37 2 Vol. 37, No.2 2013 3 ACTA HYDROBIOLOGICA SINICA Mar., 2 0 1 3 doi: 10.7541/2013.33 1 杨 移 斌 2 夏 永 涛 3 郑 卫 东 1 胡 鲲 1 杨 先 乐 (1., 201306; 2., 100039; 3., 430223) ISOLATION AND IDENTIFICATION OF YERSINIA RUCKERI FROM ACIPENSER BAERII AND ITS ANTIBIOTIC SENSITIVITY YANG Yi-Bin 1, XIA Yong-Tao 2, ZHENG Wei-Dong 3, HU Kun 1 and YANG Xian-Le 1 (1. State Collection Center of Aquatic Pathogen, Shanghai Ocean University, Shanghai 201306, China; 2. Chinese Academy of Fishery Sciences, Beijing 100039, China; 3. Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 4310223, China) : ; 16S rdna; ; Key words: Yersinia Ruckeri; 16S rdna; Antibiotic sensitivity; Acipenser baerii 中 图 分 类 号 : S941 文 献 标 识 码 : A 文 章 编 号 : 1000-3207(2013)02-0393-06,,, (Acipenseriformes), 26 [1], 2 3 8, (Acipenser sinensis) (Psephurus gladius) (Acipenser dabryanus); (Acipenser schrenckii) (Huso dauricus); (Acipenser nudiventris) (Acipenser ruthenus) (Acipenser baerii) [2],,,, 18% 29%,,, 1992,, 1 [2],,, (Aeromonas hydrophila) (Aeromonas carvia) (Plesimonas shigelloides) [3 6], [7,8] 2012 6,,,,,,,,,,,,, 16S rdna,,, 收 稿 日 期 : 2012-08-06; 修 订 日 期 : 2012-12-27 基 金 项 目 : - (ZD-2012-345- 6); ( ) (201203085); 863 (2011AA10A216); (31172430); ( (2011) 4-8 ) 作 者 简 介 : (1988 ),, ; ; E-mail: yangyb988@126.com 通 信 作 者 : (1948 ), E-mail: xlyang@shou.edu.cn
394 37 1 1.1,, 5% (MH) ; ; ; DNA PCR 16S rdna, TaqDNA ; 1.2,,,,, 75%,,, 28 24 48h,,,, 4, 1 1 6, 4, 20 22, HD0923 28 24h,, 4 10 7 CFU/mL,, 0.2 ml,,, HD0923, 28 24h,,, [9] HD0923 16S rdna (1) 16S rdna : (BHI), 28 18h, 12000 r/min, DNA DNA, PCR DNA (2)16S rdna : 16S rdna PCR 27F: 5 -AGAGTTTGATC(C/A)TGGCTC- AG-3 ( E. coli 16S rrna 8 27 bp ) 1492R: 5 -GGTTACCTTGTTACGACTT-3 ( E. coli 16S rrna 1492 1510 bp ) PCR : 95 3min, 94 35s, 55 35s, 72 1min, 35, 72 10min PCR 1.0% PCR, PCR (3) : HD0923 16S rdna NCBI 16S rdna, 17 5, Clustal w, MEGA5.1 Neighbor-Joining, 1000 Bootstrap K-B, HD0923 (BHI), 28 18h (MH) 17, 5, 28 24h [10] 2 2.1 2012 6 8 3,, 80% 20 22,,,,,,,,,,,,,,, 3 cm,,,,,,,,
2 : 395 2.2 5, 28 24h, HD0923,,,,, 1 mm, 2.3 24h,,,,, 66.7%,,,,,,,,,,, HD0923 16S rdna, HD0923 2.4 HD0923,,,,,,, 0.7 1 mm;,,,, 1 1.3 mm ; 5% 2.5 HD0923 18 36, 24 31, 28 29 ph 4 10, ph 5.0 8.0, ( 1) 2.6 16S rdna PCR PCR HD0923 16S rdna 1500 bp ( 1) 1416 bp, GenBank 16S rdna, 16S rdna (Y. ruckeri), 100%, 16S rdna Neighbor- Joining, ( 2) HD0923 (Y. ruckeri), 100%, 16S rdna, (Y. ruckeri) Tab. 1 表 1 HD0923 菌 株 的 生 理 生 化 特 征 Physiological and biochemical characteristics of HD0923 Test item HD0923 Y. ruckeri Indole test Methyl red + + V-P Voges-Proskauer test H 2 S Gelatin liquefaction + + Motility + (+) KCN Growth in KCN d Urea Lysine + (+) Arginine Ornithine + + Citrate Esculoside Malonic acid Xylose Maltose + + Arabinose (+) Trehalose + + Fructose Sucrose Raffinose Cellobiose Rhamnose Inositol Sorbitol + Mannitol + + D- D-adonitol Melibiose Salicin Lactose D- D- glucose aerogenesis D- D glucose acid production + + : + - ; - ; (+) - 24h, ( ) - 24h; d - 11% 89% Note: + -positive; - negative; (+) The time of Positive reaction exceed 24h; ( ) The time of negative reaction exceed 24 h; d -11% 89% strain is positive 2.7 HD0923 17 ( 2), HD0923 V 10,,
