Development and evaluation of a Mycobacterium bovis interferon-γ enzyme-linked immunospot (ELISpot) assay for detection of bovine tuberculosis.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important zoonotic disease. This infection is difficult to control because of the limited ability of the tuberculin skin test (TST) and ancillary IFN-γ release assay to detect all infected animals. In this study, we aimed to develop an efficient assay based on the enzyme-linked immunospot (ELISpot) technique for the diagnosis of bTB, with IFN-γ monoclonal antibodies 3E9 and Bio-labeled 6F8 used as capture and detection antibodies, respectively. As expected, there were significantly more M. bovis-specific spot-forming units (SFU) in bTB-infected cattle than in healthy cattle when an M. bovis-specific antigen, CFP-10-ESAT-6 fusion protein (CE protein), was used. The M. bovis IFN-γ ELISpot assay demonstrated a high level of agreement (90.83%) with the BOVIGAM ELISA test (Thermo Fisher Scientific) for detecting bTB. Furthermore, 3 of 109 cattle tested negative by both the TST and the BOVIGAM ELISA tests, but positive by the ELISpot assay (TST- ELISA- ELISpot+). During subsequent long-term monitoring, these 3 cattle became TST+ ELISA+ ELISpot+. These results suggest that the M. bovis IFN-γ ELISpot assay we established could detect infected cattle earlier than the BOVIGAM ELISA test.

Rapid loss of early antigen-presenting activity of lymph node dendritic cells against Ag85A protein following Mycobacterium bovis BCG infection.

BACKGROUND: Control of Mycobacterium tuberculosis (Mtb) infection requires CD4+ T-cell responses and major histocompatibility complex class II (MHC II) presentation of Mtb antigens (Ags). Dendritic cells (DCs) are the most potent of the Ag-presenting cells and are central to the initiation of T-cell immune responses. Much research has indicated that DCs play an important role in anti-mycobacterial immune responses at early infection time points, but the kinetics of Ag presentation by these cells during these events are incompletely understood. RESULTS: In the present study, we evaluated in vivo dynamics of early Ag presentation by murine lymph-node (LN) DCs in response to Mycobacterium bovis bacillus Calmette-Guérin (BCG) Ag85A protein. Results showed that the early Ag-presenting activity of murine DCs induced by M. bovis BCG Ag85A protein in vivo was transient, appearing at 4 h and being barely detectable at 72 h. The transcription levels of CIITA, MHC II and the expression of MHC II molecule on the cell surface increased following BCG infection. Moreover, BCG was found to survive within the inguinal LN DC pool, representing a continuing source of mycobacterial Ag85A protein, with which LN DCs formed Ag85A peptide-MHCII complexes in vivo. CONCLUSIONS: Our results demonstrate that a decrease in Ag85A peptide production as a result of the inhibition of Ag processing to is largely responsible for the short duration of Ag presentation by LN DCs during BCG infection in vivo.