Profiling host- and parasite-derived miRNAs associated with Strongylus vulgaris infection in horses

The equine bloodworm, Strongylus vulgaris, is a common and highly pathogenic parasite in horses due to its migratory life cycle involving the intestinal arteries. Current diagnostic techniques cannot detect the prepatent migrating stages of S. vulgaris, highlighting the need for new biomarkers. Parasites release microRNAs (miRNAs) into their environment, which could potentially be detectable in host blood samples. Additionally, host miRNA expression patterns may change in response to infection. This study aimed to identify miRNAs associated with S. vulgaris infection by profiling the horse's miRNA response in the larval predilection site, the Cranial Mesenteric Artery (CMA) and examining the circulating parasite and horse-derived miRNAs in plasma of S. vulgaris-infected horses. Plasma samples were collected from 27 horses naturally infected with S. vulgaris and 28 uninfected horses. Arterial tissue samples from the CMA and Aorta were collected from a subset (n = 12) of the infected horses. Small RNA sequencing (small RNAseq) of a subset of the plasma samples (n = 12) identified miRNAs of interest, followed by quantitative real-time PCR (qPCR) evaluation of selected miRNAs in plasma from a larger cohort of horses. Small RNAseq detected 138 parasite-derived and 533 horse-derived miRNAs in the plasma samples. No difference in parasite-derived miRNA abundance was found between the infected and uninfected horses, but 140 horse-derived miRNAs were significantly differentially abundant between the two groups. When evaluated by qPCR, none of the selected parasite-derived miRNAs were detectable in plasma, but seven horsederived miRNAs were confirmed differentially abundant in plasma between the two groups. Seven horsederived miRNAs were differentially expressed in CMA tissue affected by migrating S. vulgaris compared with unaffected aortic tissue, with Eca-Mir-223-3p (Log2FC: 4.74) and Eca-Mir-140-3p (Log2FC: -3.64) being most differentially expressed. A receiver operating characteristic curve analysis suggested that Eca-Mir-486-5p and Eca-Mir-140-3p had the best diagnostic performance for distinguishing between infected and uninfected horses, with areas under the curve (AUC) of 0.78 and 0.77, respectively. Notably, Eca-Mir-140-3p was associated with age, and correcting for interaction with age increased the AUC to 0.96. In conclusion, several horse-derived miRNAs were associated with S. vulgaris infection and could differentiate between infected and uninfected horses based on their plasma abundance. However, the levels of these miRNAs were influenced by other factors (i.e age, breed), complicating their use as biomarkers. Parasite-derived miRNA abundance did not differ between S. vulgaris infected horses and those infected with other parasites using small RNAseq and were below detection limits of qPCR.