Equine atypical myopathy in the UK: Epidemiological characteristics of cases reported from 2011 to 2015 and factors associated with survival.

BACKGROUND: Equine atypical myopathy (AM) is a toxic rhabdomyolysis associated with ingestion of hypoglycin A, derived typically in Europe, from Acer pseudoplatanus tree. Despite the wide distribution of this tree species in the UK, the number of cases reported annually varies, and there has been an apparent increase in prevalence in recent years. Although AM was first recognised in the UK, epidemiological studies have never been conducted focused solely on this country. OBJECTIVES: To describe the spatiotemporal distribution, presentation, treatment and outcome of AM cases reported in the UK. STUDY DESIGN: Retrospective case series. METHODS: British AM cases reported to the atypical myopathy alert website, between 2011 and 2015 were included (n = 224). Data were obtained via standardised epidemiological questionnaires from owners and veterinarians. Factors associated with survival were assessed using logistic regression. RESULTS: Most cases reported were from England (87.9%). Survival was 38.6% (n = 73/189). Clinical factors associated with reduced odds of survival included, hypothermia (odds ratio [OR] 0.18; 95% confidence interval [CI] 0.06-0.57; P = 0.01), bladder distension (OR 0.11; CI 0.02-0.59; P = 0.01), tachycardia (OR 0.97; CI 0.94-0.99; P = 0.04) and serum creatine kinase activity >100,000 IU/L (OR 0.17; CI 0.04-0.68; P = 0.01) in the univariable analysis as well as recumbency. The latter was the only sign retained in multivariable analysis (OR = 0.19; CI 0.06-0.62; P = 0.006). Administration of vitamins during the disease was associated with survival (OR 3.75; CI 1.21-11.57; P = 0.02). MAIN LIMITATIONS: Reporting cases to the Atypical Myopathy Alert Group is voluntary; therefore, under-reporting will result in underestimation of AM cases; furthermore, direct owner-reporting could have introduced misdiagnosis bias. CONCLUSION: Some areas of the UK reported AM cases more commonly. Clinical signs such as recumbency, rectal temperature, distended bladder and serum creatine kinase activity might be useful prognostic indicators though should be considered in the context of the clinical picture. Treatment with vitamins increases odds of survival.

Samaras and seedlings of Acer pseudoplatanus are potential sources of hypoglycin A intoxication in atypical myopathy without necessarily inducing clinical signs.

REASONS FOR PERFORMING STUDY: Ingestion of sycamore seeds (Acer pseudoplatanus) is the likely source of hypoglycin A in atypical myopathy (AM) but ingestion of seedlings in spring might also contribute to intoxication. OBJECTIVES: To test for hypoglycin A in seeds and seedlings collected on pastures where AM cases were reported and compare its concentration in serum of affected and healthy horses. STUDY DESIGN: Field investigation of clinical cases. METHODS: Whenever present, samaras (the winged nuts that each contain one seed) and/or seedlings were collected from pastures of 8 AM cases and 5 unaffected horses from different premises. Two AM cases were each co-grazing with an apparently healthy horse. Acylcarnitines and hypoglycin A were quantified in blood samples of all horses involved in the study. RESULTS: Hypoglycin A was detected in serum of AM (5.47 ± 1.60 µmol/l) but not in healthy controls pasturing where A. pseudoplatanus trees were not present. However, hypoglycin A was detected at high concentrations (7.98 µmol/l) in serum of a clinically healthy horse grazing a pasture with seedlings and samaras and also in the 2 healthy horses co-grazing with AM cases (0.43 ± 0.59 µmol/l). Hypoglycin A was detected in all samples of seeds and spring seedlings of A. pseudoplatanus. CONCLUSIONS: Atypical myopathy can be associated with the ingestion of sycamore samaras and also ingestion of seedlings. Hypoglycin A can be detected in the blood of horses with no detectable clinical signs at pasture in which there is A. pseudoplatanus. Determination of hypoglycin A concentration in blood is useful for screening for exposure in suspected cases of AM.

Detection of hypoglycin A in the seeds of sycamore (Acer pseudoplatanus) and box elder (A. negundo) in New Zealand; the toxin associated with cases of equine atypical myopathy.

