Identification Of Genes Transcribed By Riemerella Anatipestifer In Vivo By Selective Capture Of Transcribed Sequences

The Riemerella anatipestifer disease is one of the most important diseases in duck production, which is characterized by an acute or chronic septicemia. It accounts for significant economic losses to the duck industry. To date, little is known about the molecular basis of the virulent factors and the pathogenesis of this organism. Selective capture of transcribed sequences (SCOTS) has been used to identify genes that enable RA to survive and multiply within liver tissue of ducks, and try to research the mechanism of RA infection in vivo. This will provide powerfull foundation to prevent and control RA disease.The genome of RA was abstracted, labelled by photobiotin acetate. The 16S rRNA and 23S rRNA plasmids were constructed using pMD18-T vector( pMD-16r,pMD-23-1r,pMD-23-2r and pMD-23-3r). Mix the biotinylated chromosome DNA and the 16S rRNA and 23S rRNA plasmids according to specific ratio. Then sonicated the mixture, we obtained 100～2000bp DNA fragnents.Infected 7-day-old white Pekin duck, total RNA isolated from infected duck liver tissue and RA culture in TSB, the synthesized cDNAs were named infection cDNAs pool in vivo and culture cDNAs pool in vitro respectively. SCOTS had been used to nomorlize the two cDNA pools, and enrichement of the specific sequences expressed in vivo based on PCR. The specific sequences were connected to the pMD18-T vector and sequencing. The 16 genes specific expressed in vivo obtained by bioinformatic analysis, which can be divided into five functional groups: metabolism, regulatory, stress, transport, proteinase, and genes with unknown function.Bacterial proteinases are important virulent factors of pathogen. In this study, two proteinases, DPPIV and ZDP were identified. DPPIV of Streptococcus gordonii can degrade P substractance, also cooperate extracellular Arg aminopeptidases to enzymolysis bradykinin. The combined effect of these modifications may result in local changes in vascular permeability and smooth muscle contraction at the infected endothelium. Fibrous exudate is a main pathological change of RA infection, therefore, DPPIV may play a critical role in virulence of RA.