Acta Tropica 136 (2014) 118–122
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Experimental infection of Opisthorchis viverrini cercariae to the cyprinid fish, Barbonymus gonionotus Chalermlap Donthaisong a , Patpicha Arunsan a , Kulwadee Suwannatrai a , Sattrachai Prasopdee a , Jutharat Kulsantiwong b , Sutee Wongmaneeprateep c , Apiporn Suwannatrai a , Smarn Tesana a,∗ a
Food-borne Parasite Research Group, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Department of Biology, Faculty of Science, Udon Thani Rajabhat University, Udon Thani 41000, Thailand c Department of Fisheries, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand b
a r t i c l e
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Article history: Received 22 October 2013 Received in revised form 21 March 2014 Accepted 19 April 2014 Available online 26 April 2014 Keywords: Opisthorchis viverrini cercariae Metacercariae Infectivity Barbonymus gonionotus
a b s t r a c t Opisthorchis viverrini is an important public health problem, a major cause of cholangiocarcinoma in the Greater Mekong subregion including Lao PDR, Cambodia, Vietnam and Thailand. Humans acquire the infection by consumption of raw, fermented or partially cooked freshwater cyprinid fish containing infective metacercariae. This study examined the effect of cercarial infection dosage (25–200 cercariae), age (1–60 day) and size (1–24 mm) of Barbonymus gonionotus fish on infection success of O. viverrini cercariae. Additionally, the site of cyst formation of O. viverrini cercariae and subsequent development to the metacercariae was examined. The result showed that cercarial infection dose (F4,95 = 8.52, P < 0.001) age 2 2 (P < 0.001, ¯ LR = 954.72) and size (P < 0.001, ¯ LR = 1204.84) were significantly associated with number of O. viverrini metacercariae recovery with in every additional fish age, the O. viverrini metacercarial recovery rate increased by 5.4% (P < 0.001). Most metacercariae were found in the body of the fish (81.4%). We concluded that O. viverrini cercarial infection dosage, and age and size of fish were important determinants for a successful infection to fish. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Fish-borne trematodiasis of Opisthorchiasis and clonorchiasis caused by the liver flukes in the family Opisthorchiidae: Opisthorchis felineus, O. viverrini and Clonorchis sinensis which are endemic in Siberia and East Europe, Southeast, and East Asia, respectively. O. viverrini and C. sinensis have been classified as group 1 carcinogens, which are a risk factor for inducing cholangiocarcinoma in infected humans. O. viverrini is mostly found in endemic areas in the Greater Mekong subregion including Lao PDR, Cambodia, Vietnam and Thailand (Jongsuksuntigul and Imsomboon, 2003; Sayasone et al., 2007; Dang et al., 2008; IARC, 2011). O. viverrini has a complex life cycle, involving bithynid snails as the first intermediate host, cyprinid fish as the second intermediate host, and cats, dogs, and humans as final hosts (Sithithaworn and HaswellElkins, 2003; Upatham and Viyanant, 2003). Cyprinid fish infected with O. viverrini (90.4%) have 800 folds higher prevalence than in
∗ Corresponding author. Tel.: +66 43 363434; fax: +66 43 202475. E-mail addresses: smarn [email protected], [email protected] (S. Tesana). http://dx.doi.org/10.1016/j.actatropica.2014.04.022 0001-706X/© 2014 Elsevier B.V. All rights reserved.
bithynid snails (0.11%) in the same endemic area (Vichasri et al., 1982; Brockelman et al., 1986). Cyprinid fish accumulate infectious metacercariae and enhance transmission of this parasite to the final host. Many species of cyprinid fish serve as intermediate hosts of O. viverrini. High infection rates have been reported in many species of native fish such as Amblyrhynchichthys truncates (100.0%), Puntius brevis (92.0%), Hampala dispar (44.4–100.0%), Puntioplites falcifer (33.3%), P. proctzysron (26.8%), Esomus metallicus (75.0%), Cyclocheilichthys armatus (43.1–100%), C. repasson (58.5%), Dangila lineata (69.6%), Henicorhynchus lineatus (42.9%), Osteochilus waandersii (30.5%), Baybonymus schwanenfeldii (66.0%), Thynnichthys thynnoides (59.7%), Labiobarbus siamensis (51.3%) and Mystacoleucus marginatus (50.0%) (Rim et al., 2008; Manivong et al., 2009; Touch et al., 2013). The highest density of O. viverrini metacercariae was in the head region, followed by the caudal fin and muscles of fins and the lowest was found in the anal fin (Tesana et al., 1985). High rate of fish infection were found in the late rainy season continuing to the cold season (July to January), while low burdens rate were found in the hot-dry season (April–June) (Sithithaworn et al., 1997). Barbonymus gonionotus (silver barb) is a native cyprinid species which is commonly reared in freshwater aquaculture. It is
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widely consumed in Thailand, Lao PDR and Cambodia. The maximum length of mature B. gonionotus is 50 cm. B. gonionotus in natural water resources has been shown to be infected with various parasite species such as Haplorchis taichui (80.3%), Centrocestus spp. (11.3%), and O. viverrini (1.0%) in Chiang Mai Province, northern Thailand, has been found (Sukontason et al., 1999) and 16.1% of O. viverrini infected B. gonionotus in Cambodia (Touch et al., 2013). The present study examined of affect O. viverrini cercarial infection namely, cercarial dosages, and age and size of fish host as determinants. 2. Materials and methods This study was approved by the animal ethics committee of Khon Kaen University, Khon Kaen Province, Thailand (Ethical Clearance No. AEKKU25/2555). 