Studies On The Immunodiagnostic Methods And Differential Expressed Genes Of Riemerella Anatipestifer In Infected Duck Livers

The Riemerella anatipestifer infection is one of the most important diseases to duck industry throughout the world,which usual occurs as an acute,contagious,septic disease in ducks.Infected ducks have fibrinous pericarditis,perihepatitis,meningitis and peritonitis.R.anatipestifer causes increased mortalities,decreased weight gains,and increased condemnations at slaughter and is estimated to cause substantial economic losses to the commercial duck industry each years.R.anatipestifer have complex serotypes,among which lack of effective cross-protection,leading to the difficulty of using inactivated bacterins,live or cell-free filtrates vaccines to immunize ducks against infection.Characterizaton of the field isolates and differentiation of theirs serotype, respectively,and monitoring of antibodies against R.anatipestifer,which play a very important role in understanding epidemic status of R.anatipestifer and formulating immunized procedure and evaluating immunized effect.R.anatipestifer firstly causes local inflammation at the site of entry,then rapidly break through the defensive organizations into blood.After the proliferation in blood,R.anatipestifer located mainly in serous membrane all over the body,cause inflammation in here and result in septicemia. Howerer,at present,little is known about the survive and multiply while avoiding the host immune system within a host organism of R.anatipestifer during infection.Therefore,we explored that development of OmpA-ELISA detecting antibodies against R.anatipestifer and differential expressed genes of R.anatipestifer in infected duck livers by selective capture of transcribed sequence technique.The research contents are summarized as follows:1.Preparation and preliminary of stain plate agglutination disgnostic antigen of R. anatipestiferThe serotype 1 field isolate,R.anatipestifer RA-YM was used to prepare diagosis antigen of stain plate agglutination.The results showed that has a good specificity, sensitivity and repeatability.It has no cross-reaction with five cluck diseases sera,and titer dectected by stain plate agglutination is higher than agar immune diffusion.This work showed stain plate agglutination could be used for antibodies detection and diagnosis for R.anatipestifer,which will play an active role in prevention and control the R. anatipestifer disease.Ducks were immunized with serovar 1,R.anatipestifer RA-YM emusified with Frund's adjuvant,and sera of duck against RA-YM were acquired after four inoculations,and the titers of plate agglution reach 1:32.Twenty-three R. anatipestifer isolated from Hubei province were identificated by antisera prepared above, the result showed thet are serovar 1.At the same time,purified serum of rabbit anti-duck IgG(AGID:1:64) from rabbit immunized with purified duck IgG was labeled with horseradish peroxidase(HRP).The conjugate is characteristic of high activity of immunity and its work concentration reached 1:4000.This laid a foundation for the development of indirect enzyme-linked immunosorbent assay(ELISA) for R. anatipestifer.2.Cloning,expression of the OmpA gene of Riemerella anatipestifer and protection test of rOmpA in ducksThe OmpA gene of RA-YM was amplified by PCR technique and cloned into pMD 18-T,and recombinant plasmid of pT-OmpA was sequenced by Sanger's sequencing techqique.Then,the OmpA from pT-OmpA was inserted into the pGEX-KG vector,to yield the recombinant plasmid pGEX-OmpA.After induced by IPTG,a high expression of fusion proteins was obtained.SDS-PAGE analysis showed that the fusion proteins was 68 kD in size.rOmpA existed mainly in form of inclusion bodies and was specific to antisera against R.anatipestifer by western blot analysis.The rOmpA was used to immunize ducks,the result showed rOmpA couldn't protect vaccinated ducks from challenge with RA-YM.3.Development and preliminary of OmpA-ELISA diagnostic methodThe OmpA-ELISA was developed using expressed rOmpA in E.coli.The suitable concentration of coated antigen,0.81μg/mL,and the serum dilutions,1:160,were selected by checkerboard.The cut-off value determined by testing 84 negative sera was 0.22.In the analysis of indirect ELISA cross specificity,it revealed a negative reaction with positive sera of duck influenza,duck hepatitis,duck plague,s.anatum,E.coli(O₇₈),serum of duck uninfected with R.anatipestifer and a positive reaction with standard positive serum of or R.anatipestifer.In order to know its reliability,a repetitive experiment was conducted in different ELISA in the same time and in the different time that result in less than 10%variable coefficient of absoprotion.To