Isolation and identification of Brachyspira pilosicoli from laying hens flocks, using conventional culture and molecular methods in Mashhad, Iran

Avian intestinal spirochaetosis (AIS) is an intestinal infection caused by anaerobic spirochaetes of the genus Brachyspira, including B. pilosocoli. The purpose of this study was isolation and identification of B. pilosocoli from laying hen flocks, located in Mashhad suburb, Khorasan Razavi province, Iran, and investigating the frequency of the infection. One hundred and eighty cloacal swab samples from 18 randomly selected flocks (10 samples /flock) were cultured anaerobically on selective agar and confirmed as intestinal spirochaete by its spirichaetal form using phase contrast microscopy. Then, the samples were subjected to PCR amplification followed by DNA sequencing. A total of 24 samples from 8 flocks were selected as suspected cases by culture and phase contrast microscopy. Upon PCR amplification by specific primers, only 9 cultures belonged to 3 flocks appeared to be B.pilosicoli. Sequence analysis of the amplicons confirmed the identity of all isolated ones. Based on the results obtained, it was concluded that B. pilosicoli might be strongly involved in AIS among laying hen flocks of this geographical region. The results could also be considered as an indicator for large scale investigation into the true prevalence of the infection. This study is the first report of infection in laying hens flocks of Iran.