Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/21/2010
Publication Date: 9/17/2010
Citation: Tabatabai, L.B., Zimmerli, M.K., Zehr, E.S., Briggs, R.E., Tatum, F.M. 2010. Ornithobacterium rhinotracheale North American field isolates express a hemolysin-like protein. Avian Diseases. 54(3):994-1001. Interpretive Summary: Ornithobacterium rhinotracheale (ORT) is a Gram-negative bacterium responsible for the sporadic outbreaks of airsacculitis in poultry. This organism causes millions of dollars in economic losses to the poultry industry not just in North America but world-wide. Losses are due to lowered egg production, decreased growth rates and post-harvest condemnation due to airsacculitis. Commercial vaccines are not readily available, although one commercial vaccine has been used in Western Europe with mixed results. In this report we describe the observation that North American isolates of ORT, unlike the reference strain, ATCC 51463, causes hemolysis of sheep blood agar. The hemolytic protein component is considered a virulence factor of bacterial pathogens. We further analyzed this hemolytic activity in an outer membrane protein extract by comparative mass spectrometry, Western blotting and several in vitro hemolysis assays. Based on the in vitro kinetic assays we conclude that the hemolytic protein of ORT is a pore-forming cytolytic protein. The significance of this first report of evidence of hemolytic activity of the North American field isolates compared to the reference strain remains unknown and awaits the completion of the genomic sequence of the hemolytic phenotype.
Technical Abstract: 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 airsacs indicated hemolytic activity on blood agar plates upon extended incubation for 48 h at room temperature in air after initial incubation at 37 deg C for 48 h 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, red blood cell membrane binding assays followed by Western blotting, Western blotting with leukotoxin-specific monoclonal antibodies, isobaric tagging and relative and absolute quantitative (iTRAQ) analysis of membrane preparations. Additional characterization employed 2-D gel electrophoresis followed by electrospray tandem mass spectrometry (ES/MS/MS) and matrix-assisted time-of-flight mass spectrometry (MALDI-TOF) analysis of protein digests. 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 acetemcomitans leukotoxin. The significance of this first report of evidence of hemolytic activity of the North American field isolates compared to the reference strain, ATCC 51463 remains unknown and awaits genomic sequencing of the hemolytic phenotype.