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

Abstract

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 beta (TGF-beta); 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-gamma) 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-beta had minimal impact on IFN-gamma production. IL-2 and GM-CSF promoted IFN-gamma 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.

FIG. 1.

Modulation of IFN-γ production by anti-IL-10 in naturally infected cattle from Great Britain. Blood samples from 31 field reactor cattle from herds with culture-confirmed bovine tuberculosis were stimulated as described in the text for New Zealand cattle with PPD-a, PPD-b, and the ESAT-6/CFP-10 fusion protein in the absence (−) or presence (+) of MAb neutralizing bovine IL-10. Antigen-specific IFN-γ responses, determined by BOVIGAM ELISA, are shown, with medium control values subtracted (ΔIFN-γ).

FIG. 2.

Antigen-specific IL-10 and IFN-γ production in field reactors from Great Britain. PBMC, prepared from 24 naturally infected cattle, were incubated with PPD-a and PPD-b (10 μg/ml), a cocktail of ESAT-6 and CFP10 proteins (5 μg/ml each), or staphylococcal enterotoxin B (SEB) (1 μg/ml). Supernatants were collected after 48 h of incubation and assessed for antigen-specific IL-10 (A) or IFN-γ (B) production. The results are expressed as mean antigen-specific responses plus standard errors of the mean with background (medium control) values subtracted (ΔIL-10 and ΔIFN-γ).