| Aeromonas is Gram-negative bacteria which exists frequently in soil,fresh water,sea water and aquatic environment.As opportunistic pathogen that is common Infection among human and aquatic animals.The content of this research is mainly to investigate the correlation between virulence genes and pathogenicity of Aeromonas species,173 strains of Aeromonas were isolated from different diseased freshwater fish from 2009 to 2018 were taken as the research,to evaluate the main biological characteristics of different virulence genotypes Aeromonas veronii strains and to explore the distribution of ompAⅡ gene,the antigenicity and immune effect of rOmpAⅡ from Aeromonas veronii GZ09007.The main contents are as follows:1.Distribution of virulence genes in Aeromonas species isolated from diseased freshwater fish and the pathogenicity to Carassius auratus gibelioTo investigate the correlation between virulence genes and pathogenicity of Aeromonas species,173 strains of Aeromonas were isolated from different diseased freshwater fish from 2009 to 2018 were taken as the research.Virulence genes including aer,act,fla,lip,gca T,exu,ast,alt,epr CAI and ahy B were detected and the bacterial pathogenicity with different virulence genotypes was assessed through intraperitoneal(IP)injection challenged Gibel carp.Molecular identification revealed that A.veronii(119/173,68.8%)and A.hydrophila(50/173,28.9%)were the prevalent species based on gyrB gene sequencing.The distribution of 10 virulence genes including aer(162/173,93.64%),act(131/173,75.72%),ast(55/173,31.79%),alt(58/173,33.53%),lip(152/173,87.86%),exu(154/173,89.02%),fla(143/173,82.66%),gcaT(148/173,85.55%),eprCAI(41/173,23.70%)and ahyB(51/173,29.48%)were determined and these strains were sorted into 7 clusters(Ⅰ-Ⅶ)and formed 53 genotypes according to the virulence distribution profile.8-10 virulence genes were present in 24.86%(43/173)Aeromonas stains,among which 38 strains were identified as A.hydrophila and 4 stains A.veronii.epr CAI(0/119,0%),ahy B(19/119,15.13%),ast(7/119,5.88%)and alt(24/119,20.17%)were detected from the A.veronii isolates.Most of the Aeromonas strains(94.22%,163/173)had hemolytic activity.At the challenge dose of 3.0×10~6CFU per fish,3 strains of A.veronii(Ⅳ-5、Ⅴ-1 andⅥgenotype)and 16 strains of A.hydrophila(Ⅰ、Ⅱ-1、Ⅱ-3、Ⅱ-5、Ⅲ-1、Ⅲ-2、Ⅲ-3、Ⅲ-5、Ⅳ-1 and Ⅳ-2 genotype)were highly pathogenic and the mortality reached 80%-100%.The study suggested that A.veronii was the most prevalent species in the diseased fish.A.veronii isolates had fewer virulence genes and lower pathogenicity comparing to A.hydrophila ones.The result has important reference significance for epidemiological investigation of motile aeromonad septicemia and vaccine research.2.Biological characteristics of different Aeromonas veronii strainsThe pathogenicity and biological characteristics from different Aeromonas veronii strains culture characteristics were evaluated by detecting extracellular product activity,virulence gene,antigenicity of outer membrane protein and drug sensitivity.The optimum growth temperature,growth salinity and pH from A.veronii GZ09007strainwas 24～40℃,5‰and 7.0,respectively.All strains were positive for hemolytic activity,elastase activity and lipase activity,and carried aer,act,lip,exu and other virulence genes.Western blotting suggested that the Omps with molecular masses 36kDa in A.veronii strains were recognized by rabbit antiserum of GZ09007 Omps.The drug sensitivity result of 6 strains of Aeromonas veronii were consistent to the same kind of drug.Florfenicol and enrofloxacin could be used to prevent aquatic animals from Aeromonas veronii strain infecting.3.Recombinant expression and antigenicity of OmpAⅡ of Aeromonas veronii strain GZ09007The antigenicity of recombinant protein OmpAⅡ of A.veronii GZ09007 were assessed through gene detection,cloning expression,western blotting.The result showed that ompAⅡgene is common in A.verionii strains(33/39,84.61%).Cloning and sequencing of ompAⅡ revealed that it contains an open reading frame(ORF)of999 bp nucleotides encoding 332 aa with molecular mass 35k Da and isoelectric point(p I)4.91.Western blotting suggested that rOmpAⅡwas recognized by rabbit antiserum of A.veronii GZ09007 rOmpAⅡ,whole bacteria cell and Omps,as well as the Omps with molecular masses 36kDa of A.veronii strains were recognized by rabbit antiserum of rOmpAⅡ.4.Immune effect of rOmpAⅡ of Aeromonas veronii strain GZ09007The immune effect of recombinant protein OmpAⅡof A.veronii GZ09007 were assessed by serum antibody titer,SOD activity and LZM activity,challenged fish with different Aeromonas strains.ELISA demonstrated that Carassius auratus gibelio immunized with rOmpAⅡ produce significant antibody response.The serum antibody titers were significantly high at 7d,14d,21 d,28 d,and 42 d and 56d post-immunization,which antibody titers were 1:100,1:800,1:1600,1:800 and 1:800,respectively.The lysozyme(LZM)activity of immunized group was significantly higher than the control group at 7d,14d,21d,28d,42d and 56d post-vaccination,and the superoxidase(SOD)activity was significantly higher than the control group at the14d post-immunization.The relative percent survival(RPS)after challenged by A.veronii(GZ09007,YC170511 and XZ180501)and A.hydrophila(Hn099)was 100%,100%,38%and 89%at the 28d,respectively.These results indicate that the recombinant protein OmpAⅡof A.veronii has positive antigenicity and is a subunit vaccine candidate. |
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