A glycogen synthase 1 mutation associated with equine polysaccharide storage myopathy and exertional rhabdomyolysis occurs in a variety of UK breeds

Reasons for performing study: A glycogen synthase (GYS1) mutation has been described in horses with histopathological evidence of polysaccharide storage myopathy (PSSM) in the USA. It is unknown whether the same mutation is present in horses from the UK. Objectives: To determine whether the GYS1 mutation occurs in UK horses with histopathological evidence of PSSM and exertional rhabdomyolysis. Hypothesis: The R309H GYS1 mutation is present in a variety of UK horse breeds and that the mutation is commonly associated with exertional rhabdomyolysis. Methods: DNA was extracted from 47 muscle or blood samples from UK horses with histories of exertional rhabdomyolysis in which muscle biopsy diagnosis had been pursued. The proportions of GYS1 mutation positive cases were compared among histopathologically defined groups. In addition, breeds that carried the GYS1 mutation were identified from a total of 37 grade 2 (amylase‐resistant) PSSM cases. Results: Of 47 horses with exertional rhabdomyolysis in which a muscle biopsy diagnosis was pursued, 10 (21%) carried the GYS1 mutation. The mutation was only found in horses with grade 2 PSSM (i.e. not in horses with normal, idiopathic myopathy or grade 1 PSSM biopsy samples). In total, the GYS1 mutation was found in 24/37 (65%) of grade 2 PSSM cases. A variety of breeds, including Quarter Horse, Appaloosa, Warmblood, Connemara‐cross, Cob, Polo Pony and Thoroughbred cross carried the mutation. Conclusions: The GYS1 mutation is an important cause of exertional rhabdomyolysis of UK horse breeds but does not account for all forms of PSSM. Potential relevance: Genotyping is recommended in cases of exertional rhabdomyolysis, prior to or in combination with, muscle biopsy. However a significant proportion of horses with histopathological evidence of PSSM and/or exertional rhabdomyolysis have different diseases.     

Polysaccharide storage myopathy – the story so far

Polysaccharide storage myopathy (PSSM) was first described in 1992 in Quarter Horses, Appaloosa and Paint‐related breeds with clinical signs of exertional rhabdomyolysis. The disease is characterised by the accumulation of excessive glycogen and diastase‐resistant amylopectin polysaccharide inclusions within skeletal muscle fibres. The discovery of a mutation in the glycogen synthase 1 (GYS1) gene in some, but not all, horses with the disease suggested that PSSM represents a group of diseases with similar pathology but different aetiologies and that the pathogenesis is more complex than initially thought. Type 1 PSSM (PSSM1) refers to horses with the GYS1 mutation and has subsequently been identified in a large number of breeds found in Europe and North America. Clinical presentations associated with PSSM1 can vary and increased muscle enzyme activity at rest or following exercise often accompanies PSSM1; however, such changes may not be present in all cases. A diagnosis of PSSM is made on the basis of histopathology or specifically PSSM1 is diagnosed by genotyping horses for the GYS1 mutation. Cases usually respond well to management changes, in particular a diet low in starch and high in fat when it is accompanied by regular exercise.     

Glycogen synthase 1 (GYS1) mutation in diverse breeds with polysaccharide storage myopathy

Background: A missense mutation in the GYS1 gene was recently described in horses with polysaccharide storage myopathy (PSSM).
Objectives: The first objective was to determine the prevalence of the GYS1 mutation in horses with PSSM from diverse breeds. The second objective was to determine if the prevalence of the GYS1 mutation differed between horses diagnosed with PSSM based on grade 1 (typically amylase-sensitive) or grade 2 (typically amylase-resistant) polysaccharide.
Animals: Eight hundred and thirty-one PSSM horses from 36 breeds.
Procedures: Horses with PSSM diagnosed by histopathology of skeletal muscle biopsy samples were identified from the Neuromuscular Disease Laboratory database. Eight hundred and thirty-one cases had blood or tissue that was available for DNA isolation; these 831 cases were genotyped for the GYS1 mutation by restriction fragment length polymorphism.
Results: The PSSM mutation was identified in horses from 17 different breeds. The prevalence of the GYS1 mutation in PSSM horses was high in Draft- (87%) and Quarter Horse-related breeds (72%) and lower in Warmbloods (18%) and other light horse breeds (24%), when diagnosis was based on grade 2 diagnostic criteria. Overall, the PSSM mutation was present in 16% of grade 1 and 70% of grade 2 PSSM horses.
Conclusions and Clinical Importance: GYS1 mutation causes PSSM in diverse breeds and is the predominant form of PSSM in Draft- and Quarter Horse-related breeds. False-positive diagnosis, as well as the possibility of a second glycogenosis in horses with neuromuscular disease (type 2 PSSM), might explain the absence of the GYS1 mutation in horses diagnosed with excessive glycogen accumulation in muscle.     

