Xin Ting, Gao Xintao, Yang Hongjun, Li Pingjun, Liang Qianqian, Hou Shaohua, Sui Xiukun, Guo Xiaoyu, Yuan Weifeng, Zhu Hongfei, Ding Jiabo, Jia Hong
Institute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.
Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Jinan, China.
Front Vet Sci. 2018 Mar 6;5:28. doi: 10.3389/fvets.2018.00028. eCollection 2018.
Bovine tuberculosis (bTB) is primarily caused by infection with , which belongs to the complex. The airborne route is considered the most common for transmission of , and more than 15% of cattle with bTB shed the , which can be detect by nested PCR to amplify mycobacterial 70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally -infected, experimentally 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate -infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate -infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of infection in cattle that were confirmed positive for infection by nested PCR. Furthermore, the levels of PPD-B- or CE-induced IP-10 and IL-17A protein were lower than IFN-γ in -infected cattle. Therefore, IL-17A and IP-10 protein are not suitable biomarkers for bTB. Antigen-induced IP-10 mRNA should be analyzed further for their potential to be used in the diagnosis of bTB.
牛结核病（bTB）主要由感染牛分枝杆菌引起，牛分枝杆菌属于牛分枝杆菌复合群。空气传播途径被认为是牛分枝杆菌传播最常见的途径，超过15%的患牛结核病的牛会排出牛分枝杆菌，可通过巢式PCR从奶牛的鼻拭子中扩增分枝杆菌70来检测。为了筛选有助于早期准确检测牛结核病的细胞因子，从自然感染、实验感染68002的牛和未感染的牛中分离外周血单核细胞，然后用纯化蛋白衍生物B（PPD-B）、CFP-10-ESAT-6（CE）或磷酸盐缓冲盐水（PBS）刺激这些细胞6小时。使用实时PCR测量干扰素γ（IFN-γ）、IFN-γ诱导蛋白10（IP-10）、白细胞介素6（IL-6）、白细胞介素12（IL-12）、白细胞介素17A（IL-17A）和肿瘤坏死因子α mRNA的水平。为了探索与牛分枝杆菌感染不同时期相关的细胞因子，使用结核菌素皮肤试验、基于CFP-10/ESAT-6/TB10.4蛋白混合物的皮肤试验、IFN-γ释放试验（IGRA）、基于CFP-10/ESAT-6（CE）的IGRA和巢式PCR将牛分为三组：PCR阳性、PCR阴性和未感染。通过酶联免疫吸附测定（ELISA）检测PPD-B、CE或PBS诱导的IP-10、IL-17A和IFN-γ蛋白的表达。结果表明，自然或实验感染牛分枝杆菌的牛中，PPD-B刺激的IL-17A和IP-10（mRNA和蛋白）以及CE诱导的IP-10（mRNA和蛋白）水平显著高于未感染的牛。PPD-B或CE诱导的IL-17A和IP-10（蛋白）水平可用于区分感染后6至30周感染牛分枝杆菌的犊牛和未感染的犊牛，而PPD-B和CE诱导的IP-10和IL-17A mRNA表达可用于区分感染后6至58周感染牛分枝杆菌的犊牛和未感染的犊牛。然而，CE诱导的IL-17A（蛋白）不是通过巢式PCR确诊感染的牛中牛分枝杆菌感染的可靠指标。此外，在感染牛分枝杆菌的牛中，PPD-B或CE诱导的IP-10和IL-17A蛋白水平低于IFN-γ。因此，IL-17A和IP-10蛋白不是牛结核病合适的生物标志物。抗原诱导的IP-10 mRNA在牛结核病诊断中的潜在用途应进一步分析。
