Why is it difficult to develop a test for bovine tuberculosis?
Mycobacteria such as M. tuberculosis, which is the main cause of TB in humans, and M. bovis, the main cause in cattle, have a very complex interaction with their host and its immune system and are very successful pathogens. In fact about one third of the world’s human population are considered by the WHO to be infected with human TB.
Controlling and eradicating the disease in cattle and other species, caused by M bovis, is proving very difficult, there are many reasons for this, but the foremost being due to complex and not fully understood interrelationships in infection transmission between cattle and other host species and their environment and the complexity of the host response to disease, which in turn, leads to problems with developing accurate diagnostic tests.
Testing for disease can be based on either detecting the immune responses resultant from infection, these immune responses coming either from cellular responses, cell mediated immune responses (CMI) or antibody response, or via detecting the organism responsible, M bovis, itself.
It is very difficult to develop a ‘gold standard test for bTB which will sensitively and specifically detect all stages of infection, from just after the organism invades the body right up until the late stages of advanced disease. Tests for immune responses to TB, rather than directly for the organism itself, have the greatest flexibility and utility for detecting TB. However, immune responses to TB are very complex making good tests difficult to develop.
The main reasons for this are:
The immune responses to M. bovis, are complicated and variable throughout the course of infection. CMI responses are considered the principal response however antibody (properly termed “humoral”) responses are increasingly being recognised as important. How the immune system decides whether to deploy cell mediated responses or antibody responses, and how the bacteria modulates this, is discussed here.
There are at least 120 Mycobacterial species in the environment and they are very homogenous with very similar structural components. The genome has about 4000 genes and these show a high degree of similarity with those of M bovis, and the proteins recognised by the immune system can be very similar too. Differentiation between types is very difficult for the immune system to achieve, and this creates difficulties for developing good diagnostic tests. Recent studies have shown that to make an effective diagnostic test for TB you need to be able to test for responses to several TB antigens at the same time. Conventional testing methods do not permit this due to reasons of cost and complexity.
The biology / pathology of mycobacteria – M bovis is very slow growing, clinical signs can take months/years to develop and the bacteria are thought to be able to become latent (where infection remains in a dormant state). During this phase immune responses are at a low level and to test for the infection means that you have to stimulate the immune system to amplify the response. This has been a cornerstone of CMI testing which routinely involves stimulation with antigen, either directly injected into the skin or incubated with cells in vitro. This ‘recall’ method of testing has not been routinely used with tests for antibody.
Cell Mediated and Antibody Humoral Immune responses in TB infections
The immunological responses to infection by Mycobacterium bovis (M bovis) are highly complex. The following is a basic highly simplified explanation of how the immune response is modulated.
Following infection by M bovis, the bacteria is attacked by specific white blood cells termed phagocytes. Phagocyte is a term to describe different types of white cells that essentially share the same function, which is to absorb breakdown harmful antigens or other dead cells in the body. White cells that fall within this classification include neutrophils, monocytes, macrophages, dendritic and mast cells.
Phagocytes are drawn to the infected areas by chemicals that come from either the antigen or other phagocytes already present in the area of infection. The way phagocytes move in response to chemical stimulation is known as chemotaxis. Chemokines are a type of small signalling protein secreted by these cells to activate other nearby white cells, their function is to direct the movement of cells to the point of invasion. This is the basis of the skin test (SICCT) where the presentation of antigen, in the form of tuberculin within the skin, results in the attraction of white blood cells to the site of injection and thereby creates a lump.
When a phagocyte detects M bovis it will bind to it and then it begins a process of wrapping around the bacteria until it is completely engulfed inside the phagocyte, in doing this the phagocyte create a membrane around the antigen, this structure being termed a phagosome. The bacteria are able to and like to live in the phagosome, however then the host starts to secrete acid to lower the pH in the phagosome and the phagosome fuses with lysosomes to form phagolysosomes.
Protein dissolving enzymes which are optimally functional at low pH are secreted into the phagolysosome, and the bacteria antigens are digested into short peptides. The phagolysosome then fuses with another cell vesicle which contains newly synthesised MHC class II molecules. The bacterial peptides bind to the MHC class II groove to form a peptide:MHC class II complex. These complexes are taken to the cell surface in these vesicles and expressed. Available T helper cells recognise these peptide/MHC class II complexes and become activated.
If these complexes are expressed to T-helper cells where the bacteria has been killed within the phagocyte, then the T-helper cells will “assume” that the infection is under control and direct the immune response to further cell mediated responses by releasing cytokines to the phagocytes and other immune cells that perform this cell-mediated immune function (these cells collectively termed T-cells). The principal cytokine T-helper cells release to affect this response being gamma-interferon. The gamma-interferon test is therefore based on detecting this release of this cytokine when M bovis antigens are added to white blood cells.
However if antigens to M bovis, that have been detected and picked up from outside the phagocytes, (i.e. the bacteria / antigen is extra-cellular), are presented to T-helper cells by macrophages (especially B-cells) then the T-helper cells “assume” that if infection is occurring in a free uncontrolled manner extra-cellularly then it is “of control” and will need to deploy antibodies to fight this extra-cellular infection. Therefore, instead of promoting cell mediated responses by release off gamma-interferon, it will release different cytokines (interleukins) that stimulate B-cells to produce antibodies. This antibody production is then what can be detected by serological tests as with the Enferplex Bovine TB test.
How M Bovis subverts this response
As stated, M bovis likes to live within the phagocytes’ phagosome. It is in essence its target habitat. However, it is it to survive (in a state of dormancy) and or replicate (and thereby subsequently cause disease) within the phagocyte it needs to prevent the processes described above that allow the phagocyte to “dissolve” the bacteria. The principal means it does this is by preventing the acidification of the phagosome and, also, its subsequent fusion with lysosomes.
Furthermore, as M bovis wants to live in macrophages the bacteria “fishes” for them. It does this by stimulating macrophage chemotaxis towards it by inducing the formation of ‘antigen export vesicles’ containing mycobacterial antigens, these vesicles bud off the phagosome and are carried to the cell surface where the antigens are released.
Once released these extracellular antigens can be taken up by B cells and presented to T helper cells which will in turn stimulate further macrophages towards the infected macrophage, this mechanism being the basis for the classic tubercle formation. However, a further effect is that, as the antigen is detected extra-cellularly it also results in activation of B cells for antibody production.
Disease progression and antibody production
It is well accepted, within tuberculosis and other mycobacterial infections, that as antibodies are produced as a response to free uncontrolled infections as opposed controlled intra-cellular infections, then as disease progresses them the greater the levels of antibody responses that are seen. The most likely familiar example of this is Johne’s disease where the high antibody animals are considered to have the most advanced disease.
Therefore in advanced infections this explains why we tend to see high antibodies but low responses to cell-mediated tests. This dynamic is the basis for how the Enferplex test can identify animals that are most likely to have lesions.
The boosting effect of Tuberculin
The effect of prior administration of tuberculin on the sensitivity of the test
It is well understood and recognised that prior administered prior to sampling results in a memory response that causes an increase in production of antibody in animals that are infected with M bovis, such that sensitivity of the test is enhanced if sampling occurs after a skin test. This boosting effect is technically termed the ‘anamnestic response’ and is observed between 5 and 30 days post injection of the tuberculin.
Does tuberculin testing affect the specificity of the test?
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 on 160 cattle to check whether this concern has any basis and have found NO effect of the multiple administration of tuberculin (4 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. This work has not been performed in any other species and therefore we cannot definitively confirm this conclusion in other species.