S. M. MahanUniversity of Florida/USAID/SADCC
Heartwater Research Project,
Box 8101 Causeway, Harare, Zimbabwe.
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Summary Heartwater has an economically significant impact on livestock. The disease is caused by the rickettsial organism Cowdria ruminantium and is transmitted by ticks of the genus Amblyomma. In Africa heartwater is the second major tick-borne disease after theileriosis and its occurence spans most of the sub-Saharan region and the neighbouring islands. In southern Africa it is considered to be the main tick-borne disease. The principal objective of the University of Florida/USAID/SADCC Heartwater Research Project is to develop new vaccines and diagnostic tests to replace the current cumbersome methods of immunisation and diagnosis for heartwater. The Project's goal is to understand the molecular and immunological aspects of heartwater with respect to developing improved vaccines which would contain a repertoire of protective immunodominant antigens Of the approaches being pursued, the first is to develop a tissue culture-derived vaccine, and the second involves cloning of genes encoding antigens to C. ruminantium which are recognised by the immune responses and which may be useful as vaccine candidates. These approaches are discussed in this paper. |
The prevalence of heartwater has a significant economic impact on livestock. Caused by the rickettsial organism Cowdria ruminantium and transmitted by ticks of the genus Amblyomma in Africa, heartwater is the second mayor tick-borne disease after theileriosis. Its occurrence spans most of the sub-Saharan region and the neighbouring islands (Uilenberg, 1983). In southern Africa it is considered to be the main tick-borne disease.
Economic losses due to heartwater are a result of the high mortality rate it causes, which ranges between 20 and 90% (Uilenberg, 1987), and the reduced productivity it engenders in both clinically ill and surviving/recovered animals which become chronic carriers of the disease.
Heartwater is routinely controlled by acaricides and artificial immunisation. However, tick control is expensive and ticks build up a resistance to acaricides. Hence complete disease control using this method is not really achievable. In addition, intensive dipping against ticks is difficult to sustain in many African countries.
In the artificial immunisation method, young animals (less than six months old) are inoculated with infected blood to maximise their age-related innate immunity to heartwater. This process has given varied results, as it does not reproducibly induce immunity in the vaccinated animals. In older animals, artificial immunisation is achieved by infection with blood infected with the Ball-3 heartwater strain (Oberem and Bezuidenhout, 1987) followed by treatment with tetracyclines (Uilenberg, 1990; van der Merwe, 1987). The infection-and-treatment method has been used with a degree of success as animals that have recovered from heartwater infections have been shown to be immune to homologous and, in most cases, heterologous challenge (Uilenberg et al, 1983; van Winkelhoff and Uilenberg, 1981). However, the infection-and-treatment method has limitations. The vaccine has to be stored in liquid nitrogen or dry ice up to the time of inoculation of the animals; the vaccine has to be administered intravenously; vaccinated animals have to be monitored clinically and treated with a curative dose of tetracyclines during the febrile reaction; some vaccinated animals may die due to acute reactions to the vaccine despite administration of tetracyclines; other disease-causing organisms may be accidentally transmitted in the blood vaccine; and lastly, overall this method is cumbersome and cannot be applied on a wide scale.
Clinically ill animals in the field may be treated with tetracyclines. However, the course of the disease is rapid and by the time the clinical reaction is detected it is usually too late for antibiotic therapy.
These control methods against heartwater have been used for many years and currently there are no suitable alternatives.
The principal objective of the University of Florida/USAID/SADCC Heartwater Research Project was to develop new vaccines and diagnostic tests to replace the current cumbersome methods of immunisation and diagnosis against heartwater. The project's initial goal was to propagate C. ruminantium organisms in vitro.
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 first is to develop a tissue-culture-derived vaccine. The second involves cloning genes encoding antigens of C. ruminantium that are recognised by the immune system and that may be useful as vaccine candidates.
In the mammalian host, C. ruminantium is found both 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 stage, and the organisms are found to be freely circulating in the blood looking to invade new 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 antibody-mediated immune responses. In this report the strategy of developing vaccines based on the identification of antigens recognised by the antibody responses will be discussed.
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 recovery 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 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 electro-phoresed 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 substrate (Kirkegaard and Perry, USA).
The analysis revealed the recognition of several C. ruminantium proteins by the immune antisera and also showed that the 32 kD protein was the most immunodominant. Furthermore, the immunodominant proteins of C. ruminantium from geographically different strains of heartwater 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-titred immune antisera are being used to screen genomic DNA libraries to isolate genes encoding the immunodominant proteins of C. ruminantium. A gene 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 far yielded encouraging results and is being evaluated in greater detail.
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