Microsoft Word - 邴旭文.doc

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1 40 6 Vol.40, No OCEANOLOGIA ET LIMNOLOGIA SINICA Nov., 2009 (Misgurnus anguillicaudatus) (Vibrio cholerae) * ( ; ),, ; PCR 16S rrna gyrb, 16S rrna gyrb,,, ; 16S rrna gyrb (LD081008B-1) 16S rrna gyrb GenBank, gyrb ; 16S rrna 1446bp (GenBank : GQ205447), gyrb 1207bp (GenBank : GQ205452);, (Vibrio Pacini 1854)(Vibrio cholerae) DNA, 7%β, 49, 48,,, 16S rrna, gyrb Q346 (Vibrio cholerae) (Vibrionaceae V ron 1965) (Vibrio Pacini 1854), (type species), (cholera) O1, Muroga (1979) Kiiyukia (1992), Amano ; Reddacliff (1993) ; (1986) ; (1997) ; (1993) ; 劼 (2006) 鲀, O1,, ,, 3, 1, 16S rrna gyrb * BZ CN0826 [email protected] : , :

2 6 : (Misgurnus anguillicaudatus) (Vibrio cholerae) 693,, (Misgurnus anguillicaudatus), ;, 1.2, (28 48h ), (28 24h), LD081008B LD081008B-1, 28 ( CFU/ml),, 6, 0.2ml; ;,, h, ;, 28 24h,, 1 Formvar, 1min, 0.5% 1 1min,, JEM-100CX (TCBS), 28 24h,, ( ) H 2 S MR V-P OF (Simmons), (, 2001) (, 2003) 1.5, 4 (10%) (10%) 80(1.0%) (1%) DNA (0.5%), (8%), 28 24h 80 DNA ; 28 24h, 4 ( 7%), 28 24h, S rrna gyrb PCR DNA LB 28 16h, DNA ( ) DNA PCR DNA S rrna PCR 16S rrna PCR 27F( ) 5 -AGA GTT TGA TC(C/A) TGG CTC AG-3, 1492R( ) 5 -GGT TAC CTT GTT ACG ACT T-3 (Martin et al, 1998) 20μl 14.4μl, 10 PCR 2μl, 1.5mmol/L MgCl 2 1.6μl, 4 dntp 0.4μl, 0.2μl, 2.5U/μl Taq DNA 0.2μl, DNA 1μl PCR 95 3min 94 1min 55 1min 72 2min, min PCR gyrb PCR gyrb PCR UP1( ) 5 -GAA GTC ATC ATG ACC GTT CTG CAY GCN GGN GGN AAR TTY GA-3, UP2r( ) 5 -AGC AGG GTA CGG ATG TGC GAG CCR TCN ACR TCN GCR TCN GTCAT-3 (Yamamoto et al, 1995) 20μl

3 μl, 10 PCR 2μl, 1.5m mol/l MgCl 2 1.6μl, 4 dntp 0.4μl, 0.2μl, 2.5U/μl Taq DNA 0.2μl, DNA 1μl PCR 94 5min, 94 1min 57 1min 72 2min 30, 72 7min PCR S rrna gyrb 16S rrna gyrb NCBI Blast ( gov/blast/), Clustal X GenBank (Multiple Alignments), MEGA3(Molecular Evolutionary Genetics Analysis, MEGA), (neighbor joining method), Bootstrap (1000 ) 1.7, Bergey s Manual of Determinative Bacteriology. 9th ed (Holt et al, 1994) Bergey s Manual of Systematic Bacteriology (Krieg et al, 1984), 16S rrna gyrb, 1.8, (K-B) 49, (, 1997) , 7, 22000kg/, ,,, 40kg 3 38, 1,,, ;, 2.2, ( ) μm ( )μm, 24h 1.0mm 48h 1.5mm 2.3 (LD081008B-1) CFU/ml, 48h, 14 ;, (LD081008B-1) 2.4, (LD081008B-1) EM, ( 1) 4 ; TCBS, 24h, 48h ( ) mm ; ( ), 28 24h,, 4 DNA,, 7% β, ( 1) S rrna gyrb 1 LD081008B-1 (LD081008B-1) Fig.1 Electron micrograph of LD081008B-1 16S rrna

4 6 : (Misgurnus anguillicaudatus) (Vibrio cholerae) bp(GenBank GQ205447); gyrb 1207bp(GenBank GQ205452) (LD081008B-1) 16S rrna NCBI Blast, 16S rrna, (V. cholerae)(v. mimicus), 98% 99%; (V. vulnificus) (V. fluvialis), 94%; 16S rrna ( DQ233654), 2 (LD081008B-1) gyrb NCBI Blast, gyrb, (V. cholerae), 97% 98% ( 94%)( 82% 84%) ( 81% 82%) gyrb ( FJ608552), 3 表 1 分离菌的理化特性结果 Tab.1 The physiological and biochemical characteristics of isolates a a β d O-F F F d V-P d H 2 S + + α- -D- + + NaCl 0% % + + 3% + 6% d O/129 10μg S S 150μg S S +,, F, S, d 11% 89%, a Bergey s Manual of Determinative Bacteriology. 9th ed. (Holt et al, 1994) Bacterial Fish Pathogens: Disease of Farmed and Wild Fish (Austin et al, 1999)

