Pedigree Analysis and Exclusion of Alpha‐Tocopherol Transfer Protein (TTPA) as a Candidate Gene for Neuroaxonal Dystrophy in the American Quarter Horse

Background

Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (NAD/EDM) is a neurodegenerative disorder affecting young horses of various breeds that resembles ataxia with vitamin E deficiency in humans, an inherited disorder caused by mutations in the alpha‐tocopherol transfer protein gene (TTPA). To evaluate variants found upon sequencing TTPA in the horse, the mode of inheritance for NAD/EDM had to be established.

Hypothesis

NAD/EDM in the American Quarter Horse (QH) is caused by a mutation in TTPA.

Animals

88 clinically phenotyped (35 affected [ataxia score ≥2], 53 unaffected) QHs with a diagnosis of NAD/EDM with 6 affected and 4 unaffected cases confirmed at postmortem examination.

Procedures

Pedigrees and genotypes across 54,000 single nucleotide polymorphism (SNP) markers were assessed to determine heritability and mode of inheritance of NAD/EDM. TTPA sequence of exon/intron boundaries was evaluated in 2 affected and 2 control horses. An association analysis was performed by 71 SNPs surrounding TTPA and 8 SNPs within TTPA that were discovered by sequencing. RT‐PCR for TTPA was performed on mRNA from the liver of 4 affected and 4 control horses.

Results

Equine NAD/EDM appears to be inherited as a polygenic trait and, within this family of QHs, demonstrates high heritability. Sequencing of TTPA identified 12 variants. No significant association was found using the 79 available variants in and surrounding TTPA. RT‐PCR yielded PCR products of equivalent sizes between affected cases and controls.

Conclusions and Clinical Importance

NAD/EDM demonstrates heritability in this family of QHs. Variants in TTPA are not responsible for NAD/EDM in this study population.     

Equine Degenerative Myeloencephalopathy: A Vitamin E Deficiency That May Be Familial

Two horse farms, on which there was a high incidence of proven and suspected equine degenerative myeloencephalopathy (EDM), were studied. Symmetric ataxia and paresis, along with laryngeal adductor, cervicofacial, local cervical, and cutaneous trunci hyporeflexia, characterized the syndrome. Serum vitamin E concentration reflected a deficient state in affected and unaffected horses on both farms when compared with selected reference groups and with published values. A high incidence of the disease was evident in offspring of two particular sires on one farm. Vitamin E supplementation resulted in correction of the deficient state in most horses and was associated with a drastic reduction in the incidence of EDM on one farm from 40% to less than 10% the year following vitamin E supplementation. In addition, during the last year, the severity of signs in the few cases was dramatically reduced. This information substantiates the hypothesis that EDM is a vitamin E-responsive disorder of Equidae with a possible familial predisposition.     

Plasma and liver copper values in horses with equine degenerative myeloencephalopathy.

Equine degenerative myeloencephalopathy (EDM) is a common spinal cord disease in the horse. The etiology of EDM currently is unknown. In other species, there are similarities in the clinical signs and neuropathological changes observed in EDM and in copper deficiency. The objective of this study was to determine if horses affected with EDM had low levels of plasma or liver copper. Plasma copper values were determined in 25 EDM affected horses and 35 normal horses. Liver copper levels were determined on 13 EDM affected horses and 22 normal horses. Plasma and liver copper values were not significantly lower in EDM affected horses than in control horses.     

Clinical, viral, and genetic evaluation of equine degenerative myeloencephalopathy in a family of Appaloosas

A clinical, viral, hematologic , and genetic study was conducted over a 4-year period on a family of Appaloosas with high incidence of clinical ataxia and pathologic features of equine degenerative myeloencephalopathy. Marginal to deficient serum vitamin E (alpha-tocopherol) and blood selenium values were the only other consistent antemortem abnormalities in the affected horses. Members of this family were all descendants of a clinically normal mare and were raised in 3 separate environments with variable quality of feed. All horses had access to pasture grasses. Normal chromosomal karyotypes were found in 11 affected and/or related horses examined. Equine herpesvirus type 2 was isolated from 4 of the horses, but evidence for a role of this virus in the pathogenesis of the disease was not found. The role of antioxidant deficiency in the pathogenesis of neurologic dysfunction in this equine family and in others reported to be affected with equine degenerative myeloencephalopathy remains speculative.     

