Doctoral Dissertations

Date of Award

5-1995

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

David A. Bemis

Committee Members

Gary Stacey, Beth Mullin, Robert Moore

Abstract

Bordetella bronchiseptica is a Gram negative respiratory tract pathogen which infects a wide variety of animal species including dogs, pigs, and guinea pigs. Infection and colonization of ciliated respiratory epithelial cells by B. bronchiseptica has been associated with atrophic rhinitis in pigs and acute tracheobronchitis (kennel cough) in dogs. The first step in colonization is attachment of bacteria to the host cells. B. bronchiseptica, like the human pathogen, Bordetella pertussis, produces several proteins which could act as adhesins, including filamentous hemagglutinin (FHA), pertactin, and fimbriae. It is believed that attachment by B. pertussis is mediated primarily through the action of FHA in concert with pertactin and pertussis toxin. B. bronchiseptica, however, in order to infect a wider range of animal species, may utilize different adherence mechanisms than B. pertussis. Despite experiments suggesting that fimbriae are not necessary for attachment by B. pertussis, there has been data suggesting that fimbriae may play a role in adherence by B. bronchiseptica. Therefore, experiments were undertaken to further explore the role of fimbriae in attachment by B. bronchiseptica. Two approaches were used to examine the involvement of fimbriae in attachment. The first approach involved determination of relative amounts of fimbrial proteins expressed on the surface of B. bronchiseptica cells and comparison of these results with attachment ability. A monoclonal antibody specific for non-denatured serotype 2 fimbrial filaments was developed and characterized in this laboratory. This antibody was used in a pre-adsorption ELISA to measure serotype 2 fimbrial expression levels on B. bronchiseptica cells. Thirty-six strains of B. bronchiseptica from pigs, dogs, guinea pigs, and four other species were tested using this assay. Strains originally isolated from pigs exhibited significantly more reactivity with monoclonal antibody CPS than those from guinea pigs. Strains originally isolated from dogs, however, were highly variable in reactivity suggesting that dogs are capable of being infected with a wider variety of B. bronchiseptica strains than pigs or guinea pigs. Levels of reactivity in this assay were compared with attachment ability in an in vitro attachment assay using Vero cells. No correlation was seen between attachment ability and reactivity with monoclonal antibody CF8. However, a correlation was observed between attachment ability and expression of a 24 kD fimbrial subunit protein which reacts in Western blots with antibody specific for serotype 2 fimbriae, the same fimbrial serotype as recognized by CF8. The second approach to exploring the role of fimbriae in attachment involved cloning a gene affecting expression of fimbriae and examining attachment ability of strains carrying this cloned gene. A genomic library was constructed from B. bronchiseptica strain 110H and screened with oligonucleotide probes derived from each end of the B. pertussis fim2 gene. A cosmid clone reacting with both of these probes and designated pGB710 was introduced into B. bronchiseptica strains 17640 and R-5. Expression of serotype 2 fimbriae, as measured by ELISA with monoclonal antibody CF8 and examination of fimbrial protein profile on SDS-PAGE, increased in each of these strains after introduction of pGB710. Attachment ability of strain R-5 also doubled when cosmid pGB710 was present. This increase in attachment ability could be inhibited by pre-incubation of the bacteria with monoclonal antibody against serotype 2 fimbriae but not with antibodies against FHA or pertactin. In addition, recombinational inactivation of the gene reactive with the serotype 2 fimbrial oligonucleotide probes prevented the increase in attachment in strain R-5. These data suggest that serotype 2 fimbriae are involved in attachment by B. bronchiseptica.

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