Construction Of Three Lipopolysaccharide Mutants Of Brucella Abortus A19 And Evaluation Of Their Potential Use As Marked Vaccine Candidates

Brucellosis is a bacterial zoonotic infectious disease that can be transmitted to humans through animals,resulting in significant economic losses and endangering human health.Therefore,it is crucial to prevent and control the occurrence and spread of brucellosis.Vaccination of animals against brucellosis can effectively prevent the occurrence of brucellosis in animals and thereby reduce the incidence of brucellosis in humans.The attenuated live vaccine 19 against Brucella abortus(S19 vaccine)and the attenuated live vaccine Rev.1 against Brucella melitensis(Rev.1 vaccine)are widely used in the world for immune prevention of brucellosis in cattle and sheep.Due to the prolonged serological response caused by these two vaccines,it is difficult to distinguish vaccinated animals from highly virulent Brucella infected animals in clinical practice,which hinders early diagnosis of brucella infected animals and eradication of livestock for brucellosis.Therefore,it is very important and urgent to study novel Brucellosis marker vaccines that can differentiate and diagnose vaccinated animals.In China,the Brucella abortus vaccine strain A19 is widely used to immunize cattle and achieve good preventive effects.However,the A19 vaccine can stimulate host animals to produce anti-smooth lipopolysaccharide(LPS)antibodies,interfere with serum diagnosis,and have residual virulence to immunized animals,making it unsuitable for vaccination in pregnant and young animals.In this study,we used the acetyltransferase gene wbd R of Escherichia coli O157 to replace the formyltransferase gene wbk C related to LPS synthesis of Brucella A19 vaccine strain and constructed a vaccine candidate strain A19mut2 with modified lipopolysaccharide characteristics.Animal experiments showed that compared with the inoculated A19 vaccine strain,the lipopolysaccharide antibody produced by immunization with A19mut2 was significantly less reactive to the wild smooth lipopolysaccharide,can be used for differential diagnosis between immunization and wild virulent Brucella infections! Based on A19mut2,a three gene mutant strains A19mut2Δery CDΔvce C(A19mut5)with deletion of ery CD and vce C genes related to erythritol metabolism and inflammatory response,and a dual gene mutant A19mut2Δvdt R(A19mut6)with vdt R deletion were constructed respectively,and their immunoprotective and differential diagnostic significance of these mutant strains were investigated.Research has found that compared to the A19 strain,A19mut2 has a decreased resistance to acidic p H values,and the initial in vivo bacterial load in mice after immunization is significantly reduced.It also induces significant humoral and cellular immune responses,leaning towards Th1 type immune responses.The protective effect of A19mut2 immunization on wild-type Brucella S2308 Nal R challenge in mice is similar to that of A19 immunization,showing no significant difference.The protective effect of A19mut2 immunization on wild-type Brucella S2308 Nal R challenge in mice is similar to that of A19 immunization,but there is no significant difference.Indirect ELISA based on wild-type LPS coated ELISA can detect higher levels of A19 induced antibodies,but the detection titer for mutant A19mut2 induced antibodies is significantly reduced.In further research,the virulence and immune protection of the Brucella A19mut5 were evaluated in mice.It was found that compared with the A19 vaccine strain,A19mut5 and A19mut6 showed a decrease in resistance to acidic p H values and a decrease in residual virulence to mice during the early vaccination stage.In immunized mice,A19mut5 induced significant humoral and cellular immune responses,similar to A19 immunity,with a bias towards Th1 type immune responses.The protective effect of mice immunized with A19mut5 against wildtype Brucella S2308 Nal R infection is similar to that of mice immunized with A19,and there is no significant difference.It can be foreseen that indirect ELISA coated with wild-type LPS can be used as a method for differential diagnosis between A19mut5 immunized mice and A19 immunized mice.The research results have laid a good foundation for the development and application of the novel Brucella differential diagnosis vaccine candidate.