CASE HISTORY AND CLINICAL FINDINGS: During April and May 2014 four horses aged between 5 months and 9 years, located in the Canterbury, Marlborough and Southland regions, presented with a variety of clinical signs including recumbency, stiffness, lethargy, dehydration, depression, and myoglobinuria suggestive of acute muscle damage. Two horses were subjected to euthanasia and two recovered. In all cases seeds of sycamore maple (Acer pseudoplatanus) or box elder (A. negundo) were present in the area where the horse had been grazing. LABORATORY INVESTIGATION: The samaras (seeds) of some Acer spp. may contain hypoglycin A, that has been associated with cases of atypical myopathy in Europe and North America. To determine if hypoglycin A is present in the samaras of Acer spp. in New Zealand, samples were collected from trees throughout the country that were associated with historical and/or current cases of atypical myopathy, and analysed for hypoglycin A. Serum samples from the four cases and four unaffected horses were analysed for the presence of hypoglycin A, profiles of acylcarnitines (the definitive diagnosis for atypical myopathy) and activities of creatine kinase and aspartate aminotransferase.Markedly elevated serum activities of creatine kinase and aspartate aminotransferase, and increased concentrations of selected acylcarnitines were found in the case horses. Hypoglycin A was detected in the serum of those horses but not in the healthy controls. Hypoglycin A was detected in 10/15 samples of samaras from sycamore maple and box elder from throughout New Zealand. DIAGNOSIS: Cases of atypical myopathy were diagnosed on properties where samaras containing hypoglycin A were also found. CLINICAL RELEVANCE: Sycamore and box elder trees in New Zealand are a source of hypoglycin A associated with the development of atypical myopathy. If pastured horses present with clinical and biochemical signs of severe muscle damage then the environment should be checked for the presence of these trees. Horses should be prevented from grazing samaras from Acer spp. in the autumn.

Identification of methylenecyclopropyl acetic acid in serum of European horses with atypical myopathy.

REASONS FOR PERFORMING STUDY: It is hypothesised that European atypical myopathy (AM) has a similar basis as seasonal pasture myopathy in North America, which is now known to be caused by ingestion of hypoglycin A contained in seeds from the tree Acer negundo. Serum from horses with seasonal pasture myopathy contained the conjugated toxic metabolite of hypoglycin A, methylenecyclopropyl acetic acid (MCPA). STUDY DESIGN: Retrospective study on archived samples. OBJECTIVES: 1) To determine whether MCPA-carnitine was present in serum of European horses confirmed to have AM; 2) to determine whether Acer negundo or related Acer species were present on AM pastures in Europe. METHODS: Concentrations of MCPA-carnitine were analysed in banked serum samples of 17 AM horses from Europe and 3 diseased controls (tetanus, neoplasia and exertional rhabdomyolysis) using tandem mass spectrometry. Atypical myopathy was diagnosed by characteristic serum acylcarnitine profiles. Pastures of 12 AM farms were visited by experienced botanists and plant species were documented. RESULTS: Methylenecyclopropyl acetic acid-carnitine at high concentrations (20.39 ± 17.24 nmol/l; range 0.95-57.63 nmol/l; reference: <0.01 nmol/l) was identified in serum of AM but not disease controls (0.00 ± 0.00 nmol/l). Acer pseudoplatanus but not Acer negundo was present on all AM farms. CONCLUSIONS: Atypical myopathy in Europe, like seasonal pasture myopathy in North America, is highly associated with the toxic metabolite of hypoglycin A, MCPA-carnitine. This finding coupled with the presence of a tree of which seeds are known to also contain hypoglycin A indicates that ingestion of Acer pseudoplatanus is the probable cause of AM. This finding has major implications for the prevention of AM.

Traditional and quantitative assessment of acid-base and shock variables in horses with atypical myopathy.

