해수사육무지개송어에서분리한 Vibrio anguillarum 특성연구 255, , ,,, (Lee, 2013)., (infectious hematopoietic necrosis virus; IHNV) (infectious pancreatic

Size: px

Start display at page:

Download "해수사육무지개송어에서분리한 Vibrio anguillarum 특성연구 255, , ,,, (Lee, 2013)., (infectious hematopoietic necrosis virus; IHNV) (infectious pancreatic"

명길 도
6 years ago
Views:

1 한수지 51(3), , 2018 Original Article Korean J Fish Aquat Sci 51(3), ,2018 해수사육무지개송어 (Oncorhynchus mykiss) 에서분리된 Vibrio anguillarum 의특성분석 천혜진 김위식 1 조미영 정승희 한현자 * 국립수산과학원병리연구과, 1 전남대학교수산생명의학과 Characteristics of Vibrio anguillarum Isolated from Seawater Cultured Rainbow Trout Oncorhynchus mykiss in Korea Hye-Jin Chun, Wi-Sik Kim 1, Mi-Young Cho, Sung-Hee Jung and Hyun-Ja Han* Pathology Division, National Institute of Fisheries Science, Busan 40683, Korea 1 Department of Aqualife Medicine, Chonnam National University, Yeosu 61186, Korea From 2014 to 2017, mortalities of seawater-cultured rainbow trout Oncorhynchus mykiss, were observed in the Goheung and Jeju areas of Korea, with Vibrio anguillarum (seven strains: RT1, 2, 3, 4, 5, 6, and 7) identified as the etiological agent. The phenotypic (based on API 20NE, API ZYM, and E-test kits), serotypic (slide agglutination tests with O1, O2, O3, O4, and O7 antisera), and genotypic (16S rrna and ompu sequencing) characteristics of the seven RT strains were analyzed and compared to those of seven additional V. anguillarum stains (SF, isolated from sweet fish; FM, isolated from flathead mullet; 43305; 43311; 43307; 43308; and KCTC2711). The phenotypes of the RT strains showed variance, while the slide agglutination tests of the RT1-7, SF, and FM strains all showed positive reactions with serotype O1 antiserum. The 16S rrna and ompu sequences of the RT1-7, SF, and FM strains were affiliated with V. anguillarum (Serotype O1), but the ompu sequence of the SF strain differed from those of the RT1-7, FM, and strains, including one amino acid substitution. We thus confirmed that serotype O1 V. anguillarum, with multiple phenotypes, continues to infect seawater-cultured rainbow trout in Korea. Key words: Seawater-cultured, Rainbow trout, Oncorhynchus mykiss, Vibrio anguillarum, Serotype O1 서론 (Oncorhynchus mykiss) (Salmoniformes) (Salmonidae), (Behnke, 1992).., (Landless, 1976; Gall and Crandell, 1992) % (KOSIS, 2017).,,,,, (Lee, 2013; Kang et al., 2014), (Lee, 2013). Korean J Fish Aquat Sci 51(3) , June 2018 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 6 February 2018; Revised 7 March 2018; Accepted 20 April 2018 *Corresponding author: Tel: Fax: address: hjhan77@korea.kr Copyright 2018 The Korean Society of Fisheries and Aquatic Science 254 pissn: , eissn:

