Bergmann Andreas, Trefz Phillip, Fischer Sina, Klepik Klaus, Walter Gudrun, Steffens Markus, Ziller Mario, Schubert Jochen K, Reinhold Petra, Köhler Heike, Miekisch Wolfram
Department of Anaesthesia and Intensive Care, University Medicine Rostock, Rostock, Germany.
Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Jena, Germany.
PLoS One. 2015 Apr 27;10(4):e0123980. doi: 10.1371/journal.pone.0123980. eCollection 2015.
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.
副结核分枝杆菌鸟亚种（MAP）是反刍动物慢性肠道疾病的病原体。现有的诊断测试复杂且耗时。在体外，MAP培养物释放的挥发性有机化合物（VOC）模式反映了细菌的生长情况，并能够区分不同的菌株。本研究旨在在动物模型的可控环境中测定体内VOC。通过针阱微萃取（呼吸）和固相微萃取（粪便）从42只山羊（16只对照动物和26只接种MAP的动物）的呼吸和粪便中预浓缩VOC，并通过气相色谱/质谱进行分析。在接种后18、29、33、41和48周进行分析。从血液中测定MAP特异性抗体和MAP特异性干扰素-γ反应。通过分析纯参考物质确认了所有标记VOC的身份。基于高皮克每升的检测限和两个数量级以上的线性范围，在呼吸和粪便顶空中可检测到100多种VOC。接种动物和未接种动物之间有28种物质存在差异。尽管在感染过程中，大多数突出物质（如呋喃、氧化物质和碳氢化合物）的模式发生了变化，但在任何时候，粪便中的16种物质和呼吸中的3种VOC在接种动物和未接种动物之间仍可检测到差异。粪便中VOC浓度的差异反映了MAP细菌的存在。呼吸中VOC谱的差异与干扰素-γ反应方面的宿主反应有关。从长远来看，体内VOC分析可能有助于克服现有测试的局限性。
