Detection of microRNA in cattle serum and their potential use to diagnose severity of Johne's disease

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, which is characterized by chronic progressive granulomatous enteritis. The infection leads to wasting and weight loss in the animals and eventually death, causing considerable production losses to the agricultural industry worldwide. Currently available ELISA- and PCR-based diagnostic tests have limited sensitivity and specificity during early MAP infection in cattle, suggesting that there is an urgent demand for alternative diagnostic tests. Circulating microRNA (miRNA) have recently gained attention as potential biomarkers for several diseases in humans. However, knowledge and use of miRNA as biomarkers in diseases of ruminants, including Johne's disease, are very limited. Here we used NanoString nCounter technology (NanoString, Seattle, WA), a digital platform for amplification-free and hybridization-based quantitative measurement of miRNA in the sera of noninfected and naturally MAP-infected cattle with different severity of infection. Using probes developed against human miRNA, 26 miRNA were detected in cattle serum; 13 of these miRNA were previously uncharacterized for cattle. Canonical discrimination analysis using 20 miRNA grouped animals into 4 distinct clusters based on their disease status, suggesting that the levels of these miRNA can reflect disease severity. A model was developed using a combination of 4 miRNA (miR-1976, miR-873-3p, miR-520f-3p, and miR-126-3p), which distinguished moderate and severely infected animals from noninfected animals. Our study demonstrated the ability of the NanoString nCounter technology to detect differential expression of circulating miRNA in cattle and contributes to widely growing evidence that miRNA can be used as biomarkers in infectious diseases in cattle.

Application of Transcriptomics to Enhance Early Diagnostics of Mycobacterial Infections, with an Emphasis on Mycobacterium avium ssp. paratuberculosis

“…These results entirely correspond to another study that analyzed miRNA expression in calves [52]. Gupta and colleagues isolated serum from older (three to five year old) uninfected and naturally MAP infected cows and performed a human-based miRNA measurement [113]. This approach exposed 26 differentially expressed miRNAs, of which 13 miRNAs had not yet been characterized in cattle.…”

Identification of microRNAs in bovine faeces and their potential as biomarkers of Johne’s Disease

“…Using an ileal loop model in neonate calves, Liang et al 10 identified 9 miRNAs that showed differential expression between infected and control tissues. At the level of serum, Gupta et al 8 used Nanostring human miRNA panels to detect 26 miRNA in bovine serum. Quantifying miRNA abundance in serum from cattle stratified based on JD presentation (control, mild, moderate, severe) they revealed that a model employing the expression of just 4 miRNA could separate moderate and severely infected animals from the control and mild animals.…”

“…Previous studies in MAP infected cattle have measured miRNA expression in serum 8 , whole blood 9 and ileal tissues 10 . We have previously investigated circulating miRNA expression in peripheral blood at silent and subclinical MAP infection stages but detected minimal changes that did not provide diagnostic benefit 11,12 .…”

Identifying Differentially Expressed MicroRNAs, Target Genes, and Key Pathways Deregulated in Patients with Liver Diseases

“…Currently, there are a number of methods for miRNA profiling: hybridization (microarray), small RNA sequencing (miRNAseq), and reverse-transcription quantitative PCR (RT-qPCR) [ 17 ]. In order to identify possible roles of miRNAs and to better understand the molecular mechanisms of liver diseases, we integrated miRNA and mRNA analysis.…”