한수지 51(1), 23-30, 2018 Original Article Korean J Fish Aquat Sci 51(1),23-30,2018 넙치 (Paralichthys olivaceus) 근육채취방법에따른 Kudoa septempunctata 진단효율비교 송준영 정승희 1 최혜승 2 * 국립수산과학원남동해수산연구소, 1 국립수산과학원병리연구과, 2 국립수산과학원남해수산연구소 Evaluation of a Non-destructive Diagnostic Test for Kudoa septempunctata in Farmed Olive Flounder Paralichthys olivaceus Jun-Young Song, Sung Hee Jung 1 and Hye-Sung Choi 2 * Southest Sea Fisheries Research Institute, Tongyeong 53085, Korea 1 Pathology Division, National Institute of Fisheries Science, Busan 46083, Korea 2 Aquaculture Industry Division, South Sea Fisheries Research Institute, Yeosu 59780, Korea Kudoa septempunctata, a myxosporean parasite that infects olive flounder Paralichthys olivaceus is known to cause Kudoa food poisoning. Entire trunk muscle (ETM) is used for diagnosis of the parasite in fish and this method demands sacrifice of the host, causing a loss of commercial value. We developed a non-destructive method that uses a plastic syringe-style implanter to draw the sample, called the part-point muscle (PPM) sampling technique. We validated the PPM method in fish infected with K. septempunctata at the level detectable by the ETM method. We confirmed that the PPM method is equally sensitive in comparison to the ETM method for diagnosing K. septempunctata spores in olive flounder muscle. Our study also confirmed that the parasite is uniformly distributed in the dorsal muscle of infected fish. Over a period of 1 month, we observed no mortality of the host fish used for sampling by the PPM method. Thus, our studies demonstrate that the PPM sampling technique is an efficient, non-destructive method for diagnosing K. septempunctata in olive flounder. Key words: Kudoa septempunctata, Olive flounder, Paralichthys olivaceus, Diagnostic test 서론 Kudoa septempunctata (Matsukane et al., 2010), K. septempunctata (Harada et al., 2012a; Kawai et al., 2012), (Song et al., 2013; 2014), (MOF, 2015).,, (Moran et al., 1999), Kudoa thyrsites, Kudoa musculoliquefaciens, Kudoa paniformis, Kudoa clupeidae, Kudoa miniauriculata ( ) (Kudo et al., 1987, Moran et al., 1999, Yokoyama et al., 2004)., K. septempunctata (Matsukane et al. 2010),. K. septempunctata,, 60, 30., https://doi.org/10.5657/kfas.2018.0023 Korean J Fish Aquat Sci 51(1) 23-30, 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 October 2017; Revised 27 November 2017; Accepted 1 December 2017 *Corresponding author: Tel: +82. 61. 690. 8970 Fax: +82. 61. 685. 9073 E-mail address: choihs@korea.kr Copyright 2018 The Korean Society of Fisheries and Aquatic Science 23 pissn:0374-8111, eissn:2287-8815
24 송준영ㆍ정승희ㆍ최혜승 (Song et al., 2013; 2014), 1 kg,.,,. 재료및방법 근육채취방법에따른 K. septempunctata 의검출효능비교 실험어 2013 PCR 5 (Table 1) K. septempunctata. 부분근육 (Part-point muscle; PPM) 채취방법 (PPM 방법 ) PPM plastic syringe-style MK7 implanter (Biomark, USA) needle (Fig. 1). 20-25 mg 2 50 mg. 2 ml tube containing 2 mm ziruconia and 5 mm stainless beads (WATSON), 1X TE buffer (Promega) 5, FastPrep -24 (MP, USA) 20. 100 µl ( 20 mg ) quantitative polymerase chain reaction (qpcr) conventional polymerase chain reaction (PCR) DNA, 10 L. 전체근육 (Entire trunk muscle; ETM) 채취방법 (ETM 방법 ) ETM (Song et al., 2013) (1-5 g). 200 mg 2mL tube containing 2 mm ziruconia and 5 mm stainless beads (WATSON), 1X TE buffer (Promega, USA) 5, Fast- Prep -24 (MP, USA) 20. 100 L ( 20 mg ) qpcr PCR DNA, 10 L. 넙치등근육에서의쿠도아분포조사 PPM, K. septempunctata 2. 6 point point PPM 6 (Fig. 2). point 3 (qpcr, conventional PCR) triplicate, 3. 사육넙치에대한 PPM 방법의적용 PPM, 100 ( 500 g). PPM MS-222 (Sigma, Germany). plastic syringe-style MK7 implanter (Biomark, USA), (a) (b) (c) Fig. 1. Taking of the samples by part-point muscle (PPM) method in the fish Paralichthys olivaceus using a plastic syringe-style implanter on a polystyrene board (a). 20 to 25 mg of muscle is taken by the PPM method (b, c).
