ISSN (Print) 0023-4001 ISSN (Online) 1738-0006 ORIGINAL ARTICLE Korean J Parasitol Vol. 55, No. 6: 631-641, December 2017 https://doi.org/10.3347/kjp.2017.55.6.631 Zoonotic Trematode Metacercariae in Fish from Yangon, Myanmar and Their Adults Recovered from Experimental Animals Jong-Yil Chai 1,2, Woon-Mok Sohn 3, *, Byoung-Kuk Na 3, Jong-Bok Park 1, Hoo-Gn Jeoung 1, Eui-Hyug Hoang 1, Thi Thi Htoon 4, Htay Htay Tin 4 1 Korea Association of Health Promotion, Seoul 07653, Korea, 2 Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, Seoul 03080, Korea; 3 Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju 52727, Korea; 4 National Health Laboratory, Yangon 11191, Myanmar Abstract: A survey was performed to investigate the infection status of zoonotic trematode (ZT) metacercariae in fish from a local market in Yangon City, Myanmar. A total of 264 fish (12 species) were collected through 4 times from December 2013 to June 2015. All collected fish were transferred to our laboratory on ice and examined by the artificial digestion method. More than 7 species of ZT metacercariae, i.e., Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus spp., Stellantchasmus falcatus, Pygidiopsis cambodiensis, and Procerovum sp. were detected. Metacercariae of H. taichui were collected in 58 (42.3%) out of 137 fish (5 species), and their average density was 42.9 per fish infected. Metacercariae of H. pumilio were detected in 96 (49.0%) out of 196 fish (9 species), and their average density was 23.6 per fish infected. H. yokogawai metacercariae were found in 40 (50.0%) out of 80 fish (5 species), and Centrocestus spp. metacercariae in 91 (50.8%) out of 179 fish (8 species), and their densities were 306 and 25.8 per fish infected, respectively. Metacercariae of S. falcatus and P. cambodiensis were detected only in mullets, Chelon macrolepis. A total of 280 Procerovum sp. metacercariae were found in 6 out of 12 climbing perch, Anabas testudineus. Morphological characteristics of adult flukes recovered from experimental animals were described. It has been first confirmed that fish from Yangon, Myanmar are commonly infected with various species of ZT metacercariae. Key words: Haplorchis taichui, Haplorchis pumilio, Haplorchis yokogawai, Stellantchasmus falcatus, Centrocestus spp., Pygidiopsis cambodiensis, Procerovum sp., zoonotic trematode metacercaria INTRODUCTION Received 24 September 2017, revised 7 December 2017, accepted 14 December 2017. * Corresponding author (wmsohn@gnu.ac.kr) 2017, Korean Society for Parasitology and Tropical Medicine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Zoonotic trematode (ZT) infections are an important public health problem in many Asian countries, including Lao People's Democratic Republic (Lao PDR), Vietnam, Cambodia, Thailand, the Philippines, China, Taiwan, and the Republic of Korea (Korea). Especially, fishborne trematodes (FBT) provoke a remarkable morbidity in residents of these countries and cause a serious economic damage in the industry of fish aquaculture [1-3]. Human FBT infections are mainly localized in riverside areas, where the riparian populations are infected by habitual consumption of raw and/or fermented fish containing infective larvae, i.e., metacercariae. It has been known that riverside areas in Southeast Asia, especially the Mekong river basin in Vietnam, Lao PDR, Cambodia, and Thailand, are highly endemic with FBT infections [4-8]. The Republic of the Union of Myanmar (Myanmar) is a sovereign state in the region of Southeast Asia, and bordered by India and Bangladesh to its west, Thailand and Lao PDR to its east, and China to its north and northeast. Administratively, it is divided into 7 states and 7 regions (formerly called divisions). Yangon City is the capital of the Yangon Region of Myanmar. It is the largest city and the most important socioeconomic center in Myanmar. Geographically, this city is located in the lower and coastal region of Myanmar, and it has diverse ethnic people [9]. We have been performing a health promotion project for the elementary schoolchildren of the vulnerable areas around Yangon area [10]. A survey on metacercarial infections in the second intermediate host, in combination with a survey on adult worm infec- 631
632 Korean J Parasitol Vol. 55, No. 6: 631-641, December 2017 tions in humans, can be a useful index in the trematode epidemiology in a particular area. However, fecal examinations are not suitable to know the exact infection status in humans, since the egg identification in feces is very difficult in cases of mixed infections with small egg-sized trematodes, especially the liver flukes, as well as heterophyid, gymnophallid, and lecithodendriid flukes [5-7,11,12]. Therefore, investigation of metacercarial infections in the second intermediate host can provide a more valuable information on the trematode epidemiology. On the other hand, there are not so many studies on helminthic infections among the residents in Myanmar. Most of the previous studies were surveys of soil-transmitted helminthiases [13-15] except for one, Aung et al. [16], which reported the presence of the liver fluke (Opisthorchis viverrini) in Myanmar. Therefore, in the present study, we surveyed on the infection status of ZT metacercariae in fish purchased from a local market in Yangon, Myanmar. In addition, the morphological characteristics of adult flukes recovered from animals experimentally infected with these ZT metacercariae were described. MATERIALS AND METHODS We purchased a total of 264 fish (12 species) in a local fish market of Yangon, Myanmar, through 4 times (December 2013, June and December 2014, and June 2015). All collected fish were transferred on ice to the laboratory of Department of Parasitology and Tropical Medicine, Gyeongsang National University College of Medicine, Jinju, Korea. The length and weight of fish were individually measured and the species of fish identified with the aid of the FishBase website (http:// www.fishbase.org/search.php) [17] (Table 1). Individual fish was finely ground with a mortar with pestle. 