A survey was performed to investigate the infection status of fishborne trematode (FBT) metacercariae in freshwater fish from Guangxi Zhuang Autonomous Region, China. A total of 307 freshwater fish of 31 species were collected from 5 administrative regions of Guangxi Zhuang Autonomous Region. They were examined by artificial digestion method from July 2003 to August 2004. No metacercariae were detected in fish from Fusui-xian. In fish from Mashan-xian and a market in Nanning, 3 species of metacercariae, Haplorchis taichui, Haplorchis pumilio, and Centrocestus formosanus, were mainly detected. Metacercariae (8 in number) of Clonorchis sinensis were found in 1 Chanodichthys dabryi purchased from a market in Nanning. In fish from Yangshuo, Metagonimus yokogawai metacercariae were detected from all 18 fish species examined. Total 13 C. sinensis metacercariae were found in 3 out of 10 Hemibarbus maculatus from Yangshuo. All 7 Zacco platypus from Yangshuo were infected with 8-112 Echinochasmus perfoliatus metacercariae. In fish from Binyang-xian, H. pumilo metacercariae were mainly detected in all 5 fish species examined, and only 1 metacercaria of C. sinensis was found in a Hemiculter leucisculus. From the above results, it was confirmed that some species of freshwater fish play a role of second intermediate hosts for FBT in Guangxi Zhuang Autonomous Region, China. In particular, 4 species of intestinal flukes, M. yokogawai, H. taichui, H. pumilio, and C. formosanus, were prevalent in fish hosts, whereas C. sinensis metacercariae were detected only in 3 fish species.
Fishborne trematode (FBT) infections affect the health of more than 18 million people around the world, particularly in Asian countries. These flukes provoke remarkable morbidity and cause serious damage to aquaculture in developing countries [1,2]. The liver flukes, Clonorchis sinensis and Opisthorchis spp., have been known as the representative FBT. Many species of intestinal flukes, mainly belonging to the family Heterophyidae and Echinostomatidae are also infected by eating raw fish .
In the Republic of Korea, many species of FBT including C. sinensis have been reported. C. sinensis is still prevalent in riverside areas and is the most important helminth species of public health importance. About 10 species of the Heterophyidae and 3 of the Echinostomatidae have been reported as the intestinal flukes infected by eating raw fish meat [3-6]. On the other hand, C. sinensis infections are found in 22 out of 30 PRM (Province/Autonomous Region/Municipality) in China. Especially, prevalences in 4 provinces, Guangdong, Guangxi, Anhui and Heilongjiang, are much higher than those of the other regions [7-9]. As the intestinal FBT, 8 species of the Heterophyidae (Metagonimus yokogawai, M. takahashii, Stellantchasmus falcatus, Centrocestus formosanus, Haplorchis pumilio, H. taichui, and H. yokogawai) and 5 species of the Echinostomatidae (Echinostoma angustitestis, Echinochasmus japonicus, E. perfoliatus, E. liliputanus, and E. fujianensis) are distributed in China .
Guangxi Zhuang Autonomous Region (GZAR) locates in the southern part of China, on the subtropical zone and its southwest area contact with Vietnam. This province has many minority people. They have some different food habit. Some monority people like to eat raw fish and easily infect with FBT. Furthermore, they have engaged in aquaculture in the fishponds with latrines that contribute as an important source of contamination . It has been revealed that many minority peoples are infected with FBT including C. sinensis in GZAR, China . However, the major source of infection has not yet been obvious. Therefore, we performed epidemiological surveys to know the second intermediate hosts and the infection status of FBT metacercariae in freshwater fish from 5 administrative regions of GZAR, China.
MATERIALS AND METHODS
A total of 307 freshwater fish of 31 species was collected from 5 administrative regions, Fusui-xian, Mashan-xian, Nanning City, Yangsuo County, and Binyang-xian, in GZAR, China from July 2003 to August 2004 (Table 1). All collected fish were transferred to the laboratories (Division of Helminthiasis Prevention and Control, Guangxi Centre for Disease Prevention and Control, Nanning, China, and Department of Parasitology, Gyeongsang National University School of Medicine, Jinju, Korea), individually measured the length and weight according to the species, and identified the fish species with the aid of ichthyologists in Nanning, GZAR, China, and FishBase site in internet . They all were 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℃ for 2-3 hr. The digested material was filtered with 1 × 1 mm of mesh, and washed with 0.85% saline untill the supernatant is clear. The sediment was carefully examined under a stereomicroscope. The metacercariae were separately collected by the general feature, and they were identified species level based on the detail morphologies and their demensions under a light microscope. Identified metacercariae were experimentally infected to mice, rats, hamsters, chicks and cats to obtain adult worms.
