Duplex PCR methods for the molecular detection of Escherichia fergusonii isolates from broiler chickens.

Escherichia fergusonii is an emerging pathogen that has been isolated from a wide range of infections in animals and humans. Primers targeting specific genes, including yliE (encoding a conserved hypothetical protein of the cellulose synthase and regulator of cellulose synthase island), EFER_1569 (encoding a hypothetical protein, putative transcriptional activator for multiple antibiotic resistance), and EFER_3126 (encoding a putative triphosphoribosyl-dephospho-coenzyme A [CoA]), were designed for the detection of E. fergusonii by conventional and real-time PCR methods. Primers were screened by in silico PCR against 489 bacterial genomic sequences and by both PCR methods on 55 reference and field strains. Both methods were specific and sensitive for E. fergusonii, showing amplification only for this bacterium. Conventional PCR required a minimum bacterial concentration of approximately 10(2) CFU/ml, while real-time PCR required a minimum of 0.3 pg of DNA for consistent detection. Standard curves showed an efficiency of 98.5%, with an R(2) value of 0.99 for the real-time PCR assay. Cecal and cloacal contents from 580 chickens were sampled from broiler farms located in the Fraser Valley (British Columbia, Canada). Presumptive E. fergusonii isolates were recovered by enrichment and plating on differential and selective media. Of 301 total presumptive isolates, 140 (46.5%) were identified as E. fergusonii by biochemical profiling with the API 20E system and 268 (89.0%) using PCR methods. E. fergusonii detection directly from cecal and cloacal samples without preenrichment was achieved with both PCR methods. Hence, the PCR methods developed in this work significantly improve the detection of E. fergusonii.

Optimization of furin inhibitors to protect against the activation of influenza hemagglutinin H5 and Shiga toxin.

Proprotein convertases (PCs) are crucial in the processing and entry of viral or bacterial protein precursors and confer increased infectivity of pathogens bearing a PC activation site, which results in increased symptom severity and lethality. Previously, we developed a nanomolar peptide inhibitor of PCs to prevent PC activation of infectious agents. Herein, we describe a peptidomimetic approach that increases the stability of this inhibitor for use in vivo to prevent systemic infections and cellular damage, such as that caused by influenza H5N1 and Shiga toxin. The addition of azaß(3)-amino acids to both termini of the peptide successfully prevented influenza hemagglutinin 5 fusogenicity and Shiga toxin Vero toxicity in cell-based assays. The results from a cell-based model using stable shRNA-induced proprotein convertase knockdown indicate that only furin is the major proprotein convertase required for HA5 cleavage.