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1 47 3 Vol.47, No OCEANOLOGIA ET LIMNOLOGIA SINICA May, 2016 (Mylopharyngodon piceus) (Plesiomonas shigelloides) * ( ; ; ; ), (Mylopharyngodon piceus), (HZ2015), 16S rrna, (Plesiomonas shigelloides),, ; (LD 50 ) CFU;, HZ2015, ; ; S943 doi: /hyhz (Mylopharyngodon piceus),,, 70kg,,,, (Li et al, 1995), (Aeromonas hydrophila) (Pseudomonas fluorescens) (Wang et al, 2009;, 2010),,,, ,,,,, ;, ;,,, 5% 1,,,, ; Premix Ex Taq, PCR TaKaRa ( ); Qiagen ( ); ( g),,, ; 40 SPF BALB/c *, ;, 2013C32045 ; 2014B70044,, yejian1015@163.com :, @163.com : , :

2 %,, LB ; LB 37 C 36 h,, 20% (V/V), 80 C 1.3,,, JSM-6360LV API ID32E (Biomerieux, ) S rdna 的 PCR 扩增 测序和分析, 16S rrna (8F: 5 -AGAGTTTGATCCTGGCTCAG-3 ; 1492R: 5 - AAGGAGGTGATCCAGCCGCA-3 ) PCR PCR PCR, Invitrogen 16S rrna NCBI Blast 16S rrna,, MEGA4.0,, 药敏实验 (K-B ), 24h, CFU/mL, LB,, 37 C 24h 1.6 动物实验 LB, 30 C 18h, : CFU/mL, h, mL ; 0.1mL 28 C,,, mL, CFU/mL 0.1 ml, LD h,, 1 2mm,, ( 1),,,, HZ2015,,, ( ) μm 1.5μm ( 2) 1 Fig.1 Arrangement of the colonies shown on nutrient agar 2 Fig.2 TEM image of the strain 2.2 ATB ID 32E ( 1), HZ2015, 99.9%

3 3 : (Mylopharyngodon piceus) (Plesiomonas shigelloides) 635 表 1 病原菌的生化特征 Tab.1 Major biochemical characteristics of the pathogenic bacteria strains Item HZ2015 Item HZ2015 L- + L- L- + + L- + D- D- L- D- + L βd- 4- -βd D- L αd N- 1-βD- 4- -αd- + D- L- D- + + D- D- 4- -βd αd S rrna NCBI Blast, HZ2015, 98.5% 99.9% 16S rrna, (Salmonella) (Edwardsiella) (Enterobacter),, 药敏实验 29 ( 2), HZ Fig.3 3 HZ S rdna Phylogenetic tree of strain HZ2015 based on 16S rrna gene sequence

4 表 2 药物敏感性试验 Tab.2 Results of test for antibiotic susceptibility (mm) (mm) (P) R(10) (OX) R(7) (AMP) R(6) (PRL) I(20) (CTRX) R(0) (CXM) S(27) (CFP) S(24) (MID) I(13) (EM) I(14) (GM) S(25) (OFLX) I(13) (NOR) I(12) (DA) I(16) (VA) R(10) (TE) I(16) (CL) I(17) (SXT) S(22) (CB) R(9) (CN) R(10) VI(PVI) S(22) V(PV) S(21) (ZOX) S(30) (KA) S(26) (N) S(22) (DO) S(21) (LR) S(24) (GR) I(14) B(PB) S(15) (AOZ) S(18) B ; , 28 C, 36h,, 48, 100%, 0( 3), ( 4),,,, 16S rdna, 表 3 回归感染试验 Tab.3 Results of artificial infection test 12h 24h 36h 48h HZ2015 0/10 0/10 7/10 10/10 Control 0/10 0/10 0/10 0/10

5 3 : (Mylopharyngodon piceus) (Plesiomonas shigelloides) Fig.4 Ulcerated body surface of diseased fish A: 自然感染 ; B, C: 人工感染 h, 24h, 48h ( 4), HZ2015 LD CFU/mL Tab.4 (CFU/mL) 表 4 HZ2015 细菌毒力实验 Pathogenic test of strain HZ2015 in mice (ml) 1d 2d 3d 4d (%) PBS ,,,,,,,,,,,, HZ2015,, 16S rdna,, 98.5% 99.9%,,,,, 16S rdna, HZ2015,,,, (Brenner et al, 2005), 3536 (Chen et al, 2013), 7.3% (Matsuyama et al, 2015),, 鲀 (Takifagu obscurus) (Ctenopharygodon idellus) (Trionyx sinensis) (Pelteobagrus fulvidraco) (Huso huso Acipenser baeri ) (Tilapia nilotica) (Carassius auratus gibelio Cyprinus carpio ) (Macrobrachium rosenbergiideman) (Ietalurus punetaus) (Scleropages macrocephalus) (Clarias leather) (Anguilla japonica) (Oncorhynchus mykiss) (Miller et al, 1986;, 2004; 劼, 2005; 璠, 2007;, 2009;, 2009; Salgado-Miranda et al, 2010; Jun et al, 2011; Parvez et al, 2011; Joh et al, 2012; Lingham et al, 2012;, 2013;, 2013;, 2014;, 2015), (, 2014),,,,, C

