The Gamma Interferon (IFN-γ) Assay
The gamma interferon (IFN-γ) assay is one of the diagnostic tests for bovine tuberculosis approved by the OIE (Wood and Jones 2001; Gormley et al. 2006). The IFN-γ assay is based on the same cell-mediated immune response as the TSTs and measures the production of IFN-γ by sensitized lymphocytes exposed in vitro to PPD or similar cocktails of antigens.
Compared to individual animal Se, the test also has a higher herd Se (Praud et al. 2015). The results vary accordingly when the test is used as a serial test to enhance Sp or done in parallel to enhance Se (Schiller et al. 2010a). Serial testing is advised in those areas where the prevalence of BTB is low, and frequent cross-reactions occur (Praud et al. 2015). Cross-reactions are caused by environmental mycobacteria other than M. avium that is the usual component of avian PPD used to detect non-specific, false-positive reactors (Gormley et al. 2013). Inclusion of the ESAT6/CFP10 cocktail antigen (Vordermeier et al. 2001; Pollock et al. 2003), PPD-N (M. nonchromogenicum), tuberculin derived from Mycobacterium fortuitum, Rv3615 (ESpC), and Rv0287 (esxG) and ESAT6/ CFP10 were immunogenic in the infected cattle and distinguished infected cattle from the non-infected NTM-exposed animals. Recombinant mycobacterial antigens (R-Mags) derived from NTM mycobacteria and heat shock proteins (HSP) may also be beneficial in attempting to more accurately identify true positive reactors and to reduce the number of false positives (Jenkins et al. 2018).Briefly, the assay consists of two phases. Heparinized whole blood is incubated with different sets of antigens (normally bovine PPD and avian PPD for comparative purposes) and a control for about 18-24 h, and the amount of IFN-γ released by sensitized lymphocytes is assessed with a sandwich ELISA (Wood et al. 1991) or purified lymphocytes from peripheral blood, using the enzyme-linked immunospot (ELISPOT) (Maas et al.
2013). The test is easier to use than the skin test as it is necessary to immobilize cattle only once for the purpose of blood collection. The main problem with using the test is its cost (40-60 ˆ per test), and the need to process the blood in a laboratory within at least 8 but not more than 30 h after collection because of the progressive decrease in secretion of IFN-γ by lymphocytes during the course of that time.The IFN-γ assay has been approved for diagnostic and trade purposes in cattle by the OIE. It has been used as an ancillary test in Australia since the 1980s and in Europe from 2000 onward. It suffers from the same limitations as the SIT and the CCT, in that it lacks Sp and Se. In comparison to the SIT, the two tests detect different subpopulations of infected animals, and doing the two tests in parallel improves the Se and more positive reactors are detected than when used in isolation. The IFN-γ assay is more sensitive than the TSTs with a range of 96.2-98.1% (Wood et al. 1991; Wood and Jones 2001); it is also more Se than the CCT and is as specific (Schiller et al. 2010b).
The assay is commonly used as an ancillary test in most countries in which it is applied, either as a parallel or as a serial test. As with the TSTs, its specificity is influenced by a number of factors, including infection with nonpathogenic mycobacteria. It is also more specific (96.9%) in a TB-endemic zone compared to a TB-free zone (≤90.8%) (Palmer and Waters 2006; Anon 2007). The assay should not be used as a screening test for routine surveillance in low prevalence areas as it lacks sufficient Se, and with an increasing herd size, there is an increase in sensitization by environmental mycobacteria and consequently the number of non-specific reactors.
As with the SIT and the CCT, interpretation of the test remains somewhat subjective, and one should expect a degree of variation in interpretation depending on different operators, the variations in cut-offs used, the antigens used, varying test interpretation criteria, and epidemiological variables (Praud et al.
