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1 37 5 Vol. 37, No ACTA HYDROBIOLOGICA SINICA Sep., doi: 0.754/ 黄 钧 黄 艳 华 2 胡 大 胜 3 罗 华 平 施 金 谷 彭 民 毅 禤 均 成 覃 丽 芬 滕 忠 作 3 曾 桂 忠 (. /, ; 2., ; 3., 53000) :,, API 20NE 6S rrna, K-B, PCR 6, 3, 9, 4 9, 5 Aeromonas hydrophila ATCC 7966, 4 Aeromonas hydrophila QDC0 ; 4 Aeromonas sobria ATCC 43979, 3, 6, Aer Act ahp 00%, hly Alt 92.3%, ahal 76.92%; 4, hly Aer Alt Act ahal ahp, hly Aer Alt Act ahal ahp, hly : ; ; ; ; 中 图 分 类 号 : S947. 文 献 标 识 码 : A 文 章 编 号 : (203) (Truogx sinensis),, (Trionyx sinensis),,,,,,,,, 200,,,,,,, [ 8],, [9], (Aeromonads),, [0 9],,, hly Ae Alt Act ahal ahp, 收 稿 日 期 : ; 修 订 日 期 : 基 金 项 目 : (20GXNSFA08065); ([200]58 ; [20]52 ) 通 信 作 者 : (957 ),, ;, ; [email protected]

2 5 : , 20d API 20NE pmd8-t, BamHⅠ HincⅡ HindⅢ TaKara ; DNA, DNA ; ; Solarbio ; PCR, 20.2, 75%,,, h,,, 4 :, 37 24h, h API 20NE, API 20NE [20] 6S rrna 4 ml LB, 28, 200 r/min 6h, 0.8 ml.5 ml EP, 2000 r/min 2min TIANamp Bacterial DNA Kit DNA, 20 6S rrna PCR : fd: 5 -AGAGTTTGATCCTGGC TCAG-3, rp2: 5 -ACGGCTACCTTGTT ACGACTT-3, (25 µl) : 0 PCR 2.5 µl, 0 mmol/l dntps 0.5 µl, 25 mmol/l MgCl µl, 0 μmol/l µl, Taq DNA (5U) 0.3 µl, DNA 3 µl, ddh 2 O : 94 5min; 94 30s, 56 min, 72 2min, 30 ; 72 0min PCR.0% (00V 35min),, PCR.3,, 37 24h,, 0.2 ml/, PBS, 5d,, h, PBS,,,,, PBS 5d.4 DNA 4 ml LB, 28, 200 r/min 6h, 0.8 ml.5 ml EP, 2000 r/min 2min TIANamp Bacterial DNA Kit DNA, 20 PCR, hly [7] Aer [8] Alt [2] Act [9] ahal [0] ahp [22] PCR, (25 µl) : 0 PCR 2.5 µl, 0 mmol/l dntps 0.5 µl, 25 mmol/l MgCl µl, 25 pmol/l µl, Taq DNA (5U) 0.3 µl, DNA 3 µl, ddh 2 O PCR.0% (00V 35min),, PCR %

3 Tab. Virulence gene hly Aer Alt Act aha ahp 表 PCR 反 应 引 物 及 退 火 温 度 Primers and annealing temperatures for PCR Primer sequences (5-3 ) F: ggc cgg tgg ccc gaa gat acg gg R: ggc ggc gcc gga cga gac ggg F: cac agc caa tat gtc ggt gaa g R: gtc acc ttc tcg ctc cag gc F: atg acc cag tcc tgg cac gg R: gcc gct cag ggc gaa gcc gc F: aga agg tga cca cca aga aca R: aac tga cat cgg cct tga act c F: cta tga aaa aga caa ttc tgg ct R: agg cta gat tag aag ttg tat tg F: att gga tcc ctg cct atc gct tca gtt ca R: gct aag ctt gca tcc gtg ccg tat tcc Denatured temperature ( ) Fragements (bp) ,, TIANgel Midi Purification Kit, pmd8-t, EZ Spin Column Plasmid Mini-Preps Kit.5 K-B,,, h 2 2.,, (),,,,, , 8 β, 4 α, ( 2) Tab. 2 表 2 细 菌 分 离 结 果 与 溶 血 性 Pathogens isolated and their hemolytic properties Infected Truogx sinensis farms A Liuzhou A B Liuzhou B C Nanning C D Nanning D E Nanning E E Nanning E Isolates LZG, LZX LZG2, LZX2, LZP2 NNG, NNX NNX2, NNG2 NNE, NNE2, NNE4 NNE3 Hemolytic properties β β α α β h PBS 5d (28.5±.5) 5d 3 3, 24h, 8d NNE %, 00%, 5d 00%, 3,, 2 3d, ; ;,, ; ;,, 2.4 API 20NE, 3 NNE NNE2 NNE3 NNE4 (Aeromonas sobria), 9 (Aeromonas hydrophila) 4

