The importance of secreted virulence factors in Actinobacillus pleuropneumoniae bacterin preparation: a comparison.

Current bacterins provide only partial protection against morbidity and mortality in swine following infection by Actinobacillus pleuropneumoniae. We compared the efficacy of a cell-free concentrate from mid-log phase growth cultures of Actinobacillus pleuropneumoniae (APP) serotype 1 to four commercial bacterins. This cell-free preparation contained carbohydrate, endotoxin, and protein, and had hemolytic and cytotoxic activity. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis indicated the presence of one major 110,000-molecular-weight protein. This protein band also stained by the periodic acid Schiff method, indicating the presence of carbohydrate. Cell-free concentrates of APP serotypes 5 and 7 had identical profiles following electrophoresis and staining with either Coomassie blue for protein or Schiff reagent for carbohydrate. Lipopolysaccharide profiles for the cell-free concentrates of serotypes 1 and 5 were semi-rough while the LPS profile for serotype 7 was smooth. Five A. pleuropneumoniae-free SPF pigs per group were vaccinated on days 0 and 21 with cell-free concentrate of serotype 1 plus adjuvant, or one of four commercial bacterins according to the manufacturer's directions. Control pigs were vaccinated with PBS mixed with adjuvant. All pigs were challenged intranasally on day 35 with serotype 1 and necropsied on day 50. Protection was greatest in the cell-free concentrate group, as compared with all other groups, in that no deaths occurred, clinical scores were less severe, and percent lung affected was significantly reduced (P < 0.05). In addition, whole-cell ELISA titers were significantly increased (P < 0.05) postvaccination in the cell-free concentrate group, and postvaccination and postchallenge sera neutralized the hemolytic activity of the cell-free concentrate from serotypes 1 and 5 (P < 0.05), as compared with all other groups. No serum neutralization to the hemolysin of serotype 7 was observed. Immunoblot analysis using antisera derived from gnotobiotic pigs indicated that the cell-free vaccine generated a response that was identical to the response observed following live challenge. Similar, but not identical, responses were observed when antisera generated against the bacterins was used. This study indicates that an acellular vaccine containing multiple virulence factors can provide complete protection from mortality and significantly reduced morbidity to homologous challenge.