Development of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae infection

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia which leads to high economic losses in the swine industry worldwide. Vaccination against this pathogen is hampered by the occurrence of 15 serotypes, and commonly used whole cell bacterin vaccines are not sufficiently cross-serotype protective. In addition, for generating and maintaining specified pathogen-free herds it is desirable to use DIVA (differentiating infected from vaccinated animals) vaccines. Based on a detergent wash extraction of outer membrane associated proteins and secreted proteins we developed a DIVA vaccine using the immunogenic ApxII toxin which is present in 13 of the 15 A. pleuropneumoniae serotypes as the DIVA antigen. The apxIIA gene was deleted in one strain each of serotypes 1, 2, and 5 using a single-step transconjugation system, and equal parts of detergent washes from these strains served as the vaccine antigen. After intramuscular immunisation all pigs developed a strong humoral immune response to the vaccine antigen and showed no reactivity in an ApxIIA ELISA. Upon challenge all pigs were completely protected from clinical symptoms in trials with a homologous (serotype 2) as well as with a heterologous strain (serotype 9); in addition, colonisation of the challenge strain was clearly reduced but not abolished completely. As a result of the highly efficient protection, however, immunised pigs did not develop antibodies to the DIVA-antigen at levels detectable by ELISA but only by a more sensitive Western blotting approach, thereby demonstrating the challenge in developing appropriate marker vaccines for the livestock industry.