Serum vitamin E and blood glutathione peroxidase values of horses with degenerative myeloencephalopathy

Serum vitamin E and blood glutathione peroxidase values were determined in 40 horses with a histologically confirmed diagnosis of degenerative myeloencephalopathy and in 49 age-matched control horses with normal neurologic function. Significant differences were not detected in serum vitamin E or blood glutathione peroxidase values between horses affected with degenerative myeloencephalopathy and control horses. These findings fail to support a reported role of vitamin E deficiency as a cause of equine degenerative myeloencephalopathy.     

Abnormal synaptic protein expression in two Arabian horses with equine degenerative myeloencephalopathy

Numerous swollen neurons and multiple dystrophic axons were observed in the gracillis and cuneatus nuclei of two male Arabian horses, aged six and 12 months of age, with equine degenerative myeloencephalopathy. Swollen neurons and dystrophic axons showed synaptophysin, synaptosomal-associated protein of 25 kDa, syntaxin-1 and alpha-synuclein immunoreactivity. Moreover, dystrophic axons were strongly immunopositive against the ubiquitin protein and against the anti-phosphorylated 200 kDa neurofilament protein. Abnormal expression of integral synaptic vesicle, synaptic vesicle-associated presynaptic plasma membrane and cytosolic proteins, which participate in the trafficking, docking and fusion of the synaptic vesicle to the plasma membrane, suggest that severe disruption of axonal transport plays a crucial role in the pathogenesis of dystrophic axons in equine degenerative myeloencephalopathy (EDM).     

Serially determined plasma alpha-tocopherol concentrations and results of the oral vitamin E absorption test in clinically normal horses and in horses with degenerative myeloencephalopathy

Plasma alpha-tocopherol (vitamin E) values were monitored serially in 9 foals sired by a stallion with equine degenerative myeloencephalopathy (EDM) and in 5 age-matched control foals (sired by a clinically normal stallion) raised in the same environment for the first year of life. Clinical evaluation determined that 8 of the 9 foals sired by the stallion with EDM had neurologic deficits consistent with the disease on one or more occasions during the study period, whereas control foals had normal gait. From 6 weeks to 10 months of age, plasma alpha-tocopherol values in foals with signs of EDM were significantly (P less than 0.001) lower than those in control foals. An oral vitamin E absorption test was performed, and results for 8 of the affected horses and the affected stallion were compared with results for 4 of the monitored control horses and 4 additional control horses. Significant differences were not evident in any of the absorption indices. On the basis of data from this study and supported by reported prophylactic and therapeutic benefits of supplemented vitamin E, low plasma concentration of vitamin E is concluded to be a factor in the development of EDM in the first year of life of hereditarily predisposed foals. It was also concluded that the significantly lower alpha-tocopherol values seen in the foals in this study did not reflect a primary gastrointestinal tract absorption problem.     

Horses on pasture may be affected by equine motor neuron disease

REASONS FOR PERFORMING STUDY: Equine motor neuron disease (EMND) was diagnosed in 3 horses maintained on lush, grass-based pasture. This contrasted with North American studies which identified limited or no access to green herbage as an important risk factor for EMND. HYPOTHESIS: Grazing horses that have an apparently adequate intake of pasture herbage to meet normal equine vitamin E requirements can develop EMND. METHODS: Owners of 32 European horses diagnosed with EMND completed a questionnaire regarding intrinsic, managemental, nutritional and environmental factors that could potentially be risk factors for EMND, and also regarding clinical signs, treatments and case outcome. Plasma/serum vitamin E data for these horses were supplied by the veterinarians. No control population was studied. RESULTS: Thirteen of 32 horses (termed the 'grazing' group) had part- or full-time access to grass-based pasture at the onset of EMND (median duration at pasture 12 h/day, range 3-24 h). Five of these horses were at pasture for at least 235 h/day at the onset of EMND, 2 of which were at pasture for at least 23.5 h/day throughout the year. Despite grazing, all these horses had a low vitamin E status. The remaining 19 horses resembled those cases reported from North America, in that they had no or limited access to pasture. CONCLUSIONS AND POTENTIAL RELEVANCE: A diagnosis of EMND should not be discounted on the basis that a horse has access, even full-time, to lush grass-based pasture. Inadequate vitamin E intake was probably not the sole cause of either the EMND or the low vitamin E status in the grazing horses; the latter was probably the result of abnormal bioavailability or excessive utilisation of vitamin E.     