BACKGROUND: Descriptions of acid-base disturbances in atypical myopathy (AM) are limited. OBJECTIVES: Describe and compare traditional and quantitative acid-base abnormalities and cardiovascular shock status in horses with AM at admission. ANIMALS: 34 horses with AM, 15 healthy controls. METHODS: Retrospective case-control study. Records were searched for shock variables (packed cell volume [PCV], blood urea nitrogen [BUN], heart and respiratory rate) and acid-base variables (venous blood gas analysis, electrolytes, total protein, lactate) on admission. Base excess (BE) of free water (BEfw), chloride (BEcl), total protein (BEtp), and unidentified anions (BEua), anion gap (AG), measured strong ion difference (SIDm), and concentration of total nonvolatile weak acids ([Atot]) were calculated. Acid-base classifications, using simplified strong ion model and traditional approach, and shock grades were assigned. A 2-sample Wilcoxon rank-sum test and Bonferroni correction compared variables in AM cases versus control horses. Significance was P < .05/16 for acid-base and P < .05/5 for shock variables. RESULTS: Tachycardia, tachypnea, and normal to increased PCV and BUN were common in AM cases. Respiratory, metabolic acid-base alterations, or both were mainly caused by respiratory alkalosis, lactic acidosis, and SIDm alkalosis, alone or in combination. Evaluated variables (except pH, potassium concentration, total protein, and related calculations) were significantly different (P < .001) between AM cases and control horses. The strong ion model provided a more accurate assessment than the traditional approach and identified mixed derangements. CONCLUSIONS AND CLINICAL IMPORTANCE: Acid-base derangements should be evaluated in horses with AM and this preferably with the strong ion model.

European outbreaks of atypical myopathy in grazing equids (2006-2009): spatiotemporal distribution, history and clinical features.

REASONS FOR PERFORMING STUDY: Improved understanding of the epidemiology of atypical myopathy (AM) will help to define the environmental factors that permit or support the causal agent(s) to exert toxicity. OBJECTIVES: This European survey of AM aimed to describe spatiotemporal distribution, survival, clinical signs, circumstances in which AM develops and its different expressions between countries and over time. METHODS: The spatiotemporal distribution, history and clinical features of AM cases reported to the Atypical Myopathy Alert Group from 2006 to 2009 were described. Comparisons of data from the most severely affected countries and from the large outbreaks were made with Fisher's exact and Welch's tests with Bonferroni correction. RESULTS: Of 600 suspected cases, 354 met the diagnostic criteria for confirmed or highly probable AM. The largest outbreaks occurred during the autumns of 2006 and 2009 in Belgium, France and Germany. For the first time, donkeys, zebras and old horses were affected, and clinical signs such as gastrointestinal impaction, diarrhoea, penile prolapse, buccal ulceration and renal dysfunction were observed. Affected horses spent >6 h/day on pastures that almost always contained or were surrounded by trees. The latency period was estimated at up to 4 days. Overall survival rate was 26%. Although differences between countries in affected breeds, body condition, horse management and pasture characteristics were recognised, the common presenting clinical signs and mortality were similar between countries. CONCLUSIONS AND POTENTIAL RELEVANCE: This study describes new data on case details, history and clinical course of AM that is of preventive, diagnostic and therapeutic value. However, the true impact of the findings of this study on the development of or severity of AM should be tested with case-control studies.

European outbreaks of atypical myopathy in grazing horses (2006-2009): determination of indicators for risk and prognostic factors.

REASONS FOR PERFORMING STUDY: Appropriate management of atypical myopathy (AM) requires the establishment of an accurate diagnosis and prognosis. Furthermore, preventive measures to avoid AM need to be refined. OBJECTIVES: The aims of the study were as follows: 1) to improve the diagnosis of AM; 2) to identify prognostic predictors; and 3) to refine recommended preventive measures based on indicators of risk factors. METHODS: An exploratory analysis of cases in Europe between 2006 and 2009 reported to the Atypical Myopathy Alert Group was conducted. Based on clinical data, reported cases were allocated into 2 groups: confirmed or highly probable AM (AM group; further divided into survivors and nonsurvivors); and cases with a low probability of having AM or with another final diagnosis (non-AM group). Using Welch's test and odds ratios corrected for multiple comparisons, the AM vs. non-AM groups were compared to identify indicators for diagnosis and risk factors, and survivors vs. nonsurvivors in the AM group were compared to identify prognostic factors. Sensitivity, specificity and positive and negative predictive values were calculated for specific clinical signs related to final diagnosis and outcome. RESULTS: From 600 reported cases, 354 AM cases (survival rate of 26%) and 69 non-AM cases were identified, while there were insufficient data to categorise the remainder. Variables valuable for diagnosing AM compared with similar diseases were as follows: presence of dead leaves and wood and/or trees on pastures; sloping pastures; full-time pasture access; no food supplementation; normal body condition; pigmenturia; normothermia; and congested mucous membranes. Nonsurvival was associated with recumbency, sweating, anorexia, dyspnoea, tachypnoea and/or tachycardia. Survival was associated with remaining standing most of the time, normothermia, normal mucous membranes, defaecation and vitamin and antioxidant therapy. CONCLUSIONS AND POTENTIAL RELEVANCE: This study refines the list of risk factors for AM. Clinical signs valuable for diagnosis and prognosis have been identified, enabling clinicians to improve management of AM cases.

