Anschütz Nils H, Gerbig Stefanie, Ventura Alejandra M Peter, Silva Liliana M R, Larrazabal Camilo, Hermosilla Carlos, Taubert Anja, Spengler Bernhard
Institute of Inorganic and Analytical Chemistry Justus Liebig University Giessen Giessen Germany.
Institute of Parasitology Justus Liebig University Giessen Giessen Germany.
Anal Sci Adv. 2022 Aug 30;3(7-8):244-254. doi: 10.1002/ansa.202200016. eCollection 2022 Aug.
is an obligate intracellular protozoan parasite of the phylum Alveolata (subphylum Apicomplexa) which has not been studied extensively in a biochemical context. is a primary cause of reproductive disorders causing mummification and abortion not only in cattle but also in other small ruminant species resulting in a substantial economic impact on the livestock industry. In canids, which are the final hosts of , clinical disease includes neuromuscular symptoms, ataxia, and ascending paralysis. Fatal outcomes of neosporosis have also been reported depending on the host species, age and immune status, however, its zoonotic potential is still uncertain. Therefore, should be thoroughly investigated. Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) and MS imaging (MSI) were used, combined with high-performance liquid chromatography (HPLC) to investigate these intracellular parasites. The aim of this study was to identify molecular biomarkers for tachyzoite-infected host cells and to further clarify their functions. By atmospheric-pressure scanning microprobe MALDI MS(I), sections of -infected and non-infected host cell pellets were examined in order to determine potential markers. In vivo, infects different types of nucleated cells, such as endothelial cells which represent a highly immunoreactive cell type. Therefore, primary bovine umbilical vein endothelial cells were here used as a suitable infection system. For comparison, the permanent MARC-145 cell line was used as an additional, simplified in vitro cell culture model. HPLC-tandem MS (HPLC-MS/MS) experiments combined with database search were employed for structural verification of markers. The statistically relevant biomarkers found by MS and identified by HPLC-MS/MS measurements were partly also found in infected monolayers. Marker signals were imaged in cell layers of -infected and non-infected host cells at 5 µm lateral resolution.
是肺泡虫门（顶复门亚门）的专性细胞内原生动物寄生虫，尚未在生化背景下进行广泛研究。是生殖障碍的主要原因，不仅导致牛，还导致其他小型反刍动物物种出现木乃伊化和流产，对畜牧业造成重大经济影响。在作为其终末宿主的犬科动物中，临床疾病包括神经肌肉症状、共济失调和上行性麻痹。新孢子虫病的致命后果也有报道，具体取决于宿主物种、年龄和免疫状态，然而，其人畜共患病潜力仍不确定。因此，应对其进行彻底研究。采用基质辅助激光解吸/电离（MALDI）质谱（MS）和MS成像（MSI），并结合高效液相色谱（HPLC）来研究这些细胞内寄生虫。本研究的目的是鉴定速殖子感染宿主细胞的分子生物标志物，并进一步阐明其功能。通过大气压扫描微探针MALDI MS（I），检查感染和未感染的宿主细胞沉淀切片，以确定潜在标志物。在体内，感染不同类型的有核细胞，如代表高度免疫反应性细胞类型的内皮细胞。因此，原代牛脐静脉内皮细胞在此用作合适的感染系统。为了进行比较，使用永久MARC-145细胞系作为另一种简化的体外细胞培养模型。采用HPLC串联MS（HPLC-MS/MS）实验并结合数据库搜索对标志物进行结构验证。通过MS发现并经HPLC-MS/MS测量鉴定的具有统计学意义的生物标志物，部分也在感染的单层细胞中发现。在感染和未感染的宿主细胞的细胞层中，以5μm的横向分辨率对标志物信号进行成像。
