Genome-Wide Association Study and Subsequent Exclusion of ATCAY as a Candidate Gene Involved in Equine Neuroaxonal Dystrophy Using Two Animal Models.

Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder of unknown etiology. Clinical signs of neurological deficits develop within the first year of life in vitamin E (vitE) deficient horses. A genome-wide association study (GWAS) was carried out using 670,000 SNP markers in 27 case and 42 control Quarter Horses. Two markers, encompassing a 2.5 Mb region on ECA7, were associated with the phenotype (p = 2.05 × 10-7 and 4.72 × 10-6). Within this region, caytaxin (ATCAY) was identified as a candidate gene due to its known role in Cayman Ataxia and ataxic/dystonic phenotypes in mouse models. Whole-genome sequence data in four eNAD/EDM and five unaffected horses identified 199 associated variants within the ECA7 region. MassARRAY® genotyping was performed on these variants within the GWAS population. The three variants within ATCAY were not concordant with the disease phenotype. No difference in expression or alternative splicing was identified using qRT-PCR in brainstem across the ATCAY transcript. Atcayji-hes mice were then used to conduct functional analysis in a second animal model. Histologic lesions were not identified in the central nervous system of Atcayji-hes mice. Additionally, supplementation of homozygous Atcayji-hes mice with 600 IU/day of dl-α-tocopheryl acetate (vitE) during gestation, lactation, and adulthood did not improve the phenotype. ATCAY has therefore been excluded as a candidate gene for eNAD/EDM.

Treatment Options for Melanoma of Gray Horses.

All gray horses inherited a single gene mutation, STX17G, that unbalances melanocyte behavior to cause graying and propensities to develop vitiligo and melanoma. The coat color genes ASIPa and MC1RE add risk such that relative likelihood of melanoma based on pregraying coat color is black > bay > chestnut. Melanomas begin at about 4 years. Locoregional control of melanoma masses depends on surgical removal and/or intralesional chemotherapy (possibly with adjunctive hyperthermia or electroporation). Systemic treatment is not evidence based but immunomodulators (cimetidine, levamisole) and vaccines can be tried.

Effect of increased adiposity on insulin sensitivity and adipokine concentrations in different equine breeds adapted to cereal-rich or fat-rich meals.

The relationships between diet, obesity and insulin dysregulation in equids require further investigation due to their association with laminitis. This study examined the effect of dietary glycaemic load and increased adiposity on insulin sensitivity and adipokine concentrations in different equine breeds. Equal numbers of Standardbred horses, mixed-breed ponies and Andalusian horses were provided with ad libitum hay plus either cereal-rich (CHO; n = 12), fat-rich (FAT; n = 12) or control (CON; n = 9) meals over 20 weeks. The isocaloric CHO and FAT diets were fed to induce obesity by gradually increasing the supplementary feeds to provide 200% of daily digestible energy requirements by Week 20. The CON group were fed a basal ration only and maintained moderate body condition. At Week 20, the CHO and FAT groups demonstrated significantly increased body condition score, bodyweight, total body fat mass and plasma leptin concentrations compared with the CON group (P <0.001). The CHO group had lower insulin sensitivity (SI; P <0.001) and higher acute insulin response to glucose (P = 0.002) than the CON group. In contrast, the FAT group was no different to the control group. Ponies and Andalusians had lower SI values compared with Standardbreds, regardless of diet group (P = 0.001). Adiponectin concentrations were similar between the FAT and CON groups, but were significantly lower in the CHO group (P = 0.010). The provision of cereal-rich meals appeared to be a more important determinant of insulin sensitivity than the induction of obesity per se. Whether hypoadiponectinaemia is a cause or consequence of insulin dysregulation warrants further investigation.

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.

Twenty-five thousand years of fluctuating selection on leopard complex spotting and congenital night blindness in horses.

Leopard complex spotting is inherited by the incompletely dominant locus, LP, which also causes congenital stationary night blindness in homozygous horses. We investigated an associated single nucleotide polymorphism in the TRPM1 gene in 96 archaeological bones from 31 localities from Late Pleistocene (approx. 17 000 YBP) to medieval times. The first genetic evidence of LP spotting in Europe dates back to the Pleistocene. We tested for temporal changes in the LP associated allele frequency and estimated coefficients of selection by means of approximate Bayesian computation analyses. Our results show that at least some of the observed frequency changes are congruent with shifts in artificial selection pressure for the leopard complex spotting phenotype. In early domestic horses from Kirklareli-Kanligecit (Turkey) dating to 2700-2200 BC, a remarkably high number of leopard spotted horses (six of 10 individuals) was detected including one adult homozygote. However, LP seems to have largely disappeared during the late Bronze Age, suggesting selection against this phenotype in early domestic horses. During the Iron Age, LP reappeared, probably by reintroduction into the domestic gene pool from wild animals. This picture of alternating selective regimes might explain how genetic diversity was maintained in domestic animals despite selection for specific traits at different times.