Effect of intensive exercise on plasmatic neutrophil elastase level in eventing and endurance horses.

REASONS FOR PERFORMING STUDY: Intensive exercise induces a systemic inflammatory response characterised by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase (NE) could also contribute to tissues lesions by its proteinase activities. OBJECTIVE: To compare plasmatic NE concentrations before and after different forms of intensive exercise. MATERIALS AND METHODS: EDTA blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of a one or 2 star eventing competition (n = 51), or a 120 or 160 km endurance race (n = 32). Plasmatic NE and MPO were measured by a specific equine ELISA. Neutrophil counts and creatine kinase (CK) levels were also measured. A Wilcox on test for paired samples was used to compare mean values of neutrophils, CK, MPO and NE at T0 and T1 in EvH and in EndH. Correlations were calculated between all the 4 parameters in EvH and EndH. RESULTS: At T0, mean NE levels were 14.43 ± 3.63 ng/ml for EvH and 11.7 ± 2.11 ng/ml for EndH. The competition induced a significant increase of NE levels in (58.57 ± 24.06 ng/ml) EvH and (95.74 ± 22.70 ng/ml) EndH (P < 0.05). NE was significantly (P < 0.0001) correlated to MPO in EvH (r = 0.293) and EndH (r = 0.594) and to CK (r = 0.297) in EndH (P < 0.0001). Neutrophils, CK and MPO were significantly increased between T0 and T1 in both types of horses. CONCLUSIONS: Significant increase of NE (EndH) was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in MPO and NE indicates that not all horses show the same intensity of systemic inflammatory response.

Hydration and electrolyte balance in horses during an endurance season.

REASONS FOR PERFORMING STUDY: Limited information exists about the physiological training-induced changes in electrolyte balance of horses competing in long distance endurance races. OBJECTIVES: To determine the effects of endurance training and racing on hydration and electrolyte balance in horses. METHODS: Blood and urine were sampled at rest in 8 endurance horses before training and after two 11 week training periods (T1 and T2). Each training was followed by a 120 km endurance ride and horses were sampled before, during and 2 h after the rides. Blood was analysed for packed cell volume (PCV), total protein (TP), urea, creatinine and electrolyte concentrations. Urine was analysed for pH, specific gravity, creatinine and electrolyte concentrations, which allowed calculation of fractional excretion of electrolytes (FE). Changes associated with training and with the rides were assessed using a Student paired t test (P ≤ 0.05). RESULTS: Plasma TP, urea, creatinine and sodium concentrations increased during T1 and PCV decreased significantly during T2. FE(Cl) increased during T1 then decreased. FE(K) increased significantly during both training periods. Other blood and urine parameters did not show remarkable changes with training. PCV, plasma TP, urea, creatinine and total Ca concentrations increased and plasma Na(+) and Cl(-) concentrations decreased during both rides. Urine concentrations of Na(+), K(+) and Cl(-), FE(Na) and FE(Cl) decreased during the rides while urinary creatinine increased. FE(K) increased during the first part of the rides then decreased. CONCLUSIONS: These data contribute to the understanding of changes associated with training and prolonged endurance exercise.

Altered systolic left ventricular function in horses completing a long distance endurance race.

