Metabolomic analysis of Ranunculus spp. as potential agents involved in the aetiology of Equine Grass Sicknes

Introduction: Equine grass sickness (EGS) is a polyneuropathy affecting the central, peripheral and enteric nervous systems of grazing horses (esp. in Great Britain). It is very likely that EGS has a multifactorial aetiology, including intoxication with Clostridium botulinum type C, but the causes of the disease are still unknown. Our recent research has shown that a range of edaphic and botanical factors are strongly associated with EGS outbreaks including the regular occurrence of Ranunculus spp. on EGS-sites. Aim: To determine if the metabolomic variability of Ranunculus spp. could be linked to increased risk of EGS outbreaks. Methods: Ranunculus samples from twelve farms with EGS outbreaks and nine controls were extracted with methanol in triplicate. Metabolic profiling using 1H-NMR spectroscopy and multivariate data analysis in combination with principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and partial least squares (PLS) was used. Results: Metabolomic differences were found between different Ranunculus spp. PCA on R. repens methanol extracts from all Equine Grass Sickness and control sites showed a cluster of control samples, whereas there was a higher variation among the EGS samples. Seasonal variation and correlations with elevated metal levels from soil samples from the same sites were also found. Conclusion: The metabolomic composition of Ranunculus spp. on EGS and non-EGS sites differ. High levels of iron, nitrate or chromium may have an impact on the variation in the extracts' metabolomic profile. A toxic metabolite – protoanemonin (a hydrolisation product of ranunculin) may provoke lesions in the horses' gastrointestinal tract.