Early detection of immune responses against Mycobacterium avium subsp. paratuberculosis infection

约翰氏病（JD），一种由鸟分枝杆菌引起的慢性肠炎。副结核（Map）是奶牛的一种重要的经济疾病。为了有效控制JD，重要的是在感染动物开始脱落之前发现并隔离它们。然而，目前的诊断测试（粪便培养和抗体ELISA）对检测低脱毛动物的敏感性较差。已经确定，针对Map感染的细胞免疫反应（CIR）先于可检测的抗Map抗体的产生。这种Map特异性反应可以在干扰素释放试验中测量。血液需要在采集后12小时内在实验室进行分析，这是该测试的一个主要缺点，影响了其一般用途。修改这个测试，使它可以很容易地在农场进行，将大大提高JD控制。由于可测量的CIR随着时间的推移而降低，因此将其检测与补充试验中特定抗体的检测结合起来很重要。由于目前没有农场JD诊断方法，我们的目标是开发一套农场分子检测方法，可以检测与Map感染所有阶段相关的免疫反应。1)第一类试验将检测受感染牛的CIR。一种生物传感器将被生产出来，它能将受Map抗原刺激的血液中存在的干扰素转化为比色输出。2)第二种类型的分析将测量血清中的Map特异性抗体，抗体/抗原结合触发酶比色反应的激活。3)在此之前，我们开发了不同的检测方法，基于在实验感染的小牛中通过转录组学鉴定的生物标志物来检测Map感染。这些检测方法将在大量自然感染的动物身上得到进一步验证。4)与此同时，我们将使用数字液滴PCR来精确量化为生物传感器和生物标志物测试开发提供血液样本的动物的脱落水平，以将测试结果与传染性水平联系起来。综上所述，针对感染的几个阶段开发少量的检测方法将显著提高检测感染动物的敏感性，并改善JD控制程序。

Johne's disease (JD), a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (Map), is an economically important disease in dairy cattle. For effective control of JD it is important to detect and isolate infected animals before they become infectious (=start shedding). However, current diagnostic tests (fecal culture and antibody ELISA) have poor sensitivity for detecting low shedding animals. It has been well established that a cellular immune response (CIR) against Map infection precedes the production of detectable anti-Map antibodies. This Map specific response can be measured in an interferon-gamma release assay. Blood needs to be analysed in the lab within 12 h of collection, which is a major disadvantage of this test compromising its general use. Modifying this test so that it can be performed easily on-farm would dramatically improve JD control. Because the measurable CIR decreases over time it is important to combine its detection with that of specific antibodies in complementary tests. As currently no on-farm JD diagnostics exist, we aim to develop a set of on-farm molecular assays which can detect immune responses which are associated with all stages of Map infection. 1) A first type of assay will detect the CIR in infected cattle. A biosensor will be produced which converts the presence of interferon-gamma in blood stimulated with Map antigens into a colorimetric output. 2) A second type of assay will measure Map specific antibodies in serum with antibody/antigen binding triggering the activation of an enzymatic colorimetric reaction. 3) Previously, we developed different assays to detect Map infection based on biomarkers identified in experimentally infected calves by transcriptomics. These assays will be further validated on large numbers of naturally infected animals. 4) In parallel, we will use digital droplet PCR to precisely quantify shedding levels in the animals that provide blood samples for the biosensor and biomarker test development to link the test outcomes to levels of infectivity. In summary, the development of a small array of assays, together targeting several stages of infection will significantly increase the sensitivity to detect infected animals and improve JD control programs.