Construction Of GL01019 Gene Deletion Mutant Of Rimerella Anatipestifer And Analysis Of Its Biological Characteristics

Riemerella anatipestifer infection,also known as Duck infectious serositis,is a kind of contact infectious disease of domestic ducks,geese,turkeys and other birds,and also a major disease confronting the duck industry worldwide.It accounts for significant economic losses due to high mortality,weight loss,condemnations,downgrading,and salvage.There are at least 21 RA serotypes with low or no cross protection.At present,some commercial oil emulsion vaccines have been used to prevent the disease at home and abroad,but the oil emulsion vaccine is easy to cause stress reaction in ducklings,and there may be oil emulsion residues in ducks when meat ducks are on the market;while the live attenuated vaccine does not have the above risks.Gene deleted live vaccine has attracted much attention because of its good safety,retained immunogenicity of the strain,and not easily returned virulence.That selection of candidate target deletion gene from about 2000genes of one R.anatipestifer strain is the key point of constructing gene deletion vaccine strain.Our previous study showed that the GL01019 gene(in this study,it is referred to as 1019gene)mutant by Tn4351 transposon insertion significantly reduced the pathogenicity of ducklings.The protein encoded by 1019 gene may be involved in the biosynthesis of bacterial biotin.In order to analyze whether the 1019 gene can be used as the deletion target gene for the construction of R.anatipestifer gene deletion vaccine strain,the 1019gene deletion mutant of R.anatipestifer serotype 1 strain WJ4 were constructed.The effect of 1019 gene deletion on the pathogenicity of strain WJ4 and the bacterial load in blood,liver and brain tissues of ducks infected by the deletion mutant were analyzed.In this study,the upstream and downstream homologous arms of 1019 gene were ligated into suicide plasmid 313 to construct recombinant suicide plasmid 313-1019-LR,and then the recombinant suicide plasmid 313-1019-LR was introduced into R.anatipestifer strain WJ4 with Escherichia coli S17-1(313-1019-LR)strain as donor strain and the wild strain WJ4 as receptor strain by conjugation.Then,the 1019 gene deletion mutant WJ4?1019was identified by the resistance screening,sucrose screening and PCR identification.The ORF of 1019 gene was inserted into E.coli-R.anatipestifer shuttle expression plasmid p RES2 to construct the recombinant shuttle plasmid p RES2-1019.Then S17-1(p RES2-1019)was used as the donor strain and WJ4?1019 as the recipient strain,the recombinant plasmid p RES2-1019 was introduced into WJ4?1019 by conjugation,and the complemented strain c WJ4?1019 was constructed.The biological characteristics of WJ4,WJ4?1019 and c WJ4?1019 were analyzed.The results of growth curves showed that the deletion of 1019 gene had no significant effect on the growth of R.anatipestifer,and the capability of adhesion and invasion of the deletion strain WJ4?1019 to Vero cells was significantly lower than that of the wild strain WJ4.The LD₅₀ of the deletion strain WJ4?1019 to ducklings was more than 10¹⁰ CFU,and the bacterial load in blood,liver and brain tissues of the ducks infected by the deletion mutant WJ4?1019 was little or zero,indicating that the 1019 gene was closely related to the virulence of R.anatipestifer.On the other hand,that the bacterial load in infected ducks was little or zero,indicated that further animal experiments and determination of the minimum immune dose were needed to determine whether 1019 gene could be used as a candidate deletion target for the construction of R.anatipestifer gene deletion vaccine strain.This study is of great significance for the development of attenuated vaccine against R.anatipestifer infection and the study of molecular pathogenesis of R.anatipestifer.