The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis.
The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis.
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DOI：
10.3389/fvets.2022.921160
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发表时间：
2022
影响因子：
3.2
通讯作者：
中科院分区：
文献类型：
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副结核分枝杆菌（Mycobacterium avium subspecies paratuberminum，MAP）是约讷氏病（Johne’s Disease）的病原体，约讷氏病是一种在世界范围内流行的反刍动物慢性肠炎。据估计，大约50%的英国奶牛群感染了MAP，但这可能低估了真实的患病率。感染可导致产奶量减少、不育和过早扑杀动物，导致农业经济的重大损失，并对动物福利产生负面影响。了解MAP与宿主之间的初始相互作用对于开发改进的诊断工具和新型疫苗至关重要。在这里，我们描述了三种不同的多细胞在体外模型来自牛肠组织的特点，以及它们的使用与MAP的细胞相互作用的研究。除了先前描述的基底向外的3D牛类肠外，我们还建立了可行的2D单层和3D顶端向外的类器官。顶出肠不同于先前描述的牛肠，因为顶表面暴露在3D结构的外表面上，使得能够在上皮表面研究宿主-病原体相互作用而不需要显微注射。我们已经使用RT-qPCR来检测预测的细胞类型特异性基因表达，并使用共聚焦显微镜来检测细胞类型特异性蛋白表达，从而表征了每个模型系统中存在的细胞类型。每个模型都含有原始牛肠组织中存在的细胞，确认它们代表牛肠道。将三个模型系统暴露于MAP K10的K10参考菌株和最近的苏格兰分离株（称为C49），导致通过共聚焦显微镜观察细胞内细菌。通过定量基因组拷贝数对细菌进行计数，表明在所有模型系统中，在感染的早期时间点，K10的侵袭性低于C49。这项研究表明，牛肠模型是允许感染MAP，这些模型可能是有用的，在调查早期阶段的MAP发病机制在生理相关的体外系统，同时减少使用动物在科学研究中。牛：urn：lsid：zoobank.org：act：4C90C4FA-6296-4972-BE6A-5EF578677D64
Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's Disease, a chronic enteritis of ruminants prevalent across the world. It is estimated that approximately 50% of UK dairy herds are infected with MAP, but this is likely an underestimate of the true prevalence. Infection can result in reduced milk yield, infertility and premature culling of the animal, leading to significant losses to the farming economy and negatively affecting animal welfare. Understanding the initial interaction between MAP and the host is critical to develop improved diagnostic tools and novel vaccines. Here we describe the characterisation of three different multicellular in vitro models derived from bovine intestinal tissue, and their use for the study of cellular interactions with MAP. In addition to the previously described basal-out 3D bovine enteroids, we have established viable 2D monolayers and 3D apical-out organoids. The apical-out enteroids differ from previously described bovine enteroids as the apical surface is exposed on the exterior surface of the 3D structure, enabling study of host-pathogen interactions at the epithelial surface without the need for microinjection. We have characterised the cell types present in each model system using RT-qPCR to detect predicted cell type-specific gene expression, and confocal microscopy for cell type-specific protein expression. Each model contained the cells present in the original bovine intestinal tissue, confirming they were representative of the bovine gut. Exposure of the three model systems to the K10 reference strain of MAP K10, and a recent Scottish isolate referred to as C49, led to the observation of intracellular bacteria by confocal microscopy. Enumeration of the bacteria by quantification of genome copy number, indicated that K10 was less invasive than C49 at early time points in infection in all model systems. This study shows that bovine enteroid-based models are permissive to infection with MAP and that these models may be useful in investigating early stages of MAP pathogenesis in a physiologically relevant in vitro system, whilst reducing the use of animals in scientific research. Bos taurus: urn:lsid:zoobank.org:act:4C90C4FA-6296-4972-BE6A-5EF578677D64
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影响因子：
4.6
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4.6
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通讯作者：
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影响因子：
2.2
作者：
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