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1 热带海洋学报 JOURNAL OF TROPICAL OCEANOGRAPHY 2010 年第 29 卷第 5 期 : 海洋生物学夏小安 1, 吴清洋 1, 李远友 1, 王树启 1, 游翠红 1, 林尤顺 (1., ; 2., ) 2 : Scylla serrata 2, TCBS, HY, HG,,, DNA, 16S rrna PCR, HY 1509bp HG 1532bp,, HY HG Aeromonas hydrophila Vibrio parahaemolyticus,,, 3, 100% : Scylla serrata; Aeromonas hydrophila; Vibrio parahaemolyticus; ; ; 中图分类号 : S947.9 文献标识码 : A 文章编号 : (2010) Isolation and identification of two bacterial pathogens from mixed infection mud crab Scylla serrata and drug therapy XIA Xiao-an 1, WU Qing-yang 1, LI Yuan-you 1, WANG Shu-qi 1, YOU Cui-hong 1, LIN You-shun 2 (1. Guangdong Provincial Key Laboratory of Marine Biology, Shantou University, Shantou China; 2. Shantou Dashun Industry Co., Limited, Shantou China) Abstract: Two preponderant bacterial strains were isolated from the infected mud crab (Scylla serrata) in Niutianyang aquaculture area of Shantou. One displayed yellow color and the strain was named as HY, the other showed green color and the strain was named as HG after growing on the TCBS agar plate. Both of them were rod shape and displayed gram negative. After healthy S. serrata individuals were infected with HY or HG by intramuscular injection, obvious histopathological changes could be observed in their tissues as those in the naturally diseased S. serrata, which lead them to their death. These results demonstrate that both HY and HG are pathogens of S. serrata. With the total HY or HG DNA as a template and bacterial 16S rrna gene universal primers as primers, PCR amplification could obtain a partial HY sequence with 1509bp and HG fragment with 1532bp. Based on the blasting of 16S rrna gene sequence and phylogenetic tree, together with the physiological and biochemical characteristics analysis, HY and HG were identified as Aeromonas hydrophila and Vibrio parahaemolyticus, respectively. To our knowledge, this is the first report that A. hydrophila is a pathogen of S. serrata. Both HY and HG showed high sensitivity to Ofloxacin, Gentamicin, streptomycin and Amoxicillin, medium sensitivity to Sulfadiazine and Thiamphenicol, and no sensitivity to Benzylpenicillin Sodium. After the healthy mud crabs were infected with A. hydrophila by immersion or intramuscular injection and displayed typical symptoms, drug therapy shows 100% success rate after the infected crabs were bathed in Amoxicillin at 15μg/mL for three days. 收稿日期 ; 修订日期基金项目 (2006A ) (2008B ) (cgzhzd0812)(2007b )(a200905j01) 作者简介 (1982 ),,,, xia @163.com 通信作者 : yyli@stu.edu.cn

2 104 Vol. 29, No. 5 / Sep., 2010 Key words: Scylla serrata; Aeromonas hydrophila; Vibrio parahaemolyticus; histopathology; isolation and identification of pathogens; drug therapy Scylla serrata,,,,,,,,, [1 2] [1,3] [1] [3] ; [1,3 4] ; [1,3,5] [1, 5] [1] [6 7] ; [1,3] ; [8 10],, 3, 80%, 2005, 9 11,, ( 44%) (, 46%), 7%, 3%,, :,,,, ;,,,, ;,,, [11], ;, 100 g, Zobell 2216E ; TCBS MH ; PCR () ; 1.2,, Zobell 2216E TCBS, h, TCBS HY, HG, 15% Zobell 2216E HY HG Zobell 2216E, h, 0.85%, 10, 5 : 3 HY ( CFU ml 1 ), 1 HG ( CFU ml 1 ), 1 ( 0.85% NaCl) 7 8, 0.2 ml, 3, 21 23, 9 00 ;, 3,, Zobell 2216E TCBS, 28 24h,, 2, 1.4,, 4 Bouin s ( :40% : 15:5:1, v/v/v) 12 24h, 70%, ; 5 μm, HE, (Carl ZEISS Axioplan 2)

[3] ; [1,3 4] ; [1,3,5] [1, 5] [1] [6 7] ; [1,3] ; [8 10],, 3, 80%, 2005, 9 11,, ( 44%) (, 46%), 7%, 3%,, :,,,, ;,,,, ;,,, [11], 1 1.1 2006 9 11 ;, 100 g, Zobell 2216E ; TCBS MH ; PCR () ; 1.