The Interplay of Genetics,Exercise, and Nutrition in Polysaccharide Storage Myopathy

Polysaccharide storage myopathy (PSSM), identified in 1992 in a subset of horses with exertional rhabdomyolysis, is a glycogenosis characterized by amylase-resistant polysaccharide in a small number of skeletal muscle fibers along with 1.5 to 4 times normal muscle glycogen. Extensive biochemical and physiological analyses failed to identify defects in glycogenolysis and glycolysis. In 2008, a genome-wide association analysis detected a locus on equine chromosome 10 that was strongly associated with the PSSM in Quarter Horses. Glycogen synthase 1 (GYS1), which encodes the skeletal muscle isoform of glycogen synthase (GS), was a strong candidate gene for PSSM based on its location on equine chromosome 10. Sequencing of the GYS1 gene in PSSM and control Quarter Horses identified only one single base-pair change that resulted in an amino acid substitution in the GS enzyme. Mean GS activity was higher in PSSM than control muscle homogenates in both the presence and absence of the allosteric activator glucose 6-phosphate, suggesting that the GS enzyme in horses with PSSM is constitutively active. High-grain diets increase serum insulin concentrations which further act to stimulate GS activity. An restriction fragment length polymorphism assay for the GYS1 mutation showed that 10% of the Quarter Horse breed and a minimum of 20 other breeds have the GYS1 mutation. Muscle biopsies obtained after 20 minutes of aerobic exercise revealed much higher inosine monophosphate concentrations and lower adenosine monophosphate in whole muscle and single fibers from PSSM as compared with control horse muscle. Thus, the GYS1 mutation responsible for PSSM seems to cause an energy imbalance exacerbated by high-grain diets, which results in adenine nucleotide degradation in individual muscle fibers of horses with PSSM during submaximal exercise.     

An epidemiological study of myopathies in Warmblood horses

REASON FOR PERFORMING STUDY: There are few detailed reports describing muscular disorders in Warmblood horses. OBJECTIVES: To determine the types of muscular disorders that occur in Warmblood horses, along with presenting clinical signs, associated risk factors and response to diet and exercise recommendations, and to compare these characteristics between horses diagnosed with polysaccharide storage myopathy (PSSM), those diagnosed with a neuromuscular disorder other than PSSM (non-PSSM) and control horses. METHODS: Subject details, muscle biopsy diagnosis and clinical history were compiled for Warmblood horses identified from records of biopsy submissions to the University of Minnesota Neuromuscular Diagnostic Laboratory. A standardised questionnaire was answered by owners at least 6 months after receiving the muscle biopsy report for an affected and a control horse. RESULTS: Polysaccharide storage myopathy (72/132 horses) was the most common myopathy identified followed by recurrent exertional rhabdomyolysis (RER) (7/132), neurogenic or myogenic atrophy (7/132), and nonspecific myopathic changes (14/132). Thirty-two biopsies were normal. Gait abnormality, 'tying-up', Shivers, muscle fasciculations and atrophy were common presenting clinical signs. Forty-five owners completed questionnaires. There were no differences in sex, age, breed, history or management between control, PSSM and non-PSSM horses. Owners that provided the recommended low starch fat supplemented diet and regular daily exercise reported improvement in clinical signs in 68% (19/28) of horses with a biopsy submission and 71% of horses diagnosed with PSSM (15/21). CONCLUSIONS: Muscle biopsy evaluation was a valuable tool to identify a variety of myopathies in Warmblood breeds including PSSM and RER. These myopathies often presented as gait abnormalities or overt exertional rhabdomyolysis and both a low starch fat supplemented diet and regular exercise appeared to be important in their successful management. POTENTIAL RELEVANCE: Warmbloods are affected by a variety of muscle disorders, which, following muscle biopsy diagnosis can be improved through changes in diet and exercise regimes.     

