| Riemerella anatipesifer(RA)is a contagious disease which mainly infects ducks,geese,turkeys and other poultry.The disease is widely distributed all over the world,with high morbidity and mortality.It is one of the major infectious diseases seriously endangering duck industry.With the in-depth study of bacterial virulence factors,bacterial secretion system plays an important role in the study of bacterial pathogenicity.In recent years,with the discovery of Ⅸsecretion system(T9SS),also known as Por secretion system,in members of Bacteroidetes,the core components of T9SS were found on the genome of all Riemerella anatipestifer strains,which full genome sequences were published on GenBank.It is particularly noteworthy to study the interaction between bacteria and hosts and the role of the secretion system in the pathogenesis of bacteria.In this study,we constructed the core component of T9SS,gldL gene-deletion mutant and a gldL+gldN double-deletion mutant of Riemerella anatipestifer Yb2 strain,and analyzed the biological characteristics of the deletion mutant.In addition,the immunogenic proteins in the supernatant of Yb2 strain were identified by 2-D and Western Blot.1.Construction and biological characteristics of gldL and gldL+gldN gene deletion mutants of Riemerella anatipestifer Yb2 strainIn this study,the left and right homologous arms of gldL gene were amplified and inserted into suicide plasmid 313,generating suicide plasmid 313-gldL-R.The gldL gene deletion mutant Yb2△gldL was constructed after the recombinant suicide plasmid 313-gldL-R was introduced into wild type Yb2 by conjugation transduction.The double deletion mutant Yb2△gldL-gldN was constructed after 313-gldL-R was introduced into deletion mutant Ybl△gldN.Then the gldL gene ORF of wild strain Yb2 was linked to the E.coli-Riemerella anatipestifer shuttle expression plasmid pRES1.The generated recombinant plasmid pRES1-gldL was introduced into the deleted strain Yb2△gldL by conjugation transduction,and the complemented strain cYb2△gldL was obtained.The results of biological characteristics of wild type Yb2 and the deletion mutants showed that,compared with that of wild type Yb2,there was no significant difference in the growth curve of deletion mutants Yb2△gldL,Yb2△gldL-gldN and the complemented strain cYb2△gldL.In addition,the protease hydrolysis test showed that the deletion mutants Yb2△gldL and Yb2△gldL-gldN could not hydrolyze protease on TSA plate containing skimmed milk for 24h.The results of pathogenicity to ducklings showed that the LD50 of Yb2△gldL and Yb2△gldL-gldN were increased by 48 and 110 folds respectively,compared with wild strain Yb2.The bacterial loads of liver,brain and blood in Yb2AgldL,Yb2AgldL-gldN or cYb2AgldL infected ducklings were also decresed significantly.The results showed that Yb2AgldL and Yb2△gldL-gldN were less invasive in these tissues than wild type Yb2.Therefore,the above results showed that the deletion of gldL or gldL+gldN genes impaired the function of Ⅸ secretory system and affected the virulence of Riemerellaanatipestifer to ducklings.2.Identification of immunogenic protein in the culture supernatant of Riemerella anatipestifer strain Yb2TCA precipitation method was used to precipitate the proteins from culture supernatant by adding 10%TCA into the supernatant of Yb2 strain.Then the obtained proteins in the culture supernatant were identified by two-dimensional electrophoresis(2-D)and western blot methods.12 immunogenic proteins in the culture supernatant were identified.Three immunogenic proteins P1,P2 and P3 were expressed in E.coli.Animal experiments showed that P1 protein,but not P2 or P3,could protect some immunized ducklings against challenge with virulent Yb2.This study laid a foundation for the development of a new vaccine against Riemerella anatipestifer infection. |