Allergen-specific IgE in Icelandic horses with insect bite hypersensitivity and healthy controls, assessed by FcepsilonR1alpha-based serology.

Insect bite hypersensitivity (IBH) and atopy can both be causes of pruritus in horses and are associated with allergen-specific IgE to biting insects and environmental allergens respectively. Information with respect to differences in IgE levels in diseased and healthy animals is crucial in enabling an understanding of the clinical relevance of results of allergen-specific IgE tests. The aim of this study was (i) to evaluate and compare levels of allergen-specific IgE, using an ELISA method, in Icelandic horses, with and without IBH, from Iceland and Sweden respectively; (ii) to investigate patterns of allergen-specific IgE to insects, pollens, moulds and mites in those groups of horses; and (iii) to investigate the clinical significance of employing two different cut-off levels for the ELISA. The study compromised a total number of 99 horses from Iceland and Sweden, with and without IBH, divided in 5 groups. Sera from the horses were analysed blindly with the use of Allercept , a non-competitive, solid-phase ELISA-test, designed to detect the presence of allergen-specific IgE in sera using the recombinant alpha chain of the high-affinity IgE receptor (FcepsilonR1alpha). The distribution of the ELISA values was shown for each insect, mould, mite and pollen allergen, in the different groups using 10th, 50th and 90th percentiles. The use of two cut-off levels, 150 EA and 300 EA, did not eliminate the false positives. Horses with IBH had a higher number of positive reactions, counting all the 29 allergens, than healthy controls and this was borderline significant (P=0.053). In this study it was shown that serological testing with an ELISA that uses the high-affinity IgE receptor (FcepsilonR1alpha) is presently not suitable as a tool for establishing a diagnosis of IBH or equine atopy. The importance of establishing a correct cut-off level for the ELISA for the different allergens is emphasised.

[Animal welfare in prevention and therapy of laminitis]. / Tierschutzaspekte bei der Prophylaxe und Therapie der Hufrehe.

Laminitis is a systemic disease which is manifested as a non infectious condition in the foot. The management of feeding and housing conditions is necessary to treat the endocrinological and metabolic disturbances of laminitic horses. The Equine Metabolic Syndrome (EMS) is predisposing for developing laminitis, and it is characterised by obesity, insulin resistance, hypertension and dyslipidaemia. A genetical predisposition is supposed and EMS is accompanied by a lack of exercise and inadequate energy intake. Laboratory examinations are of great importance for diagnosis. Analyses of insulin, glucose and ACTH are of interest. Several approaches to treat laminitis are available, including pharmacological and orthopaedic strategies as well as the management of the feeding and housing conditions. However, the prophylaxis to prevent laminitis has to be emphasised. Predisposed horses should be detected and adequately treated; especially weight reduction in obese horses is in the focus of interest. Horses in the acute stage of laminitis have to be stabled. Furthermore redistributing weight from the most stressed wall is necessary to prevent pain and to minimise laminar damage and displacement of the distal phalanx. In cases of displacement of the distal phalanx a close communication between the veterinarian and the authorised farrier is necessary, in these cases treatment should be supported by x-ray diagnosis. Horses have to be treated with NSAISs to ensure a proper therapy to consider animal welfare. Horses have to be fed with hay and supplemented with minerals and vitamins. Feeding exclusively straw and feed restriction has to be avoided.

The effect of age on serum antibody titers after rabies and influenza vaccination in healthy horses.