Comparison of gluteus medius muscle activity in Haflinger and Noriker horses with polysaccharide storage myopathy

Type 1 polysaccharide storage myopathy caused by genetic mutation in the glycogen synthase 1 gene is present in many breeds including the Noriker and Haflinger horses. In humans, EMG has already been used to document changes in the muscle activity patterns of patients affected by human glycogen storage disorders. Therefore, the aim of the present study was to describe gluteus muscle activity with surface electromyography (sEMG) in Haflinger and Noriker horses with known GYS1 mutation status during walk and trot. Thirty-two horses (11 Haflinger and 21 Noriker horses) with homozygous non-affected (GG), heterozygous affected (GA) and homozygous affected (AA) status of GYS1 mutation without overt clinical signs of any myopathy were selected for the current study. Using surface electromyography gluteus medius muscle activity at walk and at trot was measured, and muscle activity was described in relation to the maximum observed value at the same sensor and the same gait. In order to further describe the signals in detail comprising both frequencies and amplitudes, the crossings through the baseline and the 25, 50 and 75 percentile lines were determined. The result of the relative muscle activity did not show a consistent difference between affected and non-affected horses. Genetically affected (GA and AA) horses showed significantly less density of muscle activity for both gaits and horse breeds except for the crossings per second at the baseline and 75 percentile at walk in the Haflinger horses and 75 percentile at trot in the Noriker horses. The medians of all calculated density values were significantly lower in the GA Haflingers compared to the GG Haflingers (p = 0.012) and also in the AA Norikers compared to the GG Norikers (p = 0.011). Results indicate that the GYS1 mutation reduces the number of functional muscle fibres detected by sEMG measurements even in the absence of overt clinical signs.     

Epidemiologic characteristics and management of polysaccharide storage myopathy in Quarter Horses

OBJECTIVE: To characterize onset and clinical signs of polysaccharide storage myopathy (PSSM) in a well-defined population of affected Quarter Horses, identify risk factors for PSSM, determine compliance of owners to dietary and exercise recommendations, and evaluate the efficacy of dietary and exercise recommendations. ANIMALS: 40 Quarter Horses with PSSM and 37 unaffected control horses. PROCEDURES: Owners of horses with PSSM completed a retrospective questionnaire concerning their horse's condition. RESULTS: Between horses with PSSM and control horses, no significant differences were found in sex distribution (21 vs 15 females and 16 vs 22 males, respectively), temperament, muscle build, diet, or amount of turnout. In horses with PSSM, signs of muscle stiffness, muscle fasciculations, sweating, exercise intolerance, weakness, muscle wasting, reluctance to move, colic, abnormal gait, recumbency, lameness, and swollen muscles began between the age of 1 day and 14 years (mean age, 4.9 +/- 3.5 years). Five horses with PSSM developed acute muscle atrophy. Sixty-three percent (25/40) of owners fed the recommended diet, 55% (22/40) provided regular exercise, and 40% (16/40) followed both dietary and exercise recommendations. Owners of affected horses for which a decrease in severity or frequency of PSSM was not found did not follow the exercise, dietary, or both recommendations. All horses for which both dietary and exercise recommendations were followed had improvement in signs of PSSM. CONCLUSIONS AND CLINICAL RELEVANCE: n addition to exertional rhabdomyolysis, signs of PSSM include acute muscle atrophy and gait abnormalities. It appears that PSSM can be managed by following dietary recommendations combined with gradual increases in daily exercise.     

