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

约翰氏病(JD)是由禽分枝杆菌亚种引起的一种慢性肠炎。副结核(MAP)是奶牛的一种重要的经济疾病。为了有效地控制JD，重要的是在感染动物开始脱落之前对它们进行检测和隔离。然而，目前的诊断试验(粪便培养和抗体酶联免疫吸附试验)对低脱落动物的检测灵敏度较低。已经证实，针对MAP感染的细胞免疫反应(CIR)先于可检测到的抗MAP抗体的产生。这种MAP特异性反应可以在干扰素-伽马释放试验中测量。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。修改这项测试，使其可以在农场上轻松执行，将极大地改善JD控制。由于可测量的CIR随着时间的推移而减少，因此将其检测与互补试验中的特定抗体相结合是很重要的。由于目前还没有农场上的JD诊断方法，我们的目标是开发一套农场上的分子检测方法，可以检测与MAP感染的所有阶段相关的免疫反应。1)第一类检测将检测感染牛的CIR。将生产一种生物传感器，将MAP抗原刺激的血液中存在的干扰素-伽马转化为比色输出。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来检测MAP感染，这是基于通过转录组学在实验感染的小牛中鉴定的生物标志物。这些检测将在大量自然感染的动物身上进一步验证。4)同时，我们将使用数字液滴聚合酶链式反应精确量化为生物传感器和生物标记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。总而言之，开发一小批检测方法，共同针对感染的几个阶段，将显著提高检测受感染动物的敏感度，并改进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.