REASONS FOR PERFORMING STUDY: It is unknown whether or not exercise-induced cardiac fatigue (EICF), as demonstrated in human athletes performing long duration exercise, occurs in endurance horses. OBJECTIVE: To examine the effects of a long distance endurance race on left ventricular systolic function in horses. METHODS: Echocardiography was performed before, and after, a 2 or 3 star international endurance race (106-132 km) in 11 horses. Systolic (s) and diastolic (d) interventricular and left ventricular free wall thickness (IVS and LVFW, respectively), left ventricular, left atrial and aortic internal diameter (LVID, LA and Ao, respectively), fractional shortening (FS) and ejection fraction (EF) were measured by echocardiography. Heart rate (HR), peak flow velocity (Vmax), flow velocity integral (FVI), ejection time (ET), pre-ejection period (PEP), velocity of circumferential fibre shortening (Vcf), stroke volume (SV) and cardiac output (CO) were measured from aortic Doppler wave recordings. RESULTS: After the race, LVIDd, Ao, LA, EF, FS, FVI, SV, ET and ET indexed for HR were significantly lower and IVSd, LVFWd, HR, PEP, PEP/ET and Vcf significantly higher as compared with prerace values. Pre- to post exercise changes in those parameters were not significantly correlated with changes in HR or in LVIDd. CONCLUSIONS: Results suggest that EICF, with a decrease in left ventricular systolic function, could occur post exercise in horses performing long duration endurance races. However, a multanecus effect of altered preload and heart rate on the studied variables cannot be discounted.

Alterations in mitochondrial respiratory function in response to endurance training and endurance racing.

REASONS FOR PERFORMING STUDY: Limited information exists about the muscle mitochondrial respiratory function changes that occur in horses during an endurance season. OBJECTIVES: To determine effects of training and racing on muscle oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities in horses with high resolution respirometry (HRR). METHODS: Mitochondrial respiration was measured in microbiopsies taken from the triceps brachii (tb) and gluteus medius (gm) muscles in 8 endurance horses (7 purebred Arabians and 1 crossbred Arabian) before training (T0), after two 10 week training periods (T1, T2) and after 2 CEI** endurance races (R1, R2). Muscle OXPHOS capacity was determined using 2 titration protocols without (SUIT 1) or with pyruvate (SUIT 2) as substrate. Electrons enter at the level of Complex I, Complex II or both complexes simultaneously (Complexes I+II). Muscle ETS capacity was obtained by uncoupling Complexes I+II sustained respiration. RESULTS: T1 improved OXPHOS and ETS capacities in the tb as demonstrated by the significant increase of oxygen fluxes vs. T0 (Complex I: +67%; ETS: +37%). Training improved only OXPHOS in the gm (Complex I: +34%). Among horses that completed the race, a significant decrease in OXPHOS (Complex I: ∼ -35%) and ETS (-22%) capacities was found in the tb with SUIT 2 indicating a reduced aerobic glycolysis. Significant correlations between CK activities and changes in OXPHOS were found suggesting a relationship between exercise-induced muscle damage and depression of mitochondrial respiration. CONCLUSIONS: For the first time, OXPHOS and ETS capacities in equine muscle at different steps of an endurance season have been determined by HRR. Significant alterations in mitochondrial respiratory function in response to endurance training and endurance racing have been observed although these changes appeared to be muscle group specific.

Effect of a 120 km endurance race on plasma and muscular neutrophil elastase and myeloperoxidase concentrations in horses.

REASONS FOR PERFORMING STUDY: Intense physical exercise can induce the degranulation of neutrophils leading to an increase in plasma concentration of the neutrophil marker enzymes myeloperoxidase (MPO) and elastase (ELT). These enzymes have pro-oxidative and pro-inflammatory properties and may play a role in the exercised-induced muscular damage. OBJECTIVES: To measure MPO and ELT concentrations in plasma and muscles of endurance horses and to correlate them to the extent of exercise-induced muscular damage. METHODS: Seven endurance horses qualified on 120 km races were tested in this study. Neutrophil count, serum creatine kinase (CK), plasmatic and muscular MPO and ELT concentrations were measured before and 2 h after a 120 km endurance race. RESULTS: The race produced a significant increase of neutrophils, CK, and plasma MPO and ELT levels. A significant correlation was observed between the MPO and ELT values in plasma (r(2) = 0.92, P < 0.01) and in muscles (r(2) = 0.89, P < 0.01) while plasmatic concentrations of MPO and ELT were not significantly correlated to muscular ones. An increase of mean concentrations (± s.e.) of MPO (T0: 9.85 ± 3.9, T1: 228.9 ± 95.9 ng/mg proteins) and ELT (T0: 8.4 ± 2.4, T1: 74.5 ± 39.7 ng/mg proteins) in the muscles were observed after the race. Interestingly, the individual data showed large differences between the horses. Muscular MPO and ELT concentrations were significantly correlated to plasma CK levels. The coefficient of correlation (r(2)) was 0.69 (P < 0.01) for MPO and 0.66 (P < 0.01) for ELT, respectively. CONCLUSIONS: Results underline the possible role of MPO and ELT in exercise-induced muscular damage. POTENTIAL RELEVANCE: Further studies should investigate the effect of exercise type and intensity, as well as the role of the training state on MPO and ELT involvement in muscular damage. The assessment of the intensity of exercise-induced neutrophilic degranulation may have a potential role in the monitoring of the athletic career.

