Immunity and Immune-Based Diagnostic Tests

20.4.1 Immunological responses

Specific immune responses to MAP infections can be divided into proinflammatory and anti­inflammatory reactions (see Chapter 17, this volume). The proinflammatory process is char­acterized by the production of IFN-γ and other cytokines involved in the cell-mediated responses, which attempt to control the infection by destroy­ing MAP in activated macrophages (Coussens, 2001).

The speed of progression between disease stages is poorly characterized. Some infected cows produce antibodies several years prior to con­tinuous shedding of detectable amounts of MAP, while antibodies may not be detectable in other animals during the early stages of infection, when MAP shedding is minimal. This phenomenon is il­lustrated in Fig. 20.1. It should be noted that this example cannot be considered a definitive under­standing of the antibody response, because the ap­pearance of antibodies is yet to be fully elucidated.

20.4.2 Antigens and antibodies

The choice of antigen for an immune-based test has a major impact on the test result. Antibody reactivity is not well characterized for the ma­jority of MAP-specific antigens available. Koets et al.

Table 20.2. Examples of reported sensitivities and specificities, where the intended target condition and the given meaning differ considerably.

^aThe reported diagnostic sensitivities (Se) and specificities (Sp) are for an antibody enzyme-linked immunosorbent assay (ELISA) based on the MAP021 Oc antigen (Li et al., 201 7), while estimates for more antigens were presented in the paper.

336 S. Saxmose Nielsen

Fig. 20.1. Probability of testing positive using a commercial antibody enzyme-linked immunosorbent assay (ELISA) at various time points relative to the start of MAP shedding.

MAP-derived protein purified derivative (PPD-P) known as johnin, which is the MAP tuberculin. IgGl reactivity to all four antigens was elevated in cows classified as MAP shedders when com­pared with non-infected animals. Only PPD-P was associated with high levels of IgG2 in ‘non- shedders’, ‘MAP shedders’ and animals with ‘clinical paratuberculosis’. PPD-P was also as­sociated with very high levels of IgGl in MAP affected animals. These results are complex and underline the importance of knowing the cen­tral components of a given immune-based test.

20.4.3 Types of tests and sample material

One of the most widely used immune-based tests is the indirect antibody enzyme-linked immuno­sorbent assay (ELISA), which detects antibodies in serum and milk samples. There is a consist­ent level of accuracy across different ELISAs, irrespective of whether they are used with milk or serum samples (Sweeney et al., 1995; Nielsen et al., 2002b). However, differences in the prob­ability of testing positive during lactation exist between the two sample types. A serum ELISA is more likely to yield a positive reaction towards the end of lactation, whereas a milk ELISA is more likely to be positive at the beginning of lac­tation (Nielsen et al., 2002a). These differences can be used to optimize sampling but will not be discussed further in this chapter.

The IFN-γ response after stimulation of a whole-blood sample with MAP-specific antigens (e.g. PPD-P) has also been used in an ELISA- formatted test. Detection of IFN-γ depends on the processes for successful antigen presentation and presence of viable T cells, which are stimu­lated by the antigen to secrete IFN-γ.

20.4.4 Advantages and disadvantages of immune-based tests

The main advantages of immune-based diagnos­tic tests include that they are relatively inexpen­sive, can easily be adapted to high-throughput testing and the results can be available within 1-2 weeks in routine settings. Since antibody ELISA testing is cheaper than most agent­detecting tests, farmers are more likely to test their animals frequently, if frequent testing is required (see Section 20.6.2). The advantage of sampling milk over serum is that the milk has often already been collected for other purposes in milk-recording schemes and is easy to obtain from the animal. Since immune-based tests de­tect host responses to a MAP infection, they should be able to determine that an infection has already occurred and may have persisted. A prerequisite is that the performance of the test has been characterized at the various stages of infection. Repeated testing makes determination of the stage of infection easier.

A major disadvantage of immune-based tests is that they do not provide a direct meas­ure of a MAP infection, infectiousness or of being affected by a MAP infection (see Section 20.2). Therefore, there is a need to correlate the immune response profiles to relevant stages of infection. False-positive reactions occur be­cause in some cases the positive test results do not relate to the purpose of testing. For exam­ple, antibodies can occur prior to shedding of MAP. If the testing aims to determine whether the animal is infectious prior to this shedding, the detection of antibodies will be considered false positive, whereas the animal is actually infected.

False-positive reactions may also occur due to cross-reacting antibodies or laboratory er­rors. It should be noted that vaccinated animals can give a positive reaction to immune-based tests for a considerable period of time (Muskens et al., 2002) without necessarily having a MAP infection. These disadvantages make communi­cation of test results more complex. None the less, with appropriate interpretation, immuno­logical tests may be more reliable than micro­biological tests, depending on the purpose of testing.

20.5