Genomic evidence for host expansion and respiratory dysbiosis associated with Riemerella anatipestifer infection in laying hens

Abstract Background Riemerella anatipestifer ( R. anatipestifer ) is a Gram-negative pathogen that primarily infects waterfowl and causes substantial losses in poultry production. Increasing reports of R. anatipestifer infections in chickens suggest ongoing host expansion, but the genomic features associated with adaptation to chickens remain unclear. In this study, we isolated R. anatipestifer from the infraorbital sinus of diseased laying hens, indicating involvement of the upper respiratory tract during infection. However, the relationship between R. anatipestifer infection and respiratory microbiome disruption in chickens remains poorly understood. Here, we integrated comparative genomics of 273 genomes with metagenomic profiling of the infraorbital sinus and trachea to investigate putative cross-host dissemination, host-associated genomic variation, and respiratory dysbiosis. Results Core-genome phylogeny, single-nucleotide polymorphism distances, and average nucleotide identity identified closely related duck- and chicken-derived lineages, providing genomic evidence supporting cross-regional and cross-host dissemination. Pan-genome and HGT analyses revealed a three-state stable–divergent–stable pattern in accessory-gene structure, with the divergent state showing increased total gene counts, inferred HGT events, and HGT/gene proportions. Chicken-derived isolates showed an inferred HGT proportion of 7.92% and greater numbers of predicted integrative and conjugative elements and insertion sequences. A chicken-associated genomic region showed signatures of acquisition from Flavobacteriaceae and Weeksellaceae. Functional profiling further revealed enrichment of genes related to regulation, genome maintenance, cell-envelope biogenesis, metabolism, and carbohydrate-active enzymes in chicken-derived genomes. Chicken-derived genomes also contained more pseudogenes and higher pseudogene dN/dS ratios, consistent with host-associated shifts in selective regimes. In naturally infected laying hens, R. anatipestifer was detected in the infraorbital sinus but not the trachea and was associated with multi-organ pathology, impaired immunoglobulin responses, elevated inflammatory cytokines, reduced infraorbital-sinus microbial diversity, enrichment of potential respiratory co-pathogens, and increased antibiotic resistance gene burden. Conclusions These findings provide genomic evidence supporting R. anatipestifer host expansion into chickens and link natural infection with respiratory dysbiosis and increased resistome burden. Integrated genomic, epidemiological, and microbiome-based surveillance is warranted to monitor R. anatipestifer emergence and support targeted disease-control strategies in poultry production systems.