嗜水气单胞菌引致的金钱鱼细菌性疾病

Research on Aeromonas hydrophila caused bacteria disease in spotted scat (Scatophagus argus)

  • 摘要: 为了分离鉴定实验室养殖的金钱鱼暴发性疾病的病原,利用传统病原分离的方法,从病鱼肝脏分离得到一株G-短杆菌(Ah201416),对其进行电镜观察和生理生化鉴定,对病鱼组织进行病理切片分析,并根据科赫法则,用分离株对健康金钱鱼进行人工感染。结果显示,该菌株电镜下观察细菌大小为0.8~1.0μm×1.0~2.0μm(宽×长),无芽孢和荚膜,生理生化特性与嗜水气单胞菌基本一致;16S rRNA序列(登录号:KR006248)与GenBank中嗜水气单胞菌(Aeromonas hydrophila,AhATCC7966)的16S rRNA基因序列的同源性高达99%,系统进化树与Ah聚为一支。病理切片分析发现,发病金钱鱼与正常金钱鱼相比,鳃小片细胞不同程度地脱落,伴有白细胞浸润;肠绒毛结构消失,肌肉层疏松明显;肾脏中肾小管细胞脱落,管腔中有坏死细胞,并出现不同程度的颗粒变性;肝脏细胞形态不规则,伴有血细胞浸润等病理损伤。人工感染实验表明,其对健康金钱鱼96 h的半数致死剂量(LD50)为7.35×107 CFU/mL。研究表明,发病金钱鱼中分离的Ah201416菌株为嗜水气单胞菌,丰富了金钱鱼细菌性病原的研究,此结果为金钱鱼细菌性疾病的防治和养殖业的健康发展提供了重要的理论依据。

     

    Abstract: Spotted scat Scatophagus argus is popularized in South China as a new resource of marine aquaculture and ornamental fish in recent years. The research of S. argus was focused on biological characteristics, gonad development and reproduction, composition of muscle, artificial culture and the construction of a cell line, and so on. Until now, only three parasites, including Lenolopa huizhouensis, Metahaliotrema mizellei and M. gemiratohamula have been recorded to exist in the gut and gill of S. argus. In aquaculture, priority is given only to bacterial diseases caused by various aquatic pathogenic bacteria, which led to enormous economic loss in the aquatic animals, but the bacteria diseases were not reported in the S. argus. Therefore, the tasks of comprehensive study on the bacterial disease in S. argus become more urgent and important. In order to separate and identify the pathogen of fulminant disease in S. argus cultured in a laboratory in Shanghai Ocean University, we used the traditional method of pathogen isolation, virus and parasite were not detected, but we obtained a bacterial strain isolated from the diseased fish liver. And then, we observed the isolate strain by the electron microscope and tested the physiological and biochemical identification, and histopathological analysis was made in the natural incidence of fish, in addition, artificial infection was also implemented according to Koch's postulates. The results show that the isolate is a Gram-negative staining, cells straight with rounded ends, 0.8-1.0 μm ×1.0-2.0 μm, while observed by electron microscope, and consistent with A. hydrophila through physiological and biochemical characteristics. 16S rRNA gene sequence analysis by Blast in GenBank indicates that the isolate (accession number:KR006248) showed high levels of similarity to Ah ATCC7966 (99%). A phylogenetic tree of the isolate and other Ah strains formed a tight cluster. Histopathological analysis of diseased fish shows the pathological damages, including gill lamellae cells denudation and leukocytes infiltration in gills, abscission of intestinal villus, muscles loosening in intestine and a marked increase of pyknosis of glomerulus in kidney, infiltrating of hemocytes and inflammatory cells in liver. The healthy S. argus could be infected through artificial infection using the isolated bacteria. The half of lethal dose (LD50) to S. argus in 96 h is 7.35×107 CFU/mL. To conclude, the Ah201416 strain was identified as A. hydrophila which is the pathogen for S. argus. This study provides a theoretical basis for disease control and prevention and calls on people to pay more attention to the potential dangers caused by A. hydrophila against S. argus.

     

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