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 gr own 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 deoxycho late. Using the iron-repressible transferrin-binding lipoprotein TbpB and t he 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 deoxycholat e increased the lipoprotein contents in the CFS, but did not affect the int egrity of the OM. This was demonstrated by the absence of the iron-repressi ble integral OM transferrin-binding protein TbpA. Furthermore, the absence of periplasmic and cytoplasmic proteins in CFS after extraction was determi ned in immunoblot analyses with anti-bacterial alkaline phosphatase and ant i-Hsp60 antisera, demonstrating that there was no rupture of the OMs or the plasma membranes due to the extraction procedure. Antigen preparations fro m A. pleuropneumoniae serotype 2 and 9 grown under iron restrictive conditi ons were combined, emulsified, and tested for their ability to confer prote ction in pigs. Pigs immunized with CFS from sodium deoxycholate extracted c ultures developed a strong antibody response and, upon challenge with A. pl europneumoniae serotype 2, the immunized pigs showed no or only mild clinic al signs of disease and had a significantly lower degree of lung damage tha n the control pigs. (C) 2000 Elsevier Science Ltd. All rights reserved.