Development of a counterselection system for efficient marker-free genetic manipulation in Avibacterium paragallinarum

Avibacterium paragallinarum is the etiological agent of infectious coryza, a significant respiratory ailment associated with growth inhibition and diminished egg production in poultry. Given the increasing incidence of antibiotic resistance in bacteria, there is an imperative need for the development of genetically engineered vaccines; however, the lack of effective tools for genetic manipulation in Av. paragallinarum has limited the scope of related research. Herein, we investigated the employment of the gene pheSm as a counterselectable marker for the screening of marker-free mutants of Av. paragallinarum. We report the successful and precise deletion of multiple genes in Av. paragallinarum and an instance of the effective seamless integration of the exogenous gene lpxE using this method. This study presents a valuable genetic tool for investigating the virulence genes and associated mechanisms of pathogenicity of this bacterium, as well as aiding the development of genetically engineered vaccines.