Actinobacillus pleuropneumoniae is the etiological agent of
contagious swine pleuropneumonia, an economically important
disease of the swine industry worldwide. Improved control of
this disease requires enhanced understanding of the factors
contributing to pathogenesis. The objectives of this study
were to investigate the immune response and virulence
properties of the 110-kilodalton (110-KDa) hemolysins
[hemolysin I (HlyI) and hemolysin II (HlyII)] of A. pleuropneumoniae. Several monoclonal antibodies (MAb) to the
hemolysins were developed. An IgGl. MAb (8C2) specific for
HlyII, as determined by immunoblotting, was cross-linked to
Protein A-Sepharose, and HlyII was purified from serotypes 1
and 5 by immunoaffinity chromatography. An indirect enzyme-linked
immunosorbent assay (ELISA) using MAb 8C2, or affinitypurified
rabbit IgG to both hemolysins, was developed for
detection of swine antibody to one or both hemolysins,
respectively. In comparison with the complement fixation test,
the ELISA was highly sensitive and specific, and was able to
identify animals infected with or exposed to most, if not all,
serotypes of A. pleuropneumoniae. Several nonhemolytic mutants
of A. pleuropneumoniae serotype 5 were isolated following
electroporation of the parent with an hemolysin gene whose
open-reading-frame was disrupted with a kanamycin resistance
gene. One mutant was characterized for phenotypic and
pathogenic properties. Biochemical profiles, growth rate,
capsule content, and lipopolysaccharide and whole cell protein electrophoretic profiles of the parent and one of the mutants
were similar. The nonhemolytic mutant lacked both HlyI and
HlyII proteins in culture supernatant and in whole cell
lysates as determined by immunoblot analysis; extracellular
and intracellular hemolytic and cytotoxic activity was also
absent. The mutant was avirulent in mice and pigs at doses
greater than 10 times the lethal dose of the parent. Unlike
the parent, the nonhemolytic mutant failed to confer
protection against lethal challenge in mice following
immunization. Thus, one or both hemolysins are essential for
virulence and immunoprotection in A. pleuropneumoniae serotype 5.
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