36 6 Vol.36, No.6 2012 6 JOURNAL OF FISHERIES OF CHINA June, 2012 文章编号 : 1000-0615(2012)06-0937-07 DOI:10.3724/SP.J.1231.2012.27674 虾夷扇贝脓胞病病原的分离 鉴定与致病性, *,,,,,, (,,, 116023) :, 16S rrna 19 1.0910 5 CFU/mL, 16S rrna : ; ; : S 944.3 : A 20 80 (Patinopecten yessoensis) [1], [2] 2009 2010,,, 80%,, [3-5], 16S rrna,,, 1 1.1, 1.2, 5,, 2216E [6] 25 48 h ;, [7-13] 1.3 病原菌的培养与定量, 15 000g 30 min, 10 2216E, 25 24 h,, 30, 600 nm, 1.09 10 7 CFU/mL 浓度梯度感染实验 2010 5, 8 cm 2 300, 10 70 L, 50%, 1 7 d, 0.5~1.0, 19, 7 d 150, 10, 15, 5 ( 1 ), 4 0.1 ml, 10 0 10 1 10 2 10 3, 5, 收稿日期 : 2011-8-10 修回日期 : 2011-11-1 资助项目 : (200805037); (2008203004) 通讯作者 :, E-mail:wenji1016@yahoo.com.cn
938 36,, 温度梯度感染实验 2010 10, 8 cm 2 240, 12, 20 4, 1517 1921 3, 2 7 d, 0.5~1.0,, 7 d 10, 1.09 10 5 CFU/mL 0.1 ml,,, 1.4 形态特征检查, 生理生化鉴定 [3-5], [8] [9] 分子测序及比对 16S rrna PCR, DNA 16S rrna Gen- Bank BLAST,, DNAStar 1.5 4 Vibrio chagasii, HX2 1 2 2.1,,,,, ( 1), 2.2 6, XJ01-XJ06, XJ02 图 1 患病虾夷扇贝外观 Fig. 1 Appearance of sick P. yessoensis 2.3 浓度感染试验结果 31 d, 100%, 1, 1 ( 1.0910 7 CFU/mL) 24 h,,, 3 ( 1.0910 5 CFU/mL), 40%, ( 12 ) ( 4 ), 1.09 10 5 CFU/mL, 温度感染试验结果 28 d,, 2 2, 19,,, / delivery volume / dead volume / symptoms volume 表 1 浓度感染试验结果 Tab. 1 Concentration infection s of the experiment injection group 1 (10 0 ) 2 (10 1 ) 3 (10 2 ) 4 (10 3 ) group 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 1 0 0 4 3 6 6 1 2 0 0
6, : 939 /N delivery volume /N dead volume //N symptoms volume 1(10 0 ) 2(10 1 ) Tab. 2 表 2 温度感染试验结果 Temperature infection s of the experiment 15 17 19 21 1(10 0 ) 2(10 1 ) 1(10 0 ) 2(10 1 ) 1(10 0 ) 2(10 1 ) 10 10 10 10 10 10 10 10 10 10 10 10 10 10 0 10 10 0 10 10 1 10 10 1 0 0 0 0 1 0 3 2 0 1 1 0 19,,, 病原菌的再分离,, XJ02 2.4 病原菌的外观形态特征 XJ02 TCBS 25 24 h ( 2),,,,,,, ( 3) 图 2 XJ02 在 TCBS 培养基上生长状况 Fig. 2 Growth situation on TCBS of XJ02 病原菌的理化性状 XJ02, L-D-D, L-, ( 3) 菌株 XJ02 的 16S rrna 扩增 测序及分析, 1 1 438 bp 16S rrna ( 4) GenBank BLAST,,, 图 3 XJ02 电镜图片 Fig. 3 Electron microscopy images of XJ02 2.5 22, 20, ( 4) 3,,, [15], 20 [16],, 20~30 [17], [18] (V. fluvialis),
940 36 cell morphology nitrate reduction α- α-cyclodextrin dextrin glycogen Tween40 Tween80 N- N-acetylgalactosamine N- N-acetylglucosamine ribitol L- L-arabinose D- D-a sugar alcohol D- D-cellobiose i- I-erythritol D- D-fructose L- L-fructose D- D-galactose gentiobiose α-d- α-d-glucose m- m-inositol α-d- α-d-lactose lactulose Tab. 3 short bar 表 3 分离菌株的生理生化指标 Separate strains physiological and biochemical indexes gram stain amylohydrolysis D- D-melibiose methyl glucoside D- D-psicose D- D-gossypose L- L-rhamnose D- D-sorbitol sucrose D- D-trehalose turanose xylitol acid methyl ester methyl succinic acid ethanoic acid - trans-aconic acid citric acid formic acid D- D-galactose acid lactone D- D-galactose uronic acid D- D-sour grapes β- β-methyl Glucoside D- D-glucuronic acid catalase Vp Vp p- p-mandelic acid itaconic acid α- α-4-oxobutanoic acid α- α-oxygen glutaraldehyde α- α-oxopentanoic acid D, L- D, L-lactate malonic acid propionic acid quetiapine in acid D- D-hexonic acid sebacic acid succinic acid bromide succinate succinic acid glucuronic acid amide L- L-Acrylic amide D- D-alanine L- L-alanine L- L-alanyl glycine L- L-asparagine L- L-aspartic acid oxidase hydrolyzed gelatin L- L-histidine hydroxy proline L- L-leucine L- L-ornithine L- L-phenylalanine L- L-proline L- L-pyroglutamic acid D- D-serine L- L-serine L- L-threonine D, L- D, L-carnitine g- g-amino butyric acid irocanic acid inosine uridine thymidine phenyl ethylamine putrescine 2-2-amino ethanol 2, 3-2, 3-butanediol glycerine maltose α- α-butyric acid L- L-glutamic acid D, L- D, L-glycerol phosphate D- D-mannitol β- β-butyric acid glycyl aspartic acid D--1- D - glucose-1-phosphate D- D-mannose : +; Notes: + is positive; is negative. g- g-butyric acid glycyl glutamate D--6- D-glucose-6-phosphate