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 1 1 mm of mesh, and washed with 0.85% saline until the supernatant became clear. The sediment was carefully examined using a stereomicroscope and then metacercariae were separately collected by their general morphological features. These collected metacercariae were categorized according to the size and morphological characteristics, and then the infection rate (%) and the intensity of infection (no. of metacercariae per fish infected) were calculated for each species of fish. The identified metacercariae were experimentally infected to cats and hamsters to obtain adult flukes. At days 7-10 after infection, cats and hamsters were killed under anesthesia, and their small intestines were isolated and longitudinally opened with a pair of scissors in a beaker with 0.85% saline. The experimental animals were treated according to the guidelines of Institutional Animal Care and Use Committee (IACUC) in Gyeongsang National University, Jinju, Korea. 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 equipped with a micrometer (OSM-4, Olympus Co., Tokyo, Japan). Table 1. Fish a purchased in a local market in Yangon, Myanmar Fish species No. of fish collected Length (cm) Weight (g) Range Average Range Average Thynnichthys thynnoides 67 6.5-9.7 8.3 6.1-19.8 12.0 Chelon macrolepis 58 11.2-18.0 13.8 26.7-86.0 46.0 Puntius aurotaeniatus 32 4.2-9.5 7.2 2.2-23.8 10.5 Esomus altus 23 7.6-10.0 8.9 7.7-16.9 11.9 Channa striata 20 12.5-27.3 19.3 37.2-249 108.9 Rhynogobius sp. 17 10.3-14.4 11.7 17.8-38.6 24.4 Anabas testudineus 12 12.0-14.0 12.8 44.6-67.7 57.6 Trichogaster pectoralis 10 6.5-7.2 6.8 10.3-16.4 12.7 Mystacoleucus sp. 10 7.3-9.9 8.2 10.7-26.6 16.0 Notopterus notopterus 7 11.4-17.1 15.6 13.7-45.1 35.3 Labeo sp. 5 6.6-8.1 7.4 4.6-9.1 6.8 Puntioplites proctozysron 3 9.0-10.1 9.7 12.3-18.6 15.8 a A total of 264 fish (12 species) were examined.
Chai et al.: Zoonotic trematode metacercariae from Yangon, Myanmar 633 RESULTS Infection status of ZT metacercariae in fish from Yangon City More than 7 species of ZT metacercariae, i.e., Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus spp., Stellantchasmus falcatus, Pygidiopsis cambodiensis, and Procerovum sp., were detected. The metacercariae of H. taichui were collected in 58 (42.3%) out of 137 fish (5 species), i.e., Thynnichthys thynnoides, Puntius aurotaeniatus, Esomus altus, Mystacoleucus sp., and Labeo sp., with their average density of 42.9 per fish infected. The infection status of H. taichui metacercariae by the fish species is revealed in Table 2. The metacercariae of H. pumilio were detected in 96 (49.0%) out of 196 fish (9 species), with their average density of 23.6 per fish infected. Their infection status by the fish species is designated in Table 3. H. yokogawai metacercariae were found in 40 (50.0%) out of 80 fish (5 species), and their density was 306 per fish infected. Centrocestus spp. metacercariae were found in 91 (50.8%) out of 179 fish (8 species), and their density was 25.8 per fish infected. The infection status of H. yokogawai and Centrocestus spp. metacercariae by the fish species is shown in Tables 4 and 5. The metacercariae of S. falcatus were detected in 15 (34.1%) out of 44 mullets, Chelon macrolepis, examined in December 2013 and 2014, and their density was 2.4 per fish infected. Total 38 metacercariae of Pygidiopsis cambodiensis were detected from only 1 mullet examined in December 2013. Total 280 (2-245: 46.7 in average) metacercariae of Procerovum sp. were detected Table 2. Infection status of fish from Yangon, Myanmar with Haplorchis taichui metacercariae Fish species examined No. of fish examined No. (%) of fish infected No. of metacercariae detected Total Range Average Thynnichthys thynnoides 67 40 (59.7) 2,250 1-480 56.3 Puntius aurotaeniatus 32 9 (28.1) 79 1-20 8.8 Esomus altus 23 1 (4.4) 2-2.0 Mystacoleucus sp. 10 3 (30.0) 23 1-16 7.7 Labeo sp. 5 5 (100) 134 12-69 26.8 Total 137 58 (42.3) 2,488 1-480 42.9 Table 3. Infection status of fish from Yangon, Myanmar with Haplorchis pumilio metacercariae Fish species examined No. of fish examined No. (%) of fish infected No. of metacercariae detected Total Range Average Thynnichthys thynnoides 67 43 (64.2) 1,157 1-116 26.9 Puntius aurotaeniatus 32 13 (40.6) 105 1-21 8.1 Esomus altus 23 6 (26.1) 19 1-10 3.2 Channa striata 20 14 (70.0) 913 1-650 65.2 Anabas testudineus 12 7 (58.3) 42 1-25 6.0 Rhynogobius sp. 17 2 (11.8) 2-1.0 Trichogaster pectoralis 10 9 (90.0) 16 1-3 1.8 Mystacoleucus sp. 10 1 (10.0) 8-8.0 Labeo sp. 5 1 (20.0) 1-1.0 Total 196 96 (49.0) 2,263 1-650 23.6 Table 4. Infection status of fish from Yangon, Myanmar with Haplorchis yokogawai metacercariae Fish species examined No. of fish examined No. (%) of fish infected No. of metacercariae detected Total Range Average Puntius aurotaeniatus 80 28 (87.5) 9,183 1-1, 750 32.8 Esomus altus 23 1 (4.4) 1-1.0 Trichogaster pectoralis 10 1 (10.0) 1-1.0 Mystacoleucus sp. 10 9 (90.0) 3,050 30-930 33.9 Labeo sp. 5 1 (20.0) 1-1.0 Total 137 40 (50.0) 12,236 1-1, 750 30.6
