College of Veterinary Medicine, University of Florida, 2015 SW 16th AV, Box 100103, Gainesville, FL 32608-1166, USA.
Vet Microbiol. 2011 Dec 15;153(3-4):264-73. doi: 10.1016/j.vetmic.2011.05.035. Epub 2011 May 26.
Continuous culture of Anaplasma marginale in endothelial cells and the potential implications for vaccine development heightened interest in determining the importance of endothelial cells in the A. marginale life cycle. A. marginale-infection trials were performed to determine if endothelial cells are an in vivo host cell in cattle and if A. marginale from in vitro endothelial cells were infective to cattle. Adult, immunocompetent steers were infected by tick-feeding transmission and were euthanized at different points in the parasitemic cycle. Based on quantitative PCR, the tissue distribution of A. marginale DNA during peak and trough parasitemia was variable with higher quantities observed in spleen, lung, hemal nodes, and abomasum. A. marginale was not conclusively identified in tissue endothelial cells from the steers' tick-bitten dermis or post-mortem tissues using three microscopy techniques (dual indirect immunofluorescence, transmission electron microscopy, and in situ DNA target-primed rolling-circle amplification of a padlock probe). Intravenous inoculation of spleen-intact or splenectomized calves with endothelial cell culture-derived VA isolate A. marginale did not cause seroconversion or clinical anaplasmosis regardless of whether the endothelial culture-derived bacteria were inoculated as host cell-free organisms or within endothelial cells and regardless of the type of endothelial cell culture used - RF/6A primate endothelial cells or primary bovine testicular vein endothelial cells. Data presented here suggest that endothelial cells are likely not a pivotal component of the A. marginale life cycle in vivo.
连续培养边缘无浆体在血管内皮细胞中的应用及其对疫苗开发的潜在影响，使人们对确定内皮细胞在边缘无浆体生命周期中的重要性产生了浓厚的兴趣。为了确定内皮细胞是否是牛体内的一种宿主细胞，以及体外培养的内皮细胞中的边缘无浆体是否对牛具有感染力，进行了边缘无浆体感染试验。通过蜱传播感染成年、免疫功能正常的阉牛，并在寄生虫血症周期的不同时间点对其进行安乐死。基于定量 PCR，在寄生虫血症高峰期和低谷期，边缘无浆体 DNA 的组织分布是可变的，在脾脏、肺、血液节点和皱胃中观察到的数量较高。使用三种显微镜技术（双重间接免疫荧光、透射电子显微镜和原位 DNA 靶标引物滚环扩增检测锁式探针），均未从牛的被蜱叮咬的真皮或死后组织的内皮细胞中明确鉴定出边缘无浆体。无论内皮细胞培养衍生的细菌是否作为无宿主细胞的生物体或在内皮细胞中接种，也无论使用何种类型的内皮细胞培养物（恒河猴内皮细胞 RF/6A 或原代牛睾丸静脉内皮细胞），对完整脾脏或脾切除术牛进行静脉内接种内皮细胞培养衍生的 VA 分离株边缘无浆体均不会导致血清转化或临床无浆体病。这里提出的数据表明，内皮细胞可能不是体内边缘无浆体生命周期的关键组成部分。
