A detailed analysis of 16S rRNA gene sequencing and conventional PCR-based testing for the diagnosis of bacterial pathogens and discovery of novel bacteria.

This study represents the first analysis of the bacterial community in chickens affected by swollen head syndrome, utilizing 16S rRNA gene sequencing. Samples were obtained from clinical laying chickens and were examined for the presence of Avibacterium paragallinarum (APG) and Ornithobacterium rhinotracheale (ORT) using conventional polymerase chain reaction (PCR). From the samples, five APG-positive (APG) and APG-negative (N-APG) samples were chosen, along with five specific pathogen-free chickens, for 16S rRNA gene sequencing. Results showed that APG and ORT were widely detected in the chicken samples with swollen head syndrome (SHS, 9/10), while APG was detected in all five specific pathogen-free (SPF) samples. In contrast, conventional PCR sensitivity was found to be inadequate for diagnosis, with only 35.7% (5/14) and 11.1% (1/9) sensitivity for APG and ORT, respectively, based on 16S rRNA gene sequencing data. Furthermore, 16S rRNA gene sequencing was able to quantify the bacteria in the samples, revealing that the relative abundance of APG in the APG group ranged from 2.7 to 81.3%, while the relative abundance of APG in the N-APG group ranged from 0.1 to 21.0%. Notably, a low level of APG was also detected in all 5 SPF samples. The study also identified a significant number of animal and human common bacterial pathogens, including but not limited to Gallibacterium anatis, Riemerella columbina, Enterococcus cecorum, Mycoplasma synoviae, Helicobacter hepaticus, and Staphylococcus lentus. In conclusion, 16S rRNA gene sequencing is a valuable tool for bacterial pathogen diagnosis and the discovery of novel bacterial pathogens, while conventional PCR is not reliable for diagnosis.

Pmorf0222, a Virulence Factor in Pasteurella multocida, Activates Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase via Toll-Like Receptor 1/2.

Pasteurella multocida primarily causes hemorrhagic septicemia and pneumonia in poultry and livestock. Identification of the relevant virulence factors is therefore essential for understanding its pathogenicity. Pmorf0222, encoding the PM0222 protein, is located on a specific prophage island of the pathogenic strain C48-1 of P. multocida. Its role in the pathogenesis of P. multocida infection is still unknown. The proinflammatory cytokine plays an important role in P. multocida infection; therefore, murine peritoneal exudate macrophages were treated with the purified recombinant PM0222, which induced the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) via the Toll-like receptor 1/2 (TLR1/2)-nuclear factor kappa B (NF-κB)/mitogen-activated protein kinase (MAPK) signaling and inflammasome activation. Additionally, the mutant strain and complemented strain were evaluated in the mouse model with P. multocida infection, and PM0222 was identified as a virulence factor, which was secreted by outer membrane vesicles of P. multocida. Further results revealed that Pmorf0222 affected the synthesis of the capsule, adhesion, serum sensitivity, and biofilm formation. Thus, we identified Pmorf0222 as a novel virulence factor in the C48-1 strain of P. multocida, explaining the high pathogenicity of this pathogenic strain.