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1 微生物学通报 JAN 20, 2009, 36(1): 71~77 Microbiology 2009 by Institute of Microbiology, CAS 研究报告引起文蛤暴发性死亡病原菌的分离和鉴定刘连生 1,2 闫茂仓 2 林志华 2 柴雪良 2 汤洁 1 赵海泉 1* ( ) ( ) 摘要 : 本文从江苏吕泗文蛤养殖区发病文蛤和养殖水体中分离到 5 株优势菌 ( 病蛤中分离到 3 株优势菌, 养殖水体中分离到 2 株优势菌 ), 人工感染实验结果显示, 菌株 WG1702 能引起供试文蛤发病, 死亡率为 100%, 且发病症状与原发病症状相同, 提示该菌是引起文蛤大面积死亡的主要致病菌, 对其形态生理生化特征分类地位及致病性进行了初步研究, 菌株 WG1702 为革兰氏阴性菌, 直杆状, 生理生化指标为 : 赖氨酸脱羧酶鸟氨酸脱羧酶硝酸盐还原甘露糖氧化酶甲基红阳性, 精氨酸脱羧酶精氨酸双水解酶水杨素 V.P 阴性为确定该致病菌的分类学地位, 进行了 16S rrna 分子鉴定, PCR 扩增获得了大小约 1.5 kb 的 16S rrna 部分基因片段, 对其序列进行了测定, 并进行同源性分析和系统进化树构建结果表明, 该菌株与哈氏弧菌 (DQ146937) 同源性最高, 为 97.4%, 结合形态生理生化鉴定结果, 最后鉴定菌株 WG1702 为哈氏弧菌 (Vibrio.harveyi) 关键词 : 文蛤, 哈氏弧菌, 16S rrna, 系统发育 Pathogens Isolation and Identification of Eruptive Epidemic Disease in Meretrix meretrix LIU Lian-Sheng 1, 2 YAN Mao-Cang 2 LIN Zhi-Hua 2 CHAI Xue-Liang 2 TANG Jie 1 ZHAO Hai-Quan 1* (1. Faculty of Life Science, Anhui Agricultural University, Hefei, Anhui , China) (2. Zhejiang Mariculture Reseach Institute, Wenzhou, Zhejiang , China) Abstract: Five kinds of bacteria were isolated from disease Meretrix meretrix and culture water (two isolated from the M. meretrix, three from the culture water) in Lvsi harbor, Jiangsu province. The results of infection showed that strain WG1702 can cause disease with the mortality rate 100 percent and the same symptoms as original. It was cued that strain WG1702 was the pathogenic bacteria and caused the death in large scare, a preliminary research was done on the bacteria including morphological, physiological, biochemical characteristics, phylogenetic position and pathogenic. Strain WG1702 was a straight-rod gram negative bacterium, the physiological and biochemical indicators were lysine decarboxylase, ornithine decarboxylase, reduction of NO 3 to NO 2, mannitose, oxidase and methylred reaction positive, arginine decarboxylase, arginine dihydrolase, salicin and voges-proskauer negative. In order to identify the bacteria phylogenetic position and a 16S rrna molecular identification was done, PCR amplification got a size of * 通讯作者 : : swjs12@ahau.edu.cn 收稿日期 : ; 接受日期 :

