Objectives: This research is done for isolation and molecular identification of bacteriaOrnithobacterium rhinotracheale(ORT) in Broiler Chicken flocks in Razavi Khorasan Province in Iran and characterize it using biochemical tests and polymerase chain reaction assays. Materials &Methods:This study was conducted on 150 lung tissue and tracheal swabs samples from Broiler Chicken with respiratory tract diseases signs. Samples were obtained from private clinics and slaughterhouse of province. Allof samples transported to the laboratory in sterile conditions and were culturedon sheep blood agar with5μg gentamicin at 37 C in the presence of 5% CO2. Results &Conclusion: In total, 150 pooled samples were obtained from Broiler Chicken flocks. After 48h of incubation , four suspected bacterial samples (2.66%) could be grown and isolated on blood agar plates and did not grow on MacConkey agar, was oxidase positive, catalase negative, were gram-negative, small, rod-shaped, pleomorphic, grey-white, opaque, non hemolytic and differ in diameter (1-3 mm) and nonmotile, After DNA extraction, a specific Polymerase chain reaction ( PCR) performed using the primer combination OR16S-F1 (5\'-GAG AAT TAA TTT ACG GAT TAA G) and OR16S-R1 (5\'-TTC GCT TGG TCT CCG AAG AT). This combination amplifies a 784 bp fragment on the 16S rRNA gene of O. rhinotracheale.This work is the first report of the presence of O.rhinotrachelae by culture and molecular method in Razavi Khorasan Province in Iran .The results confirm that ORT is present in this region and finally, it is recommended if needed Vaccination of Broiler Chicken flocks be done to protect progenies.

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