半滑舌鳎源维罗纳假单胞菌的分离鉴定及药敏试验

Isolation,identification and drug sensitivity of Pseudomonas veronii from diseased half smooth tongue-sole(Cynoglossus semilaevis Günther)

为探究导致半滑舌鳎(Cynoglossus semilaevis)疾病发生的病原,从患病半滑舌鳎肝脏中分离纯化出一株细菌,命名为CLPV-114,结合细菌形态学观察、生化鉴定和16S rRNA序列分析进行病原鉴定,对其进行药敏试验和生长曲线测定,人工感染试验后测定半数致死剂量(LD50),并进行组织病理学观察。结果表明:菌株CLPV-114为革兰氏阴性杆菌,在系统发育树上与GenBank登录号分别为AF064460、AY144583的维罗纳假单胞菌(Pseudomonas veronii)亲缘关系最近,自然聚为一支;综合生理生化特征和16S rRNA基因序列分析鉴定其为维罗纳假单胞菌;药敏试验显示,CLPV-114对阿莫西林、氨苄西林和头孢哌酮等18种抗菌药物高度敏感,对呋喃妥因、阿奇霉素和四环素等11种抗菌药物中度敏感,对头孢克肟、磺胺异恶唑和甲氧嘧啶等4种抗菌药物耐药;人工感染试验显示,CLPV-114对半滑舌鳎具有致病性,人工感染后的患病鱼与自然患病鱼症状相似,CLPV-114菌株对半滑舌鳎的LD_(50)为1.3×10^(7)CFU/mL;组织病理学观察显示,CLPV-114可引起明显的组织学损伤,表现为肝细胞组织坏死,鳃血管中心充血,脾脏出现含铁血黄素沉积、出血,肠绒毛脱落。研究表明,本次从患病半滑舌鳎肝脏中分离的菌株CLPV-114为维罗纳假单胞菌,对半滑舌鳎具有较强的致病性,可引起不同组织出现明显病理变化,本研究结果可为半滑舌鳎维罗纳假单胞菌病的防控提供科学参考。

In order to investigate the pathogenesis of diseased half smooth tongue-sole(Cynoglossus semilaevis Günther),a bacterial strain was isolated from the liver of the diseased half smooth tongue-sole with body weight of 20 to 40 g,designed as CLPV-114,and identified by morphological observation,physiological,and biochemical features and the sequence analysis of 16S rRNA.The drug sensitivity,artificial infection,histopathological examination and determination of growth curve were examined in the strain CLPV-114.The median lethal dose was determined and histopathological observations were conducted in the artificial infection test.The results showed that the strain CLPV-114 was gram-negative bacterium,and closely related to Pseudomonas veronii(AF064460)and Pseudomonas veronii(AY144583),so identified as Pseudomonas veronii.The drug sensitivity test showed that CLPV-114 was highly sensitive to 18 kinds of drugs including amoxicillin,ampicillin,cefoperazone,moderately sensitive to 11 antibiotics such as nitrofurantoin,azithromycin,tetracycline,and resistant to antibiotics such as cefixime,sulfafurazole and sulfamethoxydiazine.Infection test showed that the clinical symptoms were similar to those of naturally diseased fish,with the LD_(50) of 1.3×10^(7)CFU/mL in CLPV-114 against half smooth tongue-sole.Histopathological observations found that CLPV-114 strain caused obvious histological damage,such as hepatocellular tissue necrosis,vascular congestion of the central filament vessel of gill,haemorrhage and deposition of hemosiderin in the spleen,and the shedding of the intestinal villi.The finding indicates that the strain CLPV-114 isolated from the liver of diseased tongue sole is Pseudomonas veronii,which has strong pathogenicity to tongue sole and can cause significant pathological changes in different tissues,and provides an effective scientific reference for control of Pseudomonas veronii disease in half smooth tongue-sole.