Development of a real-time PCR for Escherichia coli based on gadE, an acid response regulatory gene.

Increasingly, molecular methods have become important in identification and confirmation of bacteria at the species level. Rapid molecular methods provide sensitivity and specificity while reducing cost and resources. The primary goal of this study was to develop a real-time PCR assay for identification of Escherichia coli from an agar plate. GadE (gadE) directly regulates the glutamate-dependent acid response system (GDAR) in E. coli and is responsible for survival of at pH 2. Based on gene sequence data, a real-time PCR assay targeting gadE was developed for this purpose. Seventy bacterial isolates recovered from ground beef enrichments and 714 isolates from caecal contents were identified biochemically and tested with the real-time PCR assay developed in this study. The PCR assay and the biochemical identification had 100% agreement on the tested isolates. The gadE real-time PCR assay was demonstrated in this study to be an inexpensive, reliable method for confirming E. coli colonies within 1.5 h from an agar plate, thereby saving on final identification time.

Pathogenicity of two Egyptian H5N1 highly pathogenic avian influenza viruses in domestic ducks.

Domestic ducks have been implicated in the dissemination and evolution of H5N1 highly pathogenic avian influenza (HPAI) viruses. In this study, two H5N1 HPAI viruses belonging to clade 2.2.1 isolated in Egypt in 2007 and 2008 were analyzed for their pathogenicity in domestic Pekin ducks. Both viruses produced clinical signs and mortality, but the 2008 virus was more virulent, inducing early onset of neurological signs and killing all ducks with a mean death time (MDT) of 4.1 days. The 2007 virus killed 3/8 ducks with a MDT of 7 days. Full-genome sequencing and phylogenetic analysis were used to examine differences in the virus genes that might explain the differences observed in pathogenicity. The genomes differed in 49 amino acids, with most of the differences found in the hemagglutinin protein. This increase in pathogenicity in ducks observed with certain H5N1 HPAI viruses has implications for the control of the disease, since vaccinated ducks infected with highly virulent strains shed viruses for longer periods of time, perpetuating the virus in the environment and increasing the possibility of transmission to susceptible birds.