4 5 : 表 3 分 离 菌 株 的 人 工 感 染 试 验 结 果 Tab. 3 Animal challenged with the isolates ( ) ( ) Bacteria con- Duraton (d) Total numbers Morta- Volume Isolates Animal individuals tested tion(cfu/ml) sinensis (%) centri- of dead Truogx lity rate (ml) LZG LZX LZG LZX LZP NNG NNX NNX NNG NNE NNE NNE NNE PBS 表 4 分 离 菌 株 的 API 20 NE 鉴 定 结 果 Tab. 4 API 20 NE assay for the isolates Isolates Test items LZG LZX LZG2 LZX2 LZP2 NNG NNX NNX2 NNG2 NNE NNE2 NNE3 NNE4 KNO TRP GLU ADH URE ESC GEL β-d PNPG GLE ARA MNE MAN N- - NAG MAL GNT CAP ADI MLT CIT PAC + - OX : + ; Note: + positive; negative

5 848 水 生 生 物 学 37 卷 报 6S rrna 基因序列分析结果 3 株病原菌扩增出大小一致的 6S rrna 基因片 将此序列进行 BLAST, 与 GenBank 中已报道的 6S 段, 大小约为.5 kb 基因测序结果, 菌株 LZG 进行多重序列比对分析, 并用 MEGA 5.0 软件构建系统 LZX LZG2 LZX2 和 LZP2 的 6S rrna 基因 发育树 根据系统发育树, 菌株 LZG LZX LZG2 序列长度为 457 bp (登录号 JX JX478240), 菌 LZX2 LZP2 与嗜水气单胞菌 ATCC 7966 亲源关系最 株 NNG NNX NNX2 NNG2 的 6S rrna 基因 NNX NNX2 NNG2 近, 相似性达 99.9 ; 菌株 NNG 序列长度为 452 bp(登录号 JX47824-JX478244), 与嗜水气单胞菌北京株 QDC0 亲源关系最近, 相似性 NNE NNE2 NNE3 和 NNE4 得到的 6S rrna 基 达 99.8 ; NNE NNE2 NNE3 NNE4 与温和气单胞 因序列长度为 426 bp (登录号 JX JX478248) 菌的 ATCC 亲源关系最近, 相似性达 99.9%(图 ) 2.5 rrna基因序列进行同源性比较, 采用DNA Star 5.0 软件 2.6 六种毒力基因的 PCR 检 测结果 3 株 病 原 的 基 因 组 DNA 经 PCR 扩增之后, 6 种 毒力基因扩增到的片段均与 预期片段大小相吻合, 毒力 基因的检测结果见表 5 从表 5 可见, 6 种毒力基因 在嗜水气单胞菌和在温和气单 胞菌中的检出阳性率和分布特 点都不尽相同 在 9 株嗜水气 单胞菌中, 毒力基因的阳性率 hly Aer Alt Act 和 ahp 均为 00%, ahal 为 88.89%, 而在 4 株温和气单胞菌中, Aer Act ahal和ahp的阳性率均为00%, hly 为 75.00%, Alt 为 25.00% 毒力基因型共 4 种, 嗜水气单 胞菌和温和气单胞菌各 2 种, 其分布情况在嗜水气单胞菌中 hly+aer+alt+act+ahal+ahp+ 基 因 型占 88.89%, hly+aer+alt+act+ ahal ahp+基因型占.%; 在 温和气单胞菌中 hly+aer+alt Act+ahal+ ahp+基因型占 75.00%, hly Aer+Alt+ Act+ahal+ahp+基因 型占 25.00% 2.7 病原菌的药敏试验结果 根据 3 株病原菌对 26 种药物的药敏试验结果, 不 图 Fig. 3 株病原菌的 6S rrna 基因序列系统发育树 Phylogenetic tree based on the 6S rrna genes of the 3 isolates 同菌株对同一种药物的药敏 试验结果不尽相同, 有的甚

