Title page for ETD etd-01062011-153805


Document Type Doctoral Thesis
Author Liebenberg, Junita
Email liebenbergj@arc.agric.za
URN etd-01062011-153805
Document Title Ehrlichia ruminantium : genome assembly and analysis with the identification and testing of vaccine candidate genes
Degree PhD
Department Veterinary Tropical Diseases
Supervisor
Advisor Name Title
Dr N E Collins Co-Supervisor
Prof B A Allsopp Supervisor
Keywords
  • Ehrlichia ruminantium
  • vaccine candidate genes
Date 2010-11-26
Availability unrestricted
Abstract

A shotgun genome sequencing project was undertaken in the expectation that access to the entire protein coding potential of E. Ruminantium (Welgevonden) will facilitate the identification of vaccine candidate genes against heartwater. The 1,516,355 bp sequence is predicted to encode 888 proteins and 41 stable RNA species. The most prominent feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number. These repeats have mediated numerous translocation and inversion events and seem to be responsible for the generation of both new full and partial protein coding sequences. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Of the 13 members of the order Rickettsiales compared in this study, E. Ruminantium has the lowest coding capacity (62%), lowest GC content (27.5%), but the highest proportion of repetitive sequences, which comprise 8.5% of the genome. Metabolic reconstruction of E. Ruminantium revealed the metabolic and biosynthetic capabilities typical of an obligate intracellular organism. We identified a number of genes unique to E. Ruminantium, most of which are not functionally characterised in any organism, and those shared with 12 other members of the Rickettsiales. Bioinformatic tools were used to identify possible vaccine candidates from the annotated genome sequence. The protective properties of seven open reading frames (ORFs), which induced cellular immune responses in vitro, were tested in vivo Only 20% survival was obtained in sheep immunised with a DNA formulation consisting of three ORFs. We found that the levels of peripheral blood mononuclear cell proliferation and interferon-gamma (IFN-γ) production did not correlate with each other, nor with the levels of protection, suggesting that the current assays are just not reliable and that IFN-γ expression alone is not an indicator of protection. Therefore more cytokines and different assays will have to be investigated to define in detail what constitutes a protective immune response against E. Ruminantium infection. However, the data generated from the genome sequence will continue to facilitate novel approaches to study the organism and to develop an efficacious vaccine against heartwater.

2010, 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.

Please cite as follows:

Liebenberg, J 2010, Ehrlichia ruminantium : genome assembly and analysis, with the identification and testing of vaccine candidate genes , PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-01062011-153805/ >

D10/906/gm

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