Enhancement of the Sensitivity of the Whole-Blood Gamma Interferon Assay for Diagnosis ofMycobacterium bovisInfections 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); granulocytemacrophage colony-stimulating factor (GM-CSF); antibodies neutralizing IL-10 and transforming growth factor ␤ (TGF-␤); 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-␤ 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.

Neutralization of Interleukin-10 from CD14+Monocytes Enhances Gamma Interferon Production in Peripheral Blood Mononuclear Cells fromMycobacterium aviumsubsp.paratuberculosis-Infected Goats

“…In the present study, neutralization of IL-10 resulted in enhanced IFN-␥ release in infected and exposed goats, confirming the expected suppressive effect of IL-10 on Th1 im- (10) and M. bovis infection (15). It was speculated that Tregs might be the source of IL-10, but this was not confirmed (15).…”

“…It was speculated that Tregs might be the source of IL-10, but this was not confirmed (15). We found that the main IL-10-producing cell type in both infected and healthy goats was CD14 ϩ MHCII ϩ monocytes.…”

“…paratuberculosis-or Mycobacterium bovis-infected cattle, and it has been suggested that the neutralization of IL-10 could be a way of increasing the sensitivity of the IFN-␥ assay since this leads to an antigen-specific increase in the level of IFN-␥ production (10,15). The cells producing IL-10 in those studies were not identified, but it was speculated that regulatory T cells (Tregs) were involved (15). Lately, the role of Tregs in suppressing immune responses has been a focus, and regulatory IL-10-producing CD4 ϩ CD25 ϩ cells from M. avium subsp.…”

Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS

“…Although whole-blood gamma interferon assays have been developed for diagnostic of bovine tuberculosis 32,33 , and depending on the antigens used, these tests can effectively differentiate vaccinated from infected animals 13,14,16 A key aim of this work was to identify proteins that could be used to generate an immunological reagent that could be used to differentiate M.…”

IP-10 release assays in the diagnosis of tuberculosis infection: current status and future directions

“…We have studied MCP-2 and found a lower sensitivity of 71% for active TB at 97% specificity in healthy controls, but no significant added effect by combination with either IP-10 or IFN-g [52]. In a subsequent eval uation in HIV-infected patients with confirmed TB, we observed significant lower sensitivity of MCP-2 compared with IFN-g and IP-10, and found no added effect by combination Tweaking of the incubation step, for example by increasing incubation temperature [49], prolonging the incubation period [23,92,93], improving culture conditions with nutrients [201] or addition of proinflammatory cytokines (e.g., IL-12 [85]), blocking of anti-inflammatory cytokines (e.g., IL-10 [49,94]) or adding 'survival' cytokines (e.g., IL-7 [95,96]), have also shown potential for augmentation of the antigen-specific immune response.…”