Novel platforms for detection of Mycobacterium avium subsp. paratuberculosis infection in dairy cattle.

约翰氏病（JD）是由鸟分枝杆菌（Mycobacterium avium subsp.）副结核病（地图）是乳制品行业的主要健康问题之一，造成重大的经济影响。预防和控制计划受到当前诊断测试性能不佳的严重阻碍，特别是在感染的早期阶段。他们无法在感染动物传播疾病之前可靠地检测到它们。目前用于抗体（Ab）测试的ELISA具有未针对早期检测优化的粗抗原（Ag）混合物。粪便脱落和抗体反应相关性较差，但这些组合测试增加了测试的灵敏度。 该研究计划的目标是改进目前的JD诊断方法，并开发一种补充的农场诊断方法，可以以更高的灵敏度检测活的Map细菌或特定Ab，从而通过检测和剔除策略进行剔除/隔离并减少疾病的传播。我们提出的两个测试平台是兼容的综合控制程序的多种测试要求。 第一个平台将测量针对Map的Ab响应。第一个平台的关键创新是病原体特异性Ab恢复了被分成两半的报告酶的活性。这是通过将抗原融合到这种酶的每一半上，并通过包含一种蛋白质-内含肽-将片段连接在一起来实现的。由于海藻糖酶具有较高的活性，且与廉价的葡萄糖检测试剂相容，因此被选为新型报告基因。在分层方法中，我们将首先在该测定中掺入充分表征的免疫原性表位，然后是已知的Map抗原肽，接下来我们将使用该平台发现新的Map Ag，用于更早、更灵敏和更特异地检测Map感染。 第二个平台将使用工程噬菌体来检测活的Map细胞。活Map的检测与感染性直接相关，并消除了与当前qPCR方法的低水平检测相关的不确定性。这项技术依赖于这样一个事实，即DNA只在活细胞内扩增。我们正在通过引入一种在复制过程中被激活的基因开关来改进这种方法。这种标记基因开关可以很容易地检测和定量，并区分复制的DNA和添加到样品中的DNA。这也将允许更准确的定量，并增加基于噬菌体的活性MAP检测的灵敏度。 总之，我们的新平台为阻碍当前JD诊断的所有关键困难提供了解决方案。开发可在农场进行的补充检测方法将大大提高检测受感染动物的灵敏度，从而改善JD控制程序。这些诊断平台在检测动物和人类的其他传染病方面具有巨大的潜力。

Johne’s disease (JD), a debilitating chronic enteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map), is one of the main health issues in the dairy industry, resulting in significant economical impact. Prevention and control programs are severely hampered by the poor performance of current diagnostic tests, especially in the early stages of the infection. They fail to reliably detect infected animals before they can spread the disease. Current ELISAs for antibody (Ab) testing have crude antigen (Ag) mixtures that are not optimized for early detection. Fecal shedding and Ab responses correlate poorly, but combined testing for these increases testing sensitivity. The goal of this research program is to improve current JD diagnostics and develop a complementary panel of on-farm diagnostics that can detect viable Map bacteria or specific Abs with higher sensitivity, allowing culling/segregation and reducing the spread of the disease by test-and-cull strategies. Our two proposed test platforms are compatible with multiple testing requirements of comprehensive control programs. The first platform will measure Ab responses against Map. The key innovation of the first platform is that pathogen specific Ab restore activity of a reporter enzyme that was split in half. This is accomplished by fusing Ags to each half of this enzyme and by including a protein –an intein- that can ligate the fragments back together. Trehalase was selected as novel reporter because of its high activity and compatibility with inexpensive glucosemeters for detection. In a tiered approach, we will first incorporate a well characterized immunogenic epitope in this assay, then known Map antigenic peptides and next we will use the platform to discover new Map Ags for earlier and more sensitive and specific detection of Map infections. The second platform will use engineered bacteriophages to detect viable Map cells. Detection of viable Map directly correlates with infectivity and eliminates uncertainty associated with low level detection by current qPCR methods. The technology relies on the fact that phages only amplify within living cells. We are improving this method by introducing a genetic switch that is activated during replication. This signature genetic switch can be easily detected and quantified and distinguishes replicated phages from phages added to the sample. This will also allow more accurate quantification and increase the sensitivity of phage-based detection of viable MAP. In summary, our new platforms provide solutions to all the crucial difficulties hampering current JD diagnostics. The development of complementary assays that can be performed on-farm, will dramatically increase the sensitivity of detecting infected animals and thereby improve JD control programs. The diagnostic platforms have a great potential for detecting other infectious diseases in animals and humans.