2 해수사육무지개송어에서분리한 Vibrio anguillarum 특성연구 255, , ,,, (Lee, 2013)., (infectious hematopoietic necrosis virus; IHNV) (infectious pancreatic necrosis virus; IPNV) (Park et al., 1993, Kim et al., 2015). Lepeophtheirus salmonis, viral hemorrhagic septicaemia (VHS), pancreas disease (Nolan et al., 2000; Taksdal et al., 2007; Dale et al., 2009) Vibrio anguillarum (Kim et al., 2014). (Kim et al., 2015). V. anguillarum µm (Colorni et al., 1981) (vibriosis) (Actis et al., 2011). V. anguillarum,. red pest red disease,. V. anguillarum 23, O1, O2, O3 (Stelnum et al., 2016) , V. anguillarum, V. anguillarum,,. 재료및방법 시험균주 V. anguillarum 7 7 Table 1. 1% NaCl tryptic soy agar (TSA, Becton Dickinson, USA) brain heart infusion agar (BHIA, Becton Dickinson, USA), 생화학적특성분석 V. anguillarum 1% NaCl TSA 25, 24 API 20NE kit (BIOMERIEUX, France) API ZYM (for semi- Table 1. Bacterial strains used in this study Strain Isolated Year Host/Source Geographical location RT Rainbow trout Oncorhynchus mykiss Jeju, South Korea RT Rainbow trout Oncorhynchus mykiss Jeju, South Korea RT Rainbow trout Oncorhynchus mykiss Goheung, South Korea RT Rainbow trout Oncorhynchus mykiss Goheung, South Korea RT Rainbow trout Oncorhynchus mykiss Goheung, South Korea RT Rainbow trout Oncorhynchus mykiss Jeju, South Korea RT Rainbow trout Oncorhynchus mykiss Jeju, South Korea SF Sweet fish Plecoglossus altivelis South Korea FM Flathead mullet Mugil cephalus Hadong, South Korea Rainbow trout Oncorhynchus mykiss Denmark European eel Anguilla anguilla Denmark Rainbow trout Oncorhynchus mykiss Denmark Cod Gadus macrocephalus Denmark KCTC Cod Gadus macrocephalus Norway RT1-7, isolated from rainbow trout; SF, isolated from sweet fish; FM, isolated from flathead mullet;, American type culture collection; KCTC, Korean collection for type cultures.

3 256 천혜진ㆍ김위식ㆍ조미영ㆍ정승희ㆍ한현자 quantitation of enzymatic activities) kit (BIOMERIEUX, France), oxidase test., , (RT1, RT3) (43305) NaCl (0.5-7%) tryptic soy broth (TSB, Becton Dickinson, USA) 항생제최소억제농도측정 8 (gentamicin, tetracycline, doxycycline, amoxicillin, erythromycin, ciprofloxacin, chloramphenicol clindamycin) (minimum inhibitory concentration; MIC) E-test (AB Biodisk) disk diffusion. Clinical and Laboratory Standards Institute (CLSI 2005, 2011) Vibrio spp. 유전적특성분석 1% NaCl TSB 24 13,000 rpm 2, pellet. pellet promega genomic DNA purification kit (Promega, USA) DNA. 16S rrna (27F, AGAGTTTGATCMTGGCTCAG; 1,492R, TACGGYTACCTTGTTACGAC) (Kim et al., 2005), ompu gene (encoding outer membrane protein, ompu-f, ATGAA- CAAAACTCTGATTGCTT; ompu-r, TAGAAGTCGTAAC- GTAGACC) polymerase chain reaction (PCR). 16S rrna 95 pre-denaturation 5, 95 (30 )-55 (30 )-72 (30 ) 30cycles 72 5 post-extension. ompu gene 95 pre-denaturation 5, 95 (30 )-50 (30 )-72 (60 ) 25cycles 72 5 post-extension. PCR EtBr 1.5% agarose gel (Bioneer, Korea) UV transilluminator (Alpha Innotech, USA) band,. MEGA6 program GENETYX Ver. 8.0 (SDC Software Development, Japan), national center for biotechnology institute (NCBI) basic local alignment search tool (BLAST) V. anguillarum. Blast search bioedit Ver multiple alignments, MEGA6 program( megasoftware.net; Tamura et al., 2013) (NJ, neighbor-joining analysis; 1,000 rounds of boostrap) (phylogenetic tree). 혈청형분석, (V. anguillarum 43305, serotype O1; KCTC2711, serotype O2; 43309, serotype O3; 43308, serotype O , serotype O7). V. anguillarum 5 70 L, V. anguillarum 9 (RT1-7, SF, FM) McFarland no. 6 standard 10 L 30.,. 결과및고찰 Kim et al. (2014) V. anguillarum,,,. V. anguillarum,,, Kim et al. (2014) V. angillarum. V. anguillarum,,, (Grisez et al., 1991) % NaCl TSA colony, V. anguillarum 14 Table 2. API ZYM kit akline phosphatase, leucine arylamidase 2, lipase (C14), valine arylamidase, crystine arylamidase, trypsin, -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, -mannosidase -fucosidase. API 20NE kit indole, urease, capric acid, adipic acid phenylacetic acid. Vibrio glucose, TCBS, oxidase catalase (Pazos et al., 1993). V. anguillarum oxidase catalase indole

