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1 微生物学通报 OCT 20, 2009, 36(0): 57~576 Microbiology 2009 by Institute of Microbiology, CAS 简报青虾软壳综合症病原及其特性潘晓艺 * 沈锦玉 * 李建应 2 贺宝祥 2 尹文林郝贵杰徐洋姚嘉赟 (. 3300) ( ) 摘要 : 从患软壳综合症的濒死青虾体内分离到一株细菌 QXL07B, 经人工感染试验, 其对青虾的半数致死浓度 (LC 50 ) 为 CFU/mL, 具有较强毒力 API 32E 系统鉴定及 6S rrna 序列分析, 该病原菌为维罗纳气单胞菌温和生物变种 (Aeromonas veronii biovar sobria, 登录号 : FJ808727) 其系统发育分析表明, 菌株 QXL07B 与维罗纳气单胞菌 ( 登录号 : X720) 和维罗纳气单胞菌温和生物变种 ( 登录号 : AY987729) 的亲缘关系最近, 其同源性都为 99% 药敏实验结果表明 : 菌必治复达欣环丙沙星恩诺沙星氟哌酸等 5 种化学疗剂对该病原菌有较强的抑菌作用关键词 : 青虾, 软壳综合症, 维罗纳气单胞菌, 日本沼虾 Identification and Biological Characteristics of the Pathogen Causing Macrobrachium nipponense Soft-shell Syndrome PAN Xiao-Yi * SHEN Jin-Yu * LI Jian-Ying 2 HE Bao-Xiang 2 YIN Wen-Lin HAO Gui-Jie XU Yang YAO Jia-Yun (. Key Laboratory of Fish Health and Immunology, Chinese Academy of Fishery Science, Zhejiang Institute of Freshwater Fisheries, Huzhou, Zhejiang 3300, China) (2. Deqing Fishery Technical Extension Station, Huzhou, Zhejiang 3300, China) Abstract: A pathogenic bacterial strain QXL07B was isolated from freshwater shrimp (Macrobrachium nipponense) suffering with soft-shell syndrome. The 50% lethal concentration (LC50) of strain QXL07B was CFU/mL, which showed that strain QXL07B was rather strong virulent to Macrobrachium nipponense. Strain QXL07B was gram negative and had β-hemolytic activity on sheep blood agar. By means of API 32E identification and 6S rrna sequence analysis, strain QXL07B was identified as Aeromonas veronii biovar sobria (locus number: FJ808727), which was the closest relative to Aeromonas veronii strain (locus number: X720) and Aeromonas veronii biovar sobria strain (locus number: AY987729) with 99% homology. In addition, strain QXL07B was highly sensitive to ceftriaxone ceftazidime ciprofloxacin enrofloxacin and norfloxacin. Keywords: Freshwater shrimp, Soft-shell Syndrome, Aeromonas veronii, Macrobrachium nipponense, (Macrobrachium nipponense),, (Decapoda), (Palaemonidae), (Macrobrachium), [ 3] 基金项目 :(No. 2008C22058); (No. 2007YN22); * 通讯作者 :Tel: ; : panxiaoyi@36.com, sjinyu@26.com 收稿日期 : ; 接受日期 :

2 572 微生物学通报 2009, Vol.36, No.0, , 20.92, 73 [4] 300,,, [5], [6] 2005,,,,, 4 cm, 3~5 0~2, 4.6%~60%, 7.4%, 90%,,,, 材料与方法. 材料, 4 g~5 g,,.2 病原菌分离,,, 70%,,, (TSA), 28 C 24 h,,.3 病原菌的人工感染试验 60 cm 50 cm 40 cm, 4 g~5 g 0,, 5 d 28 C 8 h~24 h, CFU/mL, (0. ml/ ), 0, 9 C~ 22 C 7 d,,, g:0 ml, 0000 r/min, 0.22 μm,.4 病原菌毒力测定 CFU/mL CFU/mL CFU/mL CFU/mL,, 0. ml 0, 9 C~22 C 7 d, [7] (LC 50 ).5 病原菌鉴定.5. 菌株形态观察和 API 32E 系统鉴定 : 28 C 8 h~24 h,,, (Biomerieux) API32E, [8] Bergey s Manual of Systematic Bacteriology [9].5.2 6S rrna 鉴定 : [0], 6S rrna, 27F: 5 -AGAGTTTGATCCTGGCTCAG-3 (Escherichia coli 8~27) 54R: 5'-AAGGAGGTGATCCA GCCGCA-3' (Escherichia coli 54~ 522) PCR.0%,.6 序列分析 6S rrna NCBI Blastn, DNAStar Megalign GenBank 6S rrna,.7 药敏试验 TSA,

