似鲶高原鳅温和气单胞菌的分离鉴定及其感染的病理损伤

Isolation,Identification and Pathogenicity of Aeromonas sobria from Triplophysa siluroides

2013年9月,四川雅安某似鲶高原鳅养殖场发生以体表出血、皮肤溃疡、内脏器官肿大、出血与坏死为特点的疾病。从自然发病的似鲶高原鳅肝与肾分离到1株G-短杆菌（XJJ130928）,其在BHI平板上28℃培养24 h,形成灰白色、表面光滑、边缘整齐、半透明状微隆的圆形菌落,人工感染证实其为本次似鲶高原鳅发病的病原菌。根据分离菌株的形态学和生理生化检测结果初步判定其为温和气单胞菌Aeromonas sobria;进一步的16S rRNA和gyrB基因序列分析表明,分离株的16S rRNA与gyrB基因序列（GenBank登录号：KF761305与KJ139988）在GenBank中进行Blast比对与A.sobria同源性最高。在以分离株16S rRNA和gyrB基因序列及GenBank中同源性较高的序列构建的系统发育树上,分离株与A.sobria聚为一族,其同源性分别为96.0%-99.0%与94.5%-98.0%,结合生理生化特性鉴定分离菌为A.sobria。该菌对头孢西丁、亚胺培南、强力霉素、氟苯尼考和氧氟沙星等敏感,对磺胺甲基异恶唑、洛美沙星、环丙沙星中度敏感,对氨苄青霉素、链霉素、卡那霉素耐药。组织病理学观察发现,A.sobria感染似鲶高原鳅对多组织器官都造成明显的病理损伤,尤其是肝、肾、脾、肠的损伤较为严重,表现为明显的淤血、出血,变性,坏死及炎症细胞浸润。

In September 2013, a serious infectious disease characterized by surface bleeding, cutaneous ulcers, swelling, bleeding and necrosis of visceral organs occurred in the Triplophysa sihtroides farm in Ya＇ an, Sichuan province. One Gramnegative, short, rod-shaped bacterial strain （XJJ130928） was isolated from the liver and kidney of sick T. siluroides. The phenotype of the colonies were offwhite, circular, smooth-surface, regular and translucent after incubation at 28% for 24 h on BHI medium. The strain was considered as the etiological agent as confirmed by artificial infection test. The strain XJJ130928 was primarily identified as Aeromonas sobria according to the morphology, physiological and biochemical charac- teristics. Furthermore, sequence analysis of the 16S rRNA and gyrB gene by aligning in the GenBank indicated that the 16S rRNA （KF761305） and gyrB gene sequences （KJ139988） of the isolated strain were highly similar to A. sobria. Phyloge- netic trees were then constructed by comparing the 16S rRNA and gyrB gene sequences with other homogeneous sequences in GenBank, respectively. In the phylogenetic trees, the isolated strain and other A. sobria strains were clustered into the same branch with 96.0% ~ 99.0% and 94.5% ~ 98.0% sequence similarity. Based on the phenotypic characteristics and phylogenetic analysis of the 16S rRNA and gyrB gene, the isolated strain was confirmed to be A. sobria. The result of drug susceptibility test showed that the strain was sensitive to cefoxitin, imipenem, doxycycline, florfenicol and ofloxacin. Meanwhile, the strain was moderately sensitive to sinomin, iomefloxacin and ciprofloxacin, but resistant to ampiciuin, streptomycin, and kanamycin. Histopathologic observation showed that A. sobria could cause obvious pathological changes in several tissues and organs of T. siluroides, such as serious pathogenicity of congestion, bleeding, degeneration, necrosis and inflammatory cell infiltration, especially in liver, kidney, spleen and intestines.