Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne's Disease using MAP Protein Microarrays.

Considerable effort has been directed toward controlling Johne's disease (JD), a chronic granulomatous intestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in cattle and other ruminants. However, progress in controlling the spread of MAP infection has been impeded by the lack of reliable diagnostic tests that can identify animals early in the infection process and help break the transmission chain. To identify reliable antigens for early diagnosis of MAP infection, we constructed a MAP protein array with 868 purified recombinant MAP proteins, and screened a total of 180 well-characterized serum samples from cows assigned to 4 groups based on previous serological and fecal test results: negative low exposure (NL, n = 30); negative high exposure (NH, n = 30); fecal-positive, ELISA-negative (F + E-, n = 60); and both fecal- and ELISA-positive (F + E+, n = 60). The analyses identified a total of 49 candidate antigens in the NH, F + E-, and F + E+ with reactivity compared with the NL group (p < 0.01), a majority of which have not been previously identified. While some of the antigens were identified as reactive in only one of the groups, others showed reactivity in multiple groups, including NH (n = 28), F + E- (n = 26), and F + E+ (n = 17) groups. Using combinations of top reactive antigens in each group, the results reveal sensitivities of 60.0%, 73.3%, and 81.7% in the NH, F + E-, and F + E+, respectively at 90% specificity, suggesting that early detection of infection in animals may be possible and enable better opportunities to reduce within herd transmission that may be otherwise missed by traditional serological assays that are biased towards more heavily infected animals. Together, the results suggest that several of the novel candidate antigens identified in this study, particularly those that were reactive in the NH and F + E- groups, have potential utility for the early sero-diagnosis of MAP infection.

A defined antigen skin test for the diagnosis of bovine tuberculosis.

Bovine tuberculosis (bTB) is a major zoonotic disease of cattle that is endemic in much of the world, limiting livestock productivity and representing a global public health threat. Because the standard tuberculin skin test precludes implementation of Bacille Calmette-Guérin (BCG) vaccine-based control programs, we here developed and evaluated a novel peptide-based defined antigen skin test (DST) to diagnose bTB and to differentiate infected from vaccinated animals (DIVA). The results, in laboratory assays and in experimentally or naturally infected animals, demonstrate that the peptide-based DST provides DIVA capability and equal or superior performance over the extant standard tuberculin surveillance test. Together with the ease of chemical synthesis, quality control, and lower burden for regulatory approval compared with recombinant antigens, the results of our studies show that the DST considerably improves a century-old standard and enables the development and implementation of critically needed surveillance and vaccination programs to accelerate bTB control.