Specific immuno-extraction followed by enzymatic detection (SIEFED) of myeloperoxidase and mitochondrial complex I in muscular microbiopsies: preliminary results in endurance horses.

REASONS FOR PERFORMING STUDY: Intense exercise in horses induces an increase of creatine kinase (CK) and stimulation of neutrophils which release the strong oxidant enzyme, myeloperoxidase (MPO) into the blood. It is not known whether active MPO is found in equine muscles and whether oxidant activity of neutrophils could affect muscular tissues and mitochondrial activity. OBJECTIVES: Specific immuno-extraction followed by enzymatic detection (SIEFED) methods will be employed for the first time to assess both the normal range of MPO and mitochondrial complex I (MCI) activities in equine muscular microbiopsies and to study the variation of these activities induced by endurance races. MATERIALS AND METHODS: Forty-six microbiopsies were taken from 8 endurance Arabian horses (age: 10 ± 2 years) in the triceps brachii (n = 23) or in the gluteus medius muscle (n = 23). Myeloperoxidase and MCI activities were measured in muscle extracts by enzyme immunocapture assays or SIEFED methods. Further, 7 endurance horses were sampled in the triceps brachii muscle before (T0) and after (T1) a 120 km endurance race (mean speed: 15.4 ± 1.4 km/h). RESULTS: The 46 microbiopsies from 8 horses revealed mean values for active MPO concentration and MCI activity of 21 ± 14 ng/mg proteins and 0.0172 ± 0.0066 mOD/min/µg proteins, respectively. No significant difference was observed between the 2 muscles. In 3 out of the 7 horses sampled after exercise, the 120 km endurance race induced a severe increase of muscle MPO activity (+118 ± 45% vs. T0), a large decrease of MCI activity (-63 ± 18% vs. T0) associated with a high mean plasma CK level (4642 ± 658 iu/l). In the 4 remaining horses, the 120 km endurance race did not modify the MPO and MCI activities and moderately increased the plasma CK level. CONCLUSIONS: Preliminary observations showed a possible link between MPO activity and mitochondrial functions.

Acquired multiple Acyl-CoA dehydrogenase deficiency in 10 horses with atypical myopathy.

The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, (iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly short-chain acylcarnitines. Furthermore, measurement of dehydrogenase activities in lateral vastus muscle from one horse with atypical myopathy indeed showed deficiencies of short-chain acyl-CoA dehydrogenase (0.66 as compared with 2.27 and 2.48 in two controls), medium-chain acyl-CoA dehydrogenase (0.36 as compared with 4.31 and 4.82 in two controls) and isovaleryl-CoA dehydrogenase (0.74 as compared with 1.43 and 1.61 nmol min(-1) mg(-1) in two controls). A deficiency of several mitochondrial dehydrogenases that utilize flavin adenine dinucleotide as cofactor including the acyl-CoA dehydrogenases of fatty acid beta-oxidation, and enzymes that degrade the CoA-esters of glutaric acid, isovaleric acid, 2-methylbutyric acid, isobutyric acid, and sarcosine was suspected in 10 out of 10 cases as the possible etiology for a highly fatal and prevalent toxic equine muscle disease similar to the combined metabolic derangements seen in human multiple acyl-CoA dehydrogenase deficiency also known as glutaric acidemia type II.

Morphological alterations in oxidative muscles and mitochondrial structure associated with equine atypical myopathy.

