M. paratuberculosis infection in sheep causes a chronic granulomatous enteritis,
characterised by a massive cellular infiltration of macrophages, epithelioid cells and
lymphocytes. Like other mycobacterial infections the predominant immune response is
cell-mediated and this response centres on a complex interaction between T cells and
macrophages infected with M. paratuberculosis organisms. T cells have been identified
as the predominant inducers of the cell mediated immune response to mycobacteria.
Stress proteins have been shown to be immunodominant antigens in immune responses
to other pathogenic mycobacteria. T cells reactive to stress proteins have been isolated
from infected animals. For these reasons, the 60-kDa stress protein of
M. paratuberculosis was chosen for the investigation of the immune response to
M. paratuberculosis infection in sheep.
In this thesis the DNA encoding the 60-kDa stress protein of M. paratuberculosis was
amplified by PCR, sequenced and the ORF of this protein expressed as a fusion protein
with glutathione S-transferase. Recombinant M. paratuberculosis 60-kDa stress protein
was obtained by cleavage of the fusion protein with the proteolytic enzyme thrombin.
Recombinant M. paratuberculosis 60-kDa stress protein was used to generate polyclonal
T and B cell responses in sheep which were assessed by in vitro proliferation and
enzyme-linked immunosorbent assays, respectively. In addition, the recombinant
protein was used to generate a monoclonal antibody. The assays and reagents generated
in this thesis were subsequently used to investigate the immune response to
M. paratuberculosis 60-kDa stress protein in ovine paratuberculosis.
This investigation provides preliminary evidence that paratuberculosis-infected animals
have T cells that recognise and are capable of responding by proliferation to
M. paratuberculosis 60-kDa stress protein in vitro, and that antibodies to this protein
could be detected in sera from these animals. Furthermore, M. paratuberculosis 60-kDa
stress protein was detected in tissues from infected animals by immunocytochemical
analysis. These preliminary findings support a role for M. paratuberculosis 60-kDa
stress protein in the ovine immune response to M. paratuberculosis infection.