E.coli DH5 Á Taq DNA , 1.5%, ( ), Giemsa CsCl % TSB 28 o C 24~48 h 3 80 o C API32E 0% 1% 3% 5% 7% 10% ph

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1 , , Scophthalmus maximus H1 H1, LD CFU/mL CFU/mL 1.5% API32E H1 99.9% 16S RNA H1 99% (Edwardsiella tarda) Scophthalmus maximus (Edwardsiella tarda) S965 A (Scophthalmus maximus) (Bothidae) (Scophthalmus),,, [1] 1992, + [2], [3] [4~10] ~9 10 cm 100% 500~600 H1 API32E 16SrRNA cm - 13 cm±1 cm 48 g±2 g pmd18-t Marine Sciences/Vol.33,No.7/

2 E.coli DH5 Á Taq DNA , 1.5%, ( ), Giemsa CsCl % TSB 28 o C 24~48 h 3 80 o C API32E 0% 1% 3% 5% 7% 10% ph o C 10 o C 15 o C 20 o C 30 o C 35 o C 40 o C 45 o C 24 h 600 nm, S rrna PCR DNA DNA S rrna PCR 16S rrna PCR 27 F 5 ä-agagtttgatc(c\a)tgg- CTCAG-3 ä( E.coli 16S rrna 8 27 ) R : 5 ä- TACGG (C/T) TACCTTGTTACGACTT-3 ä( E.coli 16S rrna ) [11] 25 ÌL PCR 2.5 ÌL 10 PCR 2 mol/l MgSO 4,0.5 ÌL 10 mol/l 4 dntp 0.5 ÌL 10 Ìmol/L 0.2 ÌL TaqDNA U/mL 21 ÌL PCR 94 o C 5 min 94 o C 1 min 52 o C 1 min 72 o C 1 min o C 10 min PCR DNA S rrna pmd18-t Vector 16 o C E.coli DH5 Á LB 37 o C LB 37 o C 170 rpm 6~8 h PCR rrna GenBank BLAST 20 H1 16 rrna BIOEDIT Clustalw H1 GenBank rrna PHYLIP3.6a3 [12] (Neighbor - joining), (Bootstraping), Accession number H1 16S rrna GenBank FJ /2009 / 33 / 7

3 1 16S rrna Tab.1 The strains for phylogenetic dendrogram constructed from GenBank and their accession number Edwardsiella ictaluri AF E.tarda LTB EU E.ictaluri CGX DQ E.tarda WY28 EU E.ictaluri HSN-1 EF E.ictaluri EU E.ictaluri HSS-1 EF E.ictaluri EU E.tarda SMW7 EF E.ictaluri EU E.tarda TX1 EF E.tarda 0304 FJ E.ictaluri GDH1 EF E.tarda K3 FJ E.tarda FV2 EF E.tarda K5 FJ E.tarda EH-202 EU E.tarda K7 FJ E.ictaluri TCCC11053 EU E.tarda K20 FJ h 1.5% CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL 100 cm 100 cm cm± 1 cm 48 g±2 g 17 o C 0.2 ml/ 1.5% [13] H1 17 o C 13 cm±1 cm 48 g± 2 g LgLD 50 =X K d( ²P i 0.5) X K d P i i CsCl 48 h TSB H1 2.2 H1 TSB h 1~2 mm 1 Fig.1 1 H1 Electronic micrograph of the isolated strain H1 A. ; B. A. rod-shaped organism with flagella; B. fissiparism 2.3 0%~4% ph 5.5~9.5 15~40 API32E 99.9% S rrna 16S rrna 16S rrna 2 Marine Sciences/Vol.33,No.7/

4 2 H1 API32E Tab.2 Bio-chemical characteristics of strain H1 by API32E test E.tarda H1 E.tarda H1 ODC + + +James IND + + ADH - - N- - Â- ÂNAG - - LDC + + Â- ÂGAL - - URE - - GLU + + L- LARL - - SAC - - GAT + + L- LARA KG - - D- DARL - - LIP - - a- aglu - - RP + + a- agal - - Â- ÂGLU - - TRE - - MAN - - RHA - - MAL + + INO - - ADO - - CEL - - PLE - - SOR - - Â- ÂGUR - - a- amal - - MNT - - L- ASPA Fig.2 2 H1 16SrRNA Amplification product of 16SrRNA gene of strain H bp E.coli DH5 Á DNA bp NCBI GenBank BLAST 3 H1 16S rrna 16S rrna H1 EU % d CFU/mL 4 16 d CFU/mL 80% CFU/mL 4 API32E lgld 50 =lg( ) 1 (1+9/10+8/10+7/10+ 5/10+3/10 0.5)=5.45 LD 50 = CFU/mL 3 H1 90% 60 /2009 / 33 / 7

