大口黑鲈白皮病病原菌的分离鉴定及药物敏感性试验

Isolation, identification, and antimicrobial susceptibility test of the pathogen from largemouth bass (Micropterus salmoides) with white skin disease

摘要:

【背景】近年，广东、广西等多地养殖的大口黑鲈(Micropterus salmoides)在苗种期间极易暴发白皮病，并且与已报道的症状不同，严重危害大口黑鲈苗种的生产。【目的】确定大口黑鲈苗种白皮病的病原，通过分析生长特性、毒力因子、致病性、组织病理与敏感药物筛选试验，为该病的研究与防控提供科学参考。【方法】从患白皮病大口黑鲈的病灶处分离细菌，结合形态观察、生理生化指标、16S rRNA基因和gyrB基因序列分析等鉴定菌株；绘制菌株的生长曲线，分析温度、盐度及pH对生长的影响；采用平板法检测菌株毒力因子活性，PCR方法检测携带毒力基因的情况；通过浸泡感染试验验证菌株的致病性及其组织病理特征；采用微量稀释法测定菌株对水产常用的10种抗菌药物和6种消毒剂的敏感性。【结果】从患病的大口黑鲈白皮处肌肉分离纯化到优势菌株ZJS18004，经形态特征、生理生化特性、16S rRNA基因和gyrB基因序列分析，鉴定为维氏气单胞菌(Aeromonas veronii)；菌株ZJS18004的最适生长温度为30℃，最适pH值为8.0，最适盐度为5‰；在30℃时的生长曲线显示0−1 h是迟缓期，1−5 h是对数期；菌株ZJS18004具有溶血性、蛋白酶和脂肪酶活性，携带aer、act、fla、ahyB、exu和lip等6种毒力基因；感染试验证明菌株ZJS18004在23、28和33℃对健康大口黑鲈均具有较强的致病性，与自然发病症状相似，致病力强弱与细菌浓度和水温密切相关；菌株ZJS18004感染造成大口黑鲈肌肉组织溶解，肝、脾、肾脏组织细胞坏死等明显病理损伤；菌株ZJS18004对盐酸多西环素、硫酸新霉素、甲砜霉素、氟苯尼考等4种抗菌药物敏感；三氯异氰尿酸对菌株ZJS18004的体外抑杀菌效果最好，其次是苯扎溴铵。【结论】维氏气单胞菌是大口黑鲈苗种白皮病的主要病原菌，通过分析该菌的生长特性、致病力、组织病理和敏感药物筛选试验，为进一步解析维氏气单胞菌的致病机制和开展精准防控研究打下坚实的基础。

关键词:大口黑鲈;维氏气单胞菌;生长特性;毒力基因;药物敏感性

Abstract:

[Background] In recent years, largemouth bass (Micropterus salmoides) has been prone to white skin disease during seed stage in Guangdong, Guangxi, and other places, with the symptoms obviously different from those reported clinically, which seriously endangers the production of largemouth bass seeds. [Objective] To provide scientific reference for the research and treatment of white skin disease of largemouth bass, we identified the pathogen and analyzed its growth characteristics, virulence factors, pathogenicity, and antibiotic susceptibility as well as the fish histopathological changes caused by it. [Methods] The pathogen was isolated from the focus of largemouth bass suffering from white skin disease and identified based on the morphological characteristics, physiological and biochemical indexes, and 16S rRNA and gyrB gene sequence analysis. Further, the growth curve of the strain was established, and the effects of temperature, pH, and salinity on the growth were analyzed. The activities of virulence factors were detected by plate method, and the virulence genes were screened out by PCR method. The pathogenicity of the strain and histopathological changes caused by it were confirmed by immersion challenge experiments. The susceptibility of strain to 10 antibiotics and 6 disinfectants commonly used in aquaculture was determined with microdilution method. [Results] The dominant strain ZJS18004 was isolated from the largemouth bass with white skin disease, which was identified as Aeromonas veronii. The optimum growth conditions of ZJS18004 were 30 °C, pH 8.0, and salinity 5‰. The growth curve at 30 °C showed that 0–1 h and 1–5 h were the retardation phase and logarithmic phase, respectively. Strain ZJS18004 caused hemolysis, had protease and lipase activities, and carried six virulence genes including aer, act, fla, ahyB, exu, and lip. The results of challenge experiments showed that ZJS18004 had strong pathogenicity to healthy largemouth bass at 23 °C, 28 °C, and 33 °C. The tested fish exhibited disease signs similar to those in natural infections. The pathogenicity was closely correlated with the bacterial concentration and water temperature. The infection of ZJS18004 caused obvious pathological damage such as muscle tissue lysis and necrosis of the liver, spleen, and kidney. The antimicrobial susceptibility assay revealed that ZJS18004 was susceptible to doxycycline hyclate, neomycin sulphate, thiamphenicol, and florfenicol. Six commonly used aquatic disinfectants had certain bactericidal effects on ZJS18004 in vitro, among which trichloroisocyanuric acid ranked first, followed by benzalkonium bromide. [Conclusion] A. veronii was the major pathogen of white skin disease attacking largemouth bass. The results of the present study will build a solid foundation for further analyzing the pathogenic mechanism of A. veronii and carrying out the research on accurate prevention and control.

Key words:Micropterus salmoides;Aeromonas veronii;growth characteristics;virulence gene;antibiotic susceptibility