Isolation And Identification Of Aeromonas Veronii From Micropterus Salmoides And Construction Of Mutant Strain Of OmpA Gene

In March 2019,a serious disease broke out in a farm in Henan Province,resulting in a large number of deaths of Largemouth Bass.The main symptoms were muscle ulceration,bleeding on the body surface and fins,liver swelling,ascites,etc.In order to find out the cause of the disease and explore its pathogenetic mechanism,the pathogeny and pathology of the diseased Largemouth Bass were studied.The pathogen was identified as Aeromonas veronii.And the OmpA gene of Aeromonas veronii was cloned.The sequence,protein structure and B cell antigen epitope were analyzed by bioinformatics.In addition,OmpA gene-deleted strains were also prepared to provide a basis for the functional study of OmpA gene in Aeromonas veronii.1.Identification and analysis of the pathogen of Largemouth Bass.Virus and parasite pathogens were detected in the diseased tissues of Largemouth Bass,and lernaea was detected,but virus was not detected.At the same time,a dominant bacterium HN1903 was isolated from the liver,kidney and fester of Largemouth Bass,and its morphological characteristics,physical and chemical characteristics,molecular characteristics,virulence gene carrier,regression infection experiment and histopathological changes were studied.The results showed that HN1903 was a gram-negative Brevibacterium,and its sequence,phylogenetic analysis and physiological and biochemical detection showed that HN1903 had the highest similarity with Aeromonas veronii.When HN1903 was returned to infect healthy Largemouth Bass,the symptoms of fish were consistent with the clinical symptoms of natural disease,and the median lethal dose of HN1903 to Largemouth Bass was5.01×10~6 cfu/m L.Based on the above results,indicating that the pathogen of Largemouth Bass was Aeromonas veronii,and the strain carried eight virulence genes including aer A,gca T,act,fla,lip,Ser,hly A,ahy B,withβhemolytic activity and protease activity.Among the 12antimicrobial agents tested,HN1903 was highly sensitive to gentamicin and cefotaxime,moderately sensitive to florfenicol and streptomycin,and insensitive to eight drugs,such as compound sulfamethoxazole,norfloxacin and tetracycline.The pathogen can cause lesion to many tissues and organs of Largemouth Bass,mainly manifested as congestion of hepatic sinuses,indistinct outline of hepatocytes,dissolved necrosis,enlargement of glomerular cavity,enlargement of renal tubule capsule cavity,dissolved necrosis of renal tubule epithelial cells,increase of hemosiderin granules in spleen,proliferation of cells at the base of gill lamellae,detachment of cells in gill lamellae,and shortening of gill lamellae.Based on the above results,it can be concluded that the pathogeny of the disease in this farm may be the lernaea parasitized on the body surface,causing damage to the body surface,and then Aeromonas veronii took the opportunity to invade the body from the damaged part,and finally caused the disease.2.Cloning and bioinformatics analysis of OmpA gene of Aeromonas veronii.The outer membrane protein A(OmpA)gene of Aeromonas veronii HN1903 was amplified by PCR.The physicochemical property,architectural feature and conservative B cell antigen epitopes of OmpA were predicted and analyzed.From the phylogenetic diagram,we can see that the OmpA amino acid sequence of HN1903 strain is the same as that of Aeromonas veronii,with the highest homology.The total length of OmpA is 1035 bp,encoding 344 amino acids.The molecular weight of the protein is predicted to be 36.9 k Da,and the theoretical isoelectric point is 5.43.OmpA encodes a signal peptide,which has the uniqueβbucket structure of Gram-negative bacteria,and the structure is composed of six reverse parallelβfolding sheets.OmpA is a transmembrane protein with topological structure.There are 10 major linear B cell epitopes in OmpA protein of HN1903 strain of Aeromonas veronii,among three of which were exposed to the bacterial surface and could be used as vaccine candidate antigens.3.Preparation of the OmpA gene deletion strain of Aeromonas veronii.In this study,the upstream and downstream homologous arms of OmpA gene were amplified using the DNA of the HN1903 strain of Aeromonas veronii as the template.The two gene arms were overlapped into a fragment by PCR and connected to the p DM4 suicide vector.The plasmid p DM4-?OmpA for knockout was successfully constructed.The knockout plasmids in Escherichia coli were transferred to Aeromonas veronii by conjugation of the two bacteria,and homologous recombination with OmpA gene took place for the first time.Using10%sucrose plate to screen the second time successfully recombinant Aeromonas veronii,and finally get the OmpA gene knockout Aeromonas veronii deletion strain.The knockout strain of OmpA gene obtained in this study provides a basis for the study of the function of OmpA gene.