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1 2011 3, 18(2): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 朱成科 1, 周晓扬 1, 张其中 1,2 1.,,,, ; 2., 摘要 : (Silurus meridionalis Chen) 1, SCL1, SCL1,,,, 16S rdna (Aeromonas hydrophila),,,, ;,, ;,, [, 2011,18(2): ] : ; ; ; 16S rdna; 中图分类号 : S94 文献标识码 : A 文章编号 : (2011) (Silurus meridionalis Chen), (Siluriformes) (Siluridae) (Silurus), [1-2], [3],,, 1985,,, [4-5],, (Aeromonas caviae) [4] (Proteus vulgaris) [6] (Flexibacter columnaris) [7] 1986, 20,,, (Carassius auratus gibelio) [8] (Cyprinus carpio) [9] (Megalobrama amblycephala) [10] (Hypophthalmichthys molitrix) (Aristichthys nobilis) [11] (Ictalurus punctatus) [12] (Siniperca chuatsi) [13] (Channa maculata) [14] (Acipenser baeri) [15] (Scophthalmus maximus) [16] (Kareius bicoloratus) [17] (Trionyxsi nensis) [18],, 收稿日期 : ; 修订日期 : 基金项目 : (CSTC, 2005AB1009); ; (CSTC, 2010AC1116). 作者简介 : (1981 ),,,. zhuck2003@163.com. 通讯作者 :,,. zhangqzdr@126.com

2 2 : 361,, [19], (A. hydrophila) [8, 14, 18] (A. sobria) [8, 10] (Yersinia ruckeri) [11] (Edwardsiella tarda) [16] (A.salmonicida) [17],,,,,, ;, 黏,,,, 15%~40%, 45%~80%,,,, 16S rdna,,, : , (19.5±1.9) g, (10.7±1.6) cm;, (22.0±1.6) g, (12.3±1.3) cm, 5 d,,, (24±1), (1) : (Tryptone) (Yeast extract) (Agarose) ; Taq dntp PMD-19T DNA Marker DL2000 (TaKaRa) ; DNA BioFlux ; (2) : SW-CJ-1F ( ); YSEI-150L ( ); DU730 (Beckman Coulter ); ABI 9700 PCR (Applied Biosystems ) 1.2 : 5.0 g; 10.0 g; NaCl 5.0 g; K 2 HPO g, ml 2 %(W/V) min, 4 1.3, 70%,,, h,,, 4,, 28 24~28 h, 1.4, 1,,, (24±1), 1/3, h,, CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL 0.2 ml, 0.2 ml0.65%,,,,,,,,

3 ,, [20], DADE Behring MicroScan 4, 1.6 ph [21] w(nacl)(0 14.0%) ph( ),, (28 ) (180 r/min) 24 h, nm OD [w(nacl)=0, ph 7.0] (3~47 ) 24 h, nm OD S rdna SCL1 DNA SCL1, r/min 24 h, 2 ml r/min 4 min,, DNA Sambrook - DNA [22] DNA PCR 16S rdna fd1/rd1 [23], fd1: 5 -AGA GTT TGA TCC TGG CTC AG-3, (Escherichia coli) 16S rdna 8~27 ; rd1: 5 -TAC GGY TAC CTT GTT ACG ACT T-3 E. coli 16S rdna 1 492~1 510 PCR 50 μl, 2 μl DNA (50 ng/μl), 5 μl 10 PCR Buffer, 3 μl MgCl 2 (25 mmol/l), 1 μl dntp(2.5 mmol/l), 0.4 μl Taq (5 U/μL), 1 μl(10 μmol/l), 50 μl : 95 3 min, 30 (94 30 s, s, s), min, 1.0% TBE, PCR : TaKaRa pmd 19-T μl,,, PCR, ABI SCL1 16S rdna GenBank Blast, 16S rdna, DNA star 5.0 MEGA , (K-B ) [24] CFU/mL,,, h, ( 3 ), 3, ± ( x ±SD) [24] (minimal inhibition concentration, MIC) ( minimum bactericidal concentration, MBC) 500 μg/ml, 28, 24 h, MIC h, MBC 1.9, Bouin s 48 h( ), 70% 80% 90% 100%,.. ( 1.. 1), (2 ),, 6 μm,, -,,,, NIKON 80i [25] :