REASONS FOR PERFORMING STUDY: There is a lack of well documented studies about muscular lesions in equine atypical myopathy (EAM). OBJECTIVES: To characterise morphopathological changes of striated muscles and myocardium, to progress understanding of this disease. METHODS: Thirty-two horses age 0.5-7 years kept on pasture were referred for a sudden ataxia/myoglobinuria syndrome. Clinical examination (stiffness, muscle pain, muscle fasciculations, abnormal gait, recumbency, myoglobinuria, tachycardia, sweating) and plasma CPK, LDH and AST levels were consistent with extensive myonecrosis and, together with anamnestic data, with so-called 'equine atypical myopathy' (EAM), a disease of unknown aetiology reported since 1939. Macroscopic and microscopic (histology, histoenzymology, ultrastructure) lesions were evaluated. RESULTS: Necropsic examination revealed large areas of muscle necrosis, the extent and severity of which varied between cases and muscles, but which were clearly more constant and severe in respiratory and postural muscles and in the myocardium. Histology highlighted a multifocal and monophasic process compatible with Zenker degeneration/necrosis that mostly and segmentally affected type 1 fibres. Histochemical evaluation revealed a weak and disorganised pattern of NADH tetrazolium reductase staining, the absence of calcium salts precipitates and a dramatic accumulation of lipid droplets. Ultrastructural examination often revealed fibres of which the sole modifications were altered mitochondria and sarcoplasmic lipidosis. CONCLUSIONS: Taken together, the data suggest that a primary alteration of mitochondria should be considered, although secondary mitochondrial abnormalities have yet to be ruled out. POTENTIAL RELEVANCE: The morphological features gathered here reveal that EAM shares most of the characteristics of toxic myopathies.

Qualitative and quantitative evaluation of equine respiratory mechanics by impulse oscillometry.

REASONS FOR PERFORMING STUDY: The long-established conventional reference technique (CRT) for measuring respiratory mechanics in horses lacks sensitivity and there is a need for further refinement in new technology, such as the impulse oscillometry system (IOS). OBJECTIVES: To evaluate the potential use of the IOS as a clinical respiratory function test and compare it to the current CRT in horses suffering from common upper and lower airway dysfunctions. METHODS: Six healthy horses were tested before and after induction of a unilateral nasal obstruction (UNO) or transient left laryngeal hemiplegia (LLH). Six heaves-affected horses were tested in clinical remission and during a heaves crisis, before and after nebulisation of cumulative doses of a bronchodilator therapy (ipratropium bromide; IPB). RESULTS: As opposed to the CRT, the IOS was able to detect partial upper airway obstruction (UAO) caused by UNO or LLH in resting horses, without differentiating both conditions. Upper airway obstruction caused an upward shift of resistance (R(rs)) from 5 to 35 Hz without altering reactance (X(rs)). As for the CRT, IOS respiratory parameters measured in heaves-affected horses in crisis differed significantly from values measured during remission. The difference in frequency-dependent behaviour of R(rs) and X(rs) allowed discrimination between upper and lower airway obstructions. Bronchodilator treatment induced significant dose-dependent changes in X(rs) at 5 and 10 Hz, from the first dose. Total pulmonary resistance (RL) and R(rs) at 5 Hz were affected from the second dose and displayed similar sensitivity. Although post treatment RL values were comparable to remission, R(rs) and X(rs) remained significantly different, characterising persistent peripheral obstruction. CONCLUSIONS: The IOS was more sensitive than the CRT in detecting partial UAO in resting horses and persistent post treatment peripheral dysfunction in heaves-affected horses. The IOS is a sensitive test that provides graded quantitative and qualitative information on disease-induced respiratory dysfunctions as well as on treatment efficiency in horses. POTENTIAL RELEVANCE: The IOS could represent a practical and sensitive alternative respiratory function test for routine clinical investigations of common airway obstructive diseases and therapy in horses.

Plasma concentrations of myeloperoxidase in endurance and 3-day event horses after a competition.

