Flow Cytometric Detection of Gamma Interferon Can Effectively DiscriminateMycobacterium bovisBCG-Vaccinated Cattle fromM. bovis-Infected Cattle

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that is increasing in incidence in United Kingdom cattle herds. In addition to increasing economic losses, the rise in bovine tuberculosis poses a human health risk. There is an urgent requirement for effective strategies for disease eradication; this will likely involve vaccination in conjunction with current test and slaughter policies. A policy involving vaccination would require an accurate diagnosis of M. bovis-infected animals and the potential to distinguish these animals from vaccinates. Currently used diagnostic tests, the skin test and gamma interferon (IFN-gamma) blood test, have a sensitivity of up to 95%. A further complication is that M. bovis BCG-vaccinated animals are also scored positive by these tests. We tested the hypothesis that the quantification of IFN-gamma-producing lymphocytes by flow cytometric analysis of intracellular IFN-gamma expression would provide a more accurate discrimination of M. bovis-infected animals from BCG vaccinates. Significant numbers of IFN-gamma-expressing CD4+ T cells were detected following culture of heparinized blood from M. bovis-infected animals, but not from BCG vaccinates, with purified protein derived from M. bovis (PPD-B) or live mycobacteria. Only 1 of 17 BCG-vaccinated animals had a significant number of CD4+ T lymphocytes expressing IFN-gamma, compared with 21/22 M. bovis-infected animals. This assay could allow an accurate diagnosis of M. bovis and allow the discrimination of BCG-vaccinated cattle from infected cattle.