ABSTRACT Avibacterium paragallinarum , the causative agent of infectious coryza (IC), is an important respiratory pathogen of chickens with growing prevalence in commercial and backyard flocks. Current strain-typing methods, including classical serotyping and molecular approaches, such as ERIC-PCR or single-locus HPG2 typing, lack sufficient discriminatory power to investigate the epidemiology or population structure. To address this limitation, we developed a genome-guided multilocus sequence typing (MLST) scheme as a robust and portable tool for A. paragallinarum strain differentiation. Housekeeping genes were identified from 42 whole-genome sequences (WGS); 18 candidates were evaluated; and six were selected for the final MLST scheme. We used the scheme to differentiate 75 A. paragallinarum samples and compared its performance against classical HPG2-based typing, ad hoc core genome MLST (cgMLST), and the MLST scheme published by M. Guo, Y. Jin, H. Wang, X. Zhang, and Y. Wu (Vet Sci 11:208, 2024, https://doi.org/10.3390/vetsci11050208 ). The new MLST showed higher discriminatory power than HPG2 and outperformed Guo’s scheme with higher discriminatory power, particularly for characterizing the samples originating from North and South America. It also showed strong concordance with cgMLST clustering while being more practical for routine use. Overall, the six-locus MLST identified 31 sequence types across 75 samples, revealing epidemiologically meaningful clustering at regional and national scales and capturing temporal persistence of lineages. All allele definitions and sequence types have been deposited in PubMLST, ensuring standardized nomenclature and global accessibility. This scheme represents a reproducible, cost-effective, and globally applicable tool that enhances outbreak investigation, surveillance, and population studies of A. paragallinarum , bridging the gap between low-resolution traditional methods and resource-intensive whole-genome sequencing. IMPORTANCE Infectious coryza (IC) caused by Avibacterium paragallinarum is a major respiratory disease of poultry that causes acute infection, reducing egg production and growth and resulting in significant economic losses in poultry production worldwide. Controlling IC depends on understanding how different strains spread and persist, yet current methods to differentiate strains are either unreliable or too costly for routine use. In this study, we developed a standardized multilocus sequence typing system that provides a simple, accurate, and globally accessible way to identify and compare strains of A. paragallinarum . This scheme identified important links between outbreaks at local and regional levels and showed that certain strains persisted over time. By making the scheme available through PubMLST, laboratories worldwide can use a common tool to track and investigate the pathogen. This accessible tool improves disease surveillance, supports outbreak investigations, and helps poultry producers and veterinarians respond more effectively to IC.
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