Immune response to and pathogenic mechanisms of contagious bovine pleuropneumonia infection: investigation of the importance of the capsular polysaccharide and assessment of its vaccine potential

Abstract

Mycoplasma mycoides subsp. mycoides small colony type {MmmSC) is the causative agent of contagious bovine pleuropneumonia (CBPP), a major disease of cattle in Africa for which current vaccines exhibit a poor efficacy. MmmSC possesses a capsular polysaccharide (CPS) believed to be an important virulence factor. Antibodies directed against CPS are bactericidal in an in vitro growth inhibition test (GIT). Therefore, CPS is a good vaccine candidate.

The aim of this thesis was to investigate the vaccine potential of MmmSC CPS.

Before using CPS as a vaccine, the immunogenic structure of CPS needed to be studied. MmmSC strains were tested with rabbit antisera (raised against different MmmSC strains) in a GIT. The results showed that CPS was conserved between the strains. Purified CPS from different MmmSC strains were then investigated with monoclonal antibodies (mAbs) in an enzyme linked immunosorbent assay (ELISA). It appeared that CPS from all the strains were recognised by the mAbs, except the strain PG1 that had a low signal with two of the mAbs. GIT was used with mAbs that recognised MmmSC CPS to test their growth inhibiting (GI) activity; all of the mAbs used in the GIT were bactericidal.

The specificity of these mAbs was then investigated, using an ELISA, against other mycoplasmas, Mycoplasma mycoides subsp. mycoides large colony and Mycoplasma mycoides subsp. capri. The results obtained suggested that the mAbs recognised at least three different epitopes on CPS.

A competitive ELISA was performed to clarify the number of epitopes that these mAbs recognised. No conclusion could be drawn from this experiment since the results were unclear.

Since antibodies to CPS are bactericidal in vitro, this suggested they were protective against MmmSC. Passive immunisation of mice with a bactericidal mAb directed against CPS was used to investigate the protective efficacy of anti-CPS antibodies in vivo. After injection with the antibody, mice were challenged with an MmmSC strain. The incidence and the duration of mycoplasmaemia were used to assess the protection given by this antibody. No significant difference could be seen between mice passively immunised with anti-CPS antibody and the control group, suggesting that a bactericidal antibody was not protective in vivo in mice against challenge with MmmSC.

The type of immune response to CPS was examined in three groups of cattle: M/nmSC-intubated animals, CBPP-vaccinated and unvaccinated animals in contact with MwrzSC-intubated cattle. The results showed that only a quarter of CBPPvaccinated cattle had an immune response against CPS. The immune response against CPS in the three different groups was of IgM type even after a second exposure to the pathogen where it could have been expected an IgG type immune response.

The lack of immune response to CPS in cattle might be due to cross-reactions with bovine lung. Cross-reactions between bovine lung and MmmSC CPS were confirmed by western-blot with anti-MwwSC or anti-CPS sera from rabbits, mice and mAbs. It was not possible to know whether antibodies recognising both CPS and bovine lung were present in anti-AfrwwSC cow sera because of a background signal due to the secondary antibody specific for cow immunoglobulins.

To minimise the cost of CPS vaccine production, polysaccharides from other sources could be used as a vaccine. A western-blot was then performed to examine the cross-reactions between MmmSC CPS and carbohydrates from cereals with A/mmSC-infected cow sera, MmmSC-immunised rabbit sera and mAbs. The results showed that CPS shared epitopes with the polysaccharides from the cereals used in this experiment.