2015). Advantages of the use of IFN-γ in Egypt were confirmed. Here there were indications that using the test in parallel with the CCT was more effective, but that under their circumstances, financial constraints forced the use of it as a serial test (Abdellrazeq et al. 2014).Disadvantages of the IFN-γ test include inadequate specificity, the logistical demands such as the time limit within which the test must be done, access to laboratory facilities and trained staff, the presence of non-specific reactions, the variation of potency of the PPDs used, and its high cost (Schiller et al. 2010a).
The advantages of using the IFN-γ assay include its increased sensitivity, being able to do more repeat testing, no need to visit the farm and handle the animals for a second time to read the test, and more objective procedures when reading the test results. Further advantages of the IFN-γ assay over TST include that, as there is no interference with the immune status of the host, there is no waiting period required between IFN-γ assays, thereby creating the opportunity for more rapid repeat testing. The test performance is also not substantially affected by the experience of field staff, and more objective testing schemes and interpretation of results are possible in comparison to the TSTs. Finally, the IFN-γ assay offers improved sensitivity and has the capability of detecting early infections (parallel use with TST therefore results in increased overall diagnostic sensitivity) (DAFF 2016; Schiller et al. 2010a; Wood and Jones 2001).
However, one significant drawback with regard to the use of the IFN-γ assay compared to the TSTs, is its high cost. With the exception of South Africa where the use of the IFN-γ assay is an integral part of the BTB control strategy in buffaloes (DAFF 2016), its high cost has led to the limited use of the test in Africa, and for this reason, it has never been used in many parts of Sub-Saharan Africa.
9.4.1 The Use ofIFN-γ in Wildlife in Africa
Against the background of the known M. bovis infection in a range of wildlife species in Africa, few tests have been validated to diagnose the disease or to do surveillance to determine their role in the epidemiology of the disease. Reference in the section on tuberculin skin tests has been made to buffaloes and lions, but there are no published data about any of the other species. Some work has been done in a number of species to assess the use of the INF-γ assay or its modifications in an attempt to validate them according to the OIE guidelines.
In South Africa, the assay has been used fairly extensively for detecting BTB in free-ranging African buffaloes and lions. In buffaloes the IFN-γ assay and the CCT had a comparable Se and Sp (Raath et al. 1995; Grobler et al. 2002). Due to the misclassification as positive reactors (as much as 9.3%) due to presensitization with environmental mycobacteria in certain areas of South Africa, the assay was modified by the additional stimulation with crude proteins extracted from M. fortuitum cultures. When used as a stand-alone test for the detection of infected buffaloes, the results obtained appeared to have satisfactory Se under field conditions (Michel et al. 2011). The BOVIGAM® 1G assay had the highest Se, and when used with a number of ancillary tests, its Se approaches 100% (van der Heijden et al. 2016). Recent improvements increased the Sp of the assay by targeting specific antigens such as ESAT-6 and CFP-10. Currently different assays, such as BOVIGAM® EC and HP, are available commercially, and they appear to be more Se than the CCT. The modified QuantiFERON-TB Gold assay, a modified human assay that utilizes ESAT-6, CFP-10, and TB 7.7 (Parsons et al. 2011), has also been assessed, but it is less sensitive than the other tests that are now used (Goosen et al. 2014).
Recently a sensitive diagnostic gene expression assay was developed for lions allowing discrimination between M. bovis-infected and -uninfected lions. This test measures the amount of monokine induced by IFN-γ mRNA measuring the degree of sensitization by ESAT-6/CFP10 (Olivier et al. 2017).
A lot of work needs to be done, particularly in those African wildlife species that are vulnerable and face extinction. In Africa, rhinoceroses are on the verge of extinction, and since the more recent confirmation of M. bovis infections in this species (Espie et al. 2009), it has become even more important to develop a reliable ante-mortal diagnostic test for them. Recent work has gone a long way in adapting the IFN-γ assay for them by developing a rhino-specific IFN-γ, the RhIFN-γ (Morar et al. 2007). This test in BTB-negative white rhinos was 100% Sp (Morar et al.
2013), but further work is required to confirm its Se and Sp in BTB-infected rhinos.
9.5