396 37 3,, (Aeromonas hydrophila) (Aeromonas caviae) (Plesiomonasshigelloides) [3 6], [8],, 1 HD0923 16S rdna PCR Fig.1 The result of 16SrDNA PCR amplification of HD0923 strain A: PCR PCR amplification products; M: DNA Marker DL5000 2 16S rdna Fig. 2 The phylogenetic tree based on 16S rdna sequence homolog HD0923,,,, ;, HD0923, HD0923 HD0923 16S rdna (Y. ruckeri), [11],,,,,,,, [12],,,,, DNA rdna (G+C) mol% 16S rdna Gyrb 16S rdna, [13, 14] 16S rdna NCBI,, 16S rdna,, HD0923 100%,, HD0923 (Enterobacteriaceae) (Yersinia), [15],, [16], 1952 [15],,,,, 20 22, [17,18],,
2 : 397 Tab.2 表 2 HD0923 药 敏 试 验 结 果 Antibiotic sensitivity test of strain HD0923 Drug The judgement standard of inhibition zone diameter (mm) Resistant Medium sensitivity Sensitive Dose (μg/disc) Inhibition zone diameter (mm) Sensitivity Deoxycycline 12 13 15 16 30 25 Amikacin 14 15 16 17 30 24 Florfenicol 12 13 17 18 75 30 Streptomycin 11 12 14 15 10 18 Chloramphenicol 12 13 17 18 30 17 Claforan 14 15 22 23 30 15 V Xianfeng Meisu V 14 15 17 18 30 19 Penicillin 19 20 27 28 10U 0 Erythromycin 13 14 22 23 15 21 Clindamycin 14 15 20 21 2 0 Amoxicillin 13 14 17 18 10 21 Azithromycin 13 14 17 18 15 22 Rifampicin 16 17 19 20 5 19 Levofloxacin 13 14 16 17 5 34 Furazolidone 14 15 16 17 300 20 Gentamycin 12 13 14 15 10 21 Neomycin 12 13 16 17 30 15,,, [12],, 20,, 28, [19] 5%, [12],,,, HD0923 V 10,, [12],, [20],, HD0923, HD0923,, HD0923,,,,,,, [21], [22],,,,, : [1] Ruban G I. Species structure, contemporary distribution and status of the siberian sturgeon, Acipenser baerii [J]. Envi-
398 37 ronmental Biology of Fishes, 2002, 17: 221 230 [2] Cui H, He J X, Zheng W Z. Analysis and development proposals about the status of the our sturgeon industry [J]. China Fisheries, 2006, (6): 8 10 [,,.., 2006 (6): 8 10] [3] Yang Z G. Isolation and identification of the Aeromonas hydrophila pathogen the sturgeon [J]. Freshwater Fisheries, 2001, (5): 40 41 [.., 2001, (5): 40 41] [4] Meng Y, Xiao H B, Zhang L, et al. Isolation and identification of the hemorrhagic septicemia pathogen of Amur Sturgeon, Acipenser schrenckii Brandt [J]. Journal of Huazhong Agricultural University, 2007, 26(6): 822 826 [,,,.., 2007, 26(6): 822 826] [5] Li Y Y, Cao H P, He S, et al. Isolation and identification of Aeromonas hydrophila strain X1 from Acipenser baerii and its antibiotic sensitivity [J]. Microbiology, 2008, 35(8): 1186 1191 [,,,. X1., 2008, 35(8): 1186 1191] [6] Cao H P, Yang X L, Gao P, et al. Preliminary study of the pathogens isolated from bacterial septicaemia syndrome of sturgeons [J]. Freshwater Fisheries, 2007, 37(2): 53 56 [,,,.., 2007, 37(2): 53 56] [7] Roy P E, Yanong Ruth. Francis-Floyd. Streptococcal infections of fish [J]. Circular, 2006, 57: 1 6 [8] Pna H J, Liu X Y, Chang O Q, et al. Isolation,identification and pathogenicity of Streptococcus dysgalactiae from Siberian Sturgeon, Acipenser baerii [J]. Fishery Sciences of China, 2009, 16(6): 891 904 [,,,.., 2009, 16(6): 891 904] [9] Dong X Z, Cai M Y. General Manual of Systematic and Determinative Bacteriology [M]. Beijing: Science Press. 