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Cellular immune responses induces in vitro by secreted proteins of Ehrlichia ruminantium
Dissertation (MSc)--University of Pretoria, 2008.
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University of Pretoria
2013
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| _version_ | 1867613474418327553 |
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| access_status_str | Open Access |
| author2 | Van Kleef, Mirinda |
| author_browse | Van Kleef, Mirinda |
| author_facet | Van Kleef, Mirinda |
| collection | Thesis |
| dc_rights_str_mv | © 2008, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Dissertation (MSc)--University of Pretoria, 2008. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/30590 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:36:43.511Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/30590 Cellular immune responses induces in vitro by secreted proteins of Ehrlichia ruminantium Van Kleef, Mirinda ThemaN@arc.agric.za Pretorius, Alri Thema, Nontobeka Cellular immune Ehrlichia ruminantium UCTD Dissertation (MSc)--University of Pretoria, 2008. Ehrlichia ruminantium is an obligate intracellular pathogen and, as such, cell mediated immunity plays a key role in the control of bacterial replication and subsequent protection against heartwater in ruminants. IFN-γ has been shown to be a potent inhibitor of E. ruminantium growth in endothelial cells in vitro. Thus, identification of antigens that preferentially activate T cells to proliferate and secrete IFN-γ needs to be done so that they can be evaluated as vaccine candidates. Previously, a cocktail of four open reading frames (ORFs) that induce 100% protection after needle challenge have been identified. However, only 20% protection was obtained after tick challenge in the field, when administered as a DNA vaccine in sheep. Because only limited protection was obtained during a field vaccine trial, our research focused on the identification of additional ORFs as a mean to improve the efficacy of this vaccine. Because secreted proteins are reported to be major targets in a specific immune response we hypothesize that they may be potential heartwater vaccine candidates. Five ORFs (Erum5000, Erum5010, Erum7760, Erum8060 and Erum8610) encoding secreted E. ruminantium proteins were selected from the Welgevonden stock genome sequence using bioinformatics tools. The ORFs were cloned into a pET 102/D-TOPO® vector. The corresponding recombinant (r) proteins were expressed in a bacterial expression system and the expression was confirmed by immunoblots using anti-His antibodies and sheep sera. Proteins were purified by immobilized metal ion affinity chromatography and resulted in a yield of 200 μg-2000 μg per 100 ml and 1L cultures respectively. Four of the five recombinant proteins (rErum5000, rErum7760, rErum8060 and rErum8610) could be expressed. Recombinant proteins were assayed to determine whether they induce recall cellular immune responses in vitro. The peripheral blood mononuclear cells used in the assays were obtained from a naïve and four heartwater immune sheep. Significant proliferative responses (p ≤ 0.01) were evident for 3/4 recombinant proteins (rErum5000, rErum8060 and rErum8610). IFN-γ production was determined using an ELISPOT assay and 3/4 recombinant proteins (rErum5000, rErum8060 and rErum8610) induced IFN-γ production. Each recombinant protein had its own optimum concentration for inducing immune responses and the responses differed between animals. In addition, real-time PCR was used to measure IFN-γ and IL-4 gene expression by antigen stimulated immune PBMC. The real-time PCR results correlated with the ELISPOT assay results. Based on the results obtained, it can be concluded that a Th1 type immune response was elicited. Thus these proteins that induced proliferation and IFN-γ production may be important in protection against heartwater and will be tested in future vaccine studies. Veterinary Tropical Diseases Unrestricted 2013-09-07T19:22:35Z 2009-04-15 2013-09-07T19:22:35Z 2008-11-28 2011-03-17 2009-02-23 Dissertation Thema, N 2008, Cellular immune response induces in vitro by secreted proteins of Ehrlichia ruminantium, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/30590 > E1265/gm http://hdl.handle.net/2263/30590 http://upetd.up.ac.za/thesis/available/etd-02232009-125116/ © 2008, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | Cellular immune Ehrlichia ruminantium UCTD Cellular immune responses induces in vitro by secreted proteins of Ehrlichia ruminantium |
| title | Cellular immune responses induces in vitro by secreted proteins of Ehrlichia ruminantium |
| title_full | Cellular immune responses induces in vitro by secreted proteins of Ehrlichia ruminantium |
| title_fullStr | Cellular immune responses induces in vitro by secreted proteins of Ehrlichia ruminantium |
| title_full_unstemmed | Cellular immune responses induces in vitro by secreted proteins of Ehrlichia ruminantium |
| title_short | Cellular immune responses induces in vitro by secreted proteins of Ehrlichia ruminantium |
| title_sort | cellular immune responses induces in vitro by secreted proteins of ehrlichia ruminantium |
| topic | Cellular immune Ehrlichia ruminantium UCTD |
| url | http://hdl.handle.net/2263/30590 http://upetd.up.ac.za/thesis/available/etd-02232009-125116/ |