Abstract
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.
-
Key words: Opisthorchis viverrini, Haplorchis taichui, Haplorchis yokogawai, Haplorchis pumilio, Centrocestus formosanus, Procerovum varium, fishborne trematode (FBT) metacercaria, Lao PDR
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.
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.
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 MgSO
4 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.
Notes
-
The authors have no conflicts of interest concerning the work reported in this paper.
This work was implemented through a Korea-Lao PDR collaborative project for control of foodborne trematode infections (especially opisthorchiasis) in Lao PDR (2007-2011) which was supported by Korea Foundation for International Healthcare (KOFIE). We thank the staff of the Center for Laboratory and Epidemiology, Department of Hygiene and Prevention, Ministry of Public Health, Vientiane, and the staff of the Champasak Provincial Health Department, Lao PDR, for their help in collecting the fish specimens. We also thank Jung-A Kim and Hee-Joo Kim (Department of Parasitology and Tropical Medicine, Gyeongsang National University School of Medicine, Jinju, Korea), for their help in the examination of fish.
Fig. 1.Surveyed areas of Vientiane Municipality (VM) and Champasack Province (CP), Lao PDR.
Fig. 2.A metacercaria of Procerovum varium detected in a climbing perch, Anabas testudineus, from a local fish market of Vientiane Municipality, Lao PDR. It is elliptical and 175×136 μm in average size, and has brownish pigment granules, a muscular oral sucker (OS), a pair of eyespots (arrow) lateral to pharynx, a ventral sucker deflectively located from median, a thick-walled expulsor, and a D-shaped excretory bladder (EB) with grouped granules. Scale bar is 50 μm.
Fig. 3.An adult of P. varium recovered in the small intestine of cat experimentally infected with metacercariae at day 7 after infection. Its body is small, 304×186 μm in average size, has a muscular oral sucker (OS) and pharynx (P), a small ventral sucker (VS), a long and thick-walled expulsor (E), a long saccular seminal receptacle (SR), a spherical ovary (O), single globular testis (T), and follicular vitellaria distributing in post-ovarian fields. Scale bar is 100 μm.
Table 1.Infection status of heterophyid fluke metacercariae in fishes from Vientiane Municipality, Lao PDR
Table 1.
|
Fish species examined |
No. of fish examined |
Haplorchis taichui
|
Haplorchis yokogawai
|
Haplorchis pumilio
|
Centrocestus formosanus
|
|
No (%) of fish infected |
Average (range)a
|
No (%) of fish infected |
Average (range)a
|
No (%) of fish infected |
Average (range)a
|
No (%) of fish infected |
Average (range)a
|
|
Cyclocheilichthys repasson
|
25 |
6 (24.0) |
2.5 (1-4) |
20 (80.0) |
22 (1-80) |
0 |
- |
0 |
- |
|
Cyclocheichthys armatus
|
20 |
6 (30.0) |
2.3 (1-5) |
20 (100) |
2,450 (860-5,500) |
0 |
- |
4 (20.0) |
6.3 (1-9) |
|
Anabas testudineus
|
16 |
0 |
- |
3 (18.8) |
4 (1-7) |
7 (43.8) |
5.6 (1-21) |
10 (62.5) |
5.3 (1-18) |
|
Henicorhynchus siamensis
|
15 |
15 (100) |
25 (5-47) |
0 |
- |
3 (20.0) |
1.3 (1-2) |
4 (26.7) |
1.3 (1-2) |
|
Cyclocheilichthys apogon
|
15 |
0 |
- |
15 (100) |
1,667 (187-3,835) |
0 |
- |
0 |
- |
|
Puntius brevis
|
10 |
5 (50.0) |
1.2 (1-2) |
10 (100) |
93 (7-275) |
0 |
- |
10 (100) |
32.8 (5-80) |
|
Cirrhinus molitorella
|
10 |
8 (80.0) |
13 (5-28) |
4 (40.0) |
52 (1-195) |
0 |
- |
0 |
- |
|
Osteochilus hasseltii
|
2 |
1 (50.0) |
5 |
1 (50.0) |
3 |
1 (50.0) |
1 |
0 |
- |
|
Osteochilus lini
|
2 |
2 (100) |
3.0 (1-5) |
2 (100) |
1,940 (260-3,620) |
0 |
- |
0 |
- |
|
Puntius orpoides
|
2 |
0 |
- |
2 (100) |
226 (5-446) |
0 |
- |
0 |
- |
|
Total |
117 |
43 (36.8) |
12 (1-47) |
77 (65.8) |
1,038 (1-5,500) |
11 (9.4) |
4.0 (1-21) |
28 (23.9) |
14.6 (1-80) |
Table 2.Infection status of Opisthorchis viverrini metacercariae in fishes from Vientiane Municipality in Lao PDR
Table 2.
