Development and Application of Optimized Isolation Methods and Diagnostic PCR Protocols for the Detection of Pathogenic Isolated from Broiler Chickens.

(EC) is a dominant enteric commensal in broiler chickens. However, pathogenic strains of EC cause increased morbidity and mortality from septicemic disease in broiler production worldwide. EC infections can present as pericarditis and paralytic spinal lesions from which pathogenic EC can be isolated. However, the inability to distinguish between commensal and pathogenic EC strains has confounded the search for the source of pathogenic EC in environmental or hatchery samples. This issue is exacerbated by poor sensitivity of standard sampling and culture methods. Comparative genomic analysis of EC isolates previously identified a conserved capsule region in pathogenic EC strains that is absent or variable in commensal strains. Based on a capsular synthesis gene, O, and EC species-specific A primers, we designed a standard multiplex PCR to distinguish pathogenic EC from commensal EC strains. To allow for increased sample throughput, a real-time PCR protocol was developed in tandem based on detection of these genes. To increase the culture sensitivity, a selective enrichment protocol using Todd-Hewitt Broth with 1% yeast extract and four antibiotics enabled the isolation of pathogenic EC from egg transfer residue and culled eggs at hatcheries. Pulsed-field gel electrophoresis was used to genotype recovered hatchery isolates, which identified clonal pathogenic EC strains isolated from hatchery residue and a spinal lesion of a broiler. The ability to distinguish pathogenic EC from commensal EC coupled with modified culture methods will facilitate improved surveillance of pathogenic EC throughout broiler production, ideally leading to decreased incidence or eradication of this disease.