Regidor-Cerrillo Javier, García-Lunar Paula, Pastor-Fernández Iván, Álvarez-García Gema, Collantes-Fernández Esther, Gómez-Bautista Mercedes, Ortega-Mora Luis M
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain.
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain.
Vet Parasitol. 2015 Sep 15;212(3-4):92-9. doi: 10.1016/j.vetpar.2015.08.020. Epub 2015 Aug 21.
Pathogenesis of bovine neosporosis is determined by different host- and parasite-dependent factors, including isolate virulence. A previous study identified that several Neospora caninum tachyzoite proteins were more abundant in virulent isolates, Nc-Liv and Nc-Spain7, compared with the low-virulent isolate Nc-Spain1H. Herein, we explored differences in the immunomes of these three isolates. Protein extracts from the Nc-Liv, Nc-Spain1H and Nc-Spain7 isolates were analysed in a 3×3 design by 2-DE immunoblot using sera from experimentally infected mice with these same three isolates. All isolates displayed a highly similar antigenic pattern when they were assessed using the same serum. Most of the reactive spots were located in the acidic region (pH 3-7) and grouped in 3 antigenic areas (250-70, 45-37 and 35-15 KDa). Differences found in the immunome depended on the sera used, regardless of the extract employed. In this sense, sera from Nc-Liv and Nc-Spain7 infected mice recognized the highest number of antigens, followed by Nc-Spain1H infected mice sera. In fact, 4 proteins identified by MS were not consistently detected in each isolate extract by sera from low-virulent Nc-Spain1H-infected mice: serine-threonine phosphatase 2C and superoxide dismutase (related to metabolism), gliding associated protein GAP45 (related to tachyzoites invasion), and NcGRA1 (located on dense granules). Similarly, 4 non-identified spots and another 2 spots chains located in 45-37 kDa area were not detected by this pooled sera. Variations between virulent Nc-Spain7 and Nc-Liv were limited to the absence of recognition by sera from Nc-Spain7-infected mice of GAP45 and the spot chains located in the 45-37 kDa area. These results suggest that variations in the immunome profiles rely on the immune response induced by each isolate and that these differentially recognized antigens could be investigated as putative virulence markers of neosporosis.
牛新孢子虫病的发病机制由不同的宿主和寄生虫相关因素决定，包括分离株的毒力。先前的一项研究表明，与低毒力分离株Nc-Spain1H相比，几种犬新孢子虫速殖子蛋白在强毒株Nc-Liv和Nc-Spain7中更为丰富。在此，我们探究了这三种分离株免疫组的差异。使用来自经这三种相同分离株实验感染小鼠的血清，通过二维电泳免疫印迹法，以3×3设计分析了Nc-Liv、Nc-Spain1H和Nc-Spain7分离株的蛋白提取物。当使用相同血清评估时，所有分离株均呈现高度相似的抗原模式。大多数反应性斑点位于酸性区域（pH 3 - 7），并分为3个抗原区域（250 - 70、45 - 37和35 - 15 kDa）。免疫组中发现的差异取决于所用血清，而与所采用的提取物无关。从这个意义上讲，来自感染Nc-Liv和Nc-Spain7小鼠的血清识别的抗原数量最多，其次是感染Nc-Spain1H小鼠的血清。事实上，低毒力的Nc-Spain1H感染小鼠血清在每种分离株提取物中未能一致检测到质谱鉴定的4种蛋白质：丝氨酸 - 苏氨酸磷酸酶2C和超氧化物歧化酶（与代谢相关）、滑行相关蛋白GAP45（与速殖子入侵相关）以及NcGRA1（位于致密颗粒上）。同样，该混合血清未检测到4个未鉴定斑点以及位于45 - 37 kDa区域的另外2个斑点链。强毒株Nc-Spain7和Nc-Liv之间的差异仅限于感染Nc-Spain7小鼠的血清未识别GAP45以及位于45 - 37 kDa区域的斑点链。这些结果表明，免疫组图谱的差异依赖于每种分离株诱导的免疫反应，并且这些差异识别的抗原可作为新孢子虫病假定毒力标志物进行研究。