4 해수사육무지개송어에서분리한 Vibrio anguillarum 특성연구 257 Table 2. Phenotypic characteristics of 7 Vibrio anguillarum strains isolated from rainbow trout Oncorhynchus mykiss in this study Test Strains RT1 RT2 RT3 RT4 RT5 RT6 RT7 SF FM Serotype O1 O1 O1 O1 O1 O1 O1 O1 O1 O1 O7 O3 O4 O2 API ZYM API 20NE Esterase(C4) Esterase Lipase(C8) Valine arylamidase Acid phosphatase β-galactosidase Naphthol-AS-BIphosphohydrolase N-acetyl-βglucosaminidase Nitrate reduction Glucose fermentation Arginine dihydrolase Gelatinase β-galactosidase Assimilation of : Glucose Arabinose Mannose Mannitol Esculin hydrolysis N-Acetylglucosamine Maltose Potassium gluconate Malate Trisodium citrate RT1-7, isolated from rainbow trout; SF, isolated from sweet fish; FM, isolated from flathead mullet;, American Type Culture Collection; KCTC, Korean Collection for Type Cultures. KCTC 2711, arabinose (Buller, 2004; Jee et al., 2011). Santos et al. (1996) V. anguillarum, (n=45) nitrate, glucose., nitrate glucose (n=1). Vibrio,, (Pazos et al., 1993). V. anguillarum RT1, RT3 (43305), 0.5-5% NaCl 24

5 258 천혜진ㆍ김위식ㆍ조미영ㆍ정승희ㆍ한현자, % (data not shown)., (43305) V. anguillarum. V. anguillarum Vibrio NaCl, 0.5-7% (Mehrnaz and Davar, 2010) % (Eguchi et al., 2000), 15-25, ph 5-9 (Larsen, 1984), V. anguillarum MIC, Table 3. CLSI Vibrio spp., gentamicin, tetracycline, doxycycline, chloramphenicol ciprofloxacin, clindamycin, amoxicillin. (SF) (43305) erythromycin, erythromycin. 12 Vibrio anguillarum cloxacillin, ampicillin, sulfamethoxazol-thrimethoprime erythromycin 100% (Parin et al., 2017). V. anguillarum. 7 (RT1-7) 5 7 O1 type, (Table 2). Sørensen (1986) Larsen (1986) 585 V. anguillarum O, 71% O1, 78% O2. O1 O2, O1 (Salmonidae) (Tranzo and Barja, 1990; Larsen et al., 1994). Lee et al. (1988), 26 V. anguillarum serotype C, serotype C Sørensen Larsen (1986) serotype O1. V. anguillarum (RT1-7) serotype O1, V. anguillarum (SF, FM) O1 type. Table 3. The MIC values for the Vibrio anguillarum strains and MIC interpretive standards (μg/ml) according to CLSI (2005, 2011) Breakpoint (μg/ml) 4 MIC Antimicrobial Agent Bacterial Strains S 1 I 2 R 3 KCTC RT1 RT2 RT3 RT4 RT5 RT6 RT7 SF FM Gentamicin AMINOGLY COSIDES Tetracycline Doxycycline TETRACY- CLINES Amoxicillin >256 >256 >256 > >256 > BETA- LACTAMS: PENICILLINS Vibrio anguillarum (n=14) MACROLIDES Erythromycin QUINOLONESCiprofloxacin <0.047 <0.004 < OTHER Chloramphenicol ANTIBIOTICS Clindamycin ? Susceptible. 2 Intermediate. 3 Resistant. 4 Breakpoints recommended by the clinical laboratory standards institute in M45-A(2011), M45-P(2005).RT1-7, isolated from rainbow trout; SF, isolated from sweet fish; FM, isolated from flathead mullet;, American Type Culture Collection; KCTC, Korean Collection for Type Cultures.