634 Korean J Parasitol Vol. 55, No. 6: 631-641, December 2017 Table 5. Infection status of fish from Yangon, Myanmar with Centrocestus spp. metacercariae Fish species examined No. of fish examined No. (%) of fish infected No. of metacercariae detected Total Range Average Thynnichthys thynnoides 67 46 (68.7) 1,696 2-530 36.9 Puntius aurotaeniatus 32 19 (59.4) 485 1-142 25.5 Esomus altus 23 1 (4.4) 9-9.0 Channa striata 20 4 (20.0) 52 3-31 13.0 Anabas testudineus 12 2 (16.7) 3 1-2 1.5 Trichogaster pectoralis 10 9 (90.0) 41 1-13 4.6 Mystacoleucus sp. 10 7 (70.0) 40 1-11 5.7 Labeo sp. 5 3 (60.0) 25 3-18 8.3 Total 179 91 (50.8) 2,351 1-530 25.8 in 6 out 12 climbing perch, Anabas testudineus, examined in June 2015. Morphology of metacercariae detected (all measurement unit; μm) Haplorchis taichui metacercariae (n=10) were elliptical, 192-218 (203 in average) 162-188 (172 in average) in size, had a baseball glove-shaped ventrogenital sac with 11-18 rodlets and an O-shaped excretory bladder occupying large portion of posterior body (Fig. 1A). Haplorchis pumilio metacercariae (n= 10) were elliptical, 162-193 (176) 143-173 (160) in size, had 35-43 deer horn-like minute spines arranged in 1-2 rows around the ventrogenital complex, and an O-shaped excretory bladder occupying large portion of posterior body (Fig. 1B). Haplorchis yokogawai metacercariae (n=10) were round or elliptical, 175-225 (196) 163-218 (188) in size, had an U- shaped ventrogenital sac with 70-74 min spines, and an O- shaped excretory bladder occupying large portion of posterior body (Fig. 1C). Procerovum sp. metacercariae (n = 10) were elliptical, 168-205 (185) 138-165 (155) in size, had yellow brownish pigment granules scattering in body area of intestinal bifurcation, a ventral sucker deflectively located from median, a thinwalled bulb-like expulsor, and a D-shaped (half-moon shaped) excretory bladder with grouped granules (Fig. 1D). Centrocestus formosanus metacercariae (n=10) were elliptical, 152-195 (171) 122-157 (142) in size, had 32 circumoral spines around the oral sucker arranged in 2 rows, and a X- shaped excretory bladder occupying greater portion of posterior body (Fig. 1E1, 1E2). Stellantchasmus falcatus metacercariae (n=10) were elliptical, 198-242 (225) by 135-192 (165) in size. Brownish pigment granules scattered in the worm body. Excretory bladder was O- shaped or not obviously seen in the posterior body (Fig. 1F). Pygidiopsis cambodiensis metacercariae (n=10) were elliptical, 222 260 (242) 177-212 (194) in size, and had an oral sucker, a pair of eyespots, ventral sucker, ventrogenital sac, and X- shaped excretory bladder (Fig. 1G). Morphology of heterophyid adults recovered from experimental animals H. taichui (n= 20, Fig. 2A): Body small, pear-shaped, 490-660 (591 in average) long, and 240-370 (298) wide, with greatest width at middle, the ovarian level. Oral sucker subterminal, 40-63 (48) by 53-75 (58). Pharynx subglobular or elliptical, 25-45 (34) by 18-38 (24). Esophagus short, 80-138 (106) in length. Ventrogenital sac small with 12-18 rodlets, baseball glove-shaped, 50-80 (64) by 40-63 (53). Seminal vesicle saccular, bipartite. Ovary spherical or subspherical, 45-95 (64) by 43-88 (63), dextral to midline. Seminal receptacle ellipsoidal, lying the right-side of ovary. One testis globular or subglobular, 113-188 (146) by 120-183 (148), lying posterior 1/4 of body. Uterus with eggs occupying from anterior 1/3 to posterior end, most of hind-body. Vitellaria follicular, distributing in post-ovarian fields. Eggs small, yellow, and 23-28 (25) by 11-14 (13). H. pumilio (n= 20, Fig. 2B): Body small, pear-shaped, 400-600 (485) long, and 200-260 (223) wide, with greatest width at middle, the ovarian level. Oral sucker subterminal, 43-53 (45) by 50-63 (55). Pharynx subglobular or elliptical, 30-38 (33) by 18-30 (21). Esophagus short, 50-113 (73) in length. Ventrogenital sac small with 36-42 deer horn-like minute spines, 48-75 (62) by 30-63 (47). Seminal vesicle saccular. Ovary spherical or subspherical, 40-68 (56) by 50-80 (61), slightly dextral to midline. Seminal receptacle elliptical, lying
Chai et al.: Zoonotic trematode metacercariae from Yangon, Myanmar 635 A B C D E1 E2 F G Fig. 1. The metacercariae of zoonotic trematodes detected in fish from a local market of Yangon, Myanmar. (A) H. taichui: elliptical, 203 172 in average size (μm), had a baseball glove-shaped ventrogenital sac with 11-18 rodlets and an O-shaped excretory bladder. (B) H. pumilio: elliptical, 176 160 in average size, had 35-43 deer horn-like minute spines arranged in 1-2 rows around the ventrogenital complex, and an O-shaped excretory bladder. (C) H. yokogawai: round or elliptical, 196 188 in average size, had a U-shaped ventrogenital sac with 70-74 min spines, and an O-shaped excretory bladder. (D). Procerovum sp.: elliptical, 185 155 in average size, had yellow brownish pigment granules scattering in body area of intestinal bifurcation, a thin-walled bulb-like expulsor, and a D-shaped excretory bladder with grouped granules. (E1, E2) Centrocestus spp.: elliptical, 171 142 in average size, had 32 circumoral spines around the oral sucker arranged in 2 rows, and a X-shaped excretory bladder. (F) S. falcatus: elliptical, 225 165 in average size, had brownish pigment granules scattered in the body, and an O-shaped excretory bladder. (G) P. cambodiensis: elliptical, 242 194 in average size, and had an oral sucker, a pair of eyespots, ventral sucker, ventrogenital sac, and X-shaped excretory bladder (G). All scale bar is 50 μm.
