40 5 Vol.40, No.5 2009 9 OCEANOLOGIA ET LIMNOLOGIA SINICA Sep., 2009 (Litopenaeus vannamei) (Vibrio parahaemolyticus) * 1 2 1 2 1 1 (1. 222005; 2. 066600),, (Vibrio Pacini 1954) (Vibrio parahaemolyticus), 4,, KP, K ; (JGB080708-1 ) 16S rrna 1456bp, GenBank GQ205448 gyrb 1195bp, GenBank GQ205453 98%, 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) 1.2 3 *, 2008HS016 ;, CN0826 ;, KQ07106,,, E-mail: zxj9307@163.com : 2008-09-15, : 2008-11-23
5 : (Litopenaeus vannamei)(vibrio parahaemolyticus) 655, (JGB080708-1), 28 ( 1 10 8 CFU/ml), 25ml 50ml 100ml 1000ml 2.5 10 6 CFU/ml 5 10 6 CFU/ml 1 10 7 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 JGB080708-1, (OK)(121 1h) (O), 28 18h OK, JGB080708-1 OK O ( 1 0.025ml ), OK O (++), O K ( 2min ); OK O, O K ( + K );, () (20ml/ ) 28 18h, 10ml/ 2, 1 OK, 1 121 1h O, JGB080708-1 OK O (, 1998) 0.5% 1.. 2 ( 1 ) 1.. 65536 ( 16 )( 1log2 16log2)0.2ml/, OK O ( 0.4ml/ ),, 24h, (++) 1.6 16S rrna gyrb 1.6.1 PCR DNA LB 28 16h, DNA ( ) DNA PCR DNA 1.6.2 16S rrna PCR 16S rrna PCR : 27F(
656 40 ): 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, 30 72 6min PCR 1.6.3 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 1.6.4 16S 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, (0.8 1.0) μm (1.2 2.0) μm ( ), 4, : JGB080708-1 4 2.2 (JGB080708-1), 48h, 3 48h ;, (JGB080708-1), 1 2.3 4 (), (JGB080708-1) EM, ( 1) 2.4 4 2216E 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 2.5 10 6 30% 50% 100% 100 5 10 6 50% 100% 100 1 10 7 50% 100% 100 2.5 10 6 20% 50% 100% 100 5 10 6 50% 100% 100 1 10 7 70% 100% 100 2.5 10 6 20% 40% 80% 100% 100 5 10 6 35% 50% 100% 100 1 10 7 50% 100% 50 0 50 0 50 0 Fig.1 1 JGB080708-1 Electron micrograph of JGB080708-1 500nm 2.6 16S rrna gyrb (JGB080708-1) 16S rrna 1456bp(GenBank : GQ205448); gyrb 1195bp(GenBank : GQ205453) (JGB080708-1) 16S rrna NCBI Blast, 16S rrna, (V. parahaemolyticus) (V. alginolyticus)(v. campbellii)(v. natriegens), (JGB080708-1) 16S rrna 98% 99%, 16S rrna ( : DQ233654), 2 (JGB080708-1) gyrb NCBI Blast, gyrb, (V. parahaemolyticus), 98% 99%; (V. alginolyticus) (V. campbellii) (V. natriegens)(v. rotiferianus), gyrb 86% 89%, gyrb ( : FJ608553), 3 2.7 16S rrna gyrb, (Vibrio Pacini 1854) [Vibrio parahaemolyticus (Fujino et al 1951) Sakazaki Nakamura and Takizawa 1963] 2.8 4 49 4 3,,,,, (, 2002;, 1998);,
658 40 (, 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 ) 1 2 3 4 a 1 2 3 4 a 37 + + + + + + + + + + + + + + + + + + + H 2 S + + α--d- O-F F F F F F + + + + + + + + + + : + + + + + [+] V-P + + + + + + + + + + + + + + d + + + + + + + + + + β- NaCl : 0% + 1% + + + + + 3% + + + + + 6% + + + + + + + + + O/129 : 10 μg + + + + + + + 150 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 (JGB080708-1) OK O OK- ++ 7log2 (1 : 128X) ** 2log2 (1 : 4X) O- +( ) 3log2 (1 : 8X) 7log2 (1 : 128X) 0log2 (< 1 : 2X) 0log2 (< 1 : 2X) : ++ ; ; **