근육채취방법에따른 Kudoa septempunctata 진단효율 25 Point 1 Point 2 Point 3 Point 4 Point 5 Point 6 Fig. 2. Sampling points for testing Kodoa septempunctata distribution in the dorsal muscle of fish Paralichthys olivaceus. From each point, part-point muscle (PPM) was obtained six times for qpcr, PCR and microscopy.., (qpcr, PCR ). 시료분석 DNA 추출 100 µl ( 20mg ) DNA (qpcr, PCR). DNA Song et al. (2014) QIAamp DNA mini kit (Qiagen, Japan), 200 µl volume elusion. DNA -20. qpcr DNA K. septempunctata 18S rdna (AB553293) real-time PCR. Primers probe (MHLW, 2011) Kudoa-F (5 -CATGGGATTAGCCCGGTTTA-3 ), Kudoa-R (5 - ACTCTCCCCAAAGCCGAAA-3 ), Kudoa-P (5 -FAM-TC- CAGGTTGGGCCCTCAGTGAAAA-TAMRA-3 ). PCR DNA 4 µl plasmid positive control 4 µl, Taqman 2X Universal master mix II (AB, USA) 10 µl, forward reverse primer 0.4 µm, probe 0.25 M 20 µl volume. PCR 95 10 1 cycle, 95 15 60 60 45 cycles duplicate. Plasmid positive control Song et al.(2014) Nanodrop ND-1000 spectrophotometer DNA plasmid copy. 2.5 10 7 ( 10 8 ), 2.5 10 5 ( 10 6 ), 2.5 10 3 ( 10 4 ), 2.5 10 1 ( 10 2 ) copies/µl plasmid, realtime PCR plasmid positive control. PCR PCR primer K. septempunctata 28S rdna primer set (forward, 5 -gtgtgtgatcagacttgatatg-3, reverse, 5 -aagccaaaactgctggccattt -3 ) (MAFF, 2012). 4 µl template primer AccuPower PCR premix (Bioneer, Korea) 20 µl PCR. PCR ethidium bromide 1.5% agarose gel MultiImage II (Alphainnotech, USA) PCR (target size 356 bp). 현미경검사 TE buffer (Promega, USA) 5 Loffler s methylene blue solution 1:1 cell-counting chamber 1 g. PPM ( 3), PPM slide glass, Loffler s methylene blue solution 1. 결과 근육채취방법에따른 Kudoa septempunctata 의검출효능비교 5 PPM ETM
26 송준영ㆍ정승희ㆍ최혜승 Table 1. Comparison of PPM and ETM methods for detecting Kudoa septempunctata in the fish Paralichthys olivaceus infected with the parasite, by qpcr, PCR and microscopy Fish no. Fish size (cm) PCR qpcr (log rdna copy no./g) Microscopy (log spore no./g) PPM 1 ETM 2 PPM ETM PPM ETM 1 43 P P 9.72 9.56 5.57 6.48 2 35 P P 9.20 9.31 5.27 4.98 3 38 P P 8.20 7.89 5.91 6.02 4 42 N P 5.75 6.28 nd nd 5 33 P P 7.32 7.77 5.24 5.10 1 PPM, part-point muscle. 2 ETM, entire trunk muscle. P, positive ( 7.0 log rdna copy); N, negative (<7.0 log rdna copy); nd, not detected. (Table 1). qpcr, 5 4 ( 1, 2, 3, 5 ) PPM ETM (7.00 log rdna copy no./g) (PPM 7.32-9.72 log rdna copy no./g, ETM 7.77-9.56 log rdna copy no./g )., 4 PPM ETM. PCR, qpcr 4 ( 1, 2, 3, 5 ) PPM ETM, 4 PPM, ETM., qpcr PCR 4 PPM ETM (4.98-6.48 log spore no./g), 4.,,, PPM ETM. 넙치등근육에서 Kudoa septempunctata 의분포조사 2 (Point 1-6) PPM, 1 6 point PCR, qpcr point (point 2 1 ) 7.72-9.73 log rdna copy no./g., 6 point 5.06-6.59 log spores/g., 2, point 1 3 (triplicates) PCR, point 2 3 1, point 2 3 2 PCR, point 4-6 PCR. qpcr, point 1-3 (nd 5.45-6.87 log rdna copy no./g ), point 4-6. (Table 2)., 6 point. 사육넙치에서의쿠도아검사를위한 PPM 방법의적용 100 ( 500g) PPM, (data not shown). 100, 7 (PCR, qpcr, ), 4 PCR qpcr, 1 PCR (Table 3)., PPM 고찰 Kudoa septempunctata, K. septempunctata (MOF, 2015). K. septempunctata, (Song et al., 2013; 2014),., K. septempunctata., K. septempunctata 5