6 5 : Tab. 5 Isolates 表 5 3 株 菌 株 毒 力 基 因 检 测 结 果 Detection of six known virulence genes in thirteen strains Types of bacteria hly Aer Alt Act ahal ahp LZG LZX LZG2 LZX2 LZP2 NNG NNX NNX2 NNG2 NNE NNE2 NNE3 NNE ,, ( 7) 7, 70% B 7, 2 70% B 8, , 26,, 6 ( 8) 8, B,, , , [6] [] ;, [2] ;,,,, [3, 5, 7] ;,, [4] ; [6] ;, [23],,,,,, 5d,, 3 ATCC 7966 QDC0 ATCC % 99.9%,,,

7 Drug 表 6 药 物 敏 感 性 试 验 结 果 Tab. 6 Tests of pathogen sensitivity to drug Isolates tested and sizes in diameter of bacterium-inhibited circles (mm) LZG LZX LZG2 LZX2 LZP2 NNG NNX NNX2 NNG2 NNE NNE2 NNE3 NNE4 Fortum VI Cephalospotin VI Ceftriaxone Streptomycin Kanamycin Cotrimoxazole Neomycin Cefoperazone Albamycin Norfloxacin Enrofloxacin Ciprofloxacin B Polymyxin B Levoflxacin Enoxacin Ofloxacin Cefobid Piperacillin Doxycycline Erythromycin Florfenicol Sulfamethazine Sarafloxacin Sulfamonomethoxine Sulfamethoxydiazine Compound Sulfadiazine : d 20.0 mm ; 0.0 mm d 20.0 mm ; 0 mm d 0.0 mm ; d=0 mm B d 9.0 mm, 0 mm d 9.0 mm ; d=0 mm Note: Under definition when diameter(d) 20.0 mm, the drug sensitivity was considered to be high; when 0.0 mm d 20.0 mm, the drug sensitivity, medium; when 0 mm d 0.0 mm, the drug sensitivity, light; when d=0 mm, the drug sensitivity, none. However, for Polymyxin B d 9.0 mm is high, 0 mm d 9.0 mm is medium, and d=0 mm is insensitivity,,,, 2,,,,,,, [2, 3], [5], [6],, 3.2 PCR 3 hly Aer Alt Act ahal ahp, hly Aer Alt

8 5 : 6 85 Drug names Fortum VI Cephalospotin VI Ceftriaxone Streptomycin Kanamycin Cotrimoxazole Neomycin Cefoperazone Albamycin Norfloxacin Enrofloxacin Ciprofloxacin B Polymyxin B Aeromonas hydrophila High sensitivity No sensitivity 表 7 不 同 病 原 菌 对 26 种 药 物 的 敏 感 率 Tab. 7 Pathogens sensitive to 26 drugs (%) Aeromonas sobria High sensitivity No sensitivity Drug names Levoflxacin Enoxacin Ofloxacin Cefobid Piperacillin Doxycycline Erythromycin Florfenicol Sulfamethazine Sarafloxacin Sulfamonomethoxine Sulfamethoxydiazine Compound Sulfadiazine Aeromonas hydrophila High sensitivity No sensitivity Aeromonas sobria High sensitivity No sensitivity Tab. 8 The source of isolates obtained A Liuzhou A B Liuzhou B C Nanning C D Nanning D 表 8 来 源 于 不 同 养 殖 场 嗜 水 气 单 胞 菌 的 药 敏 感 结 果 比 较 Comparisons of drug sensitivities for isolates obtained from different farms (mm) High effective drugs B B B B Low or no effective drugs VI VI Act ahp 9 00%, ahal 88.89%; 4, Aer Act ahal ahp 00%, hly Alt 75.00% 25.00%; hly + Aer + Alt + Act + ahal + ahp + hly + Aer + Alt + Act + ahal ahp + hly + Aer + Alt Act + ahal + ahp + hly Aer + Alt + Act + ahal + ahp + 4, hly + Aer + Alt + Act + ahal + ahp +,

