Studies On Ulcerative Disease Syndrome Pathogen Of Pelteobagrus Fulvidraco And Its Major Virulence Factors

Pelteobagrus fulvidraco, which blongs Siluriformes, Bagridae, Pelteobagrus is in the taxonomy, and widely distributed in China's Yangtze River, Pearl River, Heilongjiang River and the Pacific River system, is one of the important economic freshwater fish of our country, and one of the admirable fingerling export, of which economic value beyond of four domestic fishes. In China, catfish has successfully achieved the domestication of artificial breeding, and the production of artificial breeding catfish reached50million tons, the output value over1billion yuan per year. Because of changes of the breed model and the breeding scale expand ceaselessly. There have been new infectious disease outbreak and epidemic. In recent years, a large number of P. fulvidraco occurred an infectious disease in many Chongqing's intensive fish farms, of which prevalence rate and mortality rate exceed30%and15%, respectively. The disease characterized swelling of fins, bleeding, muscle ulcers and septicemia. The prevalence of disease have caused huge economic losses, and seriously jeopardized the survival and development of fish farming. However, the infectious etiology is unknown; there is no effective methods for prevention and treatment it. Therefore, determination of the etiology and pathogenic mechanism can provide clinical basis for effective prevention and treatment ulcerative disease syndrome of P. fulvidraco. This has important theoretical and practical significance for P. fulvidraco farming.The etiology, which cause ulcerative disease syndrome of P. fulvidraco, at present, is not clear. Its pathogenic mechanism could be studied. Because of more than10species of pathogenic bacteria can cause fish ulcerative disease syndrome, it is necessary to clear the pathogen of P. fulvidraco ulcerative disease syndrome and understand its pathogenesis for effectively control ulcerative disease syndrome. This study focuses on the isolation and identification of pathogenic bacteria from the prevalence of P. fulvidraco,by using Bacterial biochemistry and molecular identification methods. The virulence associated gene aerolysin, hemolysin, extracellular serine proteases and lipases were cloned and sequenced. Furthermore, the recombinant hemolysin is obtained by heterologous expression in Escherichia coli. This provides a basis for establishment of A. sobria detection technology and subunit vaccine research by use of recombinant hemolysin as antigen. The main results of this research are as follows:1. Isolation, identification of the pathogen and its pathogenicityTwo isolates of A. sobria, named as strain RC-07-KA and strain RC-07-XB, were isolated from heart blood of P. fulvidraco. Both of them can be cultured on LB medium, MacConkey's agar and fresh blood agar. The biochemical tests results showed that the oidase test and arginine test demonstrate positive, the O/F fermentation, KCN growth test, aescin glycosides test and salicin test demonstrate negative. These results addressed that the biochemical properties consistent with that of A. sobria. The16S rRNA gene fragment was amplified by polymerase chain reaction and the sequence was aligned with the25sequences of bacteria published on GenBank. The results revealed that the isolates of ours showed close phylogenetic relationship with the other6strains of A. sobria, which shared96.4-99.8%homology with each other.Animal regression experiment results show that A. sobria strain RC-07-KA can kill yellow catfish, carp and crucian by muscle inoculation with6.15×107CFU/fish in7days. Histopathological observation showed that the challenged fish characterized as renal glomerular enlargement, glomerular capillary endothelial cell and renal tubular epithelial cell swelling, the visible filamentous fibrin exudation within some of the lumen, visible inflammatory leukocyte infiltration in renal tubule interstitial; the portion of the cardiac muscle fiber swelling, ambiguous grosgrain; hepatic cellular degeneration and necrosis, derangement, fibrin exudation at central vein, neutrophil infiltration; part of intestinal mucosa epithelial shedding, a large number of neutrophils and a small amount of macrophages infiltrating in enteral. The results demonstrated that A. sobria strain RC-07-KA can cause obvious pathological injury of multiple organ of crucian.A. sobria strain RC-07-KA was inoculated and cultured in LB medium. The cultures were filtered, and the filtration was employed to detect the hemolytic activity on the LB agar which contained10%fresh blood. The results showed that the cell-free filtration can lysis red blood cells, and the hemolytic titer reached1×25. The results Indicates that the A. sobria strain RC-07-KA posses strong βhemolysin production characteristics. Extracellular crude extracts were obtained by two step salting-out method. The hemolytic test results demonstrated that extracellular crude extracts address hemolytic activity. This suggest that hemolytic activephemolysin can be obtained by ammonium sulfate precipitation method, despite its low yields.2. Cloning and sequence analysis of main virulence gene of strain RC-07-KAThere are many virulence factors associated with Aeromonas pathogenicity, including aerolysin, hemolysin, extracellular serine proteases and lipases, ect. In order to study the virulence factor of A. sobria strain RC-07-KA, the hemolysin, aerolysin, extracellular serine protease and lipase gene were amplified by polymerase chain reaction. The four gene fragments were cloned and sequenced. The obtained gene sequences were analyzed.2.1Aerolysin