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

* * * *** * 正常* 0 * * * * * 假*假*假* * en - us *是* X-NONE * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * **** /* 样式定义* / *表。MsoNormalTable* {mso-style-name:"Table Normal"；* mso-tstyle-rowband-size: 0;* mso-tstyle-colband-size: 0;* mso-style-noshow:是的;* mso-style-priority: 99;* mso-style-parent:“”;* mso-padding-alt:0cm 5.4pt；* mso-para-margin: 0厘米;* mso-para-margin-bottom: .0001pt;* mso-pagination: widow-orphan;*字体大小:12.0分;*字体类型:威尔士;* mso-ascii-font-family:威尔士;* mso-ascii-theme-font: minor-latin;* mso-hansi-font-family:威尔士;* mso-hansi-theme-font: minor-latin;* mso-ansi-language: en - us;*******约翰氏病（JD），一种在反刍动物中由鸟分枝杆菌引起的使人衰弱的慢性肠炎。副结核（Map）是乳制品行业的主要健康问题之一，造成重大的经济影响。目前诊断检测的不良表现严重阻碍了预防和控制规划，特别是在感染的早期阶段。它们无法在被感染的动物传播疾病之前可靠地发现它们。目前用于抗体（Ab）检测的elisa是粗糙的抗原（Ag）混合物，未优化用于早期检测。粪便脱落和Ab反应相关性较差，但联合测试这些增加了测试的敏感性。***本研究计划的目标是改进现有的JD诊断方法，并开发一套补充性的农场诊断方法，能够以更高的灵敏度检测活的Map细菌或特异性抗体，从而实现剔除/分离，并通过测试和剔除策略减少疾病的传播。我们提出的两种测试平台可兼容综合控制方案的多种测试需求。***第一个平台将根据Map测量Ab响应。第一个平台的关键创新是病原体特异性Ab恢复了被一分为二的报告酶的活性。这是通过将Ags融合到这种酶的每一半上，并包括一种蛋白质——一种可以将片段连接在一起的蛋白——来完成的。海藻糖酶因其高活性和与廉价血糖仪的相容性而被选为新的报告基因。在分层的方法中，我们将首先在该检测中纳入一个具有良好特征的免疫原性表位，然后是已知的Map抗原肽，然后我们将使用该平台发现新的Map Ags，以便更早，更敏感和特异性地检测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的检测与传染性直接相关，并消除了目前qPCR方法低水平检测带来的不确定性。这项技术依赖于噬菌体只在活细胞内扩增的事实。我们正在通过引入在复制过程中被激活的基因开关来改进这种方法。这种标记遗传开关可以很容易地检测和量化，并区分复制噬菌体和添加到样品中的噬菌体。这也将允许更准确的定量和提高基于噬菌体的活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。可以在农场进行的补充检测的发展将大大提高检测感染动物的灵敏度，从而改善JD控制程序。诊断平台有一个

* * * *** * Normal* 0* * * * * false* false* false* * EN-US* JA* X-NONE* * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * **** /* Style Definitions */*table.MsoNormalTable* {mso-style-name:"Table Normal";* mso-tstyle-rowband-size:0;* mso-tstyle-colband-size:0;* mso-style-noshow:yes;* mso-style-priority:99;* mso-style-parent:"";* mso-padding-alt:0cm 5.4pt 0cm 5.4pt;* mso-para-margin:0cm;* mso-para-margin-bottom:.0001pt;* mso-pagination:widow-orphan;* font-size:12.0pt;* font-family:Cambria;* mso-ascii-font-family:Cambria;* mso-ascii-theme-font:minor-latin;* mso-hansi-font-family:Cambria;* mso-hansi-theme-font:minor-latin;* mso-ansi-language:EN-US;}*******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