Infection status in fish from Fusui-xian and Mashan-xian
No metacercariae were detected in fish from Fusui-xian. However, in fish from Mashan-xian, 3 species of metacercariae, H. taichui, H. pumilio, and C. formosanus, were detected. H. taichui metacercariae were found in 7 fish species and their infection status is presented in Table 2. H. pumilio metacercariae were detected in 10 fish species. Among them, Opsariichthys bidens, Puntius semifasciolatus, Pseudohemiculter dispar, and Carassius auratus were heavily infected (Table 3). C. formosanus metacercariae were found in 3 fish species. Especially, Microphysogobio fukiensis was infected with more than 300 metacercariae of C. formosanus (Table 4).
Infection status in fish from Nanning City
In freshwater fish from a market in Nanning, 3 species of metacercariae, H. taichui, H. pumilio, and C. formosanus, were mainly detected. H. taichui metacercariae were found in 11 fish species. Among them, Hemiculter leucisculus, Toxabramis houdemeri, and Cyprinus carpio were heavily infected with H. taichui metacercariae (Table 5). H. pumilio metacercariae were detected in 11 fish species, and their infection status is shown in Table 6. C. formosanus metacercariae were found in 7 fish species and their infection status is shown in Table 7. One Chanodichthys dabryi was infected with 3 metacercariae of C. sinensis. Four metacercariae of Metagonimus sp. were detected in 1 Hemibarbus maculatus.
Infection status in fish from Yangshuo County
In freshwater fish from Yangshuo, Metagonimus sp. metacercariae were detected from all 18 fish species examined. Among them, Zacco platypus, H. maculatus, P. semifasciolatus, and Abbottina rivularis were infected with more than 100 metacercariae per infected fish (Table 8). A total of 13 C. sinensis metacercariae were found in 3 out of 10 Hemibarbus maculatus examined. All 7 Z. platypus examined were infected with 8-112 metacercariae of E. perfoliatus.
Infection status in fish from Binyang-xian
In freshwater fish from Binyang-xian, H. pumilo metacercariae were mainly detected in all 5 fish species examined, and their infection status is presented in Table 9. Metorchis taiwanensis metacercariae were found in 4 Ctenopharyngodon idella (40%) and their numbers were ranged in 1-425. Only 1 metacercaria of C. sinensis was detected in H. leucisculus.
Overall infection status of fish from GZAR, China
Metacercariae of Metagonimus sp. were detected in 18 species of fish, and their fish hosts are revealed in Table 8 (see full names of fish). Those of H. taichui were found in 15 fish species, such as H. molitrix, C. auratus, H. maculatus, T. houdemeri, P. dispar, O. bidens, S. argentatus, C. carpio, A. rivularis, P. semifasciolatus, H. leucisculus, C. recurviceps, S. dabryi, M. lineate, and C. dabryi. H. pumilio metacercariae were detected in 18 fish species, i.e., C. auratus, A. tonkinensis, H. maculatus, H. molitrix, H. leucisculus, C. idella, T. houdemeri, M. fukiensis, P. dispar, O. bidens, S. argentatus, M. lineata, C. carpio, P. semifasciolatus, S. dabryi, C. recurviceps, C. dabryi, and P. parva. C. formosanus metacercariae were found in 10 fish species, such as M. fukiensis, A. tonkinensis, S. argentatus, C. carpio, H. molitrix, A. rivularis, H. leucisculus, M. lineata, S. dabryi, and P. parva (Table 10). On the other hand, metacercariae of C. sinensis were demonstrated in low infection rates and intensities in only 3 fish species, H. maculatus, C. dabryi, and H. leucisculus.
