Biologia ( IF 1.5 ) Pub Date : 2020-11-02 , DOI: 10.2478/s11756-020-00639-z Chinmayee Muduli , Gayatri Tripathi , Kurcheti Paniprasad , Kundan Kumar , Rajeev Kumar Singh , Gaurav Rathore
Aeromonas hydrophila is considered an important pathogen of fish causing substantial economic loss in aquaculture worldwide. The objective of the current study was to assess the virulence potential of A. hydrophila strains isolated from gills of apparently healthy fish using phenotypic assays, PCR based genotypic tests, and in-vivo pathogenicity assays in Indian catfish, Clarias magur. For this, motile aeromonads (n = 50) were isolated from gills of apparently healthy freshwater food fish (n = 50) collected from domestic fish market. Gill swabs were incubated for primary enrichment in alkaline peptone water supplemented with cephalothin (10 mg/mL) for 12 hours followed by plating on Starch Ampicillin Agar. A single big yellow colony was selected from each sample, purified, and presumptively identified as Aeromonas with the help of Aerokey-II. Out of the fifty isolates of Aeromonas, five isolates of diverse origin (9C, 10G1, 7C, 10C and 4P) were confirmed to be A. hydrophila by automated bacterial identification system VITEK 2 with confidence interval of 90–98%. In the phenotypic assays, strains 9C and 10G1 showed high serum resistance, swimming and swarming activity and low biofilm producing capabilities, which were indicative of putative virulence. Both the strains belonged to genotype aah+act+ alt+ascV+eno+lip+ast+ on the basis of detection of virulence genes by PCR, i.e. extracellular haemolysin (aah), cytolytic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), type-III secretion system (ascV), enolase (eno), lipase (lip) and heat-stable cytotonic enterotoxin (ast). In-vivo pathogenicity assays confirmed that both the strains were pathogenic to magur fish. The LD50 for 9C strain was 6.81 × 104 CFU/mL and 7.62 × 105 CFU/mL for 10G1strain. In conclusion, our phenotypic and genotypic findings showed that A. hydrophila isolated from apparently healthy fish harbour number of important virulence genes/factors and could have important implications in triggering disease in farmed fish under stress.
中文翻译:
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从看似健康的淡水食用鱼中分离出嗜水气单胞菌的毒力
嗜水气单胞菌被认为是鱼类的重要病原体,导致全世界水产养殖的大量经济损失。本研究的目的是使用表型测定,基于PCR的基因型检测以及印度cat鱼的体内致病性检测来评估从明显健康的鱼g中分离的嗜水链球菌菌株的毒力潜力。。为此,从从国内鱼市场中收集的看似健康的淡水食用鱼(n = 50)的isolated中分离出了运动型气单胞菌(n = 50)。将ill拭子在补充了头孢菌素(10毫克/毫升)的碱性蛋白water水中孵育12小时,然后在淀粉氨苄青霉素琼脂上平板接种。从每个样品中选出一个大的黄色菌落,纯化,并在Aerokey-II的帮助下假定鉴定为气单胞菌。在五十种气单胞菌菌株中,五种不同来源的菌株(9C,10G1、7C,10C和4P)被确认为嗜水链球菌。通过自动细菌识别系统VITEK 2,置信区间为90–98%。在表型分析中,菌株9C和10G1显示出较高的血清抗性,游泳和成群活动以及较低的生物膜产生能力,这表明假定的毒力。在通过PCR检测毒力基因的基础上,这两个菌株均属于基因型aah + act + alt + asc V + eno + lip + ast +,即细胞外溶血素(aah),溶细胞性肠毒素(act),热不稳定的细胞张力性肠毒素(alt),III型分泌系统(ascV),烯醇酶(eno),脂肪酶(lip)和热稳定的细胞紧张性肠毒素(ast)。体内致病性分析证实,这两个菌株均对玛格鱼有致病性。9C菌株的LD 50为6.81×10 4 CFU / mL,10G1菌株为7.62×10 5 CFU / mL。总之,我们的表型和基因型分析结果表明,嗜水气单从重要的毒力基因/显然因素健康的鱼港号分离并可能在压力下养殖鱼类疾病引发重要的意义。