47 0 6 CFU/mL, Devesa [], QXL07B QXL07B 表人工回归感染结果 Table Results of artificial infection test of strain QXL07B ( ) Death number( ) Strain Concentration (CFU/mL) Number d 2 d 3 d 4 d 5 d 6 d 7 d

3 : 573 ( 6 mm), 28 C 24 h 2 结果 2. 病原菌的分离与致病性试验 2, QXL07B QXL07M( 6S rrna GenBank : FJ808728,, Vogesella indigofera 98% ),, QXL07B,, 7 d, 00% ( ) :, QXL07B QXL07M 7 d, 0%,,, 20 d (%) [ log(cfu/ml)] : y=24.5x 0.( ), QXL07B (LC 50 ) CFU/mL, Devesa [], QXL07B QXL07B 表人工回归感染结果 Table Results of artificial infection test of strain QXL07B ( ) Death number( ) Strain Concentration (CFU/mL) Number d 2 d 3 d 4 d 5 d 6 d 7 d Mortality (%) QXL07B % QXL07M % PBS / 图青虾死亡率 (%) 与菌液浓度对数 [log(cfu/ml)] 的关系曲线 Fig. The relation curve of M. nipponense mortality with logarithm of bacterial concentration 2.2 病原菌的鉴定 2.2. 病原菌形态特征和生化鉴定 : QXL07B,,,, β ( 2),,,, API 32E 图 2 菌株 QXL07B 在羊血琼脂平板上产生的 β- 溶血圈 Fig. 2 The beta hemolysis zone of strain QXL07B on sheep-blood agar plate, ATCC506, (A. veronii biovar sobria)( 2) 病原菌 6S rrna 基因序列及系统发育分析 : 6S rrna, QXL07B 500 bp ( 3), 537 bp DNA, GenBank FJ NCBI Blastn, 32

GAT D- DARL 5-5KC α- αglu LIP α- αgal RP TRE β βglu RHA MAN INO MAL CEL ADO SOR PLE α- αmal β- βgur L- ASPA MNT ESC CIT SAL : +: ; : ; g+:.

4 574 微生物学通报 2009, Vol.36, No.0 Item Table 2 QXL07B 表 2 API 32E 生化鉴定 The results of biochemical experiment with API 32E Stain ATCC506 Item QXL07B Stain ATCC506 OX IND ODC N-β βnag ADH β- βgal LDC GLU g+ g+ URE SAC L- LARL L- LARA GAT D- DARL 5-5KC α- αglu LIP α- αgal RP TRE β βglu RHA MAN INO MAL CEL ADO SOR PLE α- αmal β- βgur L- ASPA MNT ESC CIT SAL : +: ; : ; g+:. Note: +: Denotes positivity; : Denotes negativity; g+: Gas produced. 6S rrna, QXL07 (A. veronii biovar sobria) 2.3 药敏结果 QXL07B 4, QXL07B,, 3 3 QXL07B 6S rrna Fig. 3 The electrophoresis photograph of the 6S rrna sequence from strain QXL07B ( Aeromonas spp.), 97%~00% 30 (A. veronii), 4 (A. veronii biovar sobria), (A. veronii bv. veronii), ( 4) QXL07B (A. veronii) (A. veronii biovar sobria) 3 讨论 (Aeromonas veronii) 987 Hickman-Brenner [3], 77 :, [2,4], [5],,

5 : 575 Fig. 4 图 4 基于 6S rrna 序列的系统发育树 The phylogenetic tree based on 6S rrna sequence Antibiotics Diameter of inhibiting ring (mm) 表 3 菌株 QXL07B 的药物敏感性 Table 3 The antibiotic sensitivity of strain QXL07B Antibiotics Diameter of inhibiting ring (mm) Antibiotics Diameter of inhibiting ring (mm) Kanamycin 8 Furazolidone 5 Ceftazidime 29 Compound sulfamethoxazole 5 Norfloxacin 25 Doxycycline 8 Gentamycin 8 Neomycin 9 Enrofloxacin 26 V Cefalin V 7 Tetracycline 8 B Aerosporin B 4 Penicillin 0 Erythromycin 3 Ofloxacin 22 Vancomycine 8 Chloramphenicolum 26 Spectinomycine 2 Ciprofloxacin 26 Streptomycin 9 Ceftriaxone 38 Fosfomycin Novobiocin 0 Vancomycin 0 Nitrofurantoin 5 Rifampicin 7 Sulperazon 22, [6 20], 2005~2008 4, 2,, QXL07B,,,, ATCC506,,, 6S rrna 6S rrna 27F 54R QXL07B 6S rrna,, NCBI Blastn, QXL07B, 6S rrna QXL07B, [2],,, CFU/g 00%, LD CFU/g [22] QXL07B (LC 50 ) CFU/mL,