4 2 : 363,, ;, ;,,,,, 2.2 1, SCL1, 1 SCL1,,,, SCL1,, SCL2, SCL1 LD 50 : CFU/mL [26] 2.3 SCL h,, 2~4 mm,,,,, SCL h,,, 2~4 mm,, SCL1 2.4 SCL1 SCL2,, ; ; ; ; SCL1 SCL ph 752, SCL1 0~4, 0( 1); ph 4.0~10.0, ph 7.0( 2); SCL1 w(nacl)=0, ph7.0, 18.0~38.0, 23.0~33.0 ( 3) S rdna SCL1, PCR 16S rdna, bp( 4),, bp, GenBank( GU295963) SCL1 group Tab.1 /(CFU ml 1 ) bacterial concentration 表 1 分离菌株对南方鲇幼鱼人工感染实验结果 Results of healthy juvenile southern catfish infected with strains SCL1, SCL2 /ml dosage fish nos. 7 d death number in each day total death number /% mortality rate SCL control 0.65% NaCl SCL control 0.65% NaCl

5 Tab.2 表 2 病原菌 SCL1 SCL2 生理生化特征 Physiological and biochemical characteristics of pathogenic bacterium strains SCL1 and SCL2 item SCL1 SCL2 ** Aeromonas hydrophila ** cell shape gram stain growth control oxidase glucose raffinose inositol urease lysine decarboxylase + + d phenylalanine deaminase citrate utilization tartrate utilization fermentation of glucose oxidation sucrose rhamnose adonitol H 2 S H 2 S production salicin sodium Malonate sorbitol arabinose maltose indole ornithine decarboxylase esculine utilization V-P Voges-Proskauer reaction ONPG ONPG hydrolysis xylose nitrate reduction : - : ; + : ; d:,, ; ** : [27 28]. Note: - : negative, + : positive, d: some strains are positive or negative. ** Data are from references 27 and 28. Fig.1 1 SCL1 Effects of salinity on growth of strain SCL1 n=5; x ±SD 2 ph SCL1 Fig.2 Effects of ph on growth of strain SCL1

2 : 365 2.7 14 SCL1 3 n=5; x ±SD 3 SCL1 Fig.3 Effects of temperature on growth of strain SCL1 GenBank (Aeromonas hydrophila) 16S rdna, 98.7%~99.5% SCL1 A. hydrophila ( 5) 4 SCL1 16S rdna PCR 1.

6 2 : SCL1 3 n=5; x ±SD 3 SCL1 Fig.3 Effects of temperature on growth of strain SCL1 GenBank (Aeromonas hydrophila) 16S rdna, 98.7%~99.5% SCL1 A. hydrophila ( 5) 4 SCL1 16S rdna PCR 1. SCL1; 2. ; M. DNA marker DL2000. Fig.4 Result of 16S rdna amplification of SCL S rdna of strain SCL1, 2. Blank control, M. DNA marker DL S rdna SCL1 ( ) bootstrap. GenBank. Fig.5 Phylogenetic tree of SCL1 and Aeromonas hydrophila based on 16S rdna sequence The number at each branch point is the percentage supported by bootstrap with repetitions. Sequence accession numbers in GenBank are in brackets.

7 ; TMP 3 ; 5 ( 3) SCL1, MIC MBC 0.23 mg/l 0.47 mg/l; TMP, MIC MBC mg/l mg/l( 4) 2.8,,,, ( I 1), ;,,,,,, ( I 2),, ( I 3),,, ( I 4),,, ( I 5),, drug 表 3 菌株 SCL1 对抗菌药物的敏感性 Tab.3 Sensitivity of the strain SCL1 to some antibacterial drugs /(μg disc 1 ) concentration /mm diameters of inhibitory zone n=5; x ±SD sensitivity oxytetracycline ±3.56 S streptomycin ±2.04 S ofloxacin ±3.56 S norfloxacin ±2.48 S kanamycin ±3.12 S gentamycin ±1.80 S tetracycline ±1.96 M TMP 23.75/ ±2.60 M rifampicin ±2.86 M penicillin sodium 10IU 0 R midecamycin 30 0 R roxithromycin 15 0 R trimethoprim 5 0 R acetylspiramycin 30 0 R : S ; M ; R. Note: S-High sensitive; M-Medium sensitive; R- Resistant. 表 4 敏感抗菌药物对病原菌的最小抑菌浓度和最小杀菌浓度 Tab. 4 MIC and MBC of different antimicrobial agents against the pathogen(scl1) mg L 1 MIC MBC drug name drug name MIC MBC oxytetracycline streptomycin ofloxacin norfloxacin gentamicin kanamycin tetracycline rifampicin TMP

