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

Abstract

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