Efficacy of Cytokine Flow Cytometric Assay to Differentiate Between Tuberculosis Infected and BCG Immunized Cattle.

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that is increasing in developing countries. 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-γ) 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 assessed the possibility of using the quantification of IFN-γ -producing CD4+T lymphocytes by Cytokine Flow Cytometric analysis of intracellular IFN-γ expression for discrimination of M. bovis-infected animals from BCG- immunized in Egypt. Heparinized blood was collected from 2 infected dairy cattle, 2 BCG-immunized cattle and 2 control cattle. Blood was stimulated in the presence of anti-CD49d, anti-CD28 (1 μg/ml) and activated with PPDb (20 ug/ml) and PPDa (20 ug/ml). PMA (50 ng/ml) and ionomycin (1μg/mL) were used as a positive control. Blood was incubated at 37°C in 5% CO2 for 6 hours. After 2 hours, Brefeldin A (10μg/mL) was added. Cells were stained with CD3, CD4, CD45R0, CD69 and IFN-γ antibodies. Two flow cytometer (FC) gating strategy were designed for data acquisition and data were analyzed using the De Novo Software. Significant numbers of IFN-γ -expressing CD4+ T cells were detected following culture of heparinized blood from M. bovis-infected animals, but not from BCG –immunized with purified protein derived from M. bovis (PPDb). The finding suggested that this assay could allow the discrimination of BCG-immunized cattle from infected cattle. It is recommended to evaluate this discrimination technique on large number of animals to deduce dependable results.