Objectives: Brachyspira spp. are the etiologic agents of intestinal disorders in a broad spectrum of animals and also humans. The aim of the current study was to investigate molecular identification of avian intestinal spirochetes based on sequence analysis of nox gene (NADH oxidase) on our isolates from previous work. huge diversities in biochemical tests and highly conserved sequence in 16S rDNA of these bacteria yields limitations to count on them only for species differentiation. Phylogenetic analysis of our results confirms previous finding on our isolates taken from Ostrich, turkey, laying hens and dog. Materials & Methods: The fecal samples were shaken and prepared to detect the spirochetes using phase contrast. Bacterial culture was done based on standard procedures as follow: TS-blood agar (tryptose soy), incubation temperature 40°C and spectinomycin and vancomycine as selective materials. β-hemolysis was classified as strong or weak. The sequences of the NADH oxidase from different Brachyspira spp. deposited in Genbank were selected. Based on these sequence data analysis, a set of primers were designed to amplify a segment of containing the most variable part of the target gene among all species and PCR was done by boiling extraction method on single colonies. Results & Conclusion: Sequence analysis of 793 bp PCR products revealed that the avian isolates were B. pilosicoli and Brachyspira canis in dog’s fecal sample. Reported partial sequence of nox gene for B. canis to Gene Bank was the first documented report in the world.

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