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Aufarbeitung und Praxisbeispiele für die online Lehrplattform EquiSurf

The transcriptomic profile of a cell population can now be studied at the cellular level using single-cell mRNA sequencing (scRNA-seq). This novel technique provides the unprecedented opportunity to explore the cellular composition of the bronchoalveolar lavage fluid (BALF) of the horse, a species for which cell type markers are poorly described. Here, scRNA-seq technology was applied to cryopreserved equine BALF cells. Analysis of 4,631 cells isolated from three asthmatic horses in remission identified 16 cell clusters belonging to six major cell types: monocytes/macrophages, T cells, B/plasma cells, dendritic cells, neutrophils and mast cells. Higher resolution analysis of the constituents of the major immune cell populations allowed deep annotation of monocytes/macrophages, T cells and B/plasma cells. A significantly higher lymphocyte/macrophage ratio was detected with scRNA-seq compared to conventional cytological differential cell count. For the first time in horses, we detected a transcriptomic signature consistent with monocyte-lymphocyte complexes. Our findings indicate that scRNA-seq technology is applicable to cryopreserved equine BALF cells, allowing the identification of its major (cytologically differentiated) populations as well as previously unexplored T cell and macrophage subpopulations. Single-cell gene expression analysis has the potential to facilitate understanding of the immunological mechanisms at play in respiratory disorders of the horse, such as equine asthma.

Tick-borne encephalitis is an important viral tick-borne zoonosis in Europe and Asia. The disease is induced by tick-borne encephalitis virus (TBEV). This report describes a 16-year-old Warmblood gelding presenting with sudden onset of lethargy, ataxia, and muscle fasciculations on the nostrils, the lips, and the eye lids as the most important clinical findings. The horse further had a mild facial nerve paralysis with drooping of the right upper and lower lip. Diagnosis was based on paired serum samples using TBEV-ELISAs revealing high serum IgM in the first sample with normal IgM in the second sample and an increase in serum IgG and neutralizing antibodies, indicating acute and recent infection. TBEV was confirmed by a virus-neutralization test, revealing a fivefold increase in antibodies 32 days after of the onset of clinical signs. Although the specific PCR on cerebrospinal fluid (CSF) was negative, TBEV-specific IgG and IgM were identified in the CSF of the horse. Treatment consisted of anti-inflammatory and anti-oxidative treatment and the horse recovered with a mild drooping of the right nostril as the only remaining clinical sign. TBEV infection is a potential differential diagnosis of neurological disease in horses living in endemic areas and this is the first report to describe the diagnostic criteria in a horse as recommended in humans with suspected TBEV infection.

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 [OD ] = 3.54 [0.48-15.07]) and group IBH/EA (OD = 4.55 [0.46-17.15]) had significantly (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

Additional file 3: Figure S2. Full length original image of western blot analyses shown in Fig. 2B & C. On the left is the original western blot image and on the right is a photo of the membrane taken after ECL detection with protein ladder seen. These image are directly exported from the software (Bio-Rad Image lab version 5.2.1) without any manipulation. The membrane was cut into two pieces after protein transfer, one was used for anti-his-tag antibody and the other one was used for anti-TK1 antibody. They were put together just before ECL detection. The membrane is outlined with black lines

BACKGROUND Equine asthma (EA) causes airflow impairment, which increases in severity with exercise. Electrical impedance tomography (EIT) is an imaging technique that can detect airflow changes in standing healthy horses during a histamine provocation test. OBJECTIVES To explore EIT-calculated flow variables before and after exercise in healthy horses and horses with mild-to-moderate (MEA) and severe equine asthma (SEA). ANIMALS Nine healthy horses 9 horses diagnosed with MEA and 5 with SEA were prospectively included. METHODS Recordings were performed before and after 15 minutes of lunging. Absolute values from global and regional peak inspiratory (PIF, positive value) and expiratory (PEF, negative value) flows were calculated. Data were analyzed using a mixed model analysis followed by Bonferroni's multiple comparisons test to evaluate the impact of exercise and diagnosis on flow indices. RESULTS Control horses after exercise had significantly lower global PEF and PIF compared to horses with SEA (mean difference [95% confidence interval, CI]: 0.0859 arbitrary units [AU; 0.0339-0.1379], P

Additional file 2: Figure S1. Original full length image of SDS-PAGE analysis shown in Fig. 2A. The SDS-gel was stained with Page Blue protein staining solution (Thermo Scientific) and distained with water. The wet gel was scanned directly. Half of the gel image is shown here because the other half is not relevant.

The aims of this study were to better characterize the adrenal response to i.v. adrenocorticotropic hormone (ACTH) in horses with and without gastric disease and to validate and simplify the ACTH stimulation test by determining the diagnostic accuracy of six consecutive sampling time points after ACTH administration for equine glandular gastric disease (EGGD) and equine squamous gastric disease (ESGD). Twenty-six endurance and eventing horses without clinical disease [Sport Horse Population (SHP)] and an independent population of 62 horses [General Population (GP)] were grouped by gastroscopic findings (no/mild vs. moderate/severe EGGD, grade 0–1 vs. 2–4 ESGD, respectively) and underwent an ACTH stimulation test. Salivary cortisol (ng/mL) was analyzed before and 30, 60, 90, 120, and 150 min after i.v. injection of 1 µg/kg BW synthetic ACTH1-24. The association between having moderate or severe EGGD or ESGD and the amount of salivary cortisol was analyzed by means of receiver-operating characteristic (ROC) analysis. The following explanatory variables were considered: cortisol values for every time point, the area under the curve (AUC)—including all time points and corrected for the baseline—and the partial areas under the curve AUC0-90 and AUC90-150. Sampling after 60 min had highest association with moderate/severe EGGD. The diagnostic potential of the ACTH test was higher for the SHP [sensitivity 100% (95% CI 54% to 100%), specificity 75% (95% CI 51% to 91%), ROC-AUC 91% (95% CI 69% to 98%), 1-sided P-value < 0.001] than for the GP [sensitivity 75% (95% CI 48% to 93%), specificity 52% (95% CI 37% to 67%), ROC-AUC 68% (95% CI 51% to 79%), 1-sided P-value = 0.0064]. There were, however, no significant associations with ESGD. The superiority of sampling after 60 min suggests that the initial release of cortisol rather than its peak or the AUC are relevant regarding EGGD. Even though the wide confidence intervals and thus the lack of diagnostic accuracy do not presently support clinical use, characterization of the adrenal response to an ACTH stimulus improves the understanding of EGGD pathophysiology and its relation to stress.

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.

Background: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. Results: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of similar to 5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of similar to 2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of similar to 670 thousand SNPs (MNEc670k), was designed for genotype imputation. Conclusions: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.

Circulating miRNAs in body fluids, particularly serum, are promising candidates for future routine biomarker profiling in various pathologic conditions in human and veterinary medicine. However, reliable standardized methods for miRNA extraction from equine serum and fresh or archived whole blood are sorely lacking. We systematically compared various miRNA extraction methods from serum and whole blood after short and long-term storage without addition of RNA stabilizing additives prior to freezing. Time of storage at room temperature prior to freezing did not affect miRNA quality in serum. Furthermore, we showed that miRNA of NGS-sufficient quality can be recovered from blood samples after >10 years of storage at -80 °C. This allows retrospective analyses of miRNAs from archived samples.