6 해수사육무지개송어에서분리한 Vibrio anguillarum 특성연구 259 (A) Vibrio anguillarum, 19264T, [X ], serotype O2, cod Vibrio anguillarum, 43313, [X7817.1], serotype O10, cod Listonella anguillarum, 43307, [FJ ], serotype O3, rainbow trout Listonella anguillarum, 43312, [FJ ], serotype O9, cod Vibrio anguillarum, KCTC2711, [LC365684], serotype O2, cod Vibrio anguillarum, 43311, [LC365683], serotype O7, european eel Vibrio anguillarum, 43308, [LC365682], serotype O4, cod Vibrio anguillarum, 43307, [LC365681], serotype O3, cod Vibrio anguillarum, 43305, [LC192806], serotype O1, rainbow trout Vibrio anguillarum, FM, [LC192806], serotype O1, flathead mullet Vibrio anguillarum, SF, [LC192806], serotype O1, sweet fish Vibrio anguillarum, RT7, [LC192806], serotype O1, rainbow trout Vibrio anguillarum, RT6, [LC192806], serotype O1, rainbow trout Vibrio anguillarum, RT5, [LC192806], serotype O1, rainbow trout Vibrio anguillarum, RT4, [LC192806], serotype O1, rainbow trout Vibrio anguillarum, RT3, [LC192806], serotype O1, rainbow trout Vibrio anguillarum, RT2, [LC192806], serotype O1, rainbow trout Vibrio anguillarum, RT1, [LC192806], serotype O1, rainbow trout Listonella anguillarum, 43309, [FJ ], serotype O5, cod Listonella anguillarum,43314, [FJ ], serotype O8, cod Listonella anguillarum, 43310, [FJ ], serotype O6, cod Vibrio cholerae, 14035, [NR ] 16S rrna (B) Vibrio anguillarum, FM, [LC192808], serotype O1, flathead mullet Vibrio anguillarum, 43305, [LC192808], serotype O1, rainbow trout Vibrio anguillarum, RT7, [LC192808], serotype O1, rainbow trout Vibrio anguillarum, RT6, [LC192808], serotype O1, rainbow trout Vibrio anguillarum, RT5, [LC192808], serotype O1, rainbow trout Vibrio anguillarum, RT4, [LC192808], serotype O1, rainbow trout Vibrio anguillarum, RT3, [LC192808], serotype O1, rainbow trout Vibrio anguillarum, RT2, [LC192808], serotype O1, rainbow trout Vibrio anguillarum, RT1, [LC192808], serotype O1, rainbow trout Vibrio anguillarum, SF, [LC192807], serotype O1, sweet fish Vibrio anguillarum, [ABY ] Listonella anguillarum, [EU ] Vibrio anguillarum, [CDQ49447], serotypeo1 Vibrio anguillarum, [AAN78349], serotypeo1 Vibrio anguillarum, [ACL97683] Listonella anguillarum, [AY157299], serotypeo1 Listonella anguillarum, [FJ ], serotype O1 Listonella anguillarum, [AY ] Vibrio anguillarum, 43308, [LC365686], serotype O4, cod Vibrio anguillarum, KCTC2711, [LC365688], serotype O2, cod Listonella anguillarum, [AY ] Vibrio anguillarum, 43307, [LC365685], serotype O3, cod Vibrio anguillarum, 43311, [LC365687], serotype O7, european eel ompu Fig. 1. Phylogenetic trees constructed based on the analyses of the 16S rrna (a), ompu (b) genes using the neighbor-joining method.

