Krasteva Ivanka, Luciani Mirella, D'Onofrio Federica, Di Febo Tiziana, Di Pancrazio Chiara, Perletta Fabrizia, Maggetti Marta, Ulisse Simonetta, Sonsini Luigina, Orsini Gianluca, Caporale Marco, Ponsart Claire, Djokic Vitomir, Vicente Acacia Ferreira, Freddi Luca, D'Alterio Nicola, Tittarelli Manuela, De Massis Fabrizio, Sacchini Flavio
Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise, Teramo, Italy.
Department of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy.
Front Microbiol. 2025 Jul 30;16:1641710. doi: 10.3389/fmicb.2025.1641710. eCollection 2025.
The brucellin skin test (BST) detects brucellosis in animals through a cell-mediated immune response to a protein extract from strain 115, which is almost free of lipopolysaccharide. It is highly specific and used to confirm suspected false positive serology results in small ruminants and swine, but not recommended for screening due to low sensitivity. Despite its diagnostic significance, the protein composition of brucellin has not been fully characterized. This study used nLC-ESI-MS/MS analysis and bioinformatics tools to evaluate brucellin's protein composition and identify immunoreactive proteins. An allergen suspension of purified proteins (free of S-LPS) of EU Standard Brucellin, produced by ANSES, IZS-Teramo (IZSAM) and the former commercialised brucellergene OCB were used. Proteomic analysis identified 247 (ANSES), 542 (IZSAM) and 183 (OCB) proteins. Two hundred and six proteins (ANSES), 458 proteins (IZSAM) and 156 (OCB) were predicted as potential antigens, and 123 proteins are common to all 3 brucellins examined. Among the 123 proteins common to all three brucellin formulations examined, several key immunodominant proteins previously identified in Brucella research-such as ribosomal L7/L12, outer membrane protein BP26/OMP28, GroEL, and Bacterioferritin-were consistently detected. Their presence across all formulations supports their important role in inducing delayed hypersensitivity and contributing to Brucella pathogenesis. These findings underscore the importance of introducing mass spectrometry analyses as quality control for brucellin batches production and the potential of these proteins as candidates for detecting cellular immunity against . Developing recombinant -allergenic proteins could help in standardizing skin tests, providing reliable allergens favoring disease control and eradication. Moreover, a serological test using these recombinant proteins could improve specificity of current indirect tests for and eliminate false-positive results associated with LPS-based diagnostics.
布鲁菌素皮肤试验（BST）通过对来自115菌株的蛋白质提取物的细胞介导免疫反应来检测动物的布鲁氏菌病，该提取物几乎不含脂多糖。它具有高度特异性，用于确认小反刍动物和猪中疑似假阳性血清学结果，但由于灵敏度低，不推荐用于筛查。尽管布鲁菌素具有诊断意义，但其蛋白质组成尚未完全表征。本研究使用nLC-ESI-MS/MS分析和生物信息学工具来评估布鲁菌素的蛋白质组成并鉴定免疫反应性蛋白。使用了由法国食品安全局（ANSES）、意大利泰拉莫动物卫生与食品质量研究所（IZS-Teramo，IZSAM）生产的欧盟标准布鲁菌素的纯化蛋白（不含S-LPS）过敏原悬浮液以及以前商业化的布鲁氏菌基因OCB。蛋白质组学分析鉴定出247种（ANSES）、542种（IZSAM）和183种（OCB）蛋白质。预测有206种蛋白质（ANSES）、458种蛋白质（IZSAM）和156种（OCB）为潜在抗原，并且在所有检测的3种布鲁菌素中共有123种蛋白质。在所检测的所有三种布鲁菌素制剂共有的123种蛋白质中，一致检测到了布鲁氏菌研究中先前鉴定的几种关键免疫显性蛋白，如核糖体L7/L12、外膜蛋白BP26/OMP28、GroEL和细菌铁蛋白。它们在所有制剂中的存在支持了它们在诱导迟发型超敏反应和布鲁氏菌发病机制中的重要作用。这些发现强调了引入质谱分析作为布鲁菌素批次生产质量控制的重要性，以及这些蛋白质作为检测针对布鲁氏菌细胞免疫候选物的潜力。开发重组变应原蛋白有助于标准化皮肤试验，提供有利于疾病控制和根除的可靠过敏原。此外，使用这些重组蛋白的血清学试验可以提高当前布鲁氏菌间接试验的特异性，并消除与基于脂多糖诊断相关的假阳性结果。
