Isolation And Identification Of Aeromonas Veronii From A Ietalurus Punetaus And Its Preliminary Immunogenicity Evaluation Of Lateral Flagella

Aeromonas veronii disease of Ietalurus Punetaus is a kind of acute infectious disease with severe ascites,enteritis and extensive blood stasis of internal organs.The disease has a high infection rate,high mortality rate,poor water quality and fish body injured.Immunized ponds with low immunity are extremely serious threats.In mid-November2017,I’ve isolated a Gram-negative bacterium from multiple tissues and organs such as liver,spleen and kidney of diseased spotted scorpionfish in Jingyan County,Sichuan Province,and confirmed its pathogenicity through regression experiments.Morphological,physiological and biochemical characteristics,16S and gyrB genes were constructed by phylogenetic tree and identified as Aeromonas veronii,named XH.VA.2.Scanning electron microscopy showed short rod-shaped bacteria with a smooth surface with an average length of 2.777±0.61μm and an average width of 0.843±0.098μm.The growth curve measurement showed that the bacteria entered the plateau after the 12th hour.The pathogen is sensitive to six drugs such as ofloxacin,florfenicol,ciprofloxacin,kanamycin,norfloxacin,and cefradine.Histopathological observation showed that sputum,heart,liver,spleen,kidney and intestine had different degrees of inflammatory cell infiltration by neutrophils,and the lesions of spleen and kidney were the most serious.In order to further study the pathogenicity of pathogens,this experiment performed genome-wide sequencing and analysis of A.veronii XH.VA.2.Whole genome sequencing showed that the XH.VA.2 genome size was 4.91 Mb,the GC content was 58%,and 4640genes were encoded.The genome sequence was uploaded to NCBI to obtain the accession number:NZ_QQOQ01000000.The XH.VA.2 whole genome was compared with the environmental isolate A.veronii B565（GenBank:CP002607）for comparative genomic analysis.A specific gene cluster encoding the lateral flagella（36,778 bp）was found in the XH.VA.2 genome.At the same time,XH.VA.2 contained 35 lateral flagellation-related genes through the VFDB virulence factor database,while the related genes in B565 were deleted.Based on the results of comprehensive comparative genomics,a specific side flagellar gene cluster was found on XH.VA.2 compared to B565,which may be the molecular basis of the virulence of pathogen XH.VA.2.In order to verify the results of comparative genomics analysis,it is explored whether this lateral flagella gene cluster is the molecular basis of the virulence of pathogen XH.VA.2.Therefore,two lateral flagellin genes（lafA1 and lafA2）which are most correlated with lateral flagellar immunogenicity and antigenicity in the XH.VA.2 side flagellum gene cluster were selected.The expression of E.coli prokaryotic expression system was constructed and expressed.The results showed that the recombinant side flagellin proteins LafA1 and LafA2 appeared in the form of inclusion bodies.SDS-PAGE results showed that both proteins were approximately 50.7 kDa in size.Western blot analysis showed that rabbit anti-histidine tagged protein serum can specifically bind LafA1recombinant protein and LafA2 recombinant protein.Then,the LafA1 and LafA2 proteins were emulsified in equal proportions with Freund’s incomplete adjuvant,and the healthy Ietalurus Punetaus were injected intraperitoneally.Serum was collected at seven time points within seven weeks after immunization,and serum non-specific immune indicators（serum total protein,serum albumin,alkaline phosphatase,complement C3,superoxide dismutase,lysozyme）were detected.In order to understand whether LafA1 and LafA2proteins are immunogenic.At the same time,the challenge was performed at 28 days after immunization(50 LD₅₀),and the mortality of each group was recorded within 14 days and the relative protection rate of the two protein groups to the channel catfish was calculated.The results showed that the content or activity of immune factors in both immunoassay groups increased first and then decreased,and the other indicators except lysozyme were not significantly different from the control group（P>0.05）.The content or activity level of each immunological factor in the two experimental groups was approximately equal throughout the experimental period.The total protein content of the two protein groups was significantly increased at the fourth week（P<0.01）.The albumin content of the two protein groups was significantly higher than that of the control group at the third week（P<0.01）.The alkaline phosphatase content of the two groups from the first week to the fifth week was significantly higher than that of the control group（P<0.01）;the C3 content of the complement at the fourth week was compared with the control.There was a significant increase in the group（P<0.01）.The T-SOD content in the experimental period from the second week to the fifth week was significantly higher than that in the control group（P<0.01）.During the whole experimental period,The serum lysozyme levels of the two protein groups were significantly increased compared with the control group（P<0.01）.The relative protection rates of the LafA1 and LafA2 groups were 36.67%and 33.33%,respectively,indicating that the two groups of immunized groups had a channel catfish Has a certain immune protection effect.Studies have shown that LafA1 and LafA2 proteins have certain immunogenicity,and the lateral flagella gene cluster may also be the molecular basis of the pathogenic bacteria XH.VA.2 virulence.At the same time,LafA1and LafA2 proteins can also help the Ietalurus Punetaus to resist the infection of Aeromonas veronii,and can be used as a candidate protein for the development of the vaccine subunit vaccine.