한수지 51(1), 47-53, 2018 Original Article Korean J Fish Aquat Sci 51(1),47-53,2018 2016 년소양호에서채집한자연산잉어과어류와갑각류의법정전염병및기생충성질병모니터링 문성희 1 허준욱 2 차승주 3 황성돈 4 손맹현 4 권준영 1 권세련 1,5 * 1 선문대학교수산생명의학과, 2 생물모니터링센터, 3 국립수산과학원내수면양식연구센터, 4 국립수산과학원수산방역과, 5 유전체기반바이오 IT 융합연구소 Monitoring Pathogen Infection of Freshwater Cyprinid Fish and Crustacean in Soyang Lake in 2016 Seong Hee Mun 1, Jun Wook Hur 2, Seung Joo Cha 3, Seong Don Hwang 4, Maeng-Hyun Son 4, Joon Yeong Kwon 1 and Se Ryun Kwon 1,5 * 1 Department of Aquatic Life Medical Sciences, Sunmoon University, Asan 31460, Korea 2 Bio-Monitoring Center, Sejoung 30121, Korea 3 Inland Aquaculture Research Center, National Institute of Fisheries Sciences, Busan 51688, Korea 4 Aquatic Disease Control Division, National Institute of Fisheries Sciences, Busan 46083, Korea 5 Genome-based BioIT Convergence Institute, Asan 31460, Korea The presence of pathogens in wild fish is a potential threat to the fish being raised nearby fish farm. Surveillance of these pathogens in the wild is, thus, highly important to keep fish in the farm safe from serious communicable diseases. Fish and crustacean were sampled 4 times at Soyang Lake in 2016. Pathogens for five reportable communicable diseases including KHVD (koi herpesvirus disease), SVC (spring viraemia of carp), EUS (epizootic ulcerative syndrome) and WSD (white spot disease), and parasites were investigated. In Soyang Lake, pale chub Zacco platypus was the most abundant. Forty seven pale chub, 8 Korea piscivorous chub Opsarichthys uncirostris, 3 oily shiner Sarcocheilichthys variegatus wakiyae and 2 crucian carp Cyprinus carassius were investigated for detection of target diseases of fish. Seventeen Caridina denticulata denticulata, 12 Oriental river prawn Macrobrachium nipponense and 1 Chinese mitten crab Eriocheir sinensis were used for detection of WSD. As the result, reportable communicable diseases were not detected in all the freshwater animals. Dactylogyrus was detected in Korea piscivorous chub. Copepoda was also detected in pale chub and Korea piscivorous chub. Metacercaria was detected in the pale chub sampled in September and October. Those were not metacercaria of liver fluke Clonorchis sinensis. Key words: Soyang Lake, Cyprinid fish, wild crustacean, reportable communicable diseases, parasite 서론... 2007 3 (WSD, white spot disease) (WSSV, white spot syndrome virus) (Baumgartner et al., 2009). 1997 1998, (SVCV, spring viremia of carp virus) (Miller et al., 2007). 2002 North Carolina https://doi.org/10.5657/kfas.2018.0047 Korean J Fish Aquat Sci 51(1) 47-53, February 2018 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 23 November 2017; Revised 21 December 2017; Accepted 10 January 2018 *Corresponding author: Tel: +82. 41. 530. 2289 Fax: +82. 41. 530. 2917 E-mail address: srkwon@sunmoon.ac.kr Copyright 2018 The Korean Society of Fisheries and Aquatic Science 47 pissn:0374-8111, eissn:2287-8815