2 72 微生物学通报 2009, Vol.36, No.1 about 1.5 kb gene fragment, The whole 16S rrna sequences were determined, analyzed the homology of sequences and built the phylogenetic tree. Sequences analysis revealed that strain WG1702 exhibited the highest level of 97.4% similarity to Vibrio harveyi(dq146937), combining morphological, physiological and biochemical identification results, the isolated strain WG1702 was identified as Vibrio harveyi. Keywords: Meretrix meretrix, Vibrio Harveyi, 16S rrna, Phylogenetic analysis (Meretrix meretrix Linnaeus),,,,,,, hm 2, 6.0 l0 5 t, 2.0 l0 5 t [1],,,, 20~30 [2] (Vibrio),,,, [3], (Vibrio parahaemolyticus) [4 7] (Vibrio furnissii) [8], [9] (Vibrio alginolyticus),,,,, 1 材料与方法 1.1 发病文蛤症状 , ( 2 cm ),,,,,,,, 1.2 分离方法, 70%,,, zobell 2216E TCBS, 28 C 24 h,, 4 C 1.3 人工感染试验 20 L, 28 C, 26.4, ph 8.2,, 16 2 cm 1, 10 8 CFU/mL, 1 ;, ; 12 d,, 1.4 细菌鉴定传统的细菌生理生化方法鉴定 : [10], ( ) 分子生物学方法鉴定 : 24 h 10 8 CFU/mL, 1 ml, r/min 1 min, DNA, DNA; 16S rrna 16S rrna PCR 27F 5 -AGAGTTTGAT CCTGGCTCAG-3, E. coli 16S rrna 8-27, 1492R 5 -TACGGCTA CCTTGTTACGACTT-3, E. coli 16S rrna PCR (20 μl):sterilized ddh 2 O 13.6 μl, 10 PCR Buffer 2 μl, 2 mmol/l dntps 0.3 μl, 0.4 μl, 5 U/50 μl Taq DNA 0.1 μl, 25 mmol/l MgCl μl, DNA 2 μl PCR 96 C 6 min; 94 C 1 min, 55 C 1 min, 72 C 2 min, 30 ; 72 C 6 min, 1.0% PCR 序列分析与数据处理 : 16S

3 : 73 rrna Blast GenBank, Clustal V DNA Star 1.5 药敏试验, zobell 2216E 24 h, ( ), 19 2 结果与分析 2.1 细菌分离及其形态特征, 2 WS1501 WS WG1701 WG1501 WG1702,,,, TCBS( - - ) 18 h, WG1702, 18 h, 3 mm~7 mm; WS1502 WG1501, 1 mm~4 mm; WS1501 WG1701, 3 mm~5 mm; 5 zobell 2216E 2.2 人工感染试验 2, 5, Group 表 1 人工感染试验结果 Table 1 Results of infection The time and the number of death Numbers 1 d~6 d 7 d 8 d 9 d 10 d 11 d 12 d Mortality WS WS WG WG WG Contral items Table 2 表 2 菌株 WG1702 药敏试验结果 Results of the strain WG1702 antibiotics experiments Chemical Diameter(mm) Sensitivity (AN) S (NOR) M (SXT) S (RIF) M (CEF) M (CED) S (CRO) S (CFP) S (STR) S (GEN) S (CEX) M G(PCN) R (TE) 9.21 R (NEO) S (KAN) S (AMP) M S: ; M: ; R: ; :. Note S: Highly sensitive; M: Middlely sensitive; R: Restrain; : No-inhibitory zone.

4 74 微生物学通报 2009, Vol.36, No.1 Table 3 表 3 分离菌株的生理生化特性 Physiologlcal and biochemical characteristics of bacteria isolated Test items WG1702 [11] (V. harveyi) Gram stain V TCBS Test items / / Prgment O/F Oxidation/Fermentation F F O/129(10 μg)o/129 Sensitivity R S O/129(150 μg)o/129 Sensitivity S S 4 C Growth at 4 C 10 C Growth at 10 C 30 C Growth at 30 C 40 C Growth at 40 C D 0%NaCl Growth in 0% NaCl 1%NaCl Growth in 1% NaCl 6%NaCl Growth in 6% NaCl 8%NaCl Growth in 8% NaCl D 10%NaCl Growth in 10% NaCl D Salicin D Oxidase Catalase Arginine Decarboxylase Arginine Dihydrolase Iysine Decarboxylase Ornithine Decarboxylase WG1702, 100%, WS1502, 12.50%; WG d,, 9 d WG 药物敏感性试验 16,, WG1702 9, 5, 2.4 分离菌株生化特性 WG C 4 C,, 8% NaCl, V.P,, O/129(150 μg),, WG1702 (V. harveyi) 2.5 PCR 扩增 16S rrna 图谱 PCR 1.0%, kb Gas from Glucose Arabinose D Cellobiose Sucrose D Lactose Mannose Inositol KCN D- D-Manicol Nitrate Reduction Gelatine Liquefication D Indole Production Methyl Red V.P Voges-Proskauer +: ; : ; R: ; S: ; F: ; D:. Note: +: Positive; : Negative; R: Resistant; S: Sensitive; F: Ferment; D: Some strains are positive and some negative. 图 1 PCR 扩增菌株 WG S rrna 序列 Fig. 1 PCR amplification of strain WG S rrna sequences 2.6 测序结果及同源性分析 GenBank, 89.2%, 5.7% WG1702 DQ S rrna, 97.4%, AJ874352, 96.0%,

