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

约内氏病（JD）是由鸟分枝杆菌（Mycobacterium avium subsp.）副结核病（MAP）是奶牛的一种经济上重要的疾病。为了有效控制JD，重要的是在感染动物具有传染性（=开始散毒）之前对其进行检测和隔离。然而，目前的诊断试验（粪便培养和抗体ELISA）检测低散毒动物的灵敏度差。已经充分确定的是，针对Map感染的细胞免疫应答（CIR）先于可检测的抗Map抗体的产生。该Map特异性反应可以在干扰素-γ释放测定中测量。血液需要在采集后12小时内在实验室进行分析，这是该测试的主要缺点，影响了其一般用途。修改此测试，使其可以很容易地在农场进行，将大大提高JD控制。因为可测量的CIR随时间降低，所以将其检测与补充试验中的特异性抗体的检测结合联合收割机是重要的。由于目前没有农场JD诊断存在，我们的目标是开发一套农场分子测定，可以检测与Map感染的所有阶段相关的免疫反应。1)第一种类型的测定将检测感染牛中的CIR。 将生产生物传感器，其将用Map抗原刺激的血液中干扰素-γ的存在转化为比色输出。2)第二种类型的测定将测量血清中的Map特异性抗体，其中抗体/抗原结合触发酶促比色反应的激活。3)以前，我们开发了不同的检测方法，以检测地图感染的基础上，通过转录组学在实验感染的小牛中鉴定的生物标志物。这些检测方法将在大量自然感染动物中进一步验证。4)同时，我们将使用数字液滴PCR来精确量化动物的脱落水平，这些动物为生物传感器和生物标志物测试开发提供血液样本，以将测试结果与感染性水平联系起来。总之，开发一系列小规模的检测方法，共同针对感染的几个阶段，将显著提高检测感染动物的灵敏度，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.