患病异育银鲫中嗜冷黄杆菌的分离鉴定及组织病理学观察

Isolation,identification,and histopathological observation of Flavobacterium psychrophilum from diseased Carassius auratus gibelio

为确定越冬期内江苏多地异育银鲫暴发性死亡的原因,本研究利用高通量测序比较了健康样品和患病样品的微生物群落多样性和结构组成的差异,分析了异育银鲫病害暴发过程中的细菌类型及特性。结果显示,在属水平上,患病样品中黄杆菌属丰度最高。在种水平上,患病样品中嗜冷黄杆菌的丰度最高,分别达到63.01%和61.31%,显著高于健康组的1.55%。根据微生物群落特征分析结果,从患病样品体表的病灶处分离出优势病原菌NJ01,通过细菌形态学、生理生化分析、16S rDNA序列比对确定NJ01菌株为嗜冷黄杆菌。人工感染NJ01菌株14 d后,1.7×10^(6)和1.7×10^(7) CFU/mL两组的死亡率达到100%,感染症状和自然发病症状一致。组织病理学观察发现,病鱼的肌细胞坏死,间质中充满了淋巴细胞;肝脏中细胞溶解坏死,细胞核消融;在脾脏中发现脾细胞散在坏死,伴随着充血的症状;肾小管上皮脱落,肾间质存在大量淋巴细胞。药物敏感实验结果显示,NJ01菌株对头孢西叮、头孢哌酮、庆大霉素和克拉霉素等敏感。研究表明,优势菌NJ01是致病菌,可通过扰乱机体正常的免疫和代谢功能导致疾病的发生。本研究首次报道了嗜冷黄杆菌在异育银鲫中的致病性,这将为大宗淡水鱼“越冬综合征”的药物防治及其致病机理研究提供参考依据。

In recent years,the diseases of Carassius auratus gibelio have occurred frequently,especially with the decrease of water temperature in winter and the decline of fish immunity,which made the pathogenic bacteria easy to invade the fish and led to the outbreak of fish disease and the decline of culture efficiency.To determine the cause of the sudden death of C.auratus gibelio in Jiangsu during the overwintering period,high-throughput sequencing was used to compare the diversity and structure of the microbial community between healthy samples and diseased samples.The types and characteristics of bacteria in the outbreak of disease were analyzed.The results showed that the abundance of Flavobacterium was the highest in the diseased samples at the genus level.At the species level,the abundance of F.psychrophilum in the diseased samples increased significantly,reaching 63.01%and 61.31%respectively,which was significantly higher than that in the healthy group(1.55%).According to the results of the analysis of microbial community characteristics,the dominant pathogen NJ01 was isolated from the lesions on the body surface of the diseased samples.Through bacterial morphology,physiological and biochemical analysis,and 16S rDNA sequence alignment,the NJ01 strain was identified as F.psychrophilum.After 14 days of artificial infection with the NJ01 strain,the mortality rate of the 1.7×10^(6) and 1.7×10^(7) CFU/mL groups reached 100%,and the symptoms of infection were consistent with those of natural disease.Histopathological observation showed that the muscle cells of the diseased fish were necrotic and the stroma was full of lymphocytes;the large cells in the liver were lytic and necrotic,and the nucleus melted;splenocytes were found with scattered necrosis in the spleen with symptoms of hyperemia;the renal tubular epithelium fell off;there were a lot of lymphocytes in the renal interstitium.The results of the drug sensitivity test showed that the NJ01 strain was sensitive to cefoxitin,cefoperazone,gentamicin,and clarithromycin.The conclusion was that the strain NJ01 was the pathogen for the death of C.auratus gibelio,which could disturb the normal immune and metabolic function,and consequently the pathological phenomena of tissues and organs or death appeared.In this study,the pathogenicity of F.psychrophilum in C.auratus gibelio is reported for the first time,which will provide a reference basis for the study of drug prevention and the pathogenesis of"overwintering mortality syndrome"in conventional freshwater fish.