Cross-host transmission of <i>Riemerella anatipestifer</i> to chickens: Genomic evolution and identification of the novel <i>vapX</i> -like- <i>vapD</i> toxin-antitoxin system

Riemerella anatipestifer (R. anatipestifer), a well-known waterfowl pathogen, increasingly threatens Chinese poultry by spreading to chickens. The genetic differentiation and host adaptation following cross-host transmission remain unclear. Here, we characterized a highly virulent, multidrug-resistant chicken-source strain (SDAU-RA1) and performed comparative genomics with global R. anatipestifer strains to elucidate population structure and evolutionary dynamics. SNP phylogeny revealed significant geographic clustering and dominant clonal groups. Strains from different hosts showed a pattern of “overall mixing with local clustering,” and ancestral state reconstruction (ASR) identified multiple independent duck-to-chicken spillover events, confirming cross-host transmission rather than strict host-specific evolution. In terms of virulence, certain virulence genes are enriched specifically in chicken-source strains. Notably, the study is the first to identify and confirm vapX-like-vapD as a functional type II toxin-antitoxin system in chicken-source R. anatipestifer, demonstrating that it enhances biofilm formation, intracellular survival, and antibiotic persistence. Analysis of the geographical distribution and temporal dynamics of antibiotic resistance genes (ARGs) reveals high heterogeneity among R. anatipestifer strains from different hosts. Pangenome analysis revealed that R. anatipestifer possesses an open pangenome, conferring high genetic plasticity. In conclusion, our study shows R. anatipestifer transmits to chickens without strict host-specific adaptation, though incipient genetic differentiation has emerged. The discovery of plasmid pRASD and its carried vapX-like-vapD system suggests key mechanisms for the adaptive evolution and enhanced pathogenicity of R. anatipestifer. These findings enhance our understanding of cross-host transmission and underscore the importance of continuous surveillance of chicken-source R. anatipestifer and its novel mobile genetic elements.