Virulence attenuation of intestinal pathogenicity via combined gene deletion in duck enteritis vaccine strain restores gut microbiota balance and enhances safety

There is an increasing need for a new generation of effective and safe vaccines, in the context of large-scale poultry farming and the prevalence of infectious diseases. With this in mind, we developed, for the first time, a duck enteritis virus (DEV) mutant, ΔTK-ΔgI/gE-ΔgG/gJ, through the deletion of multiple virulence genes. The resulting gene-deletion strain exhibited replication kinetics similar to those of the parent strain and was found to be safe in various animal models, offering a strategy for rapidly generating attenuated DEV strains. Previously, our team reported that DEV infection leads to intestinal dysbiosis; however, the impact of DEV vaccines on the gut microbiota remains unclear. This study aimed to characterize the gut microbiota of ducks, chicks, and mice immunized with DEV strains using microbiome analysis, assess the effects on microbial composition, and compare the outcomes. Both two strains caused significant shifts in gut microbiota diversity. Both strains restored the diversity of the microbiota, whereas the parental vaccine caused the enrichment of potential pathogens in chicks. Moreover, the conventional DEV vaccine disrupted gut microbiota and morphology, but the gene-deleted strain largely reversed these changes. These findings may improve the safety of vaccine through gene editing, thereby enhancing the protection of target animals.