<OL> <LI> To determine the difference between species/biovars of brucellae using molecular techniques (in silico post-genomic analysis, microarray analysis, subtractive hybridisation and proteomics).
<LI> To follow experimental B. suis infection in pigs, in order to identify biomarkers of infection, which could form the basis of future diagnostic tools.
Progress: Objective 1 <BR>
The in-silico comparative genomics has shown that the three main Brucella species, B. melitensis, B. abortus and B. suis, are remarkably similar, with <99% identity amongst most open reading frames (ORFs). Some polymorphism was identified among putative outer membrane proteins or associated with insertion/deletion events. A specific 2,750 bp sequence has been identified within an autotransporter encoding region on Chr II in B. abortus. In B. suis, a 7,738 fragment associated with metabolic capabilities appears species specific. Brucella melitensis unique ORFs were found to be associated with a 20kb pathogenicity island. However, many of these were shared with B. abortus, highlighting the greater similarity between these two species compared with B. suis. Whether these differences alone will account for the observed differential host preferences among the brucellae remains unresolved. The results support the location of these genomic insertions and deletions for B. suis and B. melitensis (- the genome has only just been released for B. abortus; Halling et al. 2005).
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An ORFeome of B. melitensis has been produced (Dricot et al. 2004) and is being utilised to prepare microarray slides through a European consortium (which includes the VLA). However, another group has now published a microarray comparison of B. abortus, B. melitensis and B. suis, thus negating the need for this work to be undertaken as part of this project. Differences between these species were found to be slight, with most differences associated with insertion/deletion events or pathogenicity islands. These appear to be stable among other isolates of the same species and thus representative.
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Work has started using subtractive hybridisation to focus on differentially possessed genes in B. melitensis, B. abortus and B. suis. However, technical difficulties have been experienced, although it is hoped it should still be possible to complete this work by the end of the project.
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Differential pathogenesis may be based on post-translational modification of gene products affecting the ability of these to interact with the immune response of the host. Two-dimensional gel electrophoresis is currently being used to compare the spot profiles of panel members. Where differentially expressed proteins are seen, spots will be excised and identified using MALDI-TOF technology. This should yield the amino acid sequence of excised spots, facilitating protein identification. This may provide insights into the proteomic basis of host pathogenicity.
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Objective 2 <BR>
An experimental B. suis infection in pigs was successfully completed during the first year of the project, with serum samples collected during acute infection and weekly thereafter. The samples have now been assessed for cytokine responses. The next step will involve the identification of biomarkers for brucellosis. The information gained will allow the assessment of the diagnostic potential of this technology, by looking at the presence of biomarkers for infection and how these biomarkers change during the course of infection. <P>