ISOLATION, IDENTIFICATION AND PATHOLOGICAL LESIONS OF PSEUDOMONAS PUTIDA FROM HYBRID CATFISH
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摘要: 为确定一起杂交鲇皮肤溃疡症的病原，实验从病鱼体内分离到几株优势菌（DYJ140914-DYJ140917），根据4株分离菌的形态、生理生化特性，结合16S rRNA和gyrB基因序列测定（GenBank登录号分别为KP693689和KP693690）与系统发育分析，将其鉴定为恶臭假单胞菌（Pseudomonas putida）。在此基础上以腹腔注射的方式进行人工感染试验，证实其为杂交鲇溃疡症的病原菌。病鱼组织器官具有典型的病理变化，其主要靶器官为肝脏、皮肤肌肉以及肾间质，分别引起多灶性坏死性肝炎、坏死性肌炎及坏死性间质性肾炎。此外，还可引起心外膜、心内膜炎及坏死性脾炎。药敏结果显示该菌对强力霉素、诺氟沙星和左氧氟沙星等药物敏感；对青霉素、氟苯尼考、磺胺甲基异恶唑、头孢西丁、阿奇霉素等药物耐药。Abstract: In order to identify the pathogens of the skin ulcer disease in hybrid catfish, several strains of dominant bacteria (DYJ140914-DYJ140917) were isolated from the diseased fish. They were identified as Pseudomonas putida based on morphology, biochemical, physiology characteristics and sequencing of 16S rRNA and gyrase B subunit (gyrB) gene (GeneBank accession number were KP693689 and KP693690 respectively). They were also proved to be the pathogen of the disease by artificial infection experiment. Histologically, liver, skin, muscle, and renal interstitial were the target organs of the disease, which characterized by multifocal necrotic hepatitis, necrotic myositis, and necrotizing interstitial nephritis. Moreover, it could also lead to mild epicardial and endocardial inflammation, and mild necrotic splenitis. The result of drug sensitivity test showed that the isolates were sensitive to the Doxycycline, Norfloxacin, and Levofloxacin, but were resistant to Ampicillin, Azithromycin, Florfenicol, Sinomin, Mefoxin, Penicillin.
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Keywords:
- Hybrid catfish /
- Pseudomonas putida /
- 16S rRNA /
- gyrB gene /
- Pathological lesion
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图 1 杂交鲇感染恶臭假单胞菌的主要剖解症状及肾脏触片观察
A.眼周溃疡灶 (★); B.背部肌肉深度溃疡 (★), 出血; C.尾鳍溃烂 (★); D.肾脏触片可见短杆菌 (→)
Figure 1. Major anatomy symptoms and kidney imprint ofPseudomonas putida infected with hybrid catfish
A. Ulceration of the skin around the eyes (★); B. Ulceration and discoloration of the dorsum skin (★); C. Caudal fin rot (★); D. Some bacterias and inflammatory cells in the Intercellular (→)
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图 2 菌株DYJ140914的16S rRNA基因序列 (A)、gyrB基因序列 (B) 及相关菌株构建的系统发育树
Figure 2. Phylogenetic trees were generated using MEGA5.0 based on alignment for 16S rRNA (A) andgyrB gene (B) sequence of the isolate strain DYJ140914 and other related species inPseudo-monas genus
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图 3 杂交鲇恶臭假单胞菌感染的病理组织学损伤 (HE×100, 200, 400, 1000, 标尺=10, 100, 200 μm)
A.肝脏多灶性坏死; B.肝细胞间隙内大量杆状菌; C.皮肤表皮坏死脱落; D.肌纤维和肌浆凝固, 间隙内大量短杆菌; E.肾间质多灶性坏死; F.间质内大量中性白细胞浸润, 大量红细胞积聚, 细胞间隙可见杆状菌; G.脾脏淋巴细胞坏死, 少量杆状菌; H.心外膜及心内膜炎性细胞浸润; I.心脏动脉球外膜空泡样极度扩张