Ataxia and weakness associated with fourth ventricle vascular anomalies in two horses

Two adult horses with progressive neurologic signs were examined clinically and at necropsy. Both horses had signs of progressive ataxia and weakness, clinically diagnosed as spinal cord in origin. Differential diagnoses for cervical spinal ataxia in horses included cervical vertebral malformation, equine degenerative myeloencephalopathy, equine herpes-virus-I myeloencephalopathy, and equine protozoal myeloencephalopathy. Necropsy findings in both horses were similar and consisted of a large hematoma in the fourth ventricle, with upward compression of the cerebellum and downward compression of the pons and rostral portion of the medulla.     

Variations of serum vitamin E, cholesterol, and total serum lipid concentrations in horses during a 72-hour period

Fluctuations of serum vitamin E (alpha-tocopherol), cholesterol, and total lipids were monitored in 12 horses at 3-hour intervals for 72 hours. Mean coefficients of variation were 12, 5, and 15%, respectively. Statistical analyses were used to conclude that instrumentation error was accountable for only a small portion of the vitamin E variation. Results indicated that a single serum sample assay is an unsatisfactory indicator of vitamin E status in horses. These data have clinical application in the evaluation of horses suspected to be affected with equine degenerative myeloencephalopathy. The large variance of serum total lipids and the lack of correlation of it with serum vitamin E over time preclude the use of vitamin E/serum total lipids ratio in assessing vitamin E status.     

First Report of Methicillin-Resistant Staphylococcus aureus ST5 and ST398 from Purebred Lusitano Horses

Methicillin-resistant Staphylococcus aureus (MRSA) was first described in horses in 1996. The frequency of MRSA colonization in horses varies among European countries, but it is unknown in Portugal. The aim of this study was to screen for MRSA nasal carriage in a sample of horses entering the Equine Unit, Large Animal Veterinary Teaching Hospital of the Faculty of Veterinary Medicine, Lisbon, Portugal. Seventy-one horses were swabbed, and MRSA was identified by selective isolation on a chromogenic medium. Two S aureus isolates showed resistance to oxacillin (minimum inhibitory concentration >4 μg/mL) and contained the mecA gene. Both strains were isolated from purebred Lusitano horses that lived in farms with more than 20 equines. These MRSA strains represented two different clones: isolate FMVA3/10 was an MRSA sequence type ST5 with a staphylococcal cassette chromosome mec VI, coresistant to erythromycin and clindamycin; and isolate FMVA16/10 was sequence type ST398, with a staphylococcal cassette chromosome mec IV, coresistant to tetracycline, gentamicin, and trimethoprim. Isolate FMVA3/10 represents a human epidemic clone not previously reported among horses in Europe, which once again reinforces the fact that transmission of MRSA clones between horses and humans occurs. Isolate FMVA16/10 represents the first report of the detection of MRSA ST398 among horses in Portugal. Lusitano horses can carry animal and human MRSA in the nostrils, acting as reservoirs, which can potentially be transmitted to humans.     

The first reported outbreak of equine herpesvirus myeloencephalopathy in New Zealand

CASE HISTORY AND CLINICAL FINDINGS: On 9 January 2014 (Day 0) a mare from a stud farm in the Waikato region presented with urinary incontinence without pyrexia. Over the following 33 days 15 mares were clinically affected with neurological signs. All but one mare had a foal at foot. The most commonly observed clinical signs were hind limb paresis and ataxia. In some cases recumbency occurred very early in the course of disease and seven mares were subject to euthanasia for humane reasons.

