한수지 48(3), 337-345, 2015 Original Article Korean J Fish Aquat Sci 48(3),337-345,2015 제주도여윔증상넙치 (Paralichthys olivaceus) 로부터분리한점액포자충의특성분석 김승민 전려진 1 박명애 2 정현도 3 정준범 * 제주대학교해양의생명과학부, 1 제주대학교수산백신연구센터, 2 국립수산과학원수산생물방역과, 3 부경대학교수산생명의학과 Characterization of the Myxosporean Parasite Isolated from Emaciated Olive Flounders Paralichthys olivaceus on Jeju Island Seung Min Kim, Lyu Jin Jun 1, Myoung Ae Park 2, Hyun Do Jeong 3 and Joon Bum Jeong* Faculty of Marine Biomedical Science, Jeju National University, Jeju 690-756, Korea 1 Fish Vaccine Resarch Center, Jeju National University, Jeju 695-814, Korea 2 Aquatic Life Disease Control Division, National Fisheries Research and Development Institute, Busan 619-705, Korea 3 Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Korea To investigate the causes of emaciation in cultured olive flounder Paralichthys olivaceus in Korea. We performed histological examinations and polymerase chain reaction (PCR) with a new primer set. In most cases, the most severe emaciation was observed in the abdominal area Using PCR on extracted livers, kidneys, spleens, gills, brains, and intestines, we found that areas around the kidneys and intestines were as almost always positive. In significantly emaciated fish, PCR was positive in all internal organs except the gills. In addition, the homology of 812-bp nucleotide sequences of the 28S rrna gene was more than 99% in emaciated fish. Partial homology with Myxobolus spp. and Cystodiscus axonis, whose data were obtained from GenBank was 86% and 88%, respectively. Histological examinations detected spores in kidneys and intestines but not in other organs. We also performed cohabitation experiments to determine whether infections could be exchanged among species or only within species. Uninfected olive flounder and red sea bream, Pagrus major, cohabitating with emaciated olive flounder showed 100% and 0% cumulative mortality, respectively. Thus the cause of emaciation in cultured olive flounder of Korea is likely due to a new parasite. Key words: Emaciation disease, Olive flounder, PCR, Spore, Cohabitation experiment 서론 1990 (Takifugu rubripes), (Pagrus major), (Oplegnathus fasciatus), (Paralichthys olivaceus),., (Tun et al., 2000). Tun et al. (2002), 3, Enteromyxum fugu, Enteromyxum leei Leptotheca fugu., (Ogawa and Yokoyama, 2001). Yasuda et al. (2005),, 3 primers PCR. 50% 95%. 700. http://dx.doi.org/10.5657/kfas.2015.0337 Korean J Fish Aquat Sci 48(3) 337-345, June 2015 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 February 2015; Revised 1 June 2015; Accepted 8 June 2015 *Corresponding author: Tel: +82. 64. 754. 3426 Fax: +82. 64. 756. 3493 E-mail address: jeongjb@jejunu.ac.kr Copyright 2014 The Korean Society of Fisheries and Aquatic Science 337 pissn:0374-8111, eissn:2287-8815
