Sensitivity – The probability that if the animal has the disease that the test will be positive.
Specificity – The probability that if the animal does not have the disease that the test will be negative.
Tests for cell mediated immune responses
The intra-dermal test utilises tuberculin which is a purified protein derivative (PPD), a complex preparation obtained from filtrates of sterilized, concentrated cultures of Mycobacterium bovis. The injection of this into the skin of animals exposed to bTB results in an inflammatory reaction in which TB-specific memory T cells are attracted into the skin and multiply in response to the PPD antigen. The use of avian and bovine tuberculin allows the differentiation between bTB and other Mycobacterial species, principally avian TB, thereby significantly improving the specificity.
The advantage of this test lies in its excellent specificity (generally considered around 99.98%) which makes it very suitable for herd level testing. However, it is now considered to have poor sensitivity. Cell mediated responses may wane and not be present during later stages of the disease (termed anergy). This is a serious issue in endemic areas of bTB, where it is recognised that the sensitivity of the test can fall considerably.
This test is based on measuring the response to PPD by T memory cells in blood. These cells are known to be critical factors in the body’s immune responses to pathogens due to their ability to retain memory of the infection over a long period of time and by fighting the infection directly. When stimulated by tuberculin T cells release interferon gamma (IFN-γ) along with a number of other cytokines to fight the infecting organism. Specific antigens on the infecting organism have to be recognized by the T cells to initiate the release of IFN-γ. The test therefore relies on artificially stimulating these cells in blood samples with these specific antigens. If the T memory cells of an animal with an M. bovis infection encounter these proteins they will respond by releasing interferon gamma which can then be measured and detected.
The test is considered to have better sensitivity than the SICCT, presumably due to its ability to detect infection earlier, but suffers from poorer specificity. In cattle, sensitivity and specificities have been estimated at 88.3% (range 88-99%) and 96.6% (range 88-99%) respectively.
Antibody responses were traditionally thought to be (a) slow to develop (b) only occurred towards the end stage of the disease process (c) were not present at high enough levels during the protracted ‘latent’ phase of the infection. However, over the last 5-10 years new methods have shown that antibodies are produced early on after infection and can be diagnostically useful if tests are sensitive enough to detect them and the range of pure TB antigens which is used is broad enough.
New technological developments have resolved us to resolve some of these problems. The whole genome of the TB organism has been sequenced and purified proteins and small peptides (fragments of large proteins) have been made in the lab and can be sourced commercially. Furthermore, research using multiplex antigen based assays, which can detect responses to a number of individual antigens at the same time, has shown that antibody activity for different antigens develops at different times during the course of infection. This finding, along with the difficulties of differentiating between species discussed previously, it is now possible to simultaneously detect antibody activity for multiple antigens and achieve the high sensitivity and specificity levels which are needed for reliable TB diagnosis.
The advantages of our serological testing system for bovine TB include:
The tests are easy to perform, the analysis can be performed by any competent appropriately equipped laboratory, and they are cheap, quick and give an objective result
- As it has no effect on the immune responses of the animal, the tests can be repeated without delay thereby allowing rapid repeat testing to detect or confirm infection
- Responses begin early in infection and tend to increase over time and correlate with pathology and shedding of bacteria.
For details of the performance of the Enferplex test click here
Boosting with antigen
When the antibody test is performed without any prior skin test, it relies on the presence of detectable antibody in already present in blood, and therefore unlike the skin and interferon gamma tests it is not being used as a memory recall test. However, it has been found that antibody responses usually receive a boost from the PPD-B given in following skin testing. This is most probably due to B memory cells (B cells are the lymphocytes that secrete antibody) becoming activated by the PPD-B “boost” to secrete antibody. In this way, the test is converted into a “memory recall test” and the sensitivity of the test can be increased accordingly.
There has been a postulated concern that the administration of tuberculin itself induces the production of antibody by the animal and therefore animals that have not met the disease could be found as positive by antibody tests. We have conducted a large trial to check whether this concern has any basis and have found NO effect of the multiple administration of tuberculin (5 short interval tests) on the likelihood that a bovine Tb free animal will test positive, i.e. there is no effect on the specificity of the test 1.
1 Rhodes, S.G., Holder, T., Clifford, D., Dexter, I., Brewer, J., Smith, N., Waring, L., Crawshaw, T., Gillgan, S., Lyashchenko, K., Lawrence, J., Clarke, J., de la Rua-Domenech, R. and M. Vordermeier. 2012. Evaluation of gamma interferon and antibody tuberculosis tests in alpacas. Clinical and Vaccine Immunology, 19(10): 1677-1683.