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A study on the characteristics, immunogenicity and pathogenicity of the pili of Moraxella bovis

Osman, Md Annuar (1993) A study on the characteristics, immunogenicity and pathogenicity of the pili of Moraxella bovis. PhD thesis, Murdoch University.

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Various physical, biochemical and immunological characteristics of the haemolysin, protease and pili produced by several strains of Moraxella bovis were studied by a number of techniques and the results are reported and discussed in the thesis. These products are factors which have been considered to affect the pathogenicity of M. bovis and their ability to produce infectious bovine keratoconjunctivitis (IBK) in cattle.

Methods were developed for the quantitative determination of the haemolysin, an alkaline casein protease and pili produced by M. bovis and used to determine the kinetics of their production during replication of M. bovis in broth culture. Differences in the pathogenicity of two highly piliated strains of M. bovis did not appear to be due to any detectable quantitative differences in the production of haemolysin or casein protease.

Pili were purified by caesium chloride density-gradient centrifugation and shown to have a density of 1.284 g ml-1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the pilus proteins of eight piliated strains of M. bovis showed that the pilin proteins were heterogenous and their molecular weight ranged from 22,000 to 16,300 daltons.

Monoclonal antibodies were produced against the pilin protein of two strains of M. bovis. Polyclonal antiserum was also produced against the purified pilin protein of eight strains strains of M. bovis. Agar gel precipitation tests (AGPT), an enzyme-linked immunosorbent assay (ELISA), an immunoblot ELISA and Western immunoblotting techniques were used to antigenically characterise the pili of eight strains of M. bovis using the polyclonal antisera and monoclonal antibodies. Four pilus serogroups were defined by the polyclonal antisera: group A comprising pili of strains 25R, 7R and R337; group B of strains 545,361 and 118; groups C comprising strain SG; group D comprising strain 510. There was no relationship between the molecular weight of the pilin proteins and the antigenic type.

Monoclonal antibodies produced against pili of M. bovis strains 25R reacted with in the ELISA and Western immunoblotting techniques with the homologous strain and with both the other two strains in serogroup A. However, a monoclonal antibody against pili of strain 545 reacted only with the homologous strain and not with any heterologous strain. The result indicated the occurrence of more than one epitope on the pilus protein of strain 545 and that pili of strain 545 could be considered an antigenic subtype of serogroup B.

Piliated strains of M. bovis adhered to in vitro cell cultures of bovine origin, viz. bovine corneal epithelium (BCE) and Madin-Darby bovine kidney (MDBK) cells, but not to cell cultures derived from other animals species. This adherence to cell cultures was considered to mimic the adhesion of bacteria to conjunctival epithelial cells in cattle, the first stage in the pathogenesis of IBK. The adhesion of piliated strains was greater than non-piliated strains and the adhesion of piliated strains was markedly reduced after the pili were detached by treatment with magnesium chloride, indicating pili were the major adhesins on the M. bovis cells. However, the ability of non-piliated strains to also adhere, albeit at a lower level than piliated strains of bacteria, suggested that adhesins other than pili are also present on M. bovis.

Adhesion of M. bovis to BCE cells was inhibited by the addition of simple sugars D-mannose and D-glucose, especially D-mannose, suggesting that the cellular receptor for the M. bovis pilus adhesin was a glycoprotein with a predominantly mannose sugar moiety. Autoagglutination, a common phemomenon of piliated strains of M. bovis, was shown to be inhibited in diluents containing simple sugars and water at pH 6.0.

Polyclonal anti-pilus serum and monoclonal pilus antibody inhibited the adhesion of the homologous strain and some heterologous strains of M. bovis to BCE cells; the extent of inhibition of the heterologous strains was correlated to the serological groups and subtypes identified by ELISA and indicates that the antigenic characterisation of the pilus antigens is of biological significance and would determine the degree of inhibition by antibody of adherence of M. bovis to the conjunctival epithelial cells of cattle. It also suggests that assessment of the extent of cross-immunity which would be induced by immunisation with pili of a single strain of M. bovis against antigenically heterologous strains, could be determined by the degree of inhibition of adherence of M. bovis to cell cultures instead of cross-immunity tests in cattle.

Two haemolytic and highly piliated M. bovis strains, 25R and 545, differed in their pathogenicity during experiments to induce IBK in cattle. Strain 545 was highly pathogenic and produced a severe form of IBK but strain 25R was of only moderate pathogenicity. Cattle immunised with purified pili produced a marked antibody response; the principal antibody type detected in serum and lacrimal was IgG. Cattle immunised with purified pili were protected against challenge with the homologous M. bovis strain but were only partially protected against challenge with the heterologous strain. The results indicate that immunisation of cattle for the prevention of IBK would require immunisation with multiple antigenic types of the pilin protein.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary Studies
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Wilcox, Graham
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