6 (Lingham et al, 2012),,, 25 C,, 28 C,,,,,,, ; (, 2004;, 2009; Matsuyama et al, 2015) (Chen et al, 2013),,,,,,, , 53(7): ,,, , 41(9): ,,, (Plesiomonas shigelloides)., 55(1): 劼,,, 鲀., 25(4): 18 21,, , 24(4): ,,, , 29(5): ,,, , 39(2): 48 53,,, , 24(3): ,,, , 54(2): 璠,,, , 28(6): Brenner D J, Krieg N R, Staley J T, Bergey s Manual of Systematic Bacteriology (Vol. 2): The Proteobacteria, Part B: The Gammaproteobacteria. New York, US: Springer, Chen X, Chen Y, Yang Q et al, Plesiomonas shigelloides infection in Southeast China. PLoS One, 8(11): e77877 Joh S J, Ahn E H, Lee H J et al, Bacterial pathogens and flora isolated from farm-cultured eels (Anguilla japonica) and their environmental waters in Korean eel farms. Veterinary Microbiology, 163(1 2): Jun J W, Kim J H, Choresca Jr C H et al, Isolation and molecular detection of Plesiomonas shigelloides containing teta gene from Asian arowana (Scleropages formosus) in a Korean aquarium. African Journal of Microbiology Research, 5(28): Li S F, Lu G Q, Zhou B Y, Evaluation on the potential capacity of the swan oxbow for the conservation of the major Chinese carps. Aquaculture, 137(1 4): Lingham T, Besong S, Ozbay G et al, Antimicrobial activity of vinegar on bacterial species isolated from retail and local channel catfish (Ictalurus punctatus). Journal of Food Processing & Technology, 4(S11): S Matsuyama R, Kuninaga N, Morimoto T et al, Isolation and antimicrobial susceptibility of Plesiomonas shigelloides from great cormorants (Phalacrocorax carbo hanedae) in Gifu and Shiga Prefectures, Japan. Journal of Veterinary Medical Science, 77(9): Miller M L, Koburger J A, Tolerance of Plesiomonas shigelloides to ph, Sodium chloride and temperature. Journal of Food Protection, 49(11): Parvez N, Rathore G, Swaminathan T R et al, Isolation and characterization of bacteria associated with ulcerative disease of the fish, Clarias gariepinus. Bulletin of Pure & Applied Sciences-Zoology, 30(2): Salgado-Miranda C, Palomares E, Jurado M et al, Isolation and distribution of bacterial flora in farmed rainbow trout from Mexico. Journal of Aquatic Animal Health, 22(4): Wang H R, Hu Y H, Zhang W W et al, Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine. Vaccine, 27(30):

7 3 : (Mylopharyngodon piceus) (Plesiomonas shigelloides) 639 ISOLATION AND IDENTIFICATION OF PATHOGENIC PLESIOMONAS SHIGELLOIDES FEOM DISEASED BLACK CARP MYLOPHARYNGODON PICEUS YE Jian 2, LIU Xiao-Ning 3, WAN Xiao 4, XU Hui 4, SHEN Wen-Ying 1, GAO Min-Jie 1, ZHANG Xiao-Dong 1 (1. College of Life Sciences, Shaoxing University, Shaoxing , China; 2. Hangzhou Aquacultural Techica Extending Centre, Hangzhou , China; 3. Hangzhou Dajiangdong Industrial District Agricultural Bureau, Hangzhou , China; 4. Shaoxing Entry-exit Inspection and Quaratine Bureau, Shaoxing , China) Abstract In a recent outbreak of massive death of black carp Mylopharyngodon piceus in a breeding farm in Hangzhou, showing ulcerated body surface, we isolated a bacterium from liver, kidney, and muscle of diseased fish. In morphology, Gram s staining, ATB ID32E bacterial biochemical identification, and sequence analysis on the 16S rrna gene, the isolate was identified as Plesiomonas shigelloides, named HZ2015. In our regress experiment, similar symptoms replayed. The same bacterium could be identified biochemically in 16S rdna homology analysis. Isolate HZ2015 was demonstrated with virulence potential in mouse model, and the median lethal dose (LD 50 ) in mice was CFU/mL. Therefore, we deduce that P. shigelloides was the pathogenic bacteria responsible for the disease. In addition, in our susceptibility test on antibiotics, the bacterial strain HZ2015 was susceptible to cefoperazone, cefuroxime, gentamicin, kanamycin, and ceftazidime. This study presents the first case report on P. shigelloides-induced massive death of black carp M. piceus in China. Key words Mylopharyngodon piceus; Plesiomonas shigelloides; strong pathogenicity