The tocopherol transfer protein mediates vitamin E trafficking between cerebellar astrocytes and neurons.

Alpha-tocopherol (vitamin E) is an essential nutrient that functions as a major lipid-soluble antioxidant in humans. The alpha-tocopherol transfer protein (TTP) binds α-tocopherol with high affinity and selectivity and regulates whole-body distribution of the vitamin. Heritable mutations in the TTPA gene result in familial vitamin E deficiency, elevated indices of oxidative stress, and progressive neurodegeneration that manifest primarily in spinocerebellar ataxia. Although the essential role of vitamin E in neurological health has been recognized for over 50 years, the mechanisms by which this essential nutrient is transported in the central nervous system are poorly understood. Here we found that, in the murine cerebellum, TTP is selectively expressed in glial fibrillary acidic protein-positive astrocytes, where it facilitates efflux of vitamin E to neighboring neurons. We also show that induction of oxidative stress enhances the transcription of the TtpA gene in cultured cerebellar astrocytes. Furthermore, secretion of vitamin E from astrocytes is mediated by an ABC-type transporter, and uptake of the vitamin into neurons involves the low-density lipoprotein receptor-related protein 1. Taken together, our data indicate that TTP-expressing astrocytes control the delivery of vitamin E from astrocytes to neurons, and that this process is homeostatically responsive to oxidative stress. These are the first observations that address the detailed molecular mechanisms of vitamin E transport in the central nervous system, and these results have important implications for understanding the molecular underpinnings of oxidative stress-related neurodegenerative diseases.

Safety and efficacy of subcutaneous alpha-tocopherol in healthy adult horses.

Vitamin E is essential for neuromuscular function. The primary treatment, oral supplementation with natural ('RRR') α-tocopherol, is not effective in all horses. The objectives of this pilot study were to evaluate the safety and efficacy of a subcutaneously administered RRR-α-tocopherol preparation. Horses were randomly assigned in a cross-over design to initially receive RRR-α-tocopherol (5000 IU/450 kg of 600 IU/mL) subcutaneously (n = 3) or orally (n = 3) or were untreated sentinels (n = 2). Tissue reactions following injection in Phase I of the study necessitated adjustment of the preparation with reduction of the RRR-α-tocopherol concentration to 500 IU/mL in Phase 2. Following an 8-week washout period, horses received the reciprocal treatment route with the new preparation (5000 IU/450 kg of 500 IU/mL). Serum, CSF and muscle α-tocopherol concentrations were determined by high-performance liquid chromatography over a 14-day period during each phase. Serum and CSF α-tocopherol concentrations increased significantly postinjection only when the 500 IU/mL product was administered (P<0.0001). There was no significant difference in the muscle concentration of α-tocopherol following either treatment. All eight horses had marked tissue reaction to subcutaneous injection, regardless of product concentration. Whilst we have demonstrated that this route may be a useful alternative to oral supplementation, the marked tissue reaction makes use of such products limited at this time to only the most refractory of cases.

Functional phenotyping of the CYP2D6 probe drug codeine in the horse.

