许氏平鲉致病菌P5W的分离鉴定及致病力分析

Isolation,identification and pathogenicity analysis of pathogenic bacterium P5W in Sebastes schlegeli

为明确养殖过程中许氏平鲉(Sebastes schlegelii)病害爆发的原因,本研究从烟台养殖区自然发病的许氏平鲉病灶处分离获得1株优势菌株P5W,综合采用形态特征观察、结合线粒体16S rDNA序列比对与系统发育进化鉴定分析,明确菌株P5W为哈维氏弧菌(Vibrio harveyi)。进一步开展了人工感染实验、组织病理学观察、胞外产物酶活性及溶血性分析、理化特性及药敏实验等研究,结果显示:人工感染证实菌株P5W为导致此次许氏平鲉患病的病原菌,且半数致死量LD50为5.7×10^(6)CFU/mL;患病许氏平鲉肝、脾、肾和肌肉等组织器官出现了不同程度的病变,主要表现在肝细胞出现明显空泡化,脾脏组织细胞发生离散,肾小管上皮细胞脱落坏死,肌纤维断裂;P5W胞外产物具有蛋白酶、脂肪酶、卵磷脂酶、淀粉酶、尿素酶及明胶酶活性,对绵羊红细胞有溶血活性;P5W的适宜生长条件为15~35℃, pH值5~10, NaCl含量(w/v%)1~8,最适生长条件为温度25℃, pH值7, NaCl含量2~4;P5W对头孢噻肟、头孢曲松、左氟沙星、四环素、复方新诺明等13种药物尤为敏感。研究结果为许氏平鲉养殖过程中弧菌类疾病的防治提供了理论和实践指导。

Sebastes schlegelii is the main fish for proliferation and release and deep-water cage aquaculture in the coastal areas of northern China.In recent years,with the expansion of the scale of S.schlegelii farming and the limitations of disease prevention and control techniques,aquaculture diseases occur frequently.To find out the cause of the disease and death of S.schlegeli during culture,a dominant strain P5W was isolated from the focus of diseased S.schlegeli in Yantai aquaculture area.Through morphological characteristics observation,mitochondrial 16S rDNA sequence comparison and phylogenetic evolutionary identification,the strain P5W was identified as Vibrio harveyi.The artificial infection experiment,histopathological observation,extracellular product enzymatic and hemolytic activity analysis,physiological biochemical and susceptibility testing were further carried out.Healthy S.schlegelii was then challenged with the strain P5W through intraperitoneal injection.The results confirmed that P5W was the pathogenic bacterium of S.schlegelii,causing ulceration and congestion on the surface of challenged S.schlegeli.The median lethal dose of P5W was 5.7×10^(6)CFU/mL.Histopathological analysis revealed that the liver,spleen,kidney and muscle showed different degrees of lesions and necrosis.For example,hepatocytes were obviously vacuolized,blood cells diffused in spleen,kidney tubule partial epithelial were shed and necrotic and muscle fiber fractured.The extracellular products of the bacterium had the activities of protease,lipase,lecithin,amylase,urease and gelatinase,and the hemolytic activity of sheep erythrocytes.P5W could survive at temperatures ranging from 15 to 35℃,acidities varying between pH 5 and 10,NaCl content(w/v%)ranging from 1 to 8.The best condition for P5W was 25C,pH 7,and NaCl content varying between 2 and 4.The susceptibility testing showed that P5W was particularly sensitive to 13 drugs including cefotaxime,ceftriaxone,levofloxacin,tetracycline and cotrimoxazole.The research results provided a theoretical and practical guidance for the prevention and control of vibriosis during the culture of S.schlegelii.As a strong pathogenic strain of V.harveyi,P5W can be used to selecting and breeding anti-disease seedlings and developing a V.harveyi vaccine for S.schlegelii.