9 hly + Aer + Alt Act + ahal + ahp + ; alt + ahal hly, alt + ahal + hly +[0] ;, ahpa, aera + hlya + ahpa + [2] ; 4 Aha AHH AerA OMP [4] 7, aer 85.7%, act 28.57%, eprcai 42.86%, alt 85.7%, ahp 57.4%, ahyb 00%, 6 [5],,,, NNE3 hly NNE NNE2 NNE Alt NNX ahal, 0,, NNE3,, 3.3,,, 4, 26, 3,, 2 0, 00%, 88.89%,,,,,,,, (MIC) [24, 25],,,,,,, [] Chen X F, Zhou C Y, Qing X. Research on pathongen of white abdominal shell disease of turtle [J]. Journal of Fisheries of China, 997, 2(3): [,,.., 997, 2(3): ] [2] Ding L, Yue Y S, Song J Y, et al. Studies on the pathogenic bacteria of white shell disease and skin fester disease from Turtle and drug treatment [J]. Freshwater Fisheries, 200l, 3(): [,,,.., 200, 3(): 46 48] [3] Ye Q Z, He J G, Qiu D Q, et al. Bacteria identification and their pathogenesis in red and white abdominal shell disease of Trionyx sinensis [J]. Microbiology, 2000, 27(6): [,,,.., 2000, 27(6): ] [4] Zhou B, Huang X H, Zheng Y Y. Study on pathogenic bacteria and pathological of the white abdominal shell disease from Trionyx sinensis [J]. Current Fisheries, 2000, (0): 7 9 [,,.., 2000, (0): 7 9] [5] Ye Q Z, He J G, Weng S P. Study on the pathogenesis of virus and bacteria in white and red abdominal shell disease of Trionyx sinensis [J]. Freshwater Fisheries, 999, 29(8): 3 7 [,,.., 999, 29(8): 3 7]

10 5 : [6] Shen J Y, Pan X Y, Yu X P, et al. Pathogen in white abdominal shell disease of soft-shelled turtle (Trionyx sinensis) [J]. Journal of Fishery Sciences of China, 2007, (5): [,,,.., 2007, (5): ] [7] Weng S P, Ye Q Z, He J G, et al. Study on the Pathogen and Histopathology of Red and White Skin Disease of Trionyx sinensis [J]. Supplement To The Journal of Sun Yatsen University, 996, (suppl): 6 65 [,,,.., 996, ( ), 6 65] [8] Zhang X J, Yang W M, Li T T, et al. The genetic diversity and virulence characteristics of Aeromonas hydrophila isolated from fishponds with disease outbreaks in Hubei Province [J]. Acta Hydrobiologica Sinica, 203, 37(3): [,,,.., 203, 37(3): ] [9] Tong G X, Wei X X, Li X Z, et al. Isolation, identification and drug sensitive test of Aeromonas sobria from Truogx sinensis [J]. Guangdong Agricultural Sciences, 200, (2): [,,,.., 200, (2): 24 26] [0] Fang B, Li J N, Wang T J, et al. Cloning and sequence analysis of virulence genes of six Aeromonas strains isolated from aquatic animals in Anhui [J]. Journal of Fishery Sciences of China, 2006, 3(6): [,,,. 6., 2006, 3(6): ] [] Pan X Y, Hao G J, Yao J Y, et al. Fusion expression of spe gene with hly gene of Aeromonas hydrophila strain TPS-30 in Escherichia coli [J]. Acta Hydrobiologica Sinica, 200, 34(3): [,, 赟,. TPS-30., 200, 34(3): ] [2] Zhu D L, Li A H, Wang J G, et al. The correlation between the distribution pattern of virulence genes and the virulence of Aeromonas hydrophila strains [J]. Acta Scientiarum Naturalium Universitatis Sunyatseni, 2006, 45(): [,,,.. ( ), 2006, 45(): 82 85] [3] He M X, Ye Q Z, Chen C, et al. Construction and high expression of an act-ompts fusion vector of Aeromonas hydrophila [J]. Acta Hydrobiologica Sinica, 2004, 28(2): [,,,.., 2004, 28(2): 69 73] [4] Wang Z Z, Zhao P P, Wang Z F, et al. Phentype and molecular indentification of virulence genes for four pathogenic AEROMONAS hydrophila isolated from a dying Trionyx sinensis [J]. Oceanologia et Limnologia Sinica, 200, 4(5): [,,,. (Aeromonas hydrophila)., 200, 4(5): ] [5] Jiang C Y, Huang J H, Chen M, et al. Isolation of Aeromonias hydrophila in some pools of Nanjing and detection of the virulence-associated genes [J]. Animal Husbandry & Veterinary Medicine, 200, 42(6): 4 7 [,,,.., 200, 42(6): 4 7] [6] Zhang X J, Yan B L, Bing X W, et al. Detection of hemolysin gene and phenotypic and molecular identification of pathogenic Aeromonas sobria from gibel carp (Carassius auratus gibelio) [J]. Journal of Hydroecology, 200, 3(4): [,,,.., 200, 3(4): 02 07] [7] Heuzenroeder M W, Wong C Y, Flower R L. Flower. Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model [J]. FEMS Microbiology Letters, 999, 74(): 3 36 [8] Vijai Singh G D, Kapoor B N, Mishra W S, et al. Detection of aerolysin gene in Aeromonas hydrophila isolated from fish and pond water [J]. Indian Journal of Microbiology, 2008, 48(2): [9] Cesar B K, Geert H, Mauro T, et al. PCR Detection, Characterization, and Distribution of Virulence Genes in Aeromonas spp [J]. American Society for Microbiology, 999, 65(2): [20] Huang J, Wen H C, Shi J G, et al. Isolation and identification of pathogenic bacteria from Pelteobagrus fulvidraco ulcerative syndrome and its drug sensitive test [J]. Journal of Southern Agriculture, 202, 43(): 07 2 [,,,.., 202, 43(): 07 2] [2] Per Einar Granum, Kristin O'Sullivan, Juan M Tomaès, et al. Possible virulence factors of Aeromonas spp. from food and water [J]. FEMS Immunology and Medical Microbiology, 998, 2: 3 37 [22] Chu W H, Lu C P. Cloning and sequence analysis of an extracellular serine-protease gene of Aeromonas hydrophila J- [J]. Journal of Fisheries of China, 2004, 28(): [,. J-., 2004, 28(): 84 88] [23] Wang H T, He L, Zhang J. Relationship on feed quality and white abdominal shell disease from Trionyx sinensis [J]. Freshwater Fisheries, 2002, 32(2): [,,

