Title: Brucellosis diagnostics and immunology
Funder: Department for Environment, Food and Rural Affairs

Abstract: Description The ambition of this project is to contribute towards the maintenance of GB’s Officially Brucellosis Free (OBF) status. This has been in place since 1985 and is of great economic value to the nation. To maintain freedom of disease the nation must rapidly identify and then resolve outbreaks of disease. This may occur via re-importation of infected animals or even re-emergence from an environmental source. The aim of this project is to improve the tools available to detect, trace and eradicate any reintroduction of disease as well as to better facilitate the efficient maintenance of disease free status. This project aims to improve the specificity of immunological assays for the indirect detection of brucellosis in livestock. The currently available tests are vulnerable to cross reactions due to infection with some non-Brucella organisms. The aim is to improve specificity through the design, synthesis and application of cutting edge oligosaccharide antigens which contain only the unique structural elements of the dominant Brucella antigen. The native antigen contains these epitopes within polymers which also contain common epitopes. The project also supports the production, purification and investigation of other abundant surface antigens, such as the rough lipopolysaccharide, that we will derive from Brucella culture. Direct detection methods have potentially greater specificity than immunological tests. The challenge is that Brucella occurs at very low levels in accessible clinical material such as milk and blood. Therefore a means to improve the direct detection of Brucella DNA will be developed and evaluated. This method will combine two powerful techniques, immuno-capture and Real Time PCR, to increase the concentration of target DNA in biological samples in a manner compatible with specific application by PCR. Anti-Brucella antibodies will be coupled to small magnetic beads which will be dispersed and incubated within biological samples. Any Brucella cells present in the samples will specifically bind to the antibodies on the beads. These beads can be concentrated and washed with the aid of magnets such that any captured Brucella cells remain surface bound. The cells, and their DNA, can then be delivered to the PCR reaction. The third challenge to be addressed is to improve the means by which outbreaks of disease can be traced back to source. Traditional classification methods have very low ability to differentiate strains from the same Brucella species and biovar. DNA based molecular methods have greatly improved resolving and identification capability. However, the DNA of different strains of Brucella frequently have such similar DNA sequences that even these methods lack power. This project will combine whole genome sequencing and bioinformatics tools to maximise the ability to differentiate between strains of Brucella. Working with samples from the APHA archive and sourced from international partners we shall construct a database of whole genome sequences such that the ability to identify the source of any disease outbreaks is improved. Objective Objective 1: Developing and applying capped synthetic oligoperosamine-BSA glycoconjugate antigens for the serodiagnosis of bovine brucellosisObjective 2: Further evaluation of antigenic potential and diversity of the rough lipopolysaccharide (rLPS) for the serodiagnosis of bovine and porcine brucellosisObjective 3: Improvement of molecular diagnostic sensitivity by immunocapture PCRObjective 4: Enhanced epidemiology using whole genome sequencing and other molecular methodsObjective 5: Maintenance of current molecular typing schemes


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION