草鱼源致病性维氏气单胞菌的鉴定及其黏附特性分析

Identification of pathogenic Aeromonas veronii isolated from grass carps(Ctenopharyngodon idella) and analysis of its adhesion characteristics

采用表型鉴定与细菌16S rRNA基因序列分析对从患病草鱼体内分离的病原菌株16-1进行分类鉴定,综合该菌株的表型特征与16S rRNA基因序列,确定其为维氏气单胞菌。随后,对该菌株的细胞黏附特性及其携带的黏附素基因进行检测与分析。结果显示,分离菌株能以聚集性方式黏附于鲤鱼上皮瘤细胞(EPC)周围,平均黏附菌数随共孵育时间延长而增加,90min达到峰值,说明该分离株具有良好的细胞黏附性。黏附因子检测表明,该菌株同时携带omp AI、omp AII和ahl13种黏附素基因,这些黏附素基因在分离菌株与不同来源参考株之间的核苷酸序列和氨基酸序列同源性分别介于95.8%~99.5%和95.0%~100%(omp AI),96.3%~99.1%和97.9%~100.0%(omp AII),78.6%~99.6%和75.5%~99.4%(aha1),说明所携带的omp AI和omp AII黏附素基因在不同来源的维氏气单胞菌之间具有较高的保守性。

The taxonomy of pathogenic strain 16-1 recovered from the diseased grass carps was identified by phenotype examination combined with 16 S rRNA sequence analysis.The strain 16-1 was identified as A.veronii based on morphological and structure features,culture characteristics,physiological and biochemcal characteristics,as well as 16 S rRNA gene sequence.Subsequently,the adhesin-carrying genes and the adhesion characteristics to EPC cells of the strain were also detected.The results showed that the strain 16-1 carried three kinds of adhesin genes including Omp AI,Omp AII and aha1.The nucleotide and amino acid homology of the adhesin genes among strain 16-1 and different sources reference strains respectively ranged from 95.8%to 99.5%and 95.0%to 100%(Omp AI),96.3%to 99.1%and 97.9%to 100.0%(Omp AII),78.6%to 99.6%and 75.5%to 99.4%(aha1).The strain 16-1 could adhere to the EPC cells mainly in aggregative pattern,and the average adhesive bacterial number around each cell increased steadily with the extension of co-incubation time,peak value reached 51.77±1.17 at 90 min post co-incubation,while cells almost completely ruptured and fell off due to the pathogenicity of bacteria at 120 min post co-incubation.These results indicated that the pathogenic A.veronii strain 16-1 isolated from the diseased grass carps exhibited a good adhesion capability to EPC cells.The adhesion genes OmpAI and OmpAII were highly conservative among different sources of A.veronii.