Cowdria ruminantium (Cowdria) is an obligate intracellular pathogen that causes heartwater in ruminants. Cellular immunity and the type I cytokine IFN-γ have been implicated in protective immunity to heartwater. The aim of this thesis was to identify proteins of the Welgevonden isolate of Cowdria that induce lymphocyte proliferation and IFN-γ production. Differential centrifugation was found to be the simplest and most efficient method of Cowdria purification. Cowdria organisms were fractionated into their constituent proteins of between 11 and 168 kDa by continuous flow electrophoresis. The resulting fractions were tested for their ability to stimulate lymphocyte proliferation in vitro. In an attempt to simulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with the proteins fractions. In a parallel study, four cattle were immunised with inactivated Cowdria to determine whether their lymphocytes responded similarly. Cowdria-specific proliferation was detected for only a brief period after immunisation by infection with live organisms. This response was only detected again two to three years later. In contrast, PBMC from animals immunised with inactivated organisms were continuously responsive for at least three years. Only Cowdria proteins with molecular masses of 11, 12, 14 to 17 and 19 to 23 kDa induced proliferative responses in PBMC obtained from all six animals. Cell surface phenotypic analysis of Cowdria specific T-cell lines indicated that CD4⁺ lymphocytes were enriched over time with a concomitant increase in antigen-specific proliferation and IFN-γ production. Proteins of molecular masses 13 to 18 kDa induced CD4⁺ lymphocyte proliferation and IFN-γ production by T-cell lines from all the animals tested. Antibodies raised in a chicken and in rabbits to these low molecular weight proteins had low titres and specificity. Two-dimensional electrophoresis indicated that proteins within a single molecular weight range comprised several components with different pIs, revealing the complexity of the Cowdria proteome. This complicates the search for potentially protective antigens. Nevertheless, since they cause proliferation and IFN-γ production by lymphocytes from immunised cattle, these low molecular weight proteins merit further investigation as potential vaccine antigens. / Author: Mirinda van Kleef neé Rossouw
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:rhodes/vital:4061 |
| Date | January 2002 |
| Creators | Van Kleef, Mirinda |
| Publisher | Rhodes University, Faculty of Science, Biochemistry, Microbiology and Biotechnology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Doctoral, PhD |
| Format | 226 leaves, pdf |
| Rights | Van Kleef, Mirinda |
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