Immunization with Temperature-Sensitive Mutants of Actinobacillus pleuropneumoniae Induces Protective Hemolysin-Neutralizing Antibodies in Mice

The immunogenicity and protective potential of three temperature-sensitive mutants of Actinobacillus pleuropneumoniae were evaluated in mice with respect to antibodies against the capsular polysaccharide, lipopolysaccharide, outer membrane proteins, and hemolysin protein. Antibodies to the capsular polysaccharide and lipopolysaccharide could not be correlated with protection in the mice; there were no significant differences among the anti-capsular and anti-lipopolysaccharide antibody titers regardless of the severity of infection. Sera from mice immunized with the mutants and challenged with the wild type contained antibodies that reacted in immunoblots to four major outer membrane proteins (66, 39, 29, and 16 kDa) regardless of the severity of infection after challenge. Both the tight and coaster mutants synthesized and secreted the 105-kDa hemolysin protein exotoxin in vitro and in vivo; hemolysin protein neutralization titers and the blotting intensity of the sera, however, varied inversely with the severity of infection. Sera from mice surviving challenge with little to no lung involvement stained the hemolysin band more intensely and had significantly higher neutralization titers (P < 0.05) than sera from mice that either died or survived with severe pulmonary hemorrhage. These results confirm the importance of the hemolysin in pathogenesis and the need for including it in any vaccine preparation. Received: 26 August 1996 / Accepted: 3 September 1996