Identification Of LPS Synthesis Genes Of Salmonella Pullorum And Primary Immunobiological Analysis Of Mutants

Salmonella Pullorum(SP)is an adaptive pathogen of the host(avian),which can spread both horizontally and vertically,easily causing high pathogenicity and high mortality of chickens,and serious economic losses to the poultry industry.So pullorum disease is one of the main diseases of priority prevention and control in Chinese poultry farms.However,SP prevention and control measures by current quarantine and elimination method have not achieved the expected results in China,new drugs and vaccines were hoped to carry out SP clearance in chinese poultry farms as soon.Salmonella lipopolysaccharide is one of important pathogenic factors and its synthetic genes may be potential target genes for new vaccines and drugs.Lipopolysaccharide is composed of lipid A,core polysaccharide and O antigen.Rough Salmonella convers from Smooth Salmonella because of the loss of O antigen,but less study is worked on the pathogenic mechanism in SP.Some genes of SP invovled in lipopolysaccharide biosynthesis was find by establishing a transposon mutant library with Tn5 transposon,screening for rough mutants and primary immunobiological analysis of mutants,which would help to understood lipopolysaccharide synthesis genes on the pathogenicity of SP.1.Screening of rough SP mutants and identification of functional genesBy transposon mutagenesis,one mutant library of Salmonella Pullorum S06004 was constructed.High-throughput screening was performed for mutants which could not agglomerate with O9 monoclonal antibody by PAT and verified by the acridine orange method.Four mutants were selected out from 10,022 strains,and transposon-flanking sequence were amplified by PCR from genomic DNA.Sequences blast in NCBI showed rfbE,rfbM and rfc genes were identified,and the rfbE and rfbM genes encode CDP-tyrosine-2-epimerase and mannose-1-phosphate guanosyltransferase respectively,and rfc gene encodes 0 antigen polymerase.These results revealed that rfbE,rfbM and rfc genes were involved in lipopolysaccharide biosynthesis and provided target genes for further studies on the pathogenicity of SP.2.Primary immunobiological analysis of rfbE,rfbM and rfc mutantsS06004?rfbE,S06004?rfbM,and S06004?rfc mutants were constructed and primary analyzed their immunobiological features.The results of the LPS silver staining,the self-agglutination and western blotting showed that mutants were rough Salmonella.Cell assay displaied mutants of the rfbE,rfbM and rfc genes reduced the adhesion,invasion and intracellular proliferation ability of SP to HD-11 cells compared to S06004.Virulence test showed the virulence of S06004?rfbE,S06004?rfbM and S06004?rfc decreased by 100.0 folds,120.0 folds and 18.0 folds,respectively,which revealed the rfbE and rfbM genes were closely related to virulence,and the rfc gene had little effect on virulence.The in vivo dynamic anslysis showed that loads of mutants were lower than that of wild strains in liver and spleen,which indicated that they were more easily eliminated.Immune ptotection assay showed,sera antibody titers induced by mutants were higher than that of the blank group,which could cause a good humoral immunity,and provide high immune protection against lethal dose challenge of S06004.The cytokine detection by qRT-PCR showed that expression of IL-2,IFN-? and IL-1? were high level in the early stage,while the expression of IL-4 was significantly up-regulated at 14 days,which proved that these mutants could also induce good cellular immunity.3.Primary immunobiological analysis of rfbElspiC mutantS06004?rfbE?spiC was constructed,which was rough type with the loss of LPS.S06004?rfbE?spiC also had no effect on the biochemical features,and the virulence decreased by about 405.1 folds compared with the wild group.But the S06004?rfbE?spiC could not be isolated in the liver and spleen.The sera antibody titer of S06004?rfbE?spiC was higher than that of the blank group after 3 weeks.S06004?rfbE?spiC could also provide good immune protection.In summary,three genes,rfbE,rfbM and rfc were identified by transposon mutagenesis,they were invovled in LPS synthesis and closely related to pathogenicity of SP.These results could also provide the selection of the target genes of vaccine against SP.