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

Abstract

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.

Evaluation of needle trap micro‐extraction and solid‐phase micro‐extraction: Obtaining comprehensive information on volatile emissions from in vitro cultures

“…This could be related to their spatial conformation and thus worse binding ability to the CAR/PDMS fiber compared with linear alkanes. LODs in the same range were reported by Bergmann et al ().…”

“…Furans and esters were calibrated with low LODs (median 0.018 and 0.012 nmol L −1 , respectively). Similar ranges for SPME and NTME were reported in former studies (Bergmann et al, ; Mesarchaki et al, ; Trefz, Koehler, et al, ). As 2‐hydroxybenzoic acid methyl ester has an hydroxyl group, its LOD for SPME is not within the expected concentration range, corresponding to the LOD of other alcohols assessed with SPME.…”

“…According to this basic difference, the properties of the extraction materials and the analytes, efficiency of VOC extraction may differ if different packing materials are applied. In this study packing material was therefore selected with respect to optimal bottom‐up properties for VOC profiling validated in previous studies (Bergmann et al, ; Küntzel et al, ; Trefz et al, ; Trefz, Koehler, et al, ) rather than to best comparable material combinations. General benefits and drawbacks of both micro‐extraction methods are summarized in Table .…”

“…In this study we used micro‐extraction devices and chromatographic columns reported to be the best suited for VOC profiling studies using SPME (Bergmann et al, ; Trefz, Koehler, et al, ) or NTME (Trefz et al, ; Trefz, Rösner, et al, ). Chromatographic methods were adapted with respect to the applied micro‐extraction method.…”

“…Both methods can be applied with various types of adsorption materials. As both extraction methods require specially adapted materials and appropriate analytical setups we chose adsorption materials and analytical conditions which had already been applied and optimized in other studies (Bergmann et al, ; Küntzel et al, ; Trefz et al, ; Trefz, Koehler, et al, ). The following questions were addressed in detail: Are sensitivity and selectivity of both techniques comparable? Do both techniques allow a bottom‐up approach to characterize emitted VOC profiles from bacterial cultures? What additional information is revealed by applying both sampling techniques in parallel? …”