Grazing horses have a very rich and diverse GI mycobiota
This is the first reported molecular characterisation of the mycobiota throughout the GI tract of grazing horses. All horses, at all 5 GI sites, had a very rich and diverse mycobiota, evidenced by the overall detection of 13,204 OTUs and 2816 phylotypes. The majority of taxa were identified to genus level, but only 40.1% were identified to species level. Dominant phyla, in terms of abundance, were Ascomycota, Basidiomycota and Neocallimastigomycota. Dominant classes were Dothideomycetes, Eurotiomycetes, Leotiomycetes, Saccharomycetes, Sordariomycetes (Ascomycota), Tremellomycetes, Wallemiomycetes (Basidiomycota) and Neocallimastigomycetes. Dominant orders were Capnodiales, Eurotiales, Pleosporales, Saccharomycetales, Thelebolales (Ascomycota), Filobasidiales (Basidiomycota) and Neocallimastigales. Dominant families were Aspergillaceae, Phaffomycetaceae, Sporormiaceae, Thelebolaceae (Ascomycota), Bulleribasidiaceae, Filobasidiaceae, Wallemiaceae (Basidomycota) and Neocallimastigaceae. Dominant genera were Aspergillus, Preussia, Thelebolus, Wickerhamomyces (Ascomycota), Naganishia, Vishniacozyma Wallemia (Basidiomycota) and Piromyces (Neocallimastigomycota). Dominant species were Aspergillus proliferans, Wickerhamomyces anomalus (Ascomycota) and Vishniacozyma victoriae, Wallemia muriae, W. sebi (Basidiomycota).
FUNGuild analysis parsed 87% of the 2816 phylotypes into 20 growth morphologies, 26 ecological guilds and 3 tropic modes. Most were classified as environmental microfungi, agaricoids or yeasts, comprising wood, soil, plant, dung and undefined saprotrophs, plant pathogens, endophytes, animal pathogens, fungal parasites and ectomycorrhizal fungi. These fungi are typical of those colonising grassland soils and plants  and show considerable overlap with those identified in a metagenomic study of grass endophytes .
The GI mycobiota of grazing horses appears to be richer and more diverse than that of humans and mice, which typically comprise nearly 70 genera and more than 184 species of fungi, but with 10 or fewer taxa typically accounting for the vast majority of fungi detected [35,36,37,38]. The marked richness and diversity of the equine GI mycobiota likely reflects the richness and diversity of the environmental fungi present in the plants, soil and water that are ingested by grazing horses. Consistent with this, the diversity of fungal species in equine faeces was considered to reflect the different forage types fed to stabled horses . Similarly, most of the fungi detected in human faeces are derived from the consumption of different foods, which contain, as a whole, more unique fungi than the population colonising the GI tract . Many of the fungi commonly considered to represent the human core GI mycobiota, including Candida, Saccharomyces, Penicillium, Aspergillus, Cryptococcus, Malassezia, Cladosporium and Trichosporon [36, 41, 42], were detected in the equine GI tract. The predominant fungal phyla in both equine and human GI tracts are Ascomycota and Basidiomycota, while Neocallimastigomycota, GI adapted anaerobic fibre degrading endosymbionts, are abundant only in the horse [43, 44]. Neocallimastigomycota are an essential part of the core mycobiota colonising the equine GI tract, but the composition of the remainder of the equine core GI mycobiota is unknown and cannot be determined from this study alone. Indeed it is currently unclear whether the aerobic fungi detected in the GI tract of horses and other animals, including man, are true endosymbionts, opportunistic pathogens which colonise the GI tract only under particular circumstances, or are ingested fungi from food, water, environment, and nasal or oral cavities, and which are simply transiting through the GI tract [36, 42]. While the majority of aerobic fungi detected in the equine GI tract are likely to be transient non-colonising ingested environmental fungi, because these can maintain metabolic activity during GI transit , they could potentially contribute to EGS aetiology by producing extrolites in vivo. Opportunistic fungal pathogens which have colonised the equine GI tract could also contribute to EGS aetiology by producing toxic extrolites in vivo.
Mycobiota richness (Chao1) varied throughout the equine GI tract, being higher distally (caecum, colon, faeces) than proximally (stomach, ileum). PLS-DA and weighted UniFrac distance analysis (beta-diversity) identified significant differences in mycobiota structure among GI sites in both EGS and CTRL groups. Neocallimastigomycota, Neocallimastigomycetes, Neocallimastigales and Neocallimastigaceae were more abundant in distal than proximal GI sites, consistent with previous findings . In contrast, Wallemiomycetes, Wallemiales, Wallemiaceae and Tremellales were more abundant proximally.
