Cowdria ruminatium is the causative agent of heartwater, an important tick-borne disease of ruminants in sub-Saharan Africa.
Animals which recover after natural or experimental infections of heartwater are solidly immune to homologous challenge but, the
immune responses responsible for protection against heartwater are poorly characterised.
This study sought to identify antigens involved in protective immune responses to Cowdria by Western blotting using immune sera
and surface labelling of elementary body (EB) proteins using biotin to identify which of these are outer membrane proteins.
Antigens which could be considered as potential vaccine candidates were identified.
Immunisation of goats with live Cowdria or with inactivated elementary bodies (IEBs) leads to development of antibodies to at
least six antigenic components of the EB of 24kDa, 27kDa, 31kDa, 58kDa and 66kDa. In contrast immunisation of goats with
detergent extracted soluble antigens stimulated production of antibodies to only four antigens of 24kDa, 27kDa, 28kDa and
31kDa. Six surface exposed antigens of the Cowdria elementary body were identified by biotin labelling, with molecular masses
of 21kDa, 28kDa, 31kDa, 62kDa, 74kDa and 115kDa and are therefore considered outer membrane proteins. These proteins
reacted with antibodies in sera raised by immunisation of goats with live or inactivated EBs. The 58kDa heat shock protein
(GroEL) of C. ruminantium is an immunodominant antigen. The immune responses to 58kDa antigen expressed as a recombinant
in E. coli were investigated by immunisation of mice and sheep. The immunised animals stimulated specific antibody which
reacted with the native homologue on the EB 58kDa.
In order to investigate the possibility that immunisation with live or inactivated organisms induces qualitatively different immune
responses , IgG isotyping was undertaken using sera from natural and experimental infections of C. ruminantium or immunisation
with IEBs. Production of IgG, and IgG2 antibodies in animals has been associated with T helperl and T helper 2 (Thl and Th2)
responses respectively. Antigen specific IgG, but not IgG2 was detected in the sera of goats and sheep experimentally infected
with C. ruminantium.. In contrast a low but detectable concentration of IgG2 and a high concentration of IgG, was detectable in
field sera from cattle naturally exposed to heartwater. Specific IgG2 was induced in goats after first priming the immune response
by immunisation with IEBs. IgG, was dominant in sera of goats after immunisation with IEBs however, immunisation of cattle
with killed organisms induced a high level of specific IgG, and IgG2. Live infections of Cowdria in mice induce a dominant IgG2a
antibody response unlike the case in goats and sheep. This indicated that mice respond differently from small ruminants to live C.
ruminantium. The results suggest that live organisms suppress a Thl type antibody profile (IgG2 production) except following
immunisation with killed organisms. The suppression of protective Thl responses of immunised goats aids the transmission of the
parasite. Production of IgG2 after challenge of IEB immunised goats may provide a method for monitoring vaccination trials in the
Immunisation with recombinant 58kDa heat shock protein of C. ruminantium led to development a of specific antibody response
of the IgG, isotype. Challenge of immunised sheep showed a highly significant reduction (p<0.005) in infection rates of brain
capillary endothelial cells in the immunised group compared to control animals. However the incubation period and clinical
outcome were not significantly different in controls and immunised sheep. Mice immunised with recombinant 58kDa were
partially protected against virulent homologous challenge. The lack of protection in sheep was attributed to failure of the antigen
to induce a detectable lymphocyte response.
IFN-y is produced by PBMC from I/T and IEB immunised goats in response to stimulation with Cowdria. In contrast, no IFN-y
response was detected with PBMC from sheep immunised with recombinant 58kDa hsp.
Protection of ruminants against cowdriosis in the future will depend on the development of save and easy to use vaccines. This
study identified at least 6 antigens for further study to which dominant antibody responses were made by animals immunised with
live or inactivated antigens. The use of alternative antigen delivery system for induction of T helper responses to Cowdria antigens
are required for assessment of candidate vaccines for immunisation.