Exploring alternative cytokines as potential biomarkers for Mycobacterium bovis infection in cattle

<i>Mycobacterium bovis</i> (<i>M. bovis</i>) is the primary agent of Bovine tuberculosis (bTB) in cattle. It represents both a threat to human health and the cattle industry worldwide. Improving bTB diagnostic performance in cattle represents a key step in eradicating the disease. The interferon-gamma (IFN-γ) release (IGRA) blood assay is routinely used in the diagnosis of <i>M. bovis</i> infection, but additional cytokines might be useful as biomarkers of this infection in cattle. In our study, we evaluated the utility of sixteen immune cytokines as diagnostic biomarkers of <i>M. bovis</i> infection. Fifty-five cattle were used in this study: healthy animals (N = 19), infected (IFN-γ test positive, no post-mortem lesions; N = 17), and affected (IFN-γ test positive, visible post-mortem lesions; N = 19). Heparin blood samples were stimulated <i>in vitro</i> with bovine purified protein derivative (PPD-B), alongside controls. After 18-24 h of incubation, plasma were collected and levels of 16 key cytokines were measured: IL-1α, IL-4, IL-6, IL-10, IL-17, IL-36Ra, MIP-1α, IP-10, MCP-1, TNF, VEGF-A, IFN-γ, IL-23, IL-27, IL-35, and THBS-1. We observed that both <i>M. bovis</i> exposed cattle (both infected and affected) released higher levels of PPD-B specific IFN-γ and IP-10. On the contrary, only cattle belonging to the affected group released higher levels of PPD-B specific IL-4, IL-17, and TNF compared to healthy subjects. Canonical discriminant analyses (CDA) indicated that IP-10, IL-4, IL-17, and TNF could be useful biomarkers for infection status. In particular, our data suggest that the parallel measurement of IFN-γ and IP-10 might improve the diagnosis of <i>M. bovis</i> infection in cattle in terms of sensitivity and specificity, although this should be validated on a larger set of animals. In the CDA analysis, only a modest separation between infected and affected cattle was observed. Nevertheless, our data suggested that IL-4, IL-10, and TNF might improve, at least in part, the differentiation of cattle in diverse stages of TB infection. Overall, the data generated in our study provide a foundation to improve the diagnosis and staging of <i>M. bovis</i> in cattle.