EGS is associated with changes in the richness, diversity and structure of the GI mycobiota
EGS is associated with significant alterations in the GI mycobiota. Mycobiota richness (Chao1) was higher in the caecum and colon of EGS horses compared with CTRL horses, while mycobiota diversity (Inverse Simpson) was higher in EGS colon and faeces, compared with CTRL and CoG horses, respectively. Indices of beta-diversity demonstrated inter-group differences in mycobiota structure at all taxonomic levels. Analysis with the Bioconductor software package DESEq2 identified a large number of phylotypes that were differentially abundant between EGS and the 2 control groups, most of which had increased abundance in EGS horses. PLS-DA and VIP scores (> 1.5) were used to identify those taxa that were the most important contributors to the inter-group differential mycobiota structure. Key phylotypes (n = 56) associated with EGS, and which could have a potential role in EGS aetiology, were then identified which had a) high abundance and high prevalence in EGS samples, b) significantly increased abundance in EGS samples, and c) a VIP score > 1.5 indicating they contributed significantly to inter-group differences in mycobiota structure. Key phylotypes comprised fungi of diverse taxonomy. FUNGuild analysis parsed the key phylotypes as predominantly environmental microfungi, classified as soil, dung, wood, plant and undefined saprotrophs, fungal parasites, plant pathogens, endophytes and animal pathogens. Some key phylotypes were macrofungi; Entoloma sericeum, G_Coprinopsis, O_Agaricales and C_Agaricomycetes.
The increased abundance of key phylotypes in the GI tract of EGS horses could reflect increased GI colonisation by opportunistic pathogenic fungi, but more likely reflects ingestion of increased numbers of these fungi in plants, litter and soil while grazing. Indeed there is evidence to suggest that EGS horses are exposed to increased numbers of a wide range of diverse environmental microbes. In addition to key fungi, EGS horses had increased abundance of K_Rhizaria (G_Cercozoa), heterotrophic protists that predate bacteria thereby substantially changing the structure and function of microbial communities on plant surfaces [47,48,49]. Further, a previous study identified increased numbers of cyanobacteria, filamentous green algae, unicellular green algae, diatoms, motile algal flagellates and desmids (Closterium sp.) on plants growing on EGS pastures during disease outbreaks . It is likely that this increased abundance of a wide range of diverse microbes on EGS fields reflects favourable environmental conditions for microbial growth, including suitable vegetation, soil organic matter content, pH, conductivity, temperature and availability of water and macronutrients . Conditions which favour fungal growth and/or extrolite elaboration on the pasture could potentially account for some of the environmental risk factors for EGS. These factors include spring and early summer season, cool, dry weather with irregular ground frosts, faecal contamination, sand and loam rather than chalk soils, high soil nitrogen and low Cu and Zn, and pasture disturbance [15, 20, 52]. Many key phylotypes are extremophilic fungi, including Pseudeurotium, Thelobolus (psychrophilic), Thermomyces lanuginosus (thermophilic), Wallemia (xerophilic) and extremophilic or polyextremophilic yeasts including Apiotrichum, Bannozyma, Cystobasidium, Nagashinia, O_Saccharomycetales, Saitozyma, Tausonia and Vishniacozyma [51, 53, 54]. Increased abundance of these extremophiles likely reflects their ability to survive the potentially adverse environmental conditions associated with EGS, including cold and dry weather. The increased abundance of some soil yeasts may also be attributable to extracellular polysaccharide capsules that facilitate survival in sandy soils  which are associated with EGS . Many key phylotypes are dung saprotrophs, including Acremonium spp. O_Agaricales, Coprinus spp., Coprinopsis spp., Pleospora spp., Preussia spp., Thelebolus spp. , Cleistothelebolus nipigonensis, O_Coniochaetales and Kernia retardata, potentially explaining the reduction in EGS risk when faeces are collected manually from pastures .
Alternatively, the alterations in mycobiota associated with EGS could be a consequence, rather than a cause of EGS, perhaps attributable to the GI stasis which characterises the disease. Consistent with this possibility, experimental murine antimycotic drug-induced intestinal fungal dysbiosis resulted in increased abundance of Wallemia , one of the key phylotypes associated with EGS. Further work is therefore required to determine whether any of the key phylotypes contribute to EGS aetiology or whether their association with EGS is a consequence of the disease. None of the key phylotypes has been previously associated with a pasture neuromycotoxicosis resembling EGS, however many of them are predicted to produce cytotoxic and/or neurotoxic extrolites, including brefeldin, communiols, cytochalasans, d-lysergic acid amide, gliotoxin, L-DOPA, polyketides, preussins, sesquiterpenoids, trichothecenes, tyrosinase and walleminol [39, 57,58,59]. In addition, antibacterial and antifungal activities of fungal extrolites within the GI tract  could potentially induce the marked GI bacterial dysbiosis which occurs in EGS  and contribute to the changes in mycobiota reported herein. Examination of GI contents from EGS horses for those extrolites produced by key phylotypes is therefore warranted to further test the hypothesis that they contribute to EGS aetiology.
