An official website of the United States government.
<ol><li>To further develop, optimize and evaluate an indirect ELISA to detect antibodies within serum that react to the rough LPS of Brucella. Pilot work conducted as part of the previous ROAME funded project has demonstrated the potential of this approach to resolve FPSRs in cattle and swine.</li>
<li>To adapt and apply the multiplex indirect ELISA developed in the previous ROAME funded project SE0312 for use on sera from swine. This multiplex detects antibodies against eight different types of antigen (lipopolysaccharides and proteins) within a single test well. The assay will be formally evaluated using a panel of sera from swine that have been infected with Brucella, that are free of the disease and from those that are free of disease but present FPSRs.</li>
<li>Develop a multiplex indirect ELISA for serum antibody detection using peptide antigens derived from the Brucella protein BP26. The protein sequence will be evaluated in-silico to eliminate sequences that lack specificity and theoretical antibody binding properties. The remaining overlapping peptide sequences from this protein will be obtained by bespoke commercial synthesis. These will then each be evaluated for their diagnostic potential using a serum panel from Brucella infected and non-infected cattle and swine. The most useful sequences will be combined in a multiplex indirect ELISA that will be evaluated using a panel of sera from animals (cattle and swine) that have been infected with Brucella, that are free of the disease and from those that are free of disease but present FPSRs.</li>
<li>To develop an indirect ELISA to detect antibodies within serum that react to a synthetic oligosaccharide designed to mimic the structure of the Brucella specific ‘M’ epitope that occurs within the Brucella OPS. The ELISA will be evaluated using a panel of sera from animals (cattle and swine) that have been infected with Brucella, that are free of the disease and from those that are free of disease but present FPSRs. The same samples will be evaluated with an indirect ELISA using the OPS from Y. enterocolitica O:9 and the two outputs for each sample will be expressed as a proportion of each other to discover if the proportional outputs offer superior resolution of true and false positive samples.</li>
<li>To use a chemically modified OPS from cross reactive Escherichia hermannii strains to evaluate the relative serum antibody response to the different structural features within the Brucella OPS. The modified E. hermannii OPS will be a homopolymer with the same monosaccharide constituent as Brucella OPS the majority of which are linked together to create epitopes specific to Brucella and eliminate the common epitopes that are also found in other organisms. This structure and the native form will be used to develop indirect ELISAs which will be evaluated using a panel of sera from animals (cattle and swine) that have been infected with Brucella, that are free of the disease and from those that are free of disease but present FPSRs.</li></ol>
<p>The overarching aim of this project is to develop novel serodiagnostic tools to significantly aid in the resolution of false positive serological reactions (FPSRs) for brucellosis in cattle and swine. Serodiagnostic techniques underpin veterinary surveillance for brucellosis as well as facilitating the international trade in livestock. Whilst serology is a critical tool it is known that infection with gram negative bacteria possessing an O-polysaccharide with sufficient structural similarity to that of Brucella can give rise to cross reactive antibodies which generate FPSRs. The effective detection and tracing of future incursions of brucellosis would be significantly enhanced by the ability to eliminate FPSRs from further enquiry. This would reduce surveillance costs and hasten a transition back to disease freedom. Alternative detection methods for Brucella either through culture or DNA identification from material obtained from live animals (with the exception of tissues related to abortions) are currently insufficiently reliable to aid in diagnosis. Furthermore both approaches are considerably more expensive than serology. Therefore reliable serological confirmatory tests are very desirable.</p>
<p>The proposed project contains five separate objectives, each building on the intellectual and physical assets generated though our previous research. The first two extend the application of immunoassays developed in the previous ROAME project to enhance the validation data for cattle and extend their use to swine. The first assay is an indirect ELISA using the Brucella rough lipopolysaccharide (LPS). The second is a multiplex indirect ELISA which simultaneously measures the antibody response to eight separate antigens. These are: the smooth LPS from both serotypes of Brucella as well as the cross reacting organisms Y. enterocolitica O:9 and E. coli O:157, the Brucella rough LPS, two Brucella recombinant proteins and a commercial protein extract from Brucella. The multiplex format enables the efficient extraction of the combined diagnostic attributes of all eight antigens.</p>
<p>Two further objectives are to develop and validate entirely new serological assays which use single epitope antigens designed by the Brucella Immunology Group. The aim is to apply the specific antigen structures required for assay sensitivity whilst eliminating superfluous structures that may compromise specificity. The first will be a multiplex indirect ELISA using up to eight different peptides derived from a Brucella periplasmic protein that demonstrates good diagnostic sensitivity but poor specificity. The peptides will be selected from a population of 48 overlapping peptides that span the entire sequence of the protein by using bioinformatic tools and in-vitro screening against a serum panel of defined status. The second will use an oligosaccharide with an added functional group to enable it’s conjugation to a solid phase and permit the development of an indirect ELISA. Once developed, both these assays will undergo preliminary validation.</p>
<p>The final objective is to create a novel OPS antigen which is enriched in Brucella specific epitopes with reduced non-specific epitopes. This will be prepared through the chemical modification of a structurally related OPS from a different organism. The native and modified OPS will be used in indirect ELISA and evaluated to assess their effectiveness in resolving FPSRs. </p>