The incidence of bovine tuberculosis (bTB) in GB has been increasing despite the use of a control strategy based on tuberculin skin testing and slaughter of animals that react positively to the test. There is therefore a need for the development of suitable ante-mortem test for bovine tuberculosis in cattle. This is of particular relevance for the development of cattle vaccines to allow the continuation of test and slaughter policies alongside vaccination. Therefore, development of an assay that Distinguishes Infected from Vaccinated Animals (DIVA tests) is an urgent research requirement.
Since 2006, an ancillary test to the tuberculin skin test, the Bovigam interferon-gamma (IFN) assay, is being used in defined and prescribed circumstances to maximise overall sensitivity (parallel testing) or to increase specificity in circumstances where the skin test specificity is doubtful (serial testing). This assay is based on the detection of IFN in whole blood cultures stimulated by antigens such as tuberculin. It involves several critical steps, like transport of viable blood at ambient temperatures to the laboratory within a day of sampling, a cell culture step and finally an ELISA assay to measure IFN. Although the advantages in identifying significant numbers of infected cattle that escaped skin test detection are clear and demonstrable, the conditions summarised above make this test to some degree technically challenging as well as relatively resource-intensive and expensive.
Serological tests have logistical and financial advantages over assays based on cell-mediated immunity. Yet, in the past they have suffered from poor sensitivity and were therefore not considered the method of choice to deliver highly sensitive tests for the detection of bovine TB. However, recent advantages in antigen discovery and the development of novel and more sensitive detection systems have led to serological assays that show promise in delivering sensitive tests. One recently described assay is based on a chemiluminescent-based multiplex system that can simultaneously detect and analyze antibody responses to multiple antigens spotted in a single well of a 96-well plate (EnferplexTB). Data recently using field samples in an un-blinded study conducted in Ireland, demonstrated that this assay is both sensitive and specific. We have recently extended this original observation in a blinded study using sera collected in GB from skin test-positive animals with bTB, from TB-free animals and from experimentally with BCG vaccinated cattle. Our results indicate that the test has a relative (to skin testing) sensitivity range of 77.0% to 86.5% at corresponding specificity levels of 100.0% to 77.6 %. Importantly, this serological multiplex assay does not react with sera from BCG vaccinated calves and could therefore be suitable as DIVA test alongside BCG-based vaccine strategies. Our study therefore largely confirmed and extended the original unblinded study conducted in Ireland.
However, neither study was aimed at the group of animals that an ancillary test should target, i.e. animals that escape skin test detection (= skin test negative cattle with confirmed bTB) and further studies need to directly target this class of animals. Unfortunately, samples from such animals are logistically very difficult to obtain and in most scenarios also require them to be bled under a project license granted under the Animals (Scientific Procedures) Act 1984 (ASPA), which further increases the logistical and legal challenges associated with such sampling efforts. Further, a scientific imperative in validating novel tests is to compare assay performances against current tests in use, in our case the Bovgiam IFN assay, thus requiring additional sampling and experimentation increasing the costs and lowering the feasibility of such studies.
To overcome these legal and logistical impediments to sampling and testing, we propose to collect an additional serum sample from each animal sampled for the routine IFN assay. This project, whose relevance and importance is acknowledged by Defra, is only viable through the active collaboration of Animal Health who are currently undertaking the Bovigam sampling and have joined this project as sub-contrator . Another important component is our collaboration with Enfer Scientific. The project is therefore a collaborative proposal of different VLA departments (SEB, LTD, CERA), Animal Health and Enfer Scientific. It has several crticial advantages: Firstly, it would avoid ASPA implications; secondly, it will target the animal category this project is aimed at (tuberculous cattle escaping the skin test); thirdly, it would allow the direct comparison of the ancillary test in routine use (Bovigam IFN test) with the EnferplexTB serum test. In addition, the serum bank thus generated could be used to validate other, novel btB serum assays (for example the assay currently under development by IDDEXX), or to test serum and biomarker assays for other diseases (e.g. Johne's disease).
In summary, this proposed project has the following objectives:
1. Serum collection from animals bled for the routine Bovigam IFN assay.
2. Preparation and maintenance of serum bank and database of epidemiologincal background data including comparative IFN test results.
3. Testing serum samples with the Enferplex TB test. Testing to be done in parallel at Enfer in Ireland and at VLA Weybridge to establish reproducibilty and robustness of assay.
4. Compare performance of Bovigam and Enferplex TB assay.