Respiratory infections particularly those in which bacterial pathogens are involved (Glisson, 1998), are some of the most serious diseases of poultry (van Empel and Hafez, 1999). Increased mortality, increased costs of medication, increased condemnation rates, drops in egg production, reduction in shell quality, and decreased hatchability directly associated with these respiratory infections in poultry cause heavy economic losses (van Empel and Hafez, 1999). Ornithobacterium rhinotracheale infection, also known as ornithobacteriosis, is a contagious disease of avian species, primarily turkeys and chickens, causing respiratory distress, decreased growth, and mortality (Chin and Charlton, 2008; Chin et al., 2008). 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 (Chin et al., 2008; Hoerr, 2010). After its identification and characterization in 1994 by Charlton et al, O. rhinotracheale has been isolated throughout the world (Hinz et al., 1994; 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; 2001; 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; 2012).

1 History                                                                                                                   
The first report related to the characterization of O. rhinotracheale was that of Charlton et al (1993). Then, Vandamme et al (1994) described the phylogenetic position and various genotypic, chemotaxonomic, and classical phenotypic characteristics of 21 strains were then described, and assigned the name O. rhinotracheale (Vandamme et al., 1994). However, this bacterium appears to have been isolated before 1993 (Chin et al., 2008). In 1991 a new respiratory disease in 28-day-old broiler chickens was observed in South Africa. The birds suffered sneezing, associated with increased mortality and poor performance parameters. At postmortem examination, pneumonia and foamy white and “yoghurt-like” exudate were observed in the abdominal air sacs and the bacteriological study revealed a slow growing, pleomorphic, Gram-negative rod unknown among the bacterial species previously reported (van Empel, 1998; van Empel and Hafez, 1999; Bisshop, 2003). A bacterium similar to Pasteurella spp. isolated from 10-week-old Pekin ducks with respiratory disease in Hungary in 1987 (van Empel, 1998; van Empel and Hafez, 1999; Pyzik, 2007; Chin et al., 2008), as well as Riemerella anatipestifer-like strains isolated from turkeys suffering respiratory disease in Germany in 1991 and 1992 showed appearance and biochemical properties similar to those of the South African isolates (van Empel, 1998; van Empel and Hafez, 1999; Pyzik, 2007). This bacterium was named Pasteurella-like, Kingella-like, TAXON 28, or pleomorphic Gram-negative rod before the name Ornithobacterium rhinotracheale gen. nov. sp. nov. was suggested (van Empel and Hafez, 1999; Chin et al., 2008). Investigations of German culture collections revealed that O. rhinotracheale had been already isolated from the respiratory tract of 5-week-old turkeys with nasal discharge, facial edema, and fibrinopurulent airsacculitis in 1981 and from rooks in 1983 (van Empel, 1998). In Belgium, France and Israel, O. rhinotracheale had also been isolated before 1990 (van Empel, 1998). No isolates of O. rhinotracheale had been reported before 1981 (van Empel, 1998).

2 Etiology
2.1 Morphology, staining, growth requirements, antigenic structure, and colony morphology
Ornithobacterium rhinotracheale is a Gram-negative, non-motile, highly pleomorphic, rod-shaped, and non-sporulating bacterium of the rRNA superfamily V within the Cytophaga-Flavobacterium-Bacteroides phylum. When cultured on solid media, the bacterium appears as short, and plump rods measuring 0.2~0.9 µm in width and 0.6~5 µm in length (van Empel and Hafez, 1999; Chin et al., 2008), and less frequently as long filamentous rods or club-shaped rods (Chin and Charlton, 2008). No structures such as pili, fimbriae, and plasmids or properties such as specific toxic activities have been reported for the species (van Empel and Hafez, 1999; Chin et al., 2008). The use of 5%~10% sheep blood agar plate is recommended for isolation and optimal growth of the causing agent. Ornithobacterium rhinotracheale does not grow on MacConkey agar, Endo agar, Gassner agar, Drigalski agar, or Simmons citrate medium (Chin et al., 2008). The bacterium grows aerobically, microaerobically, and anaerobically, but the best growth occurs in air enriched with 7.5~10 % CO₂ at 37? (Chin and Charlton, 2008). Under these conditions and 24 h post-incubation, O. rhinotracheale develops pin-point colonies smaller than 1 mm in diameter. After 48 h, the colonies are approximately 1~2 mm in diameter, gray to gray-white, circular, and convex with an entire edge, and some isolates from chickens have a reddish glow. Cultures of O. rhinotracheale have a distinct smell similar to that of butyric acid (van Empel and Hafez, 1999; Chin and Charlton, 2008). Because of the resistance to gentamicin and polymyxin B observed in 90% of O. rhinotracheale field isolates (Vandamme et al., 1994), 5 µL/mL of each antibiotic is recommended to be added to blood agar media for selective isolation of this bacterium (van Empel and Hafez, 1999; Chin and Charlton, 2008; Chin et al., 2008). The use of 10 µg of gentamicin per mL of blood agar medium has also been suggested to isolate O. rhinotracheale from contaminated samples (Chin et al., 2008). 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; Chin et al., 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. 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. In search for the optimal dilution to differentiate the pathogenicity of the isolates, these authors compared mortality patterns for 8 days post-inoculation and found the most drastic differences in the 10³ dilution groups. The results showed higher mortality numbers in an embryo model caused by hemolytic O. rhinotracheale isolates, which appear to be more virulent than non-hemolytic ones.

