Microsoft Word - 祝璟琳.doc

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1 41 4 Vol.41, No OCEANOLOGIA ET LIMNOLOGIA SINICA July, 2010 (Oreochromis niloticus) * 璟 ( ) API 20 strep, 2009,, 2, 16S rrna (GenBank GU GU363870) 16S rrna 96.5% 100%, 2 16S rrna 100% 16S rrna, 2, 27, 11,,, 16S rrna, S942 (Oreochromis niloticus),, 85,, 50%,,,, (, 2007;, 2001; Chern et al, 1994; Suanyuk et al, 2008) (Streptococcus agalactiae)(streptococcus inia),, 1.5 (Shoemaker et al, 1997) HN , HN ,, API 20 strep 16S rrna 2, (Oreochromis niloticus), ( ), BHIA, 28 24h,, ( HN HN ) UNIQ-10 Taq Takara, * 2007 (), 3-49 ;, nycytx-48 ; 2010 (948 ), 2010-C26 ;, 2006BAD01A1202 璟,, zhujl@ffrc.cn :,, yangh@ffrc.cn : , :

2 4 璟 : (Oreochromis niloticus) 591, (100±10)g, 1.2 BHIA 28 24h, 0.85%, (, 1),,, 32, 9,,, 1.3 API (, 2001; Palleroni, 1990) API Strep S rrna DNA HN HN BHIA h, 2ml ddh 2 O, 1.5ml 1.5ml, 8000r/m 2min, / DNA (, 2002) S rrna PCR 16S rrna 27F: 5 -AGAGTT TGATCCTGGCTCAG-3 ( E. coli 16S rrna 8 27 ); 1492R: 5 -TAC GGCTACCTTGTTACGACTT-3 ( E. coli 16S rrna ) (Martin et al, 1998) 50μl PCR : 10 PCR 5μl, 25mmol/L MgCl 2 4μl, dntp( 2.5μmol/L) 4μl, 10μmol/L 2μl, 5U/μl Taq DNA 0.4μl, DNA 2μl, ddh 2 O 30.6μl PCR : 94 4min; 94 1min, 56 1min, 72 2min, 30 ; 72 5min PCR 1.0%, UNIQ-10, GenBank Blast, GenBank 17, Clustalx (1.83) (multiple alignments), 1, -, N-J, (bootstrap), , (K-B), ,,,,, 4,,, 8, 86.7%, 2, 1,,,, 2,,, ( 1) 2.2 API 20 strep HN HN BHIA,,,,,, API 20 strep, HN HN %, ( 2) S rrna PCR 2, 16S rrna DNA, 27F 1492R 16S rrna, 1513, GenBank GU GU S rrna, HN HN S rrna 16S rrna 96.5% 100%, 2 16S rrna 100% GenBank 16S rrna Clustalx (1.83)( 1), 2,

3 表 1 回归感染试验结果 Tab.1 Results of the recurrent infection experiments (d) (CFU/ml) (ml) (%) * * * : 1 HN , 2 HN , * 30min 表 2 2 株链球菌的 API 20 strep 生化反应表 Tab.2 API 20 strep biochemical identification of two strains of Streptococcus / HN HN VP HIP + + ESC β- PYRA -2- α-gal 6-2- α- α- β-gur AS-B1β-D- β- β-gal 2- -β-d- β- PAL LAP L ADH + + RIB + + ARA L- MAN SOR LAC TRE + + INU ( ) RAF AMD + + GLYG : +, : ( ) G :

4 4 璟 : (Oreochromis niloticus) 593 B2, HN , HN S rrna Fig.1 Phylogenetic tree of Streptococcus built based on the structure of 16S rrna genes : GenBank, 0.01, 1000 (Streptococcus spp.),,,,, (Eldar et al, 1995; Evans et al, 2000, 2009a; Duremdez et al, 2004;, 2008; Suanyuk et al, 2008; Hernandez et al, 2009; Mian et al, 2009),,, 10 ( a b ), (Slotved et al, 2007),,, 26, (Mian et al, 2009) 26 37, 32,,,,, 2 3,,,, API 20 Strep ID 32 strep,, (Suanyuk et al, 2008) API 20 strep, API 20 Strep Evans (2000) API B, VP, VP,

5 Tab.3 表 3 药敏纸片名称和规格及病原无乳链球菌的药物敏感性 Names, types of discs and antimicrobial sensitivity of pathogenic Streptococcus agalactiae (μg/ ) (mm) HN HN (μg/ ) (mm) HN HN R HS HS S HS HS S S S R 6 G HS 30 75/ HS / R HS S HS HS HS HS R HS HS HS HS S S R HS S 39 B R HS HS R R HS HS HS HS HS R HS R HS S HS HS R : HS, S, R 16S rrna 16S rrna, 16S rrna (, 2008;, 2008) 16S rrna (, 2008; Mian et al, 2009) HN HN S rrna 16S rrna 96.5% 100%, 2 16S rrna 100% GenBank 16S rrna 2,,,,, (Creeper et al, 2006),,,,

