Dominique Votion

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.

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.

Measurement of respiratory function by impulse oscillometry in horses

REASONS FOR PERFORMING STUDY Due to technical implementations and lack of sensitivity, pulmonary function tests are seldom used in clinical practice. Impulse oscillometry (IOS) could represent an alternative method. OBJECTIVES To define feasibility, methodology and repeatability of IOS, a forced oscillation technique that measures respiratory resistance (Rrs) and reactance (Xrs) from 5 to 35 Hz during spontaneous breathing, in horses. METHODS Using 38 healthy horses, Rrs and Xrs reference values were defined and influence of individual biometrical parameters was investigated. In addition, IOS measurements of 6 horses showing clinical signs of heaves were compared to those of 6 healthy horses. RESULTS Airtightness and minimal dead space in the facemask were prerequisites to IOS testing and standardisation of head position was necessary to avoid variations in Rrs due to modified upper airway geometry. In both healthy and diseased animals, measurements were repeatable. In standard-type breeds, the influence of the horses size on IOS parameters was negligible. An increase in R5Hz greater than 0.10 kPa/l/sec and R5Hz>R10Hz, combined with negative values of Xrs between 5 and 20 Hz, was indicative of heaves crisis. CONCLUSIONS IOS is a quick, minimally invasive and informative method for pulmonary function testing in healthy and diseased horses. POTENTIAL RELEVANCE IOS is a promising method for routine and/or field respiratory clinical testing in the equine species.

Airway response of horses with COPD to dry powder inhalation of ipratropium bromide.

To determine the effects of the dry powder inhalation (DPI) of ipratropium bromide (ipratropium) on the airways of health horses and the dose-response curve in horses suffering from chronic obstructive pulmonary disease (COPD) by means of pulmonary function tests, five healthy horses were first studied. Ipratropium (2400 micrograms ipratropium horse-1) was contained in gelatine capsules and administered using a dry powder device connected to an adapted face mask. Pulmonary function tests were recorded before inhalation and 15 and 60 min after inhalation. No modification of pulmonary function was observed. The airway response to ipratropium DPI was then determined in six horses suffering from COPD. To induce airway obstruction, the horses were bedded on straw and fed hay. When the maximal change in pleural pressure during tidal breathing exceeded 1.96 kPa, pulmonary function tests were recorded before DPI, and 15 and 60 min post-inhalation. Placebo (six capsules horse-1) or ipratropium (600, 1200 and 2400 micrograms horse-1) was administered in a randomized order to each horse using the dry powder device and the adapted face mask. Neither ipratropium nor placebo DPI affected respiratory frequency (f) or tidal volume (VT). Inhalation of 600 micrograms ipratropium horse-1 resulted in a delayed decrease of total pulmonary resistance (RL) whereas dynamic compliance (Cdyn) was improved (although not significantly) at both times of measurement when compared with values following placebo inhalation. Simultaneous decreased RL and increased Cdyn, was observed within 15 min after DPI of 1200 micrograms ipratropium horse-1 and persisted for the 1 h duration of the experiment. Doubling the dose also improved pulmonary function but not significantly more than following inhalation of 1200 micrograms ipratropium. No systemic side effects were observed in either group of horses.

Aerosol therapy in the equine species

Inhalation therapy plays an increasing role in the management of equine respiratory disorders. This alternative to systematic treatment permits a high concentration of medication to act locally while minimizing side effects and residues. In human medicine, literature in this field is prolific and continuously renewed, whereas in veterinary medicine, applications of aerosol therapy are less extensive. This review considers the principles of action of the different types of devices used for inhalation, i.e., nebulization, metered-dose inhalation and dry powder inhalation, describes the technical and practical requirements for their use in the equine species and considers the advantages and disadvantages of each inhalation device. The pharmacological agents currently administered to horses by inhalation are also discussed. Perspectives of aerosol therapy in the equine species, including aerosols already used in human medicine and their potential applications for horses are described.

Analysis of scintigraphical lung images before and after treatment of horses suffering from chronic pulmonary disease.

Scintigraphical analysis of the ratio of inhalation (i) to perfusion (Q) was designed to determine whether chronic pulmonary disease in horses induced mismatches in i/Q and to assess whether medical treatment would restore an I/Q distribution pattern identical to that of control horses. In addition, the results of the I/Q analysis were correlated with the alveolar-arterial PO2 difference (AaDO2). The I/Q matching found in a group of control horses was compared with the I/Q analysis of a group of diseased horses before and after their clinical signs had been treated. The analysis indicated that there was mismatching between I and Q before they were treated. The treatment improved the diseased horses, pulmonary function but there was still heterogeneity in the I/Q distribution after they had been treated. The i/Q analysis parameters were well correlated with the ratio of ventilation (VA) and pulmonary blood flow (Q), evaluated by the determination of AaDO2.

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.

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.

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.

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.