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1 45 2 Vol.45, No OCEANOLOGIA ET LIMNOLOGIA SINICA Mar., 2014 (Pseudosciaena crocea) * ( ; ; ; ) (Pseudosciaena crocea) NZBD9 NZBD11, C, 7 10 C C, 16S rdna, NZBD9 NZBD11 NZBD9 7, ; ; ; S941 doi: /hyhz (Pseudosciaena crocea), (, 2007),,, 2013, 1 4, 20 C, 0.5 3mm, (2006) (Nocardia seriolea)(2008) (2012)(Pseudomonas putida), 1 1.1, 100g, 150g 1.2, TSA, 18 C 24h, TSA 18 C 24h 20%, 80 C , 10, 1 3d *(), ;, , ,, @qq.com :,, yanqp@jmu.edu.cn : , :

2 C 24h, 0.2mL, 10 3 cfu/g, 0.2mL 7d, 3,, C 1, 3 3, 7 10 C C C, 0.2mL, 1, 6 1, cfu/g, 0.2mL, C, , 3min, 10000r/min 70%, 3min, 10000r/min,, Microflex LRF S rdna E.Z.N.A BacterialDNA Kit( Omega ) NZBD9 NZBD11 DNA 2 ( ), 27F: 5 -AGAGTTTGAT CCTGGCTCAG-3, 1492R: 5 -TACGGCTACCTTGTT ACGACTT-3 25 L PCR : 1 L, 0.5 L, Mg 2+ 2 L, DNTP 2 L, 10 Buffer 2.5 L, Taq 0.2 L, 15.3 L: 94 C 5min; 94 C 50s; 54 C 50s; 72 C 90s; 30 ; 72 C 10min 1% 100mA, 110V, 20min PCR PCR : E.Z.N.A GelExtraction Kit ( Omega ) PCR PCR S rdna NCBI Blast ( Blast/), ClustalX 1.83 GenBank,, Bootstrap (1000 ) 1.6 TSA 18 C 24h cfu/ml, 0.1mL TSA, 10min ( ), 18 C 24h 1.7, Bouin, Leica RM2128 ( 3 5 m), Leica AUTOSTAINER XL H.E, Leica DM 4500B , NZBD1 NZBD16, NZBD1 NZBD9 NZBD11 NZBD14 NZBD15 18 C 48h, NZBD9 NZBD11 TSA 1 2mm C 1 6, NZBD9 NZBD11 Tab.1 表 1 6 株分离菌人工感染结果 The results of artificial infection of 6 isolations ( ) 1d 2d 3d 4d 5d 6d 7d ( ) (%) NZBD NZBD NZBD2 0 0 NZBD3 0 0 NZBD NZBD % NaCl 0 0

2 : (Pseudosciaena crocea) 411, NZBD9 NZBD11,, NZBD9 NZBD11 NZBD9 2 10 cfu/g, ; 10 2 cfu/g, ; 1 10 5 cfu/g 100%; NZBD9 3 3 7 10 C 24 27 C, ; 16 19 C, 50%; Tab.

3 2 : (Pseudosciaena crocea) 411, NZBD9 NZBD11,, NZBD9 NZBD11 NZBD cfu/g, ; 10 2 cfu/g, ; cfu/g 100%; NZBD C C, ; C, 50%; Tab.2 表 2 菌株 NZBD9 人工感染结果 The results of artificial infection of strain NZBD9 ( ) (cfu/g) 1d 2d 3d 4d 5d 6d 7d ( ) (%) % NaCl 0 0 Tab.3 表 3 不同温度下菌株 NZBD9 人工感染结果 The results of artificial infection of strain NZBD9 under different temperatures ( C) ( ) (cfu/g) 1d 2d 3d 4d 5d 6d 7d ( ) (%) % NaCl % NaCl % NaCl 0 0 1, mm, NZBD9 NZBD11 1 Fig.1 Symptoms of internal organs white-spots disease of P. crocea S rdna NZBD9 NZBD11 16S rdna S rdna 1459bp,, 2.4 NZBD9 NZBD , NZBD9 NZBD Blast 2 16S rdna 100%, NZBD9, NZBD9, NZBD9 19

412 45 2 NZBD9 NZBD11 16S rdna Fig.2 Sequence of 16S rdna genes of strain NZBD9 and NZBD11 Fig.

4 NZBD9 NZBD11 16S rdna Fig.2 Sequence of 16S rdna genes of strain NZBD9 and NZBD11 Fig.3 3 NZBD9 NZBD11 Results of strain NZBD9 and NZBD11 identified by Time of Flight Mass Spectrometer 4, 9 B 11 ; ,,,, ( 7a),,, ( 7c), ;

5 2期胡娇等: 大黄鱼(Pseudosciaena crocea)内脏白点病病原分离鉴定及致病性研究图4 时间飞行质谱鉴定仪对 NZBD9 的具体鉴定结果 Fig.4 Results in detail of strain NZBD9 identified by Time of Flight Mass Spectrometer Fig.5 Results in detail of strain NZBD11 identified by Time of Flight Mass Spectrometer 图5 时间飞行质谱鉴定仪对菌株 NZBD11 的具体鉴定结果 413

6 Fig.6 6 NZBD9 16S rdna Phylogenetic tree based on 16S rdna sequence of strain NZBD9 GenBank,, ( 7b) 2.7.2,, ( 7d),, ( 7e);,, ( 7f) 2.7.3,,,,,, ;,, H.E, ( 7g),,,,

2期胡娇等: 大黄鱼(Pseudosciaena crocea)内脏白点病病原分离鉴定及致病性研究图7 415 患病大黄鱼肾脏脾脏肝脏组织病变 Fig.7 Pathological changes of kidney, spleen and liver tissues of diseased P. crocea a. 健康大黄鱼肾脏图; b.