BACKGROUND: In humans, the drug metabolizing enzyme CYP2D6 is highly polymorphic resulting in substantial differences in the metabolism of drugs including anti-arrhythmics, neuroleptics, and opioids. The objective of this study was to phenotype a population of 100 horses from five different breeds and assess differences in the metabolic activity of the equine CYP2D6 homolog using codeine as a probe drug. Administration of a probe drug is a common method used for patient phenotyping in human medicine, whereby the ratio of parent drug to metabolite (metabolic ratio, MR) can be used to compare relative enzyme function between individuals. A single oral dose of codeine (0.6 mg/kg) was administered and plasma concentrations of codeine and its metabolites were determined using liquid chromatography mass spectrometry. The MR of codeine O-demethylation [(codeine)/(morphine + morphine-3-glucuronide + morphine-6-glucuronide)] was determined using the area under the plasma concentration-time curve extrapolated from time zero to infinity (AUC0-∞) for each analyte and used to group horses into predicted phenotypes (high-, moderate-, and low-MR). RESULTS: The MR of codeine O-demethylation ranged from 0.002 to 0.147 (median 0.018) among all horses. No significant difference in MR was observed between breeds, age, or sex. Of the 100 horses, 11 were classified as high-MR, 72 moderate-MR, and 17 low-MR. Codeine AUC0-∞ and O-demethylation MR were significantly different (p < 0.05) between all three groups. The mean ± SD MR was 0.089 ± 0.027, 0.022 ± 0.011, and 0.0095 ± 0.001 for high-, moderate-, and low-MR groups, respectively. The AUC for the morphine metabolites morphine-3-glucuronide and morphine-6-glucuronide were significantly different between high-and low-MR groups (p < 0.004 and p < 0.006). CONCLUSIONS: The MR calculated from plasma following codeine administration allowed for classification of horses into metabolic phenotypes within a large population. The range of codeine metabolism observed among horses suggests the presence of genetic polymorphisms in CYP2D82 of which codeine is a known substrate. Additional studies including CYP2D82 genotyping of high- and low-MR individuals are necessary to determine the presence of CYP2D polymorphisms and their functional implications with respect to the metabolism of therapeutics.

Warmblood fragile foal syndrome type 1 mutation (PLOD1 c.2032G>A) is not associated with catastrophic breakdown and has a low allele frequency in the Thoroughbred breed.

BACKGROUND: Catastrophic fractures are among the most common cause of fatalities in racehorses. Several factors, including genetics, likely contribute to increased risk for fatal injuries. A variant in the procollagen-lysine, 2-oxoglutarate 5-dioxygenase1 gene (PLOD1 c.2032G>A) was shown to cause Warmblood fragile foal syndrome type 1 (WFFS), a fatal recessive defect of the connective tissue. Screening of multiple horse breeds identified the presence of the WFFS allele in the Thoroughbred. PLOD1 is involved in cross-linking of collagen fibrils and thus could potentially increase the risk of catastrophic breakdown. OBJECTIVES: Estimate the frequency of the WFFS allele (PLOD1 c.2032G>A) and determine if it is a risk factor for catastrophic breakdown in the Thoroughbred. STUDY DESIGN: Case-control genetic study. METHODS: Genomic DNA from hair and/or tissue samples was genotyped for the WFFS allele. Fisher's Exact tests were performed to compare allele and carrier frequencies between the case cohort (catastrophic breakdown, n = 22) and several cohorts with no record of injury (n = 138 raced/trained at same track and season and n = 185 older than 7 years and raced during same season), nonracers (n = 92), and a random sample without consideration for racing history (n = 279). RESULTS: The frequency of the PLOD1 c.2032G>A variant in the Thoroughbred breed is low (1.2%). Seventeen of 716 Thoroughbreds tested were carriers (2.4%) and no WFFS homozygotes were detected. Only one catastrophic breakdown case carried the WFFS allele. No statistically significant difference in allele or carrier frequency was identified between case and control cohorts (P>0.05 in all comparisons performed). MAIN LIMITATIONS: This study evaluated cases from one single track. CONCLUSIONS: This study demonstrated that the PLOD1 c.2032G>A associated with WFFS is present at very low frequency in Thoroughbreds and is not a genetic risk factor for catastrophic breakdown.

Ten years of the horse reference genome: insights into equine biology, domestication and population dynamics in the post-genome era.

The horse reference genome from the Thoroughbred mare Twilight has been available for a decade and, together with advances in genomics technologies, has led to unparalleled developments in equine genomics. At the core of this progress is the continuing improvement of the quality, contiguity and completeness of the reference genome, and its functional annotation. Recent achievements include the release of the next version of the reference genome (EquCab3.0) and generation of a reference sequence for the Y chromosome. Horse satellite-free centromeres provide unique models for mammalian centromere research. Despite extremely low genetic diversity of the Y chromosome, it has been possible to trace patrilines of breeds and pedigrees and show that Y variation was lost in the past approximately 2300 years owing to selective breeding. The high-quality reference genome has led to the development of three different SNP arrays and WGSs of almost 2000 modern individual horses. The collection of WGS of hundreds of ancient horses is unique and not available for any other domestic species. These tools and resources have led to global population studies dissecting the natural history of the species and genetic makeup and ancestry of modern breeds. Most importantly, the available tools and resources, together with the discovery of functional elements, are dissecting molecular causes of a growing number of Mendelian and complex traits. The improved understanding of molecular underpinnings of various traits continues to benefit the health and performance of the horse whereas also serving as a model for complex disease across species.

