Biological Characteristics Of Riemerella Anatipestifer RA-YM ΔKYF39＿09285 And ΔKYF39＿09945 Single Gene Deletion Strains And Their Effects On Virulence In Ducklings

Duck infectious serositis is an acute septicemic infectious disease caused by Riemerella anatipestifer（RA）infection,with high mortality and rapid spread after infection in ducklings or goslings to great harm.The research group discovered the Pho P/Pho R two-component regulatory system in Riemerella anatipestifer in the early stage,and the virulence of the constructedΔPho P/Pho R,ΔPho P andΔPho R deletion strains were significantly reduced.Further study found that both KYF39＿09285 and KYF39＿09945 were down-regulated genes inΔPho P andΔPho R.Therefore,it is speculated that KYF39＿09285 and KYF39＿09945 may be related to the virulence of Riemerella anatipestifer.In this experiment,KYF39＿09285 and KYF39＿09945 single gene deletion strains and their corresponding gene-replenishing strains were constructed respectively,and their biological characteristics and effects on duckling virulence were further studied.The main results are as follows:1.Bioinformatics analysis of KYF39＿09285 and KYF39＿09945 genes and their encoded proteinsThe size of the KYF39＿09285 gene is 483 bp,and its protein has a TPR（tetrapeptide repeat）domain and no signal peptide;the size of the KYF39＿09945 gene is 321 bp,and the conserved domain and signal peptide are not predicted;and predicted the protein interaction network of these two proteins.2.Construction of KYF39＿09285 and KYF39＿09945 gene deletion mutant strains and corresponding complementing strainsThe recombinant suicide plasmid p RE112-LSR was constructed and introduced into the parental strain RA-YM,two single-gene deletion mutants,RA-YMΔKYF39＿09285and RA-YMΔKYF39＿09945,were constructed respectively.The KYF39＿09285 and KYF39＿09945 genes were introduced into the gene deletion strains to construct their complementary strains RA-YMCΔKYF39＿09285 and RA-YMCΔKYF39＿09945.The constructed gene deletion strains and their complementing strains have good genetic stability.3.Biological characteristics of RA-YMΔKYF39＿09285 gene deletion strain and its effect on virulence in ducklingsThe expression level of KYF39＿09285 gene was significantly increased under high osmotic pressure,high temperature,penicillin or streptomycin stress.Dna K is a heat shock protein that acts as a molecular chaperone to help proteins fold correctly under physiological or stressful situations.The expression of Dna K in the RA-YMΔKYF39＿09285 deletion strain was significantly lower than that in the parental strain RA-YM.Compared with the parental strain,the tolerance of the deletion strain to high osmotic pressure,penicillin,ampicillin,gentamicin and streptomycin was weakened,and the tolerance of the gene-replenished strain was restored.The median lethal dose(LD₅₀)of the parental strains RA-YM and RA-YMΔKYF39＿09285 to 8-day-old Cherry Valley meat ducks were 3.62×10⁴CFU and 6.66×10⁴CFU,respectively,and the virulence of the deletion strain was 1.84 times lower than that of the parental strain.Compared with the RA-YM strain,the gene deletion strain and its complement strain had no significant difference in the experiments of Vero cell adhesion and invasion ability,growth characteristics,biochemical characteristics and adaptation to high concentration of phosphate.4.Biological characteristics of RA-YMΔKYF39＿09945 gene deletion strain and its effect on virulence in ducklingsCompared with the parental strain RA-YM,the growth rate of the RA-YMΔKYF39＿09945 deletion strain was slowed down in the basal medium containing high concentration of phosphate,and the growth rate of the complemented strain was not significantly different from that of RA-YM.The LD₅₀of the RA-YMΔKYF39＿09945deletion strain to 8-day-old Cherry Valley meat ducks was 1.33×10⁴CFU,and its virulence was 2.71 times stronger than that of the parent strain.Compared with RA-YM,RA-YMΔKYF39＿09945 and its complementing strain had no significant difference in phenotype experiments such as adhesion and invasion ability,growth characteristics and biochemical characteristics to Vero cells.In conclusion,the RA-YMΔKYF39＿09285 and RA-YMΔKYF39＿09945 gene deletion strains and their gene complementing strains were constructed in this experiment.Compared with the parental strain,the virulence of RA-YMΔKYF39＿09285 was reduced by 1.84 times,and the virulence of RA-YMΔKYF39＿09945 was enhanced.KYF39＿09285 can affect the expression of Dna K,and is also closely related to the tolerance of RA to high osmotic pressure,high temperature and toβ-lactams and aminoglycoside antibiotics,KYF39＿09945 is involved in the adaptation of RA to high concentrations of phosphate Sexual regulation.It lays a foundation for further research on the pathogenic mechanism of Riemerella anatipestifer.