Cytokine and Chemokine Responses of Peripheral Blood Mononuclear Cells from Dogs Infected with Mycobacterium bovis

Mycobacterial infections are an important emerging zoonosis in companion animals for which diagnostic options remain imperfect, and the canine immunological response to these infections has been poorly investigated. We sought to further define the cellular response of peripheral blood mononuclear cells (PBMCs) from dogs infected with <i>Mycobacterium bovis</i>, as determined using a commercial interferon-gamma response assay (IGRA). To this end, PBMCs from healthy or infected dogs were collected. Serum samples were tested to further classify dogs as seropositive or seronegative for circulating antibodies against <i>M. bovis</i> using the DPP^® VetTB Assay, Idexx <i>M. bovis</i> antibody ELISA, and a novel purified protein derivative ELISA. Isolated PBMCs were stimulated with mycobacterial proteins (PPDB or ESAT-6/CFP-10), and 13 cytokines/chemokines were measured in the supernatant. These concentrations were determined using the CYTOMAG-90K MILLIPLEX MAP Canine Cytokine/Chemokine system. PBMCs from infected dogs released IFN-γ in response to stimulation, but this response was reduced in those that had seroconverted. Similarly, cells stimulated with PPDB secreted increased amounts of TNF-α when dogs were seronegative, but cells taken from seropositive dogs did not. Finally, the IL-18 response of seropositive dogs was reduced compared to those that were seronegative in response to PPDB, potentially suggesting that these dogs have a reduced macrophage functionality. This work demonstrates that the inflammatory cytokine response may wane following seroconversion with deleterious consequences for the host response. Overall, combining IFN-γ and TNF-α assessment during diagnosis may increase IGRA sensitivity, whilst further work is needed to better understand the prognostic and diagnostic implications of seroconversion in dogs.