Generation of an equine biobank to be used for Functional Annotation of Animal Genomes project.

The Functional Annotation of Animal Genomes (FAANG) project aims to identify genomic regulatory elements in both sexes across multiple stages of development in domesticated animals. This study represents the first stage of the FAANG project for the horse, Equus caballus. A biobank of 80 tissue samples, two cell lines and six body fluids was created from two adult Thoroughbred mares. Ante-mortem assessments included full physical examinations, lameness, ophthalmologic and neurologic evaluations. Complete blood counts and serum biochemistries were also performed. At necropsy, in addition to tissue samples, aliquots of serum, ethylenediaminetetraacetic acid (EDTA) plasma, heparinized plasma, cerebrospinal fluid, synovial fluid, urine and microbiome samples from all regions of the gastrointestinal and urogenital tracts were collected. Epidermal keratinocytes and dermal fibroblasts were cultured from skin samples. All tissues were grossly and histologically evaluated by a board-certified veterinary pathologist. The results of the clinical and pathological evaluations identified subclinical eosinophilic and lymphocytic infiltration throughout the length of the gastrointestinal tract as well as a mild clinical lameness in both animals. Each sample was cryo-preserved in multiple ways, and nuclei were extracted from selected tissues. These samples represent the first published systemically healthy equine-specific biobank with extensive clinical phenotyping ante- and post-mortem. The tissues in the biobank are intended for community-wide use in the functional annotation of the equine genome. The use of the biobank will improve the quality of the reference annotation and allow all equine researchers to elucidate unknown genomic and epigenomic causes of disease.

Investigation of Known Genetic Mutations of Arabian Horses in Egyptian Arabian Foals with Juvenile Idiopathic Epilepsy.

BACKGROUND: The carrier status of lavender foal syndrome (LFS), cerebellar abiotrophy (CA), severe combined immunodeficiency (SCID), and occipitoatlantoaxial malformation (OAAM1) in foals with juvenile idiopathic epilepsy (JIE) is unknown. HYPOTHESIS/OBJECTIVES: To determine the carrier status of LFS, CA, SCID, and OAAM1 in foals with JIE. ANIMALS: Ten foals with JIE. MATERIALS AND METHODS: Archived DNA samples were tested for known genetic mutations causing LFS, CA, SCID, and OAAM1. The inclusion criteria consisted of having been diagnosed with JIE by ruling out other causes of seizures in foals and supported by electroencephalographic examination. RESULTS: Ten Egyptian Arabian horses (5 females and 5 males) were phenotyped as foals with JIE by electroencephalography (EEG). All foals were negative for the genetic mutations that cause LFS, CA, SCID, and OAAM1 except for 1 foal that was a carrier of CA. CONCLUSIONS AND CLINICAL IMPORTANCE: Juvenile idiopathic epilepsy of Egyptian Arabian foals and LFS appear to be phenotypically and genetically distinct disorders. There was no apparent association between JIE and LFS, CA, SCID, and OAAM1.

Variation in MUTYH expression in Arabian horses with Cerebellar Abiotrophy.

Cerebellar Abiotrophy (CA) is a neurodegenerative disease in Arabian horses affecting the cerebellum, more specifically the Purkinje neurons. Although CA occurs in several domestic species, CA in Arabian horses is unique in that a single nucleotide polymorphism (SNP) has been associated with the disease. Total RNA sequencing (RNA-seq) was performed on CA-affected horses to address the molecular mechanism underlying the disease. This research expands upon the RNA-seq work by measuring the impact of the CA-associated SNP on the candidate gene MutY homolog (MUTYH) and its regulation, isoform-specific expression and protein localization. We hypothesized that the CA-associated SNP compromises the promoter region of MUTYH, leading to differential expression of its isoforms. Our research demonstrates that the CA-associated SNP introduces a new binding site for a novel transcription factor (Myelin Transcription Factor-1 Like protein, MYT1L). In addition, CA-affected horses show differential expression of a specific isoform of MUTYH as well as different localization in the Purkinje and granular neurons of the cerebellum.