2.1. O. viverrini cercarial preparation Bithynia siamensis goniomphalos snails were collected manually in various freshwater bodies such as rice paddy fields, ponds, canals and roadside ditches from Khon Kaen Province, Thailand. They were transported to the laboratory of Khon Kaen University, Khon Kaen in plastic bags. These, they were washed with dechlorinated tap-water and examined for O. viverrini infection using the cercarial shedding method (stimulated by electric light in the daytime for 3 h). When the cercariae were released from snails, they were identified under light microscope using morphological characteristics of tobacco-pipe shaped when briefly hanged head down or laid on the bottom, a pair of eye-spots and long tail with fin on both lateral sides (Wykoff et al., 1965; Schell, 1970; Frandsen and Christensen, 1984). O. viverrini cercariae were confirmed using specific PCR primers (forward primer OV-6F 5 -CTG AAT CTC TCG TTT GTT CA-3 ; reverse primer OV-6R 5 -GTT CCA GGT GAG TCT CTC TA-3 , Wongratanacheewin et al., 2001). 2.2. Preparation of fish samples Females and males of B. gonionotus were obtained from the stock farm of the Department of Fisheries, Faculty of Agriculture, Khon Kaen University. Spawns of B. gonionotus were induced by intramuscularly injecting in both sexes with synthetic hormones (Luteinizing Hormone-Releasing Hormone analog, LHRHa). In additions, females were injected intramuscularly with 20 g of Buserelin acetate (Suprefact® , nasal solution, Aventis Pharma Deutschland GmbH, Frankfurt am Main, Germany) mixed with 5 mg of domperidone maleate (Motilium® , tablet, Janssen Korea Ltd., Kyunggi-Do, Republic of Korea) per kg body weight. Males were injected with 10 g of Suprefact mixed with 5 mg of Motilium per kg body weight. Females and males were stocked together in a spawning cement tank at a ratio of 1:2 and fed with floating fish food. The eggs were fertilized naturally. After 8–12 h of fertilization the eggs were hatched at a water temperature of 26–28 ◦ C. Fish fry were transported to the laboratory, reared in aquaria and fed with soft husk mixed fish meal under standard conditions to maintain them parasite free. 2.3. Experimental infection in B. gonionotus 2.3.1. Infection process of cercarial infection dosage One hundred and twenty B. gonionotus fingerlings (30 days old, 0.5–1.0 cm in length) were divided into 6 groups (20 per group). Group 1, 2, 3, 4, 5 and 6 were exposed to 0 (control), 25, 50, 100, 150 and 200 cercariae (fresh shedding) per fish for 24 h, respectively. The procedure of infection for each fingerling was done in 5 ml dechlorinated tap-water in a plastic cup (3 cm diameter, and 2.5 cm
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height) at room temperature (25 ± 3 ◦ C) and given the electric light throughout the infection period. Thus, the fingerlings were killed in 4 ◦ C water. Then, they were observed for O. viverrini infection by compressed between two slides. The number of O. viverrini metacercariae was counted under a light microscope (40 magnification), and recorded. 2.3.2. Infection success in age- and size-groups of fish Fish were divided into 16 age-groups of 1–2, 3–4, 5–6, 7–8, 9–10, 11–14, 15–19, 20–24, 25–29, 30–34, 35–39, 40–44, 45–49, 50–54, 55–59 and 60–64 days (20 fish per age-group, total of 320, Fig. 1). From day 1 to 8 after hatching fish fry presented with yolk sac. On day 9 the yolks disappeared and they started active feeding. All fry was exposed to 50 O. viverrini cercariae in the conditions described above. After 24 h of exposure, the fish were killed in 4 ◦ C water and their length, width and weight were recorded. They were examined on O. viverrini infection by compressing these between two slides. The number of O. viverrini metacercariae were counted under a light microscope (40 magnification) and recorded. 2.3.3. Fish body part of O. viverrini metacercarial infection Forty B. gonionotus fingerlings (30 days old, 0.5–1.0 cm in length) were exposed to 100 O. viverrini cercariae per fish in the same conditions as described above. After 24 h of exposure fish were killed in 4 ◦ C water. Then, each part of the fingerlings (head, gills, body, fins, and tail) was examined on the present of O. viverrini metacercariae. The fingerlings were compressed between two slides and the number of O. viverrini metacercariae were counted in each body part of fingerling under a light microscope (40×). 2.4. Data analyses Statistical analyses was conducted with SPSS software version 17.0. The association between cercarial infection dosages in each fish group and the number of O. viverrini metacercariae in B. gonionotus fingerlings was analyzed using post hoc Tukey’s HSD tests in ANOVA. The association of fish age-groups and size-groups and number of O. viverrini metacercariae was examined with Poisson regression. O. viverrini metacercariae counts in fish were not normally distributed. 3. Results 3.1. Infection success of cercarial infection dosage O. viverrini metacercariae were found in all B. gonionotus fingerlings except in the group exposed to 25 cercariae where 19 of 20 (95%) B. gonionotus fingerlings were infected. The highest metacercarial recovery was in group of 200 cercariae infection dose found in 1 of 20 B. gonionotus fingerling (87.5%, 175 metacercariae from 200 cercariae infection dose). The average metacercariae recovery was also high in cercariae infection dose of 200 cercariae gave 42.9% recovery whereas a low dose of 25 cercariae resulted in 13.8% (3.4 of 25) metacercarial recovery. Analysis of variance revealed significant differences between groups (F4,95 = 8.527, P < 0.001). Post hoc comparisons (Tukey’s HSD) showed the lowest (25 cercariae) differed from the highest two groups (150 and 200 cercariae) (diff25VS150 = −0.17, P = 0.028, diff25VS200 = −0.29, P < 0.001), and the second level of dosage (50 cercariae) differed from the highest dosage group (diff50VS200 = −0.26, P < 0.001, Fig. 2). 3.2. Infection success in age- and size-groups of fish We found that fish fry developed and increased their size grew rapidly in the early period of life and reached a plateau after 50 days. The fish fry at early development was small. Only the length and
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Fig. 1. B. gonionotus fry. Age-group of 1–2 days of age (A), 9–10 days of age (B), and 55–59 days of age (C). Scale bars: A&B = 1 mm, C = 5 mm.