Prevalences and clinical signs of polysaccharide storage myopathy and shivers in Belgian draft horses

OBJECTIVE: To determine prevalences of polysaccharide storage myopathy (PSSM) and shivers in Belgian Draft Horses (BDHs) and determine whether there was an association between these 2 conditions. DESIGN: Prospective cohort study. ANIMALS: 103 BDHs > 1 year old. PROCEDURE: Owners were questioned regarding clinical signs of PSSM, shivers, and hindquarter weakness, defined as poor hindquarter muscling and lack of propulsion. Blood samples were collected for determination of serum creatine kinase and aspartate transferase activities and serum selenium and vitamin E concentrations. A biopsy sample from the gluteus medius muscle was submitted for histologic, histochemical, and biochemical analysis. A diagnosis of PSSM was made if abnormal amylase-resistant polysaccharide inclusions were seen histologically. RESULTS: 37 (36%) horses had PSSM and 19 (18%) had shivers, but only 6 (6%) had both PSSM and shivers, whereas 31 (30%) had PSSM alone, 13 (13%) had shivers alone, and 53 (51%) had neither, and a significant association between PSSM and shivers was not detected. Hindquarter weakness was found in 30 horses. Only 13 of 37 (35%) horses with PSSM and 11 of 19 (58%) horses with shivers had hindquarter weakness. Serum creatine kinase and aspartate transferase activities and serum selenium and vitamin E concentrations were not significantly different between horses with and without PSSM or between horses with and without shivers. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that PSSM and shivers are common but unrelated disorders in BDHs.     

In vitro contractile responses and contracture testing of skeletal muscle from Quarter Horses with exertional rhabdomyolysis.

OBJECTIVE: To determine whether increased sensitivity to pharmacologic agents was a general property of equine exertional myopathies, including polysaccharide storage myopathy (PSSM) in Quarter Horses. ANIMALS: 5 adult Quarter Horses with exertional rhabdomyolysis and abnormal polysaccharide accumulation in skeletal muscle and 4 clinically normal adult Quarter or Quarter-type horses. PROCEDURES: Twitch time course measurements and contracture responses to various concentrations of caffeine and halothane for small bundles of intact external intercostal muscle fibers were measured in all horses. RESULTS: Caffeine contracture threshold of muscles from Quarter Horses with PSSM was not different from that of clinically normal horses (5 mM in both groups). Muscles from horses with PSSM and from clinically normal horses did not have contracture in response to up to 2% halothane. CONCLUSIONS AND CLINICAL RELEVANCE: Results were in contrast to the increased sensitivity to caffeine and halothane for muscles from Thoroughbreds with recurrent exertional rhabdomyolysis (RER). Although clinical signs of muscular stiffness after exercise are similar between Quarter Horses with PSSM and Thoroughbreds with RER, these breeds appear to have 2 distinct myopathies with different pathophysiologic bases. Unlike RER in Thoroughbreds, PSSM in Quarter Horses does not appear to be accompanied by a defect in regulation of muscle contraction.     

Presence of the glycogen synthase 1 (GYS1) mutation causing type 1 polysaccharide storage myopathy in continental European draught horse breeds

The purpose of this study was to determine which continental European draught horse breeds harbour a mutation in the glycogen synthase 1 gene (GYS1) that is known to be responsible for type 1 polysaccharide storage myopathy in quarter horses and North American draught horses. Of a non-random selection of continental European draught horses belonging to 13 breeds, 62 per cent (250 of 403) tested were found to carry the mutant allele. The horses were located in Belgium, France, Germany, The Netherlands, Spain and Sweden. The mutation was identified in animals from each of the breeds examined. In the breeds in which more than 15 animals were available for testing, the highest percentages of GYS1-positive horses were found in the Belgian trekpaard (92 per cent; 35 of 38 horses tested), Comtois (80 per cent; 70 of 88), Netherlands trekpaard (74 per cent; 17 of 23), Rheinisch-Deutsches kaltblut (68 per cent; 30 of 44) and Breton (64 per cent; 32 of 51).     

