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.

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.

mentor in the aspects of both science and life. Throughout my graduate career, we have encountered difficult events-both separately and together-and it was the example of her grace that allowed me to move through some very difficult situations in stride. She has challenged me and encouraged me. I feel blessed to have known her as a person and for the opportunity to work with her. In addition to Louisa, I'd like to extend gratitude to the BBMB department. I was fortunate to work with faculty and students alike through various teaching assistantships. Out of those experiences, I discovered a love for teaching. I am especially grateful for Desi Gunning who has been a wonderful companion throughout my teaching career. Overall, I could not have asked for a better graduate career or a better department in which to complete this study. With that being said, I would like to thank my Co-major Professor, Amy Andreotti, and my POS committee members, Gustavo MacIntosh, Alan DiSpirito, and Emily Smith for their assistance during my academic career. I would also like to thank Emilie Zehr for showing me the ropes when I first entered the Tabatabai lab. I would like to give special recognition to my older brother, Joe, for bringing levity to some of the worst of times. I am fortunate to have a brother who is also one of my closest friends. His encouragement, along with the support of my dad, has made every step of this process possible. I have a great support network of friends and family. To name everyone would take an eternity, so I will keep this short. To everyone who has supported me on this journey in any way and for those who continue to support me: thank you.

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