근육채취방법에따른 Kudoa septempunctata 진단효율 27 Table 2. Distribution of Kudoa septempunctata from the 6 points of the dorsal muscle in the fish Paralichthys olivaceus P1 P2 P3 P4 P5 P6 Fish 1 (38 cm, 877 g) Fish 2 (43 cm, 949 g) PCR qpcr 1 Microscopy 2 PCR qpcr a Microscopy b P P (9.19) P (5.30) nd N (6.58) nd P P (8.89) P (5.85) nd N (5.49) nd P P (7.72) P (6.18) nd N (5.45) nd P N (6.51) P (5.90) nd nd nd P P (9.03) P (6.59) P N (6.87) nd P P (7.90) P (5.63) nd N (5.65) nd P P (9.04) P (6.29) nd N (6.57) nd P P (8.86) P (6.00) P N (6.60) nd P P (9.15) P (5.85) P N (6.77) nd P P (9.48) P (6.36) P P (7.09) nd P P (9.15) P (5.57) P N (6.84) nd P P (9.73) P (5.74) P P (7.48) nd P P (9.09) P (6.36) P P (7.42) nd P P (9.32) P (6.23) P P (8.37) nd P P (9.20) P (6.20) P P (7.42) nd P P (9.30) P (6.37) P P (7.30) nd P P (9.36) P (6.26) P P (7.59) nd P P (9.24) P (5.06) P N (6.96) nd 1 qpcr, The letter indicates log rdna copy no./g of fish muscle. 2 Microscopy, The letter indicates log spore no./g of fish muscle. P, positive ( 7.0 log rdna copy); N, negative (<7.0 log rdna copy); nd, not detected. ETM, PPM., (7 log rdna copy/g ) ETM PPM PCR,.,, ETM PCR, PPM PCR., PPM ETM., PPM, 2 6 point,,. Iijima (2012) /, Harada (2012b), 1.1 10 6 spores/g rdna copy, 5.6 10 4-8.4 10 5 spores/g rdna copy., PPM,., PPM, 100., 100, 7 (PCR, qpcr), 5. Kudoa thyrsites (Fillets) hyohyoid ventralis, 79% ( 93%) (St-Hilaire et al., 1997).
28 송준영ㆍ정승희ㆍ최혜승 Table 3. Kodoa septempunctata detection from the 100 fish Paralichthys olivaceus for non-destructive diagnostic test Fish no. PCR qpcr 1 Microscopy Fish no. PCR qpcr 1 Microscopy 1 N nd nd 51 N nd nd 2 N nd nd 52 N N (4.87) nd 3 N nd nd 53 N nd nd 4 N nd nd 54 N nd nd 5 N nd nd 55 N nd nd 6 N nd nd 56 N nd nd 7 N nd nd 57 N nd nd 8 N nd nd 58 N nd nd 9 N nd nd 59 N nd nd 10 N nd nd 60 N nd nd 11 P P (7.06) nd 61 N nd nd 12 N nd nd 62 N nd nd 13 P P (7.24) P 63 N nd nd 14 N nd nd 64 N N (4.67) nd 15 N nd nd 65 N nd nd 16 N nd nd 66 N nd nd 17 N nd nd 67 N nd nd 18 N nd nd 68 N N (5.35) nd 19 N nd nd 69 N nd nd 20 N nd nd 70 N N (5.23) nd 21 P P (8.73) P 71 P N (6.53) nd 22 P P (7.29) P 72 N nd nd 23 N nd nd 73 P P (7.22) P 24 N nd nd 74 N nd nd 25 N N (5.05) nd 75 N nd nd 26 N nd nd 76 N nd nd 27 P P (7.96) nd 77 N nd nd 28 N N (5.02) nd 78 N nd nd 29 P P (8.15) nd 79 N nd nd 30 N nd nd 80 N nd nd 31 N nd nd 81 N nd nd 32 P P (8.24) P 82 N nd nd 33 N nd nd 83 P P (7.11) nd 34 N nd nd 84 N nd nd 35 N N (4.94) nd 85 N nd nd 36 N N (5.37) nd 86 N nd nd 37 N nd nd 87 N nd nd 38 N nd nd 88 N nd nd 39 N nd nd 89 N nd nd 40 N nd nd 90 N nd nd 41 P P (7.16) P 91 N nd nd 42 N N (5.00) nd 92 N nd nd
근육채취방법에따른 Kudoa septempunctata 진단효율 29 Table 3. Continued Fish no. PCR qpcr 1 Microscopy Fish no. PCR qpcr 1 Microscopy 43 N nd nd 93 N nd nd 44 N nd nd 94 N nd nd 45 P P (7.79) P 95 N N (4.79) nd 46 N nd nd 96 N nd nd 47 N nd nd 97 N nd nd 48 N nd nd 98 N nd nd 49 N N (4.27) nd 99 N nd nd 50 N nd nd 100 N nd nd 1 qpcr, The letter indicates log rdna copy no./g of fish muscle. P, positive ( 7.0 log rdna copy); N, negative (<7.0 log rdna copy); nd, not detected. PPM., PPM,., PPM. 