SureFarm providing Bovine Tuberculosis Diagnostics using the Enferplex test to the animal health industry

Sensitivity and Specificity Bovine Tuberculosis Tests

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

 SICTT

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 bovine TB (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 99.5%) which makes it very suitable for herd level testing. However, in camelids in particular, it suffers from very poor sensitivity, estimated at around 10-15%. 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. Why camelids are particularly poor in responding to the test is not understood. A final important factor in the use of the test in the camelids is the difficulty in administering the PPD and reading the test, particularly in thin skinned younger animals.

 Gamma-Interferon (IFN)

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. In camelids, sensitivities of up to 80% were observed in the BAS trial.  However at this level of sensitivity the specificity was only 80.9%, that is in a population of 100 TB-free animals about 20 would wrongly be classified as positive by the test Setting the specificity higher resulted in a significant detriment of sensitivity (34.6% (Rhodes et al, 2012). This, and the fact that the test is expensive and requires specialised laboratory facilities, sample collection, storage and transport makes it impractical in camelids.

 Serological Tests

The use of serology using the conventional methods which have long been available for the diagnosis of TB has been of limited value. 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 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. The availability of these proteins/peptides has allowed a resurgence in TB antibody testing in recent years. 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.

Other advantages of serology testing 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.

To understand why the Enferplex test has been chosen for testing in camelids, please click here.

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.