5 : 系统发育分析 GenBank, 100, 86%, WG S rrna 16S rrna, WG1702 DQ 图 2 菌株 WG1702 核苷酸序列同源性和遗传距离分析结果 Fig. 2 Homology and Genetic Relationship results of the strain WG1702 nucleotide sequences 图 3 16S rrna 序列的系统发育树 Fig. 3 Phylogenetic tree of 16S rrna sequences

6 76 微生物学通报 2009, Vol.36, No.1, 16S rrna 97.4%, WG1702, 3 讨论 16S rrna, WG1702, WG1702 [11], WG1702 (Vibrio harveyi), [12] [13] [14] ; 16S rrna NCBI Blast, 16S rrna, WG1702 DQ146937, 97.4%, WG1702 (V. harveyi) WG S rrna, AJ874352(V. natriegens),, TCBS ;, TCBS, 24 h,,,,, [15], GenBank 16S rrna,, [16 20],,, ,,,,,,,,,,,, [12], 100%,, WG1702,, WG1702 9, 5, G,,,,,, ( ),, 参考文献 [1],,,.., 2005, 24(2): [2].. :, 1998, pp [3],,,.., 2000, 21(3): [4] Sen YL, Yu YS. Pathogenicity of vibrio paraheamolyticus to healthy clam Meretrix meretrix and the prevention and treatment of vibrio infections. Fisheries China, 1993, 17(3): [5]. vibrao paraheamolyticus. ( ), 1978, pp [6],,,.., 1996, 16(4): 1 5. [7].., 1996, 23(2): [8],,,.., 1992, 19(4): [9],,,. ( )., 1991, 15(2): [10] John GH, Noel RK, Peter HAS, et al. Bergey s manual of

7 : 77 determinative bacteriology. Baltimore: Williams & Wilkins Press, 1994, pp [11] Baumann P. Genus Vibrio In: Holt JC. Bergey s Manual of Systematic Bacteriology. Wilkins: Baltimore & Williams, 1984, pp [12],,,.., 2003, 9(5): [13],,,.., 2005, 15(3): [14],,,.., 2008, 15(2): [15] Bruno Gomez-Gil, Sonia Soto-Rodriguez, Alejandra Garcia-Gas-ca, et al. Molecular of identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms. Microbiology, 2004, 150(6): [16],,.., 1993, 8(2): [17],,,.. ( ), 1999, 18(4): [18],,,.., 2000, 18(3): [19].., 2001, 40(1): [20],,.. ( ), 2005, 24(2): 中国科学院微生物研究所期刊广告部成立编辑部公告 , ( 8107 ),,,, (CA)(BA) (MEDLINE ) (AJ), () ( PCR ) ( ),,,, , 提示 : 2007, 收款单位 : 开户银行 : 帐号 : 联系电话 : ; 联系人 : 电子信箱 :gg@im.ac.cn 网址 :

5 : 1061, 1.0 10 8 CFU/mL, 100 200 g,, 0.4 ml/, 5, 28 30, 10d 1.4 (NA) 30 18h,, JEM-1011 ( ) 1.5 16SrDNA [11] (8f: 5 -AGAGTTTGAT- CCTGGCTCAG-3, 1492r:

5 : 1061, 1.0 10 8 CFU/mL, 100 200 g,, 0.4 ml/, 5, 28 30, 10d 1.4 (NA) 30 18h,, JEM-1011 ( ) 1.5 16SrDNA [11] (8f: 5 -AGAGTTTGAT- CCTGGCTCAG-3, 1492r: 34 5 Vol. 34, No.5 2010 9 ACTA HYDROBIOLOGICA SINICA Sep, 2 0 1 0 DOI: 10.3724/SP.J.1035.2010.01060 1,2 沈文英 2 李卫芬高 2 研 (1., 312000; 2., 310029) ISOLATION AND DRUG SENSITIVITY STUDY OF PATHOGENIC BACTERIA