齐口裂腹鱼无乳链球菌的分离鉴定及其感染的病理损伤

2012–2013年四川齐口裂腹鱼(Schizothorax prenanti)养殖场流行一种临床特征为突眼、体表出血和神经症状的传染病, 在肝、肾涂片检查中发现链状G⁺球菌。从自然发病齐口裂腹鱼分离到2株形态与涂片检查一致的G⁺球菌(DML120817, YZH130830), 其在脑心浸液培养基(BHI)平板上28℃培养48 h, 形成白色圆形、表面光滑、边缘整齐的针尖大小菌落。经人工感染实验证实分离菌株为该病的病原菌。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无乳链球菌(Streptococcus agalactiae); 16S rRNA基因序列分析表明, 2分离株 (GenBank登录号分别为KF773744和KF761304) 与GenBank中无乳链球菌(S. agalactiae)16S rDNA序列同源性达96.5%以上。在以分离菌16S rDNA序列 (GenBank登录号分别为KF773744和KF761304) 及其GenBank中同源性较高的链球菌16S rDNA序列构建的系统发育树上, 分离菌与无乳链球菌聚为一族; 同时在基于无乳链球菌cfb基因进行的特异性PCR检测中, 分离菌均为阳性, 进而鉴定2株分离菌为无乳链球菌。2株无乳链球菌对强力霉素、阿莫西林、先锋霉素V、氧氟沙星和左氧氟沙星等均敏感, 但在新霉素与丁胺卡拉霉素上存在差异。病理学观察发现, 无乳链球菌感染齐口裂腹鱼对多组织器官都造成明显的病理损伤, 尤其是肝、肾、脑的损伤较为严重, 表现为明显的变性、坏死以及炎症细胞浸润, 且细菌侵入肝、肾、脾以及神经元细胞内, 导致线粒体、内质网等细胞器损伤。

Isolation, identification and pathogenicity of Streptococcus agalactiae from Schizothorax prenanti

In 2012-2013, a serious infectious disease characterized by exophthalmia, hemorrhage, and neurological symptoms emerged in farms in Sichuan Province. A Gram-positive, chain-forming coccus was found in liver and kidney smears. Two strains of Gram-positive, chain-forming cocci were isolated from the diseased fish. The colonies had a smooth-surface and were white, circular, regular and pin-head sized after incubation at 28 for 48 h on brain heart infusion (BHI). Artificial infection proved that the isolates were the pathogen of the disease. It was primarily identified to be according to morphological, physiological and biochemical characteristics. Furthermore, a 16S rRNA gene sequence analysis by BLAST in GenBank indicated that the two isolates showed high levels of similarity to (more than 96.5%). A phylogenetic tree was constructed comparing the 16S rDNA sequences of both isolates (GenBank accession number KF773744 and KF761304) to other related bacterial species in the GenBank database. In the phylogenetic tree the two isolates and other strains constituted a branch. In addition, the two isolates were positive in a specific PCR detection of gene. Based on the phenotypic characteristics, phylogenetic analysis of the 16S rRNA gene, and specific PCR detection, both . The sensitivity results showed that both strains were sensitive to doxycycline, amoxicillin, cephalothin V, ofloxacin and levofloxacin, but showed some difference in sensitivity to neomycin and amikacin. Histopathologically, the infection could cause serious pathological changes in multi-organs and tissues, especially in liver, kidney and brain. The main pathologic lesions were degeneration, necrosis and infiltration of the inflammation cells. Electron microscopical examination found that bacteria could invade hepatocytes, renal cells and neurons, causing destruction of mitochondria and endoplasmic reticulum.