REASON FOR PERFORMING STUDY: In man, exercise of any type has been shown to induce neutrophil degranulation and respiratory burst activity, as well as an increase in plasma myeloperoxidase (MPO), a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Until now, it is not known whether this is the same in horses. OBJECTIVES: To study whether degranulation of blood neutrophils may be induced by exercise by measuring the total concentration of MPO and assess the possible influence of type of competition on this exercise-induced adjustment. METHODS: Blood was sampled before, and 30 min after, the course, in 9 ponies performing the Phase D of a national 3-day event championship (CIC*), and in 7 endurance horses participating at the European endurance championship 2005. White blood cells and granulocytes, total plasma proteins, creatine phosphokinase (CK), and total MPO contents were determined from blood samples. In addition, blood was taken from all ponies and 4 of the endurance horses 5 min after completion of the course to give some idea of the intensity of exercise. RESULTS: The mean blood lactate was 15.8 +/- 5.8 mmol/l after the CIC* and 2.7 +/- 0.2 mmol/l after the 160 km course. Performing both competitions induced a significant increase in CK and MPO. After the endurance course, the number of granulocytes significantly increased. Whilst there was no significant correlation between the measurements in CIC* ponies, MPO was significantly correlated with granulocyte count (r2 = 0.776) and CK (r2 = 0.586) in endurance horses. CONCLUSIONS: Intense exercise induces an activation of blood granulocytes, with degranulation of neutrophils and release of MPO. The plasmatic MPO concentration after endurance was higher than the values reported in some inflammatory pathological conditions. POTENTIAL RELEVANCE: This phenomenon may partly contribute to the occurrence of an exercise-induced oxidative stress and to the alteration of muscular membrane permeability. Further studies should be conducted to assess the possible relationship between MPO concentration and markers of oxidative stress in performance horses.

Validation of a portable equine metabolic measurement system.

REASONS FOR PERFORMING STUDY: In equine sports medicine, VO2 has been measured exclusively with stationary systems, in laboratories equipped with a treadmill. Measurement during exercise in field conditions has not previously been reported because of the lack of portable equipment designed for horses. OBJECTIVES: A commercially available portable metabolic measurement system, based on breath-to-breath gas analysis and flow spirometry, was adapted to the horse's physiology and morphology (Cosmed K4b2 and Equimask) and its validity tested by (1) repeatability of the measures and (2) comparing metabolic data to those obtained by a reference method (RM). METHODS: To test the reproducibility of the measurements, 5 healthy saddle horses were subjected twice at 2 day intervals to a similar submaximal standardised incremental exercise test on a treadmill. The same horses performed twice at one week interval an incremental treadmill test to fatigue: the oxygen consumption and ventilation were measured once with the K4b2 system and once with the RM. The metabolic and ventilatory data obtained with both systems were compared. RESULTS: There was a good reproducibility of the metabolic measurements obtained by the K4b2 system at any workload. The VO2 obtained by both systems at any workload was not significantly different. However, the K4b2 expired fraction in CO2 (FETCO2) and carbon dioxide production (VCO2) were significantly lower at high and at maximal workloads. As a consequence, the values of the respiratory exchange ratio were too low and incompatible with normal physiological values. CONCLUSIONS: The good reproducibility of the metabolic and ventilatory measurements and the fact that the VO2 measurements at any workload were similar to the data obtained with the reference method suggested that this system may be used for comparison of repeated VO2 measurements in practical field conditions. POTENTIAL RELEVANCE: The K4b2 system could be used to improve knowledge of the energetic cost in different equine sports disciplines and offer the opportunity to undertake performance tests with genuine track conditions, on ridden or harnessed horses, rather than under laboratory conditions.

Influence of breathing pattern and lung inflation on impulse oscillometry measurements in horses.

The objective of this paper was to determine if changes in ventilation patterns could influence the outcome of respiratory function measurements performed with our impulse oscillometry system (IOS) in horses. In a first study, IOS tests were performed in vitro on six isolated equine lungs. Lung inflation levels were controlled by modifying depressurisation inside an artificial thorax and different ventilation patterns were imposed. In a second in vivo study, transient variations in breathing pattern were evaluated both with the IOS and a current reference technique (CRT) in five healthy mature horses after an intravenous (i.v.) injection of lobeline hydrochloride. In both studies, respiratory rate (RR, range: 7-42 breaths/min.) and tidal volume (V(T), range: 0.4-25 L) had minor or no influence on IOS parameters. The influence of lung inflation, most marked for resistance at 5 Hz (R(5 Hz)), was limited for the considered physiological range. In vivo, statistical models indicated that maximal changes in pleural pressure (Max Delta Ppl) and peak flows were the main determinants of the variability of the resistance (R(rs)) and the reactance (X(rs)) of the respiratory system. The fourfold increase in baseline Max Delta Ppl and peak flows obtained during hyperpnoea caused a significant increase in R(rs) at 5 and 10 Hz and a decrease in X(rs) at all frequencies. We conclude that IOS parameters are not influenced by tachypnoea, but will reflect alterations in respiratory mechanics caused by hyperpnoeic breathing.