Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Consistent immunoreactivities of the 31-, 40- and 78-kDa proteins of Haemophilus somnus were observed in immunoblots with bovine antisera to H. somnus. A genomic library of H. somnus 8025 DNA was constructed in plasmid pUC19 and 45 recombinants expressed immunoreactive proteins in Western blots using bovine antiserum. Ten of the recombinants expressing a 31-kDa protein caused lysis of bovine erythrocytes. Restriction endonuclease mapping indicated that the hemolytic recombinants shared an approximately 1.7-kb BglII fragment. Southern blot analysis using the BglII fragment as a probe revealed homology among the recombinants and the presence of an identically sized BglII fragment in the chromosome of all H. somnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion of this open reading frame resulted in the loss of hemolytic activity and protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium and Shigella dysenteriae, with P6 of Haemophilus influenzae and with PIII of Neisseria gonorrhoeae;To determine the protective ability of the recombinant expressing the 31-kDa protein of H. somnus, mice were vaccinated 2-3 times with the heat-killed recombinant, followed by a challenge with H. somnus. Vaccination with the recombinant expressing the 31-kDa antigen of H. somnus provided 5-fold greater protection compared to E. coli with the vector only. Potential diagnostic use of monoclonal and polyclonal antibodies raised against the recombinant was evaluated. Monoclonal mouse antibodies against a crude membrane protein preparation or formalin-killed recombinant were evaluated on ELISA and Western blot against H. somnus antigens. Polyclonal Guinea pig sera against different preparations of the recombinant showed different immunoreactivities on various immunological tests. Polyclonal Guinea pig sera against the heat-killed recombinant and formalin-killed H. somnus strongly detected H. somnus in immunohistochemical staining of formalin-fixed tissue sections of bovine lungs.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Won, Jonghwa, "Characterization of the 31-kDa antigen gene of Haemophilus somnus and its product " (1993). Retrospective Theses and Dissertations. 10569.