Table 1 Percentages of O. viverrini metacercarial recovery in each age-group of B. gonionotus (n = 20). Age-groups of fish (day)
Mean metacercariae per fish ±S.D.
Percentage of metacercarial recovery
1–2 3–4 5–6 7–8 9–10 11–14 15–19 20–24 25–29 30–34 35–39 40–44 45–49 50–54 55–59 60–64
0.7 ± 1.2 0.3 ± 0.8 0.1 ± 0.2 0.2 ± 0.4 0 0.4 ± 0.8 0.4 ± 0.7 0.3 ± 0.4 1.3 ± 1.9 7.8 ± 5.7 3.9 ± 4.2 5.2 ± 6.1 9.5 ± 4.8 6.7 ± 3.4 8.4 ± 7.3 7.7 ± 4.7
1.3 0.6 0.1 0.4 0 0.8 0.7 0.5 2.5 15.6 7.7 10.5 19 13.3 16.8 15.4
width could be measured but not be weighed until the age-group of 25–29 days old. The infection with O. viverrini was found in all agegroups except in the 9 days old. Mean metacercarial recovery in age-groups 1–29 days old was 0–2.5% which increased to 15.6% in age-group 30–34 days old. The percentage of infected fish in different age-groups varied from 0 to 100%, from no infection in the group of 9–10 days old (even after repeating the experiments twice), and 100% in age-groups of 30–34, 45–49, 50–54, 55–59 and 60–64. The percentage of metacercarial recovery in different age-groups varied from 0 to 56%. The lowest intensity of the infection of 0.05 metacercariae was in age-group of 5 days, and the highest intensity of 9.5 metacercariae was in age-group of 45–49 days (Table 1). The width of each fish in this study was nearly the same. Thus, only length of fish was analyzed to determine whether there was an association with O. viverrini metacercarial recovery. Larger fish were found with increasing mean metacercarial recovery with a peak in size-group 16–20 mm (19.86%, 9.9 of 50). The likelihood ratio test 2 revealed that fish age was a significant predictor ( ¯ LR = 954.72, df = 1, P < 0.001). The rate ratio from the Poisson regression was 1.054 (95%CI: 1.050–1.058) suggesting that for every additional fish
Fig. 2. Means and 95% confidence intervals for proportion of metacercariae recovered for different levels of cercarial infection dosage.
Percentage of infected fish 7(35) 3(15) 1(5) 4(20) 0 5(25) 5(25) 5(25) 9(45) 20(100) 12(60) 17(85) 20(100) 20(100) 20(100) 20(100)
ages, the rate of O. viverrini metacercarial recovery increased by 5.4%. The Poisson regression model for the outcome O. viverrini metacercariae recovered regressed against fish size class was 2 significant ( ¯ LR = 1204.84, df = 5, P < 0.001). When analyses the relative ratio of metacercarial recovery rate with fish size in 4.1–8.0 mm size class of fish did not differ from the smallest fish class (RR = 1.18, 95%CI = 0.68–2.04). However, the rate of O. viverrini metacercarial recovery was both significantly (all P < 0.001) and substantially higher for all of the large fish size groups (all fish >8.1 mm) relative to the smallest fish size group (0–4.0 mm). The relative ratio of O. viverrini metacercarial recovery in the larger fish was revealed to be at least 19.94 times of the smallest fish group. In addition, the fish with low metacercarial recovery were found to have ingested cercarial remnants in their intestinal tracts (Fig. 3 and Table 2).
Fig. 3. Partial digested O. viverrini cercariae in the intestine in low infection rate B. gonionotus fish. I: intestinal tract of fish, body (B), and tail (T) of O. viverrini cercariae. Scale bar = 20 m.
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Table 2 The analyses of O. viverrini metacercarial recovered by size-groups of B. gonionotus as relative ratio of recovery rate with 95% confidence interval. Fish size (mm) 0–4.0 4.1–8.0 8.1–12.0 12.1–16.0 16.1–20.0 20.1–24.0 *
N
Mean of metacercaria per fish ±S.D
92 88 54 82 14 10
0.3 0.3 5.2 6.4 9.9 5.4
± ± ± ± ± ±
0.7 0.6 5.3 5.7 4.6 3.2
Relative ratio of recovery rate 1.0 (reference) 1.2 19.9* 24.7* 38.1* 20.7*
95%CI – 0.7–2.0 13.2–30.3 16.4–37.2 24.7–58.7 12.8–33.5
Highly significant (P < 0.001).