LABORATORY FINDINGS: Equid herpesvirus (EHV) type 1 was detected using PCR in various tissues collected post mortem from two mares with neurological signs. DNA sequencing data from the DNA polymerase gene of the virus showed a nucleotide transition at position 2254, a mutation encoding amino acid D₇₅₂ that is highly associated with the neuropathogenic genotype of EHV-1. In total 12/15 mares were confirmed positive for EHV-1 on PCR. Results from a virus neutralisation test and ELISA on paired serum samples, and PCR on whole blood and nasal swabs, indicated that of four paddocks in a high-risk area where a cluster of cases had occurred, 20/21 (95%) horses were likely to have been exposed or were confirmed infected with EHV-1. Subsequent to the outbreak two mares aborted, one at 9 months and one at 10 months of gestation. The cause of abortion was confirmed as EHV-1 with the same genotype as that involved in the outbreak.

DIAGNOSIS: Equine herpesvirus myeloencephalopathy.

CLINICAL RELEVANCE: The outbreak described shows the considerable impact that can occur in outbreaks of equine herpesvirus myeloencephalopathy in New Zealand. Early biosecurity controls not only reduced the effect on the farm but mitigated the potential for the virus to spread to other horse enterprises.     

Bovine progressive degenerative myeloencephalopathy (weaver syndrome) in brown swiss cattle in Canada: a literature review and case report

A 15-month-old purebred Brown Swiss heifer was presented because of posterior paresis and ataxia. Histopathological examination of the brain and spinal cord showed evidence of a mild diffuse degenerative myeloencephalopathy. The most severe degenerative lesions were located in the white matter of the thoracic spinal cord. We believe this to be the first documented case of bovine progressive degenerative myeloencephalopathy (“weaver syndrome”) in Canada.     

Disease Associated with Equine Coronavirus Infection and High Case Fatality Rate

Background

Equine coronavirus (ECoV) is associated with clinical disease in adult horses. Outbreaks are associated with a low case fatality rate and a small number of animals with signs of encephalopathic disease are described.

Objectives

The aim of this study is to describe the epidemiological and clinical features of two outbreaks of ECoV infection that were associated with an high case fatality rate.

Animals

14 miniature horses and 1 miniature donkey testing fecal positive for ECoV from two related disease outbreaks.

Methods

Retrospective study describing the epidemiological findings, clinicopathological findings, and fecal viral load from affected horses.

Results

In EcoV positive horses, 27% (4/15) of the animals died or were euthanized. Severe hyperammonemia (677 μmol/L, reference range ≤60 μmol/L) was identified in one animal with signs of encephalopathic disease that subsequently died. Fecal viral load (ECoV genome equivalents per gram of feces) was significantly higher in the nonsurvivors compared to animals that survived (P = .02).

Conclusions and Clinical Importance

Equine coronavirus had a higher case fatality rate in this group of miniature horses than previously reported in other outbreaks of varying breeds. Hyperammonemia could contribute to signs of encephalopathic disease, and the fecal viral load might be of prognostic value in affected horses.     

