Ornithobacterium rhinotracheale North American Field Isolates Express a Hemolysin-Like Protein

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 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.

Isolation and Characterization of Small-Colony Variants of Ornithobacterium rhinotracheale

“…The pathogen was relatively recently discovered and is distributed worldwide in chickens and turkeys, as well as in other avian species (15)(16)(17). O. rhinotracheale is associated with respiratory diseases and causes significant economic loss to the poultry industry (17)(18)(19).…”

<i>Ornithobacterium rhinotracheale</i> Infection in Poultry: an Updated Review

“…However, the presence of extensive and unusual β-hemolytic activity has been recently reported among North American and Argentinean field isolates after the 48-h-period following incubation at room temperature (Walters et al, 2009;Tabatabai et al, 2010;Gornatti Churria et al, 2011). Tabatabai et al (2010) characterized and demonstrated the β-hemolytic activity of O. rhinotracheale isolates using in vitro kinetic hemolysis assays with sheep red blood cells, western blotting with leukotoxin-specific monoclonal antibodies, and isobaric tagging and quantitative analysis of O. rhinotracheale outer membrane protein digest preparation. Moreover, Walters et al (2011) have recently developed an embryo lethality assay to determine potential virulence differences between hemolytic and non-hemolytic O. rhinotracheale strains isolated from turkeys in Virginia´s Shenandoah Valley, USA.…”

“…van Empel and Hafez (1999) and Chin and Charlton (2008) also proposed the use of blood agar plates without antibiotic to prevent missing 10% of the antibiotic-susceptible isolates. Ornithobacterium rhinotracheale was first identified as a non-hemolytic microorganism (van Empel and Hafez, 1999;Hafez, 2002;Canal et al, 2005;Chin and Charlton, 2008;, such as the ATCC 51463 strain of O. rhinotracheale (Tabatabai et al, 2010;Gornatti Churria et al, 2011). However, the presence of extensive and unusual β-hemolytic activity has been recently reported among North American and Argentinean field isolates after the 48-h-period following incubation at room temperature (Walters et al, 2009;Tabatabai et al, 2010;Gornatti Churria et al, 2011).…”

“…Ornithobacterium rhinotracheale was first identified as a non-hemolytic microorganism (van Empel and Hafez, 1999;Hafez, 2002;Canal et al, 2005;Chin and Charlton, 2008;, such as the ATCC 51463 strain of O. rhinotracheale (Tabatabai et al, 2010;Gornatti Churria et al, 2011). However, the presence of extensive and unusual β-hemolytic activity has been recently reported among North American and Argentinean field isolates after the 48-h-period following incubation at room temperature (Walters et al, 2009;Tabatabai et al, 2010;Gornatti Churria et al, 2011). Tabatabai et al (2010) characterized and demonstrated the β-hemolytic activity of O. rhinotracheale isolates using in vitro kinetic hemolysis assays with sheep red blood cells, western blotting with leukotoxin-specific monoclonal antibodies, and isobaric tagging and quantitative analysis of O. rhinotracheale outer membrane protein digest preparation.…”

“…Although O. rhinotracheale is considered as a primary pathogen in poultry (van Veen et al, 2000b;Pan et al, 2012), the severity of clinical signs, duration of the disease, and mortality are extremely variable and are influenced by housing environmental stressors such as poor management, inadequate ventilation and high ammonia levels, high stocking density, poor litter conditions, poor hygiene, foodborne mycotoxins, suboptimal nutrition, and concomitant infectious diseases Hoerr, 2010). After its identification and characterization in 1994 by Charlton et al, O. rhinotracheale has been isolated throughout the world Devriese et al, 1995;van Empel et al, 1997;Goovaerts et al, 1998;Sakai et al, 2000;Sprenger et al, 2000;van Veen et al, 2000a;Ak and Turan, 2001;Devriese et al, 2001;Hung and Alvarado, 2001;Soriano et al, 2002;Malik et al, 2003;Banani et al, 2004;Canal et al, 2005;Türkyilmaz, 2005;Tsai and Huang, 2006;Marien, 2007;Chansiripornchai et al, 2007;Moreno et al, 2008;Murthy et al, 2008b;Uriarte et al, 2009;Walters et al, 2009;Gavrilović et al, 2010;Tabatabai et al, 2010;Chernyshev et al, 2011;Gornatti Churria et al, 2011;.…”