Abstract:

Heartwater is a febrile tick-borne disease of domestic and wild ruminants. The
serological tests, for example, 1FAT, ELISA and nucleic acid based tests which are
currently used for diagnosis of heartwater require sophisticated laboratory equipment
and trained personnel to be performed and are therefore not suitable for routine field
use. This means that there is need for a simple rapid and specific test that can be used
in the field for routine diagnosis of heartwater.
The possibility of detecting C. ruminantium antibodies and antigens circulating in sera
and plasma of experimentally infected goats, using latex beads coated with crude
Cowdria antigen (latex slide agglutination antibody test) and latex beads coated with
anti-MAP2 immunoglobulin fraction (latex slide agglutination antigen test) was
investigated. The crude Cowdria antigen was prepared from C. ruminantium
organisms cultivated in bovine pulmonary artery endothelial cells (BPA). The antiMAP2 immunoglobulin fraction was precipitated by use of 50% saturated ammonium
sulphate solution from sera obtained from rabbits which had been immunised with
recombinant Cowdria MAP2 antigen. Sera and plasma collected from experimentally
infected goats were tested for Cowdria antibodies and antigen respectively. Four sera
from goats that sutfered an acute and fatal heartwater disease tested negative for
Cowdria antibodies, using latex agglutination antibody test based on whole Cowdria
antigen, while 2 sera from goats that suffered a non-acute and non-fatal disease started
testing positive for Cowdna antibodies during the 6**1 week after inoculation. In
addition latex beads were coated with recombinant Cowdria MAP2 antigen and used
to test 81 random field goat serum samples for Cowdria antibodies Of the 81 random
field goat serum samples tested, 19 (23.4 %) were positive, 11 (13.6 %) weak positive
(border line) and 51 were negative. It was not possible to detect Cowdria antigen in
plasma of the infected goats with the Latex slide agglutination antigen test. The latex
slide agglutination antigen test failed to detect Cowdna antigen in the plasma of the
sick goats probably because of either low rickettsemia, low levels qf MAP2 from lysed
elementary bodies (EBs) in circulation, coating of circulating EBs with host proteins or the Latex slide agglutination antigen test was not sensitive enough. In addition, failure
to detect elementary bodies could be attributed to the fact that MAP2 is an internal and
not an integral membrane protein as was demonstrated by the phase partitioning of
MAP2 in the aqueous phase. Therefore, MAP2 in the intact elementary bodies was
inaccessible to the monospecific rabbit anti-MAP2 antibodies. To test this hypothesis,
intact and solubilized elementary bodies were examined by use of latex beads coated
with rabbit anti-MAP2 antibodies. A weak agglutination reaction was observed with
the solubilized elementary bodies and no agglutination was observed with the intact
elementary bodies, thus indicating that solubilization of elementary bodies exposed
epitopes recognised by the rabbit anti-MAP2 antibodies.
In the present study, sensitivity and specificity parameters of the latex slide
agglutination antibody test were not investigated, but it was demonstrated that the
latex slide agglutination antibody test is a potential diagnostic tool for detection of
C. rummantium antibodies in sera of animals that suffer a non-acute and non-fatal
heartwater disease. However, there is need to identity a specific Cowdria surface
protein to which antibodies can be raised and coated on latex beads to be able to
detect Cowdria antigen in the plasma of sick animals.