A novel strategy for protective Actinobacillus pleuropneumoniae subunit vaccines: detergent extraction of cultures induced by iron restriction.

We have characterized antigens from Actinobacillus (A.) pleuropneumoniae grown under iron restriction with respect to their immunogenic and protective potential. Antigens were the cell-free culture supernatants (CFS) obtained after treatment of A. pleuropneumoniae broth cultures with sodium deoxycholate. Using the iron-repressible transferrin-binding lipoprotein TbpB and the constitutively expressed outer membrane lipoprotein OmlA as markers, we have shown that the detergent extraction enriched the CFS with lipoproteins from the outer membrane (OM). Extractions with 0.05% of sodium deoxycholate increased the lipoprotein contents in the CFS, but did not affect the integrity of the OM. This was demonstrated by the absence of the iron-repressible integral OM transferrin-binding protein TbpA. Furthermore, the absence of periplasmic and cytoplasmic proteins in CFS after extraction was determined in immunoblot analyses with anti-bacterial alkaline phosphatase and anti-Hsp60 antisera, demonstrating that there was no rupture of the OMs or the plasma membranes due to the extraction procedure. Antigen preparations from A. pleuropneumoniae serotype 2 and 9 grown under iron restrictive conditions were combined, emulsified, and tested for their ability to confer protection in pigs. Pigs immunized with CFS from sodium deoxycholate extracted cultures developed a strong antibody response and, upon challenge with A. pleuropneumoniae serotype 2, the immunized pigs showed no or only mild clinical signs of disease and had a significantly lower degree of lung damage than the control pigs.