Morphology of metacercariae detected
H. taichui metacercariae (n = 20) were elliptical, 188-220 (203 in average) × 155-185 (168 in average) µm in size, had a baseball glove-shaped ventro-genital sac with 11-18 rodlets and an O-shaped excretory bladder occupying large portion of posterior body (Fig. 1A).
H. pumilio metacercariae (n = 20) were elliptical, 155-188 (172) × 138-163 (152) µm in size, had 36-42 deer horn-like minute spines arranged in 1-2 rows around ventrogenital complex, and an O-shaped excretory bladder occupying large portion of posterior body (Fig. 1B).
C. formosanus metacercariae (n = 30) were elliptical, 173-233 (208) × 135-178 (164) µm in size, had 32 circumoral spines around oral sucker arranged in 2 rows, and a X-shaped excretory bladder occupying greater portion of posterior body (Fig. 2A).
Metagonimus sp. metacercariae (n = 30) were elliptical, 138-200 (154) × 120-183 (144) µm in size, had yellow brownish pigment granules scattering in body area of intestinal bifurcation, a ventral sucker deflectively located from median, and a V-shaped excretory bladder (Fig. 2B).
C. sinensis metacercariae (n = 10) were elliptical, 158-193 (182) × 153-183 (168) µm in size, had 2 nearly equal sized suckers, brownish pigment granules scattering in the body, and an O-shaped excretory bladder occupying greater part of posterior body (Fig. 3A).
E. perfoliatus metacercariae (n = 10) were elliptical, 95-110 (105) × 75-88 (85) µm in size, had a transparent and double layered cyst wall, 24 dorsally interrupted collar spines, and a ventral sucker lying median on posterior 1/3 of body and as large as oral sucker (Fig. 3B).
M. taiwanensis metacercariae (n = 10) were globular, 163-170 (166) × 150-170 (162) µm in size, had a double layered cyst wall (transparent and very thick outer layer: 43-52 µm and inner layer: 5.0-7.5 µm in thickness), 2 nearly equal sized suckers, and an O-shaped excretory bladder (Fig. 4).
Morphological characters of Metagonimus yokogawai adults (n = 20)
Adults of M. yokogawai were recovered from experimentally infected dogs and cats with metacercariae collected from fish from Yangshou county. They were 0.882 × 0.437 mm in average size, had the larger ventral sucker (av. 90 × 66 µm), deviated to right side from median, than oral sucker (av. 51 × 62), muscular pharynx (av. 37 × 32), moderately long esophagus (av. 88) and ceca. The round or elliptical ovary (av. 70 × 77) was located in median portion of body, and 2 adjacent testes (right: av. 139 × 107; left av. 127 × 98) were obliquely situated near the posterior end of body. The uterine loop passed through anterior border of both testis, and the uterine eggs were elliptical, yellow in color, 24.0-26.8 (av. 25.4) long and 15.0-17.5 (av. 15.6) wide (Fig. 5).
World Health Organization (WHO) estimated that more than 18 million people are infected with fishborne trematodes (FBT), and more than half of billion people are at risk of FBT infections around the world [1,13]. FBT infections are classified as zoonoses because they are viewed as animal diseases transmitted to human. Some infections appear to be rare while others are common and cause serious disease. It has been known that liver flukes, C. sinensis and Opisthorchis spp., and some groups of intestinal flukes are representative FBT, and they are mainly infected by eating raw meat of freshwater and estuarine fish in low- and middle-income countries. On the other hand, it has been revealed that so many peoples are infected with FBT including C. sinensis in GZAR, China by the work of "Korea-China Collaborative Project of Control Strategies for Helminthiasis" . However, it has not been obvious that what kinds of fish play a role of second intermediate host of FBT in this area. Therefore, present study was performed to investigate the infection status of FBT metacercariae in freshwater fish from GZAR, China.
Through this study, it was confirmed that some species of FBT metacercariae are prevalent in freshwater fish from GZAR, China. Especially, four species of intestinal flukes, H. taichui, H. pumilio, M. yokogawai and C. formosanus, were prevalent in fish hosts while C. sinensis metacercariae were detected from only 3 species of fish. Besides aforementioned 5 species, E. perfoliatus and M. taiwanensis metacercariae were also found in this study. Moreover, it was proved for the first time that 4 trematode species, H. pumilio, M. yokogawai, E. perfoliatus and M. taiwanensis, are distributed in GZAR, China .
