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

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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.