6 4 璟 : (Oreochromis niloticus) 595,,, (2008), 2,,,,, (Locke et al, 2007),,,,,,,,,,,, 20 90,, (ECP) (US Patent ),,,,,,,,, 致谢海南省水产研究所李向民所长和王德强主任在病原菌采集过程中给予了大力支持, 谨致谢忱, 璟,, (Pseudosciaena crocea)3., 39(2): ,, :, ,,, , 26(4): ,, , (5): 30,,,,, ( ). :, Chern R S, Chao C B, Outbreaks of a disease caused by a rickettsia-like organism in cultured tilapias in Taiwan. Fish Pathol, 29: Creeper J H, Buller N B, An outbreak of Streptococcus iniae in barramundi (Lates calcarifera) in freshwater cage culture. Aust Vet J, 84(11): Duremdez R, Al-Marzouk A, Qasem J A et al, Isolation of Streptococcus agalactiae from cultured silver pomfret, Pampus argenteus (Euphrasen), in Kuwait. J Fish Dis, 27(5): Eldar A, Bejerano Y, Livoff A et al, Experimental Streptococcal meningo-encephalitis in cultured fish. Vet Microbiol, 13(9): Evans J J, Shoemaker C A, Kelsius P H, Experimental Streptococcus iniae infection of hybrid striped bass (Morone chrysops M. saxatilis) and tilapia (Oreochromis niloticus) by nares inoculation. Aquaculture, 198: Locke J B, Colvin K M, Datta A K et al, Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish. J Bacteriol, 189(4): Martin F P, Collen M C, Bias in template-to-product ratios in multitemplate PCR. Appl and Environ Microbiol, 64(10): Mian G F, Godoy D T, Leal C A G et al, Aspects of the natural history and virulence of S. agalactiae infection in Nile tilapia. Vet Microbiol, 136(1): Palleroni N J, Genus I Pseudomonas. Berger s manual of systematic bacteriology. Vol.3. Baltimore: Williams and Wilkins, Shoemaker C, Klesius P, Streptococcal disease problems and control: a review. In: Fitzsimmons K ed. Tilapia quaculture. Vol.2. Northeast Regional Agricultural Engineering Service-106, Ithaca, NY, USA, Slotved H C, Kong F, Lambertsen L et al, Serotype IX, a proposed new Streptococcus agalactiae Serotype. J Clin Microbiol, 45(9): Suanyuk N, Kong F, Ko D et al, Occurrence of rare genotypes of Streptococcus agalactiae in cultured red tilapia Oreochromis sp. and Nile tilapia O. niloticus in Thailandrelationship to human isolates? Aquaculture, 284(1): 35 40

7 ISOLATION, IDENTIFICATION AND DRUG SENSITIVITY TEST OF PATHOGENIC STREPTOCOCCUS FROM TILAPIAS OREOCHROMIS NILOTICUS CULTURED IN HAINAN ZHU Jing-Lin, YANG Hong, ZOU Zhi-Ying, LI Da-Yu, XIAO Wei (Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, ) Abstract Two strains of bacteria, HN and HN , were isolated from diseased tilapia Oreochromis niloticus cultured in Hainan, and were confirmed to be pathogenic by recurrent infections. They were identified as Streptococcus in regular physiological and biochemical analyses. With the API 20 strep technique, they were recognized as Streptococcus agalaciate, which were of 99.9% homology to S. agalaciate. In order for further confirmation on the molecular level, 16S rrna gene were sequenced, their sequence accession number in GenBank being GU and GU respectively. Sequences analysis of 16S rrna gene showed that they were very similar to other Streptococcus, and their nucleotide homology were between 96.5% and 100%. The nucleotide homology of 16S rrna gene between the two isolates were 100%. Phylogenetic tree of Streptococcus based on 16S rrna gene showed that strains HN and HN clustered together with S. agalaciate. Therefore, strains HN and HN were identified as S. agalaciate. Their sensitivities to 48 kinds of antimicrobial drugs were determined. The result showed that the two isolates were both highly sensitive to 27 antibiotics, such as Cephalosporin Drugs, amoxicillin and doxycycline, and were both resistant to 11 antibiotics, such as cotrimoxazole and kanamycin; while they showed different sensitivity to gentamicin. Key words Tilapia Oreochromis niloticus, Streptococcus agalaciate, 16S rrna, Drug sensitivity test

Microsoft Word - 张晓君.doc

Microsoft Word - 张晓君.doc 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),