An Optimized Protocol for Molecular Screening of Avian Pathogenic Escherichia Coli From Broiler Chickens in South East Queensland, Australia

Abstract

Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colibacillosis and causes localized and/or systemic infections in poultry. The presence of various virulence genes (VGs) may be a useful marker for the detection of APEC directly from fecal samples. The objectives of this study were to evaluate and compare 3 different DNA extraction methods from cloacal swabs and fecal samples of broiler chickens and determine if APEC can be detected directly from feces. The DNA extraction methods were assessed by measuring DNA yield and purity, absence of DNA shearing, 16S ribosomal DNA amplification, and reproducibility. Repeated bead beating plus column (RBB+C) was the preferred extraction method, as it yielded an adequate amount of quality DNA for PCR directly from feces. The DNA extracted from feces, with RBB+C method and DNA extracted from E. coli isolates of organs and feces, taken from 23 broiler chickens (10 healthy, 9 with colibacillosis, and 4 unhealthy with other infections), were screened with a pentaplex-PCR for the prevalence of APEC-associated VGs: iroN, ompT, iutA, iss, and hlyF. There was a statistically significant correlation between the presence of the 5 VGs in E. coli cultured from the cloaca, fecal, and organs samples from chicken affected with colibacillosis. However, screening extracted DNA from the feces for the selected VGs was not an effective diagnostic tool to detect APEC as all of the VGs were detected in the extracted fecal DNA from all chickens.