Doctoral Dissertations

Date of Award

5-2002

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

David A Bemis

Committee Members

Robert N Moore, Stephen A Kania, Albert T Ichiki

Abstract

Bordetella bronchiseptica colonizes the upper respiratory tract of a wide variety of mammals and is involved in diseases such as kennel cough in dogs, atrophic rhinitis in pigs and upper respiratory tract infections of laboratory animals. Studies were focused on developing a heterologous antigen expression system in B. bronchiseptica and to evaluate the potential of this organism as a candidate mucosal vaccine vector. Since the role of Bordetella bronchiseptica and Pasteurella multocida toxin (PMT) in the disease atrophic rhinitis of pigs was well documented, this research was focused in the possibility of developing a refined vaccine to control this disease. A promoter region related to heat shock protein genes was identified using a green fluorescent protein reporter system. This promoter drove high- level expression of GFP compared to a tac promoter or B. bronchiseptica fim N gene promoter. A nontoxic protective P. multocida toxin fragment and GFP were expressed in B.bronchiseptica in a broad-host-range plasmid vector PBBR1MCS2 under the control of the promoter region identified. Colonization kinetics, plasmid stability, and immune responses generated following intranasal inoculation of recombinant B. bronchiseptica were evaluated. While wild type and recombinant B. bronchiseptica colonized the mouse respiratory tract effectively, the plasmid was completely lost from the organism after 72 hours post- inoculation. After a single intranasal inoculation, B. bronchiseptica specific IgM, IgA and IgG responses were detected in serum and respiratory lavage. However, PMT-specific antibodies were not detected. Four intranasal inoculations with B. bronchiseptica expressing green fluorescent protein (GFP) induced a GFP-specific systemic and mucosal immune response, while similar inoculations with B. bronchiseptica expressing PMT fragment did not induce a PMT-specific immune response.

This study also evaluated the immune response to a chimeric protein generated by combining a gene fragment encoding neutralizing epitopes of Mannheimia haemolytica leukotoxin and a fimbrial protein gene (fim N) from B. bronchiseptica. Immunization of mice with the recombinant chimeric protein elicited a significantly stronger anti-leukotoxin antibody response than comparable immunizations with fusion proteins lacking FIM N. The chimeric protein exhibited more stability. Leukotoxin is an important virulence factor in shipping fever pneumonia in feedlot cattle and is a critical protective antigen. This chimeric protein may be a candidate for inclusion in new generation vaccines against shipping fever pneumonia. The results of these studies strongly support the potential for developing B. bronchiseptica as a candidate mucosal vaccine vector and FIM N as a carrier protein for heterologous antigens.

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