Identification of Novel Seroreactive Antigens in Johne's Disease Cattle by Using the Mycobacterium tuberculosis Protein Array.

Johne's disease, a chronic gastrointestinal inflammatory disease caused by subspecies , is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relationship between subsp. and the human pathogen , here, we applied a whole-proteome protein array to identify seroreactive and diagnostic subsp. antigens. A genome-scale pairwise analysis of amino acid identity levels between orthologous proteins in subsp. and showed an average of 62% identity, with more than half the orthologous proteins sharing >75% identity. Analysis of the protein array probed with sera from subsp. -infected cattle showed antibody binding to 729 proteins, with 58% of them having ≥70% identity to subsp. orthologs. The results showed that only 4 of the top 40 seroreactive antigens were orthologs of previously reported subsp. antigens, revealing the existence of a large number of previously unrecognized candidate diagnostic antigens. Enzyme-linked immunosorbent assay (ELISA) testing of 20 subsp. recombinant proteins, representing reactive and nonreactive orthologs, further confirmed that the array has utility as a screening tool for identifying candidate antigens for Johne's disease diagnostics. Additional ELISA testing of field serum samples collected from dairy herds around the United States revealed that MAP2942c had the strongest seroreactivity with Johne's disease-positive samples. Collectively, our studies have considerably expanded the number of candidate subsp. proteins with potential utility in the next generation of rationally designed Johne's disease diagnostic assays.