6, : 941 图 4 查氏弧菌 16S rrna 基因序列 Fig. 4 16S rrna gene sequence of V. chagasii 表 4 分离菌株的药物敏感性试验结果 Tab. 4 The sensitivity of the isolated strains to test chemotherapentants drug name /(μg/ml) dose sensitivity drug name /(μg/ml) dose sensitivity benzylpenicillin 8 S gentamicin 4 S ampicillin 8 S amikacin 16 S cefuroxime 8 S ciproflxacin 1 S cefotacime 8 S ofloxacin 4 S ceftazidime 8 S norfloxacin 4 S cefepime 8 S levofloxacin 2 S imipenem 4 S SXT 2/38 S meropenem 4 S tetracycline 16 R / PIZ 64/8 S doxyxycline 16 R / SAM 1 S chloramphenicol 8 S / CPS 16/16 S erythromycin 0.5 S : R ; S Notes: R is resist; S is sensitive.,,, TCBS, TCBS, 16S rrna,,,,,,,,
942 36 19, 79, 19~21 2009 7, 80%, 2 20.1,,, 19~21, (19, 1.0910 5 CFU/mL), 56 1011,,,, [14],,, ;,,,,,,,,,, : [1],,,. [J]., 2008, 27(1): 912. [2],. [J]., 2009, 28(10): 609612. [3]. [M]. :, 1978: 98193. [4],,,. [M]. :, 1992: 10110. [5] Holt J G, Krieg N R, Sheath P H, et al. Bergey S manual of determinative bacteriology(9th edition)[m]. Baltimore: William and Wilkins, 1994: 183217. [6],. [M]. :, 1985: 3536. [7]. [M]. :, 1992: 34. [8],. [M].:, 2001: 162171. [9] R E, N E. [M]. 8. :, 1984: 482486. [10] Buller N B. Bacteria from fish and other aquatic animals: A practical identification manual[m]. UK:CABI Publishing, 2004: 243 [11],. [J]., 2003, 38(4): 2427. [12],,,. [J]., 2005, 27(5): 137 144. [13],,,. [J]., 2006, 28(5): 90100. [14],,,. [J]., 2006, 21(1): 1318. [15],,,. [J]. :, 2008, 27(3): 327334. [16],,. [J]., 2002, 21(3): 372376. [17]. [C]/[M]. :, 1998: 670679. [18],. [J]., 1998, 22(3): 6166.
6, : 943 Isolation, identification and pathogenicity of Vibrio chagasii from Patinopecten yessoensis TENG Wei-ming, LI Wen-ji *, ZHANG Ming, YU Zuo-an, LI Shi-lei, LIU Xiang-feng, LI Hua-lin, FU Cheng-dong (Liaoning Open Laboratory of Applied Marine Biotechnology, Liaoning Key Laboratory of Marine Fisheries Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China) Abstract: In recent years cultured sea scallop (Patinopecten yessoensis) dead on a large-scale in seas near Changhai County, Dalian, China, in summer. The main symptom is the abscess on adductor muscle. The transmission experience was improved to indicate that a bacterium isolated from natural infected focus, named XJ02 displayed intense pathological activity to scallops, and the symptom is the same to natural intention. The bacterium XJ02 identification test through morphological specificity, physical and chemical properties and 16S rrna gene sequence analysis showed the bacterium is Vibrio chagasii. There has been no any research on the pathogenicity of V. chagasii. This article for the first time to find a more comprehensive's vibrio physicochemical properties of research. The dose-and temperature-dependent test indicated that the bacterium had the most pathogenicity at 19 while the concentration was 1.09 10 5 CFU/mL. The medicine sensitivity analysis that s to check Ampicillin, Benzylpenicillin, Ciproflxacin etc is sensitive during 20 drugs sensitive test, to Tetracycline, Doxycycline is not sensitive element. Key words: Patinopecten yessoensis; Vibrio chagasii; abscess Corresponding author: LI Wen-ji. E-mail: wenji1016@yahoo.com.cn