Circulating microRNA profiles as diagnostic and prognostic biomarkers for acute and carrier Theileria equi infection

Equine piroplasmosis caused by Theileria equi exhibits a wide clinical spectrum ranging from acute disease to lifelong asymptomatic carriage, posing significant diagnostic and epidemiological challenges. In this cross-sectional case-control study, equines were classified as acutely infected, asymptomatic carriers, or uninfected controls based on clinical examination and quantitative PCR. Hematological and biochemical alterations were assessed, and plasma miRNA profiles were characterized using small RNA sequencing, followed by qRT-PCR validation of selected miRNAs. Cytokine gene expression in peripheral blood mononuclear cells was quantified, and integrated miRNA-mRNA target prediction, pathway enrichment, and network analyses were performed. Diagnostic performance was evaluated using receiver operating characteristic (ROC) curve analysis. Acutely infected equines exhibited high parasite burden, hemolytic anemia, and pronounced upregulation of pro-inflammatory cytokines, accompanied by widespread dysregulation of circulating miRNAs. In contrast, asymptomatic carriers maintained near-normal hematological parameters and displayed a distinct miRNA signature associated with immune regulation rather than overt inflammation. Several miRNAs showed significant correlations with parasite load, erythrocytic indices, and cytokine expression. Enriched pathways included NF-κB, JAK/STAT, TGF-β signaling, apoptosis, and oxidative stress. Selected circulating miRNAs demonstrated strong diagnostic accuracy and effectively discriminated acute infection from asymptomatic carriage. These findings indicate that circulating miRNA signatures reflect disease severity and immune status in T. equi infection, highlighting their potential as diagnostic and prognostic biomarkers and providing insight into miRNA-mediated immune modulation underlying equine piroplasmosis.