New Pathophysiological Insights from Serum Proteome Profiling in Equine Atypical Myopathy.

Equine atypical myopathy (AM) is a severe environmental intoxication linked to the ingestion of protoxins contained in seeds and seedlings of the sycamore maple (Acer pseudoplatanus) in Europe. The toxic metabolites cause a frequently fatal rhabdomyolysis syndrome in grazing horses. Since these toxic metabolites can also be present in cograzing horses, it is still unclear as to why, in a similar environmental context, some horses show signs of AM, whereas others remain clinically healthy. Label-free proteomic analyses on the serum of 26 diseased AM, 23 cograzers, and 11 control horses were performed to provide insights into biological processes and pathways. A total of 43 and 44 differentially abundant proteins between "AM vs cograzing horses" and "AM vs control horses" were found. Disease-linked changes in the proteome of different groups were found to correlate with detected amounts of toxins, and principal component analyses were performed to identify the 29 proteins representing a robust AM signature. Among the pathway-specific changes, the glycolysis/gluconeogenesis pathway, the coagulation/complement cascade, and the biosynthesis of amino acids were affected. Sycamore maple poisoning results in a combination of inflammation, oxidative stress, and impaired lipid metabolism, which is trying to be counteracted by enhanced glycolysis.

Untargeted Metabolomics Profiling Reveals Exercise Intensity-Dependent Alterations in Thoroughbred Racehorses' Plasma after Routine Conditioning Sessions.

Thoroughbred (TB) racehorses undergo rigorous conditioning programs to optimize their physical and mental capabilities through varied exercise sessions. While conventional investigations focus on limited hematological and biochemical parameters, this field study employed untargeted metabolomics to comprehensively assess metabolic responses triggered by exercise sessions routinely used in TB conditioning. Blood samples were collected pre- and post-exercise from ten racehorses, divided into two groups based on exercise intensity: high intensity (n = 6, gallop at ± 13.38 m/s, 1400 m) and moderate intensity (n = 4, soft canter at ± 7.63 m/s, 2500 m). Intensity was evaluated through monitoring of the speed, heart rate, and lactatemia. Resting and 30 min post-exercise plasma samples were analyzed using ultraperformance liquid chromatography coupled with high-resolution mass spectrometry. Unsupervised principal component analysis revealed exercise-induced metabolome changes, with high-intensity exercise inducing greater alterations. Following high-intensity exercise, 54 metabolites related to amino acid, fatty acid, nucleic acid, and vitamin metabolism were altered versus 23 metabolites, primarily linked to fatty acid and amino acid metabolism, following moderate-intensity exercise. Metabolomics confirmed energy metabolism changes reported by traditional biochemistry studies and highlighted the involvement of lipid and amino acid metabolism during routine exercise and recovery, aspects that had previously been overlooked in TB racehorses.

Tissue Specific Distribution and Activation of Sapindaceae Toxins in Horses Suffering from Atypical Myopathy.

Equine atypical myopathy is caused by hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG), the known protoxins of sycamore maple (Acer pseudoplatanus). Various tissues from five atypical myopathy cases were analyzed but only HGA was found. Whether deamination of MCPrG has already occurred in the intestine as the first stage of metabolization has not been investigated. Activation of the protoxins to methylenecyclopropylacetyl (MCPA)-CoA and methylenecyclopropylformyl (MCPF)-CoA, respectively, occurred mainly in the skeletal muscles, as evidenced by very high concentrations of MCPA-carnitine and MCPF-carnitine in this tissue. Inhibition of the acyl-CoA dehydrogenases of short- and medium-chain as well as branched-chain fatty acids by the toxins led to a strong increase in the corresponding acylcarnitines, again preferentially in skeletal muscles. An accumulation of the long-chain acylcarnitines beyond the level of the control samples could not be detected in the tissues. As a high amount of HGA was always found unmetabolized in the organs, we speculate that targeting the interruption of further metabolization might be a way to stop the progression of intoxication. Inhibition of the mitochondrial branched-chain amino acid aminotransferase, i.e., the first enzyme responsible for the activation of sycamore maple protoxins, could be a therapeutic approach.

Acer pseudoplatanus: A Potential Risk of Poisoning for Several Herbivore Species.

Acer pseudoplatanus is a worldwide-distributed tree which contains toxins, among them hypoglycin A (HGA). This toxin is known to be responsible for poisoning in various species, including humans, equids, Père David's deer and two-humped camels. We hypothesized that any herbivore pasturing with A. pseudoplatanus in their vicinity may be at risk for HGA poisoning. To test this hypothesis, we surveyed the HGA exposure from A. pseudoplatanus in species not yet described as being at risk. Animals in zoological parks were the major focus, as they are at high probability to be exposed to A. pseudoplatanus in enclosures. We also searched for a toxic metabolite of HGA (i.e., methylenecyclopropylacetyl-carnitine; MCPA-carnitine) in blood and an alteration of the acylcarnitines profile in HGA-positive animals to document the potential risk of declaring clinical signs. We describe for the first instance cases of HGA poisoning in Bovidae. Two gnus (Connochaetes taurinus taurinus) exposed to A. pseudoplatanus in their enclosure presented severe clinical signs, serum HGA and MCPA-carnitine and a marked modification of the acylcarnitines profile. In this study, even though all herbivores were exposed to A. pseudoplatanus, proximal fermenters species seemed less susceptible to HGA poisoning. Therefore, a ruminal transformation of HGA is hypothesized. Additionally, we suggest a gradual alteration of the fatty acid metabolism in case of HGA poisoning and thus the existence of subclinical cases.

Metabolomic Signatures Discriminate Horses with Clinical Signs of Atypical Myopathy from Healthy Co-grazing Horses.

Atypical myopathy (AM) is a severe rhabdomyolysis syndrome that occurs in grazing horses. Despite the presence of toxins in their blood, all horses from the same pasture are not prone to display clinical signs of AM. The objective of this study was to compare the blood metabolomic profiles of horses with AM clinical signs with those of healthy co-grazing (Co-G) horses. To do so, plasma samples from 5 AM horses and 11 Co-G horses were investigated using untargeted metabolomics. Metabolomic data were evaluated using unsupervised, supervised, and pathway analyses. Unsupervised principal component analysis performed with all detected features separated AM and healthy Co-G horses. Supervised analyses had identified 1276 features showing differential expression between both groups. Among them, 46 metabolites, belonging predominantly to the fatty acid, fatty ester, and amino acid chemical classes, were identified by standard comparison. Fatty acids, unsaturated fatty acids, organic dicarboxylic acids, and fatty esters were detected with higher intensities in AM horses in link with the toxins' pathological mechanism. The main relevant pathways were lipid metabolism; valine, leucine, and isoleucine metabolism; and glycine metabolism. This study revealed characteristic metabolite changes in the plasma of clinically affected horses, which might ultimately help scientists and field veterinarians to detect and manage AM. The raw data of metabolomics are available in the MetaboLights database with the access number MTBLS2579.

Grazing Mares on Pasture with Sycamore Maples: A Potential Threat to Suckling Foals and Food Safety through Milk Contamination.

Equine atypical myopathy (AM) is seasonal intoxication resulting from the ingestion of seeds and seedlings of the sycamore maple (Acer pseudoplatanus) that contain toxins, among them, hypoglycin A (HGA). Literature mentions several cases of AM among gravid mares and in unweaned foals. The objective of this study was to determine whether HGA and/or its metabolite are present in milk from grazing mares exposed to sycamore maple trees as confirmed by detection of HGA and its metabolite in their blood. Four mare/foal couples were included in the study. Both HGA and its metabolite were detectable in all but one of the milk samples. To our knowledge, this is the first study describing transfer of HGA to the milk. This unprecedented observation could partially explain cases of unweaned foals suffering from AM. However, a transplacental transfer of the toxin cannot be excluded for newborn foals. Besides being a source of contamination for offspring, milk contamination by toxins from fruits of trees of the Sapindaceae family might constitute a potential risk for food safety regarding other species' raw milk or dairy products.

Answers to the Frequently Asked Questions Regarding Horse Feeding and Management Practices to Reduce the Risk of Atypical Myopathy.

In 2014, atypical myopathy (AM) was linked to Acer pseudoplatanus (sycamore maple) in Europe. The emergence of this seasonal intoxication caused by a native tree has raised many questions. This manuscript aims at answering the five most frequently asked questions (FAQs) regarding (1) identification of toxic trees; reduction of risk at the level of (2) pastures and (3) equids; (4) the risk associated with pastures with sycamores that have always been used without horses being poisoned and (5) the length of the risk periods. Answers were found in a literature review and data gathered by AM surveillance networks. A guide is offered to differentiate common maple trees (FAQ1). In order to reduce the risk of AM at pasture level: Avoid humid pastures; permanent pasturing; spreading of manure for pasture with sycamores in the vicinity and avoid sycamore maple trees around pasture (FAQ2). To reduce the risk of AM at horse level: Reduce pasturing time according to weather conditions and to less than six hours a day during risk periods for horses on risk pasture; provide supplementary feeds including toxin-free forage; water from the distribution network; vitamins and a salt block (FAQ3). All pastures with a sycamore tree in the vicinity are at risk (FAQ4). Ninety-four percent of cases occur over two 3-month periods, starting in October and in March, for cases resulting from seeds and seedlings ingestion, respectively (FAQ5).