Prevalence of Radiographic Signs of Osteoarthritis in Lusitano Purebred Horses

Radiographic prevalence and correlation of radiographic findings has not been performed in Lusitano Purebred horses. The aim of this study was to (1) evaluate the prevalence of primary osteoarthritis radiographic findings in Lusitano Purebred horses; (2) to assess correlations between radiographic findings in different joints of the same limb and different limbs; and (3) elucidate the effect of age in the radiographic findings. A radiographic protocol of the stifle, tarsi, fetlocks and distal limbs was done in 98 Lusitanos and the classification of the radiographs was performed using a 0-4 scale developed and applied blindly by three veterinarians. The distal interphalangeal, proximal interphalangeal, metacarpophalangeal, metatarsophalangeal, tarsometatarsal, distal intertarsal, proximal intertarsal/, tibiotarsal and femorotibial-patellar joints were evaluated. Most joints presented no abnormal findings or minor abnormal radiographic findings (82.86% grade ≤1). The most affected joint was tarsometatarsal and more severe lesions were found in tarsometatarsal and distal intertarsal. Femorotibial-patellar radiographic changes were rare (2.13%). A strong/moderate correlation was found between contralateral joints with exception hindlimb fetlocks. A moderate correlation was found between fore and hindlimbs for distal limb joints. When analyzing ipsilateral as well as diagonal distal limbs, a strong/moderate correlation was also found. The total score progressed in 0.2 score points per each year of age, revealing that age can be a statistically significant predictor for radiographic changes. Overall, Lusitano horses presented a low prevalence of severe radiographic sings of primary osteoarthritis. Findings in contralateral joints tend to be correlated.     

Equine herpesvirus myeloencephalopathy in a 14-year-old quarter horse stallion

A 14-year-old, quarter horse stallion was presented in lateral recumbency, unable to rise. Equine herpesvirus myeloencephalopathy was diagnosed, based on presentation, clinical signs, and the ruling out of other possibilities. After initial rapid improvements, ataxia remained, as did chronic cystitis secondary to bladder paralysis. He was euthanized after 2 months.     

Quality Control of Compounded Crystalloid Fluids for Intravenous Delivery to Horses

Background

Periodic lack of availability and high cost of commercially produced isotonic fluids for intravenous (IV) use in horses have increasingly led to use of home‐made or commercially compound fluids by veterinarians. Data regarding the quality control and safety of compounded fluids would be of benefit to equine veterinarians.

Objectives

To compare electrolyte concentrations, sterility, and endotoxin contamination of commercially available fluids to 2 forms of compounded isotonic crystalloid fluids intended for IV use in horses.

Methods

Prospective study. Two methods of preparing compounded crystalloids formulated to replicate commercial Plasma‐Lyte A (Abbott, Chicago, IL) were compared. One formulation was prepared by a hand‐mixed method involving chlorinated drinking water commonly employed by equine practitioners, and the other was prepared by means of ingredients obtained from a commercial compounding pharmacy. The variables for comparison were electrolyte concentrations, sterility, and presence of endotoxin contamination.

Results

Electrolyte concentrations were consistent within each product but different between types of fluids (P < 0.0001). Hand‐mixed fluids had significantly more bacterial contamination compared to commercial Plasma‐Lyte A (P = 0.0014). One of the hand‐mixed fluid samples had detectable endotoxin contamination.

Conclusions and Clinical Importance

Chlorinated drinking water is not an acceptable source of water to compound isotonic fluids for IV administration. Equine practitioners should be aware of this risk and obtain the informed consent of their clients.     

Adiposity,Plasma Insulin,Leptin, Lipids,and Oxidative Stress in Mature Light Breed Horses

Background

Increased blood insulin levels are associated with an increased risk of pasture‐associated laminitis in equids.

Objective

To determine the relationship between plasma insulin, leptin, and lipid levels, and measures of oxidative stress with adiposity in mature light breed horses.

Animals

300 randomly selected light breed horses, aged 4–20 years.

Methods

A random sample of horses (140 mares, 151 geldings, and 9 stallions) was drawn from the VMRCVM Equine Field Service practice client list. Evaluations occurred June 15 – August 15, 2006, with all sampling performed between 0600 and 1200 hours. Concentrate feed was withheld for at least 10 hours before sampling. Plasma was analyzed for insulin, glucose, leptin, triglycerides, nonesterified fatty acids, and measures of oxidative stress. Body condition score was determined as the average of 2 independent investigators.

Results

Overconditioned and obese horses had higher plasma insulin (P < .001) and leptin (P < .01) levels than optimally conditioned horses. Obese horses had higher triglyceride levels (P = .006) and lower red blood cell gluthathione peroxidase activities (P = .001) than optimally conditioned horses.

Conclusions and Clinical Importance

Maintaining horses at a BCS <7 might be important for decreasing the risk of pasture‐associated laminitis.