5 LD081008B-1 16S rrna Fig.2 Phylogenetic tree based on LD081008B-1 16S rrna gene sequences EF DQ NCBI, S rrna gyrb, (Vibrio Pacini 1854)(Vibrio cholerae) ,,,,,,,,, ( 劼, 2004;, 2006;, 2008),,, 48h,, 3 LD081008B-1 gyrb Fig.3 Phylogenetic tree based on LD081008B-1 gyrb gene sequences EF FJ NCBI,,,, PCR 16S rrna gyrb, 16S rrna gyrb Blast gyrb 97% 98%,, 94%, ; 16S rrna 98% 99%,, gyrb 16S rrna,

6 6 : (Misgurnus anguillicaudatus) (Vibrio cholerae) 697 Tab.2 (μg/ ) 表 2 药敏纸片名称和规格及病原霍乱弧菌的药物敏感性 Name and type of discs and antimicrobial sensitivity of pathogenic V. cholerae (mm) (μg/ ) (mm) 1 G HS HS HS S HS HS S HS HS HS HS HS HS HS HS HS S HS HS HS HS HS HS HS / HS HS HS HS HS HS 16 75/75 36 HS HS HS HS HS HS HS HS HS HS HS HS R HS HS HS HS HS HS, O1 O1 DNA, 16S rrna gyrb, O1 O1, O1 O1 O,, 49, 45 ( 22 48mm),,,,,,,, ( O1 )., 4(1): 45 51,, :, ,, ( ). :, ,, :, 劼,,, (), 9(1): 劼,,, 鲀 O1., 30(4): , O1., 10(2): ,,, , 22(11): ,,, , 24(2): ,,, 1993.

7 , 20(3): Austin B, Austin D A, Bacterial Fish Pathogens: Disease of Farmed and Wild Fish. Third (Revised) Edition. Praxis Publishing Ltd, Chichester, UK, 30: Holt J G, Krieg N R, Sneath P H A et al, Bergey s Manual of Determinative Bacteriology. 9th Ed. Williams and Wilkins, Baltimore, , Kiiyukia C, Nakajima A, Nakai T et al, Vibrio cholera non-o1 isolated from ayu fish (Plecoglossus altivelis) in Japan. Applied and Environmental Microbiology, 58: Krieg N R, Holt J G, Bergey s Manual of Systematic Bacteriology. Volume 1. London: Williams and Wilkins, Baltimore, Martin F Polz, Collen M Cavanaugh, Bias in template to product ratios in multitemplate PCR. Appl Environ Microbiol, 64(10): Muroga K, Takahashi S, Yamanoi H, Non-cholera Vibrio isolated from diseased ayu. Bulletin of the Japanese Society of Scientific Fisheries, 45: Reddacliff G L, Hornitsky M, Carson J et al, Mortalities of goldfish, Carassius auratus (L.), associated with Vibrio cholera non-o1. Journal of Fish Diseases, 16: Yamamoto S, Harayama S, PCR amplification and direct sequencing of gyrb genes with universal primers and their application to the detection and taxomonic analysis of Pseudomonas putida strains. Appl and Environ Microbiol, 61(3): PHENOTYPIC AND MOLECULAR IDENTIFICATION OF PATHOGENIC VIBRIO CHOLERAE ISOLATED FROM MISGURNUS ANGUILLICAUDATUS BING Xu-Wen 1, YAN Bin-Lun 2, ZHANG Xiao-Jun 2, QIN Lei 2, BI Ke-Ran 2 (1. Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Science, Wuxi, ; 2. College of Ocean, Key Laboratory of Oceanic Biotechnology of Jiangsu, Huaihai Institute of Technology, Lianyungang, ) Abstract A serious mortality of cultured loach Misgurnus anguillicaudatus occurred in some farms in Donghai County, Jiangsu Province in October Syndrome included bleeding in head, opercula, lower jaw, fins, and fin bases, swollen muscle, anus, liver, and spleen, and empty intestines. Tissues were sampled from liver, bleed, and ascitic fluid of diseased fish, from which the pathogen were isolated, and the biological characteristics were examined, including characteristics in morphology, physiology, and biochemistry, as well as the activity of extracellulase and hemolysin. The 16S rrna and gyrb were amplified by PCR, with which sequences deposited in databases were compared, and molecular phylogenetic trees were constructed. The result indicates high homogeneity between the isolates and Vibrio cholerae from GenBank database, showing that gyrb gene analysis is a more useful tool. The sequenced 16S rrna gene of strain LD081008B-1 (GenBank accession No.GQ205447) is 1446bp in length, the sequenced gyrb gene of strain LD081008B-1 (GenBank accession No.GQ205452) is 1207bp in length. The isolates were identified as V. cholerae (Pacini 1854) in terms of phenotepic and molecular characteristics. Detection of the activity of extracellulase and hemolysin shows that the isolates could produced proteinase, diastase, lecithinase, gelatinase, and DNase, and exhibited -haemolysis on agar plates containing 7% defibrinated rabbit blood. Drug resistance of isolates to 49 antimicrobial agents was detected. The results show that isolates were sensitive or highly sensitive to all agents, except for bacitracin. Key words Misgurnus anguillicaudatus L., Vibrio cholerae, Biological characteristics, 16S rrna gene, gyrb gene

Microsoft Word - 16-夏小安-lb.doc

Microsoft Word - 16-夏小安-lb.doc 热带海洋学报 JOURNAL OF TROPICAL OCEANOGRAPHY 2010 年第 29 卷第 5 期 :103 110 海洋生物学 http://jto.scsio.ac.cn; http://www.jto.ac.cn 夏小安 1, 吴清洋 1, 李远友 1, 王树启 1, 游翠红 1, 林尤顺 (1., 515063; 2., 515000)