It is conceivable that EGS is more prevalent in horses when there is a reduced abundance of particular taxa that serve key beneficial functions for the host. Notable taxa with reduced abundance in EGS horses included some species of Alternaria and Cladosporium, dominant endophytic fungi on temperate grasses , Fusarium, an animal and plant pathogen, soil and wood saprotroph, endophyte, and lichen parasite, Neoascochyta, an animal and plant pathogen and saprotroph, and the plant pathogen Mycosphaerella tassiana. Consistent with these findings, Doxey et al.  isolated Fusarium and Alternaria only infrequently from EGS horses. In contrast, Robb et al.  identified Fusarium on plants from all EGS fields in Scotland and Patagonia. Rather than contributing to EGS aetiology, reduced numbers of these aforementioned taxa likely reflects reductions in the numbers of these fungi that are ingested by EGS horses, perhaps because the environmental conditions associated with EGS are unfavourable for growth of these fungi. Alternatively, reduced numbers of certain taxa may be attributable to the inhibitory effects of antifungal extrolites produced by those taxa that were present in increased abundance.
The structure of the faecal mycobiota of CoG horses differed significantly from those of EGS and CTRL horses. CoG horses were co-grazing with EGS horses at the time of disease onset. While CoG horses remain clinically healthy, as with EGS horses, they have increased serum concentrations of acute phase proteins  consistent with subclinical exposure to the toxin that causes EGS. It is possible that the quantity of causal fungi/extrolite ingested by CoG horses is sufficient to induce a self-resolving acute inflammatory response but insufficient to induce clinical EGS. Consistent with this possibility, 29 of the 56 key phylotypes associated with EGS were more abundant in faeces of EGS versus CoG groups. Alternatively CoG horses may survive exposure to the causal toxin because of host-specific immunological protection or toxin metabolism.
Targeted amplicon sequencing identified considerably many more fungi than a previous culture-based study of the GI mycobiota of EGS and control horses . However, this methodology has biases and limitations , including underestimation, or failure to detect, taxa lacking validated phylogenetic marker sequences, such as some grass endophytes  and Fusarium . This may also explain why several taxa isolated from the equine GI tract by Doxey et al.  were not identified in the present study, including Absidia, Rhizopus, Thamnidium, Geotrichum (Dipodascus) and Monilia. The limited annotation of some fungal genes can also preclude classification of taxa to fine taxonomic ranks. In the present study, most taxa were identified to genus level, but only 40.1% were identified to species level, and some taxa, including key taxa, were identified only at kingdom or phylum levels.
While all of the fungi potentially present in the equine GI tract of grazing horses will not have been identified in this study, this was not considered to be a significant limitation given that the main aim of the study was to identify key taxa associated with EGS, rather than to generate an inventory of the equine GI mycobiota.
Inclusion of mock community and negative controls is considered an essential feature of amplicon sequencing experiments, to identify biases and sample contamination, respectively . All 12 taxa in the two mock communities were identified in all mock community samples. Samples of mock community 1 had markedly lower amplicon numbers for Saccharomyces cerevisiae than for Cryptococcus neoformans. Similarly, Bakker  failed to detect S. cerevisiae in an ITS1 amplicon library, concluding that this negative bias was likely associated with amplicon length. We have noted that the number and depth of OTUs is immensely sensitive to the stringency of the parameters applied during assembly (data not shown), and thus the abundance of rRNA gene amplicons may not accurately reflect taxon biomass in samples . Some taxa, including Coniochaeta lignicola which was included in mock community 2, were represented by a single OTU. Others, including Vishniacozyma victoriae, were represented by multiple OTUs, reflecting intragenomic marker gene polymorphism . To mitigate against these observations, a phylotype approach, rather than an OTU approach, was adopted for this study .
Negative control samples are valuable for revealing rDNA contamination, but there is little or no consensus regarding how to incorporate information from negative control samples into data processing . All negative control samples had low amplicon counts, except 3 which were contaminated with rDNA from Alternaria infectoria and Mycosphaerella tassiana. As these two taxa were included in mock community 2, and were present in many GI samples, it was considered inappropriate to remove these taxa from the GI sample analysis . Importantly, these contaminant taxa were not key taxa and were not increased in abundance in EGS samples. Retrospective analysis indicated that 53 of the 13,204 OTUs were likely contaminants; none of these contributed to the key phylotypes.
EGS horses were significantly younger than controls, in part reflecting the difficulty obtaining post-mortem samples from young control horses. While age influences mycobiota structure , it seems unlikely to account for the significant inter-group differences observed herein, which are instead consistent with inter-group differences in exposure to a diverse range of environmental fungi.