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

Abstract

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-␥)

Development of an improved ESAT-6 and CFP-10 peptide-based cytokine flow cytometric assay for bovine tuberculosis

“…Of interest, they were able to detect the presence of both CD4 + and CD8 + T cells in short term cultures. Moreover, they were able to demonstrate that results were similar to those obtained using the ELISA-based IGRA applied in their laboratory, and that Mbv infected animals could be distinguished from animals vaccinated with BCG based on differences in the level of expression of IFN-␥ [7,25].…”

“…Sopp et al [27] detected CD4 + and low numbers of CD8 + T cells producing IFN-␥ in experimentally infected animals, 8-12 weeks post infection. This difference could reflect a change in the immune status of naturally infected cattle over time.…”

“…We included the costimulatory mAbs to CD28 and CD49d to enhance activation of memory T cells requiring an added signal to respond to antigens [24]. We chose 6 h post stimulation as the optimal time for detection of IFN-␥ producing cells in whole blood cultures based on the studies by Sopp et al [25]. The use of selective gating showed cells responding to antigenic stimulation were CD4 + T cells present in the small population of mononuclear cells defined by SSC vs. FSC and not the population of CD4 + T cells present in the large population of mononuclear cells, considered to be activated CD4 + T cells [12].…”

Identification of Surrogates and Correlates of Protection in Protective Immunity againstMycobacterium bovisInfection Induced in Neonatal Calves by Vaccination withM. bovisBCG Pasteur andM. bovisBCG Danish

“…Endsley et al showed that BCG vaccination of cattle induced memory CD4 ϩ T cell populations with the capacity to kill infected macrophages (12) that may play important roles in protective immunity against virulent challenge, providing additional evidence that the quality of the CD4 ϩ T cell response induced by vaccination is a key factor in vaccine success. Postchallenge, the number of antigen-specific IFN-␥-secreting effector CD4 ϩ T cells correlated with the extent of disease, suggesting that this parameter can be used as a surrogate or predictor of disease as well as vaccine failure (29). Postchallenge IFN-␥ measurements in whole blood stimulated by either PPD-B or the M. bovis-specific antigens ESAT-6 and CFP-10 also showed a correlation between extent of disease and IFN-␥ levels.…”

“…For the final 4 h of the incubation period, 10 g/ml brefeldin A (BFA) was added. The expression of CD4 and intracellular IFN-␥ was assessed as previously described (29). The cells were assayed using a flow cytometer (FACSCalibur; Becton Dickinson), and the data were analyzed using FCS Express (De Novo Software, Ontario, Canada).…”