636 Korean J Parasitol Vol. 55, No. 6: 631-641, December 2017 A E B F C G D H Fig. 2. Adult heterophyid flukes recovered from experimental cats and hamsters 8-10 days after infection. (A) H. taichui: body small, 591 298 in average size, with a muscular oral sucker (OS) and pharynx (P), a ventrogenital sac (VGS) with 11-18 rodlets, a saccular seminal vesicle (SV), a spherical ovary (O), single globular testis (T), and follicular vitellaria. (B) H. pumilio: body small, 485 223 in average size, with an oral sucker (OS), pharynx (P), a small ventrogenital sac (VGS) with 36-42 deer horn-like minute spines, a saccular seminal vesicle (SV), a spherical ovary (O), single globular testis (T), and follicular vitellaria. (C) Procerovum sp. (ventral view): body small, 435 238 in average size, with an oral sucker (OS), pharynx (P), a small ventral sucker, a long and thin-walled expulsor (E) and another seminal vesicle (SV), a spherical ovary (O), single globular testis (T), and follicular vitellaria. (D) Procerovum sp. (dorsal view). (E) C. formosanus (normal type): body very small, 389 196 in average size, with an oral sucker (OS) armed with about 32 circumoral spines, a muscular pharynx (P), a well-developed ventral sucker (VS), a spherical ovary (O), 2 globular testes (T), and follicular vitellaria distributing along extracecal margins from the pharyngeal level to the posterior end. (F) C. formosanus (plump type). (G) S. falcatus: body small, 447 233 in average size, with a muscular oral sucker (OS) and pharynx (P), a small ventral sucker and a long and thick-walled expulsor (E), a spherical ovary (O), 2 globular testes (T), and follicular vitellaria. (H) P. cambodiensis: body small, 467 277 in average size, with a muscular oral sucker (OS) and pharynx (P), a pair of eyespots, a small ventral sucker (VS), a ventrogenital complex (VGC) with a large genital sac and 2 gonotyls, a spherical ovary (O), 2 globular testes (T), and follicular vitellaria. All scale bar is 100 μm. the right-side of ovary. One testis globular or subglobular, 65113 (90) by 75-113 (97), lying posterior 1/4 of body. Uterus with eggs occupying from anterior 1/3 to posterior end, most of hind-body. Vitellaria follicular, distributing in post-ovarian fields. Eggs small, yellow, and 28-33 (30) by 14-17 (15). Procerovum sp. (n= 3, Fig. 2C, D) Body small, pear-shaped, 425-445 (435) long, and 230-245 (238) wide, with greatest width at mid-level, between the ovary and testis. Oral sucker
Chai et al.: Zoonotic trematode metacercariae from Yangon, Myanmar 637 subterminal, 38-43 (40) by 40-45 (42). Pharynx subglobular or elliptical, 25-30 (28) by 20-25 (23). Esophagus short, 50-53 (51) in length. Ventral sucker very small, 18-20 (19) by 20-23 (21), embedded in ventrogenital sac. Seminal vesicle bipartite with thin-walled chambers, long expulsor, 118-168 (148) by 18-20 (19), and saccular portion, 88-95 (90) by 25-33 (28). Ovary spherical or subspherical, 40-65 (54) by 63-75 (71), slightly dextral to midline. Seminal receptacle saccular, 45-83 (63) by 33-55 (44), lying the right-side of testis. One testis globular or subglobular, 155-163 (160) by 115-163 (147), situated in middle of hind-body. Uterus with eggs occupying 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 (26) by 13-15 (14). C. formosanus (n =20, Fig. 2E): Body very small, 310-470 (389) long, and 150-220 (196) wide. Oral sucker subterminal, 38-55 (46) by 43-55 (50), armed with about 32 circumoral spines. Prepharynx very short. Pharynx globular, 30-45 (37) by 20-38 (30). Esophagus very short, 18-35 (24). Ventral sucker round or elliptical, 30-53 (42) by 40-60 (50). Ovary elliptical, 30-63 (45) by 43-80 (64), dextral to midline. Seminal receptacle large and saccular, 38-75 (51) by 30-75 (48). Two testes ellipsoidal, side by side near the posterior end; right 25-55 (46) by 63-93 (79), left 25-53 (43) by 55-83 (68). Vitellaria follicular, distributing along extracecal margins from pharyngeal level to posterior end. Eggs small, yellow, and 31-35 (34) by 16-19 (18). The morphological characteristics of normal type were nearly equal to those of plump type (Fig. 2F) except for the ratio of BL (body length) to BW (body width) (Table 6). S. falcatus (n= 10; Fig. 2G) were small, oval to pyriform, dorsoventrally flat, 400-515 (447 in average) long and 215-245 (233) wide. Oral sucker subterminal, 35-42 (40) by 40-52 (47). Pharynx subglobular, 24-32 (28) by 15-28 (22). Esophagus slender, 45-65 (58) long. Ventral sucker small. Expulsor long and thick-walled, 103-135 (117) by 28-36 (33). Seminal vesicle saccate. Ovary spherical, 52-73 (57) by 55-88 (67). Two testes ovoid or globular, slightly oblique and widely separated; right 104-135 (115) by 68-82 (75); left 100-130 (114) by 57-75 (66). Vitellaria follicular, distributing in the post-ovarian fields. Eggs small, yellow, and 22-24 (23) by 11-13 (12). P. cambodiensis (n= 3, Fig. 2H): Body small, tapering