7 260 천혜진ㆍ김위식ㆍ조미영ㆍ정승희ㆍ한현자 7 (RT1-7) 2 (SF, FM) 16S rrna (43305, serotype O1) 100%, 4 (KCTC2711, serotype O2; 43309, serotype O3; 43308, serotype O , serotype O7) 16S rrna % (data not shown). 16S rrna, (RT1-7), (FM, SF) (43305, serotype O1) 16S rrna cluster, (Fig. 1A). Vibrio cholerae Vibrio OmpU, V. anguillarum OmpU gene mutant, biofilm (Wang et al., 2003). V. anguillarum OmpU (Sun et al., 2012)., V. anguillarum OmpU gene (ompu), (SF), 7 (RT1-7) (FM) (43305, serotype O1) 100%, serotype O1 4 (KCTC2711, serotype O2; 43309, serotype O3; 43308, serotype O , serotype O7) ompu % (data not shown). SF, serotype O1 ompu. SF ompu (RT1-7) ompu 99.9%, ompu (RT1-7), (FM, SF) (43305, serotype O1) V. anguillarum (Listonella anguillarum, serotype O1) cluster (Fig. 1B). V. anguillarum (Busschaert et al., 2015), 16S rrna ompu V. anguillarum serotype O1, 16S rrna ompu V. anguillarum. V. anguillarum. 사사 (R ). References Actis LA, Tolmasky ME and Crosa JH Vibriosis. In : Fish diseases and disorders 3(2), Woo PTK and Bruno DW, eds. CABI International, Wallingford, UK, Behnke RJ Native trout of Western North America. American Fisheries Society, Bethesda Marryland, U.S.A., Buller NB Bacteria from fish and aquatic animals: a practical identification manual. Centre for Agriculture and Bioscience International, Wallingford, UK, Busschaert P, Frans I, Crauwels S, Zhu B, Willems K, Bossier P, Michiels C, Verstrepen K, Lievens B and Rediers H Comparative genome sequencing to assess the genetic diversity and virulence attributes of 15 Vibrio anguillarum isolates. J Fish Dis 38, jfd CLSI (Clinical and laboratory standards institute) Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fasitidious bacteria; proposed guideline CLSI document M45-P. CLSI, Wayne, Pennsylvania, U.S.A., CLSI (Clinical and laboratory standards institute ) Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria; approved guideline-second edition. CLSI document M45-A2, Wayne, Pennsylvania, U.S.A., Colorni A, Paperna I and Gordin H Bacterial infections in gilt-head sea beam Sparus aurata cultured at Elat. Aquaculture 23, (81) Dale OB, Ørpetveit I, Lyngstad TM, Kahns S, Skall HF, Olesen NJ and Dannevig BH Outbreak of viral haemorrhagic septicaemia (VHS) in seawater-farmed rainbow trout in Norway caused by VHS virus genotype III. Dis Aquat Organ 85, Eguchi M, Fujiwara E and Miyamoto N Rvival of Vibro anguillarum in freshwater environments: adaptation or debilitation?. J Infect Chemother 6,