About 70 species belonging to the 14 families, i.e., Heterophyidae, Echinostomatidae, Plagiorchiidae, Lecithodendriidae, Neodiplostomidae, Nanophyetidae, Paramphistomatidae, Cathaemaciidae, Fasciolidae, Gastrodiscidae, Gymnophallidae, Microphallidae, Strigeidae and Brachylaimidae, have been found in the intestinal tract of humans around the world. Among members in these families, it has been known that Heterophyidae and Echinostomatidae are the major groups . In China, it has been revealed that Fasciolopsis buski is most important intestinal fluke by the prevalence and morbidity, and 8 species of Heterophyidae (M. yokogawai, M. takahashii, Stellantchasmus falcatus, C. formosanus, H. pumilio, H. taichui and Haplorchis yokogawai) and 5 species of Echinostomatidae (Echinostoma angustitestis, Echinochasmus japonicus, E. perfoliatus, Echinochasmus liliputanus and Echinochasmus fujianensis) are reported as the intestinal FBT .
H. taichui metacercariae have been recorded in fish from several Asian countries, i.e. India, Thailand, the Philippines, Lao PDR and China [10, 15-20]. They were detected in 4 fish species, Cirrhinus reba (= Labeo ariza), Amblypharyngodon mola, Labeo bata, and Puntius sophore, in India, 3 fish species, Puntius binotatus, Puntius palata (= Spratellicypris palata), and Ophicephalus striatus (= Channa striata), in the Philippines, 3 fish species, Barbonymus gonionotus, Puntius brevis, and Puntius orphoides, in Thailand, 11 fish species, Hampala dispar, Hampala macrolepidota, B. gonionotus, Mystacoleucus maginatus, P. brevis, Puntioplites falcifer, Cyclocheilchthys armatus, Cyclocheilchthys repasson, Onychostoma elongatum, Labiobarbus leptocheila, and Cirrhnus molitorella, in Lao PDR, and 7 fish species, Carassius auratus, Cyprinus carpio, Ctenopharyngodon idella, Pseudorasbora parva, Gambusia affinis, Rhodeus ocellatus and Puntius binotatus, in China [10,15-20]. In the present study, H. taichui metacercariae were detected in 15 fish species including 3 susceptible ones such as, H. leucisculus, C. carpio and T. houdemeri. Moreover, it was confirmed that 14 fish species, H. molitrix, H. maculatus, T. houdemeri, P. dispar, O. bidens, S. argentatus, A. rivularis, P. semifasciolatus, H. leucisculus, C. recurviceps, S. dabryi, M. lineata and C. dabryi, are recorded as new second intermediate hosts of H. taichui in the literature. On the other hand, it had been known that H. taichui is distributed in only Taiwan, China. However, H. taichui adults were collected from residents of GZAR, China after praziquantel treatment and magnesium salt purgation by the work of "Korea-China Collaborative Project of Control Strategies for Helminthiasis" .
Distribution of H. pumilio has been recorded in Guangdong and Fujian Provinces and Taiwan, China . However, it has been confirmed by this study that H. pumilio is distributed in GZAR also. Seventeen fish species except P. parva, i.e., C. auratus, A. tonkinensis, H. maculates, H. molitrix, H. leucisculus, C. idella, T. houdemeri, M. fukiensis, P. dispar, O. bidens, S. argentatus, M. lineata, C. carpio, P. semifasciolatus, S. dabryi, C. recurviceps and C. dabryi are listed as the new second intermediate hosts of this fluke in China. On the other hand, it has been reported that the prevalence of H. pumilio is 0.5% in Zhangzhou of Fujian Province by the special processing way of fish dish . Many human cases by this fluke may be present in GZAR also because of many residents like to eat raw fish and they can easily infect with FBT.