48 문성희ㆍ허준욱ㆍ차승주ㆍ황성돈ㆍ손맹현ㆍ권준영ㆍ권세련 SVC (spring viremia of carp) (Goodwin, 2002), 2004 Wisconsin SVCV (Audrey et al., 2004)., 227 (KHV, koi herpes virus), (Fujioka et al., 2015). Fabian et al. (2013) KHVD (koi herpesvirus disease) real-time PCR KHV genome, KHV. 1998 KHV.. 2010 6 SVC, 2016 5,, SVC.,,.. 1968,,,, metacercaria (Lee, 1968). 1976 21 10 metacercaria,, scalet carp, (Choi, 1976). 2003 (Park et al., 2004). 17 14, 13,,,, (Yu et al., 1994). 2014 2015 11.1% (Kim et al., 2016). 2016 5 10 4, KHVD, SVC EUS (epizootic ulcerative syndrome) WSD.. 재료및방법 생물채집방법 (cast net,, 7 7 mm), (kick net,, 5 5 mm) (fyke, 5 5 mm). 200 m. 20, 60., 24. ( ),.. 채집생물의군집분석 (fish fauna), (dominant species) (subdominant species). (community analysis) (fish species) (DI, dominance index), (H, diversity index), (E, evenness) (R, richness).. (McNaughton, 1967). D = ni/n (D, ; N, ; ni, I ), Margalef (1958) Shannon-Weaver function (Pielou, 1975). H = - Pi Ln Pi (Pi = ni/n)
소양호잉어과어류와갑각류의질병모니터링 49, Pielou (1975). E = H'/ln(S) (E, ; H', ; S, ). (S). Margalef (1958). 법정전염병조사 R = (S-1)ln (N) (R, ; S, ; N, ),, ( ),.. PCR. SVC KHV 2, EUS, WSSV. PCR 3 pooling, 5, 3. -80. PCR. RNA TRIsure (Bioline, USA), DNA high pure PCR template preparation kit (ROCHE, Germany). protocol. PCR SVC BIONEER rocketscript RT-PCR kit hotstart PCR kit, KHV EUS Promega Go Taq green mastermix, WSSV Table 1. PCR primer sets and condition for 5 reportable communicable diseases Primer PCR condition Product size SVC KHVD EUS 1 st PCR 2 nd PCR SVCV F1 5 -TCTTGGAGCCAAATAGCTCARRTC-3 (95 1 min, 55 1 min, 72 1 SVCV R2 5 -AGATGGTATGGACCCCAATACATHACNCAY-3 min)x35 cycles - 72 10 min SVCV F1 5 -TCTTGGAGCCAAATAGCTCARRTC-3 (95 1 min, 55 1 min, 72 1 SVCV R4 5 -CTGGGGTTTCCNCCTCAAAGYTGY-3 min)x35 cycles - 72 10 min Bercovier TK TK F 5 -GGGTTACCTGTACGAG-3 94 5 min - (95 1 min, 55 1 min, 72 1 min)x40 cycles - 72 TK R 5 -CACCCAGTAGATTATGC-3 10 min Gray Sph Method 1 Method 2 Method 3 GS F 5 -GACACCACATCTGCAAGGAG-3 94 30 sec - (94 30 sec, 63 GS R 5 -GACACATGTTACAATGGTCGC-3 30 sec, 72 30 sec)x40 cycles - 72 7 min Ainvad-2F 5 -TCATTGTGAGTGAAACGGTG-3 95 2 min - (95 30 sec, 56 45 sec, 72 2.5 min)x35 cycles - Ainvad-ITSR1 5 -GGCTAAGGTTTCAGTATGTAG-3 72 5 min ITS11 5 -GCCGAAGTTTCGCAAGAAAC-3 95 5 min - (94 30 sec, 65 30 ITS23 5 -CGTATAGACACAAGCACACCA-3 sec, 72 1 min)x25 cycles - 72 5 min BO73 5 -CTTGTGCTGAGCTCACACTC-3 96 5 min - (96 1 min, 58 1 BO639 5 -ACACCAGATTACACTATCTC-3 min, 72 1 min)x35 cycles - 72 5 min 714 bp 606 bp 409 bp 292 bp 234 bp 550 bp 564 bp WSD 1 st PCR 2 nd PCR 146F1 5 -ACTACTAACTTCAGCCTATCTAG-3 (94 4 min, 55 1 min, 72 2 min)- (94 1 min, 55 1 min, 1447 bp 146R1 5 -TAATGCGGGTGTAATGTTCTTACGA-3 72 2 min)x39 cycles - 72 5 min 146F2 5 -GTAACTGCCCCTTCCATCTCCA-3 (94 4 min, 55 1 min, 72 2 min)- (94 1 min, 55 1 min, 941 bp 146R2 5 -TACGGCAGCTGCTGCACCTTGT-3 72 2 min)x39 cycles - 72 5 min SVC, spring viremia of carp; KHVD, koi herpesvirus disease; EUS, epizootic ulcerative syndrome; WSD, white spot disease.