3.3. The distribution of O. viverrini metacercariae in infected B. gonionotus fingerlings The distribution of metacercariae in infected B. gonionotus fingerlings were mostly found in the body (81.4%), followed by the tail (6.9%) and head region (9.9%), but rarely found in gills (1.6%) and fins (0.3%). From our observation, cercariae penetrated predominantly passed through the fin membrane and did not remain in the fin. 4. Discussion The association between dosages of O. viverrini cercariae and subsequent metacercarial recovery was investigated in our studies. We found that with increasing infection dosages of O. viverrini cercariae a significantly increased numbers of metacercariae were recovered from B. gonionotus fingerlings. Our results are in agreement with Höglund (1995) who reported that Oncorhynchus mykiss cercarial density affected the transmission of the parasite to Diplostomum spathaceum fish. However, Boerlage et al. (2011) did not find parapleurolophocercous cercarial dosages associated with number of metacercariae recovered (Haplorchis and Centrocestus sp.). There was an effect, however on increasing of undeveloped metacercariae in Cyprinus carpio. Similar results were found by Komiya (1966) were undeveloped metacercariae corresponded to descriptions of the early metacercarial stage of Clonorchis sinensis which were not found in control group (non-infection of fish). We have shown that early stage B. gonionotus fry increased their size rapidly in age-groups of 1–2 to 11–14 days old after that they grew slowlier. We found that O. viverrini metacercarial recovery significantly increased with increasing age and size of B. gonionotus. Our study found that B. gonionotus fry could be infected with O. viverrini cercariae in laboratory but the infection rate was low. This result was different from Thien et al. (2009) who did not find fish-borne zoonotic trematode (FZT) metacercariae in fish fry from hatchery farms in the Mekong Delta which was due to the use of a cement tank for breeding, containing of filtrated water, and no infected snails. Höglund (1995) found that Oncorhynchus mykiss fish size and Diplostomum spathaceum cercarial density prevail in parasite transmission from snails to fish both in the field and laboratory. Boerlage et al. (2011) reported that small sized fish (1 g) had higher percentage of metacercariae and attack rates of cercariae than medium (25 g) and large fish (45 g). Our results are the first report to demonstrate the metacercarial recovery rate in ≤1 g body weight of fish. In the present study, the ability O. viverrini to infect fish increased with increasing age and size of fish. Possible reasons for higher attack rates in different size of fish might be attributed to differences in the quantity or composition of biochemical compounds excreted by fish that might change when fish increase in size and age (Haas, 1992). Also, body surface increased in older age-groups which can provide more surface area for cercariae to attach. However, O. viverrini infection was also related to the behavior and morphological changes of fish in each age-group, including feeding, moving or swimming, and attachment area for cercariae. Our results suggest that the size of B.
gonionotus fish influences the intensity of O. viverrini infection due to the availability of an increased area of attachment for cercariae. Additionally, we found that some parts of O. viverrini cercariae were in the digestive tract of B. gonionotus fry in the low infection groups. When B. gonionotus fry diminished their food-reserve yolk they ingested zooplanktons as their food. O. viverrini cercariae have a similar size to zooplanktons which were also ingested by B. gonionotus fry. This was especially the case in the group of 9–10 days old after yolk diminished who were observed to be actively searching for food and feeding resulting in no infection. The highest percentage of O. viverrini metacercarial recovery was found in the B. gonionotus fish body, a large area of B. gonionotus for cercarial attachment more than other parts of B. gonionotus fish. The fins of B. gonionotus were rarely infected with cercariae because the membrane in fry and fingerlings is very thin which resulted in cercariae penetration passing through the fin membranes and not remaining within the fin. In addition, constant tail movement hinders cercarial attachment. During B. gonionotus fish respiration, water containing cercariae passed the mouth through the gills and only a few cercariae were able to attach to gills or the head of B. gonionotus fish and to develop to metacercariae. In the conclusion, increasing dosage of O. viverrini cercariae, older age and bigger size of B. gonionotus fish produced a higher risk of infection in B. gonionotus. The results showed that cyprinid fish of B. gonionotus was initially infected in the early stage of their development with dependence of dose of cercarial infection. Acknowledgments We would like to thank the higher education research promotion and national research university project of Thailand, office of the higher education commission, though the health cluster (SHePGMS), Khon Kaen University. We wish to acknowledge assistance and English editing by the Faculty of Medicine, Publication Clinic, Khon Kaen University. References Boerlage, A.S., Graat, E.A.M., Verreth, J.A., de Jong, M.C.M., 2011. Effect of fish size on transmission of fish-borne trematodes (Heterophyidae) to common carps (Cyprinus carpio) and implications for intervention. Aquaculture 321, 179–184. Brockelman, W.Y., Upatham, E.S., Viyanant, V., Ardsungnoen, S., Chantanawat, R., 1986. Field studies on the transmission of the human liver fluke, Opisthorchis viverrini, in northeast Thailand: population changes of the snail intermediate host. Int. J. Parasitol. 16, 545–552. Dang, T.C., Yajima, A., Nguyen, V.K., Montresor, A., 2008. Prevalence, intensity and risk factors for clonorchiasis and possible use of questionnaires to detect individuals at risk in northern Vietnam. Trans. R. Soc. Trop. Med. Hyg. 102, 1263–1268. Frandsen, F., Christensen, N.O., 1984. An introductory guide to the identification of cercariae from African freshwater snails with special reference to cercariae of trematode species of medical and veterinary importance. Acta Trop. 41, 181–203. Haas, W., 1992. Physiological analysis of cercarial behavior. J. Parasitol. 78, 243–255. Höglund, J., 1995. Experiments on second intermediate fish host related cercarial transmission of the eyefluke Diplostomum spathaceum into rainbow trout (Oncorhynchus mykiss). Folia Parasitol. 42, 49–53. IARC, 2011. A review of human carcinogens (Biological agents: Opisthorchis viverrini and Clonorchis sinensis). IARC Monogr. Eval. Carcinog. Risks Hum. 100(B), 351–376.
