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

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease (JD) 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 for MAP infection in cattle. Circulating miRNAs have recently gained attention as potential biomarkers for a number of diseases in humans. However, knowledge and use of miRNAs as biomarkers in diseases of ruminants, including JD is very limited. Here we have used NanoString technology, a digital platform for amplification-free and hybridization based quantitative measurement of miRNAs in the sera of non-infected and naturally MAP infected cattle with different severity of infection. Using probes developed against human miRNA, 26 miRNAs were detected in cattle serum with thirteen of these miRNAs previously uncharacterized for cattle. The results also indicated that the levels of some of the miRNAs were differentially expressed in the serum of cattle at different stages of JD. Canonical discrimination analysis using 20 miRNAs grouped animals into four distinct clusters based on their disease status, suggesting that the levels of these miRNAs are differentially modulated at different stages of MAP infection. A combination of four miRNAs, namely, miR-1976, miR-873-3p, miR-520f-3p, and miR-126-3p distinguished moderate and severely infected animals from non-infected animals. Our study has demonstrated the ability of NanoString technology to detect differential expression of circulating miRNAs in cattle and contributes to widely growing evidence that miRNAs can be used as biomarkers in infectious diseases in cattle.