<OL> <LI> To evaluate the cloned open reading frames of B. melitensis 16M (ORFeome) for their efficacy as potential diagnostic antigens.
<LI> To further reduce detection times for brucellosis by development of a real-time light cycler method for detection.
<LI> To develop tandem repeat typing methods for Brucella.
<LI> To develop a multiplex abortion screen to cover a range of bacterial causes of abortion.
<LI> To evaluate/develop serological tests for the detection of rough strains of Brucella.
<LI> Parallel testing and assessment of new molecular detection techniques with current serology based approaches.
Progress: Objective 1 <br>
A bioinformatics approach has been used to select five candidate antigens, which are hypothetically surface located or secreted, and so may be accessible to the host immune system upon infection. ORFeome entry clones are being recombined into the GATEWAY compatible expression vectors in order to permit the expression of the encoded genes. So far, one of the five candidates has been recombined successfully and the other four are underway.
Objective 2 <BR>
Two targets, IS711 and Gki, have been successfully optimised. IS711 was used in the PCR assay developed in previous projects, but use of the real-time platform necessitated repeat optimisation. Cross-reactions were found at high concentrations of Ochrobactrum DNA (50ng DNA). However, it is highly unlikely that this amount of Ochrobactrum DNA would be present in materials submitted for diagnosis and so this is not perceived as a problem. Assay sensitivity for both targets is good at 10 fg (approximately 3 genomes), but often still possible at the 1 fg level. Both Gki and IS711gave comparable results, although a better fluorescence signal strength was found with Gki. In consequence, the Gki assay is the method of choice and is currently being assessed with clinical material.
Objective 3 <BR>
Variable nucleotide tandem repeat (VNTR) typing is a new approach that has substantial promise in the typing of genetically homogeneous organisms. The value of this approach as a tool for epidemiological trace-back of Brucella was assessed using a collection of isolates associated with an importation of Brucella to Scotland from Ireland in 2003. Preliminary findings suggest that most of the isolates from the Scottish outbreak are identical to one of two genotypes detected on the suspected source farm. Epidemiologically unrelated isolates from across Ireland were found to give distinct strain profiles with the exception of one isolate from the same geographical location as the source farm. Current work is focussing on ironing out some technical difficulties and examining the stability of genotypes over long time-scales. Twenty other repetitive loci that could be useful in adding to the discriminatory power of the approach have also been assessed.
Objective 4 <BR>
A bovine multiplex abortion assay, which allows the detection of B. abortus, Salmonella enterica, Bacillus licheniformis and Arcanobacterium pyogenes, has been optimised and applied to clinical material from bovine abortions.
Objective 5 <BR>
Serum from animals infected with rough strains such as B. ovis or vaccinated with the rough attenuated RB51 have been assessed, highlighting the poor performance of current serodiagnostic tests with these samples. Once the novel antigens in objective 1 have been produced, they will be assessed with this problem group of sera.
Objective 6 <BR>
Evaluation of new techniques such as PCR and IFN-gamma has been undertaken using material collected following recent introductions of brucellosis and samples following in vivo infection. This study will be expanded to include the recently optimised real-time assay (see objective 2).