anteriorly and bluntly ending posteriorly, 395-530 (467) long and 230-350 (277) wide, with a deep ventral concave. Eye-spot pigment seen in 2 lateral fields of anterior body. Oral sucker Table 6. Dimensions of Centrocestus formosanus adults recovered in the small intestines of cats experimentally infected with metacercariae from Myanmar fish Organs Normal type a Range (average) small, subterminal, subglobular, 45-48 (46) by 43-45 (43). Pharynx muscular, oval or slightly elongated, 33-38 (34) by 20-25 (23). Esophagus slender, 45-65 (58) in length. Ventral sucker small, globular, median, almost equal in size with oral sucker, 40-45 (42) by 45-50 (48). Ventrogenital complex well developed with large genital sac and 2 gonotyls. Ovary spherical, anterior to right testis, 55-68 (62) by 58-80 (68). Testes 2, separated, side-by-side, oval, transversely elongated, near the posterior body wall, right 48-60 (53) by 80-100 (87), left 48-63 (55) by 88-95 (92). Seminal vesicle bipartite, globular or transversely elongate, filled with sperms, in the mid field of body. Seminal receptacle elongates saccular, posterodorsal to ovary, 95-100 (97) by 50-55 (52). Gonotyls 2, sinistral to ventral sucker, round to elliptical, equipped with chitinous rodlets in 2 rows. Vitellaria follicular, distributing in post-ovarian fields, almost down to the posterior end of body. Eggs ovoid to elliptical, operculate, yellow, and 21-23 (22) by 10-13 (11). DISCUSSION Plump type b Range (average) Body length (BL) 310-470 (389) 270-470 (339) Body width (BW) 150-220 (196) 170-250 (205) Ratio of BL/BW 1.98 : 1 1.65 : 1 Oral sucker (OS) 38-55 (46) x 43-55 (50) Pharynx 30-45 (37) x 20-38 (30) 35-53 (45) x 43-55 (51) 30-40 (35) x 20-35 (27) Esophagus 18-35 (24) 8-38 (23) Ventral sucker (VS) 30-53 (42) x 40-60 (50) 30-50 (38) x 43-60 (49) Ratio of OS/VS 1.04 : 1 1.1 : 1 Seminal receptacle 38-75 (51) x 30-75 (48) Ovary 30-63 (45) x 43-80 (64) Right testis 25-55 (46) x 63-93 (79) Left testis 25-53 (43) x 55-83 (68) Eggs 31-35 (34) x 16-19 (18) Each of 20 a and 20 b worms were measured; unit is μm. 30-88 (53) x 35-58 (44) 30-58 (41) x 45-75 (61) 28-63 (44) x 50-108 (80) 30-55 (40) x 50-88 (69) 29-36 (33) x 16-20 (18) Although the number and species of fish examined in this study were not so much, more than 7 species of ZT metacer-
638 Korean J Parasitol Vol. 55, No. 6: 631-641, December 2017 cariae (H. taichui, H. pumilio, H. yokogawai, S. falcatus, P. cambodiensis, Procerovum sp., and Centrocestus spp. including C. formosanus) were detected in fish from a local market of Yangon City, Myanmar. They were all minute intestinal flukes and members of the Heterophyidae. These heterophyid species have never been reported in Myanmar. Therefore, by the present study, it has been first confirmed that the life cycles of more than 7 species of heterophyid flukes are existing around Yangon, Myanmar. Approximately 48 fish species have been reported as the second intermediate hosts of H. taichui in Asian countries, i.e., India, China, the Philippines, Thailand, Vietnam, and Lao PDR [18-27]. In the present study, H. taichui metacercariae were detected in 58 (22.0%) fish in 5 (41.7%) species, i.e., T. thynnoides, P. aurotaeniatus, E. altus, Mystacoleucus sp. and Labeo sp., and their density was 42.9 per fish infected. The infection rates and densities were relatively low when compared with those of previous studies performed in other countries [23-27]. The metacercariae of H. taichui detected in fish from Myanmar in this study (203 172 μm in average) were nearly the same in size with those from Vietnam (205 175) [25] and China (203 168) [24]. However, adult flukes of H. taichui in this study (591 298) were somewhat smaller than those of Chai et al. (655 305: 8-day-old in hamster) [25] and Dung et al. (756 421: recovered from residents in Nam Dinh Province, Vietnam) [7]. As the second intermediate host for H. pumilio, total 39 fish species have been recorded in China, Vietnam, and Cambodia [25,26,28]. In the present study, H. pumilio metacercariae were detected in 96 (36.4%) fish belonging to 9 (75.0%) species, i.e., T. thynnoides, P. aurotaeniatus, E. altus, C. striata, A. testudineus, T. pectoralis, Rhynogobius sp., Mystacoleucus sp., and Labeo sp., out of 264 examined fish which belong to 12 species, and their density was 23.6 per fish infected. Therefore, by the present study, 7 more fish species, i.e., T. thynnoides, P. aurotaeniatus, E. altus, C. striata, A. testudineus, and T. pectoralis, have been newly added as the fish intermediate hosts for H. pumilio. The metacercariae of H. pumilio in this study (176 160 μm in average) were nearly the same in size with those from Vietnam (179 159) [25] and China (172 152) [24]. Adult flukes in this study (485 223) were also nearly the same in size with those of Chai et al. (496 217) [25], but they were somewhat smaller than those of Dung et al. (632 291: recovered