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1 40 5 Vol.40, No OCEANOLOGIA ET LIMNOLOGIA SINICA Sep., 2009 (Litopenaeus vannamei) (Vibrio parahaemolyticus) * ( ; ),, (Vibrio Pacini 1954) (Vibrio parahaemolyticus), 4,, KP, K ; (JGB ) 16S rrna 1456bp, GenBank GQ gyrb 1195bp, GenBank GQ %, 49 6,, 16S rrna, gyrb Q346 (Litopenaeus vannamei),,, 1988,,,,,, (, 2006;, 2003;, 2004;, 2002), 2 3cm,,,,,,,, (Vibrio Pacini 1954) [Vibrio parahaemolyticus (Fujino et al 1951) Sakazaki Nakamura and Takizawa 1963], 16S rrna gyrb, 1 1.1, ; (28 48h ), (28 24h) *, 2008HS016 ;, CN0826 ;, KQ07106,,, zxj9307@163.com : , :

2 5 : (Litopenaeus vannamei)(vibrio parahaemolyticus) 655, (JGB ), 28 ( CFU/ml), 25ml 50ml 100ml 1000ml CFU/ml CFU/ml CFU/ml 3, 3, 3 ; ;, 1.3 ;, 28 24h,, 1 Formvar, 1min, 0.5% 1 1min,, JEM-100CX 2216E ---(TCBS), 28 24h,, ( )H 2 S MR V-P OF (Simmons), (, 2001) (, 2003) 1.4 KP, 4 (10%) (10%) 80(1.0%) (1%) DNA (0.5%), (8%), 28 24h 80 DNA ; 28 24h, 4, ( 3g 10g NaCl 70g K 2 HPO 4 5g 10g 0.001g 15g 1000ml, ph 8.0, 30min ; 45 7% ), 28 24h, 1.5 K JGB , (OK)(121 1h) (O), 28 18h OK, JGB OK O ( ml ), OK O (++), O K ( 2min ); OK O, O K ( + K );, () (20ml/ ) 28 18h, 10ml/ 2, 1 OK, h O, JGB OK O (, 1998) 0.5% ( 1 ) ( 16 )( 1log2 16log2)0.2ml/, OK O ( 0.4ml/ ),, 24h, (++) S rrna gyrb PCR DNA LB 28 16h, DNA ( ) DNA PCR DNA S rrna PCR 16S rrna PCR : 27F(

3 ): 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 : 14.4μl, 10 PCR 2μl, 1.5mmol/L MgCl 2 1.6μl, 4 dntp 0.4μl, 0.2μl, 2.5U/μl Taq DNA 0.2 μl, DNA 1μl PCR : 94 5min, 94 1min 57 1min 72 2min 30, 72 7min PCR S rrna gyrb 16S rrna gyrb NCBI 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) Bacterial Fish Pathogens: Disease of Farmed and Wild Fish (Austin et al, 1999), 16S rrna gyrb, 1.8, (K-B), (, 1997) 2 2.1, ( ) μm ( ) μm ( ), 4, : JGB (JGB ), 48h, 3 48h ;, (JGB ), (), (JGB ) EM, ( 1) E 24h 1mm, 48h 1.5mm, ; TCBS, 28 24h 1.2mm (48h 1.8mm ),,, ;, 28 24h, () KP, 4, 4 KP ( 2) 2.5 K K, 3

5 : (Litopenaeus vannamei)(vibrio parahaemolyticus) 657 表 1 分离菌的感染试验结果 Tab.1 Results of challenge test with isolates (CFU/ml) 6h 12h 24h 48h 100 2.

4 5 : (Litopenaeus vannamei)(vibrio parahaemolyticus) 657 表 1 分离菌的感染试验结果 Tab.1 Results of challenge test with isolates (CFU/ml) 6h 12h 24h 48h % 50% 100% % 100% % 100% % 50% 100% % 100% % 100% % 40% 80% 100% % 50% 100% % 100% Fig.1 1 JGB Electron micrograph of JGB nm S rrna gyrb (JGB ) 16S rrna 1456bp(GenBank : GQ205448); gyrb 1195bp(GenBank : GQ205453) (JGB ) 16S rrna NCBI Blast, 16S rrna, (V. parahaemolyticus) (V. alginolyticus)(v. campbellii)(v. natriegens), (JGB ) 16S rrna 98% 99%, 16S rrna ( : DQ233654), 2 (JGB ) gyrb NCBI Blast, gyrb, (V. parahaemolyticus), 98% 99%; (V. alginolyticus) (V. campbellii) (V. natriegens)(v. rotiferianus), gyrb 86% 89%, gyrb ( : FJ608553), S rrna gyrb, (Vibrio Pacini 1854) [Vibrio parahaemolyticus (Fujino et al 1951) Sakazaki Nakamura and Takizawa 1963] ,,,,, (, 2002;, 1998);,

5 (, 2001;, 2000;, 1999;, 1999;, 1998);, (, 2001;, 1996);,,,,,, 48h, 表 2 分离菌的理化特性结果 Tab.2 Physiological and biochemical characteristics of isolates (4 ) (4 ) a a H 2 S + + α--d- O-F F F F F F : [+] V-P d β- NaCl : 0% + 1% % % O/129 : 10 μg μg :,, F, d 11% 89%, a Bergey s Manual of Determinative Bacteriology. 9th ed (Holt et al, 1994) 表 3 分离菌凝集反应结果 Tab.3 Results of the agglutination test (JGB ) OK O OK- ++ 7log2 (1 : 128X) ** 2log2 (1 : 4X) O- +( ) 3log2 (1 : 8X) 7log2 (1 : 128X) 0log2 (< 1 : 2X) 0log2 (< 1 : 2X) : ++ ; ; **

