Difference Study On Immunogen And Virulence Gene IdeR Between Two Riemerella Anatipestifer Strains

Backgrounds: Riemerella anatipestifer（RA）,which belongs to Riemerella,Flavobacteriaceaeis,is a Gram-negative short bacterium exhibiting non-motile,nonspore-forming,rod-shaped properties.It can infect domestic ducks,goose,turkeys,and other wild birds.It mainly infects domestic ducks under 8 weeks and causes duck infectious serositis,which is characterized by high morbidity,high mortality,can cause such symptoms as fibrinous pericarditis,perihepatitis,airsacculitis,caseous salpingitis.RA has 21 serotypes with lacking cross protection between each other.Among the known serotypes of RA,there were huge variations of virulence between each serotype or between different strains even within same serotype.Once the bacterium infects a duck flock,it can be endemic fastly and hard to be eradicated.However,little is known about the molecular basis of RA pathogenesis except confirmed virulence proteins such as OmpA,CAMP,SIP and TbdR1.Our group isolated a virulent strain SC（RA serotypeⅠ）from one duck raising farm in Chengdu and compared it with another virulent strain AF（RA serotypeⅡ）which separated from one duck raising farm in Chongqing.The study discovered that strain SC could cause adult laying ducks morbidity and dealth,but strain AF can’t,and the morbidity and mortality of ducklings which challenged with alive strain SC were higher than ducklings which challenged with alive strain AF.To date,nine full RA genomic sequences have been submitted to the GenBank database so far,including strains ATCC11845,RA-GD,RA-SG,DSM 15868,RA-CH-1,RA-CH-2,CH3,17 and 153,and these sequences will be benifit to further study on immunoprotective antigens and pathogenesis of this bacterium.Contents and Results:1.The difference analysis of genes ideR between strain SC and AFWe design ider primers based on ider sequence’s ORF in RA SC & AF strain’s whole genome sequencing results,get SC ider and AF ider by PCR,and clone them to vector pMD18-T.By sequences analysis,we find that their ORF include 654 bp,which coding 217 amino acid residues.There are 41 different bases between them,among these different bases,6 bases induce 4 different residues which cause tertiary structures difference.AF ider sequence is same with other RA strains which are registered in GeneBank by analysis,while strain SC ider become a new branch.2.The difference between SC and AF whole-cell protein detecting ELISA antibodyThe establishment of ELISA method by coating SC and AF whole-cell protein as antigen to detect SC and AF antibody titer and non-agglutinative antibody titer,shows that the last declines 1³ titer,but serum titer doesn’t change after different strain’s absorption.Results display that postive antibody rate SC convalescent serum is low as 42% and with low titer 1:2000⁸000.While postive antibody rate AF convalescent serum is high as 42% and with high titer 1:4000¹6000.3.The difference analysis of whole-cell proteins between strain SC and AF RAUltrasonication supernatant of RA strain SC and strain AF are proceeding with SDS-PAGE and PAGE,followed with Western blot,then we find two different stripes between two strains.One is about 45 KDa and belongs to strain SC,one is about 40 KDa and belongs to strain AF.These different protein stripes are analyzed by MALDITOF-TOF.5 different immunogenic proteins which belong to AF,are probable peptidyl-prolyl cis-trans isomerase,hypothetical protein,peptidase C1 B bleomycin hydrolase,type II citrate synthase,3-hydroxyphenylpyruvate dioxygenase;4 different immunogenic proteins which belong to SC,are elongation factor Tu,glutamate dehydrogenase（NAD）and hypothetical protein RAYM₀0330.