8 해수사육무지개송어에서분리한 Vibrio anguillarum 특성연구 261 org/ /s Gall GAE and Crandell PA The rainbow trout. Aquaculture 100, Grisez L, Ceusters R and Ollevier F The use of API 20E for the identification of Vibrio anguillarum and V.ordalii. J Fish Dis 14, tb00833.x. Jee BY, Bang JD, Cho MY, Kim JW, Won KM and Cho YA Characteristics of Vibrio isolated from cultured file fish, Stephanolepis cirrhifer in Korea. J Fish Pathol 24, Kang SI, Kim KH, Lee JK, Kim YJ, Park SJ, Kim MW, Choi BD, Kim DS and Kim JS Comparison of the food quality of freshwater rainbow trout Oncorhynchus mykiss cultured in different regions. Korean J Fish Aquat Sci 47, Kim SM, Won KM, Woo SH, Li H, Kim EJ, Choi KJ, Cho MY, Kim MS and Park SI Vibrios isolated from diseased marine culturing fishes in Korea. J Fish Pathol 18, Kim SW, Kim JO, Kim WS, Kim DH and Oh MJ Vibrio anguillarum infection in rainbow trout Oncorhynchus mykiss during seawater adaption. J Fish Pathol 27, Kim WS, Jang MS, Kim JO, Jeon YH and Oh MJ Effect of fish pathogenic viruses on maricuture of rainbow trout Oncorhynchus mykiss. Kor J Ichthyol 27, doi.org/ /rg KOSIS (Korean statistical information service) Fishery production survey: Statistics by type of fishery and species. Retrived from www. kosis.kr on Jan 9, Landless PJ Acclimation of rainbow trout to sea water. Aquaculture 7, Larsen JL, Pedersen K and Dalsgaard I Vibrio anguillarum serovars associated with vibriosis in fish. J Fish Dis 17, Larsen JL Vibrio anguillarum: Influence of temperature, ph, NaCl concentration and incubation time on growth. J Appl Microbial 57, tb01388.x. Lee JY, Chun SK, Pack SI Serotyping of Vibrio anguillarum isolated from cultured marine fishes. Bull Korean Soc Fish Pathol 1, Lee NS The current situation and suggestions of the mariculture of rainbow trout in Korea. Ocean Fish 3, Mehrnaz RAD and Davar SHAHSAVANI Isolation and characterization of Vibrio (Listonella) anguillarum from catfish. Turk J Vet Anim Sci 34, org/ /vet Nolan DT, Ruane NM., Heijden YVD, Quabius ES, Costelloe J and Bonga SEW Juvenile Lepeophtheirus salmonis (Krúyer) affect the skin and gills of rainbow trout Oncorhynchus mykiss (Walbaum) and the host response to handling procedure. Aquac Res 31, j x. Park MA, Shon SG, Lee SD, Chun SK, Park JW, Fryer JL and Hah YC Infectious hematopoietic necrosis virus from salmonids cultured in Korea. J Fish Dis 16, doi.org/ /j tb00880.x. Parin U, Savasan ES, Yuksei HT, Gurpinar S and Kirkan S Antimicrobial resistance of Vibrio (Listonella) anguillarum isolated from rainbow trout (Onchrhynchus mykiss). Indian J Ani Res, B Pazos F, Santos Y, Magrinos B,Bandín I, Núňez S and Toranzo AE Phenotypic characteristics and virulence of Vibrio anguillarum-related organisms. Appl Environ Microbiol 59, Santos Y, Pazos F and Toranzo AE Biochemical and serological analysis of Vibrio anguillarum related organisms. Dis Aquat Org 26, Sørensen UB and Larsen JL Serotyping of Vibrio anguillarum. American Society for Microbiology, Appl Environ Microbiol 51, $02.00/0. Stelnum TM, Karatas S, Martlnussen NT, Melrelles PM, Thompson FL and Colquhoun DJ Multilocus sequence analysis of close relatives Vibrio anguillarum and Vibrio ordalii. Am Soc Microbiol 82, org/ /aem Sun Y, Zhang M, Liu CS, Qiu R and Sun L A divalent DNA vaccine based on Sia10 and OmpU induce cross protectin against Streptococcus iniae and Vibrio anguillarum in Japanese flounder. Fish Shellfish Immunol 32, Taksdal T, Olsen AB, Bjekas I, Hiortaas MJ, Dannevig BH, Graham DA and Mcloughlin MF Pancreas disease in farmed atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in Norway. J Fish Dis 30, Tamura K, Stecher G, Peterson D, Filipski A and Kumar S MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30, molbev/mst 197. Tranzo AE and Barja JL A review of the taxonomy and seroepizootiology of Vibrio anguillarum, with special reference to aquaculture in the northwest Spain. Dis Aquat Org 9, Wang SY, Lauritz J, Jass J and Milton Role for the major outer-membrane protein from Vibrio anguillarum in bile resistance and biofilm formation. Micobiol 149,

42(3)-11(p ).fm

42(3)-11(p ).fm The Korean Journal of Microbiology, Vol. 42, No. 3, September 2006, p. 199-204 Copyright 2006, The Microbiological Society of Korea e ³ w ³ w Á Á½ Áx Á 1 Á 2, * w w w w w w e ³ ³ w p w. e ³ w ù š» 30 cm.