Polysaccharide storage myopathy in the M. longissimus lumborum of showjumpers and dressage horses with back pain

This study was designed to investigate whether horses with clinical signs of back pain due to suspected soft tissue injuries were affected by polysaccharide storage myopathy (PSSM). Diagnosis of PSSM in muscle biopsies obtained from the M. longissimus lumborum of 5 showjumpers and 4 dressage horses with a history of back pain is reported. M. longissimus lumborum biopsies of these horses were characterised histopathologically and in 3/9 cases also by electron microscopy. Observations were compared with M. gluteus biopsies of the same horses, and with M. gluteus biopsies obtained from 6 Standardbreds with recurrent exertional rhabdomyolysis and from 6 healthy trotters. M. longissimus biopsies from horses with back pain showed pathognomonic signs of PSSM, i.e. high glycogen and/or abnormal complex amylase-resistant polysaccharide deposits. Similar features were found in M. gluteus biopsies of the same horses. Sections of horses with rhabdomyolysis had increased PAS stain when compared with healthy horses, but did not show amylase-resistant material. Qualitative observations were corroborated by quantitative histochemistry (optical densities) of sections stained with PAS and amylase PAS. This study demonstrated the presence of PSSM in the M. longissimus of showjumpers and dressage horses with back pain and indicates that epaxial muscle biopsy is an option in diagnosing back problems in horses when clinical examination and imaging techniques do not provide a precise diagnosis.     

Relationship of horse owner assessed respiratory signs index to characteristics of recurrent airway obstruction in two Warmblood families

Reasons for performing study: The horse owner assessed respiratory signs index (HOARSI‐1–4, healthy, mildly, moderately and severely affected, respectively) is based on owner‐reported clinical history and has been used for the investigation of recurrent airway obstruction (RAO) genetics utilising large sample sizes. Reliable phenotype identification is of paramount importance in genetic studies. Owner reports of respiratory signs have shown good repeatability, but the agreement of HOARSI with an in‐depth examination of the lower respiratory tract has not been investigated. Objectives: To determine the correlation of HOARSI grades 3/4 with the characteristics of RAO and of HOARSI‐2 with the characteristics of inflammatory airway disease. Further, to test whether there are phenotypic differences in the manifestation of lung disease between families. Methods: Seventy‐one direct offspring of 2 RAO‐affected Warmblood stallions (33 from the first family, 38 from the second) were graded as HOARSI‐1–4 and underwent a clinical examination of the respiratory system, arterial blood gas analysis, endoscopic mucus scoring, cytology of tracheobronchial secretion (TBS) and bronchoalveolar lavage fluid (BALF), and clinical assessment of airway reactivity to methacholine chloride. Results: HOARSI‐3/4 animals in clinical exacerbation showed signs consistent with RAO: coughing, nasal discharge, abnormal lung sounds and breathing pattern as well as increased numbers of neutrophils in TBS and BALF, excessive mucus accumulation and airway hyper‐responsiveness to methacholine. HOARSI‐3/4 horses in remission only had increased amounts of tracheal mucus and TBS neutrophil percentages. Clinical phenotypes were not significantly different between the 2 families. Conclusions and clinical relevance: HOARSI reliably identifies RAO‐affected horses in our population.     

Estimated prevalence of polysaccharide storage myopathy among overtly healthy Quarter Horses in the United States

OBJECTIVE: To estimate the prevalence of polysaccharide storage myopathy (PSSM) among Quarter Horses in the United States and evaluate possible relationships between muscle glycogen concentration, turnout time, and exercise level. DESIGN: Cross-sectional study. ANIMALS: 164 overtly healthy Quarter Horses > 2 years old from 5 states. PROCEDURES: Horses with a history of exertional rhabdomyolysis or any other muscular disease were excluded. Muscle biopsy specimens were examined histologically for evidence of PSSM and were submitted for determination of muscle glycogen concentration. A diagnosis of PSSM was made if amylase-resistant inclusions that stained with periodic acid-Schiff stain were detected. RESULTS: Prevalences of PSSM on the 2 farms with a history of PSSM were 20% (1/5) and 40.7% (11/27); mean prevalence for the other 4 farms was 6.1% (8/132). Sex was not significantly associated with a diagnosis of PSSM, and age was not significantly different between horses with and without PSSM. Total histologic score, serum creatine kinase activity, and muscle glycogen concentration were significantly higher in horses with PSSM than in horses without. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the prevalence of PSSM among overtly healthy Quarter Horses in the United States is likely to be between 6% and 12%.     

