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Background: There is limited information relating bronchoalveolar lavage (BAL) cytology and cytokine messenger ribonucleic acid (mRNA) expression in racehorses with inflammatory airway disease (IAD). Hypothesis and Objective: We hypothesize that cytokine expression in BAL cells would correlate with cytology. Thus, we evaluated the mRNA expression of selected cytokines in BAL cells in racehorses with exercise intolerance and lower airway inflammation. Animals: Thirty-one client-owned Standardbred racehorses with exercise intolerance. Methods: Prospective, observational study. Cells were obtained by BAL, and mRNA expression of interleukin (IL)-1β, IL-4, IL-8, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results: Nine horses had normal BAL cell differential cytology (Controls), while 22 horses had evidence of IAD based on BAL fluid cytology. Relative expressions of TNF-α/glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 0.0092 ± 0.010 versus 0.0045 ± 0.005, P= .034), IL-4/GAPDH (0.001 ± 0.002 versus 0.0003 ± 0.0003, P= .029), and IFN-γ/GAPDH (0.0027 ± 0.003 versus 0.0009 ± 0.001, P= .028) were greater in horses with IAD compared with controls. Furthermore, IL-4/GAPDH (0.001 ± 0.002 versus 0.0002 ± 0.0003, P < .0001) and IFN-γ/GAPDH (0.003 ± 0.003 versus 0.001 ± 0.001, P= .002) mRNA expression was increased in horses with increased metachromatic cell counts compared with horses with normal metachromatic cell counts. Only the mRNA expression of IL-1β/GAPDH (1.1 ± 0.7 versus 0.3 ± 0.3, P= .045) was increased with airway neutrophilia. Conclusions and Clinical Importance: Differences in gene expression were associated with the presence of IAD and with specific cell types present in airway secretions of Standardbred racehorses with poor performance. These findings suggest that different pathophysiological pathways are implicated in IAD.

SEL1L is a well-known protein in the ER-associated degradation (ERAD) pathway. While it is known to be expressed in platelets, SEL1L has never been shown to play an active role. Here, we present evidence that SEL1L regulates platelet function. We first identified SEL1L through the study of Atypical Equine Thrombasthenia (AET), an autosomal recessive platelet disorder found in thoroughbred horses. A missense variant in SEL1L (c.1810A>G p.Ile604Val) was found in AET-affected horses, which we show is associated with decreased protein expression. SEL1L is intracellular in equine platelets and localizes to the surface upon activation with thrombin. Platelets from homozygous horses exhibited substantially decreased spreading on immobilized collagen. Human megakaryocytes were found to have 2 SEL1L protein isoforms that increase in expression during megakaryopoiesis, although only 1 isoform was delivered to mature platelets. Studies using inducible mouse and constitutive zebrafish KOs demonstrated that SEL1L is necessary for efficient platelet or thrombocyte (fish equivalent) adhesion to sites of endothelial injury. These data reveal a previously undescribed and conserved role for the ERAD pathway in the etiology of AET and platelet function, and GWAS data suggest that it may play a role in human platelet disorders as well.

Background Equine recurrent uveitis (ERU), an inflammatory eye disease, is the leading cause of blindness among horses. Insidious uveitis, a form of ERU, is especially pervasive within the Appaloosa breed and is highly heritable (h 2  = 0.68-1.0). To date only one risk locus, leopard complex ( LP ), has been identified, and it explained 0.16–0.33 of the heritability estimate, suggesting that insidious uveitis is a complex genetic disease within the Appaloosa horse breed with multiple unknown predisposing loci. Results A genome-wide association study (GWAS) using relatedness, LP genotype, sex, and age as covariates was performed on a sample of 96 Appaloosas (36 cases and 60 controls) and identified a 9.7 Kb region of association on ECA X (chrX:14528106–14537812) as significantly associated ( P  = 2.11 × 10 −8 ). Sex stratification followed by meta-analysis provided additional support for the association on ECA X ( P  = 1.35 × 10 −8 ). A logistic regression model was performed to test for epistasis between LP and the locus on ECA X, and the results did not support an interaction between the two loci. In the second phase of the study, single-nucleotide variants (SNVs) were identified in the region on ECA X by whole genome sequencing (WGS) of 18 horses from the GWAS (9 cases and 9 controls). Five reference markers from the GWAS, two previously associated coat color loci (LP and PATN1), and 102 SNVs were further evaluated in a combined dataset of 157 horses (70 cases and 87 controls, including the original 96 horses from the GWAS). Using logistic regression, none of the SNVs identified from the WGS analysis were significantly associated with phenotype; however, LP and the top three SNP markers from ECA X (ECA X: 14.5 Mb) were significantly associated in the larger dataset (P LP = 2.34 × 10 −6 and P X = 4.06 × 10 −5 ). Conclusion In addition to the LP locus, our investigation identified a locus on chromosome X with a significant association to insidious uveitis in Appaloosas. Replication testing in an independent cohort is necessary to determine if this locus is indeed a causal risk locus.

Strongylid nematodes in large terrestrial herbivores such as great apes, equids, elephants, and humans tend to occur in complex communities. However, identification of all species within strongylid communities using traditional methods based on coproscopy or single nematode amplification and sequencing is virtually impossible. High-throughput sequencing (HTS) technologies provide opportunities to generate large amounts of sequence data and enable analyses of samples containing a mixture of DNA from multiple species/genotypes. We designed and tested an HTS approach for strain-level identification of gastrointestinal strongylids using ITS-2 metabarcoding at the MiSeq Illumina platform in samples from two free-ranging non-human primate species inhabiting the same environment, but differing significantly in their host traits and ecology. Although we observed overlapping of particular haplotypes, overall the studied primate species differed in their strongylid nematode community composition. Using HTS, we revealed hidden diversity in the strongylid nematode communities in non-human primates, more than one haplotype was found in more than 90% of samples and coinfections of more than one putative species occurred in 80% of samples. In conclusion, the HTS approach on strongylid nematodes, preferably using fecal samples, represents a time and cost-efficient way of studying strongylid communities and provides a resolution superior to traditional approaches.

Ascending placentitis is a significant cause of equine pregnancy loss, yet the upstream inflammatory triggers are poorly defined. Recently, we identified S100A8/S100A9 (S100A8/A9) alarmins as potential upstream regulators in a chronic equine placentitis model. The current study aimed to determine whether this upregulation is sustained in the acute model and in clinical cases, and to elucidate the expression of their downstream inflammatory mediators. Using an experimental model, we quantified S100A8/A9 mRNA expression in acute (n = 5) and chronic (n = 6) placentitis induced by Streptococcus equi ssp. zooepidemicus. We found mRNA expression of S100A8 and S100A9 was significantly upregulated in chorioallantois during both acute (p < 0.001) and chronic (p < 0.0001) disease compared to controls (n = 5), demonstrating their role is not limited to chronic pathology. A strong positive correlation (r = 0.945) underscored their coordinated expression. Immunohistochemistry revealed minimal staining in controls but dense infiltrations of S100A8/A9-positive neutrophils and macrophages in placentitis tissues. To define the clinical relevance of the downstream pathway, we analyzed RNA sequencing data from clinical placentitis cases (placentitis, n = 4) compared to normal postpartum placenta (control, n = 4). This confirmed upregulation of S100A8/A9 and revealed a concurrent increase in their receptors (TLR4, RAGE) and a spectrum of NF-κB-driven effectors, including pro-inflammatory cytokines (IL1β, IL6, TNF), chemokines (CXCL8, CCL2, CXCL10), and the apoptotic mediator CASP3. Our findings establish that S100A8/A9 upregulation is a sustained feature of equine placentitis and delineates a coherent S100A8/A9-TLR4/RAGE-NF-κB signaling axis that drives inflammation and tissue damage in clinical disease. These findings highlight the diagnostic potential of S100A8/A9 and position this alarmin system as a promising therapeutic target for mitigating infection-induced pregnancy loss.

