Purification, serology and pathogenic role of the 110 kilodalton rtx hemolysins of Actinobacillus pleuropneumoniae

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dc.contributor.authorMa, Jiannengen
dc.contributor.committeechairInzana, Thomas J.en
dc.contributor.committeememberBoyle, Stephen M.en
dc.contributor.committeememberSchurig, Gerhardt G.en
dc.contributor.committeememberSriranganathan, Nammalwaren
dc.contributor.committeememberDean, Dennis R.en
dc.contributor.departmentVeterinary Medical Sciencesen
dc.date.accessioned2014-03-14T21:21:25Zen
dc.date.adate2005-10-14en
dc.date.available2014-03-14T21:21:25Zen
dc.date.issued1991-12-15en
dc.date.rdate2005-10-14en
dc.date.sdate2005-10-14en
dc.description.abstract<i>Actinobacillus pleuropneumoniae</i> 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 <i>A. pleuropneumoniae</i>. 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 <i>A. pleuropneumoniae</i>. Several nonhemolytic mutants of <i>A. pleuropneumoniae</i> 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 <i>A. pleuropneumoniae</i> serotype 5.en
dc.description.degreePh. D.en
dc.format.extentxii, 125 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-10142005-135819en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-10142005-135819/en
dc.identifier.urihttp://hdl.handle.net/10919/39935en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V856_1991.M3.pdfen
dc.relation.isformatofOCLC# 25611842en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V856 1991.M3en
dc.subject.lcshActinobacillus pleuropneumoniaeen
dc.subject.lcshHemolysis and hemolysinsen
dc.subject.lcshPleuropneumonia -- Pathogenesisen
dc.subject.lcshSwine -- Diseasesen
dc.titlePurification, serology and pathogenic role of the 110 kilodalton rtx hemolysins of Actinobacillus pleuropneumoniaeen
dc.typeDissertationen
dc.type.dcmitypeTexten
thesis.degree.disciplineVeterinary Medical Sciencesen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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