BACKGROUND: The proportion of geriatric horses within the equine population has increased in the past decade, but there is limited information on the immune function of these animals. HYPOTHESIS: Aged horses will have a lesser increase in serum antibody response to vaccination. ANIMALS: Thirty-four aged healthy horses (> or = 20 years) and 29 younger adult horses (4-12 years) of various breeds. METHODS: All horses were vaccinated with vaccines of killed rabies and influenza virus. Horses in each age group were allocated to receive either rabies or influenza booster vaccine 4 weeks after the initial vaccination. Serum samples were taken at 0, 4, 8, and 24 weeks. Rabies serum neutralization titers and equine influenza virus specific antibody sub-isotypes (IgGa, IgGb, IgG(T), and IgA) as well as single radial hemolysis (SRH) titers were determined. RESULTS: Rabies antibody titers were similar in the 2 age groups at all sampling times. Aged horses had higher IgGa and IgGb influenza antibody titers before vaccination than younger horses but similar titers after vaccination (P= .004 and P= .0027, respectively). Younger horses had significantly greater increases in titer than aged horses at all sampling times for IgGa (P= .001) and at 8 and 24 weeks for IgGb (P= .041 and .01, respectively). There was no detectable serum IgG(T) at any time point. A significant booster vaccine effect was seen for both antirabies and anti-influenza titers. Anti-influenza titer before vaccination also had a significant effect on subsequent antibody response. CONCLUSIONS AND CLINICAL IMPORTANCE: Healthy aged horses generated a primary immune response to a killed rabies vaccine similar to that of younger adult horses. Aged horses had a significantly reduced anamnestic response to influenza vaccine.

Early laminar events involving endothelial activation in horses with black walnut- induced laminitis.

OBJECTIVE: To determine proinflammatory gene expression, endothelial adhesion molecule gene expression, and matrix metalloproteinase (MMP) concentrations in laminar specimens at 1.5 hours after administration of black walnut extract (BWE) and to compare these values with later time points. ANIMALS: 25 horses. PROCEDURES: After nasogastric administration of BWE, anesthesia was induced at 1.5 hours in early time point (ETP) horses (n = 5), between 3 and 4 hours in developmental time point horses (5), and between 9 and 10 hours in acute onset of lameness time point horses (5). Anesthesia was induced at 3 and 10 hours after nasogastric administration of water in 2 groups of control horses (3-hour control group, n = 5; 10-hour control group, 5). Real-time quantitative PCR assay was performed on laminar specimens from control and ETP horses for cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1beta, tumor necrosis factor-alpha, IL-6, IL-8, IL-10, MMP-2, and MMP-9 gene expression; and on laminar specimens from all groups for endothelial adhesion molecules, intercellular adhesion molecule (ICAM)-1, and E-selectin gene expression. Leukocyte emigration was assessed via CD13 immunohistochemistry, and gelatinase accumulation was determined by gelatin zymography. RESULTS: Laminar concentrations of IL-1beta, IL-6, IL-8, COX-2, ICAM-1, and E-selectin mRNA were significantly increased in ETP horses, compared with control horses. Concentrations of IL-1beta, IL-8, ICAM-1, and E-selectin mRNA peaked at 1.5 hours. In ETP horses, leukocyte emigration was present in 3 of 5 horses and pro-MMP-9 was detected in 2 of 5 horses. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that endothelial activation and laminar inflammation are early events in laminitis; MMP accumulation likely is a downstream event.

Equine motor neuron disease is not linked to Cu/Zn superoxide dismutase mutations: sequence analysis of the equine Cu/Zn superoxide dismutase cDNA.

The cDNA encoding the equine copper/zinc superoxide dismutase (SOD1) was cloned from leukocyte total RNA from healthy horses and its nucleotide (nt) sequence was determined. We further sequenced the SOD1 gene from 16 horses diagnosed with equine motor neuron disease (EMND) and eight unrelated, clinically normal horses to determine if this disease, similar to amyotrophic lateral sclerosis (ALS) in humans, is linked to SOD1 mutations. The 465-bp SOD1 coding region in the horse encodes 153 amino acid (aa) residues. Equine SOD1 exhibited 81.8 and 79.9% sequence identity to the human homolog at the nt and aa levels, respectively, with only five distinct aa in the two loops that constitute the active site of the enzyme. None of the human SOD1 mutations found in the familial form of ALS were detected in SOD1 of the 16 affected horses. Although DNA sequence analysis identified three potential polymorphisms in equine SOD1, these were silent and were found in both normal and EMND-afflicted horses. At this time, there is no conclusive evidence for EMND linkage to SOD1 mutations.

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.

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.

Abnormal flexion of the corono-pedal joint or "contracted tendons" in unweaned foals.

A condition frequently misnamed "contracted tendons" is described in unweaned foals. Various theories regarding its aetiology are examined. Overfeeding and lack of exercise are suggested as being the most likely causes, leading to excessive growth of the long bones. An effective method of treatment is described. "Contracted tendons" in yearlings are also discussed.