|
Fish species examined |
No. of fish examined |
No. (%) of fish infected |
No. of metacercariae detected
|
|
Total |
Range |
Average |
|
Cyclocheilichthys apogon
|
15 |
15 (100) |
4,562 |
26-1,509 |
304.1 |
|
Cyclocheilichthys armatus
|
20 |
20 (100) |
2,901 |
1-359 |
145.1 |
|
Puntius brevis
|
10 |
10 (100) |
239 |
1-85 |
23.9 |
|
Cyclocheilichthys repasson
|
25 |
3 (12.0) |
16 |
2-10 |
5.3 |
|
Anabas testudineus
|
16 |
1 (6.3) |
2 |
- |
2 |
|
Henichorhynchus siamensis
|
15 |
1 (6.7) |
1 |
- |
1 |
|
Total |
101 |
50 (49.5) |
7,721 |
1-1,509 |
154.4 |
Table 3.Infection status of heterophyid fluke metacercariae in fishes from Champasak Province, Lao PDR
Table 3.
|
Fish species examined |
No. of fish examined |
Haplorchis taichui
|
Haplorchis yokogawai
|
|
No (%) of fish infected |
Average (range)a
|
No (%) of fish infected |
Average (range)a
|
|
Anabas testudineus
|
10 |
1 (10.0) |
1 |
2 (20.0) |
1 |
|
Mystacoleucus marginatus
|
10 |
10 (100) |
68.1 (6-218) |
8 (80.0) |
8.6 (2-23) |
|
Barbonymus gonionotus
|
8 |
4 (50.0) |
29.0 (2-62) |
4 (50.0) |
4.0 (1-11) |
|
Puntioplites proctozysron
|
3 |
2 (66.7) |
22.5 (11-34) |
1 (33.3) |
3 |
|
Henicorhynchus siamensis
|
2 |
1 (50.0) |
2 |
0 |
- |
|
Barbonymus schwanefeldi
|
2 |
2 (100) |
45.0 (3-87) |
2 (100) |
12.0 (1-23) |
|
Cyclocheilichthys repasson
|
2 |
2 (100) |
22.5 (11-34) |
2 (100) |
217 (9-425) |
|
Puntioplites falciper
|
1 |
1 (100) |
18 |
0 |
- |
|
Hampala dispar
|
1 |
1 (100) |
130 |
1 (100) |
3 |
|
Probarbus labeaminor
|
1 |
1 (100) |
1 |
0 |
- |
|
Total |
40 |
24 (60.0) |
47.0 (1-218) |
20 (50.0) |
27.6 (1-425) |
References
- 1. Chai JY, Murrell KD, Lymbry A. Fishborne parasitic zoonoses: status and issues. Int J Parasitol 2005;35:1233-1254.
- 2. Lao National Geographic Department. Lao geographic atlas. Vientiane, Lao PDR. 2000.
- 3. Kobayashi J, Vannachone B, Sato Y, Manivong K, Nambanya S, Inthakone S. An epidemiological study on Opisthorchis viverrini infection in Lao villages. Southeast Asian J Trop Med Public Health 2000;31:128-132.
- 4. Rim HJ, Chai JY, Min DY, Cho SY, Eom KS, Hong SJ, Sohn WM, Yong TS, Deodato G., Standgaard H, Phommasack B, Yun CY, Hoang EH. Prevalence of intestinal parasite infections on a national scale among primary schoolchildren in Laos. Parasitol Res 2003;91:267-272.
