A safe and molecular-tagged Brucella canis ghosts confers protection against virulent challenge in mice

•The combination of homologous recombinant (HR) and bacteria ghost (BG) generated a genetically stable, non-drug resistant Brucella canis ghost strain as a vaccine candidate.•To produce BGs, the ghost strain can be 100% activated upon induction and no adverse effect was detected when administered to mice.•The BGs is absent of B0419, a lipopolysaccharide associated protein, which was proved to be a diagnostic molecular tag to distinguish natural infection.•BGs confers protection against B. canis and B. melitensis. Canine brucellosis, caused by Brucella canis, is a persistent infectious reproductive disease in dogs. The absence of effective treatment to the intracellular pathogen and the irreversible consequence of infection makes the need of a specific vaccine urgent. Bacterial ghosts (BGs) are the empty envelopes of bacteria with no genome content inside, which emerge as a proper vaccine candidate due to its intact outer antigen. It is generally derived from a genetically engineered strain, through the expression of Bacteriophage phiX174 lysis E gene upon induction. In this study, we combined the homologous recombination (HR) and bacterial ghost technologies, generating a genetically stable B. canis ghost strain which bears no drug resistance gene. When the ghost strain grows to OD600 of 0.6, 100% inactivation can be achieved under 42°C in 60h. The resultant BGs showed guaranteed safety and comparable immunogenicity to a live vaccine. The bacterial B0419 protein was depleted during HR process, which is subsequently proved to work as a molecular tag in distinguishing natural infection and BGs immunization through ELISA. Additionally, the BGs also conferred protection against B. canis RM6/66 and B. melitensis 16M. Therefore, the application of current BGs as a vaccine candidate and the corresponding serological diagnostic approach may provide better B. canis prevention strategy.