Identification of long non-coding RNA in the horse transcriptome.

BACKGROUND: Efforts to resolve the transcribed sequences in the equine genome have focused on protein-coding RNA. The transcription of the intergenic regions, although detected via total RNA sequencing (RNA-seq), has yet to be characterized in the horse. The most recent equine transcriptome based on RNA-seq from several tissues was a prime opportunity to obtain a concurrent long non-coding RNA (lncRNA) database. RESULTS: This lncRNA database has a breadth of eight tissues and a depth of over 20 million reads for select tissues, providing the deepest and most expansive equine lncRNA database. Utilizing the intergenic reads and three categories of novel genes from a previously published equine transcriptome pipeline, we better describe these groups by annotating the lncRNA candidates. These lncRNA candidates were filtered using an approach adapted from human lncRNA annotation, which removes transcripts based on size, expression, protein-coding capability and distance to the start or stop of annotated protein-coding transcripts. CONCLUSION: Our equine lncRNA database has 20,800 transcripts that demonstrate characteristics unique to lncRNA including low expression, low exon diversity and low levels of sequence conservation. These candidate lncRNA will serve as a baseline lncRNA annotation and begin to describe the RNA-seq reads assigned to the intergenic space in the horse.

Clinical and histopathological features of myofibrillar myopathy in Warmblood horses.

BACKGROUND: To report a novel exertional myopathy, myofibrillar myopathy (MFM) in Warmblood (WB) horses. OBJECTIVES: To 1) describe the distinctive clinical and myopathic features of MFM in Warmblood horses and 2) investigate the potential inheritance of MFM in a Warmblood family. STUDY DESIGN: Retrospective selection of MFM cases and prospective evaluation of a Warmblood family. METHODS: Retrospectively, muscle biopsies were selected from Warmblood horses diagnosed with MFM and clinical histories obtained (n = 10). Prospectively, muscle biopsies were obtained from controls (n = 8) and a three generation WB family (n = 11). Samples were assessed for histopathology [scored 0-3], fibre types, cytoskeletal and Z disc protein aggregates, electron microscopic alterations (EM) and muscle glycogen concentrations. RESULTS: Myofibrillar myopathy-affected cases experienced exercise intolerance, reluctance to go forward, stiffness and poorly localised lameness. Abnormal aggregates of the cytoskeletal protein desmin were found in up to 120 type 2a and a few type 2x myofibres of MFM cases. Desmin positive fibres did not stain for developmental myosin, α actinin or dystrophin. Scores for internalised myonuclei (score MFM 0.83 ± 0.67, controls 0.22 ± 0.45), anguloid atrophy (MFM 0.95 ± 0.55, controls 0.31 ± 0.37) and total myopathic scores (MFM 5.85 ± 2.10, controls 1.41 ± 2.17) were significantly higher in MFM cases vs. CONTROLS: Focal Z disc degeneration, myofibrillar disruption and accumulation of irregular granular material was evident in MFM cases. Muscle glycogen concentrations were similar between MFM cases and controls. In the Warmblood family, desmin positive aggregates were found in myofibres of the founding dam and in horses from two subsequent generations. MAIN LIMITATIONS: Restricted sample size due to limited availability of well phenotyped cases. CONCLUSIONS: A distinctive and potentially heritable form of MFM exists in Warmblood horses that present with exercise intolerance and abnormal hindlimb gait. Muscle tissue is characterised by ectopic accumulation of desmin and Z disc and myofibrillar degeneration.

Effects of feeding two RRR-α-tocopherol formulations on serum, cerebrospinal fluid and muscle α-tocopherol concentrations in horses with subclinical vitamin E deficiency.

