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

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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.