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Experimental infection of Opisthorchis viverrini cercariae to the cyprinid fish, Barbonymus gonionotus Chalermlap Donthaisong a , Patpicha Arunsan a , Kulwadee Suwannatrai a , Sattrachai Prasopdee a , Jutharat Kulsantiwong b , Sutee Wongmaneeprateep c , Apiporn Suwannatrai a , Smarn Tesana a,∗ a
Food-borne Parasite Research Group, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Department of Biology, Faculty of Science, Udon Thani Rajabhat University, Udon Thani 41000, Thailand c Department of Fisheries, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand b
a r t i c l e
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Article history: Received 22 October 2013 Received in revised form 21 March 2014 Accepted 19 April 2014 Available online 26 April 2014 Keywords: Opisthorchis viverrini cercariae Metacercariae Infectivity Barbonymus gonionotus
a b s t r a c t Opisthorchis viverrini is an important public health problem, a major cause of cholangiocarcinoma in the Greater Mekong subregion including Lao PDR, Cambodia, Vietnam and Thailand. Humans acquire the infection by consumption of raw, fermented or partially cooked freshwater cyprinid fish containing infective metacercariae. This study examined the effect of cercarial infection dosage (25–200 cercariae), age (1–60 day) and size (1–24 mm) of Barbonymus gonionotus fish on infection success of O. viverrini cercariae. Additionally, the site of cyst formation of O. viverrini cercariae and subsequent development to the metacercariae was examined. The result showed that cercarial infection dose (F4,95 = 8.52, P < 0.001) age 2 2 (P < 0.001, ¯ LR = 954.72) and size (P < 0.001, ¯ LR = 1204.84) were significantly associated with number of O. viverrini metacercariae recovery with in every additional fish age, the O. viverrini metacercarial recovery rate increased by 5.4% (P < 0.001). Most metacercariae were found in the body of the fish (81.4%). We concluded that O. viverrini cercarial infection dosage, and age and size of fish were important determinants for a successful infection to fish. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Fish-borne trematodiasis of Opisthorchiasis and clonorchiasis caused by the liver flukes in the family Opisthorchiidae: Opisthorchis felineus, O. viverrini and Clonorchis sinensis which are endemic in Siberia and East Europe, Southeast, and East Asia, respectively. O. viverrini and C. sinensis have been classified as group 1 carcinogens, which are a risk factor for inducing cholangiocarcinoma in infected humans. O. viverrini is mostly found in endemic areas in the Greater Mekong subregion including Lao PDR, Cambodia, Vietnam and Thailand (Jongsuksuntigul and Imsomboon, 2003; Sayasone et al., 2007; Dang et al., 2008; IARC, 2011). O. viverrini has a complex life cycle, involving bithynid snails as the first intermediate host, cyprinid fish as the second intermediate host, and cats, dogs, and humans as final hosts (Sithithaworn and HaswellElkins, 2003; Upatham and Viyanant, 2003). Cyprinid fish infected with O. viverrini (90.4%) have 800 folds higher prevalence than in
∗ Corresponding author. Tel.: +66 43 363434; fax: +66 43 202475. E-mail addresses: smarn [email protected], [email protected] (S. Tesana). http://dx.doi.org/10.1016/j.actatropica.2014.04.022 0001-706X/© 2014 Elsevier B.V. All rights reserved.
bithynid snails (0.11%) in the same endemic area (Vichasri et al., 1982; Brockelman et al., 1986). Cyprinid fish accumulate infectious metacercariae and enhance transmission of this parasite to the final host. Many species of cyprinid fish serve as intermediate hosts of O. viverrini. High infection rates have been reported in many species of native fish such as Amblyrhynchichthys truncates (100.0%), Puntius brevis (92.0%), Hampala dispar (44.4–100.0%), Puntioplites falcifer (33.3%), P. proctzysron (26.8%), Esomus metallicus (75.0%), Cyclocheilichthys armatus (43.1–100%), C. repasson (58.5%), Dangila lineata (69.6%), Henicorhynchus lineatus (42.9%), Osteochilus waandersii (30.5%), Baybonymus schwanenfeldii (66.0%), Thynnichthys thynnoides (59.7%), Labiobarbus siamensis (51.3%) and Mystacoleucus marginatus (50.0%) (Rim et al., 2008; Manivong et al., 2009; Touch et al., 2013). The highest density of O. viverrini metacercariae was in the head region, followed by the caudal fin and muscles of fins and the lowest was found in the anal fin (Tesana et al., 1985). High rate of fish infection were found in the late rainy season continuing to the cold season (July to January), while low burdens rate were found in the hot-dry season (April–June) (Sithithaworn et al., 1997). Barbonymus gonionotus (silver barb) is a native cyprinid species which is commonly reared in freshwater aquaculture. It is
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widely consumed in Thailand, Lao PDR and Cambodia. The maximum length of mature B. gonionotus is 50 cm. B. gonionotus in natural water resources has been shown to be infected with various parasite species such as Haplorchis taichui (80.3%), Centrocestus spp. (11.3%), and O. viverrini (1.0%) in Chiang Mai Province, northern Thailand, has been found (Sukontason et al., 1999) and 16.1% of O. viverrini infected B. gonionotus in Cambodia (Touch et al., 2013). The present study examined of affect O. viverrini cercarial infection namely, cercarial dosages, and age and size of fish host as determinants. 2. Materials and methods This study was approved by the animal ethics committee of Khon Kaen University, Khon Kaen Province, Thailand (Ethical Clearance No. AEKKU25/2555). 