338 김승민ㆍ전려진ㆍ박명애ㆍ정현도ㆍ정준범 viral hemorrhagic septicemia (VHS) Scuticocilate,,. 2007 20 cm, 1-3.,,.,, cohabitation test. 실험어 재료및방법 2010 (E03), 2011 (E06), 2012 (E03) 2013 (E12)., tryptic soy agar (Difco Co., USA), thiosulfate citrate bile salts sucrose agar (Difco), Salmonella-Shigella (Difco),,, 25. Cho et al. (2007) viral hemorrhagic septicemia virus (VHSV), viral nervous necrosis virus (VNNV), hirame rhabdovirus (HRV) red seabream iridovirus (RSIV) 4 PCR. DNA 추출,,,,, DNA DNeasy Blood & Tissue Kit (Qiagen Hilden, Germany). 10 mg ATL buffer 180 L proteinase K 20 L 56., AL buffer 200 L ethanol 200 L spin column 6,000 g 1. Column tube AW1 buffer 500 L AW2 buffer 500 L, AE buffer 50 L DNA. DNA -20. Primer 제작, E. leei small subunit ribosomal DNA gene (SSU rdna) MM18Sf/MM18Sr primer set (1,589 bp) (Table 1) DNA PCR, PCR. GenBank (NCBI, USA) (Myxidium sp.) degenerated primers, DNA PCR, DNA sequencing EM-F/EM-R primer set (Table 1). PCR 및 DNA sequencing PCR microtube 1 M primer, 2.5 mm dntp, 10 G-Taq Buffer, 2.5 U G-Taq DNA polymerase (Gene Pro Themal Cycler Cosmo, Korea) template DNA distilled water PCR volume 20 L. PCR 95 3 predenaturation, 95 30 denaturation, 55 30 annealing, 72 30 extension 1 cycle, 35 cycles., 72 7 postextension. PCR 1 TAE buffer (40 Mm Tris-acetate, 1 mm EDTA), 0.5 g/ L ethidium bromide 1% agarose gel, UV band. PCR gel purification kit (Bioneer, Korea) Table 1. PCR primers used for the detection of new pathogen MM18S-F MM18S-R 28S-F 28S-R EM-F EM-R Primer Oligonucleotide sequence (5'-3') Expected sizes Function CTGGTTGATTCTGCCAGTGGTC CGGTACTAGCGACGGGCG CTAGGGGAATCCGACTGT ATCAAACTAGAGTCAAGC CAACCGCAATGTGTTTACTC CCAAACAACCTGCCACAATG 1,589 bp 313 bp 812 bp Detection of E. leei (Palenzuela et al., 2002) House keeping gene (28S ribosomal RNA) Detection of new pathogen (in this study)
원인불명여윔증진단 339, ToPo TA cloning kit (Invitrogen, USA) cloning (Solgent, Korea). 조직병리학적분석, Edwardsiella tarda, Vibrio harveyi Streptococcus sp.. Viral hemorrhagic septicemia virus (VHSV), viral nervous,,,,, Bouin's solution 24 70% EtOH. (Leica EG 1150HC, Germany) (Leica Jung 820, Germany). 4-5 m. haematoxylin eosin (H&E) (Zeiss LT60, Germany). 어류간의전이실험 Ishimatsu et al., (2007) cohabitation. PCR donor group, recipient group. (16.3 0.4 cm, 40 5.23 g) 15 (16.7 0.9 cm, 53.4 8.47 g) 15 cohabitation. 100 L recipient group.. 100 L, 10 (6.5 0.5 cm, 3.5 0.7 g) 10 cohabitation. 19 1 1 1,., PCR. Fig. 1. The external (A) and internal (B) signs of the emaciated olive flounder Paralichthys olivaceus. 결과 여윔증의원인체검출,, (Tun et al., 2000) (Fig. 1A, 1B)., 5-8 m, 7-9 m (Fig. 2), Fig. 2. The spore from the intestine of emaciated olive flounder Paralichthys olivaceus. Bar=10 μm.
340 김승민ㆍ전려진ㆍ박명애ㆍ정현도ㆍ정준범 necrosis virus (VNNV), hirame rhabdovirus (HRV) red seabream iridovirus (RSIV) 4 viruses (data not shown). EM-F/EM-R primer set PCR (Table 1),, (Fig. 3). PCR,,,,., PCR (Fig. 3). PCR products, 99%, (Fig. 4). GenBank database, 150 bp Myxobolus sp. (Accession No. JN616264.1) Cystodiscus axonis (Accession No. JN977605.1) 86% 88%, GenBank. 조직병리학적검사,, Fig. 3. PCR amplification from nucleic acids of internal organs from the emaciated olive flounder Paralichthys olivaceus in the level stages of infection. Lanes 1, 7, 13 and 19, Kidney; lanes 2, 8, 14 and 20, Intestine; lanes 3, 9, 15 and 21, Spleen; lanes 4, 10, 16 and 22, Liver; lanes 5, 11, 17 and 23, Brain ; lanes 6, 12, 18 and 24, Gill; M, 1 kb DNA ladder.