from residents in Nam Dinh Province, Vietnam) [7]. The metacercariae of H. yokogawai were reported in Asian countries such as India, Thailand, Egypt, Lao PDR, and Cambodia. They were found in about 43 fish species in these countries [4,18,20,21,23,28]. In the present study, H. yokogawai metacercariae were found in 5 fish species, i.e., P. aurotaeniatus, E. altus, T. pectoralis, Mystacoleucus sp., and Labeo sp., from Yangon, Myanmar. Among these fish, 2 species, P. aurotaeniatus and Mystacoleucus sp., were heavily infected with H. yokogawai metacercariae. The size of these metacercariae (175-225 163-218) were nearly the same as those of Rim et al. (170-240 150-230) from Lao PDR [23]. However, we failed to obtain the adult flukes from a cat and hamsters experimentally infected with the metacercariae. Centrocestus formosanus has been distributed in China, Taiwan, Japan, Philippines, India, Lao PDR, Vietnam, and Cambodia [18,19,22-26,28,31-34]. The metacercariae of this fluke were detected in 9 fish species from Lao PDR [22,23,26,29,33, 34], in 16 species from Vietnam [8,25], in 10 species from Zhuang Autonomous Region, China [24], and in 2 fish species from Cambodia [28]. In the present study, Centrocestus spp. metacercariae including C. formosanus were detected in 8 (66.7%) fish species, i.e., T. thynnoides, P. aurotaeniatus, E. altus, C. striata, A. testudineus, T. pectoralis, Mystacoleucus sp., and Labeo sp., and their density was 25.8 per fish infected. The size of C. formosanus metacercariae in this study (171 142) were nearly the same with those from Vietnam (169 138) [25] and Lao PDR (150-200 100-120, and 180 107) [23,33,34], but somewhat smaller than those from China (208 164) [24]. Adult flukes in this study (389 196) were also nearly the same in size with those from Vietnam (367 207) [25], but they were somewhat smaller than those of Chai et al. (460 180: recovered from residents in Xiengkhouang Province, Lao PDR) [34]. In the present study, Procerovum sp. metacercariae were detected only in the climbing perch, Anabas testudineus, examined in June 2015. These fish were previously reported as the second intermediate host of Procerovum varium in Vietnam and Lao PDR [8,25,29,35]. The metacercariae of Procerovum varium were found in 100%, 30.0%, and 37.5% of the climbing perch from Hanoi City and Nam Dinh Province in Vietnam and Vientiane Municipality in Lao PDR, and their mean densities were 466, 13.5, and 12.7 per fish infected, respectively [25,29]. Whereas, Procerovum sp. metacercariae were found in 50.0% of the climbing perch from Yangon in Myanmar, and the density was 46.7 per fish infected in this study. Additionally, 9 fish species, i.e., L. rohita, H. molitrix, C. mrigala, C. idella, S. curricu-
Chai et al.: Zoonotic trematode metacercariae from Yangon, Myanmar 639 lus, P. brachypomum, C. batrachus, B. gonionotus, and M. cephalus, were recorded as the fish intermediate hosts of P. varium in Vietnam and Lao PDR [8,25,29,35]. Procerovum sp. metacercariae (185 155) detected in this study were similar in size with P. varium metacercariae (187 147) in the climbing perch from Hanoi City [25], Vietnam, but they were slightly larger than the P. varium metacercariae (175 136) in same fish hosts from Vientiane Municipality in Lao PDR [29]. However, their general morphologies, i.e., elliptical in shape, presence of brownish pigment granules in the worm body, a prominent expulsor, and a D-shaped excretory bladder with grouped excretory granules, were almost identical to those of the previous studies [25,29]. Trematode members in the genus Procerovum (Family Heterophyidae) are characterized by possessing a single testis and a long prominent seminal vesicle modified into an expulsor. Among the 5 species listed, 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 (435 238) were similar in size with those of Chai et al. [25] (434 223), which were recovered from a hamster experimentally infected with the metacercariae from fish of Vietnam, but they were somewhat larger than those of Eom et al. [29] (304 186), which were recovered from a cat experimentally infected with the metacercariae from fish of Lao PDR. The length of expulsor (148 μm) is more or less longer than that in Chai et al. [25] (115) and Eom et al. [29] (123). Moreover, the expulsor in our specimens has a bipartite seminal vesicle with thin-walled chambers like that in P. cheni. However, the expulsor is saccular and thick-walled in P. varium and P. calderoni. Collectively, the length of expulsor is less than 100 μm in P. cheni, less than 160 μm in P. varium, and more than 200 μm in P. calderoni [25,36]. The morphological characteristics of the expulsor in our specimens are mixed with those of P. varium and P. cheni. It is seriously needed to clarify the valid species name of Procerovum sp. with further precise studies in Myanmar. The metacercariae of S. falcatus were detected only in the