- 5. Chai JY, Park JH, Han ET, Guk SM, Lin A, Kim JL, Sohn WM, Yong TS, Eom KS, Min DY, Hwang EH, Phommasack B, Insisiengmay B, Rim HJ. Mixed infections with Opisthorchis viverrini and intestinal flukes in residents of Vientiane Municipality and Saravane Province in Laos. J Helminthol 2005;79:283-289.
- 6. Chai JY, Han EK, Guk SM, Shin EH, Sohn WM, Yong TS, Eom KS, Lee KH, Jeong HG, Ryong YS, Hoang EH, Phommasack B, Insisiengmay B, Lee SH, Rim HJ. High prevalence of liver and intestinal fluke infection among residents of Savannakhet Province in Laos. Korean J Parasitol 2007;45:213-218.
- 7. Chai JY, Han ET, Shin EH, Sohn WM, Yong TS, Eom KS, Min DY, Um JY, Park MS, Hoang EH, Phommasack B, Insisiengmay B, Lee SH, Rim HJ. High prevalence of Haplorchis taichui, Prosthodendrium molenkampi, and other helminth infections among people in Khammouane Province, Lao PDR. Korean J Parasitol 2009;47:243-247.
- 8. Eom KS, Yong TS, Sohn WM, Chai JY, Min DY, Rim HJ, Jeon HK, Banouvong V, Insisiengmay B, Phommasack B. Prevalence of helminthic infections among inhabitants of Lao PDR. Korean J Parasitol 2014;52:51-56.
- 9. Sohn WM, Yong TS, Eom KS, Min DY, Lee DM, Jung BK, Banouvong V, Insisiengmay B, Phommasack B, Rim HJ, Chai JY. Prevalence of Haplorchis taichui among humans and fish in Luang Prabang Province, Lao PDR. Acta Trop 2014;136:74-80.
- 10. Scholz T, Ditrich O, Giboda M. Larval stages of medically important flukes (Trematoda) from Vientiane Province, Laos. Part I. Metacercariae. Ann Parasitol Hum Comp 1990;65:238-243.
- 11. Rim HJ, Sohn WM, Yong TS, Eom KS, Chai JY, Min DY, Lee SH, Hoang EH, Phommasack B, Insisengmay S. Fishborne trematode metacercariae detected in freshwater fish from Vientiane Municipality and Savannakhet Province, Lao PDR. Korean J Parasitol 2008;46:253-260.
- 12. Rim HJ, Sohn WM, Yong TS, Eom KS, Chai JY, Min DY, Lee SH, Hoang EH, Phommasack B, Insisiengmay S. Fishborne trematode metacercariae in Luang Prabang, Khammouane, and Saravane Province, Lao PDR. Korean J Parasitol 2013;51:107-114.
- 13. Chai JY, Sohn WM, Yong TS, Eom KS, Duk-Young Min DY, Lee MY, Lim H, Insisiengmay B, Phommasack B, Rim HJ. Centrocestus formosanus (Heterophyidae): human infections and the infection source in Lao PDR. J Parasitol 2013;99:531-536.
- 14. Kottelat M. Fishes of Laos. Colombo, Sri Lanka. WHT Publication. 2001, pp 7-206.
- 15. Vo DT, Murrell D, Dalsgaard A, Bristow G, Nguyen DH, Bui TN, Vo DT. Prevalence of zoonotic metacercariae in two species of grouper, Epinephelus coioides and Epinephelus bleekeri, and flathead mullet, Mugil cephalus, in Vietnam. Korean J Parasitol 2008;46:77-82.
- 16. Phan VT, Ersbøll1 AK, Nguyen KV, Madsen H, Dalsgaard A. Farm-level risk factors for fishborne zoonotic trematode infection in integrated small-scale fish farms in northern Vietnam. PLoS Negl Trop Dis 2010;4:742-750.
