Molecular interaction of Ornithobacterium rhinotracheale with eukaryotic cells

Ornithobacterium rhinotracheale (ORT) is a emerging gram negative bacterial pathogen in poultry. To facilitate the development of novel infection intervention and prevention strategies, the pathogenesis of ORT infection was investigated. In vitro infection assays demonstrated that ORT adhered to epithelial cells in a dose-dependent fashion. Comparison of the adhesive properties of different strains indicated that strain 41 lacked the ability to infect epithelial cells. This strain gained an adhesive phenotype when it was grown in suspension rather than on a solid phase medium. Bacterial adherence was inhibited in the presence of purified lipopolysaccharide (LPS). ORT also adhered to and became ingested by chicken and murine macrophages. This interaction was not blocked by LPS. The macrophage response to ORT was investigated using the production of nitric oxide (NO) as a marker. ORT induced the production of NO by macrophages. This stimulation did not require but was enhanced by rChIFN-gamma. The NO response stimulated by ORT differed from that induced by the Gramnegative bacteria E. coli and S.Enteritidis. The ORT response was slower and was not inhibited by the lipid A neutralizing agent polymixin B. LPS derived from ORT did stimulate NO production. The stimulation of NO production by ORT was specific for chicken macrophages. Both bovine and chicken serum inhibited ORT infection of epithelial cells. The inhibition was caused via an effect of serum factors on the adhesive properties of the bacteria. Biotinylated isolated serum glycoproteins showed direct binding of multiple serum glycoproteins to the bacteria. Serum inhibition was not observed in the interaction of ORT with macrophages. Competitive binding assays, in which the bacteria were first incubated with LPS or serum and subsequently with biotinylated isolated serum glycoproteins showed that LPS and serum components inhibited cellular infection via different mechanisms. Haemagglutination assays indicated variation among ORT strains to agglutinate erythrocytes. Strain 41 was unable to adhere to epithelial cells but did cause haemagglutination when the bacteria were grown onto solid phase media. When the bacteria were grown in suspension, opposite binding characteristics were observed. The apparent transition between phenotypes with different cell-type specificities (erythrocytes versus epithelial cells) thus varied with media growth conditions. To enable the use of molecualr biology in studying ORT pathogenesis, a transformation system was developed that was based on a cryptic plasmid isolated from ORT. This plasmid was completely sequenced and characterized and contained up to 14 different open reading frames of which some appear to encode the proteins involved the transport of heavy metals across the membrane. Introduction of the origin of replication and several regulatory sequences of this plasmid (designated pOR1) into the vector pGEM7, yielded a shuttle vector that replicated as an independent unit in both E. coli and ORT. An antibiotic resistance gene cfxA had been introduced into the plasmid and the transfer of this plasmid to confirm that the plasmid can be used as a genetic tool to identify novel bacterial virulence determinants and candidate vaccine antigens