青鱼病原嗜水气单胞菌分离鉴定、毒力因子检测及药敏试验

Isolation and identification,virulence factor detection,and susceptibility test of pathogen Aeromonas hydrophila isolated from Mylopharyngod piceus.

2011年8月江苏常州市武进区前黄镇某养殖场养殖的青鱼出现大量死亡,症状主要表现为病鱼口腔、头部、眼眶、鳃盖边缘、鳍条基部及鱼体尾柄处充血,肛门红肿。解剖后,可见肠道内无食物,并充血发红,腹腔内有的有淡黄色积液,肝胰脏呈紫红色、胆囊肿大。从患病青鱼肝脏、血液中分离到大量优势生长的细菌。人工感染试验证实分离菌(WJ110801-1)对健康青鱼具有很强的致病性。对分离菌进行了形态特征、理化特性等表型生物学特征检验、毒力因子检测、胞外酶分析及药敏试验,同时应用分子生物学方法测定了代表菌株的16S rRNA基因序列。结果显示,病原菌进化树与嗜水气单胞菌同源性最高,在99%以上;结合分离菌的表型及分子生物学分析,判定该菌为气单胞菌属(Aeromonas)的嗜水气单胞菌(A.hydrophila)。气溶素Aer和不耐热性细胞肠毒素alt是嗜水气单胞菌主要的毒力相关因子,利用设计的两对特异性引物对进行Aer和alt基因的PCR扩增,分离菌可以扩增出大小分别为280 bp的和250 bp的基因片段,表明本次分离的病原嗜水气单胞菌具有气溶素和细胞肠毒素毒力因子;胞外酶分析显示,分离菌具有淀粉酶、DNA酶、脲酶、明胶酶活性。分离菌进行药敏试验结果表明,供试37种抗生素中,氨曲南、氟苯尼考、庆大霉素等12种抗生素抑菌效果显著。文章报道了病原嗜水气单胞菌引起常州青鱼大量死亡,并针对病原做了详细的研究,对青鱼的疾病防控和健康养殖具有重要的参考意义。

In August 2011, a mass fish kill of the cultured Mylopharyngod piceus occurred in a farm in Qianhuang Town, Wujin District of Changzhou City, Jiangsu Province of East China. The main symptoms of the moribund fish were mainly manifested in the hyperemia of mouth, head, eye, gill cove edge, fin and caudal peduncle, and anal swelling. The postmortem observations showed that the intestine contained no food and was red from hyperemia, there were many yellow- ish effusion in abdominal, the liver was purple, and the gallbladder swollen. Large amount of bacteria with growth advantage was isolated from the liver and blood of diseased M. piceus, and through artificial infection test, the isolated strain （ WJ110801-1 ） showed very strong pathogenic to M. piceus. The phenotypic characteristics of the isolated strain, including the morphological characteristics, physiological and biochemical characteristics, virulence factor detection, extra- cellular enzyme analysis, and susceptibility test, were examined, and the 16S rRNA gene was amplified and compared with those sequences deposited in GenBank databases. The 16S rRNA sequence of the pathogen was more than 99% homology with that of Aeromonas hydrophila. The phenotype and molecular biological analyses show that the identified isolate belongs to A. bydrophila. Aerolysin （aer） and cytotoxic enterotoxin （alt） were the main virulence factors of A. hydrophila, which could produce toxins capable of causing death, By using two pairs of specific primers for the PCR amplification of aer gene and alt gene, the 280 bp and 250 bp gene frag- ments were amplified from the isolate, indicating that the isolated A. hydrophila carried virulence factors aerosol and cell toxin. The extracellular enzyme analysis showed that the isolate had amyl- ase, DNA enzyme, urease, and gelatinase activities. The drug susceptibility test by paper exten- sion methods showed that among the test 37 antibiotics, 12 antibiotics including Ammonia-South, Florfenicol, and Gentamicin had obvious inhibitory effect mass mortality of M. piceus caused by A. hydrophila in tailed study of the pathogen, which would have reference health management during M. piceus culture. on the strain. This paper reported the Changzhou City, and conducted a de- significance for the disease control and