Quantitative Proteome Analysis of Plasma Extracellular Vesicles Identifies Three Proteins with Potential Diagnostic Value for Mycobacterium bovis Infection in Cows

Bovine tuberculosis (bTB) is a zoonotic disease that affects cattle and human health. Although the tuberculin skin test (TST) is the main detection method, there is a need for simpler on-farm tests using fluid samples. This study analyzed plasma extracellular vesicles (EVs) from two cow groups (Cohort A, 15 negative, 22 positive; Cohort B, 28 negative, 40 positive) to explore bTB indicators using proteome profiling. Among the 756 proteins, 217 (Cohort A) and 233 (Cohort B) showed differences between healthy and infected cows, with 47 consistently dysregulated in both groups. These proteins were related to tuberculosis, neutrophil extracellular trap formation, and antigen processing and presentation pathways. Notably, three proteins, HSPA8, B2M, and HRG, were confirmed as bTB indicators using multiple methods, including least absolute shrinkage and selection operator (Lasso) regression selection, western blot (WB), and enzyme-linked immunosorbent assay (ELISA) validation with an independent cohort (Cohort C). This study identifies plasma EV biomarkers for bTB infection, offering insights for bTB detection.牛结核病（bovine tuberculosis, bTB）是一种危害牛只与人类健康的人畜共患病。尽管结核菌素皮肤试验（tuberculin skin test, TST）是当前主流的检测手段，但仍亟需开发采用体液样本的更简便的农场现场检测方法。本研究采用蛋白质组学分析方法，对两组牛群的血浆细胞外囊泡（extracellular vesicles, EVs）进行了分析：队列A包含15份阴性样本、22份阳性样本；队列B包含28份阴性样本、40份阳性样本，旨在筛选牛结核病的潜在标志物。在本次分析共鉴定出的756种蛋白质中，队列A与队列B分别有217种、233种蛋白质在健康牛与感染牛之间存在表达差异，其中47种蛋白质在两个队列中均呈现失调表达。上述差异蛋白质主要富集于结核病、中性粒细胞胞外陷阱形成以及抗原加工与呈递通路。值得注意的是，通过最小绝对收缩和选择算子（least absolute shrinkage and selection operator, Lasso）回归筛选、蛋白质印迹（western blot, WB）以及酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）对独立队列（队列C）进行验证后，确定HSPA8、B2M与HRG这3种蛋白质可作为牛结核病的候选标志物。本研究成功筛选出可用于牛结核病感染检测的血浆细胞外囊泡生物标志物，为牛结核病的检测提供了新的思路与理论依据。