Construction Of The Recombinant Attenuated Salmonella Gallinarum Expressing Sfm Fimbriae Of Avian Pathogenic E.coli And Investigation Of Immunoprotective Evaluation

Avian pathogenic Escherichia coli(APEC)and Salmonella enterica serovar pullorum(S.pullorum)are important pathogens responsible for the transmission of avian diseases,causing Escherichia coli disease and Salmonellosis in chickens,respectively,and leading to the farming industry to significant economic losses.Currently,therapeutic prevention and control measures for these two diseases are mainly through a combination of vaccine prevention and drug treatment,therefore,developing a dual vaccine with high immune efficacy that can prevent both Escherichia coli disease and Salmonellosis is the most effective way to solve the problem.E.coli and Salmonella diverged from a common ancestor,and the fimbriae is the major virulence factor that mediates adhesion,invasion,biofilm formation,and reciprocal regulatory interactions with other virulence factors.In addition to virulence,fimbriae can be a target of the host immune response and exert immunogenicity.Type Ⅰ fimbriae is the most common fimbriae found in E.coli and Salmonella,and researchers have focused on their structure,assembly,pathogenicity,etc.,some studies have introduced type Ⅰ fimbriae of E.coli as fimbriae antigens into vectored strains to prepare recombinant vectored vaccines.But the investigators found that the fim gene clusters encoding type Ⅰ fimbriae of E.coli and Salmonella were not homologous,at the same time,were found,Sfm fimbriae in E.coli which belong to the same γ 1 of the evolutionary branch were homologous to type Ⅰfimbriae in Salmonella,both sharing up to 70%homology.Therefore,in this study,the attenuated S.typhimurium SG01 strain was used as the host bacterium to introduce E.coli Sfm fimbrial protein into SG01,and construct a chromosome-plasmid balance system using asd nutrition as a deficiency marker,and to express the Sfm fimbrial antigen,to explore the immunoprotective efficacy of this recombinant attenuated oral vaccine.1.Construction and biological characterization of recombinant attenuated Salmonella expressing Sfm fimbriae of E.coliE.coli K88ac+ETEC C83902 genomic DNA was used as a template to amplify the sfm genome encoding Sfm fimbriae by PCR and cloned into the expression plasmid vector pYA3342 harboring the asd gene,to construct and select the pYA3342-sfm recombinant plasmid harboring the complete sfm gene,which was transformed into the asd deleted attenuated S.typhimurium SG01,designated SG01(pYA3342-sfm).After specific antibody agglutination test showed that the recombinant bacteria were able to express Sfm fimbriae at 37℃ under shaking culture conditions.Meanwhile,the growth properties of the recombinant bacteria were evaluated,and it was determined that the expression of Sfm fimbriae did not affect the normal growth level of the bacteria.To determine the duration of the anti-sfm antibody in chickens,the recombinant bacteria were orally inoculated into 40 16-week-old Isan Brown laying hens,the blood was collected weekly,and the agglutination reaction with serum was performed by target agglutination antigen S9HH(pBR3342 APEC Sfm)of Sfm fimbrial antibody,continuously monitoring for 11 weeks,both of which could monitor the expression of anti Sfm fimbrial antibody.The above results showed that the Sfm funbriae was able to stimulate chickens to produce sustained antibodies in vivo,which laid the foundation for the development of a genetically engineered vaccine using the Sfm fimbriae as an immunogen.2.Immune efficacy of the recombinant attenuated S.Typhimurium SG01 expressing APEC Sfm fimbriaeTo evaluate the expression stability of the recombinant attenuated S.Typhimurium SG01(pYA3342-sfm)constructed in study one,1-week-old non-immunized chickens were divided into five groups,including two groups orally inoculated with recombinant SG01(pYA3342sfm)as immunization group,two groups orally inoculated with SG01(pYA3342)as empty vector control group,and one healthy control group.Serum was collected every other week after the first immunization to determine the dynamic level of anti-sfm antibody in chickens of each test group,and 14 d after the first immunization,the anti-sfm antibody levels of the chickens inoculated with the recombinant SG01(pYA3342-sfm)vaccine were significantly higher than those of the empty vector control and healthy controls,and the expression of antisfm antibody was not monitored in the empty vector control and healthy controls.During the whole immune cycle(4 weeks),the level of anti-sfm antibody in chickens inoculated with recombinant SG01(pYA3342-sfm)showed an increasing trend.Two groups of chickens orally inoculated with recombinant bacteria vaccine SG01(pYA3342-sfm)were challenged with avian pathogenic Escherichia coli strain QD2 and Salmonella pullorum SP03,respectively,and the empty vector control group was used as the challenge control,and observed for 10 days after the challenge.The mortality rate of the immune group and the empty vector control group was 52.6%and 78.9%respectively in the two groups challenged by the E.coli strain QD2.In the two groups challenged with Salmonella pullorum SP03,the mortality of the immune group and the empty vector control group were 10.5%and 42.1%,respectively.The results showed that the recombinant attenuated Salmonella typhi vaccine SG01(pYA3342-sfm)constructed in this study had a certain preventive effect against both colibacillosis and salmonellosis,but had a stronger protective effect against salmonellosis.