| Aeromonas veronii is a gram-negative pathogen with strong adaptability and zoonotic diseases.In recent years,there have been outbreaks of fish diseases in Hainan,Guangdong and other places.Among them,A.veronii is the main pathogen,and the trend is increasing year by year.Due to the multi-drug resistance of A.veronii,the treatment with traditional antibiotics is not effective.This not only caused economic losses in the aquaculture industry,but also endangered people’s food safety and health.Vaccines are an effective means to solve multi-drug resistant bacteria,but the research on fish vaccines in my country started too late.The fish vaccines that have been put into production are far from being able to meet the needs of my country’s huge aquaculture industry.There is no corresponding vaccine for A.veronii,so the development of a vaccine for A.veronii is urgent.ExsA is the main regulator of the type three secretion system(T3SS)in Gram-negative bacteria,and T3 SS is responsible for mediating the virulence of the bacteria to the host.There have been related studies on exsA in other Gram-negative bacteria,but most of them are exploring its molecular mechanism.In this study,the method of homologous recombination,based on the original exsA1 knockout,constructed the A.veronii C4 strain exsA double knockout strain △exsA1 exsA2,and explored the effect of exsA on A.veronii from the following points the effect of virulence and the immune protection of tilapia after the knockout strain is weakened.Firstly,real-time fluorescent quantitative PCR is used to detect the expression of various virulence factors in A.veronii wild strains and △exsA1 exsA2 knockout strains.The results showed that after exsA was completely knocked out,the virulence effector protein genes,T3SS-related genes,and outer membrane structural genes of A.veronii were all significantly down-regulated.Comparing the half-lethal concentration of △exsA1 exsA2 knockout strains and wild strains,we found that The virulence of A.veronii△exsA1 exsA2 knockout strain to tilapia was reduced by 63.19 times.The colonization of wild strains and △exsA1 exsA2 knockout strains in tilapia was detected,and it was found that although the virulence of the △exsA1 exsA2 knockout strain was weakened,it could still exist in tilapia for a long time.Secondly,the activities of superoxide dismutase(SOD)and alkaline phosphatase(AKP)in visceral tissues of tilapia were detected by inoculating A.veronii △exsA1 exsA2knockout strain.The results showed that both enzymes were activated after inoculation.In addition,the content of immunoglobulin M(Ig M)in tilapia serum was also detected.It was found that Ig M was accumulated in large amount after inoculation with △ exsA1 exsA2knockout strain and existed for a long time.At the same time,q PCR was used to determine the expression of various non-specific immune factors in tilapia visceral cells,and it was found that the corresponding factors were activated and expressed in different viscera.The final survival rate experiment showed that the △ exsA1 exsA2 knockout strain had an immune protection rate of 85% against tilapia.In this study,the knockout strain △exsA1 exsA2 with high attenuated virulence was successfully constructed,and the molecular mechanism of the attenuated virulence of the△ exsA1 exsA2 knockout strain was explored,and it was confirmed that the attenuated virulence of the △ exsA1 exsA2 knockout strain could cause the continuous immune response of tilapia.It provided a good material for the research and development of live attenuated A.veronii vaccine.At the same time,it also laid a solid foundation for the research and development of tilapia vaccine,which is conducive to the green and sustainable development of the aquaculture industry. |
