The Construction And Biological Characterization Of GL001858 And GL001859 Gene Deletion Mutants Of Riemerella Anatipestifer

Riemerella anatipestifer infection,also known as Riemerella anatipestifer disease and infectious serositis,is a highly pathogenic and contact infectious disease that mainly infects ducks,geese,turkeys and other birds.It is also a major disease that endangers duck industry and causes great economic losses to duck industry all over the world.Duckling of 1-8 weeks old is most susceptible,with high incidence rate and mortality.Once a duck farm is infected with this disease,it is difficult to eradicate it completely.There are at least 21 serotypes of R.anatipestifer,but there is little or no cross protection among serotypes.At present,there are commercial inactivated oil emulsion vaccines against R.anatipestifer infection in China,which play an important role in the prevention and control of this disease.However,the inactivated oil emulsion vaccines have some disadvantages,such as high cost and stress response to inoculated ducklings,and more importantly,the risk of food safety due to the residue of oil emulsion vaccine in the immunized ducklings when the inoculated meat ducks,such as Cherry Valley ducks and Beijing ducks,reach the age of marketing.On the contrary,the above shortcomings and risks do not exist when vaccinated with attenuated vaccine.The development of safe and effective live attenuated vaccine,especially live gene deletion vaccine,is the future development direction of the vaccine against this disease.In our previous studies,17 tagged Tn4351 transposon mutation libraries of of R.anatipestifer serotype 1 WJ4 strain were constructed and screened by STM in a duck infection model.A total of 101transposon insertion mutants with reduced pathogenicity of ducklings were selected by STM technique.The pathogenicity of mutant WJ4(Tn::GL001858),in which GL001858 gene was inserted by transposon Tn4351,to ducklings was significantly reduced.The protective rate of challenge against virulent strain was 100%when the ducklings were immunized with the mutant for two weeks.Real-time PCR results showed that the expression of its downstream gene GL001859 was also significantly downregulated.To identify the role of GL001858,GL001859 genes in the pathogenic process of WJ4,the GL001858,GL001859 gene deletion mutant strains were constructed in this study,and the feasibility of these two genes as target gene for attenuated vaccine were evaluated.Firstly,the left and right homologous arms of GL001858 gene were amplified by PCR and then linked into suicide plasmid 313,generating the recombinant suicide plasmid 313-GL001858-LR.The recombinant suicide plasmid 313-GL001858-LR was introduced into the strain WJ4 by conjugation.Then,deletion mutant WJ4?GL001858 were screened and identified by resistance screening,sucrose screening and PCR identification.GL001858 ORF was amplified by PCR and linked into E.coli-R.anatipestifer plasmid p RES2.The complementary plasmid p RES2-GL001858 was introduced into mutant WJ4?GL001858 by conjugation,generating complemented strain c WJ4?GL001858.The GL001859 gene deletion mutant WJ4?GL001859 was constructed in the same way.Biological characteristics analysis showed that there was no significant difference in growth curves in TSB between mutant WJ4?GL001858 and the wild type(WT)WJ4.Comparison with the WT WJ4,the adhesion ability of mutant WJ4?GL001858 to Vero cells was significantly decreased,.In addition,the adhesion and invasion ability of WJ4?GL001859 to Vero cells were significantly reduced than that of the WT WJ4.The protease hydrolysis test showed that there was no significant difference between WJ4?GL001858 and the WT WJ4.The results of pathogenicity test of ducklings showed that the LD₅₀of WJ4?GL001858 and WJ4?GL001859 to ducklings were more than 10¹⁰CFU,and the bacterial load in the blood WJ4?GL001858-infected ducklings was significantly reduced than that of the WT WJ4.But there was no significant decrease in liver and brain tissue.These results indicated that both GL001858 and GL001859 genes were involved in the virulence of R.anatipestifer.The protective rates were 100%,100%,100%and 83%when the ducklings were immunized with 10⁷,10⁸,10⁹,10¹⁰CFU of WJ4?GL001858 respectively;while those were 80%,44.4%,100%and 100%respectively,when the ducklings were immunized with WJ4?GL001859.This study is of great significance for the development of the attenuated vaccine of R.anatipestifer infection and the molecular pathogenesis of R.anatipestifer.