5 : (Litopenaeus vannamei)(vibrio parahaemolyticus) 659 2 JGB080708-1 16S rrna Fig.2 Phylogenetic tree based on JGB080708-1 16S rrna gene sequences : EU660325 DQ233654 NCBI,,,, PCR 16S rrna gyrb, 16S rrna gyrb 16S rrna, (JGB080708-1)16S rrna NCBI blast, 3 JGB080708-1 gyrb Fig.3 Phylogenetic tree based on JGB080708-1 gyrb gene sequences : FM202601 FJ608553 NCBI,, 98% 99%, ( 2) JGB080708-1, 16S rrna ; DNA B (gyrb ),,,,, (, 2005), (JGB080708-1)gyrB NCBI blast,
660 40 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 26 30 25 S 2 10 0 R 27 30 15 S 3 100 12 R 28 30 17 HS 4 1 0 S 29 30 32 HS 5 30 25 HS 30 30 27 HS 6 30 37 HS 31 30 35 HS 7 30 38 HS 32 15 27 HS 8 30 16 S 33 300 22 HS 9 2 8 S 34 30 28 HS 10 300 26 HS 35 5 32 HS 11 5 35 HS 36 100 30 HS 12 30 38 HS 37 10 34 HS 13 1.25/23.75 30 HS 38 5 36 HS 14 10 34 HS 39 5 22 HS 15 30 44 HS 40 10 32 HS 16 75/75 36 HS 41 30 28 HS 17 15 32 HS 42 30 26 HS 18 15 28 HS 43 30 23 R 19 5 34 HS 44 75 25 20 2 20 R 45 15 26 HS 21 10 33 HS 46 10 36 HS 22 0.04 0 R 47 5 30 HS 23 120 30 HS 48 10 0 S 24 10 28 HS 49 30 30 HS 25 30 30 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 JGB080708-1 OK O, 4
5 : (Litopenaeus vannamei)(vibrio parahaemolyticus) 661 K,,,, 2000.., (1): 19 21,,, 2001.., 20(2): 187 192,, 2001.. :, 106 120,, 1997. ( ). :, 553 562,,, 1999.., 26(1): 7 9,,, 1996.., 16(4): 1 5,,, 2002.., 17(3): 174 177,,, 1999.., 18(4): 10 13,, 2003.. :, 1550 1610,,, 2004.., 24(1): 58 60,, 1998.. :, 68 73,, 1998.., 15(3): 24 27,,, 2001.., 20(2): 193 199, 2003. 2002 ( )., 11: 41 42,, 1998.., 22(4): 29 30,,, 2003.., 27(5): 61 65,, 2005. gyrb., 32(1): 38 41,,, 2006.., 30(4): 40 44 Austin B, Austin D A, 1999. Bacterial Fish Pathogens: Disease of Farmed and Wild Fish. Third (Revised) Edition. Praxis Publishing Ltd, Chichester, UK, 103 119 Holt J G, Krieg N R, Sneath P H A et al, 1994. Bergey s Manual of Determinative Bacteriology. Ninth Edition. Baltimore: Williams and Wilkins, 192, 253, 256 257 Krieg N R, Holt J G, 1984. Bergey s Manual of Systematic Bacteriology. Volume 1. London: Williams and Wilkins, Baltimore, 545 548 Martin F Polz, Collen M Cavanaugh, 1998. Bias in template to product ratios in multitemplate PCR. Appl Environ Microbiol, 64(10): 3724 3730 Yamamoto S, Harayama S, 1995. 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): 1104 1109
662 40 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, 222005; 2. Department of Animal Science, Hebei Normal University of Science and Technology, Qinhuangdao, 066600) 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 JGB080708-1 (GenBank accession No.GQ205448) is 1456bp in length, the sequenced gyrb gene of strain JGB080708-1 (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