gene0.7kb fragment of aerolysin gene of A. sobria strain RC-07-KA was amplified by use of PCR method and cloned into pGEM-T vector. The sequencing result showed that the gene fragment is size of692bp, which can encode a peptide of230amino acid residues. The results of Blast analysis revealed that the obtained sequence shared similarity with the aerolysin gene of Aeromonas, which belongs to the virulence factors aerolysin superfamily of Aeromonas, and containing conserved domain of pore-forming toxins.Phylogenetic analysis results showed that A. sobria strain RC-07-KA close to aerolysin gene from China, Japan and India strains(Accession:AB109093, EF034117, HM853019, AF443392, AF443394, AF443395), which located in the same cluster. In the meanwhile, the analysis result display that aerolysin gene of Chinese isolates belong to several main clusters.2.2β hemolysin geneA pair of primers were designed and synthesized according to the published hemolysin nucleotide sequence from A. sobira in GenBank. The gene fragment of (3hemolysin was amplified by PCR through the designed primers. Sequencing results of the cloned gene fragment revealed that the obtained β hemolysin gene is in size of1467bp, which can encode a peptide of487amino acid residues, an about54kDa protein. Phylogenetic analysis results showed that β hemolysin gene of A. sobria RC-07-KA close to A. hydrophila strain sb (AY611033), NLEP A-1607(AF410466),AEF (HM853019),A. sobria strain357(AY157998), A. sobria Homo sapiens isolate(EF620533) and A. salmonicida strain17-2(X65048), with shared over95%homology, and lower homology with the other strains. Phylogenetic tree analysis results demonstrated that the β hemolysin gene of Aeromonas can be divided into several distinct gene clusters, the β hemolysin gene of A. sobria strain RC-07-KA located in cluster Ⅱ, with the reference strains of AY611033, EF620533, AY157998, X6504, AF410466and HM853019.Comparative analysis the hydrophilic, antigenic index and surface accessibility of hemolysin revealed that the hemolysin have strong antigenicity, with a lower antigenic differences between A. sobria strain RC-07-KA and closed strains. This suggests that beta hemolysin may be used as a potential candidate vaccine.2.3Extracellular serine protease geneA1.9Kb gene fragment of extracellular serine protease of A. sobria strain RC-07-KA was obtained by using PCR method. The fragment was cloned and sequenced. The results addressed that the extracellular serine protease gene is1875bp, which contains an open reading frame, encoding a peptide of624amino acid residues. Phylogenetic tree analysis results showed that the extracellular serine protease gene of A. sobria strain RC-07-KA located in gene cluster Ⅱ, along with A.sobria strain288(AF253471), A. hydrophila, South Korea isolate (AF126213) and A.veronii B565(CP002607).The sequence similarity is93.6%,93.3%and92.5%, respectively. These results suggest that the extracellular serine protease gene of strain RC-07-KA was successfully cloned, an important basics for further studies.2.4Lipase geneA lipase gene fragment of A. sobria strain RC-07-KA was obtained by using PCR method. The fragment was cloned and sequenced. The results showed that lipase gene is2445bp of in size, which contains an open reading frame, encoding a peptide of814amino acid residues, an about83kDa protein. Phylogenetic tree analysis results demonstrated that the lipase gene of A. sobria strain RC-07-KA located in gene cluster Ⅱ, along with A.sobria strain AS228(AB206038), AS008(AB206037) and288(JN019936). The sequence similarity is93.6%,93.3%with AB206038and AB206037, respectively.3. construction of recombinant hemolysin expressing vector The β hemolysin gene was amplified, purified and cloned into the EcoRI and XhoI restriction enzyme digestion site to construction recombinant hemolysin expressing vector pET28a-Hly. The gained recombinant plasmid pET28a-Hly was identified by restriction enzyme digestion. The results demonstrated that pET28a-Hly be successfully constructed. The pET28a-Hly was transformed into E.coli. strain Rosetta for recombinant protein expression. SDS-PAGE analysis results demonstrated that the band of target protein appear between66.4KDa and44.3KDa, in line with expectations. The recombinant protein was confirmed by Western-blot test. The results suggest the target protein have successfully expressed in E.coli, which is an important basics for further studies.4.Optimal expression, purification and activity analysis of recombinant β hemolysinIn order to get more soluble recombinant hemolysin, the condition of inducible expression were optimized based on IPTG concentration, temperature and the time of induce. The results demonstrated that soluble target protein reached at a relatively high level when IPTG concentration is0.4mmol/L,32℃, induce period3-4hours.Amount of soluble recombinant β hemolysin were expressed at the optimized conditions, and the recombinant protein were purified by Ni2+-Sepharose6Fast Flow gel affinity chromatography. SDS-PAGE analysis results revealed a single brand between66.4KD and44.3KD, that suggest the recombinant β hemolysin have been purified. The activity of purified recombinant β hemolysin was detected by hemolysis test using crucian carp red blood cells as indicator. The results demonstrated that recombinant hemolysin have hemolysis activity at both20℃and37℃. The obvious can be detected when1.0μg recombinant hemolysin reacts with the indicator cells, and the test red blood cells can be completely dissolved in3hours. The results suggest that the recombinant hemolysin posses hemolysis activity, which is essential for further studies.