Ornithobacterium rhinotracheale is known for its hemagglutinating activity (Soriano et al., 2003; Tsai and Huang, 2006). Vega et al (2008) tested the hemagglutinating activity of serotypes A to I of O. rhinotracheale reference strains by using red blood cells from 15 different species, including avian, mammal, fish, and human erythrocytes, and concluded that rabbit erythrocytes were suitable to test O. rhinotracheale. In contrast, Chernyshev et al (2011) have recently reported the hemagglutinating activity of 19 Russian isolates with chicken and sheep erythrocytes.

2.2 Biochemical identification
The results of biochemical tests for the identification of O. rhinotracheale can be inconsistent (van Empel and Hafez, 1999; Chin and Charlton, 2008; Chin et al., 2008). Therefore, Chin and Charlton (2008) proposed the following tests as those with more consistent reactions to identify O. rhinotracheale: oxidase (+), catalase (-), β-galactosidase (+), indole (-), and triple sugar iron agar (no change). Chin et al (2008) also described a cytochrome-oxidase negative strain isolated from turkeys in Germany.                                             

On the other hand, commercial biochemical test kit such as the API-20NE identification strip (bioMérieux, France) have been found useful for the identification of O. rhinotracheale (van Empel and Hafez, 1999; Chin and Charlton, 2008; Chin et al., 2008), although this bacterium is not included in the API database (van Empel and Hafez, 1999; Chin and Charlton, 2008). In a study where a total of 1150 isolates were tested, the biocodes found in 99.5% of the strains were 0–2–2–0–0–0–4 (61%) or 0–0–2–0–0–4 (38.5%). In addition, the isolates with positive results for arginine dihydrolase test (0.5%) had the biocodes 0–3–2–0–0–0–4 and 0–1–2–0–0–0–4 (van Empel and Hafez, 1999; Chin and Charlton, 2008). Ornithobacterium rhinotracheale showed constant and negative results for five enzymatic activities in the API-ZYM system (bioMérieux, France): lipase, β-glucuronidase, β-glucosidase, α-mannosidase, and α-fucosidase reactions (Chin and Charlton, 2008; Chin et al., 2008). Another commercial system for the identification of this bacterium is the RapID NF Plus (Remel/Atlanta, USA) which was used to test 110 isolates, and found five unique biocodes: 4–7–2–2–6–4 (41.8%), 4–7–6–2–6–4 (31.8%), 6–7–6–2–6–4 (18.2%), 6–7–2–2–6–4 (7.3%) and 4–7–2–0–4–4 (0.9%), (van Empel and Hafez, 1999; Chin and Charlton, 2008).

2.3 PCR
The PCR procedure is considered a useful laboratory tool for the identification of suspected O. rhinotracheale isolates (van Empel and Hafez, 1999; Chansiripornchai et al., 2007; Hung and Alvarado, 2001; Chin et al., 2008; Gornatti Churria et al., 2011; 2012), and also for diagnostic or investigation purposes (van Empel and Hafez, 1999; Canal et al., 2003b; Eroksuz et al., 2006; Tsai and Huang, 2006; Pyzik et al., 2007). The primers OR16S-F1 (5´- GAGAATTAATTTACGGATTAAG-3´), and OR16S-R1 (5´-TTCGCTTGGTCTCCGAAGAT-3´) have allowed the amplification of a 784 bp fragment on the 16S rRNA gene of O. rhinotracheale (van Empel and Hafez, 1999; Hafez, 2002; Hung and Alvarado, 2001; Canal et al., 2003; Eroksuz et al., 2006; Tsai and Huang, 2006; Chansiripornchai et al., 2007; Pyzik et al., 2007; Uriarte et al., 2009; Gornatti Churria et al., 2011; 2012). According to van Empel and Hafez (1999) and Hafez (2002) no other bacteria closely-related to O. rhinotracheale could be confused when those primers are used.

On the other hand, Thachil et al (2007) studied the enterobacterial repetitive intergenic consensus (ERIC)-PCR and the random amplified polymorphic DNA (RAPD) assay with Universal M13 primer-based fingerprinting techniques to differentiate O. rhinotracheale isolates. To this end, they evaluated A total of 50 field strains and 8 reference strains for genetic differences using the primers ERIC 1R (5´- ATGTAAGCTCCTGGGGATTCAC-3´) and Universal M13 (5´-TTATGTAAAACGACGG CCAGT-3´). M13 fingerprinting revealed different patterns for six reference serotypes of O. rhinotracheale tested, namely, C, D, E, I, J, and K. Ornithobacterium rhinotracheale reference serotypes A and F showed indistinguishable fingerprints with M13 fingerprinting. The ERIC 1R technique discerned only five out of the eight reference serotypes. Distinct fingerprints were also found within the O. rhinotracheale serotypes with both techniques. From 58 isolates of O. rhinotracheale belonging to 8 O. rhinotracheale serotypes that were fingerprinted, 10 different fingerprints were obtained with M13 fingerprinting, whereas six different fingerprints were obtained with ERIC 1R fingerprinting. The authors concluded that M13 fingerprinting technique was more discriminative in differentiating O. rhinotracheale isolates than the ERIC 1R fingerprinting technique.

2.4 PFGE
Moreno et al (2009) genotyped O. rhinotracheale strains obtained from Spanish red-legged partridges with neurological signs, otitis and cranial osteomyelitis, following adaptations of previously reported PFGE assays. The authors incubated isolates on Columbia agar in aerobic conditions at 37? for 24 h and prepared DNA plugs from fresh colonies of bacteria suspended into 1 mL of TE1 × buffer with a transmittance of 20%. The suspension was incubated at 37? for 10 min, and 60 mL of lysozyme was added. The suspension was mixed with an equal volume of 2% molten agarose, 10% sodium dodecyl sulphate, and recombinant proteinase K. The DNA plugs were incubated at 56? for 2 h in TE 1× buffer with 30 mL of recombinant proteinase K, and lysis buf