The purpose of this study was to investigate the prevalence of the anaerobic intestinal Brachyspira spp among laying hens flocks located in Mashhad suburb of Iran. In the first stage, as a pilot study, number of 50 rectal swabs sample from five laying hen flocks (10 samples from each flock), were collected. Samples were cultured anaerobically on selective agar and confirmed primarily as intestinal spirochaete by it s spirochaetal form under phase contrast microscopy. Final confirmation as Brachyspira spp performed by PCR amplification with specific primers, nested-PCR and finally sequencing the amplified PCR product. The first isolate were used as positive control during the study. With 4% prevalence of infection in pilot study, the sample size was determined. In the second stage, number of 130 rectal swab samples from 13 flocks was collected, and Brachyspira spp were isolated by conventional culture and confirmed by PCR and nested-PCR. The prevalence of Brachyspira spp infection in laying hens determined as 5%. In this study 16.7% of flocks were determined as infected with Brachyspira spp. This is the first report of the isolation of Brachyspira spp from laying hens in Iran. Further studies are needed to clarify the sensitivity and specificity of this test, and also determining the species and presence of infection in human populations.

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