11 , 2002, 32(2): 32 33] [24] Meng X L, Chen C F, Wu Z X, et al. In vitro study on sensitivity, development of drug-resistance and reversion of acquired drug-resistance characteristics of Aeromonas hydrophila to oxytetracycline [J]. Journal of Yangtze University (Natural Science Edition) Agricultural Science Volume, 2009, 6(): [,,,.. ( ), 2009, 6(): 42 46] [25] Wang M Z, Chen C F, Liu Z X, et al. In vitro study on drug-resistance characteristics of Aeromonas hydrophila to tetracyclines and fluoroquinolones [J]. Journal of Huazhong Agricultural University, 20, 30(): [,,,.., 20, 30(): 89 93] CHARACTERIZATION OF WHITE PLASTRON DISEASE PATHOGENS AND DETECTION OF SIX KNOWN VIRULENCE GENES IN TRUOGX SINENSIS HUANG Jun, HUANG Yan-Hua, HU Da-Sheng 2, LUO Hua-Ping 3, SHI Jin-Gu, PENG Min-Yi, XUAN Jun-Cheng, QIN Li-Fen, TENG Zhong-Zuo and ZENG Gui-Zhong 3 (. Lab of Aquatic Animal Diseases Diagnosis, College of Animal Science and Technology, Guangxi University, Nanning , China; 2. Guangxi Marine Products Technical Promotion Station, Nanning , China; 3. Wuming fishery Supervision and Management Station, Wuming 53000, China) Abstract: Pathogenetic bacteria were isolated from hearts and livers of the infected individual Truogx sinensis, cultured on conventional methods, and used as challenge to determine their infectiveness. Identities of the isolated pathogens were distinguished through API 20NE, their phylogenetic status were analyzed by 6S rrna sequencing and their sensitivities to drugs were tested by K-B method. The six known virulence genes in the pathogens were detected by PCR. The results showed that, of the thirteen pathogens abtained, nine strains were Aeromonas hydrophila, and four strains were Aeromonas sobria. In phylogenetical relationship of the nine Aeromonas hydrophila, five strains were close to Aeromonas hydrophila ATCC 7966, and four strains were close to Aeromonas hydrophila QDC0, while four Aeromonas sobria were close to Aeromonas sobria ATCC Drug sesnsitive testes indicated that only thirteen strains were found to be highly sensitive to cefoperazone, and pathogens from different farms presented to be quite different in drug sensitivity. Virulence gene detection showed that Aer, Act and ahp was positive at 00%; hly and Alt, 92.3%; and Ahal, 76.92%. Four genotypes were found in pathogens isolated, being hly Aer Alt Act ahal ahp and hly Aer Alt Act + ahal ahp, mainly dominating in Aeromonas hydrophila and Aeromonas sobria, respectively, and strains with hly exhibited stronger pathogenicity. Key words: Truogx sinensis; White plastron disease; Aeromonas hydrophila; Aeromonas sobria; Virulence gene