mullet, Chelon macrolepis, like in Cambodia [37]. They were also found in several species of fish, i.e., mullet (M. cephalus and L. haematocheila), goby (A. flavimanus), wrestling halfbeak (D. pusilla), X. canciloides, giant gouramy (O. gourami), common carp (C. carpio), and grass carp (C. idella), in Japan, Hawaii, China, Korea, Thailand, Vietnam, and Lao PDR [38-45]. Among these fish intermediate hosts, the mullet is the most frequently reported in various regions [25,37-41]. The size of S. falcatus metacercariae in this study (225 165) were nearly the same with those in Cambodia (220 168), but somewhat smaller than those in Vietnam (297 232) [25,37]. The adult worms (447 233) were nearly the same in size with those of Chai et al. (450 237: 10-day-old in a hamster), but they were slightly smaller than those of Dung et al. (468 298: from residents of Vietnam) and Chai et al. (481 239: 8-day-old in a hamster) [8,25]. The metacercariae of P. cambodiensis were found only in the mullet, Chelon macrolepis, like in a previous study, and their size (242 194) and morphology were nearly equal to those of Sohn et al. (246 191) from Cambodia [46]. In the adult worms, the size of our specimens (467 277) were smaller or less than those of Sohn et al. (534 306: 10-day-old in a hamster). However, other morphological characteristics, i.e., distribution of vitellaria and uterus, and genital organs, were closely identical with those of P. cambodiensis n. sp. previously described by Sohn et al. [46]. In particular, the morphology of ventrogenital complex, i.e., a muscular, sucker-like genital sac equipped with 2 plate-like gonotyls, which are armed with chitinous rodlets (4-5 on the right side and 10-11 on the left side in 2 rows), is a species-specific character of P. cambodiensis. Conclusively, it has been first confirmed in Myanmar that more than 7 ZT species, i.e., H. taichui, H. pumilio, H. yokogawai, S. falcatus, P. cambodiensis, Procerovum sp., and Centrocestus spp. including C. formosanus, of heterophyid flukes are distributed around Yangon area, and some species of fish play the role of second intermediate hosts for these trematodes. Although there are some drawbacks in this study, such as the obscure collection sites, the small number and species of fish, and a long survey period, epidemiological trends of ZT infections in Myanmar have fairly been revealed. ACKNOWLEDGMENTS We thank Jung-A Kim and Hee-Ju Kim, Department of Parasitology, Gyeongsang National University College of Medicine, Jinju, Korea, for their help in fish examinations. We also thank the staff of the Korea Association of Health Promotion, Seoul, the Republic of Korea, who participated in the Korea-Myanmar Cooperation Project on Parasite Control in Myanmar (2013-2015).
640 Korean J Parasitol Vol. 55, No. 6: 631-641, December 2017 CONFLICT OF INTEREST The authors declare that they have no conflict of interest with this study. REFERENCES 1. Chai JY, Murrell KD, Lymbery AJ. Fish-borne parasitic zoonoses: status and issues. Int J Parasitol 2005; 35: 1233-1254. 2. Chai JY. Intestinal Flukes. In Murrell KD, Fried B eds, Food- Borne Parasitic Zoonoses. New York, USA. Springer 2007, p 53-115. 3. Hung NM, Madsen H, Fried B. Global status of fish-borne zoonotic trematodiasis in humans. Acta Parasitol 2013; 58: 231-258. 4. Radomyos B, Wongsaroy T, Wilairatana P, Radomyos P, Praevanich R, Meesomboon V, Jongsuksuntikul P. Opisthorchiasis and intestinal fluke infections in northern Thailand. Southeast Asian J Trop Med Public Health 1998; 29: 123-127. 5. Chai JY, Park JH, Han ET, Guk SM, Shin EH, Lin A, Kim JL, Sohn WM, Yong TS, Eom KS, Min DY, Hoang EH, Phommasack B, Insisienmay 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 ET, Shin EH, Sohn WM, Yong TS, Eom KS, Min DY, Um JY, Park MS, Hoang EH, Phommasack B, Insisienmay B, Lee SH, Rim HJ. High prevalence of Haplorchis taichui, Phaneropsolus molenkampi, and other helminth infections among people in Khammouane Province, Lao PDR. Korean J Parasitol 2009; 47: 243-247. 7. Dung DT, De NV, Waikagul J, Dalsgaard A, Chai JY, Sohn WM, Murrell KD. Fishborne intestinal zoonotic trematodiasis, Vietnam. Emerg Inf Dis 2007; 13: 1828-1833. 8. 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: e742. 9. Myanmar in Wikipedia-The free encyclopedia: http://en.wikipedia. org. 10. Htoon T T, Tun T, Oo KY, Thein W, Tin HH, Chai JY, Yong TS, Sohn WM. Status of infection with soil-transmitted helminths among primary school children in three selected townships of Yangon region. Myanmar Health Sci Res J 2015; 27: 221-226. 11. 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. 12. Chai JY, Han ET, Guk SM, Shin EH, Sohn WM, Yong TS, Eom KS, Lee KH, Jeong HG, Ryang 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. 