CVI 00291 - 07 ) SECOND VERSION : SEPTEMBER 7 TH 2007 1 2 3 4 Enhancement

23 In this manuscript, we determined if the sensitivity of the currently available in vitro 24 test to detect bovine tuberculosis could be enhanced by adding the following 25 immunomodulators: interleukin-2 (IL-2), granulocyte-macrophage colony stimulating 26 factor (GM-CSF), antibodies neutralising interleukin-10 (IL-10) and transforming 27 growth factor beta (TGF-β), mono-methyl-L-arginine that blocks nitric oxide (NO) 28 production, or L-methyl-tryptophan that interferes with the indoleamine dioxygenase 29 pathway. Bloods were obtained from uninfected control cattle, experimentally30 infected cattle, cattle responding positively to the skin test in tuberculosis-free areas 31 (false positives) and cattle naturally-infected with Mycobacterium bovis from New 32 Zealand and Great Britain. Interferon-gamma (IFN-γ) responses to bovine purified 33 protein derivative, avian purified protein derivative and a fusion protein of ESAT-6 34 and CFP-10 were measured. Mono-methyl-L-arginine, L-methyl-tryptophan, or an 35 antibody neutralizing TGF-β had minimal impact on IFN-γ production. IL-2 and GM36 CSF promoted IFN-γ release whether antigen was present or not. In contrast, adding 37 an antibody against IL-10 enhanced only antigen-specific responses. In particular, 38 addition of anti-IL-10 to ESAT-6/CFP-10-stimulated blood cultures enhanced the test 39 sensitivity. Furthermore, whole blood cells from field reactors produced substantial 40 amounts of IL-10 upon stimulation with PPD-b or ESAT-6/CFP-10. Testing “false 41 positive” cattle from tuberculosis-free areas of New Zealand revealed that addition of 42 anti-IL-10 did not compromise test specificity. Therefore, the use of ESAT-6/CFP-10 43 with anti-IL-10 could be useful to detect cattle potentially infected with tuberculosis, 44 which are not detected using current procedures. 45