BACKGROUND: Alpha-tocopherol (α-TP) supplementation is recommended for the prevention of various equine neuromuscular disorders. Formulations available include RRR-α-TP acetate powder and a more expensive but rapidly water-dispersible liquid RRR-α-TP (WD RRR-α-TP). No cost-effective means of rapidly increasing serum and cerebrospinal fluid (CSF) α-TP with WD RRR-α-TP and then sustaining concentrations with RRR-α-TP acetate has yet been reported. OBJECTIVES: To evaluate serum, CSF and muscle α-TP concentrations in an 8-week dosing regimen in which horses were transitioned from WD RRR-α-TP to RRR-α-TP acetate. STUDY DESIGN: Non-randomised controlled trial. METHODS: Healthy horses with serum α-TP of <2 µg/mL were divided into three groups and followed for 8 weeks. In the control group (n = 5), no α-TP was administered. In the second group (Group A; n = 7), 5000 IU/day RRR-α-TP acetate was administered. In the third group (Group WD-A; n = 7), doses of 5000 IU/day of WD RRR-α-TP were administered over 3 weeks, followed by a 4-week transition from WD RRR-α-TP to RRR-α-TP acetate, and a final 1 week of treatment with RRR-α-TP acetate. Serum samples were obtained weekly; muscle biopsies were obtained before, at 2.5 weeks and after supplementation. CSF samples were obtained before and after the 8-week period of supplementation. RESULTS: Serum α-TP increased significantly in Group WD-A at week 1 and remained significantly higher than in Group A and the control group throughout the transition, with inter-individual variation in response. Serum α-TP increased significantly by week 7 in Group A. CSF α-TP increased significantly in Group WD-A only. Muscle α-TP concentrations did not differ significantly across groups. Serum and CSF α-TP were closely correlated (r = 0.675), whereas serum and muscle-α-TP concentrations were not correlated. MAIN LIMITATIONS: The study duration was short and data on pre-transition CSF was lacking. CONCLUSIONS: The administration of 5000 IU/day of water-dispersible RRR-α-TP rapidly increases serum α-TP. Serum and CSF α-TP concentrations are sustained with a gradual transition to 5000 IU/day of RRR-α-TP acetate. Periodic evaluation of serum α-TP concentrations is recommended because responses vary among individuals.

Deletion of 2.7 kb near HOXD3 in an Arabian horse with occipitoatlantoaxial malformation.

In the horse, the term occipitoatlantoaxial malformation (OAAM) is used to describe a developmental defect in which the first cervical vertebra (atlas) resembles the base of the skull (occiput) and the second cervical vertebra (axis) resembles the atlas. Affected individuals demonstrate an abnormal posture and varying degrees of ataxia. The homeobox (HOX) gene cluster is involved in the development of both the axial and appendicular skeleton. Hoxd3-null mice demonstrate a strikingly similar phenotype to Arabian foals with OAAM. Whole-genome sequencing was performed in an OAAM-affected horse (OAAM1) and seven unaffected Arabian horses. Visual inspection of the raw reads within the region of HOXD3 identified a 2.7-kb deletion located 4.4 kb downstream of the end of HOXD4 and 8.2 kb upstream of the start of HOXD3. A genotyping assay revealed that both parents of OAAM1 were heterozygous for the deletion. Additional genotyping identified two of 162 heterozygote Arabians, and the deletion was not present in 371 horses of other breeds. Comparative genomics studies have revealed that this region is highly conserved across species and that the entire genomic region between Hoxd4 and Hoxd3 is transcribed in mice. Two additional Arabian foals diagnosed with OAAM (OAAM 2 and 3) were genotyped and did not have the 2.7-kb deletion. Closer examination of the phenotype in these cases revealed notable variation. OAAM3 also had facial malformations and a patent ductus arteriosus, and the actual malformation at the craniocervical junction differed. Genetic heterogeneity may exist across the HOXD locus in Arabian foals with OAAM.

Tissue resolved, gene structure refined equine transcriptome.