8 2 : 367,,, ( I 6) 3, (SCL1),, (SCL2), SCL1,, (Koch s postulates), SCL1 [29] [30],,,, 6, [27] [28], (A.hydrophila) SCL1 H 2 S, [31],, 16S rdna,,, [32] SCL1 16S rdna GenBank ( GQ184148) 16S rdna 99.5%, SCL1 (Aeromonas spp.) SCL1, SCL1 (A. hydrophila),, [33] (Whitmania pigra) [34] (Cyprinus carpio) [35] (Ctenopharyngodon idellus) [36] (Anguilla japonica) [37] (Trionyx sinensis) [38],,, SCL1 SCL1 23~33, ph,,,,,, (exotoxin) (exoenzyme) (adherent factor) (hemolytic toxin) (enterotoxicity) (cytotoxicity) [33] [30],,,,,,,,,,,,,, Ventura [39], ; Huizinga [40],,,,,,,,,,,,

9 (Cyprinus carpio) [35] (Carassius auratus gibelio) [8] (Siniperca chuatsi) [13] (Acipenser baerii) [15],, SCL1 6, TMP 3, (Acipense baerii) [41] (Trionyx sinensis) [42] (Plecoglossus ativelis) [43],,,, 参考文献 : [1]. [M]. :, [2]. [J]. :, 1980, 2: [3],,. [J].,1990, 25(1): [4],,,. [J]., 2008,38(2): [5],. [J]. :, 1996,21(6): [6],,,. [J]., 2007, 47(1): 1-6. [7],,. [J]., 2004, 24(4): [8],,. [J]., 1991, 15(3): [9],,. [J]., 1994, 12(2): [10],,,. [J]., 2006, 28(3): [11],,,. Yersinia ruckeri, 鱅 [J]., 1991,82(8): [12],,,. [J]., 2007,20(5): [13],. [J]., 1996, 18(4): [14],. [J]., 1998, 18(2): [15],,. [J]., 2009, 33 (2): [16],,,. [J]., 2006, 36(4): [17],,,. [J]., 2006, 30(5): [18],. [J]., 1996, 20(3): [19],,,. [J]., 1995, 4(1): [20],,. [M]. 3, :, 2004: [21],. CSS-4-2 [J]., 2004, 31(1): [22] Sambrook J, Fritsch E F, Maniatis T. Molecular Colning: A Laboratory Manual. 2 nd [M]. New York: Cod Spring Harbor Laboratory Press, [23] Wiliam G W, Susan M B, Dale A P, et al.16s ribosomal DNA amplification for phylogenetic study[j]. J Bacteriol, 1991, 173 (2): [24],,. [M]., [25]. [M]. :, [26],,,. [J]., 2004, 14(4): [27],. [M]. :, [28] R E, N E. [M]. :, [29]. [J]. :. 1992, 15 (1): [30]. [J]., 1992, 16 (3): [31],. I [J]., 2003, 30 (5): [32]. [J]., 1998, 38 (3):

