Summary Intestinal spirochetosis caused by Brachyspira spp. has been a topic of interest for many researchers in recent decades because of its economic and public health importance. The purpose of the study was to determine the occurrence of Brachyspira spp in Iran and to characterise selected isolates by biochemical and molecular methods. Fecal samples obtained from commercial layer,broiler and broiler breeder chickens, backyard and commercial turkeys ,common ostriche,zoo birds from local zoo parks from two province Khorasan Razavi & Tehran such as Guinea Fowl, Spanish Pheasant, White pea Fowl , Miniature Hen,Mallard Duck, Golden Eagle in our diagnostic laboratory and positive samples in phase contrast microscopy examination and then in selective culture media (tryptose soy agar containing 5% defibrinated ovine blood and antibiotic supplements)have been tested by standard biochemical reactions such as β—hemolysis, Indole production (Sutter and Curter, 1997) , α-galactosidase and β-glucosidase to find out Biochemical scheme . NADH (nox) gene sequence analysis was performed by using the sequences of the NADH oxidize genes from different Brachyspira spp. deposited in Genbank and Based on these sequence data analysis, two sets of primers were designed to amplify a segment of each of the gene containing the most variable part among all species. Sequencing of partial sequence of NADH oxidase gene of selected isolates revealed presence of Brachispira pilosicoli, B. innocence , B. intermedia and B. alvinipulli for the first time in iran.

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