13. Nuchprayoon S, Sanprasert V, Kaewzaithim S, Saksirisampant W. Screening for intestinal parasitic infections among Myanmar migrant workers in Thai food industry: a high-risk transmission. J Immigr Minor Health 2009; 11: 115-121 14. Htwe KT, Min TT, Mar TT, Lwin MM, Maw WW, Win MM. Detection of intestinal parasitic infestation among primary school children, Magway. Myanmar Health Sci Res J 2010; 22: 89-94. 15. Tun A, Myat SM, Gabrielli AF, Montresor A. Control of soiltransmitted helminthiasis in Myanmar: result of 7 years of deworming. Trop Med Int Health 2013; 18: 1017-1020. 16. Aung WPP, Htoon TT, Tin HH, Thinn KK, Sanpool O, Jongthawin J, Sadaow L, Phosuk I, Rodpai R, Intapan PM, Maleewong W. First report and molecular identification of Opisthorchis viverrini infection in human communities from Lower Myanmar. PLoS One 2017; 12: e0177130. 17. Search FishBase [Internet]. Available from: http://www.fishbase. org/search.php. 18. Nath D, Pande BP. Identify of the three heterophyid metacercariae infesting some of the freshwater fishes. Curr Sci 1970; 39: 325-326. 19. Velasquez CC. Observations on some Heterophyidae (Trematoda: Digenea) encysted in Philippine fishes. J Parasitol 1973; 59: 77-84. 20. Pande V, Premvati G. Development of metacercariae of Haplorchis spp. in chicks. Indian J Parasitol 1977; 1: 165-172. 21. 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 Southeast Asian J Trop Med Public Health 1982; 13: 35-60. 22. 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. 23. 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. 24. Sohn WM, 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. 25. 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. 26. Rim HJ, Sohn WM, Yong TS, Eom KS, Chai JY, Min DY, Lee SH, Hoang EH, Phommasack B, Insisengmay S. Fishborne trematode metacercariae in Luang Prabang, Khammuane and Saravane, Lao PDR. Korean J Parasitol 2013; 51: 107-114. 27. 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. 28. Chai JY, Sohn WM, Na BK, Yong TS, Eom KS, Yoon CH, Hoang
Chai et al.: Zoonotic trematode metacercariae from Yangon, Myanmar 641 EH, Jeoung HG, Socheat D. Zoonotic trematode metacercariae in fish from Phnom Penh and Pursat, Cambodia. Korean J Parasitol 2014; 52: 35-40. 29. Eom KS, Park HS, Lee D, Sohn WM, Yong TS, Chai JY, Min DY, Rim HJ, Insisiengmay B, Phommasack B. Infection status of zoonotic trematode metacercariae in fishes from Vientiane Municipality and Champasak province in Lao PDR. Korean J Parasitol 2015; 53: 447-453. 30. 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 1966; 36: 437. 31. Premvati G, Pande V. On Centrocestus formosanus (Nishigori, 1924) Price, 1932 and its experimental infection in White Leghorn chicks. Jap J Parasitol 1974; 23: 79-84. 32. Yanohara Y. On analysis of transmission dynamics of trematode infection 1. Centrocestus formosanus infection in Miyakojima. Jap J Parasitol 1985; 34: 55-70. 33. 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 34. Chai JY, Sohn WM, Yong TS, Eom KS, 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. 35. 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. 36. 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. 37. Chai JY, Sohn WM, Na BK, Jeoung HG, Sinuon M, Socheat D. Stellantchasmus falcatus (Digenea: Heterophyidae) in Cambodia: discovery of metacercariae in mullets and recovery of adult flukes in an experimental hamster. Korean J Parasitol 2016; 54: 537-541. 38. Onji Y, Nisho T. A monograph of intestinal trematodes. Chiba Igakkai Zasshi 1924; 2: 113-161 (in Japanese). 39. Alicata JE, Schattenburg OL. A case of intestinal heterophyidiasis of man in Hawaii. JAMA1938; 110: 1100-1101. 40. Kobayasi H. Studies on the trematoda in Hainan Island, South China and Vietnam (French Indochina). Reports of Scientifical Works by H. Kobayasi, 1968, pp 155-251. 41. Chai JY, Sohn WM. Identification of Stellantchasmus falcatus metacercariae encysted in mullets in Korea. Korean J Parasitol 1988; 26: 65-68. 42. Ditrich O, Scholz T, Giboda M. Occurrence of some medically important flukes (Trematoda: Opisthorchiidae and Heterophyidae) in Nam Ngum water reservoir, Laos. Southeast Asian J Trop Med Public Health 1990; 21: 482-488. 43. Sripalwit P, Wongsawad C, Chai JY, Anuntalabhochai S, Rojanapaibul A. 2003. Investigation of Stellantchasmus falcatus metacercariae in half-beaked fish, Dermogenus pusillus from four districts of Chiang Mai Province, Thailand. Southeast Asian J Trop Med Public Health 2003; 34: 281-285. 44. Thien PC, Dalsgaard A, Thanh BN, Olsen A, Murrell KD. Prevalence of fishborne zoonotic parasites in important cultured fish species in the Mekong Delta, Vietnam. Parasitol Res 2007; 101: 1277-1284. 45. Chi TT, Dalsgaard A, Turnbull JF, Tuan PA, Murrell KD. Prevalence of zoonotic trematodes in fish from a Vietnamese fish farming community. J Parasitol 2008; 94: 423-428. 46. Sohn WM, Kim DG, Jung BK, Cho J, Chai JY. Pygidiopsis cambodiensis n. sp. (Digenea: Heterophyidae) from experimental hamsters infected with metacercariae in mullets from Cambodia. Parasitol Res 2016; 115: 123-130.