2.1. O. viverrini cercarial preparation Bithynia siamensis goniomphalos snails were collected manually in various freshwater bodies such as rice paddy fields, ponds, canals and roadside ditches from Khon Kaen Province, Thailand. They were transported to the laboratory of Khon Kaen University, Khon Kaen in plastic bags. These, they were washed with dechlorinated tap-water and examined for O. viverrini infection using the cercarial shedding method (stimulated by electric light in the daytime for 3 h). When the cercariae were released from snails, they were identified under light microscope using morphological characteristics of tobacco-pipe shaped when briefly hanged head down or laid on the bottom, a pair of eye-spots and long tail with fin on both lateral sides (Wykoff et al., 1965; Schell, 1970; Frandsen and Christensen, 1984). O. viverrini cercariae were confirmed using specific PCR primers (forward primer OV-6F 5 -CTG AAT CTC TCG TTT GTT CA-3 ; reverse primer OV-6R 5 -GTT CCA GGT GAG TCT CTC TA-3 , Wongratanacheewin et al., 2001). 2.2. Preparation of fish samples Females and males of B. gonionotus were obtained from the stock farm of the Department of Fisheries, Faculty of Agriculture, Khon Kaen University. Spawns of B. gonionotus were induced by intramuscularly injecting in both sexes with synthetic hormones (Luteinizing Hormone-Releasing Hormone analog, LHRHa). In additions, females were injected intramuscularly with 20 g of Buserelin acetate (Suprefact® , nasal solution, Aventis Pharma Deutschland GmbH, Frankfurt am Main, Germany) mixed with 5 mg of domperidone maleate (Motilium® , tablet, Janssen Korea Ltd., Kyunggi-Do, Republic of Korea) per kg body weight. Males were injected with 10 g of Suprefact mixed with 5 mg of Motilium per kg body weight. Females and males were stocked together in a spawning cement tank at a ratio of 1:2 and fed with floating fish food. The eggs were fertilized naturally. After 8–12 h of fertilization the eggs were hatched at a water temperature of 26–28 ◦ C. Fish fry were transported to the laboratory, reared in aquaria and fed with soft husk mixed fish meal under standard conditions to maintain them parasite free. 2.3. Experimental infection in B. gonionotus 2.3.1. Infection process of cercarial infection dosage One hundred and twenty B. gonionotus fingerlings (30 days old, 0.5–1.0 cm in length) were divided into 6 groups (20 per group). Group 1, 2, 3, 4, 5 and 6 were exposed to 0 (control), 25, 50, 100, 150 and 200 cercariae (fresh shedding) per fish for 24 h, respectively. The procedure of infection for each fingerling was done in 5 ml dechlorinated tap-water in a plastic cup (3 cm diameter, and 2.5 cm
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height) at room temperature (25 ± 3 ◦ C) and given the electric light throughout the infection period. Thus, the fingerlings were killed in 4 ◦ C water. Then, they were observed for O. viverrini infection by compressed between two slides. The number of O. viverrini metacercariae was counted under a light microscope (40 magnification), and recorded. 2.3.2. Infection success in age- and size-groups of fish Fish were divided into 16 age-groups of 1–2, 3–4, 5–6, 7–8, 9–10, 11–14, 15–19, 20–24, 25–29, 30–34, 35–39, 40–44, 45–49, 50–54, 55–59 and 60–64 days (20 fish per age-group, total of 320, Fig. 1). From day 1 to 8 after hatching fish fry presented with yolk sac. On day 9 the yolks disappeared and they started active feeding. All fry was exposed to 50 O. viverrini cercariae in the conditions described above. After 24 h of exposure, the fish were killed in 4 ◦ C water and their length, width and weight were recorded. They were examined on O. viverrini infection by compressing these between two slides. The number of O. viverrini metacercariae were counted under a light microscope (40 magnification) and recorded. 2.3.3. Fish body part of O. viverrini metacercarial infection Forty B. gonionotus fingerlings (30 days old, 0.5–1.0 cm in length) were exposed to 100 O. viverrini cercariae per fish in the same conditions as described above. After 24 h of exposure fish were killed in 4 ◦ C water. Then, each part of the fingerlings (head, gills, body, fins, and tail) was examined on the present of O. viverrini metacercariae. The fingerlings were compressed between two slides and the number of O. viverrini metacercariae were counted in each body part of fingerling under a light microscope (40×). 2.4. Data analyses Statistical analyses was conducted with SPSS software version 17.0. The association between cercarial infection dosages in each fish group and the number of O. viverrini metacercariae in B. gonionotus fingerlings was analyzed using post hoc Tukey’s HSD tests in ANOVA. The association of fish age-groups and size-groups and number of O. viverrini metacercariae was examined with Poisson regression. O. viverrini metacercariae counts in fish were not normally distributed. 3. Results 3.1. Infection success of cercarial infection dosage O. viverrini metacercariae were found in all B. gonionotus fingerlings except in the group exposed to 25 cercariae where 19 of 20 (95%) B. gonionotus fingerlings were infected. The highest metacercarial recovery was in group of 200 cercariae infection dose found in 1 of 20 B. gonionotus fingerling (87.5%, 175 metacercariae from 200 cercariae infection dose). The average metacercariae recovery was also high in cercariae infection dose of 200 cercariae gave 42.9% recovery whereas a low dose of 25 cercariae resulted in 13.8% (3.4 of 25) metacercarial recovery. Analysis of variance revealed significant differences between groups (F4,95 = 8.527, P < 0.001). Post hoc comparisons (Tukey’s HSD) showed the lowest (25 cercariae) differed from the highest two groups (150 and 200 cercariae) (diff25VS150 = −0.17, P = 0.028, diff25VS200 = −0.29, P < 0.001), and the second level of dosage (50 cercariae) differed from the highest dosage group (diff50VS200 = −0.26, P < 0.001, Fig. 2). 3.2. Infection success in age- and size-groups of fish We found that fish fry developed and increased their size grew rapidly in the early period of life and reached a plateau after 50 days. The fish fry at early development was small. Only the length and
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Fig. 1. B. gonionotus fry. Age-group of 1–2 days of age (A), 9–10 days of age (B), and 55–59 days of age (C). Scale bars: A&B = 1 mm, C = 5 mm.
