BACKGROUND:Avian intestinal spirochetosis (AIS) is caused by spiral-shaped Gram-negative Brachyspira spp. in poultry and is known as a cause of diarrhea, low egg production, and increased occurrence of dirty eggs in layer hens. OBJECTIVES: In this study, the presence of some Brachyspira spp. was investigated in laying hens. METHODS: A total of 100 cloacal swab samples were individually collected from 20 laying hen flocks showing fecal egg staining in northeastern Iran. RESULTS: Using culture and morphologic examination, 41 samples (41%) from 20 flocks were positive; however, by using genus-specific PCR, only 37 (37%) samples were confirmed as Brachyspira spp. Using speciesspecific primers, single colonization was identified in 18 samples associated with B. pilosicoli (48.6%), while single colonization with B. intermedia was found in only two samples (5.4%). Simultaneous colonization by B. intermedia and B. murdochii was detected in 3 samples (8.1%). B. pilosicoli was the most prevalent species in concurrent colonization in 11 cases (29.7%). Finally, cocolonization by B. intermedia and B. innocens was identified in 3 samples (8.1%). CONCLUSIONS: The results of this study showed the colonization of different species of Brachyspira with dominance of B. pilosicoli in layer hens.

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