VOCs detected in biological matrixes (breath, feces, skin, etc.) contain metabolites of the infectious pathogen or compounds resulting from the interaction of the host organism with the pathogen (pathophysiological metabolism products), other metabolic products resulting from physiological processes, as well as combinations of both .
The VOCs exhaled through breath reflect a part of the body’s metabolome, and generally come from the blood [14, 15]. Their profile is dependent on the physiological status of the animal, food consumption and internal metabolism, including gastrointestinal microbiota metabolism where substances absorbed through the intestinal mucosa pass into the blood, reach the circulation and some of them are expired through the lungs . In addition to VOCs endogenously generated inside the body, VOCs of exogenous origin can also be excreted through the lungs after inhalation and distribution in the body .
Fecal VOCs are predominantly produced during bacterial fermentation in the gut and reflect both the microbial composition of the gastrointestinal tract and the microbiota-host interaction . Feces are a mixture of food residues, compounds resulting from digestive and excretory processes, epithelial cells, and fragments and metabolites of the gastrointestinal microbiota. The gastrointestinal microbiota of ruminants adapts to the physiological status of animals, food changes and environmental conditions [18, 19].
Animals’ skin releases a large number of VOCs known to be derived from skin glands, bacterial metabolites and endogenous metabolites originated during disease pathogenesis. Bacteria present on the skin of cows can metabolize compounds into volatiles, and thus bTB infected cattle aerosol residing on their skin can release potential volatile biomarkers characteristic to bTB pathogens.
Animals’ location was assessed as a confounding factor in the present study in order to counteract differences in animals’ alimentation and environmental conditions between the different farms, for focusing only on the physiological status of the animals.
Although the composition of the VOCs from different samples can be closely related, their cumulative analysis provides complementary and more complete information about the health status . Actually, in the present study, toluene was the only biomarker found in two different samples (breath and skin). The possible origin of the biomarkers found in the present study is next discussed.
Acetone was identified in a previous study as a biomarker of bovine tuberculosis in fecal samples of swine, which might be attributed to increased metabolic activity during mycobacterial infection . As acetone represents the byproduct of fat catabolism, the presence of acetone in breath samples indicates enhanced polyunsaturated fatty acids (PUFAs) β-oxidation during bacterial pathogenesis .
The methylated alkanes, such as 4-methyldecane, are generally byproducts of lipid peroxidations and subsequent degradation of long chain PUFAs by highly reactive radicals under highly oxidative stress conditions resulted from cell inflammatory reactions towards pathogens. . In bovine tuberculosis infection, the oxidative stress is caused by reactive oxygen and nitrogen species (ROS-RNS), which are important defense substances involved in the immune response against pathogens .
The presence of D-limonene in the breath of the infected animals is unclear. This aliphatic hydrocarbon of exogenous origin can be found in nature as a fragrance of oranges, but it is also used as a flavoring agent in the food industry, or as a solvent in cleaning products. In previous studies it was reported as breath biomarker for liver diseases, such as cirrhosis and non-alcoholic fatty liver disease [24, 25].
Toluene is an aromatic hydrocarbon and a common environmental pollutant. Due to the fact that the acetate metabolic pathways are sources of production of phenolic derivatives and fatty acids, the presence of toluene in feces might be also related to endogenous origins derived from enzymatic reduction of phenolic derivatives .
Phenolic compounds were shown to have positive antimycobacterial activity, producing effects such as inhibition of mycolic acid synthesis, transcription, translation and arabinogalactan synthesis . They can be produced by gut bacteria from the metabolism of aromatic amino acids . In the present study, two phenolic compounds, i.e. phenol and 2,4-di-tert-butylphenol, were identified as possible bTB biomarkers excreted through animals feces.
[(1,1-dimethylethyl)thio]acetic acid might be produced during the acid-catalyzed reaction of acetic acid with primary alkanols, and could be a potential indicator of altered activity and metabolic changes after the binding of a pathogen to an epithelial cell .
Alpha-thujene is a phytochemical that bounds well to target N protein by exhibiting high binding affinity toward its catalytic pocket . Bacteria regulates ribosomal RNA transcription and employs the phage λ N protein as a host factor , which could justify the identification of this compound as possible bTB biomarker.
Camphene is a monoterpene used in the preparation of fragrances and as a food additive for flavoring. Camphene plays a biological reactive oxygen species (ROS) scavenging role, regulating oxidative stress and lipid metabolism . It was also reported as a volatile of pulmonary tuberculosis in the breath of human patients .
O-cymene, previously reported as biomarker of bovine tuberculosis in swine breath, may be related to endogenous oxidation during aromatic metabolism, where aromatic hydrocarbons are readily adsorbed onto cell membranes after exogenous ingestion though skin and/or from food .
3-(1,1-dimethylethyl)-2,2,4,4-tetramethyl-3-pentanol is a branched alkyl alcohol. It was demonstrated that the alkyl alcohols induce changes of mitochondria at cellular level . Mitochondria appears to be among the important targets for bacterial pathogens such as Mycobacterium tuberculosis, which secretes various proteins that initiate structural changes in mitochondria to modulate its function .
2,5-dimethylhexane-2,5-dihydroperoxide is a branched alkane peroxide that was also previously reported as breath biomarker of bovine tuberculosis in swine. It could be associated with infection-induced inflammation and the occurrence of lesions in the intestinal epithelia .
Ammonia is produced by amino acids obtained from the catabolism of glutamine or from peripheral tissues, as well as from food intake. During high protein intake in animals, the catalysis of glutamine metabolism releases energy and produces a high amount of ammonia, which is then transported and stored in the liver. During disease pathogenesis, bTB pathogens induce high oxidative stress, inflammation, and enhance mitochondrial energy production from glucose and protein substrates. Thereby, glutamine catabolism tends to increase, producing excess amount of ammonia, which also indicates altered cellular metabolism .
1-methoxy-2-propanol is an alcohol derivative and its presence in the skin samples can be attributed to both exogenous origins and endogenous carbon metabolism. It might be an overlap between VOCs produced from the body cells during complex physiological processes and those available from the environment such as ketones, aldehydes and alcohols, some of which could be related to metabolites from skin microbiome. Nevertheless, most of the VOCs excreted from skin are derived from physiological processes and drastic metabolic changes due to pathogenic infection inducing high oxidative stress, lipid peroxidation and cytochrome P450 alcohol metabolism.
Finally, toluene has also been found in this study as bTB biomarker among the VOCs released by feces. In skin it was previously reported as a biomarker for tuberculosis in humans .