Table 1 Percentages of O. viverrini metacercarial recovery in each age-group of B. gonionotus (n = 20). Age-groups of fish (day)
Mean metacercariae per fish ±S.D.
Percentage of metacercarial recovery
1–2 3–4 5–6 7–8 9–10 11–14 15–19 20–24 25–29 30–34 35–39 40–44 45–49 50–54 55–59 60–64
0.7 ± 1.2 0.3 ± 0.8 0.1 ± 0.2 0.2 ± 0.4 0 0.4 ± 0.8 0.4 ± 0.7 0.3 ± 0.4 1.3 ± 1.9 7.8 ± 5.7 3.9 ± 4.2 5.2 ± 6.1 9.5 ± 4.8 6.7 ± 3.4 8.4 ± 7.3 7.7 ± 4.7
1.3 0.6 0.1 0.4 0 0.8 0.7 0.5 2.5 15.6 7.7 10.5 19 13.3 16.8 15.4
width could be measured but not be weighed until the age-group of 25–29 days old. The infection with O. viverrini was found in all agegroups except in the 9 days old. Mean metacercarial recovery in age-groups 1–29 days old was 0–2.5% which increased to 15.6% in age-group 30–34 days old. The percentage of infected fish in different age-groups varied from 0 to 100%, from no infection in the group of 9–10 days old (even after repeating the experiments twice), and 100% in age-groups of 30–34, 45–49, 50–54, 55–59 and 60–64. The percentage of metacercarial recovery in different age-groups varied from 0 to 56%. The lowest intensity of the infection of 0.05 metacercariae was in age-group of 5 days, and the highest intensity of 9.5 metacercariae was in age-group of 45–49 days (Table 1). The width of each fish in this study was nearly the same. Thus, only length of fish was analyzed to determine whether there was an association with O. viverrini metacercarial recovery. Larger fish were found with increasing mean metacercarial recovery with a peak in size-group 16–20 mm (19.86%, 9.9 of 50). The likelihood ratio test 2 revealed that fish age was a significant predictor ( ¯ LR = 954.72, df = 1, P < 0.001). The rate ratio from the Poisson regression was 1.054 (95%CI: 1.050–1.058) suggesting that for every additional fish
Fig. 2. Means and 95% confidence intervals for proportion of metacercariae recovered for different levels of cercarial infection dosage.
Percentage of infected fish 7(35) 3(15) 1(5) 4(20) 0 5(25) 5(25) 5(25) 9(45) 20(100) 12(60) 17(85) 20(100) 20(100) 20(100) 20(100)
ages, the rate of O. viverrini metacercarial recovery increased by 5.4%. The Poisson regression model for the outcome O. viverrini metacercariae recovered regressed against fish size class was 2 significant ( ¯ LR = 1204.84, df = 5, P < 0.001). When analyses the relative ratio of metacercarial recovery rate with fish size in 4.1–8.0 mm size class of fish did not differ from the smallest fish class (RR = 1.18, 95%CI = 0.68–2.04). However, the rate of O. viverrini metacercarial recovery was both significantly (all P < 0.001) and substantially higher for all of the large fish size groups (all fish >8.1 mm) relative to the smallest fish size group (0–4.0 mm). The relative ratio of O. viverrini metacercarial recovery in the larger fish was revealed to be at least 19.94 times of the smallest fish group. In addition, the fish with low metacercarial recovery were found to have ingested cercarial remnants in their intestinal tracts (Fig. 3 and Table 2).
Fig. 3. Partial digested O. viverrini cercariae in the intestine in low infection rate B. gonionotus fish. I: intestinal tract of fish, body (B), and tail (T) of O. viverrini cercariae. Scale bar = 20 m.
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Table 2 The analyses of O. viverrini metacercarial recovered by size-groups of B. gonionotus as relative ratio of recovery rate with 95% confidence interval. Fish size (mm) 0–4.0 4.1–8.0 8.1–12.0 12.1–16.0 16.1–20.0 20.1–24.0 *
N
Mean of metacercaria per fish ±S.D
92 88 54 82 14 10
0.3 0.3 5.2 6.4 9.9 5.4
± ± ± ± ± ±
0.7 0.6 5.3 5.7 4.6 3.2
Relative ratio of recovery rate 1.0 (reference) 1.2 19.9* 24.7* 38.1* 20.7*
95%CI – 0.7–2.0 13.2–30.3 16.4–37.2 24.7–58.7 12.8–33.5
Highly significant (P < 0.001).