BACKGROUND: Transcriptome interpretation relies on a good-quality reference transcriptome for accurate quantification of gene expression as well as functional analysis of genetic variants. The current annotation of the horse genome lacks the specificity and sensitivity necessary to assess gene expression especially at the isoform level, and suffers from insufficient annotation of untranslated regions (UTR) usage. We built an annotation pipeline for horse and used it to integrate 1.9 billion reads from multiple RNA-seq data sets into a new refined transcriptome. RESULTS: This equine transcriptome integrates eight different tissues from 59 individuals and improves gene structure and isoform resolution, while providing considerable tissue-specific information. We utilized four levels of transcript filtration in our pipeline, aimed at producing several transcriptome versions that are suitable for different downstream analyses. Our most refined transcriptome includes 36,876 genes and 76,125 isoforms, with 6474 candidate transcriptional loci novel to the equine transcriptome. CONCLUSIONS: We have employed a variety of descriptive statistics and figures that demonstrate the quality and content of the transcriptome. The equine transcriptomes that are provided by this pipeline show the best tissue-specific resolution of any equine transcriptome to date and are flexible for several downstream analyses. We encourage the integration of further equine transcriptomes with our annotation pipeline to continue and improve the equine transcriptome.

Defining Trends in Global Gene Expression in Arabian Horses with Cerebellar Abiotrophy.

Equine cerebellar abiotrophy (CA) is a hereditary neurodegenerative disease that affects the Purkinje neurons of the cerebellum and causes ataxia in Arabian foals. Signs of CA are typically first recognized either at birth to any time up to 6 months of age. CA is inherited as an autosomal recessive trait and is associated with a single nucleotide polymorphism (SNP) on equine chromosome 2 (13074277G>A), located in the fourth exon of TOE1 and in proximity to MUTYH on the antisense strand. We hypothesize that unraveling the functional consequences of the CA SNP using RNA-seq will elucidate the molecular pathways underlying the CA phenotype. RNA-seq (100 bp PE strand-specific) was performed in cerebellar tissue from four CA-affected and five age-matched unaffected horses. Three pipelines for differential gene expression (DE) analysis were used (Tophat2/Cuffdiff2, Kallisto/EdgeR, and Kallisto/Sleuth) with 151 significant DE genes identified by all three pipelines in CA-affected horses. TOE1 (Log2(foldchange) = 0.92, p = 0.66) and MUTYH (Log2(foldchange) = 1.13, p = 0.66) were not differentially expressed. Among the major pathways that were differentially expressed, genes associated with calcium homeostasis and specifically expressed in Purkinje neurons, CALB1 (Log2(foldchange) = -1.7, p < 0.01) and CA8 (Log2(foldchange) = -0.97, p < 0.01), were significantly down-regulated, confirming loss of Purkinje neurons. There was also a significant up-regulation of markers for microglial phagocytosis, TYROBP (Log2(foldchange) = 1.99, p < 0.01) and TREM2 (Log2(foldchange) = 2.02, p < 0.01). These findings reaffirm a loss of Purkinje neurons in CA-affected horses along with a potential secondary loss of granular neurons and activation of microglial cells.

Major Histocompatibility Complex I and II Expression and Lymphocytic Subtypes in Muscle of Horses with Immune-Mediated Myositis.

BACKGROUND: Major histocompatibility complex (MHC) I and II expression is not normally detected on sarcolemma, but is detected with lymphocytic infiltrates in immune-mediated myositis (IMM) of humans and dogs and in dysferlin-deficient muscular dystrophy. HYPOTHESIS/OBJECTIVES: To determine if sarcolemmal MHC is expressed in active IMM in horses, if MHC expression is associated with lymphocytic subtype, and if dysferlin is expressed in IMM. ANIMALS: Twenty-one IMM horses of Quarter Horse-related breeds, 3 healthy and 6 disease controls (3 pasture myopathy, 3 amylase-resistant polysaccharide storage myopathy [PSSM]). METHODS: Immunohistochemical staining for MHC I, II, and CD4+, CD8+, CD20+ lymphocytes was performed on archived muscle of IMM and control horses. Scores were given for MHC I, II, and lymphocytic subtypes. Immunofluorescent staining for dysferlin, dystrophin, and a-sarcoglycan was performed. RESULTS: Sarcolemmal MHC I and II expression was detected in 17/21 and 15/21 of IMM horses, respectively, and in specific fibers of PSSM horses, but not healthy or pasture myopathy controls. The CD4+, CD8+, and CD20+ cells were present in 20/21 IMM muscles with CD4+ predominance in 10/21 and CD8+ predominance in 6/21 of IMM horses. Dysferlin, dystrophin, and a-sarcoglycan staining were similar in IMM and control muscles. CONCLUSIONS AND CLINICAL IMPORTANCE: Deficiencies of dysferlin, dystrophin, and a-sarcoglycan are not associated with IMM. Sarcolemmal MHC I and II expression in a proportion of myofibers of IMM horses in conjunction with lymphocytic infiltration supports an immune-mediated etiology for IMM. The MHC expression also occured in specific myofibers in PSSM horses in the absence of lymphocytic infiltrates.

