Potential use an Actinobacillus pleuropneumoniae double mutant strain ΔapxIICΔapxIVA as live vaccine that allows serological differentiation between vaccinated and infected animals

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a highly contagious and often fatal disease. We have previously reported the construction and characterization of a single gene apxIIC deletion mutant HB04C− based on A. pleuropneumoniae serovar 7 which produces ApxII toxin and ApxIV. A precisely defined ΔapxIICΔapxIVA double-deletion mutant of A. pleuropneumoniae was constructed based on HB04C− by transconjugation and counterselection, and the levels of virulence of the ΔapxIIC single mutant and ΔapxIICΔapxIVA double mutant were compared in an experimental infection in mice and pigs. The results demonstrated that the ΔapxIICΔapxIVA double mutant strain was less virulent than HB04C−. Despite attenuation of virulence, the ΔapxIICΔapxIVA double mutant remains immunogenic and conferred a similar level of protective immunity to pigs against challenge with a lethal dose of a heterologous fully virulent standard serovar 1 strain of A. pleuropneumoniae. The results of the virulence study suggest that ApxIV is a critical virulence factor of A. pleuropneumoniae serovar 7 and is able to induce clinical disease, but it not required for efficient vaccination of pigs against A. pleuropneumoniae infection. Two weeks after the booster immunization, animals vaccinated with HB04C− were positive in the ApxIVAM-ELISA based on a recombinant GST-fusion protein GST-ApxIVAM as the solid-phase antigen while animals vaccinated with the ΔapxIICΔapxIVA double mutant were negative. These data demonstrate that the double mutant ΔapxIICΔapxIVA can be used as an effective live marker vaccine allowing serological differentiation between vaccinated and infected animals.