5 H1 API32E 16S RNA Edwardsiella tarda Fig.4 4 Fig 3 3 Artificially infected turbot by strain H1 16S rrna Phyogenetic tree based on 16S ribosomal gene sequences 3 Tab.3 Result of challenge test of turbot infected by H1 [19] [20] (E tarda wild type) (CFU/mL) ( ) (ml) ( ) (%) [21] Hoshina red disease [14],,,, [15] [16,17] Regalla [18], - [21] Marine Sciences/Vol.33,No.7/

6 10% 30%~40% TRBIV [22] [1],. Scophthalmus maximus [J].,1995,10 (11) : 1-3. [2]. + [J].,2002,23(4) 1-7. [3],,,. [J]., 2001,23 (2) : [4],. [J]., 2004,25(6): [5]. [J].,2004, [6]. [J].,2005, [7],,,,,.. :. :, 2003a [8],,,.. ( ). 2003b [9]. [J]. 2002, [10]. [J].,2003,24(3): [11] Regalla J. Isolation of Edwardsiella tarda from a seal[j]. Pepos Trab Inst Nac Vet,1998,14 : [12]. [M]. : [13]. [M]. :, [14] Hoshina T. On a new bacterium Paracolobactrum anguillimortif erum n.sp. Bulletin of the Japanese Society of Scientific Fisheries, 1962a, 28 : [15],,. [J] (3): [16]. [J] (4): [17]. [D] [18],. Hep-2 [J] [19] Nougayrede P H Vuillaume A Vigneulle M et a1. First isolation of Edwardsiella tarda from diseased turbot (Scophthalmus maximus)reared in a sea farm in the bay of Biscay[J] Bull Eur Assoc Fish Pathol, [20],.., 2007, [21],. [J]. 2007,31(4): [22],. [J] /2009 / 33 / 7

7 Isolation and identification of the pathogentic bacterium causing red body from cultured Turbot Scophthalmus maximus DONG Li 1,2, WANG Yin-geng 2, ZHANG Zheng, QU jiang-bo 3, CHEN Xia 4 (1. College of Life Science,Ocean University of China,Qingdao ,China; 2. Yellow Sea Fisheries Research Institute,Chinese Academy of Fisheries Sciences,Qingdao ,China; 3. Tianyuan Aquaculture Limited Company Yantai ,China;4. Sunny Oceans Aquaculture Technology Co., LTD, Qingdao ,China Received: Sep.,23,2008 Key words: Scophthalmus maximus red body identification of bacterium Edwardsiella tarda Abstract: A dominant strain of the bacteria causing red body of turbot was isolated from the diseased turbot and designated as H1.In an artificial infection test,all fish of the experimental group died in 15 days after intramuscularly being injected with a high bacterial suspention CFU/mL, and the low bacterial suspention 1.41 CFU10 3 /ml did not cause any death, while the control group showed no signs all through the experimentation. The LD 50 was calculated as CFU/mL.The moribund experimental fish had gross signs similar to the natural infected fish. The bacterium re- isolated from the challenged fish also had the same characteristics as H1, which proved that the isolated H1 was the pathogenic bacterium that triggered this red body disease. Different methods were used to identify the pathogenic bacterium. The bacterium is negative, rod in shape, peritrichous flagella under electronic microscope. Traditional biochemical identification revealed that H1 exhibited relatedness to Edwardsiella tarda, while the identification result by API 32E system indicated that H1 was E. tarda with 99.9% reliability. Its 16S ribosomal RNA gene sequences were analyzed, and it is shown from the anlysis results that this strain of bacterium has a close phylogenetic relation (99%)to E.tarda. In summary, the pathogenic bacterium was identified as E. tarda. ( ) Marine Sciences/Vol.33,No.7/

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2月5期aa 中国农学通报 212,28():2-27 Chinese Agricultural cience Bulletin 血清 11 型鸭疫里默氏菌的分离鉴定 1,2 2, 2, 2, 2, 2, 2, 杨晓辉, 程龙飞, 陈红梅, 施少华, 傅光华, 万春和, 黄瑜 ( 1 福建省农业科学院畜牧兽医研究所, 福州