10 2 : [33],,,. [J]. :, 2007, 33 (3): [34],. [J]., 2006, 33 (1): [35]. [J]., 2004, 22 (3): [36],,. [J]., 2006,22 (4): [37],,,. [J]., 2009, 16 (2): [38],,,. [J]., 1996, 20 (2): [39] Ventura M T, Grizzle J M. Lesions associated with natural and experimental infections of Aeromonas hydrophila in channel catfish, Ictalurus punctatus (rafinespue)[j]. J Fish Dis, 1988, 11 (5): [40] Huizinga H W, Eseh G W, Hazen T C, et a1. Histopathology of red-sore disease (Aeromonas hydrophila) in naturally and experimentally infected largemouth bass Micropterus salmoides (Lacepede)[J]. J Fish Dis, 1979, 2: [41],,,. [J]., 2008, 43 (6): 1-9. [42],,,. [J]., 2009,14 (5): [43],,,. [J]., 2009, 28 (7): Pathogenic bacterium identification and histopathology of septicemia of juvenile southern catfish, Silurus meridionalis Chen ZHU Chengke 1, ZHOU Xiaoyang 1, ZHANG Qizhong 1, 2 1. Key Laboratory of Aquatic Science of Chongqing, Key laboratory Eco-environments in Three Gorges Reservoir Region (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), School of Life Science, Southwest University, Chongqing , China; 2. Hydrobiology Institute of Jinan University, Guangzhou , China Abstract: The study focused on bacteriological and histopathological examination of septicemia in juvenile southern catfish, Silurus meridionalis, when the disease took place in the catfish farm in Chongqing. There were 15% 40% of cultured southern catfish suffering from the disease and the mortality rate was 45% 80%. The diseased individuals exhibited traits of hemorrhagic septicemia, and showed symptoms of emaciation, sluggishness, eye exophthalmia, and fin hemorrhages. The viscera symptoms of the diseased fish included ascites, liver swelling, and clear histopathological alterations in spleen and kidney. Histopathological examination revealed hydropic swelling of hepatocytes, degeneration and necrosis of renal tubular epithelia in kidney, tumidity in lymphocytes in spleen. One strain of a bacterial species was isolated from liver, kidney and spleen of diseased southern catfish with typical septicemia symptom, and was named SCL1. The challenge experiments were carried out with healthy juvenile southern catfish by means of injection with live SCL1 at the concentration of CFU/mL. All individuals of the catfish injected with SCL1 died in a week, and the symptom of the challenged fish was similar to those of the naturally diseased fish, and the LD 50 of SCL1 was calculated as CFU/mL in the 22 g body weight southern catfish, while the catfish injected with 0.65% sterile solution in control group had no signs in 7 d post challenge. Therefore, SCL1 was the pathogenic bacterium strain, which was gram negative, rod-shaped, and the same as Aeromonas hydrophila in physiological and biochemical indexes except for H 2 S production, and was further proved to be A. hydrophila by means of its 16S rdna sequence, which was 99.5% identical with A. hydrophila. SCL1 and was sensitive to oxytetracycline, streptomycin, ofloxacin, norfloxacin, kanamycin, and gentamycin, but resistant to penicillin sodium, midecamycin, roxithromycin, trimethoprim, and acetylspiramycin.

370 18 This paper will be helpful to disease control and health management during Southern catfish farming.

11 This paper will be helpful to disease control and health management during Southern catfish farming.[journal of Fishery Sciences of China, 2011, 18(2): ] Key words: Silurus meridionalis Chen; septicemia; Aeromonas hydrophila; 16S rdna; histopathology Corresponding author: ZHANG Qizhong. 图版 I 1:, (H), ( ). 2:,,, (H) ( ); ( ). 3:, (E) (L). 4:, (E) (L) ; 5:, (G), (RT) ; 6: : (RC) ( ); (RT) ( ). E: ; G: ; H: ; L: ; RC: ; RT: ; V:. Plate I 1: The liver of healthy southern catfish: the blood vessel is full of blood cells; hepatocytes ranking closely (arrow); 2: The liver from diseased southern catfish: tumidity, necrosis in hepatocyte (arrow); tumidity, damage in erythrocytes (arrow); 3: The tissue of spleen from healthy southern catfish: spleen is full of erythrocytes and lymphocytes; 4: The tissue of spleen from diseased southern catfish: tumidity in erythrocytes and lymphocytes; 5: The tissue of kidney from healthy southern catfish: the control group showing normal renal glomerulus and renal tubule; 6: The tissue of kidney from diseased southern catfish: expanded cavity of renal capsule, tumidity in renal tubule epithelia (arrow). E: erythrocyte; G: glomerulus; H: hepatocyte; L: lymphocyte; RC: renal capsule; RT: renal tubular; V: vein.

5 : 1061, 1.0 10 8 CFU/mL, 100 200 g,, 0.4 ml/, 5, 28 30, 10d 1.4 (NA) 30 18h,, JEM-1011 ( ) 1.5 16SrDNA [11] (8f: 5 -AGAGTTTGAT- CCTGGCTCAG-3, 1492r:

5 : 1061, 1.0 10 8 CFU/mL, 100 200 g,, 0.4 ml/, 5, 28 30, 10d 1.4 (NA) 30 18h,, JEM-1011 ( ) 1.5 16SrDNA [11] (8f: 5 -AGAGTTTGAT- CCTGGCTCAG-3, 1492r: 34 5 Vol. 34, No.5 2010 9 ACTA HYDROBIOLOGICA SINICA Sep, 2 0 1 0 DOI: 10.3724/SP.J.1035.2010.01060 1,2 沈文英 2 李卫芬高 2 研 (1., 312000; 2., 310029) ISOLATION AND DRUG SENSITIVITY STUDY OF PATHOGENIC BACTERIA