- 17. Chai JY, De NV, Sohn WM. Foodborne trematode metacercariae in fish from northern Vietnam and their adults recovered from experimental hamsters. Korean J Parasitol 2012;50:317-325.
- 18. Umadevi K, Madhavi R. Observations on the morphology and life-cycle of Procerovum varium (Onji & Nishio, 1916) (Trematoda: Heterophyidae). Syst Parasitol 2000;46:215-225.
- 19. Touch S, Komalamisra C, Radomyos P, Waikagul J. Discovery of Opisthorchis viverrini metacercariae in freshwater fish in southern Cambodia. Acta Trop 2009;111:108-113.
- 20. Sohn WM, Shin EH, Yong TS, Eom KS, Jeong HG, Sinuon M, Socheat D, Chai JY. Adult Opisthorchis viverrini flukes in humans, Takeo, Cambodia. Emerg Infect Dis 2011;17:1302-1304.
- 21. Sohn WM, Yong TS, Eom KS, Pyo KH, Lee MY, Lim H, Choe S, Jeong HG, Sinuon M, Socheat D, Chai JY. Prevalence of Opisthorchis viverrini infection in humans and fish in Kratie Province, Cambodia. Acta Trop 2012;124:215-220.
- 22. Pearson JC. A revision of the subfamily Haplorchinae Looss, 1899 (Trematoda: Heterophyidae). I. The Haplorchis group. Parasitology 1964;54:601-676.
- 23. Nath D, Pande BP. Identify of the three heterophyid metacercariae infesting some of the freshwater fishes. Current Sci India 1970;39:325-331.
- 24. Velasquez CC. Observations on some Heterophyidae (Trematoda: Digenea) encysted in Philippine fishes. J Parasitol 1973;59:77-84.
- 25. Pande V, Premvati G. Development of metacercariae of Haplorchis spp. in chicks. Indian J Parasitol 1977;1:165-172.
- 26. Sohn WM, Eom KS, Min DY, Rim HJ, Hoang EH, Yang Y, Li X. Fishborne trematode metacercariae in freshwater fish from Guangxi Zhuang Autonomous Region, China. Korean J Parasitol 2009;47:249-257.
- 27. Pearson JC, Ow-Yang CK. New species of Haplorchis from Southeast Asia, together with keys to the Haplorchis-group of heterophyid trematodes of the region. Southeast Asian J Trop Med Public Health 1982;13:35-60.
- 28. Pandey KC. Studies on metacercariae of freshwater fishes of India. 1. On the morphology of metacercaria of Haplorchis yokogawai (Katsuta, 1932) Chen, 1936. Proc Nat Acad Sci India, Sect 1966;36:437.
- 29. Kliks M, Tantachamrun T. Heterophyid (Trematoda) parasites of cats in North Thailand, with notes on a human case found at necropsy. Southeast Asian J Trop Med Public Health 1974;5:547-555.
- 30. Fahmy MA, Mandour AM, El-Naffar MK. Successful infection of dogs and cats by Prohemistomum vivax Sonsino, 1893 and Haplorchis yokogawai Katsuta, 1922. J Egyptian Soc Parasitol 1976;6:77.
- 31. Chai JY, Sohn WM, Na BK, Yong TS, Eom KS, Yoon CH, Hoang EH, Jeoung HG, Socheat D. Zoonotic trematode metacercariae in fish from Phnom Penh and Pursat, Cambodia. Korean J Parasitol 2014;52:35-40.
- 32. Han ET, Shin EH, Phommakorn S, Sengvilaykham B, Kim JL, Rim HJ, Chai JY. Centrocestus formosanus (Digenea: Heterophyidae) encysted in the freshwater fish, Puntius brevis, from Lao PDR. Korean J Parasitol 2008;46:49-53.
- 33. Sayasone S, Mak TK, Vanmany M, Rasphone O, Vounatsou P, Utzinger J, Akkhavong K, Odermatt P. Helminth and intestinal protozoa infections, multiparasitism and risk factors in Champasack Province, Lao People’s Democratic Republic. PLoS Negl Trop Dis 2011;5:e1037.