evaluate the specificity and sensitivity of the OmpA-ELISA,213 field sera samples were collected from chickens.Using flat plate agglutination test as the reference methods,the sensitivity of the OmpA-ELISA was 95.77%and coincidence 62.91%.Then a total of 472 duck sera samples were axamined that were sent from several field farms such as Hubei e.t.The results above indicated the assay to be a good method with strong specificity,high sensitivity and excellent coincidence and it could be used to fastly,accurately detect the antibodies against R. anatipestifer in the field sera samples.4.Identification of Riemerella Anatipestifer Genes Differentially Expressed in Infected Duck Livers by Selective Capture of Transcribed Sequences TechniqueThe published sequences for R.anatipestifer 16S rDNA(AY871819) were used to design primers for the amplification of 16S rDNA of R.anatipestifer RA-YM.Because the 23S rDNA sequence of R.anatipestifer is not available,the 23S rDNA sequences of S. pyogenes(NC₀02737),P.multocida(NC₀02663),E.coli(NC₀00913) and C. canimorsus strain 24231(AY661855) were aligned to identify the conservative regions for designing three sets of primers for the amplification of 23S rDNA of R.anatipestifer RA-YM.The primers were designed by using Primer premier 5.0.The amplified DNA fragments were then ligated into pMD 18-T vector and confirmed by sequencing analysis. The confirmed plasmids(pMD-16rRNA,pMD-23S1rRNA,and pMD-23S3rRNA) and biotinylated R.anatipestifer RA-YM genomic DNA were sonicated microprobe.The fragment of sample were 100-2000bp after soniacation.The sequence data of R. anatipestifer RA-YM rDNA are accessible with GenBank accession numbers:FJ031240 and FJ031241.The ducks(n=7,9-days old) were experimental infected with R.anatipestifer RA-YM and 24 hours later,the liver samples from these dusks were collected and immediately put into liquid nitrogen.Total RNA from R.anatipestifer RA-YM infected livers or from TSB controls was isolated,and double cDNAs were synthesized,which were named by in vivo and in vitro cDNAs,respectively.After three rounds of normalization,the cDNAs from infected liver-grown R.anatipestifer were enriched and cloned into pMD18-T vectors to generate R.anatipestifer RA-YM-infected liver-specific cDNA libraries. Individual SCOTS clones obtained from liver-specific cDNA libraries were verified by PCR with SCOTS01 primers,and the PCR products of these SCOTS clones were spotted on positively charged membranes in duplicate subjected to dot blot analysis.The positive clones obtained from the dot blot analysis were identified and then sequenced by Sanger's sequencing techqique.BLASTn was performed to identify sequences of genes,and Swiss-Prot database was performed to identify sequences of translated products,there were up to 48 genes that were differentially expressed in the duck livers after R. anatipestifer infection compared to the control cultures.After that,eight genes(gcvT, hmgA,aspC,cat,ptfp,dppâ…£,m28,and RA46) were random chosen and verified by real-time RT-PCR analysis.All were upregulated by R.anatipestifer in infected duck livers,with changes ranging from 1.44- to 4.62- fold compared to in vitro cultures. Totally 48 genes were identified by SCOTS analysis,which can be divided into five functional groups:metabolism,regulatory,stress,transporter and proteinase.In our study, five genes of R.anatipestifer RA-YM encode different proteinase were identified,which have the highly homology with other bacteria.DPP IV is a serine protease that cleaves X-Pro or X-Ala dipeptide from the N-terminal ends of polypeptide chains.It is indicated that DPPIV is a important virulence factor of P.gingivalis by contributing to the degradation of connective tissues,and also mediates the adhesion of P.gingivalis to fibronectin.The proteolysis of substance P by Sg-xPDPP was observed,and the concerted action of an extracellular Arg aminopeptidase and Sg-xPDPP produces a truncated form of bradykinin.The combined effect of these modifications may result in local changes in vascular permeability and smooth muscle contraction at the infected endothelium.The fibrnous exudate is the prominent pathological characterization of R.anatipestifer infection,so the DPPâ…£may serve as a critical virulence factor of this organism for pathogenicity.Inactivation of the DPPâ…£gene in R.anatipestifer RA-YM using genetic tools should make it possible to determine the contribution of this protease to bacterial growth and pathogenicity.In the future,studies will focus on the isolation,expression and knockout of this gene or more of the in vivo expressed genes in order to evaluate their relative contributions to R.anatipestifer RA-YM virulence and survival at the site of infection.