Glucose uptake in horses with polysaccharide storage myopathy

OBJECTIVE: To determine whether excessive glycogen accumulation in skeletal muscle of Quarter Horses with polysaccharide storage myopathy (PSSM) is a result of enhanced cellular uptake of glucose. ANIMALS: 6 horses with PSSM and 10 healthy (control) horses. PROCEDURE: Intravenous glucose tolerance tests (IVGTT), oral glucose tolerance tests (OGTT), and modified insulin tolerance tests (MITT) were performed. Plasma glucose and insulin concentrations were measured in blood samples collected before and for up to 8 hours after glucose or insulin administration. RESULTS: Peak glucose concentrations during IVGTT were similar for both groups of horses, but rate of glucose clearance was 1.5 times faster in horses with PSSM than in controls. Moreover, circulating concentrations of insulin before and after glucose injection were lower in the PSSM group. Blood glucose concentrations from minute 90 to minute 300 of the OGTT were lower in horses with PSSM than in controls. The MITT resulted in acute decreases in blood glucose concentrations in both groups of horses; however, horses with PSSM sustained low blood glucose concentrations for more than 3 hours after insulin injection, whereas blood glucose concentrations in controls returned to baseline values within 2 hours. CONCLUSIONS: Quarter Horses with PSSM have enhanced cellular uptake of glucose that may be, in part, caused by an increased sensitivity to insulin. CLINICAL RELEVANCE: Horses with PSSM have an increased rate of glucose clearance in response to insulin secretion. Thus, diets low in soluble carbohydrate may be the most effective way to decrease glycogen accumulation in skeletal muscle of these horses.     

Standing MRI lesions of the distal interphalangeal joint and podotrochlear apparatus occur with a high frequency in warmblood horses

Foot pain is a common presenting complaint in Warmblood horses. The aim of this retrospective, cross‐sectional study was to determine the spectrum of foot lesions detected by magnetic resonance imaging (MRI) in Warmblood horses used for dressage, jumping, and eventing. The medical records of 550 Warmblood horses with foot pain that were scanned using standing MRI were reviewed and the following data were recorded: signalment, occupation, lameness, diagnostic analgesia, imaging results, treatments, and follow‐up assessments. Associations between standing MRI lesions and chronic lameness following treatment were tested. Abnormalities of the navicular bone (409 horses, 74%), distal interphalangeal joint (362 horses, 65%), and deep digital flexor (DDF) tendon (260 horses, 47%) occurred with the highest frequency. The following abnormalities were significantly associated (P < .05) with chronic lameness following conservative therapy: moderate to severe MRI lesions in the trabecular bone of the navicular bone, mild or severe erosions of the flexor surface of the navicular bone, moderate sagittal/parasagittal DDF tendinopathies, and moderate collateral sesamoidean desmopathies. Also, identification of concurrent lesions of the DDF tendon, navicular bone, navicular bursa, and distal sesamoidean impar ligament was associated with chronic lameness after conservative therapy. Development of effective treatment options for foot lesions that respond poorly to conservative therapy is necessary.     

Proglycogen, macroglycogen, glucose, and glucose-6-phosphate concentrations in skeletal muscles of horses with polysaccharide storage myopathy performing light exercise

OBJECTIVE: To determine concentrations of proglycogen (PG), macroglycogen (MG), glucose, and glucose-6-phosphate (G-6-P) in skeletal muscle of horses with polysaccharide storage myopathy (PSSM) before and after performing light submaximal exercise. ANIMALS: 6 horses with PSSM and 4 control horses. PROCEDURES: Horses with PSSM completed repeated intervals of 2 minutes of walking followed by 2 minutes of trotting on a treadmill until muscle cramping developed. Four untrained control horses performed a similar exercise test for up to 20 minutes. Serum creatine kinase (CK) activity was measured before and 4 hours after exercise. Concentrations of total glycogen (G(t)), PG, MG, G-6-P, free glucose, and lactate were measured in biopsy specimens of gluteal muscle obtained before and after exercise. RESULTS: Mean serum CK activity was 26 times higher in PSSM horses than in control horses after exercise. Before exercise, muscle glycogen concentrations were 1.5, 2.2, and 1.7 times higher for PG, MG, and G(t), respectively, in PSSM horses, compared with concentrations in control horses. No significant changes in G(t), PG, MG, G-6-P, and lactate concentrations were detected after exercise. However, free glucose concentrations in skeletal muscle increased significantly in PSSM horses after exercise. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of the results suggests that glucose uptake in skeletal muscle is augmented in horses with PSSM after light exercise. There is excessive storage of PG and MG in horses with PSSM, and high concentrations of the 2 glycogen fractions may affect functional interactions between glycogenolytic and glycogen synthetic enzymes and glycosomes.     

Lateralised motor behaviour leads to increased unevenness in front feet and asymmetry in athletic performance in young mature Warmblood horses

Reason for performing study: Foot stance in grazing significantly influences hoof conformation and development from foal to yearling age. Objectives: To conduct a longitudinal study to establish if the relationship between motor laterality and uneven front feet persisted in 3‐year‐old horses at the time of studbook selection and to investigate if such laterality and unevenness might influence the horses' ability to perform symmetrically while trotting, cantering and free jumping. Methods: Seventeen clinically sound but untrained (with only minimal experience of handling) and sound Warmblood horses that had participated in a previous study were assessed as per the protocol reported. Laterality was tested in a preference test (PT) and z‐values were calculated for analysis purposes. Laterality and hoof unevenness were related to both relative limb length and relative head size, while the ability to perform symmetrically was tested in free trot‐canter transitions and free jumping exercises. Differences in performance between horses with and without a limb preference in the PT and those with ‘uneven’ and ‘even’ feet were tested for differences in performance metrics using Students' t test, while linearity was tested using a regression analysis (P<0.05). Results: Significant laterality was still present in 24% of the 3‐year‐old horses and the relationship between laterality and uneven feet pairs was stronger than at foal and yearling stages. Horses with significant motor laterality had almost 4 times more unevenness, a smaller head and longer limbs and the relationship between body conformation and laterality was still present. There was a strong linear relation between unevenness, laterality and a bias or side preference for trot‐canter transitions. However, this relationship was not significant during the free jumping exercise. Conclusion: Motor laterality and uneven feet pairs were still present and significantly related in the 3‐year‐old horses and both variables were also strongly related to sidedness in trot‐canter transitions. Potential relevance: Warmblood studbooks should include quantitative data on laterality at the time of studbook admission as part of the selection criteria.     

Comparison of histopathologic criteria and skeletal muscle fixation techniques for the diagnosis of polysaccharide storage myopathy in horses

The purpose of the study reported here was to determine the effect of three methods of fixation of skeletal muscle biopsy specimens on the histopathologic appearance of muscle sections and to determine criteria that were most consistently associated with a diagnosis of polysaccharide storage myopathy (PSSM) in horses. Surgically excised semimembranosus muscle biopsy specimens were obtained from nine horses previously diagnosed with PSSM and from 15 control horses. Portions of each specimen were fixed in formalin, frozen immediately, and chilled for 24 hours prior to freezing. Sections stained with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and amylase-PAS were scored for histopathologic criteria by three investigators blinded to the sample origin. The presence of amylase-resistant, abnormal polysaccharide was found to be the most sensitive and specific diagnostic indicator for PSSM, and was readily detected regardless of the fixation technique or investigator. Other less-specific features associated with PSSM included atrophy and cytoplasmic and subsarcolemmal vacuoles; however, their histologic scores varied among fixation technique and investigators. Scores for subsarcolemmal and cytoplasmic amylase-sensitive glycogen in horses with PSSM were similar to those for control horses and varied among fixation techniques. In conclusion, PSSM is most accurately diagnosed in muscle biopsy specimens on the basis of appearance of amylase-resistant, abnormal polysaccharide, not amylase-sensitive glycogen, regardless of fixation technique. In general, frozen sections appeared to be better suited for studying myopathies because many histopathologic features of skeletal muscle were obscured by formalin fixation.     

Effects of submaximal exercise on adenine nucleotide concentrations in skeletal muscle fibers of horses with polysaccharide storage myopathy

OBJECTIVE: To determine whether disruption of adenine triphosphate (ATP) regeneration and subsequent adenine nucleotide degradation are potential mechanisms for rhabdomyolysis in horses with polysaccharide storage myopathy (PSSM) performing submaximal exercise. ANIMALS: 7 horses with PSSM and 4 control horses. PROCEDURES: Horses with PSSM performed 2-minute intervals of a walk and trot exercise on a treadmill until muscle cramping developed. Control horses exercised similarly for 20 minutes. Serum creatine kinase (CK) activity was measured 4 hours after exercise. Citrate synthase (CS), 3-OH-acylCoA dehydrogenase, and lactate dehydrogenase activities prior to exercise and glucose-6-phosphate (G-6-P) and lactate concentrations before and after exercise were measured in gluteal muscle specimens. Adenine triphosphate, diphosphate (ADP), monophosphate (AMP), and inosine monophosphate (IMP) concentrations were measured before and after exercise in whole muscle, single muscle fibers, and pooled single muscle fibers. RESULTS: Serum CK activity ranged from 255 to 22,265 U/L in horses with PSSM and 133 to 278 U/L in control horses. Muscle CS activity was lower in horses with PSSM, compared with control horses. Muscle G-6-P lactate, ATP, ADP, and AMP concentrations in whole muscle did not change with exercise in any horses. Concentration of IMP increased with exercise in whole muscle, pooled muscle fibers, and single muscle fibers in horses with PSSM. Large variations in ATP and IMP concentrations were observed within single muscle fibers. CONCLUSIONS AND CLINICAL RELEVANCE: Increased IMP concentration without depletion of ATP in individual muscle fibers of horses with PSSM during submaximal exercise indicates an energy imbalance that may contribute to the development of exercise intolerance and rhabdomyolysis.     

Influenza A viruses with truncated NS1 as modified live virus vaccines: Pilot studies of safety and efficacy in horses

Reasons for performing study: Three previously described NS1 mutant equine influenza viruses encoding carboxyterminally truncated NS1 proteins are impaired in their ability to inhibit type I IFN production in vitro and are replication attenuated, and thus are candidates for use as a modified live influenza virus vaccine in the horse. Hypothesis: One or more of these mutant viruses is safe when administered to horses, and recipient horses when challenged with wild‐type influenza have reduced physiological and virological correlates of disease. Methods: Vaccination and challenge studies were done in horses, with measurement of pyrexia, clinical signs, virus shedding and systemic proinflammatory cytokines. Results: Aerosol or intranasal inoculation of horses with the viruses produced no adverse effects. Seronegative horses inoculated with the NS1‐73 and NS1‐126 viruses, but not the NS1‐99 virus, shed detectable virus and generated significant levels of antibodies. Following challenge with wild‐type influenza, horses vaccinated with NS1‐126 virus did not develop fever (>38.5°C), had significantly fewer clinical signs of illness and significantly reduced quantities of virus excreted for a shorter duration post challenge compared to unvaccinated controls. Mean levels of proinflammatory cytokines IL‐1β and IL‐6 were significantly higher in control animals, and were positively correlated with peak viral shedding and pyrexia on Day +2 post challenge. Conclusion and clinical relevance: These data suggest that the recombinant NS1 viruses are safe and effective as modified live virus vaccines against equine influenza. This type of reverse genetics‐based vaccine can be easily updated by exchanging viral surface antigens to combat the problem of antigenic drift in influenza viruses.