Institute for Infectious Diseases and Zoonoses, Department of Veterinary Sciences, Faculty of Veterinary Medicine, LMU Munich, Veterinaerstr. 13, 80539, Munich, Germany.
Department of Microbiology & Immunology Chicago Medical School Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road 2-407, North Chicago, IL, 60064, USA.
Ticks Tick Borne Dis. 2019 Feb;10(2):280-285. doi: 10.1016/j.ttbdis.2018.11.001. Epub 2018 Nov 13.
Anaplasma phagocytophilum (Ap) is a tick-transmitted obligate intracellular bacterium and the causative agent of the granulocytic anaplasmosis in various species of domestic animals and in humans. During intracellular development Ap transforms from a dense-cored cell form into a reticulate cell form and vice versa. For isolation of intracellular bacteria, a range of different purification methods is used. However, unlike other Gram-negative bacteria Ap is considered to be sensitive to mechanical stress and osmolarity changes. An updated semi-purification method using rock tumbler grit is introduced here to increase the outcome of bacteria and to facilitate the procedure of host cell lysis. The objective of this study was to evaluate the structural integrity and infectivity of Ap after lysis of the host cells using rock tumbler grit and to compare the outcome to that of the frequently used method, syringe lysis. Human promyelocytic leukemia cell lines (HL-60) were infected with Ap and following host cell-free bacteria were assessed by transmission electron microscopy. The outcome of the different purification methods was compared using live/dead-staining based on immunofluorescence to count the number of viable bacteria and real-time PCR to compare the amount of DNA. Subsequently the isolated bacteria were tested to infect naive cell cultures. We observed that both Ap dense-cored cells and reticulate cells are preserved intact after the application of rock tumbler grit. The number of viable, host cell-free bacteria was higher by factor 1.7-2.4 compared to the syringe lysis protocol. Quantitative analysis based on real-time PCR showed an increase of bacterial DNA up to 1.6-2.9 times higher using the rock tumbler grit protocol. Bacteria released from the same number of infected host cells were used for new infections. Flow cytometric analysis of the cell cultures confirmed that the number of Ap organisms recovered by using the rock tumbler grit protocol resulted in higher infection rates than the number of Ap organisms recovered by using syringe lysis protocol. Our observations indicate that the rock tumbler grit protocol can be applied as a safe, robust and convenient method to recover Ap compared to syringe lysis.
嗜吞噬细胞无形体（Ap）是一种蜱传的专性细胞内细菌，也是各种家畜和人类粒细胞无形体病的病原体。在细胞内发育过程中，Ap 从致密核心细胞形态转变为网状细胞形态，反之亦然。为了分离细胞内细菌，使用了一系列不同的纯化方法。然而，与其他革兰氏阴性菌不同，Ap 被认为对机械应激和渗透压变化敏感。本文介绍了一种使用摇滚砾石的更新半纯化方法，以提高细菌的产量，并简化宿主细胞裂解的过程。本研究的目的是评估使用摇滚砾石裂解宿主细胞后 Ap 的结构完整性和感染力，并将结果与常用的注射器裂解方法进行比较。用 Ap 感染人早幼粒细胞白血病细胞系（HL-60），然后通过透射电子显微镜评估无宿主细胞的细菌。使用基于免疫荧光的死活染色来计数活细菌数量，并通过实时 PCR 比较 DNA 量，比较不同纯化方法的结果。随后，对分离出的细菌进行测试，以感染未感染的细胞培养物。我们观察到，在应用摇滚砾石后，Ap 致密核心细胞和网状细胞都保持完整。与注射器裂解方案相比，活的、无宿主细胞的细菌数量增加了 1.7-2.4 倍。基于实时 PCR 的定量分析显示，使用摇滚砾石方案时，细菌 DNA 增加了 1.6-2.9 倍。从相同数量的感染宿主细胞中释放的细菌用于新的感染。细胞培养物的流式细胞术分析证实，使用摇滚砾石方案回收的 Ap 生物体数量比使用注射器裂解方案回收的 Ap 生物体数量更高，从而导致更高的感染率。我们的观察表明，与注射器裂解相比，摇滚砾石方案可以作为一种安全、稳健且方便的方法来回收 Ap。