3 : 制备模板 DNA HY HG Zobell 2216E, h, 0.85%, g 10 min, DNA, Moore [12] 基因序列的 PCR 扩增与测序 16S rrna PCR, P27f: 5 -AGAGTTTG ATCCTGGCTCAG-3, P1492R: 5 -TACGGCCTTTGT TACGACTT-3 [13] 25μL PCR : 2 Master Mix 12.5μL (Taq Polymerase 0.1 U μl -1, 500mmol L 1 dntp each, 20mmol L 1 Tris-HCl, ph 8.3, 100mmol L 1 KCl, 3 mmol L 1 MgCl 2 ), 1μL, DNA 1μL, ddh 2 O 8.5μL PCR : 96 6min; 94 1min, 55 1min, 72 2min, 30 ; 72 6min PCR 1%, BBI, PCR4-TOPO ; 4, HY HG 16S rrna 序列分析和系统发育树构建 HY HG 16S rrna GenBank Blastn,, Clustal X 1.81 (Multiple Alignments), PHYLIP 3.65, (neighbor joining method), Bootstrap 1000 [14] 1.6, HY Aeromonas hydrophila, HG Vibrio parahaemolyticus HY HG Zobell 2216E, 28 24h,, ; [15] 1.7, MH 7 12 ( 1), μg ml 1 2, HY HG, 10 5 CFU ml 1, (MIC) (MBC) [16 17] MH 表 1 抗菌素对分离菌 HY 和 HG 的最小抑菌 (MIC) 和最小杀菌 (MBC) 浓度 (μg ml 1 ) Tab. 1 Minimum inhibitory (MIC) and bactericidal (MBC) concentrations of antibiotics against the isolated bacteria HY and HG (μg ml 1 ) HY HG MIC MBC MIC MBC > > >100 >100 >100 >100 > HY, ;, 8, 14d 23 24, (1) 24 h HY, CFU ml 1 ;,,, 15μg ml 1, 3, 14 (2), HY ( CFU ml 1, 0.2mL 1 ), ; 15μg ml 1,, 20min, 3d, 14d (3),,, 40μg g 1,, HY TCBS, 3 mm ; Zobell

5μL PCR : 96 6min; 94 1min, 55 1min, 72 2min, 30 ; 72 6min PCR 1%, BBI, PCR4-TOPO ; 4, HY HG 16S rrna 1.5.3 序列分析和系统发育树构建 HY HG 16S rrna GenBank Blastn,, Clustal X 1.

4 106 Vol. 29, No. 5 / Sep., E, HG TCBS, 2 5mm; Zobell 2216E HY, CFU ml 1 2 7, 87.5%, ; CFU ml 1 3, %,, HG %,,, ; (), HY HG 2 1,,, 表 2 青蟹人工感染分离菌后的死亡情况 Tab. 2 Mortality of mud crab after being infected with isolated bacteria / (CFU ml 1 ) /% HY HY HY HG ,, : 肝胰腺 :,,,, ( 1a), ;,, ( 1b) 心脏 :,, ( 1c), ;, ( 1d) 鳃 : ( 1e),,, ( 1f) 肌肉 :, ( 1g), ( 1h) 2.4 HY HG DNA PCR, 16S rrna, 1509bp 1532bp, Genbank EU EU HY HG 16S rrna GenBank 16S rrna, HY, HG HY 100, 55%; 20 17, 99%; 14 16S rrna ( 2), HY (X87271), 99% HG 100, 88%, HG 96% 98%; 19 16S rrna ( 3), HG (EF203212)(AY562192), 98% HY HG HY,,,, ; HG,,,, 0/129,, 6% NaCl, 2.6 3d, 14d,,

05 10 7 8 3 2 2 7 87.5 HY 7.05 10 6 8 3 0 3 6 75 HG 2.5 10 7 7 1 3 2 6 85.7 7 0 1 0 1 14.3 2.3,, : 肝胰腺 :,,,, ( 1a), ;,, ( 1b) 心脏 :,, ( 1c), ;, ( 1d) 鳃 : ( 1e),,, ( 1f) 肌肉 :, ( 1g), ( 1h) 2.

5 : (a, b)(c, d)(e, f)(g, h) (a, c, e, g): ; (b, d, f, h): Fig. 1 Microstructure of hepatopancreas (a, b), heart (c, d), gill (e, f) and claw muscle (g, h) from healthy or diseased Scylla serrata

6 108 Vol. 29, No. 5 / Sep., S rrna Fig. 2 Phylogenetic tree of Aeromonas based on 16S rrna gene sequences 3 16S rrna Fig. 3 Phylogenetic tree of Vibrio based on 16S rrna gene sequences,, 2 2 2, 3d,,, 2,,,, 5d,,, 7d

7 : 109 表 3 分离菌株的形态特性和生理生化特征鉴定 Tab. 3 Morphological and biochemical properties of isolated strains HY HG G G G G O/F F F F F 0/129(150μg) 0/129(10μg) TCBS 6% NaCl : G ; ; ; F 3 2, ;,,, [18], [19 20] [21], ; [22],,,, ( 4 8 ), [23] V. fluvialis Moraxella nonliquefaciens [24, 25] ; V. cincinnatiensis V. alginolyticus [26] ; [3], V. anguillarum [1],, ;, [27 29],, [11], ; ;,,,,, ;,,,, 3d,

0/129(10μg) TCBS 6% NaCl 675 512 95.3 99.15 0.42893 0.

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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),