aistand south~ern afrkca - (PDF, 101 mb) - USAID

This goal has been achieved and efforts are now
concentrated towards studying the molecular
and immunological aspects of heartwater with
respect to developing improved vaccines that
contain a repertoire of protective immunodominant
antigens.
Two approaches are being pursued. The fiist
is to develop a tissue-culture-derived vaccine.
The second involves cloning genes encoding
antigens of C. ruminantiumthat are recognised
by the immune system and that may be useful as
vaccine candidates.
In the mammalian host, C. ruminantium is
fotud beth intracellularly or extracellularly. The
intracellular stages are found in macrophages,
neutrophils and endothelial cells, where they
develop into colonies containing morphologically
distinct organisms (Jongejan et al, 1991). This
stage eventually gives rise to the extracellular
staq3, and the organisms are found to be freely
circulating in the blood looking to invade rcw
ccl.atige int rceloodlularng toranivae w
cells. Antigens of intracellular organisms are
probably a target of cell-mediated immune
responses, whereas antigens of the extracellular
organisms are probably a target of antibodymediated
immune responses. In this report the
strategy of developing vaccines based on the
identification of antigens recognised by the
antibody responses will be discussed.
Methodology
Antisera
Normal and immune antisera were prepared
from the blood of 175 sheep that had been
inoculated and had recovered after experimental
infection with the Crystal Springs Heartwater
strain of Zimbabwe. After recovcry the 175 sheep
were resistant to further homologous challenge.
Propagation and isolation of Cowdria
ruminantium
Cowdria ruminantium cultures were
maintained in bovine endothelial cells using the
Byrom and Yunker (1990) method. Various
strains of Heartwater were propagated
for
analysis, namely Crystal Springs, Zwimba, Palm
River, Nyatsanga, Lemco T3, Highway (of
Zimbabwe), Ball-3, Welgevonden (of South
Africa) and a strain from Nigeria. The C.
ruminantium organisms were isolated from
References
Byrom B and Yunker C E. 1990. Improved culture
conditions for Cowdria ruminantium (Rickettsiales),
the agent of heartwater disease of domestic
ruminants. Cytotechnology 4:285-290.
Johnstone A and Thorpe R 1988. Immunochemistry in
Practice. 2nd edition. B!ackwell Scientific Publicationo,
UK.
Jongejan F,Zandbergen T A,van de Wiel P A,de Groot M
and Uilenberg G. 1991. The tick-borne rickettsia
Cowdriaruminantiumhas a Chlamydia-like development
cycle. Onderstepoort Journalof Veterinary
Research 58:227-237.
124
cell-culture supernatants after complete lysis of
the host cells. The organisms were washed three
times in phosphate buffered saline pH 7.4 (PBS)
at 30,000 G for 30 minutes each wash and
processed as necessary.
Sodium dodecyl sulphate polyacrylamide
gel electrophoresis (SDS-PAGE) and Western
blotting
Analysis of C. ruminantium proteins was
achieved by SDS-PAGE and Western blotting
(Johnstone and Thorpe, 1988). The electrophoresed
proteins were transferred to nitrocellulose,
blocked for 1 hour in 0.25% gelatin, and
probed with immune antisera. The reaction of
the antisera was developed by incubation of the
blots with peroxidase-labelled protein-G and
subatrate (Kirkegaard and Perry, USA).
Results and discussion
The analysis revealed the recognition of several
C. ruminantium proteins by the immune
antisera and also showed that the 32 kD protein
was tha most immunodominant. Furthermore,
the immunodominant proteins of C. ruminantium
from geographically different strains of
hearLwater cross-react in Western blotting
analysis with the Crystal Springs strain of
Zimbabwe, indicating antigenic conservation of
these proteins. Other investigators have also
shown cross-reactions between different strains
of heartwater by Western blotting and by
cross-protection studies (van Winkelhoff and
Uilenberg, 1981; Uilenberg et al, 1983; Rossouw
et al, 1990). High-f 'red immune antisera are
being used to screei. .enomic DNA libraries to
isolate genes encodini the immunodominant
proteins of C. ruminan
-m. Agene encoding the
21 kD protein of C. ruminantium has been
cloned and a vaccine trial using the recombinant
protein as a vaccine candidate is under way. As
other genes are cloned, their recombinant
proteins will be tested in vaccine trials and, if
necessary, used in a cocktail vaccine to optimise
the induction of protective immune responses
against heart- water.
The approach to develop a vaccine against
heartwater using tissue-culture-derived
organisms has so fer yielded encouraging results
and is being evaluated in greater detail.
Oberem PT and Bezuidenhout J D. 1987. The production
of heartwater vaccine. G ierstepoort Journal of
VeterinaryResearch 54:485-488.
Rossouw M,Neitz A WH, De Waal DT, do Plessis J L,
Van Gas L and Brett S. 1990. Identification of the
antigenic proteins of Cowdria ruminantium.
Onderstepoort Journal of Veterinary Research
57:215-221.
Uilenberg G. 1983. Heartwater (Cowdria ruminantium
infection): Current status. In: Advances in
Veterinary Science and Comparative Medicine.
pp. 427-480.