50 문성희ㆍ허준욱ㆍ차승주ㆍ황성돈ㆍ손맹현ㆍ권준영ㆍ권세련 Takara EX Taq. PCR mixture protocol. primer PCR Table 1. 기생충성질병조사,., cyst,.,,,,., 37. PS buffer (0.85% NaCl), metacercaria. Metacercaria PCR. primer PCR Cho et al. (2013) (Table 2). 결과및고찰 어류상및상대풍부도 (38.02 N, 127.57 E) 8 17 569. (family) (species) (Cyprinidae) 8, (Bagridae) (Centrarchidae) 2. (Anguillidae), (Siluridae), (Osmeridae), (Centropomidae) (Gobiidae) 1. 569 306 53.8%. 136 23.9%, 13.2% (75 ), 4.9% (28 ), 2.1%, 1.1%, Species Zacco platypus Lepomis macrochirus Hypomesus nipponensis Opsarichthys uncirostris Pseudobagrus fulvidraco Tridentiger brevispinis S. nigripinnis morii Micropterus salmoides Carassius auratus Silurus asotus Leiocassis ussuriensis Hemiculter eigenmanni siniperca scherzeri S.chankaensis tsuchigae Hemibarbus labeo Cyprinus carpio Anguilla japonica 4.9 3.9 2.1 2.1 1.9 1.9 1.1 1.1 1.1 0.9 0.4 0.2 0.2 0.2 13.2 22.0 43.1 0 10 20 30 40 50 Relative abundance (%) Fig. 1. Relative richness index of fishes sampled from the Soyang Lake. Table 2. PCR primer sets and condition for detection of Clonorchis sinensis Primer PCR condition Product size Method 1 RT5-1 5'-ACTTCATCGAGTCATTGGTCGT-3' 98 10 min - (95 30 sec, 54 1 min, RH3 5'-CGTACTGTAACGCGTTTGTGCA-3' 72 1 min)x30 cycles - 72 5 min 929 bp Method 2 Int5-2 5'-GGACATGCTAACTTCCCTCTCA-3' 98 10 min - (95 30 sec, 54 1 min, Int3-1 5'-CGTGGTGAATATCGATCAGGTT-3' 72 1 min)x30 cycles - 72 5 min 1349 bp
소양호잉어과어류와갑각류의질병모니터링 51 0.9%, 0.2%. (RA, relative abundance) (Fig. 1), (Zacco platypus) 245 43.1%, 125 22.0%. (Hypomesus nipponensis) 13.2% (75 ), (Opsarichthys uncirostris amurensis) 4.9% (28 ), (Pseudobagrus fulvidraco) 3.9% (22 ), (Tridentiger brevispinis) (Sarcocheilichthys nigripinnis morii) 2.1% (12 ), (Carassius auratus) 1.9% (11 ). (Byeon et al., 1997; Choi et al., 2003; Byeon, 2015),, 20. 10.0%. Byeon (2015).. 3, Macrobrachium nipponense 23 62.2% Neocardina denticulata 13 35.1%. 법정전염병감염현황. Table 3. 2. SVC, KHVD, EUS WSD, specific primer PCR RT-PCR (data not shown). 기생충감염현황 1 4. 1 Dactylogyrus sp. 1-2 (Fig. 2). 3 4 metacercaria. metacercaria PCR band (data not shown). metacercaria, metacercaria. Metagonimus sp., Echinostoma sp., Centrocestus armarus metacercaria PCR. Park et al. (2004) 2003 1 Table 3. Investigated fishes and crustaceans for detection of target diseases at Soyang Lake in 2016 Date of survey Species Number Mean total length (cm) Mean body weight (g) Pale chub (Zacco platypus) 6 13.3 17.8 1st 2016.05.04 Korea piscivorous chub (Opsarichthys uncirostris) 6 11.4 11.1 Oily shiner (Sarcocheilichthys variegatus wakiyae) 3 10.4 10.8 Pale chub (Z. platypus) 15 12.8 22.8 2nd 2016.06.24 Chinese mitten crab (Eriocheir sinensis) 1 5.0 51.7 Oriental river prawn (Macrobrachium nipponense) 11 3.9 0.7 Pale chub (Z. platypus) 13 12.0 16.8 3rd 2016.09.08 Crucian carp (Carassius auratus) 2 16.2 59.5 Caridina denticulata denticulate 8 0.7 0.1 Oriental river prawn (M. nipponense) 1 2.6 1.8 Pale chub (Z. platypus) 13 13.8 23.3 4th 2016.10.06 Korea piscivorous chub (O. uncirostris) 2 17.3 44.4 C. denticulata denticulate 9 1.6 0.5 Total number 90
0 10 20 30 40 50 Relative abundance (%) 52 문성희ㆍ허준욱ㆍ차승주ㆍ황성돈ㆍ손맹현ㆍ권준영ㆍ권세련 (A) (B) (C) (E) (D) (F) (G) Fig. 2. The pale chub Zacco platypus (A) and Korea piscivorous chub Opsarichthys uncirostris (B) sampled from the Soyang Lake. Dactylogyrus (C) and copepoda (D) isolated from Korea piscivorous chub. Copepoda (E) and metacercaia (F & G) isolated from pale chub. 피라미에서 흡충류 피낭유충 감염상태를 조사한 결과에서 빙 어 1마리 당 평균 0.35개, 피라미 1마리 당 평균 0.43개의 간흡 충 metacercaria가 감염되어 있다고 보고하였다. 한편 2010년 에 강원도보건환경연구원에서 소양호에서 채집한 빙어를 대상 으로 간흡충 피낭유충을 검사한 결과 모든 빙어 시료에서 피낭 유충이 검출되지 않았다고 보고하였다(NewsWire, 2010). 아울 러 2014년에 소양호에서 포획한 빙어 200마리에서 간흡충 피 낭유충을 검사한 결과 모든 빙어 시료에서 피낭유충이 검출되
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