Figure 3. Pathohistological lesions of hybrid catfish infected withPseudomonas putida (HE×100, 200, 400, 1000)
A. Multifocal necrosis in liver; B. Rod-shaped bacteria can observed in the intercellular of liver; C. Epidermis of skin necrosis and shedding; D. Muscle fibers and sarcoplasmic with solidification and honeycomb and many filamentous bacteria exist in muscle fibers; E. Severe focal necrosis in renal interstitium; F. Many erythrocytes, inflammatory cells and rod-shaped bacteria in intercellular of kidney; G. Splenocytes necrosis and with some rod-shaped bacterias; H. Infiltration of inflammatory cells in epicardium and endocardium; I. Adventitia of aortic bulb extreme expansion
表 1 分离菌的生化特性
Table 1 Biochemical characteristics of the isolated strain
测定项目Item 菌株DYJ140914-DYJ140917 P. putidaa P. putidab 精氨酸水解酶Arginine dihydrolase + + + 鸟氨酸脱羧酶Ornithine decarboxylase - - - 赖氨酸脱羧酶Lysine decarboxylase - - - 细胞色素氧化酶Cytochrome oxidase + + + 尿素酶Urease + N/A + 吲哚Indole test - - N/A 硫化氢H2S - - - 葡萄糖氧化发酵Gas from glucose + + + 明胶液化Gelatin liquefaction - - - 枸橼酸盐Citrates + N/A + 蔗糖Sucrose - - N/A 甘露醇Mannitol - - N/A 麦芽糖Maltose - N/A - 硝酸盐还原Nitrate + + + 乙酰胺Acetamidase - N/A - 甲基红试验MR test - - - V-P试验VP test - - - 木糖Xylose + N/A + 半乳糖ONPG - N/A - 注: +.阳性; -.阴性; N/A.未选用项目; a.引自参考文献[19]; b.引自参考文献[10, 11, 13] Note: “+” Positive reaction. “-” Negative reaction. “N/A” not applicable. “a” Quote from references [19], “b” Quote from references [10, 11, 13] 
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表 2 病原菌人工感染健康杂交鲇实验结果
Table 2 The mortality of hybrid catfish challenged (intraperitoneal injection) with different doses of the isolate DYJ140914
分组 (数量) Group 细菌浓度 (cfu/mL) Bacteria concentration 剂量 (mL) Dose 7天内死亡数Mortality within 7 days 死亡率 (%) Death rate 1 2 3 4 5 6 7 1(20) 3.4×108 0.1 0 1 3 5 5 3 0 85 2(20) 3.4×107 0.1 0 1 2 3 5 1 0 60 3(20) 3.4×106 0.1 0 0 2 2 4 1 0 45 4(20) 3.4×105 0.1 0 0 2 3 1 0 0 30 5(20) 3.4×104 0.1 0 0 1 0 1 0 0 10 6(20) 0 0.1 0 0 0 0 0 0 0 0
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表 3 菌株DYJ140914的药敏试验结果
Table 3 The sensitivity of isolate bacteria DYJ140914 from a disease outbreak in hybrid catfish to various antimicrobial agents
抗生素名称Antibiotic 标准Standard (mm) 抑菌圈直径及结果Diameter of inhibition zone (mm) 结果Result 耐药 (R) 中介 (I) 敏感 (S) 氨苄青霉素Ampiciuin ≤14 15-19 ≥20 0 R 青霉素Penicillin ≤19 20-22 ≥23 0 R 新霉素Neomycin ≤12 13-16 ≥17 13 I 强力霉素Doxycycline ≤12 13-15 ≥16 20 S 氟苯尼考Florfenicol ≤12 13-17 ≥18 0 R 诺氟沙星Norfloxacin ≤12 13-16 ≥17 22 S 磺胺甲基异恶唑Sinomin ≤12 13-16 ≥17 0 R 头孢西丁Cefoxitin ≤14 15-17 ≥18 0 R 阿奇霉素Azithromycin ≤13 14-17 ≥18 0 R 左氧氟沙星Levofloxacin ≤13 14-16 ≥17 24 S 利福平Rifarnpin ≤16 17-19 ≥20 13 R 注: “R”表示耐药, “I”表示中度敏感, “S”表示敏感 Note: R. Resistance. I. Middle sensitive. S. Sensitivity
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