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Improving immunodiagnosis for brucellosis - SE0310

Brucellosis is a notifiable zoonotic disease. Following an extensive and expensive eradication campaign, Great Britain (GB) has been declared Officially Brucella Free (OBF) for bovine brucellosis. In order to maintain OBF status the national herd is routinely monitored for evidence of exposure to the pathogen by a highly sensitive immunodiagnostic test which detects the presence of Brucella specific antibodies. The current surveillance practices require the sampling and testing of approximately 1,000,000 cattle sera, and 200,000 bulk milk (pooled samples from milk tankers) samples per year, in order to sample enough of the national herd to effectively assure freedom from disease. In addition, pre- and post-import testing of Brucella susceptible livestock is designed to prevent the introduction and spread of the disease. These procedures work well for the most part, and have proven adequate for the maintenance of OBF status.

Since GB is an OBF area the incidence of positive reactors in the routine surveillance tests is expected to be zero. Unfortunately, positive test reactions do occur. In the absence of epidemiological evidence to suggest exposure to Brucella these results are generally considered to be due to False Positive Serological Reactors (FPSR), and are caused by sub-optimal test specificity. However, in order to eliminate the possibility of infection a series of investigations designed to confirm the infection status of the reactor herd are necessary. During these investigations restrictions on animal trade and movements are imposed and ultimately persistent reactor animals are slaughtered as part of the control measures. The requirement for additional testing, resampling and utlimately slaughter of persistent uninfected reactors contributes significant additional costs to Defras routine surveillance programme and causes considerable inconvenience and ecomomic burden to the affected farms. At present the incidence of FPSR is estimated to be around 1.0%, resulting in approximately 10,000 samples per year which require additional testing, a small number of which will present as persistently inconclusive reactors following resampling and ultimately have to be culled.

The FPSR problem is a result of cross-reactive antibodies in the sera of uninfected animals binding with the Brucella LPS antigen used in the tests. Exposure to a number of bacteria can trigger the generation of antibodies which cross react with Brucella LPS, and as such FPSR occur randomly throughout the national herd. All the currently implemented Brucella serodiagnostic contain Brucella LPS in the antigen preparations and therefore all these tests are affected to some extent by FPSR because of the sub-optimal specificity of the antigen. The use of protein antigens unique to Brucella species in immunodiagnosis offers the promise of greater diagnostic specificity. Furthermore, the use of alternative immunological correlates of infection (e.g: the production of Brucella specific Interferon gamma (IFNg)) have also been shown to offer specificity improvements in comparison to serology based assays. However, the sensitivity and practicality of protein based serology assays and IFNg detection methods is poor in comparison to the LPS serology based assays, meaning that in their current state these assays are not good enough to replace the existing tests. Through previous research we have identified a number of specific protein antigens that have potential for improving the specificity of diagnosis. In this project we aim to develop and evaluate assays based upon specific immune responses to these proteins and to utilise fluorescent detection technologies to enhance the sensitivity and range of these assays. Due to natural variability in immunological recognition of proteins, the measurement of responses to more than one specific protein will also facilitate detection of a greater number of infected animals, and further improve the diagnostic usefulness of the tests. A number of these individual protein based tests could be implemented in the event of a suspect FPSR reaction in order to confirm freedom from Brucella infection, but this would be a costly and protracted investigation involving large volumes of sera from the suspect reactor and multiple tests. Ideally, a single assay is required that has at least equivalent sensitivity to the LPS based assay but with the enhanced specificity of multiple protein based diagnosis. Thus, in addition to the development and evaluation of sensitive and specific tests based on immune responses to single proteins we aim to combine these tests to produce a single fluorescent bead based multiplex assay. The use of fluorescent bead based technologies will allow the simultaneous but independant detection of several Brucella specific analytes, and as such provide a more comprehensive profile type test result. Results would be seen as a profile of reactivity against the selected antigens, where true positive animals would be positive for responses against the majority of the analytes. Judicious selection of protein antigens for inclusion in this multiplex test will ensure that samples classified as FPSR by monoplex LPS based assays, display a distinct profile from true positive reactors. In this project the protein specific tests and multiplex tests will be developed and evaluated for their diagnostic usefulness against a large panel of known provence sera. In addition to the development and evaluation of novel and improved immunoassays, preliminary investigations into the chemical sensor based E-nose technology will also be conducted. Analysis of the data generated in the evaluation stages of the project will determine if the novel tests provide a mechanism for reducing or eliminating the impact of FPSR on routine surveillance.
Objective 1: To assess the diagnostic potential of antigens identified and produced in previous projects (EU9901, and SE0308), for use in specific serodiagnosis or IFNg stimulation assays, and select specific assays with potential for multiplexing.

Objective 2: To evaluate the use of alternate assay read-out platforms for improving sensitivity of existing assays.

Objective 3: Combine and optimise the best assays in a multiplex format.

Objective 4: Evaluate the novel serodiagnostic assays (including multiplex test), for their potential to alleviate the FPSR issue.

Objective 5: An investigation of the potential of the Electronic Nose (EN) for use in serodiagnosis of brucellosis, and an assessment of the ability of EN to discriminate true Brucella reactors from FPSR.
Time-Scale and Cost
From: 2006

To: 2009

Cost: £327,705
Contractor / Funded Organisations
Veterinary Laboratories Agency
Animal Diseases              
Animal Health              
Plants and Animals              
Fields of Study
Animal Health