Comparative pan-genomics and in vivo validation identify genetic factors important for virulence of Salmonella enterica serovar Gallinarum and serovar Enteritidis in the avian host

Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum (SGa) and Pullorum (SPu) are avian-specific pathogens causing systemic disease, while S . Enteritidis (SEnt) is a broad host range serovar causing gastroenteritis. The genomic mechanisms underlying this difference in host range and pathogenicity remain incompletely understood. Here, we performed a large-scale pan-genome analysis of 5440 poultry-derived genomes (4927 SEnt, 106 SGa, 407 SPu) integrated with functional chicken and macrophage experiments. Compared with SEnt, avian-specific SGa and SPu exhibited extensive pseudogenization and shared 87 genes absent in SEnt, organized into four major genomic clusters (PG_1–PG_4) enriched in type VI secretion system genes and prophage-derived elements. Conserved SNPs distinguishing SGa/SPu from SEnt were enriched in carbohydrate and nitrogen metabolism pathways, suggesting potential metabolic divergences during infection. Infection experiments in chickens using deletion mutants revealed that deletions of genes in SPI-2 ( ssaE , ssaT ) and fimbrial genes ( stfA , safA ) were important for systemic infection of chicken with both SGa and SEnt, despite pseudogenization of fimbrial operons in SGa. Mutants in SPI-13 and SPI-14 were only significantly attenuated in SGa. The specific prophage region PG_3 was important for systemic infection in SGa, while a distinct prophage element (ENT_2) enhanced infection in SEnt. Together, these findings bridge comparative genomics with experimental validation, identifying genomic degradation, prophage acquisition, and serovar-specific pathogenicity islands as putative mechanisms underlying avian host specificity and systemic pathogenesis in Salmonella .