Equine myotonic dystrophy (eMD) is a rare neuromuscular disorder of undetermined origin marked by muscle hypertrophy and stiffness, dystrophic muscle histopathology, and myotonic discharges. In humans, myotonic dystrophy (DM) arises from trinucleotide repeat expansions in dystrophia myotonica protein kinase ( DMPK ) (DM1) or tetranucleotide expansions in cellular nucleic acid-binding protein ( CNBP ) (DM2), which disrupt mRNA processing and induce embryonic splicing patterns across multiple genes. In 6 eMD Quarter Horse types, (2–36 months-of-age) and 8 control Quarter Horses we determined: (1) fiber type composition of triceps , gluteal, and semimembranosus muscles; (2) differential gene (DEG) and protein (DEP) expression using transcriptomic and proteomic analyses; (3) presence of repeat expansions in transcripts of DMPK or CNBP and (4) exon 7 retention in CLCN1 or exon 22 splicing in ATP2A1 . Predominance and clustering of type 1 fibers, expression of embryonic myosin, and upregulated mitochondrial and sarcomeric DEPs characterized eMD hindlimb musculature. Gene ontology (GO) analysis of 730 upregulated DEGs identified numerous GO terms related to morphogenesis of mesoderm-derived tissues and upregulated genes impacting myoD expression in eMD muscle. Top upregulated DEG involved myogenesis ( MYOZ2, SBK2, SBK3, PAMR1 ), neurons, transcription/translation, cytoskeleton, basement/plasma membranes, and calcium binding/transport. Top upregulated proteins also impacted muscle morphogenesis (MUSTN1, CSRP3, TMSBX4, PDLIM, CALD1) as well as categories of mitochondria, sarcomere, extracellular matrix/ basement membrane, transcription, translation, cell cycle regulation, neurons amongst others. Downregulated DEP primarily impacted mitochondria, the sarcomere and glycogen metabolism. Notably, unlike human myotonic dystrophy, trinucleotide repeat expansions were not found in the DMPK 3’UTR (CTG) n nor tetranucleotide repeat expansions (CCTG) n in intron 1 of CNBP . Isoforms of CLCN1 containing fetal exon 7 were detected in equal frequency in eMD and control muscle and exon 22 was not alternatively spliced in ATP2A1 as has been found in DM1. Thus, distinct from DM1 and DM2, eMD is driven by unique molecular mechanisms impacting skeletal muscle morphogenesis, neurons and regulation of gene transcription/translation that alter fiber type composition, distribution and morphology. The origin of myotonia does not appear to be driven by a mutation in CLCN1 or retention of exon CLCN 7. Expanded splice site analysis and further research is warranted to elucidate the cause of myotonia and the distinct etiology of eMD.

Equine Juvenile Spinocerebellar Ataxia (EJSCA) is a novel autosomal recessive neurologic disease in Quarter Horses. Affected foals display a progressive proprioceptive ataxia by 1–5 weeks of age, leading to recumbency and necessitating euthanasia. Whole genome sequencing was performed on 7 EJSCA cases and unaffected horses that included 4 obligate carriers, 4 unaffected half or full-siblings, and 28 unrelated, unaffected control Quarter Horses. An 82 kb region of association was identified (EquCab3.0, chr11: 6963986–7045999), containing 9 candidate SNPs across four genes ( FADS6, FDXR, GRIN2C and TMEM104 ). Decreased FDXR mRNA expression and a cryptic exon was identified in spinal cord tissue from EJSCA cases via RNA-sequencing. One of the 9 associated SNPs ( FDXR-203 c.177 + 1778G > C) was the eighth base pair of this cryptic exon. Affected foals were all homozygous for the variant. Protein concentrations of FDXR were lower in EJSCA cases in spinal cord and liver compared to unaffected controls. The FDXR-203 c.177 + 1778G > C mutation represents the first non-coding neurological genetic variant in horses. Additionally, this is the first genetic cause of a degenerative axonopathy in the horse and a spontaneous disease model to study FDXR pathology in humans.

