Cytokines as potential biomarkers for the diagnosis of Mycobacterium bovis infection in Mediterranean buffaloes (Bubalus bubalis)

<i>Mycobacterium bovis</i> (<i>M. bovis</i>) is the primary agent of bovine tuberculosis (TB) in Mediterranean buffalo, which has a negative economic impact on buffalo herds. Improving TB diagnostic performance in this species represents a key step to eradicate efficiently this disease. We have recently shown the utility of the IFN-γ assay in the diagnosis of <i>M. bovis</i> infection in Mediterranean buffaloes (<i>Bubalus bubalis</i>), but other cytokines might be useful immunological biomarkers of this infection. We therefore investigated the utility of key immune cytokines as diagnostic biomarkers of <i>M. bovis</i> infection in this species. Thirty-six Italian Mediterranean buffaloes were used in this study: healthy animals (<i>N</i> = 11), infected (IFN-γ test positive, no post-mortem lesions, no <i>M. bovis</i> detection; <i>N</i> = 14), and affected (IFN-γ test positive, visible post-mortem lesions; <i>N</i> = 11). Heparin blood samples were stimulated with bovine purified protein derivative (PPD-B), alongside controls, and 18-24 h later plasma were collected. Levels of 14 key cytokines were measured: IFN-γ, IL-17, IL-4, IL-10, TNF, IL-1α, IL-1β, IL-6, IP-10, MIP-1α, MIP-1β, MCP-1, IL-36Ra, and VEGF-A. We observed that both infected and affected animals released higher levels of IFN-γ, IL-17, IL-10, TNF, IL-1α, IL-6, MIP-1β, in response to PPD-B compared to healthy subjects. <i>Mycobacterium bovis</i> infected animals released also higher levels of IL-1β and IP-10 in response to PPD-B compared to healthy subjects, whereas only tendencies were detected in affected animals. Affected animals only released MIP-1α in response to PPD-B compared to healthy subjects and in this group higher values of PPD-B specific TNF was also observed. Finally, canonical discriminant analysis (CDA) was used to generate predictive cytokine profiles by groups. Our data suggest that IL-10, TNF, IL-1α, IL-6, MIP-1β could be useful biomarkers of TB in Mediterranean Buffalo and can improve the TB diagnostic performance in this specie.