6 5 : (Litopenaeus vannamei)(vibrio parahaemolyticus) JGB S rrna Fig.2 Phylogenetic tree based on JGB S rrna gene sequences : EU DQ NCBI,,,, PCR 16S rrna gyrb, 16S rrna gyrb 16S rrna, (JGB )16S rrna NCBI blast, 3 JGB gyrb Fig.3 Phylogenetic tree based on JGB gyrb gene sequences : FM FJ NCBI,, 98% 99%, ( 2) JGB , 16S rrna ; DNA B (gyrb ),,,,, (, 2005), (JGB )gyrB NCBI blast,

7 Tab.4 表 4 药敏纸片名称和规格及病原副溶血弧菌的药物敏感性 Name and type of discs and antimicrobial sensitivity of pathogenic V. parahaemolyticus (μg/ ) (mm) (μg/ ) (mm) 1 G 10 0 R S R S R HS S HS HS HS HS HS HS HS S HS S HS HS HS HS HS HS HS / HS HS HS HS HS HS 16 75/75 36 HS HS HS HS HS R HS R HS HS HS R HS HS S HS HS HS, gyrb 98% 99%, 90%, ( 3) gyrb 16S rrna,, (Exotoxin) (Protease) (Capsule)(Siderophore) (Outer membrane protein, OMP) KP 4, 4 KP,, K,, 4 OK O JGB OK O, 4

8 5 : (Litopenaeus vannamei)(vibrio parahaemolyticus) 661 K,,,, , (1): 19 21,,, , 20(2): ,, :, ,, ( ). :, ,,, , 26(1): 7 9,,, , 16(4): 1 5,,, , 17(3): ,,, , 18(4): 10 13,, :, ,,, , 24(1): 58 60,, :, 68 73,, , 15(3): 24 27,,, , 20(2): , ( )., 11: 41 42,, , 22(4): 29 30,,, , 27(5): 61 65,, gyrb., 32(1): 38 41,,, , 30(4): Austin B, Austin D A, Bacterial Fish Pathogens: Disease of Farmed and Wild Fish. Third (Revised) Edition. Praxis Publishing Ltd, Chichester, UK, Holt J G, Krieg N R, Sneath P H A et al, Bergey s Manual of Determinative Bacteriology. Ninth Edition. Baltimore: Williams and Wilkins, 192, 253, 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): 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):

9 PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF PATHOGENIC VIBRIO PARAHAEMOLYTICUS ISOLATED FROM PENAEUS VANNAMEI ZHANG Xiao-Jun 1, CHEN Cui-Zhen 2, YAN Bin-Lun 1, FANG Hai 2, QIN Guo-Min 1, XU Jing 1 (1. Key Laboratory of Oceanic Biotechnology of Jiangsu, Huaihai Institute of Technology, Lianyungang, ; 2. Department of Animal Science, Hebei Normal University of Science and Technology, Qinhuangdao, ) Abstract A mass mortality of cultured Litopenaeus vannamei broke out during summer in a local farm in northern Jiangsu Province. Syndromes include swimming near water surface and the pond walls, with black gill and opaque muscle. Tissue samples were taken in the liver, and strong pathogenicity to L. vannamei, P. japonicus and P. chinensis were determined by immersion. The phenotypic characteristics of isolates (4 strains) were examined in morphology, physiology and biochemistry; the 16S rrna and gyrb were amplified by PCR and compared with sequences in databases, from which molecular phylogenetic trees were constructed. The sequenced 16S rrna gene of strain JGB (GenBank accession No.GQ205448) is 1456bp in length, the sequenced gyrb gene of strain JGB (GenBank accession No.GQ205453) is 1195bp in length. Results of phylogenetic analysis on 16S rrna and gyrb genes revealed a high homogeneity between the isolates and V. parahaemolyticus from GenBank database. Comparison in method between 16S rrna and gyrb genes confirms that gyrb gene analysis is more useful. The isolates were identified as V. parahaemolyticus [(Fujino et al 1951) Sakazaki Nakamura and Takizawa 1963] of Vibrio (Pacini 1854) in phenotypic and molecular characteristics. Test on the activity of extracellulase and hemolysin shows that the isolates could produce diastase, gelatinase, and lecithinase, but no proteinase and lipase, and KP haemolysis was negative. Examination of K-antigen and agglutination titers show that the isolates have K-antigen, and the titer of OK-agglutinogen with OK-antiserum and O-agglutinogen with O-antiserum was the same (7log2); the titer of OK-antiserum with O-agglutinogen and O-antiserum with OK-agglutinogen was 2log2 and 3log2, respectively. The drug resistance of isolates to 49 antimicrobial agents was detected, the results show that the isolates are resistant to penicillin G, ampicillin, oxacillin, lincomycin, bacitracin and amoxicillin. Key words Litopenaeus vannamei, Vibrio parahaemolyticus, 16S rrna gene, gyrb gene

Microsoft Word - 邴旭文.doc

Microsoft Word - 邴旭文.doc 40 6 Vol.40, No.6 2009 11 OCEANOLOGIA ET LIMNOLOGIA SINICA Nov., 2009 (Misgurnus anguillicaudatus) (Vibrio cholerae) * 1 2 2 2 2 (1. 214081; 2. 222005),, ; PCR 16S rrna gyrb, 16S rrna gyrb,,, ; 16S rrna