municipality , 2 species of metacercariae ( o. viverrini and h. yokogawai ) were found .
the metacercariae of o. viverrini were collected from 37 ( 50.0% ) of 74 fish ( 11 of 27 species ) , and the average number of metacercariae per fish was 18.6 . among the 11 fish species
positive for o. viverrini metacercariae , barbonymus schwanefeldi was the most heavily infected , with the average intensity of infection per fish of 74.6 metacercariae ( table 1 ) .
the metacercariae of h. yokogawai were detected from 23 ( 57.5% ) of 40 fish ( 5 of 27 species ) , and the average number of metacercariae per fish was 21.0 ( table 2 ) . in fish from pursat province , 5 species of metacercariae ( o. viverrini , h. yokogawai , haplorchis pumilio , centrocestus formosanus , and procerovum sp . )
total 3 o. viverrini metacercariae were found in 2 fish species ( henicorhynchus lineatus and puntioplites falcifer ) .
total 51 h. yokogawai metacercariae were detected in 8 ( 80% ) of 10 p. falcifer examined .
a total of 476 h. pumilio metacercariae were collected from 9 ( 90% ) of 10 h. lineatus and 1 ( 50% ) of 2 pristolepis fasciata .
only 1 c. formosanus metacercaria was detected in 1 ( 10% ) of 10 h. lineatus .
metacercariae were detected in all 9 ( 100% ) anabas testudineus examined ( table 3 ) .
metacercariae of 5 fbt species , namely , o. viverrini , h. yokogawai , h. pumilio , c. formosanus , and procerovum sp . , were detected in freshwater fish from phnom penh municipality and pursat province , cambodia . in phnom penh municipality , 2 fbt species ( o. viverrini and h. yokogawai )
were found , whereas , in pursat province , 5 fbt species were detected . from previous studies in cambodia ,
only 2 species of fbt metacercariae , o. viverrini and h. yokogawai , were reported [ 15 - 17 ] .
detected o. viverrini metacercariae in various species of freshwater fish from areas adjacent to the lake 500 near the border of kandal and takeo provinces , southern cambodia .
sohn et al .   found o. viverrini metacercariae in 1 fish species , puntioplites proctozysron , from takeo province .
sohn et al .   also detected o. viverrini and h. yokogawai metacercariae in freshwater fish from kratie province .
therefore , by the present study , it has been confirmed for the first time that the 3 additional species of fbt , namely , h. pumilio , c. formosanus , and procerovum sp . , are distributed in cambodia .
the number and species of fish examined in this study were not much compared with the study of touch et al . .
they examined a total of 1,479 fish in 30 species by the compression method . on the other hand , sohn et al .  
examined 2 fish species , p. proctozysron ( n=5 ) and cyclocheilichthys apagon ( n=10 ) , from takeo province , and sohn et al .  
, we examined a total of 184 freshwater fish ( 22 species ) from several local markets of phnom penh municipality and 36 fish ( 5 species ) from a market of pursat province by means of the artificial digestion method .
anyhow , there may be some points to be considered which include the exact origin of the fish examined in this study .
for example , fish purchased from a local market of phnom penh municipality may not have been caught from phnom penh area .
those from pursat province are not enough in the number and species of fish to show the epidemiological trend of fbt metacercariae infections in the subjected area and fish species . as the second intermediate hosts of o. viverrini , 24 species of freshwater fish ( barbonymus gonionotus , cyclocheilichthys apogon , cyclocheilichthys armatus , cyclocheilichthys enoplos , cyclocheilichthys furcatus , cyclocheilichthys repasson , esomus metallicus , hampala dispar , hampala macrolepidota , hypsibarbus lagleri , hypsibarbus pierrei , hypsibarbus wetmorei , labiobarbus lineatus , mystacoleucus marginatus , neolissochilus stracheyi , onychostoma elongatum , onychostoma fusiforme , osteochilus hasseltii , paralaubuca barroni , poropuntius dearatus , puntioplites falcifer , puntioplites proctozystron , puntius brevis , and puntius orphoides ) have been reported in thailand and lao pdr [ 18 - 22 ] . in cambodia ,
detected o. viverrini metacercariae in 10 cyprinoid fish species , i.e. , barbodes altus ( = barbonymus altus ) , c. apagon , c. enoplos , h. dispar , h. macrolepidota , henicorhynchus siamensis , p. proctozysron , p. brevis , systomus orphoides ( = puntius orphoides ) , and thynnichthys thynnoides , from kandal and takeo provinces .