Evidence of the Primary Afferent Tracts Undergoing Neurodegeneration in Horses With Equine Degenerative Myeloencephalopathy Based on Calretinin Immunohistochemical Localization.

Equine degenerative myeloencephalopathy (EDM) is characterized by a symmetric general proprioceptive ataxia in young horses, and is likely underdiagnosed for 2 reasons: first, clinical signs overlap those of cervical vertebral compressive myelopathy; second, histologic lesions--including axonal spheroids in specific tracts of the somatosensory and motor systems--may be subtle. The purpose of this study was (1) to utilize immunohistochemical (IHC) markers to trace axons in the spinocuneocerebellar, dorsal column-medial lemniscal, and dorsospinocerebellar tracts in healthy horses and (2) to determine the IHC staining characteristics of the neurons and degenerated axons along the somatosensory tracts in EDM-affected horses. Examination of brain, spinal cord, and nerves was performed on 2 age-matched control horses, 3 EDM-affected horses, and 2 age-matched disease-control horses via IHC for calbindin, vesicular glutamate transporter 2, parvalbumin, calretinin, glutamic acid decarboxylase, and glial fibrillary acidic protein. Primary afferent axons of the spinocuneocerebellar, dorsal column-medial lemniscal, and dorsospinocerebellar tracts were successfully traced with calretinin. Calretinin-positive cell bodies were identified in a subset of neurons in the dorsal root ganglia, suggesting that calretinin IHC could be used to trace axonal projections from these cell bodies. Calretinin-immunoreactive spheroids were present in EDM-affected horses within the nuclei cuneatus medialis, cuneatus lateralis, and thoracicus. Neurons within those nuclei were calretinin negative. Cell bodies of degenerated axons in EDM-affected horses are likely located in the dorsal root ganglia. These findings support the role of sensory axonal degeneration in the pathogenesis of EDM and provide a method to highlight tracts with axonal spheroids to aid in the diagnosis of this neurodegenerative disease.

Suspected myofibrillar myopathy in Arabian horses with a history of exertional rhabdomyolysis.

REASONS FOR PERFORMING STUDY: Although exertional rhabdomyolysis (ER) is common in Arabian horses, there are no dedicated studies describing histopathological characteristics of muscle from Arabian horses with ER. OBJECTIVES: To prospectively identify distinctive histopathological features of muscle from Arabian endurance horses with a history of ER (pro-ER) and to retrospectively determine their prevalence in archived samples from Arabian horses with exertional myopathies (retro-ER). STUDY DESIGN: Prospective and retrospective histopathological description. METHODS: Middle gluteal muscle biopsies obtained from Arabian controls (n = 14), pro-ER (n = 13) as well as archived retro-ER (n = 25) muscle samples previously classified with type 2 polysaccharide storage myopathy (15/25), recurrent exertional rhabdomyolysis (7/25) and no pathology (3/25) were scored for histopathology and immunohistochemical staining of cytoskeletal proteins. Glutaraldehyde-fixed samples (2 pro-ER, one control) were processed for electron microscopy. Pro-ER and retro-ER groups were compared with controls using Mann-Whitney U and Fisher's exact tests. RESULTS: Centrally located myonuclei in mature myofibres were found in significantly more (P<0.05) pro-ER (12/13) and retro-ER (21/25) horses than controls (4/14). Degenerating myofibres were not evident in any biopsies. Retro-ER horses had amylase-resistant polysaccharide (6/25, P<0.05) and higher scores for cytoplasmic glycogen, rimmed vacuoles and rod-like bodies. A few control horses (3/14) and significantly (P<0.05) more pro-ER (12/13) and retro-ER (18/25) horses had disrupted myofibrillar alignment and large desmin and αß-crystallin positive cytoplasmic aggregates. Prominent Z-disc degeneration and focal myofibrillar disruption with regional accumulation of ß-glycogen particles were identified on electron microscopy of the 2 pro-ER samples. CONCLUSIONS: In a subset of Arabian horses with intermittent episodes of exertional rhabdomyolysis, ectopic accumulation of cytoskeletal proteins and Z-disc degeneration bear a strong resemblance to a myofibrillar myopathy. While many of these horses were previously diagnosed with type 2 polysaccharide storage myopathy, pools of glycogen forming within disrupted myofibrils appeared to give the false appearance of a glycogen storage disorder.