사사 (R2017064)., Yokoyama Hiroshi Shirakashi Sho. References Harada T, Kawai T, Jinnai M, Ohnishi T, Sugita-Konishi Y and Kumeda Y. 2012a. Detection of Kudoa septempunctata 18S ribosomal DNA in patient fecal samples from novel foodborne outbreaks caused by consumption of raw olive flounder (Paralichthys olivaceus). J Clin Microbiol 50, 2964-2968. https://doi.org/10.1128/jcm.01218-12. Harada T, Kawai T, Sato H, Yokoyama H and Kumeda Y. 2012b. Development of a quantitative polymerase chain reaction assay for detection of Kudoa septempunctata in olive flounder (Paralichthys olivaceus). Int J Food Microbiol 156, 161-167. https://doi.org/10.1016/j.ijfoodmicro.2012.03.018. Iijim Y, Nakanishi N, Furusawa H, Ohnishi T and Sugita-Konishi Y. 2012. Inter-laboratory validation and applications of quantitative real-time PCR for the detection of Kudoa septempunctata in olive flounder (Paralichthys olivaceus). Jpn J Infect Dis 65, 436-438. https://doi.org/10.7883/yoken.65.436. Kawai T, Sekizuka T, Yahata Y, Kuroda M, Kumeda Y, Iijima Y, Kamata Y, Sugita-Konishi Y and Ohnishi T. 2012. Identification of Kudoa septempunctata as the causative agent of novel food poisoning outbreaks in Japan by consumption of Paralichthys olivaceus in raw fish. Clin Infect Dis 54, 1046-1052. https://doi.org/10.1093/cid/cir1040. Kudo G, Barnett H and Nelson R. 1987. Factors affecting cooked texture quality of Pacific whiting, Merluccius productus, fillets with particular emphasis on the effects of infection by the myxosporeans Kudoa paniformis and K. thyrsitis. Fish Bull 85, 745-756. https://spo.nmfs.noaa.gov/sites/default/ files/pdf-content/1987/854/kudo.pdf. MAFF (Ministry of Agriculture, Forestry and Fisheries). 2012. Retrieved from http://www.jfa.maff.go.jp/test/saibai/pdf/kudoa_notice_03.pdf on Sep 17, 2017. Matsukane Y, Sato H, Tanaka S, Kamata Y and Sugita-Konishi Y. 2010. Kudoa septempunctata n. sp. (Myxosporea: Multivalvulida) from an aquacultured olive flounder (Paralichthys olivaceus) imported from Korea. Parasitol Res 107, 865-872. https://doi.org/10.1007/s00436-010-1941-8. MHLW (Ministry of Health, Labour and Welfare). 2011. Retrieved from http://www.mhlw.go.jp/file/06-seisakujouhou- 11130500-Shokuhinanzenbu/0000124372.pdf on Sep 17, 2017 MOF (Ministry of Oceans and Fisheries) 2015. Hygienic management guidelines for domestic flounder farm. MOF, Sejong, Korea. Moran JDW, Whitaker DJ and Kent ML. 1999. A review of the myxosporean genus Kudoa Meglitsch, 1947, and its impact on the international aquaculture industry and commercial fisheries. Aquaculture 172, 163-196. https://doi. org/10.1016/s0044-8486(98)00437-2. Song JY, Choi JH, Choi HS, Jung SH and Park MA. 2013.
30 송준영ㆍ정승희ㆍ최혜승 Monitoring of Kudoa septempunctata in cultured olive flounder during 2012. J Fish Pathol 26, 129-137. https://doi. org/10.7847/jfp.2013.26.3.129. Song JY, Kim MJ, Choi HS and Jung SH. 2014. Monitoring Kudoa septempunctata in Cultured Olive Flounder Paralichthys olivaceus in Different Regions of Korea in 2013. Korean J Fish Aquat Sci 47, 611-621. https://doi.org/10.5657/ kfas.2014.0611. St-Hilaire S, Ribble C, Whitaker DJ and Kent ML. 1997. Evaluation of a nondestructive diagnostic test for Kudoa thyrsites in farmed Atlantic salmon (Salmo salar). Aquaculture 156, 139-144. https://doi.org/10.1016/s0044-8486(97)00081-1. Yokoyama H, Whipps CM, Kent ML, Mizuno K and Kawakami H. 2004. Kudoa thyrsites from Japanese flounder and Kudoa lateolabracis n. sp. from Chinese sea bass: causative myxozoans of post-mortem myoliquefaction. Fish Pathol 39, 79-86. https://doi.org/10.3147/jsfp.39.79.