Infectious bovine keratoconjunctvitis : the identification of sahred antigenic determinants in pili from divergent strains of Moraxella bovis

Author

Wallace H. Greene

Date of Award

6-1986

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Leon N. D. Potgieter

Committee Members

David Bemis, Barry Rouse, Walter Farkas

Abstract

The physical, antigenic, and functional properties of pili from four strains of Moraxella bovis were investigated. Pili were purified by alternate cycles of differential centrifugation in low and high ionic strength buffers. The homogeneity of pilus preparations was determined by electron microscopy and polyacrylamide gel electrophoresis (PAGE) and the relative molecular weights (M_r) of the pilus protein (pilin) from the various strains were determined.

The purified pilin proteins were cleaved at specific sites with cyanogen bromide under acidic conditions. Two major peptide fragments were produced. The pilus fragments and whole uncleaved pili from the four strains were inoculated into rabbits. The antisera produced were used to assay homologous and heterologous reactions with the M. bovis strains by the indirect fluorescent antibody test (IFAT), western blotting of PAGE-separated proteins, interference with the adherence of M. bovis to fetal bovine corneal epithelial cells, and by immunoelectron microscopy.

Antisera to whole pili reacted strongly with homologous pili in western blots of whole and cleaved pilin. Weaker and inconsistent reactions were detected with heterologous pili. Antisera produced to cyanogen bromidecleaved pilin reacted with antigenic determinants on homologous and heterologous pili.

Piliated forms of all the M. bovis strains adhered to the bovine corneal epithelial cells. A non-piliated variant did not attach. The antisera were assayed for their ability to block adherence of homologous and heterologous strains of M. bovis to cell cultures of fetal bovine corneal epithelial cells. Only whole-pilus antisera prevented adherence of homologous organisms in this assay.

It was determined by immunoelectron microscopy that whole pilus antisera to homologous strains caused marked agglutination of pili. Antisera to cleaved pilin fragments resulted in partial agglutination and thickening of homologous pili and thickening of heterologous pili, suggesting that heterospecific antibodies were present.

Attachment of antibodies to pili was readily detectable by IFAT and resulted in remarkably clear images of the pili. Antisera to whole-uncleaved pilin reacted primarily with homologous pili, but a weak cross reaction was seen with certain strains. Antisera to cleaved pilin fragments reacted strongly with heterologous and homologous pili.

Results of the present study indicate that cryptic immunogenic determinants were present on pili which were revealed by cleavage of the pilin proteins with cyanogen bromide. These reactions were detected best by IFAT, immunoelectron microscopy, and immunoblotting. Antibodies produced to these epitopes could recognize and bind to them in intact pili on heterologous strains of M. bovis. This is the first report of shared antigenic determinants on M. bovis pili, and may provide the basis for the first IBK vaccine which can provide protection against all strains of this bovine pathogen.

Recommended Citation

Greene, Wallace H., "Infectious bovine keratoconjunctvitis : the identification of sahred antigenic determinants in pili from divergent strains of Moraxella bovis. " PhD diss., University of Tennessee, 1986.
https://trace.tennessee.edu/utk_graddiss/12257