| Studies were focused on developing a heterologous antigen expression system in Bordetella bronchiseptica and evaluating the potential of this organism as a candidate mucosal vaccine vector. A promoter region related to heat shock protein genes was identified using a green fluorescent protein (GFP) reporter system. This promoter resulted in greater expression of GFP in B. bronchiseptica than Escherichia coli, tac or B. bronchiseptica fim N gene promoters. A non-toxic protective Pasteurella multocida toxin (PMT) fragment and GFP were expressed in B. bronchiseptica using gene fusion with this promoter on a broad-host-range plasmid vector, PBBR1MCS2. Colonization kinetics, plasmid stability, and immune responses generated following intranasal inoculation of recombinant B. bronchiseptica were evaluated in mice. Recombinant plasmids were unstable in B. bronchiseptica strains. 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 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. 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. |