6 576 微生物学通报 2009, Vol.36, No.0 28 C 36 h β, QXL07B, API 32E,, [23],, [24], Janda 98%9% 76% [25], :,, ;, QXL07B 参考文献 [],,. (). :, 990, pp. 2. [2] Cai Y, Dai AY. Freshwater shrimps (Crustacea: Decapoda: Caridea) from the Xishuangbanna region of Yunnan Province, southern China. Hydrobiologia, 999, 400: [3] Cai Y, Ng PKL. The freshwater palaemonid prawns (Crustacea: Decapoda: Caridea) of Myanmar. Hydrobiologia, 2002, 487: [4],.. :, 2007, p.204. [5],,,.. (), 2000, 9(3): [6],,,.. ( ), 2002, 2(2): [7],,.. :, 2002, pp [8],.. :, 200, pp [9] George M, Julia A, Timothy G, et al. Bergey's Manual of Systematic Bacteriology 2 nd ed Vol.2. New York: Springer, 2005, pp [0],.. :, 2002, pp [] Devesa S. First report of vibriosis in turbot (Scophthalmus maximus) cultured in Northwestern Spain. In: Fish and Shellfish Pathology, Ellis AE (ed). New York: Academic Press, 985, pp [2] Altwegg M, Steigerwalt AG, Altwegg-Bissig R, et al. Biochemical identification of Aeromonas genospecies isolated from humans. J Clin Microbiol, 990, 28(2): [3] Hickman-Brenner FW, MacDonald KL, Steigerwalt AG, et al. Aeromonas veronii, a new ornithine decarboxylase-positive species that may cause diarrhea. J Clin Microbiol, 987, 25(5): [4] Carnahan A, Altwegg M. Taxonomy. In: The Genus Aeromonas//Austin B, Altwegg M, Gosling PJ, et al. (ed). Chichester, Wiley, 996, pp. 38. [5] Nair GB, Holmes B. Subcommittee on the taxonomy of Vibrionaceae. Int J Syst Bacteriol, 999, 49: [6],,.., 994, 4(3): 33. [7],,.., 2005, 24(): [8],,.., 2008, 24(): [9],,.., 2008, 36(9): [20],,.., 2008, 24(2): [2] Chen SC, Lin YD, Liaw LL, et al. Lactococcus garvieae infection in the giant freshwater prawn Macrobranchium rosenbergii confirmed by polymerase chain reaction and 6S rrna sequencing. Dis Aquat Organ, 200, 45(): [22] Sung HH, Hwang SF, Tasi FM. Response of giant freshwater prawn (Macrobranchium rosenbergii) to challenge by 2 strains of Aeromonas spp.. J Invertebr Pathol, 2000, 76(4): [23],. mshb., 2008, 4(0): 4 6. [24] Rahman MH, Colque-Navarro P, Klihn I, et al. Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh. Appl Environ Microbiol, 2002, 68(2): [25] Janda JM. Biochemical and exoenzymatic properties of Aeromonas species. Diagn Microbiol Infect Dis, 985, 3(3):

: X Keywords: Acipenser baerii, Aeromonas hydrophila, Identification, Antibiotic sensitivity (Acipenser baerii) (Acipenserdae)(Acipenser), 26, [

: X Keywords: Acipenser baerii, Aeromonas hydrophila, Identification, Antibiotic sensitivity (Acipenser baerii) (Acipenserdae)(Acipenser), 26, [ 微生物学通报 AUG 20, 2008, 35(8): 1186~1191 Microbiology tongbao@im.ac.cn 研究报告 2008 by Institute of Microbiology, CAS 鲟源致病性嗜水气单胞菌 X1 的分离鉴定与药敏特性研究 * 李圆圆 * 曹海鹏 ** 何珊杨先乐 ( 200090) 摘要 : 从患细菌性败血症的西伯利亚鲟 (Acipenser