In Vivo Volatile Organic Compound Signatures of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests.