Development of real-time polymerase chain reaction assay for detection of Ornithobacterium rhinotracheale in poultry.

Ornithobacterium rhinotracheale (ORT) is an emerging bacterium causing severe economic losses in poultry mostly due to respiratory and locomotory disturbances. Due to the fastidious nature of the organism, ORT is often overgrown by faster-growing commensal and pathogenic bacteria. In this study we developed a real-time polymerase chain reaction (qPCR) assay for rapid and sensitive detection of ORT in samples collected from chickens and turkeys. The qPCR assay developed was able to detect 17 reference strains of ORT (serotypes A to Q) tested in this study, and no false-positive results were obtained from other organisms associated with respiratory tract infections. The qPCR assay was 100 times more sensitive than the modified conventional PCR. Using tenfold serial dilutions of the recombinant plasmid DNA containing the target gene fragment, the detection limit of the qPCR was estimated to be > or = 100 plasmid copies per reaction. Out of 42 examined poultry flocks, 26 cases were tested positive by both assays. The qPCR assay reduces turnaround time to about 2 hr, two times faster than the modified conventional PCR.

Ornithobacterium rhinotracheale North American field isolates express a hemolysin-like protein.

Ornithobacterium rhinotracheale is a gram-negative bacterium responsible for the sporadic outbreaks of airsacculitis in poultry, accounting for millions of dollars in losses to the poultry industry annually. Although the organism was originally classified as non-beta-hemolytic, recent North American field isolates of O. rhinotracheale obtained from pneumonic lungs and air sacs indicated hemolytic activity on blood agar plates upon extended incubation for 48 hr at room temperature in air after initial incubation at 37 C for 48 hr under 7.5% CO2. This report characterizes the beta-hemolytic activity of O. rhinotracheale isolates by using in vitro kinetic hemolysis assays with sheep red blood cells, western blotting with leukotoxin-specific monoclonal antibodies, and isobaric tagging and relative and absolute quantitative (iTRAQ) analysis of O. rhinotracheale outer membrane protein digest preparations. The kinetic analyses of the hemolytic activity with red blood cells indicated that the protein is a pore former. iTRAQ analysis with membrane preparations revealed four peptides with homology to Mannheimia haemolytica leukotoxin and two peptides with homology to Actinobacillus actinoacetemcomitans leukotoxin. This is the first report that North American field isolates of O. rhinotracheale may express a hemolysin-like activity.

Iron acquisition by Ornithobacterium rhinotracheale.

Ornithobacterium rhinotracheale (ORT) is an emerging respiratory pathogen of poultry in North America that is causing millions of dollars in economic losses to the poultry industry. Ornithobacterium rhinotracheale is associated with airsacculitis, pleuritis, pneumonia, and consolidation of lungs. Little is known about the molecular mechanisms of infection. In this study, the mechanism of iron acquisition by O. rhinotracheale was explored. O. rhinotracheale strains grown under iron deprivation in media containing 200 microM 2,2'-dipyridyl did not secrete siderophores as measured by the chrome azurol S (CAS) agar and CAS solution assays. Filter disks impregnated with various protein-bound iron compounds and inorganic iron salts of Fe(III) and Fe(II) placed on iron-restricted agar inoculated with a lawn of O. rhinotracheale supported growth from sheep and porcine hemoglobins, ovotransferrin, Fe(III), and Fe(II), but they did not support growth from bovine transferrin, bovine apo-transferrin, bovine lactoferrin, and hemin. However, both bovine hemoglobin and transferrin supported growth of O. rhinotracheale serotype C. Four immunoreactive proteins involved in iron acquisition were identified in an O. rhinotracheale membrane extract by using mass spectrometry. Furthermore, O. rhinotracheale field strains showed differential sensitivity to 2,2'-dipyridyl. Of the 72 field strains tested, 22 strains were resistant to the iron chelator at concentrations of 50 microM and 100 microM, suggesting this attribute may be related to disease-producing potential of these strains. This is the first report on the identification of the iron acquisition mechanism of O. rhinotracheale.

The role of outer membrane proteins of Ornithobacterium rhinotracheale in attachment to chicken tracheal epithelium.

Ornithobacterium rhinotracheale (ORT) infections cause major losses to the poultry industry. In search for factors implicated in the pathogenesis of ORT infections, the role of outer membrane proteins (OMPs) in the interaction of ORT with chicken tracheal epithelium was investigated. For this purpose, immune sera were prepared against total extracted OMPs, whole cell bacteria and three major OMPs of 45, 53 and 70 kDa and used in bacterial adherence inhibition assay. The results showed antibodies against a 53 kDa OMP significantly (p < or = 0.05) inhibited the bacterial adherence to chicken tracheal epithelium up to 78%.