Functional Identification Of LptD Gene In Riemerella Anatipestifer ATCC11845

Riemerella anatipestifer(RA)is a gram-negative pathogen that mainly infects ducklings and other poultry species.Riemerella anatipestis has a variety of serotypes and natural resistance to a variety of antibiotics.At present,little is known about the resistance and pathogenesis of Riemerella anatipestis.In this study,the function of lptD gene of RA ATCC11845 strain in drug resistance and pathogenesis was studied.The main results are as follows:1.Glutaraldehyde Tolerance of lptD Gene in Different RA IsolatesTo investigate the relationship between lptD gene expression and tolerance to organic solvents in different RA isolates,the tolerance of RA CH-1 and RA ATCC11845 strains to glutaraldehyde was tested.The results showed that the MIC values of glutaraldehyde to RA CH-1 strain and RA ATCC 11845 strain were 0.1%and 0.05%,respectively.The results showed that the transcription level of lptD gene in RA CH-1strain was about 2 times higher than that of RA ATCC 11845 strain,and the transcription level of lptD gene was increased by 3 and 10 times in RA CH-1 strain and RA ATCC11845 strain treated with 0.01%and 0.005%glutaraldehyde,respectively.In conclusion,there was a positive correlation between the expression of lptD gene and the tolerance of Riemerella anatipestis to glutaraldehyde.2.Construction of low expression strain of LptD gene of RA ATCC 11845 strain and its complementary strainIn this experiment,about 800 bp homologous arms and erythromycin resistance fragments of the lptD gene initiation site were amplified and fused by PCR.The fused fragments were naturally transformed into wild RA ATCC 11845 strain.The erythromycin resistance fragments with promoters were inserted into the front of the lptD gene initiation site,and the resistant promoters were used by lptD.The lptD sequence with promoter was cloned into pLMF03,and the recombinant plasmid was transferred into the low expression strain by conjugation transfer to construct the complement strain.The low expression strain RA ATCC 11845 lptD~- and the low expression complement RA ATCC 11845 lptD~-::pLMF03::lptD was constructed successfully by PCR and quantitative PCR.3.Identification of biological function of lptD gene in RA ATCC 11845 strain(1)The growth curve showed that compared with wild plants,the growth rate of low expression plants of lptD gene was significantly lower(2)Compared with wild type,the survival rate of RA ATCC 11845 lptD~-strain decreased by two times after glutaraldehyde treatment at induction concentration;(3)Compared with wild type,low expression plants of lptD gene had SDS table of anionic surfactant.Specifically,lptD low-expression strains have been inactivated at a concentration higher than 0.02%,and wild strains are still active at a concentration of 0.08%.(4)The film-forming rate of lptD low-expression strains is 1/2 of that of wild strains;(5)Compared with wild strains,the tolerance of lptD low-expression strains to inactivated serum is significantly weakened.4.The lptD gene of RA ATCC 11845 strain participates in its colonization in hostThe colonization of wild strains in liver,brain,spleen and heart blood was significantly higher than that of low-expression strains(p<0.05)12 and 18 hours after infection with 10~9 CFU wild strains and low-expression strains respectively.This indicated that the colonization ability of the low expression strain of RA ATCC 11845lptD gene in ducklings was reduced.In conclusion,lptD gene participates in the biological functions of RA ATCC11845,such as glutaraldehyde tolerance and hydrophobic drug tolerance,membrane permeability,membrane formation rate,inactivated serum tolerance,and affects the colonization of RA ATCC11845 in host.