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.

Seasonal pasture myopathy/atypical myopathy in North America associated with ingestion of hypoglycin A within seeds of the box elder tree.

REASONS FOR PERFORMING STUDY: We hypothesised that seasonal pasture myopathy (SPM), which closely resembles atypical myopathy (AM), was caused by ingestion of a seed-bearing plant abundant in autumn pastures. OBJECTIVES: To identify a common seed-bearing plant among autumn pastures of horses with SPM, and to determine whether the toxic amino acid hypoglycin A was present in the seeds and whether hypoglycin metabolites were present in SPM horse serum or urine. METHODS: Twelve SPM cases, 11 SPM pastures and 23 control farms were visited to identify a plant common to all SPM farms in autumn. A common seed was analysed for amino acid composition (n = 7/7) by GC-MS and its toxic metabolite (n = 4/4) identified in conjugated form in serum [tandem mass spectrometry (MS/MS)] and urine [gas chromatography (GC) MS]. Serum acylcarnitines and urine organic acid profiles (n = 7) were determined for SPM horses. RESULTS: Seeds from box elder trees (Acer negundo) were present on all SPM and 61% of control pastures. Hypoglycin A, known to cause acquired multiple acyl-CoA dehydrogenase deficiency (MADD), was found in box elder seeds. Serum acylcarnitines and urine organic acid profiles in SPM horses were typical for MADD. The hypoglycin A metabolite methylenecyclopropylacetic acid (MCPA), known to be toxic in other species, was found in conjugated form in SPM horse serum and urine. Horses with SPM had longer turn-out, more overgrazed pastures, and less supplemental feeding than control horses. POTENTIAL RELEVANCE: For the first time, SPM has been linked to a toxin in seeds abundant on autumn pastures whose identified metabolite, MCPA, is known to cause acquired MADD, the pathological mechanism behind SPM and AM. Further research is required to determine the lethal dose of hypoglycin A in horses, as well as factors that affect annual seed burden and hypoglycin A content in Acer species in North America and Europe.

Equine multiple acyl-CoA dehydrogenase deficiency (MADD) associated with seasonal pasture myopathy in the midwestern United States.

BACKGROUND: Seasonal pasture myopathy (SPM) is a highly fatal form of nonexertional rhabdomyolysis that occurs in pastured horses in the United States during autumn or spring. In Europe, a similar condition, atypical myopathy (AM), is common. Recently, a defect of lipid metabolism, multiple acyl-CoA dehydrogenase deficiency (MADD), has been identified in horses with AM. OBJECTIVE: To determine if SPM in the United States is caused by MADD. ANIMALS: Six horses diagnosed with SPM based on history, clinical signs, and serum creatine kinase activity, or postmortem findings. METHODS: Retrospective descriptive study. Submissions to the Neuromuscular Diagnostic Laboratory at the University of Minnesota were reviewed between April 2009 and January 2010 to identify cases of SPM. Inclusion criteria were pastured, presenting with acute nonexertional rhabdomyolysis, and serum, urine, or muscle samples available for analysis. Horses were evaluated for MADD by urine organic acids, serum acylcarnitines, muscle carnitine, or histopathology. RESULTS: Six horses had clinical signs and, where performed (4/6 horses), postmortem findings consistent with SPM. Affected muscle (4/4) showed degeneration with intramyofiber lipid accumulation, decreased free carnitine concentration, and increased carnitine esters. Serum acylcarnitine profiles (3/3) showed increases in short- and medium-chain acylcarnitines and urinary organic acid profiles (3/3) revealed increased ethylmalonic and methylsuccinic acid levels, and glycine conjugates, consistent with equine MADD. CONCLUSIONS AND CLINICAL IMPORTANCE: Similar to AM, the biochemical defect causing SPM is MADD, which causes defective muscular lipid metabolism and excessive myofiber lipid content. Diagnosis can be made by assessing serum acylcarnitine and urine organic acid profiles.

Serum and mucosal antibody isotype responses to M-like protein (SeM) of Streptococcus equi in convalescent and vaccinated horses.

Equine strangles, caused by the clonal pathogen Streptococcus equi, is a source of serious economic loss despite the widespread use of commercial vaccines. The anti-phagocytic 58 kDa M-like protein (SeM) is an important protective antigen. The objective of this study was to define differences, if any, between SeM-specific convalescent serum and mucosal IgA and IgG subisotypes and those induced by vaccination with commercial strangles vaccine. SeM-specific opsonophagocytic IgGb was the predominant serum antibody in horses intramuscularly vaccinated or recently recovered from infection. Infection also induced high levels of specific opsonophagocytic serum IgGa during and shortly after S. equi infection whereas vaccination stimulated only low levels of serum IgGa. Specific serum IgGc and opsonophagocytic IgA were present at very low levels following infection or vaccination. A strong specific mucosal antibody response occurred during the acute and convalescent phases of infection whereas vaccinated horses made no mucosal response. Specific IgGb was generally predominant in nasopharyngeal washings during the acute phase but was replaced by specific IgA during convalescence. SeM-specific mucosal IgGa and IgG(T) but not IgGc were detected only during the acute and early convalescent phase. The results therefore indicate that vaccination, although inducing SeM-specific serum isotype responses qualitatively and quantitatively similar to those seen in convalescence, did not induce mucosal responses. This suggests that mucosal immunity may be important in acquired resistance to strangles.