2001, 84 85 [,.. :. 2001, 84 85] [10] Wang S J. Modern Diagnostic Handbook [M]. Beijing Medical University and China Peking Union Medical College Joint Press. 1995, 511 522 [.. :. 1995, 511 522] [11] Li L, Li J N, Yu W Y. Research progress about bacterial classification and identification [J]. Anhui Agricultural Sciences, 2004, 32(3): 549 551 [,,.., 2004, 32(3): 549 551] [12] Fan F L, Wang K Y, Geng Y, et al. Isolation, identification and phylogenetic analysisof Yersinia ruckeri in channel catfish Ictalunes punctatus [J]. Oceanologia et Limnologia Sinica, 2010, 41(6): 862 868 [,,,. (Ictalunes punctatus)., 2010, 41(6): 862 868] [13] Eddy F, Powell A, Gregory S, et al. A novel bacterial disease of the European shore crab, Carcinus maenas molecular pathology and epidemiology [J]. Microbiology, 2007, 153: 2839 2849 [14] Zhou S M, Li A X, Ma Y, et al. Isolation,identification and characteristics of 16S rdna gene sequences of the pathogens responsible for the Streptococcicosis in culturedfish [J]. Acta Scientiarum Naturalium Universitatis Sunyatseni, 2007, 46(2): 68 71[,,,. 16S rdna., 2007, 46(2): 68 71] [15] Huang Q Y(Eds.). Disease of Aquatic Animals [M]. Shanghai: Shanghai Science and Technology Press. 1998, 103 139 [ ( ).. :. 1998, 103 139] [16] Xu B H, Yin Z, Chen Y, et al. A new infectious disease Yersinia ruckeri of silver carp and bighead carp, a new silver carp, bighead carp pathogens [J]. Chinese Science Bulletin, 1991, 82(8): 620 622 [,,,. Yersinia ruckeri., 1991, 82(8): 620 622] [17] Zhang X J. Yersinia ruckeri and relative infection of fish [J]. Journal of Hebei Normal University of Science & Technology, 2004, 18(3): 77 80 [.., 2004, 18(3): 77 80] [18] Danley M L, Goodwin A E, Killian H S. Epizootics infarmraised channel catfish, Ictalurus punctatus (Rafinesque), caused by the enteric redmouth bacterium Yersinia ruckeri [J]. Journal of Fish Diseases, 1999, 22: 451 456 [19] Fernandez L, Prieto M, Guijarro J A. The iron- and temperature-regulated hae molysin YhlA is a virulence factor of Yersinia ruckeri [J]. Microbiology, 2007, 153 b: 483 489 [20] Li A H, Cai T Z, Wu Y S, et al. Investigation on drug resistance of fish bacterial pathogen Aeromonas hydrophila in china [J]. Microbiology, 2001, 28(1): 58 63 [,,,.., 2001, 28(1): 58 63] [21] Ellis A E.Immunity to bacteria in fish [J]. Fish & Shellfish Immunology, 1999, 9: 291 308 [22] Martin K R, Kurt B. Innate immune response in rainbow trout (Oncorhynchus mykiss) against primary and secondary infections with Yersinia ruckeri [J]. Developmental & Comparative Immunology, 2009, 33(1): 35 455