원인불명여윔증진단 341 Fig. 4. Comparison analysis of the DNA nucleotide sequences of pathogens detected from the emaciated olive flounder Paralichthys olivaceus. Primers used in PCR are in boxes.
342 김승민ㆍ전려진ㆍ박명애ㆍ정현도ㆍ정준범,,, (plasmodium) (Fig. 5A and 5B)., (Fig. 5C and 5D)., (data not shown). 어류간의전이실험 PCR, 2 groups donor 3 100%, recipient 12 100% (Fig. 6A). recipient group donor group 7, recipient group PCR, 73.3% (Table 2),, donor fish (Fig. 7)., 3 donor group 100%, recipient group (Fig. 6B), PCR (Table 2). (data not shown). 고찰,, Fig. 5. Histological findings of kidney (A, 200 and B, 400) and intestine (C, 200 and D, 400) from the emaciated olive flounder Paralichthys olivaceus. H & E stain. Bar = 20 μm.
원인불명여윔증진단 343 (A) (B) 100 100 Cumulative mortality (%) 80 60 40 20 Cumulative mortality (%) 80 60 40 20 0 0 2 4 6 8 10 12 14 0 0 2 4 6 8 10 12 14 Day Day Fig. 6. Cumulative mortality (%) of olive flounder Paralichthys olivaceus (A) and red seabream Pagrus major (B) after cohabitated with the emaciated olive flounder., emaciated olive flounder; and, cohabitated olive flounder and red sea bream, respectively. Fig. 7. Histological findings of kidney (A, 200 and B, 400) and intestine (C, 200 and D, 400) from the recipient olive flounder Paralichthys olivaceus cohabitated with the emaciated olive flounder. H & E stain. Bar=20 μm. Table 2. Infection rate (%) of olive flounder Paralichthys olivaceus and red seabream Pagrus major after cohabitated with the emaciated olive flounder Donor fish (olive flounder) Recipient fish (olive flounder) Donor fish (olive flounder) Recipient fish (red seabream) PCR Positive 100% (15/15) 73.3% (11/15) 100% (10/10) 0% (0/10)
344 김승민ㆍ전려진ㆍ박명애ㆍ정현도ㆍ정준범.,, 30-40% (data not shown),. Choi et al. (2012) E. tarda, V. harveyi,., 5-8 m, 7-9 m (Fig. 2), (Tetsuza et al., 2004). Tun et al. (2002) E. fugu, E. leei L. fugu. SSU rdna gene MM18Sf/MM18Sr primer set (Palenzuela et al., 2002; Yasuda et al., 2005) PCR. Choi et al. (2012), primer set PCR. primer set (EM-F/EM-R) (Table 1), PCR band (Fig. 3),,., DNA nucleotide sequences 99% (Fig. 4)., GenBank Myxobolus sp. Cystodiscus axonis 80% GenBank database., DNA walking. sharpsnout sea bream Puntazzo puntazzo, red sea bream Pagrus major, red drum Sciaenops ocellatus Myxidium leei (Le Breton and Marques, 1995; Diamant, 1998; Athanassopoulou et al., 1999)., Myxosporea turbot, Scophthalmus maximus turbot (Beaman et al., 1999). Bartholomew et al. (1989) Ceratomyxa shasta., (Tun et al., 2002).,,,,.,,,. E. leei donor fish cohabitaiton, E. leei (Diamant, 1997; Yasuda et al., 2002). (Table 2), PCR.,,, cohabitation, GenBank.,,,. 사사 ( ). References Athanassopoulou F, Prapas T and Rodger H. 1999. Diseases of Puntazzo puntazzo cuvier in marine aquaculture systems in Greece. J Fish Dis 22, 215-218. Bartholomew JL, Smith CH, Rohovec JS and Fryer JL. 1989. Characterization of a host response to the myxosporean parasite, Ceratomyxa shasta (Noble), by histology, scanning electron microscopy and immunological techniques.
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