3.3. The distribution of O. viverrini metacercariae in infected B. gonionotus fingerlings The distribution of metacercariae in infected B. gonionotus fingerlings were mostly found in the body (81.4%), followed by the tail (6.9%) and head region (9.9%), but rarely found in gills (1.6%) and fins (0.3%). From our observation, cercariae penetrated predominantly passed through the fin membrane and did not remain in the fin. 4. Discussion The association between dosages of O. viverrini cercariae and subsequent metacercarial recovery was investigated in our studies. We found that with increasing infection dosages of O. viverrini cercariae a significantly increased numbers of metacercariae were recovered from B. gonionotus fingerlings. Our results are in agreement with Höglund (1995) who reported that Oncorhynchus mykiss cercarial density affected the transmission of the parasite to Diplostomum spathaceum fish. However, Boerlage et al. (2011) did not find parapleurolophocercous cercarial dosages associated with number of metacercariae recovered (Haplorchis and Centrocestus sp.). There was an effect, however on increasing of undeveloped metacercariae in Cyprinus carpio. Similar results were found by Komiya (1966) were undeveloped metacercariae corresponded to descriptions of the early metacercarial stage of Clonorchis sinensis which were not found in control group (non-infection of fish). We have shown that early stage B. gonionotus fry increased their size rapidly in age-groups of 1–2 to 11–14 days old after that they grew slowlier. We found that O. viverrini metacercarial recovery significantly increased with increasing age and size of B. gonionotus. Our study found that B. gonionotus fry could be infected with O. viverrini cercariae in laboratory but the infection rate was low. This result was different from Thien et al. (2009) who did not find fish-borne zoonotic trematode (FZT) metacercariae in fish fry from hatchery farms in the Mekong Delta which was due to the use of a cement tank for breeding, containing of filtrated water, and no infected snails. Höglund (1995) found that Oncorhynchus mykiss fish size and Diplostomum spathaceum cercarial density prevail in parasite transmission from snails to fish both in the field and laboratory. Boerlage et al. (2011) reported that small sized fish (1 g) had higher percentage of metacercariae and attack rates of cercariae than medium (25 g) and large fish (45 g). Our results are the first report to demonstrate the metacercarial recovery rate in ≤1 g body weight of fish. In the present study, the ability O. viverrini to infect fish increased with increasing age and size of fish. Possible reasons for higher attack rates in different size of fish might be attributed to differences in the quantity or composition of biochemical compounds excreted by fish that might change when fish increase in size and age (Haas, 1992). Also, body surface increased in older age-groups which can provide more surface area for cercariae to attach. However, O. viverrini infection was also related to the behavior and morphological changes of fish in each age-group, including feeding, moving or swimming, and attachment area for cercariae. Our results suggest that the size of B.
gonionotus fish influences the intensity of O. viverrini infection due to the availability of an increased area of attachment for cercariae. Additionally, we found that some parts of O. viverrini cercariae were in the digestive tract of B. gonionotus fry in the low infection groups. When B. gonionotus fry diminished their food-reserve yolk they ingested zooplanktons as their food. O. viverrini cercariae have a similar size to zooplanktons which were also ingested by B. gonionotus fry. This was especially the case in the group of 9–10 days old after yolk diminished who were observed to be actively searching for food and feeding resulting in no infection. The highest percentage of O. viverrini metacercarial recovery was found in the B. gonionotus fish body, a large area of B. gonionotus for cercarial attachment more than other parts of B. gonionotus fish. The fins of B. gonionotus were rarely infected with cercariae because the membrane in fry and fingerlings is very thin which resulted in cercariae penetration passing through the fin membranes and not remaining within the fin. In addition, constant tail movement hinders cercarial attachment. During B. gonionotus fish respiration, water containing cercariae passed the mouth through the gills and only a few cercariae were able to attach to gills or the head of B. gonionotus fish and to develop to metacercariae. In the conclusion, increasing dosage of O. viverrini cercariae, older age and bigger size of B. gonionotus fish produced a higher risk of infection in B. gonionotus. The results showed that cyprinid fish of B. gonionotus was initially infected in the early stage of their development with dependence of dose of cercarial infection. Acknowledgments We would like to thank the higher education research promotion and national research university project of Thailand, office of the higher education commission, though the health cluster (SHePGMS), Khon Kaen University. We wish to acknowledge assistance and English editing by the Faculty of Medicine, Publication Clinic, Khon Kaen University. References Boerlage, A.S., Graat, E.A.M., Verreth, J.A., de Jong, M.C.M., 2011. Effect of fish size on transmission of fish-borne trematodes (Heterophyidae) to common carps (Cyprinus carpio) and implications for intervention. Aquaculture 321, 179–184. Brockelman, W.Y., Upatham, E.S., Viyanant, V., Ardsungnoen, S., Chantanawat, R., 1986. Field studies on the transmission of the human liver fluke, Opisthorchis viverrini, in northeast Thailand: population changes of the snail intermediate host. Int. J. Parasitol. 16, 545–552. Dang, T.C., Yajima, A., Nguyen, V.K., Montresor, A., 2008. Prevalence, intensity and risk factors for clonorchiasis and possible use of questionnaires to detect individuals at risk in northern Vietnam. Trans. R. Soc. Trop. Med. Hyg. 102, 1263–1268. Frandsen, F., Christensen, N.O., 1984. An introductory guide to the identification of cercariae from African freshwater snails with special reference to cercariae of trematode species of medical and veterinary importance. Acta Trop. 41, 181–203. Haas, W., 1992. Physiological analysis of cercarial behavior. J. Parasitol. 78, 243–255. Höglund, J., 1995. Experiments on second intermediate fish host related cercarial transmission of the eyefluke Diplostomum spathaceum into rainbow trout (Oncorhynchus mykiss). Folia Parasitol. 42, 49–53. IARC, 2011. A review of human carcinogens (Biological agents: Opisthorchis viverrini and Clonorchis sinensis). IARC Monogr. Eval. Carcinog. Risks Hum. 100(B), 351–376.
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