C. formosanus has been reported in Guangdong, Fujian, GZAR, and Taiwan, and 8 fish species, Anabas testudineus, Channa formosana, Clarias fuscus, Macropodus opercularis, G. affinis, C. autatus, C. carpio, C. idella, have been recorded as the second intermediate hosts of this fluke in China . In the present study, 10 fish species including susceptible M. fukiensis were revealed to be the second intermediate host or the source of infection of C. formosanus. Among them, 9 fish species, M. fukiensis, A. tonkinensis, S. argentatus, H. molitrix, A. rivularis, H. leucisculus, M. lineate, S. dabryi and P. parva, were added as new second intermediate hosts for C. formosanus in China. Recently, metacercariae of this fluke were detected in some fish species, i.e., Esomus longimanus, P. brevis, C. repasson, O. hasseltii, and C. molitorella, from Lao PDR [15,20,21].
In China, 2 Metagonimus species, M. yokogawai and M. takahashii, have been reported. M. yokogawai has been distributed in Taiwan, Guangdong, Anhui, Hubei and Zhejiang, while M. takahashii has been reported in Anhui Province only. Twelve fish species, Abramis brama, Abramis ballerus (= Ballerus ballerus), Aspius aspius, Blicca bjoerkna, Carassius carassius, Chondrostoma nasus, Hemibarbus labeo, Leuciscus idus, Pelecus cultratus, Pseudobagrus fulvidraco (= Pelteobagrus fulvidraco), T. hakonensis and Zacco platypus, have been recorded as the second intermediate hosts of M. yokogawai in China . In the present study, M. yokogawai metacercariae were detected in 18 fish species from Yangshou county, and only one fish species, Hemibarbus maculatus, from a market in Nanning, GZAR. Among them,16 fish species, Acrossocheilus hemispinus, Discogobio tetrabarbatus, H. maculates, Puntius semifasciolatus, Sinibrama macrops, Acheilognathus tonkinensis, Abbottina rivularis, Pseudogobio esocinus, C. auratus, Coreoperca whiteheadi, Opsariichthys bidens, Squalidus wolterstorffi, C. carpio, Ancherythroculter lini, Pseudohemiculter dispar and Hemiculter leucisculus, were newly recorded as the second intermediate host of M. yokogawai in China.
Morphologically, adults of Metagonimus sp. recovered from experimentally infected animals with metacercariae, which collected in fish from Yangshou county, were identical with those of M. yokogawai except the size of eggs. They have the larger ventral sucker, deviated to right side, than oral sucker, and the uterine loop passes through anterior border of both testes, whereas it courses between both testes in M. takahashii and M. miyatai. Their eggs, 25.4 × 15.6 µm in average size, were somewhat smaller than those of 3 Metagonimus species in Korea and Japan . Biologically, the host specificity of this fluke relatively low when it is compared with those of 3 Metagonimus species, i.e. M. yokogawai, M. takahashii and M. miyatai, distributed in Korea . As their fish intermediate hosts, Plecoglossus altivelis, Tribolodon hakonensis, T. taczanowskii and Lateolabrax japonicus for M. yokogawai, C. carpio and C. auratus for M. takahashii, Zacco platypus and Z. temmincki for M. miyatai have been reported in Korea [23,24].
C. sinensis infections are distributed in 22 out of 30 PRM (Province/Autonomous Region/Municipality) in China. In a nationwide survey, the prevalence was 0.4% among about 1.5 million people examined. Especially, the prevalence in Guangxi (1.39%), with those in Guangdong (2.09%), Anhui (1.37%) and Heilongjiang (1.19%), was much higher than those of the other regions. Based on data of nationwide survey, the number of clonorchiasis patients in China may be about 6 million [7-9]. On the other hand, as the second intermediate hosts of C. sinensis, 102 fish species in 59 genera 15 families have been reported in China including Taiwan . However, metacercariae of C. sinensis were detected in only 3 fish species, H. maculatus, C. dabryi and H. leucisculus, in this study. Moreover, their infection rates and intensities were very low.
We thank Mr. Hongbo Xu and Miss Rui Lin, Division of Helminthiasis Prevention and Control, Guangxi Centre for Disease Prevention and Control, Nanning, China, for their help in our study, and Miss Jeong-A Kim and Soo-Jeong Park, Department of Parasitology, Gyeongsang National University School of Medicine, Jinju, Korea, for their help in examination of fish.
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