7 2期胡娇等: 大黄鱼(Pseudosciaena crocea)内脏白点病病原分离鉴定及致病性研究图7 415 患病大黄鱼肾脏脾脏肝脏组织病变 Fig.7 Pathological changes of kidney, spleen and liver tissues of diseased P. crocea a. 健康大黄鱼肾脏图; b. 病鱼肾脏肾小管完全失去原来的组织结构, 解体后形成大面积的坏死灶, 坏死灶中心区充满解体后的细胞碎片(箭头所示); c. 肾小管形状较不规则, 肾小管上皮细胞肿胀, 排列较紊乱(箭头所示); d. 健康大黄鱼脾脏图; e. 病变脾脏组织脾脏组织恶化充血, 坏死较严重(箭头所示); f. 髓窦内堆积大量含铁血黄素, 髓窦周围出现许多空泡(箭头所示); g. 健康大黄鱼肝脏图; h. 肝细胞坏死严重, 细胞萎缩, 排列疏松, 肝细胞界限模糊不清, 空隙较明显; i. 肝细胞实质结构遭到破坏, 干细胞崩解, 呈现空泡化(箭头所示)

8 Tab.4 表 4 菌株 NZBD9 的药敏性实验结果 Results of antibiotic sensitivity test of strain NZBD9 ( g/ ) (mm) S 10 7 R (TMP/SMZ) 1.25/ R S S 30 7 R S 5 7 R B S S S S R R 30 9 R R 30 7 R S 5 27 S R ; S ( 7h);,, ( 7h 7i) 3,,,, (2006) (Wang et al, 2005), (2004), (2002) (2008) (2012), Blanco (2002) López-Romalde (2003) Balboa (2007), 6 2, 4, 2,,,,,, (, 2002;, 2011) (Nishimori et al, 2000), (Se et al, 2000),, (LD 50 ) cfu/fish (Se et al, 2000),, 28 C, 20 C, C,,, : 8,,,, , 30(1): ,,, , 26(): 77 81,,, , 28(2): 5 7,,, (Larimichthys crocea)., 39(3): ,,, , 29(1): 1 6,,, 2007.

9 2 : (Pseudosciaena crocea) 417 (Vibrio alginolyticus)(pseudosciaena crocea)., 38(4): ,,, WBC-3., 42(4): ,,, , 32(2): Balboa S, Ferguson H W, Romalde J L, Phenotypic, serological and genetic characterization of Pseudomonas anguilliseptica strains isolated from cod, Gadus morhua L., in northern Europe. Journal of Fish Diseases, 30(11): Blanco M M, Gibello A, Vela A I et al, PCR detection and PFGE DNA macrorestriction analyses of clinical isolates of Pseudomonas anguilliseptica from winter disease outbreaks in sea bream Sparus aurata. Diseases of Aquatic Organisms, 50: L pez-romalde S, Magariños B, Ravelo C et al, Existence of two O-serotypes in the fish pathogen Pseudomonas anguilliseptica. Veterinary Microbiology, 94(4): Nishimori E, Kita-Tsukamoto K, Wakabayashi H, Pseudomonas plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis. International Journal of Systematic and Evolutionary Microbiology, 50: Se Chang Park, Ichiro Shimamura, Minoru Fukunaga et al, Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglossicida, as a candidate for disease control. Appl Environmental Microbiology, 66(4): Wang G L, Yuan S P, Jin S, Nocardiosis in large yellow croaker, Larimichthys crocea (Richardson). Journal of Fish Diseases 28: ISOLATION, IDENTIFICATION AND VIRULENCE OF THE PATHOGEN OF WHITE-SPOTS DISEASE IN INTERNAL ORGANS OF PSEUDOSCIAENA CROCEA HU Jiao 1, ZHANG Fei 1, XU Xiao-Jin 1, SU Yong-Quan 2, QIN Ying-Xue 1, MA Ying 1, ZHANG Yi 3, HAN Kun-Huang 4, YAN Qing-Pi 1 (1. Fisheries College of Jimei University, Key Laboratory in East China Sea Seawater Healthy Aquaculture of the Ministry of Agriculture, Xiamen , China; 2. College of Ocean and Earth Sciences, Xiamen University, Xiamen , China; 3. Ningde Aquatic Product Technology Promotion Department, Ningde , China; 4. Ningde Fufa Aquatic Product Co. Ltd., Ningde , China) Abstract Two dominant strains (NZBD9 and NZBD11) were isolated from internal organs of Pseudosciaena crocea suffered from white-spots disease in April Both strains could cause the disease artificially in the internal organs in but at 7 10 and The results indicate that the two strains were the causative agent of the disease in farmed P. crocea. Both were identified as Pseudomonas plecoglossicida by time-of-flight mass spectrometer in 16S rdna sequences. Experiments in susceptibility showed that NZBD9 is highly sensitive to 7 drugs including gentamicin, norfloxacin, and tetracycline. Histopathological observation showed that affected fish had clear symptoms of degeneration and necrosis in liver, kidney and spleen. Key words Pseudosciaena crocea; Pseudomonas plecoglossicida; pathogenicity; internal organs white-spots disease

Microsoft Word - 361-370 tb20110529邱杨玉

Microsoft Word - 361-370 tb20110529邱杨玉微生物学通报 Microbiology China tongbao@im.ac.cn MAR 20, 2012, 39(3): 361 370 2012 by Institute of Microbiology, CAS 研究报告大黄鱼 (Larimichthys crocea) 内脏白点病的病原分离和组织病理学观察邱杨玉 1 郑磊 2