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Clinical and Vaccine Immunology, November 2007, p. 1483-1489, Vol. 14, No. 11
1071-412X/07/$08.00+0     doi:10.1128/CVI.00291-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Enhancement of the Sensitivity of the Whole-Blood Gamma Interferon Assay for Diagnosis of Mycobacterium bovis Infections in Cattle

AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand,¹ Veterinary Laboratory Agency, Weybridge, Surrey, United Kingdom²

Received 17 July 2007/ Returned for modification 5 September 2007/ Accepted 7 September 2007

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

Published ahead of print on 19 September 2007.