Hypoglycin A (HGA) in seeds of Acer spp. is suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy (AM) in Europe, fatal diseases in horses on pasture. In previous studies, this suspicion was substantiated by the correlation of seed HGA content with the concentrations of toxic metabolites in urine and serum (MCPA-conjugates) of affected horses. However, seed sampling was conducted after rather than during an outbreak of the disease. The aim of this study was to further confirm the causality between HGA occurrence and disease outbreak by seed sampling during an outbreak and the determination of i) HGA in seeds and of ii) HGA and MCPA-conjugates in urine and serum of diseased horses. Furthermore, cograzing healthy horses, which were present on AM affected pastures, were also investigated. AM-pastures in Germany were visited to identify seeds of Acer pseudoplatanus and serum (n = 8) as well as urine (n = 6) from a total of 16 diseased horses were analyzed for amino acid composition by LC-ESI-MS/MS, with a special focus on the content of HGA. Additionally, the content of its toxic metabolite was measured in its conjugated form in body fluids (UPLC-MS/MS). The seeds contained 1.7–319.8 μg HGA/g seed. The content of HGA in serum of affected horses ranged from 387.8–8493.8 μg/L (controls < 10 μg/L), and in urine from 143.8–926.4 μg/L (controls < 10 μg/L), respectively. Healthy cograzing horses on AM-pastures showed higher serum (108.8 ± 83.76 μg/L) and urine concentrations (26.9 ± 7.39 μg/L) compared to control horses, but lower concentrations compared to diseased horses. The range of MCPA-carnitine and creatinine concentrations found in diseased horses in serum and urine were 0.17–0.65 mmol/L (controls < 0.01), and 0.34–2.05 μmol/mmoL (controls < 0.001), respectively. MCPA-glycine levels in urine of cograzing horses were higher compared to controls. Thus, the causal link between HGA intoxication and disease outbreak could be further substantiated, and the early detection of HGA in cograzing horses, which are clinically normal, might be a promising step in prophylaxis.

This placebo-controlled double-blind study was conducted to evaluate effects of Enterococcus faecium DSM 10663 NCIMB 10415 (EcF) orally given from birth to weaning on diarrhoea and performance of piglets. At the first 3 days postnatum (p.n.), piglets from 54 [verum group (VG)] and 60 [placebo group (PG)] sows got 1 g of a gel directly per mouth by a dosing device. Gel for the VG contained 2.8 x 10(9) colony forming units (CFU) EcF/g. From day 4 p.n. until weaning (24 +/- 3.2 days p.n.) a liquid additive was given that administered twice a day 1.26 x 10(9) CFU EcF to each VG piglet. In case of diarrhoea, an electrolyte solution was used which provided daily 2.9 and 5.8 (week 1 and >or= 2, respectively) x 10(8) CFU EcF per VG piglet. Diarrhoea scores were defined as follows: (i) no diarrhoea; (ii) piglets developed diarrhoea, but were vital and (iii) piglets suffered from diarrhoea and additionally looked pale, developed rough coat, showed slackening of the flank and lethargy. Counts of viable born, stillborn and weaned piglets were normal and not different between groups (p > 0.05). Placebo group vs. VG piglets suffered more frequently from diarrhoea (40.0 vs. 14.8%, p < 0.05). Duration of diarrhoea was not affected by feeding EcF (2.2 +/- 0.81 days, p > 0.05). Diarrhoea score was lower in VG vs. PG (1.2 vs. 1.5 +/- 0.54, p < 0.05) and the daily weight gain (DWG) was higher by 17 g/day (p < 0.05). Results suggest that the daily oral supplementation of EcF from birth to weaning reduces the portion of piglets suffering from diarrhoea. This may improve performance, as the higher DWG indicates. In contrast, no obvious benefit seems to result from an additional supply of EcF via electrolyte solution when diarrhoea is always present.

Weaning of the pig is generally regarded as a stressful event which could lead to clinical implications because of the changes in the intestinal ecosystem. The functional properties of microbiota inhabiting the pig's small intestine (SI), including lactobacilli which are assumed to exert health-promoting properties, are yet poorly described. Thus, we determined the ecophysiology of bacterial groups and within genus Lactobacillus in the SI of weaning piglets and the impact of dietary changes. The SI contents of 20 piglets, 4 killed at weaning (only sow milk and no creep feed) and 4 killed at 1, 2, 5, and 11 days post weaning (pw; cereal-based diet) were examined for bacterial cell count and bacterial metabolites by fluorescence in situ hybridization (FISH). Lactobacilli were the predominant group in the SI except at 1 day pw because of a marked reduction in their number. On day 11 pw, bifidobacteria and E. coli were not detected, and Enterobacteriaceae and members of the Clostridium coccoides/Eubacterium rectale cluster were only found occasionally. L. sobrius/L. amylovorus became dominant species whereas the abundance of L. salivarius and L. gasseri/johnsonii declined. Concentration of lactic acid increased pw whereas pH, volatile fatty acids, and ammonia decreased. Carbohydrate utilization of 76 Lactobacillus spp. isolates was studied revealing a shift from lactose and galactose to starch, cellobiose, and xylose, suggesting that the bacteria colonizing the SI of piglets adapt to the newly introduced nutrients during the early weaning period. Identification of isolates based on partial 16S rRNA gene sequence data and comparison with fermentation data furthermore suggested adaptation processes below the species level. The results of our study will help to understand intestinal bacterial ecophysiology and to develop nutritional regimes to prevent or counteract complications during the weaning transition.

To investigate effects of hay intake and feeding sequence on indicators of the microbial activity within the hindgut, six horses were fed 1.00 kg oats plus 0.50, 0.67, 0.83 or 1.00 kg hay/100 kg body weight (BW) · day, each for 14 days. Oats was offered either 30 min prior to hay (OA) or in the reversed sequence (HA) in a 2 · 8-week crossover design. Because typically exercised horses should be subjected to the study, faeces was used as substrate. Faecal dry matter (DM), the faecal watersÕ short-chain fatty acids (SCFA, in mmol/l) and molar percentages (mol%) of propionate and iso-butyrate were highest with OA (p < 0.01). Acetate mol%, acetate-propionate quotient (A/P) and buffering capacities 1 and 2 (BC1: current pH to pH 6; BC2: pH 6 to 5) of the faecal water were highest with HA (p < 0.01). While the hay intake rose, faecal pH, acetate mol%, A/P, BC1 and BC2 (the latter with HA only) increased (p < 0.05), but DM, SCFA and propionate mol% declined (p < 0.05). The hay-induced rise in A/P and BC1 was much higher with HA than with OA. l-Lactate and ammonia were unaffected by the feeding sequence and hay intake. In conclusion, hay intake and feeding sequence influence the microbial activity within the hindgut, although the concentrate level remains consistent. In horse rations with 1.00 kg oats/100 kg BW · day amounts of at least 0.83 kg hay/100 kg BW · day and offering the hay first seem to have the potency to protect the hindgut content from acidification. Behavioural abnormality was not observed any longer first with 1.00 kg hay/100 kg BW · day.

Atypical myopathy (AM) in horses is caused by ingestion of seeds of the Acer species (Sapindaceae family). Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A (HGA), is currently the only active toxin in Acer pseudoplatanus or Acer negundo seeds related to AM outbreaks. However, seeds or arils of various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also contain methylenecyclopropylglycine (MCPG), which is a structural analogue of HGA that can cause hypoglycaemic encephalopathy in humans. The active poison formed from MCPG is methylenecyclopropylformyl-CoA (MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that participate in β-oxidation and energy production from fat. The aim of our study was to investigate if MCPG is involved in Acer seed poisoning in horses. MCPG, as well as glycine and carnitine conjugates (MCPF-glycine, MCPF-carnitine), were quantified using high-performance liquid chromatography-tandem mass spectrometry of serum and urine from horses that had ingested Acer pseudoplatanus seeds and developed typical AM symptoms. The results were compared to those of healthy control horses. For comparison, HGA and its glycine and carnitine derivatives were also measured. Additionally, to assess the degree of enzyme inhibition of β-oxidation, several acyl glycines and acyl carnitines were included in the analysis. In addition to HGA and the specific toxic metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and MCPF-carnitine were detected in the serum and urine of affected horses. Strong inhibition of β-oxidation was demonstrated by elevated concentrations of all acyl glycines and carnitines, but the highest correlations were observed between MCPF-carnitine and isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-) glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here, for biochemical analysis of atypical myopathy of horses, it is necessary to take MCPG and the corresponding metabolites into consideration.