Equine Grass Sickness (A Multiple Systems Neuropathy) is Associated with Alterations in the Gastrointestinal Mycobiome

Abstract

Background: Equine grass sickness (EGS) is a multiple systems neuropathy of grazing horses of unknown aetiology. An apparently identical disease occurs in cats, dogs, rabbits, hares, sheep, alpacas and llamas. Many of the risk factors for EGS are consistent with it being a pasture mycotoxicosis. To identify potential causal fungi, the gastrointestinal mycobiota of EGS horses were evaluated using targeted amplicon sequencing, and compared with those of two control groups. Samples were collected post mortem from up to 5 sites in the gastrointestinal tracts of EGS horses (EGS group; 150 samples from 54 horses) and from control horses that were not grazing EGS pastures and that had been euthanased for reasons other than neurologic and gastrointestinal diseases (CTRL group; 67 samples from 31 horses). Faecal samples were also collected from healthy control horses that were co-grazing pastures with EGS horses at disease onset (CoG group; 48 samples from 48 horses). Results: Mycobiota at all 5 gastrointestinal sites comprised large numbers of fungi exhibiting diverse taxonomy, growth morphology, trophic mode and ecological guild. FUNGuild analysis parsed most phylotypes as ingested environmental microfungi, agaricoids and yeasts, with only 1% as gastrointestinal adapted animal endosymbionts. Indices of alpha-diversity indicated that mycobiota richness and diversity varied throughout the gastrointestinal tract and were greater in EGS horses. There were significant inter-group and inter-site differences in mycobiota structure. A large number of phylotypes were differentially abundant among groups. Key phylotypes (n=56) associated with EGS were identified that had high abundance and high prevalence in EGS samples, significantly increased abundance in EGS samples, and were important determinants of the inter-group differences in mycobiota structure. Many key phylotypes were extremophiles and/or were predicted to produce cytotoxic and/or neurotoxic extrolites. Conclusions: This is the first reported molecular characterisation of the gastrointestinal mycobiota of grazing horses. Key phylotypes associated with EGS were identified. Further work is required to determine whether neurotoxic extrolites from key phylotypes contribute to EGS aetiology or whether the association of key phylotypes and EGS is a consequence of disease or is non-causal.

Characterisation of the Faecal Microbiome of Foals from 0–5 Months of Age and Their Respective Mares across Five Geographic Locations

“…The primary DNA barcode for eukaryotes like fungi is the ITS1 region between the 18S rRNA and 5.8S rRNA genes [59]. The authors chose to use ITS1 primers like those used in McGorum et al [60], who collected postmortem samples from the gastrointestinal tract of 54 horses with equine grass sickness and faecal samples from control horses. Results seen in McGorum et al [60] are somewhat comparable to the current study, finding Acremonium, Preussia and Naganishia fungal species to be among the most dominant.…”

“…The authors chose to use ITS1 primers like those used in McGorum et al [60], who collected postmortem samples from the gastrointestinal tract of 54 horses with equine grass sickness and faecal samples from control horses. Results seen in McGorum et al [60] are somewhat comparable to the current study, finding Acremonium, Preussia and Naganishia fungal species to be among the most dominant. However, it is unknown why Neocallimastigomycota species or other anaerobic fungal phyla known to colonise the alimentary tract of mammalian herbivores and hindgut fermenters were not detected in faecal samples analysed in the current study [24,52,[61][62][63][64][65][66].…”

Clinical update on equine grass sickness

“…The possibility of soil‐based mycotoxin involvement in the aetiology of EGS has also been investigated using molecular techniques to type the gastrointestinal mycobiota. This showed that some key phylotypes had high prevalence and abundance in EGS cases compared to healthy control cases (McGorum and others 2021). The theory of certain fungi producing neurotoxins as a causal agent would also be consistent with the seasonality of EGS cases, as well as pasture and climate spatiality.…”