Title page for ETD etd-10142005-135819


Type of Document Dissertation
Author Ma, Jianneng
URN etd-10142005-135819
Title Purification, serology and pathogenic role of the 110 kilodalton rtx hemolysins of Actinobacillus pleuropneumoniae
Degree PhD
Department Veterinary Medical Sciences
Advisory Committee
Advisor Name Title
Inzana, Thomas J. Committee Chair
Boyle, Stephen M. Committee Member
Dean, Dennis R. Committee Member
Schurig, Gerhardt G. Committee Member
Sriranganathan, Nammalwar Committee Member
Keywords
  • Pleuropneumonia Pathogenesis.
  • Swine Diseases.
  • Actinobacillus pleuropneumoniae.
  • Hemolysis and hemolysins.
Date of Defense 1991-12-15
Availability restricted
Abstract

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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