Candidate Faecal microRNAs as Non-Invasive Biomarkers for Bovine Paratuberculosis in Marchigiana Beef Cattle

MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in the post-transcriptional regulation of gene expression. Since miRNAs modulate host immune responses, they represent promising molecular biomarkers of paratuberculosis (PTB), particularly during early or subclinical stages, when conventional diagnostic tests may lack sensitivity. In this context, faecal miRNAs could provide valuable insights into intestinal immune responses and mucosal damage associated with Mycobacterium avium subsp. paratuberculosis (MAP) infection. Although miRNAs have been extensively investigated in serum, blood, and tissues, their detection and characterization in bovine faeces remain poorly explored. The aim of this study was to evaluate the expression profiles of selected candidate faecal miRNAs in Marchigiana beef cattle naturally exposed to MAP and to assess their association with different infection phenotypes. Thirty-four cows were classified into three phenotypic groups: healthy exposed, MAP-infected, and PTB-affected based on longitudinal diagnostic records including interferon-γ assay, serological testing (ELISA), and faecal qPCR. Five candidate miRNAs were selected from previous studies and quantified in faecal samples by RT-qPCR. Four of the five selected miRNAs were consistently detected across samples. Bta-miR-92a was significantly downregulated in both MAP-infected and PTB-affected animals compared with healthy cattle, suggesting early modulation during MAP infection. Bta-miR-223 was significantly upregulated in PTB-affected animals compared with both healthy and MAP-infected groups, consistent with its established role in the regulation of intestinal inflammation. The ortholog of hsa-miR-501-5p was significantly upregulated in MAP-infected cattle, potentially reflecting early host-pathogen interactions at the intestinal mucosal level, while bta-miR-24-3p showed no significant differences among groups. Overall, these findings support the feasibility of faecal miRNA analysis as a complementary non-invasive molecular approach to support traditional diagnostic tests for PTB, especially during the early and subclinical stages of MAP infection.