sohn et al .   reported p. proctozysron as a fish host for o. viverrini in takeo province , and sohn et al .  
detected o. viverrini metacercariae in p. proctozysron , p. orphoides , and labeo chrysophekadion from kratie province , cambodia . in the present study ,
o. viverrini metacercariae were detected in 11 fish species ( b. altus , b. schwanefeldi , cirrhinus jullieni , cirrhinus microlepis , henicorhynchus lobatus , h. siamensis , l. chrysophekadion , luciosoma bleekeri , osteochilus melanopleurus , p. proctozysron , and t. thynnoides ) from phnom penh municipality and 2 fish species ( henicorhynchus lineatus and p. falcifer ) from pursat province .
therefore , in cambodia , total 18 fish species ( b. altus , b. schwanefeldi , c. jullieni , c. microlepis , c. apagon , c. enoplos , h. dispar , h. macrolepidota , h. lineatus , h. lobatus , h. siamensis , l. chrysophekadion , l. bleekeri , o. melanopleurus , p. proctozysron , p. brevis , p. orphoides , and t. thynnoides ) have been confirmed to be the second intermediate hosts for o. viverrini [ 15 - 17 ] .
the prevalence and intensity of o. viverrini metacercariae were relatively low in fish examined in the present study compared to lao pdr .
in phnom penh municipality , the average number of o. viverrini metacercariae per fish was 18.6 in 37 positive fish ( 11 species ) . among the positive fish species
. however , these results were somewhat higher than those of touch et al . .
they detected total 789 o. viverrini metacercariae ( 4.8 per fish ) in 163 positive fish ( 10 species ) collected from kandal and takeo provinces , cambodia .
some people in phnom penh like to eat raw fish , as has been reported in takeo province , and there may be a substantial number of opisthorchiasis patients in phnom penh areas of cambodia .
distribution of h. yokogawai metacercariae has been reported in asian and middle east countries such as india , thailand , lao pdr , and egypt , involving 43 fish species [ 21 - 28 ] . in the present study , h. yokogawai metacercariae
were detected in 5 fish species ( p. proctozysron , c. jullieni , l. chrysophekadion , l. rhabdoura , and b. altus ) from phnom penh municipality , and 1 species ( p. falcifer ) from pursat province .
sohn et al .   detected them in 3 fish species ( p. proctozysron , p. orphoides , and h. siamensis ) from kratie province , cambodia .
metacercariae of h. pumilio have also been found in asian and middle east countries [ 21 - 28 ] . in china , 18 fish species were found to harbour the metacercariae in guangxi zhuang autonomous region . in vietnam , the metacercariae were detected in 17 fish species from nam dinh province   and in 13 fish species from hanoi city and nam dinh province . in the present study ,
h. pumilio metacercariae were detected in only 1 fish species ( h. lineatus ) from pursat province .
c. formosanus is known to be distributed in china , taiwan , japan , the philippines , india , lao pdr , and vietnam [ 8,20 - 22 , 30 - 33 ] .
their metacercariae were detected in 9 fish species from lao pdr [ 20 - 22 ] , and in 16 fish species from vietnam . in china ,
sohn et al .   reported 10 fish species as the second intermediate host in guangxi zhuang autonomous region . in the present study ,
c. formosanus metacercariae were found in only 1 fish ( h. lineatus ) from pursat province .
( presumed to be p. varium ) metacercariae were detected in all of 9 a. testudineus from pursat province in this study .
this fish species was previously reported to be a second intermediate host for p. varium in vietnam , together with 9 more fish species ( l. rohita , h. molitrix , c. mrigala , c. idella , s. curriculus , p. brachypomum , c. batrachus , b. gonionotus , and m. cephalus ) . in conclusion ,
the present study confirmed that various species of freshwater fish play the role of a second intermediate host for various kinds of fbt ( o. viverrini , h. pumilio , h. yokogawai , c. formosanus , and procerovum sp . ) in cambodia .
epidemiological studies to detect endemic areas of human fbt infections remain to be undertaken in 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 . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION