Pathogen identification and analysis of broiler bronchial obstruction syndrome using high-throughput sequencing technology

To identify the genetic characteristics of dominant pathogens in broilers suffered from bronchial obstruction syndrome in Hebei Province, high-throughput sequencing technology was employed for pathogen detection after pathological examinations. Pathological examination revealed typical lesions consistent with bronchial obstruction syndrome. Molecular biological analyses revealed that the predominant pathogens and opportunistic pathogens in the broiler samples included H9N2 avian influenza virus (AIV), infectious bronchitis virus (IBV), Escherichia-Shigella, Pseudomonas and Enterococcus. All four H9N2 AIV isolates exhibited higher nucleotide homology in PB1, PA, NP and NS gene with A/Chicken/Shanghai/F/1998 and M gene with A/Quail/Hong Kong/G1/1997. These belonged to the Eurasian lineage, forming a quadruplex recombinant, but differed from the G57 genotype. Several H3N3, H3N8 and H10N3 AIV strains might provide internal genes contributing to the recombination of the H9N2 isolates, conferring pathogenicity enhance, antiviral drug resistance and adaptability to mammals. Complete genome analysis of three IBV isolates showed the highest nucleotide homology with YX10 strain (GI-19 lineage, QX type). Specifically, the S1, S2, 3a, 3b, E, M, 5a and N gene of the three IBV isolates exhibited the highest nucleotide identity to the YX10 strain, whereas 1a and 1b gene showed the highest nucleotide identity with I0114/14 strain (GI-29 lineage), and 5b gene displayed the highest nucleotide identity with LDT3-A strain (GI-28 lineage). Through MEGA software translation, the cleavage sites of the S protein of the three IBV isolates were determined as HRRRR, which is consistent with most QX type IBV (GI-19 lineage) cleavage sites. Phylogenetic analysis based on the S1 gene further demonstrated that all three IBV isolates were belonged to GI-19 lineage. CK/CH/HBBD/BD6 and CK/CH/HBBD/BD32 represented newly emerged recombinant variants. This study clarified the predominant pathogens causing broiler bronchial obstruction syndrome in some regions of Hebei Province and detailed the genetic profiles of H9N2 AIV and IBV. These findings could inform strategies for the prevention and management of broiler multicausal respiratory disease.