The Greying with age phenotype in horses involves loss of hair pigmentation whereas skin pigmentation is not reduced, and a predisposition to melanoma. The causal mutation was initially reported as a duplication of a 4.6 kb intronic sequence in Syntaxin 17. The speed of greying varies considerably among Grey horses. Here we demonstrate the presence of two different Grey alleles, G2 carrying two tandem copies of the duplicated sequence and G3 carrying three. The latter is by far the most common allele, probably due to strong selection for the striking white phenotype. Our results reveal a remarkable dosage effect where the G3 allele is associated with fast greying and high incidence of melanoma whereas G2 is associated with slow greying and low incidence of melanoma. The copy number expansion transforms a weak enhancer to a strong melanocyte-specific enhancer that underlies hair greying (G2 and G3) and a drastically elevated risk of melanoma (G3 only). Our direct pedigree-based observation of the origin of a G2 allele from a G3 allele by copy number contraction demonstrates the dynamic evolution of this locus and provides the ultimate evidence for causality of the copy number variation of the 4.6 kb intronic sequence.

Although several inherited ocular disorders have been extensively studied in horses, few reports of equine ectopia lentis exist and no genetic investigations have been reported. Ectopia lentis in humans and other species is reported to be caused by trauma, genetic variants, and systemic diseases. The most commonly reported genetic causes are dominant alleles in FBN1 . Here we examined a 3-day old Oldenburg x Thoroughbred colt due to concerns over bilateral ocular anomalies and hypothesized that either a recessively inherited allele or a dominant de novo allele was the genetic cause. Examination revealed bilateral microphakia and spherophakia with medioventral lens subluxation. Histopathology of the globes was consistent with ectopia lentis. Whole genome sequencing of the affected foal was conducted, and forty-six candidate genes were evaluated for SNVs and small INDELS. Testing both hypotheses, 82 variants were identified, of which 69 were present in publicly available data from 504 horses and not investigated further. Of the 13 remaining variants, two variants were found in 3’ UTRs ( ADAMTS17 and OAF ), ten were intronic, and one was a coding variant located in the FBN1 gene encoding fibrillin-1 (FBN1:p.(Ala882Val)). This variant was also computationally predicted to be deleterious to protein function, including in silico modelling of FBN1 which suggests that 882Val impacts disulfide bond formation by Van der Waals clashing in a hybrid domain of the protein. The affected foal was confirmed by Sanger sequencing to be heterozygous for this variant and his clinically unaffected dam, reportedly unaffected sire, and five paternal half-siblings were homozygous for the reference allele. Additionally, the homologous human substitution is reported to be pathogenic, causing Marfan syndrome with a dominant mode of inheritance, of which ectopia lentis is a common feature. These findings support the de novo hypothesis with FBN1 :p.(Ala882Val) as the likely cause of ectopia lentis in this foal, the first genetic explanation for this condition in the horse. Given the role of FBN1 in ectopia lentis in humans and other species, FBN1 should be evaluated as a potential candidate when other horses with this condition are identified.

Despite its critical role in protecting the fetus, the amniotic membrane remains poorly understood in the context of disease response. The equine amniotic membrane is an important physical barrier to the amniotic compartment, and there is evidence that it may contribute to surfactant synthesis. Surfactants are essential for normal fetal lung development, and disruptions in its availability may be linked to future neonatal complications. Therefore, understanding the molecular changes that occur in fetal-maternal tissues during placentitis would clarify how this condition leads to abortion, preterm delivery, and stillbirth, and identify new strategies to manage the adverse outcomes. Thus, we used RNA sequencing, bioinformatic methods, and immunohistochemistry to characterize the equine amniotic membrane gene expression during experimentally induced ascending placentitis (placentitis group, n  = 6) compared to gestationally matched control groups (control group, n  = 6) at 288 days of gestation. We identified 288 differentially expressed genes (DEG) in the placentitis group compared to the control group. Placentitis was associated with the upregulation of toll-like receptors ( TLR4 ), prostaglandin synthesis ( PTGS2 and PTGES ), apoptosis ( MMP9 and CASP3) , and hypoxia-associated genes ( SOD2 , BNIP3 , and HMOX1 ). Our RNA sequencing results were supported by the visual identification of two of those proteins (TLR4 and PTGS2) in the immunohistochemistry analysis. Functional annotation revealed significant enrichment between the DEGs and the toll receptor signaling pathway, which may be a key factor negatively affecting placental functions. In conclusion, our study revealed that the amniotic membrane is not only a physical barrier but also plays an active role in immune response during ascending placentitis.