Exosomal microRNA profiling reveals candidate biomarkers and regulatory signatures associated with paratuberculosis disease status in goats.

Paratuberculosis (Johne's disease), a chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP), is difficult to diagnose in the subclinical stage(s) because of intermittent shedding and nonspecific immune responses. Given their stability and potential as biomarkers, exosomal microRNAs (miRNAs) were investigated in this study to identify serum-derived candidates associated with Paratuberculosis-associated disease stages in goats. Exosomes were isolated from the serum of three groups: Control (ELISA- and PCR-negative), ENPP (ELISA-negative but PCR-positive), and EPPP (ELISA- and PCR-positive). After exosome characterization, small RNA sequencing and bioinformatic analyses were performed to identify differentially expressed miRNAs (DEmiRs) and their predicted regulatory targets. Functional enrichment, upstream regulator, and network analyses were then applied to interpret the biological relevance of the identified DEmiRs. Among the candidates, miR-122 and miR-21-5p were consistently upregulated in infected animals and were associated with immune modulation, epithelial integrity, and inflammation. Finally, quantitative PCR validation confirmed a significant elevation of these miRNAs in the EPPP group, supporting their potential as late-stage biomarkers. Our findings demonstrate that exosomal miRNA profiling can reflect MAP infection status and highlight their potential utility as non-invasive biomarkers for Paratuberculosis monitoring in veterinary settings.