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

Overview

• This study investigated circulating microRNAs (miRNAs) as potential biomarkers to diagnose and predict disease progression in horses infected with Theileria equi, which causes equine piroplasmosis.
• The research identified specific miRNA profiles linked to either acute infection or asymptomatic carrier states, suggesting their utility for distinguishing infection stages and understanding immune responses.

Background and Purpose

• Equine piroplasmosis is caused by Theileria equi, a parasite that infects horses and results in a spectrum of clinical outcomes:
• Acute disease with severe symptoms
• Asymptomatic carriers who harbor the parasite lifelong without obvious illness
• The disease presents diagnostic difficulties due to its varying clinical presentations.
• The study aimed to characterize circulating miRNA profiles in horses with different infection statuses to identify biomarkers for diagnosis and prognosis.

Study Design and Methods

• Study design: Cross-sectional case-control study involving three groups of horses:
  • Acutely infected (symptomatic)
  • Asymptomatic carriers
  • Uninfected controls
• Classification of groups:
  • Clinical examination combined with quantitative PCR (qPCR) to confirm parasite presence and burden
• Data collection and analysis:
  • Hematological and biochemical testing for blood parameters
  • Small RNA sequencing to profile plasma miRNAs
  • Quantitative real-time PCR (qRT-PCR) validation for selected miRNAs
  • Measurement of cytokine gene expression in peripheral blood mononuclear cells
  • Bioinformatic analyses incorporating:
    • Integrated miRNA-mRNA target prediction
    • Pathway enrichment analysis
    • Network analysis to understand interactions
  • Diagnostic evaluation using receiver operating characteristic (ROC) curves

Key Findings

• Acutely infected horses exhibited:
  • High parasite load confirmed by qPCR
  • Signs of hemolytic anemia (destruction of red blood cells)
  • Strong upregulation of pro-inflammatory cytokines, indicating an active immune response
  • Widespread dysregulation of circulating miRNAs, indicative of systemic inflammation
• Asymptomatic carriers showed:
  • Near-normal hematological and biochemical parameters
  • A unique miRNA expression signature distinct from acutely infected horses
  • miRNA patterns more associated with immune regulation and homeostasis rather than inflammation
• Specific miRNAs correlated with:
  • Parasite burden
  • Erythrocyte indices (red blood cell metrics)
  • Cytokine expression levels
• Pathway analysis revealed enrichment in key immune and cellular pathways such as:
  • NF-κB signaling (a major inflammatory pathway)
  • JAK/STAT signaling (important in cytokine signaling)
  • TGF-β signaling (involved in immune regulation and fibrosis)
  • Apoptosis (programmed cell death)
  • Oxidative stress pathways (cellular damage mechanisms)
• Diagnostic performance:
  • Selected circulating miRNAs exhibited strong diagnostic accuracy for differentiating acute infection from asymptomatic carriage
  • ROC curve analyses supported the potential of these miRNAs as reliable biomarkers

Conclusions and Implications

• Distinct circulating miRNA profiles reflect the severity of disease and immune status in horses infected with Theileria equi.
• These miRNA signatures serve as promising non-invasive biomarkers for:
  • Accurate diagnosis of acute versus carrier states
  • Prognostication of disease progression
• The study advances understanding of the molecular mechanisms underlying equine piroplasmosis, specifically how miRNAs modulate immune responses during infection.
• This knowledge could improve epidemiological surveillance and treatment decisions by enabling better identification of infected animals and monitoring their immune status.