Enhancement of the sensitivity of the whole-blood gamma interferon assay for diagnosis of Mycobacterium bovis infections in cattle

In this study, we determined if the sensitivity of the currently available in vitro test to detect bovine tuberculosis could be enhanced by adding the following immunomodulators: interleukin-2 (IL-2); granulocyte-macrophage colony-stimulating factor (GM-CSF); antibodies neutralizing IL-10 and transforming growth factor β (TGF-β); mono-methyl-L-arginine, which blocks nitric oxide production; and L-methyl-tryptophan, which interferes with the indoleamine dioxygenase pathway. Blood was obtained from uninfected control cattle, experimentally infected cattle, cattle responding positively to the skin test in tuberculosis-free areas (false positives), and cattle naturally infected with Mycobacterium bovis from New Zealand and Great Britain. Gamma interferon (IFN-γ) responses to bovine purified protein derivative (PPD-b), avian purified protein derivative, and a fusion protein of ESAT-6 and CFP-10 were measured. Mono-methyl-L-arginine, L-methyl-tryptophan, or an antibody neutralizing TGF-β had minimal impact on IFN-γ production. IL-2 and GM-CSF promoted IFN-γ release whether antigen was present or not. In contrast, adding an antibody against IL-10 enhanced only antigen-specific responses. In particular, addition of anti-IL-10 to ESAT-6/CFP-10-stimulated blood cultures enhanced the test sensitivity. Furthermore, whole blood cells from field reactors produced substantial amounts of IL-10 upon stimulation with PPD-b or ESAT-6/CFP-10. Testing "false-positive" cattle from tuberculosis-free areas of New Zealand revealed that addition of anti-IL-10 did not compromise the test specificity. Therefore, the use of ESAT-6/CFP-10 with anti-IL-10 could be useful to detect cattle potentially infected with tuberculosis, which are not detected using current procedures.