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

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.