Quantifying Mycobacterium avium subspecies paratuberculosis infection of bovine monocyte derived macrophages by confocal microscopy
Quantifying Mycobacterium avium subspecies paratuberculosis infection of bovine monocyte derived macrophages by confocal microscopy
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DOI：
10.1016/j.mimet.2019.105779
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发表时间：
2020-01-01
影响因子：
2.2
通讯作者：
Hope, Jayne C.
中科院分区：
文献类型：
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作者：
Mathie, Heather A.;Jensen, Kirsty;Hope, Jayne C.
由于目前使用的方法（如菌落计数）的局限性，在体外感染实验期间对鸟分枝杆菌副结核亚种（MAP）进行定量具有挑战性。在这里，我们描述了定量MAP感染牛巨噬细胞（M φ）使用共聚焦显微镜。牛单核细胞衍生的巨噬细胞用高或低剂量的MAP感染，并使用共聚焦显微镜在感染后2小时计算细胞内细菌的数量。通过菌落计数定量同时感染的M phi内的细菌，以将共聚焦显微镜结果与通过已建立的方法获得的结果进行比较。共聚焦显微镜检查提供了一种稳健的替代定量方法，可用于评估个体M phi水平的感染。这表明MAP感染不是同质的，并且与低剂量相比，在高剂量下存在更高数量的感染的M phi和细胞内细菌和细菌聚集体，可能影响M phi对感染的反应。因此，共聚焦显微镜可以提供其他定量方法无法获得的关于感染的详细信息。
Quantification of Mycobacterium avium subspecies paratuberculosis (MAP) during in vitro infection experiments is challenging due to limitations of currently utilised methods, such as colony counting. Here we describe quantifying MAP infection of bovine macrophages (M phi) using confocal microscopy. Bovine monocyte derived macrophages were infected with MAP at a high or low dose and the number of intracellular bacteria calculated at 2 h post infection using confocal microscopy. Bacteria within simultaneously infected M phi were quantified by colony counting in order to compare confocal microscopy results with results obtained by an established method. Confocal microscopy provided a robust alternative quantification method that allowed for assessment of the infection at the individual M phi level. This demonstrated that MAP infection was not homogeneous, and that there were higher numbers of both infected M phi and intracellular bacteria and bacterial aggregates at the high dose compared to the low dose, potentially impacting the M phi response to infection. Confocal microscopy can therefore provide a level of detail regarding the infection unobtainable by other quantification methods.