Masters Theses

Date of Award

8-1986

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

David A Bemis

Committee Members

Raymond Beck, John Kennedy, Robert Moore

Abstract

Bordetella bronchiseptica strain 110H rapidly induced ciliostasis in canine tracheal outgrowth cultures. The induction of ciliostasis was both time and dose dependent. Eighteen strains of B. bronchiseptica were examined for their ability to induce ciliostasis. Fifteen strains grown on Brucella agar induced ciliostasis. The three strains that did not induce ciliostasis were morphologically stable, non-piliated, non-hemolytic, did not produce extracellular adenylate cyclase, and were not distinguishable from rough phase B. bronchiseptica on Brucella agar. All strains which were hemolytic on Bordet-Gengou agar produced extracellular adenylate cyclase and induced ciliostasis. Two non-hemolytic strains, one with rough phase colonial morphology on Brucella agar, induced ciliostasis. The only phenotypic characteristic studied which was positively associated with the ability to induce ciliostasis was the presence of pili. Formalin-killed and Chloramphenicol-inhibited B. bronchiseptica strain 110H organisms had detectable pili and attached to cilia, but did not induce ciliostasis. Protease-treated B. bronchiseptica strain 110H organisms did not have detectable pili and in the presence of Chloramphenicol did not induce ciliostasis, nor did they attach to cilia. Concentrated (100X) supernatants from 48 hour cultures of bronchiseptica strain 110H organisms grown in Waymouth's MB 752/1 medium did not induce ciliostasis. B. bronchiseptica strain 110H organisms which were separated from canine tracheal outgrowth cultures with a parabiotic chamber were not able to induce ciliostasis. Filter sterilized supernatant fluids from ciliostatic JB. bronchiseptica infected canine tracheal outgrowth cultures were not able to induce ciliostasis in non-infected tracheal outgrowth cultures. Filter sterilized supernatant fluids from sonicated B. bronchiseptica strain 110H organisms were not able to induce ciliostasis. Hyperimmune rabbit antisera produced against whole cell B. bronchiseptica strain 110H bacterins completely inhibited, B. bronchiseptica induced ciliostasis in canine tracheal outgrowth cultures. Antisera against B. bronchiseptica did not inhibit its viability, nor cause the release of adherent organisms from cilia. Heat treatment of the antisera (56°C, 30 min) did not destroy the "protective" activity. Removal of antisera from the infected tracheal outgrowth culture resulted in complete ciliostasis (≥98%) in four hours. Infected outgrowths which had been completely ciliostatic for as long as 2 hours regained approximately 65% of their normal activity within six hours after the addition of hyperimmune sera. The ability to protect cilia from B. bronchiseptica induced ciliostasis paralleled the B. bronchiseptica agglutinin titers of the sera. Protein A-Sepharose purified IgG fractions from hyperimmune rabbit sera also protected the cilia. Absorption of sera with Staphylococcus aureus strain Cowan I, Escherichia coli strain FDA Seattle 1946, Pseudomonas aeruginosa strain PAO-1, B. bronchiseptica strain 110NH and B. bronchiseptica strain 110H removed the protective activity of serum. Boiled (100°C, 60 min) E. coli, P. aeruginosa, and B. bronchiseptica strains 100H and 100NH also absorbed out the protective activity of sera. Staphylococcus aureus strain Wood 46 and Staphylococcus epidermidis strain FDA PCI 1200 did not remove the protective activity of the hyperimmune sera. The detection of antibodies on the surfaces of boiled absorbents were determined by either ELISA, PAGE-autoradiography (with l125 labeled IgG fractions from rabbit anti-Bordetella hyperimmune serum) or imraunofluorescence tests. Immunofluorescence was used to test all absorbents; ELISA and PAGE-autoradiography was used to test only boiled E. coli and boiled B. bronchiseptica. B. bronchiseptica antigens were positive in all tests. S. aureus strain Cowan I was positive with goat anti-rabbit IgG immunofluorescence, S. aureus strain Wood 46 and S. epidermidis were negative. Antibody to boiled E. coli antigen was detected by ELISA and PAGE-autoradiography but negative for immunofluorescence assays. These data suggest that attachment may be a requirement for the induction of ciliostasis, that ciliostasis can be reversed, and that antibodies may have a role in protecting cilia against induction of ciliostasis by B. bronchiseptica.

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