Blood and Cerebrospinal Fluid α-Tocopherol and Selenium Concentrations in Neonatal Foals with Neuroaxonal Dystrophy.

BACKGROUND: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (NAD/EDM) is a neurodegenerative disorder affecting genetically predisposed foals maintained on α-tocopherol (α-TP)-deficient diet. OBJECTIVE: Intramuscular α-TP and selenium (Se) administration at 4 days of age would have no significant effect on serum or cerebrospinal fluid (CSF) α-TP in healthy foals. Serum and CSF α-TP, but not Se, would be significantly decreased in NAD/EDM-affected foals during first year of life. ANIMALS: Fourteen Quarter horse foals; 10 healthy foals supplemented with 0.02 mL/kg injectable α-TP and Se (n = 5) or saline (n = 5) at 4 days of age and 4 unsupplemented NAD/EDM-affected foals. METHODS: Complete neurologic examinations were performed, blood and CSF were collected before (4 days of age) and after supplementation at 10, 30, 60, 120, 180, 240, and 360 days of age. Additional blood collections occurred at 90, 150, 210, and 300 days. At 540 days, NAD/EDM-affected foals and 1 unsupplemented healthy foal were euthanized and necropsies performed. RESULTS: Significant decreases in blood, CSF α-TP and Se found in the first year of life in all foals, with most significant changes in serum α-TP from 4-150 days. Dam α-TP and Se significantly influenced blood concentrations in foals. Injection of α-TP and Se did not significantly increase CSF Se, blood or CSF α-TP in healthy foals. NAD/EDM-affected foals had significantly lower CSF α-TP through 120 days. CONCLUSIONS AND CLINICAL IMPORTANCE: Injection of α-TP and Se at 4 days of age does not significantly increase blood or CSF α-TP. Despite all 14 foals remaining deficient in α-TP, only the 4 genetically predisposed foals developed NAD/EDM.

Applied equine genetics.

Genome sequencing of the domestic horse and subsequent advancements in the field of equine genomics have led to an explosion in the development of tools for mapping traits and diseases and evaluating gene expression. The objective of this review is to discuss the current progress in the field of equine genomics, with specific emphasis on assembly and analysis of the reference sequence and subsequent sequencing of a Quarter Horse mare; the genomic tools currently available to researchers and their implications in genomic investigations in the horse; the genomics of Mendelian and non-Mendelian traits; the genomics of performance traits and considerations regarding genetic testing in the horse. The whole-genome sequencing of a Quarter Horse mare has provided additional variants within the equine genome that extend past single nucleotide polymorphisms to include insertions/deletions and copy number variants. Equine single nucleotide polymorphism arrays have allowed for the investigation of both simple and complex genetic traits while DNA microarrays have provided a tool for examining gene expression across various tissues and with certain disease conditions. Recently, next-generation sequencing has become more affordable and both whole-genome DNA sequencing and transcriptome-wide RNA sequencing are methodologies that are being applied to equine genomic research. Research in the field of equine genomics continues to expand rapidly as the cost of genotyping and sequencing decreases, resulting in a need for quality bioinformatics software and expertise to appropriately handle both the size and complexity of these data.