## 3 Discussion  
This is the first report of identification of volatile organic compounds obtained from the breath and feces of wild boar in order to distinguish between MTBC-positive and MTBC-negative boar. This novel approach to detect MTBC infection in free-ranging wild boar has the potential to be a reliable and efficient complement as a diagnostic screening tool for disease surveillance in this species, as well as in other affected wild animal populations [5; 11; 12]. We identified significant VOC in the breath and feces of MTBC-positive and MTBC-negative wild boar of three age classes from Donana National Park, Spain. When applying classification models to the juvenile animals, for whom several significant VOC were identified, we were able to distinguish between MTBC-positive juvenile wild boar and MTBC-negative juvenile wild boar using breath VOC or fecal VOC. During mycobacterial infection, the predominant cellular immune responses lead to cellular destruction via release of cytokines and highly reactive species resulting in tissue damage related to oxidative stress. Overproduction of highly reactive oxygen intermediates and reactive nitrogen radicals leads to disruptive cellular damage to tissue membranes, cleaving of proteins, accumulation of lipids, mainly polyunsaturated fatty acids (PUFA), and excessive mitochondrial respiration. In animals,  
Figure 3: Classification between MTBC-positive and MTBC-negative animals achieved by the discriminant functional analysis (DFA) models built with **(a)** three breath VOC biomarkers (JBVOC 11, JBVOC 13, and JBVOC 14, shown in Table 3) and **(b)** two fecal VOC biomarkers (JFVOC 01 and JFVOC 03, shown in Table 4). Left panels: Box plots of the first canonical variable (CV1) of the DFA models. Each animal is represented by one point in the box plots. The standard deviation of the CV1 values is represented by the error bars, while the boxes represent the 95% confidence interval and the dashed lines represent the threshold classification line between the MTBC-positive and MTBC-negative groups. Right panels: receiver operating characteristic (ROC) curves built with the CV1 of the DFA models. reactive oxygen molecules can be produced from arachidonic acids (such as PUFA) by lipoxygenase enzymes during disease pathogenesis [13]. The majority of compounds identified as significant in juvenile wild boar breath were alkane derivatives (nine compounds), followed by aromatic (benzene) derivatives (three compounds), one alkene, and one ester. Most of the breath VOC identified in juvenile wild boar exhibited enhanced concentrations in MTBC-positive animals, with the exception of three compounds (JBVOC 03, JBVOC 05, and JBVOC 09), whose concentrations were diminished in the breath of MTBC-positive animals. Alkanes and alkane derivatives were previously observed in human breath as VOC products of oxidative stress correlated to active pulmonary tuberculosis, while benzene derivatives were identified as volatile metabolites of _M. tuberculosis_ in vitro [6; 7]. Among the alkane derivatives, JBVOC 10 to JBVOC 14 were present in the breath of all MTBC-positive animals, but in the breath of only 30% of MTBC-negative animals. Alkane derivatives such as 3-methylpentane, decane, and heptacosane are thought to originate as byproducts of lipid peroxidation during MTBC pathogenesis [13]. A structural isomer of 3-methylpentane, 2-methylpentane, was also found in the breath samples of mice infected with _M. bovis_, but significant difference was not shown compared to uninfected controls [14]. On the other hand, although the concentration of decane in MTBC-infected animals depicts downregulation, it might be readily adsorbed onto cell membranes upon production and follow further metabolic bioconversion to lower the molecular weight VOC and/or alkane derivatives.