MicroRNAs (miRNAs) regulate post-transcriptional gene expression and may be exported from cells via exosomes or in partnership with RNA-binding proteins. MiRNAs in body fluids can act in a hormone-like manner and play important roles in disease initiation and progression. Hence, miRNAs are promising candidates as biomarkers. To identify serum miRNA biomarkers in the equine model of asthma we investigated small RNA derived from the serum of 34 control and 37 asthmatic horses. These samples were used for next generation sequencing, novel miRNA identification and differential miRNA expression analysis. We identified 11 significantly differentially expressed miRNAs between case and control horses: eca-miR-128, eca-miR-744, eca-miR-197, eca-miR-103, eca-miR-107a, eca-miR-30d, eca-miR-140-3p, eca-miR-7, eca-miR-361-3p, eca-miR-148b-3p and eca-miR-215. Pathway enrichment using experimentally validated target genes of the human homologous miRNAs showed a significant enrichment in the regulation of epithelial-to-mesenchymal transition (key player in airway remodeling in asthma) and the phosphatidylinositol (3,4,5)-triphosphate (PIP3) signaling pathway (modulator of CD4⁺ T cell maturation and function). Downregulated miR-128 and miR-744 supports a Th2/Th17 type immune response in severe equine asthma.

MiRNAs regulate multiple genes at the post-transcriptional level and therefore play an important role in many biological processes. It has been suggested that miRNA exported outside the cells contribute to inter-cellular communication. Consequently, circulating miRNAs are of particular interest and are promising biomarkers for many diseases. The number of miRNAs annotated in the horse genome is much lower compared to model organisms like human and mouse. We therefore aimed to identify novel equine miRNAs for tissue types and breed in serum. We analysed 71 small RNA-seq libraries derived from nine tissues (gluteus medius, platysma, masseter muscle, heart, liver, cartilage, bone, total blood and serum) using miRDeep2 and miRdentify tools. Known miRNAs represented between 2.3 and 62.9 % of the reads in 71 libraries. A total of 683 novel miRNAs were identified. Breed and tissue type affected the number of miRNAs detected and interestingly, affected its average intensity. A total of 50 miRNAs in serum proved to be potential biomarkers to differentiate specific breed types, of which miR-122, miR-200, miR-483 were over-expressed and miR-328 was under-expressed in ponies compared to Warmbloods. The different miRNAs profiles, as well as the differences in their expression levels provide a foundation for more hypotheses based on the novel miRNAs discovered. We identified 683 novel equine miRNAs expressed in seven solid tissues, blood and serum. Additionally, our approach evidenced that such data supported identification of specific miRNAs as markers of functions related to breeds or disease tissues.

Early recognition of lameness is crucial for injury prevention. Quantitative gait analysis systems can detect low-grade asymmetries better than the human eye and may be useful in early lameness recognition. The aims of this study were (1) to investigate the frequency of gait asymmetries based on head and pelvic movement in elite eventing horses using inertial mounted measurement units and (2) to assess the association between asymmetries and muscle enzymes and blood lactate (LA) levelspost-exercise. Movement asymmetry of the head, wither, and pelvis were quantified in 33 elite eventing horses prior to and one day after the cross-country test of three Concours Complet International (CCI3* and CCI4*) events held three weeks apart. The effects of LA concentration immediately after completion of the cross-country course and of serum levels of creatine kinase (CK) and aspartate amino-transferase (AST) four hours post-exercise on gait asymmetry parameters were tested with linear models. A total of 58% and 77% of the 33 horses exhibited gait asymmetries that exceeded published threshold values before and after the cross-country course, respectively. The magnitude of pre-existing gait asymmetries was not significantly increased after the cross-country test and no associations with post-exercise levels of CK, AST, or LA were detected. The stride duration was significantly shorter the day following the cross-country test and was associated with LA, the age and the weight of the horses. In conclusion, a majority of the horses studied presented gait asymmetries and strenuous exercise resulted in decreased stride duration but did not worsen gait asymmetries.

Background Genetic, epidemiologic, and clinical evidence suggests that, in horses, there are manifestations of hypersensitivity that can occur together. Objectives To investigate whether concurrent insect bite hypersensitivity (IBH) and severe equine asthma (EA) is associated with higher allergen‐specific and total serum immunoglobulin E (IgE) concentrations than only EA or IBH. Animals Healthy control horses (C, n = 40), horses with IBH (IBH, n = 24), severe EA (EA, n = 18), and both conditions (IBH/EA, n = 23) were included. Methods In our retrospective comparative study, sera from horses with signs of severe EA, IBH, and control animals were used. IgE specific for 15 recombinant (r) allergens as well as total serum IgE concentrations were measured by enzyme‐linked immunosorbent assay. Results Group IBH (median sum r‐Culicoides IgE: optical density at 405 nm [OD₄₀₅] = 3.54 [0.48‐15.07]) and group IBH/EA (OD₄₀₅ = 4.55 [0.46‐17.15]) had significantly (P < .001) higher IgE against Culicoides r‐allergens than groups C (OD₄₀₅ = 0.44 [0.21‐2.05]) and EA (OD₄₀₅ = 0.6 [0.2‐2.9]). There were no significant (P > .05) differences between group IBH and group IBH/EA. No significant differences among the groups were found for the other r‐allergens or total serum IgE concentration. Compared to controls, horses with severe IBH had significantly increased IgE concentration to 5 Culicoides r‐allergens (P < .05), whereas horses with moderate IBH had significantly increased IgE concentration to only 3 Culicoides r‐allergens (P < .05). Conclusions and Clinical Importance Susceptibility of IBH‐affected horses to develop EA is likely not associated with IgE‐mediated immune reactions but with other immunopathological mechanisms.

Severe equine asthma (SEA) is a complex respiratory condition characterized by chronic airway inflammation. It shares many clinical and pathological features with human neutrophilic asthma, making it a valuable model for studying this condition. However, the immune mechanisms driving SEA have remained elusive. Although SEA has been primarily associated with a Th2 response, there have also been reports of Th1, Th17, or mixed‐mediated responses. To uncover the elusive immune mechanisms driving SEA, we performed single‐cell mRNA sequencing (scRNA‐seq) on cryopreserved bronchoalveolar cells from 11 Warmblood horses, 5 controls and 6 with SEA. We identified six major cell types, including B cells, T cells, monocytes–macrophages, dendritic cells, neutrophils, and mast cells. All cell types exhibited significant heterogeneity, with previously identified and novel cell subtypes. Notably, we observed monocyte–lymphocyte complexes and detected a robust Th17 signature in SEA, with CXCL13 upregulation in intermediate monocytes. Asthmatic horses exhibited expansion of the B‐cell population, Th17 polarization of the T‐cell populations, and dysregulation of genes associated with T‐cell function. Neutrophils demonstrated enhanced migratory capacity and heightened aptitude for neutrophil extracellular trap formation. These findings provide compelling evidence for a predominant Th17 immune response in neutrophilic SEA, driven by dysregulation of monocyte and T‐cell genes. The dysregulated genes identified through scRNA‐seq have potential as biomarkers and therapeutic targets for SEA and provide insights into human neutrophilic asthma.