Microbial Composition for Dams and Foals
Samples were taken weekly for the first six weeks of life from 20 foals (nsamples=116) and 20 dams (nsamples=20) for a total of 136. There were a total of 81,365 observed operational taxonomic units (OTUs) from foal and dam samples and a total of 3,887,277 sequence counts (mean±s.d= 28,582.92±16,448.23; range= 3,469-69,307; median= 26,783.5). Average read length was 409.9628 +/- 2.552. OTUs with fewer than three reads were removed from analysis. The sequencing blank was found to have 86 OTUs and a total of 7996 sequences. Three OTUs found to be in common with the sequencing blank and 100% of the samples were subtracted from further analysis: Unassigned, Other, Ruminococcaceae; g__, and g__Bacteroides. (Additional file 1)
OTUs were classified into 19 phyla (Figure 1). The most abundant phylum present was Bacteroidetes followed by Firmicutes in both foals and dams. The average abundance of Bacteroidetes in foals and dams was 55.2% and 48.3%, respectively, and the average abundance of Firmicutes in foals and dams was 22.5% and 23.7%, respectively.
At the family level, four Bacteroidetes families were found to be significantly different between DCM and SFM dams and foals across the time course: Bacteroidaceae was enriched in SFM groups, while Paraprevotellaceae, Porphyromonadaceae, and Rikenellaceae were more abundant in DCM groups (Figure 2A and 2B). Seven Firmicutes families were found to be significantly different between DCM and SFM dams and foals across the time course: Mogibacteriaceae, Streptococcaceae, and Erysipelotrichaceae were enriched in SFM groups, while Christensenellaceae, Lactobacillaceae, and Peptostreptococcaceae were more abundant in DCM groups (Figure 2C and 2D).. Six families in other phyla were found to be significantly different between DCM and SFM dams and foals across the time course: Fusobacteriaceae and a family of Tenericutes (RF39) were enriched in SFM foals, a family of Verrucomicrobia (RFP12) and an Alphaproteobacteria family were more abundant in SFM dams, while Methanocorpusculaceae and a family of Spirochaetes were more abundant in DCM groups (Figure 2E and 2F).
Effect of Breed on Horse’s Hindgut Microbiome
OTUs picked using the combined sequence files from the dams in this study and the adult ponies and Standardbred horses in the EMP database [26] revealed no significant taxa differences with respect to breed (Kruskal-Wallis or nonparametric t-test, corrected p> 0.05), however there were 570 taxa were found to be different between the groups with respect to study [Additional files 2 and 3]. Alpha diversity differences between the EMP horses and the dams in the current study highlight that while factors inherent to each study impact microbiome composition, breed does not appear to be a major driver. [Additional file 4]
Clustering of samples by principle coordinate analysis (PCoA) of Bray-Curtis Dissimilarity point to significant beta diversity differences based on management and study (Figure 3). The Goods coverage of the EMP samples was 85.74%.
Core Microbiomes of SFM and DCM Dams and Foals
The core microbiomes of SFM and DCM foals and dams, defined as OTUs present in 95% or more of samples in each group, were different in terms of composition and numbers of OTUs comprising each taxon (Figure 4). Overall, SFM foals and dams had higher numbers of taxa in their core microbiomes, and more OTUs in almost every group. The core microbiome of SFM foals was comprised of five taxa, only one of which, Bacteroides spp., was shared with DCM foals (Figure 4A). For this shared taxa, the SFM core microbiome featured five OTUs, while the DCM core microbiome had one. Besides Bacteroides spp., the core microbiome of DCM foals contained a Rikenellaceae spp., which was not shared with the SFM foals, and the SFM foal core microbiome included four unique taxa groups: Bacteroides fragilis, Enterobacteriaceae spp., Erysipelotrichaceae spp., and Fusobacterium spp. (Figure 4A). The core microbiome of SFM dams featured 154 OTUs in 16 taxa groups, while that of DCM dams had 54 OTUs in 11 taxa groups (Figure 4B). Unique taxa groups found in the SFM core microbiome for dams included: Paulibacter spp., YRC22 spp., RFN20 spp., Oscillospira spp., Alphaproteobacteria spp., and RFP12 spp. Only one taxon, Fusobacterium spp. was unique to the DCM core microbiome for dams. This taxon was found in the core microbiome of SFM foals, but not in that of DCM foals, and was the only taxa group that overlapped between the dams and foals regardless of management.
Alpha and Beta Diversity
Foal samples were grouped into six different age groups determined by the foals’ ages in weeks at the time of sampling. Foals were also grouped by DCM or SFM, gender, access to grazing (access or no access) and where they were housed during the week of sampling (field, stall, or both). Alpha diversity (PD whole tree, Observed OTUs, Shannon, and Simpson) was calculated and compared for all foal and dam groups at each time point [Additional file 5]. There were no differences in alpha diversity (nonparametric t-test, p>0.05) between SFM and DCM when comparing dams, foals by management and time for any of these measures [Additional file 5]. When comparing foals and dams within management types, however, dams had a significantly lower mean diversity than foals (Shannon, Simpson, and PD whole tree, nonparametric t-test, p<0.05 for all measures except PD-whole tree for DCM foals and dams). (Figure 5). When comparing the six different age groups among foals, week 1 foals had a significantly lower mean diversity than all other weeks (PD whole tree, nonparametric t-test, p<0.01) (Figure 5C). The core microbiomes of SFM foals and dams were significantly more diverse than DCM dams and foals for all measures (Figure 6). The average Goods coverage for the dam and foal samples was 98.34%.
PCoA of Bray-Curtis dissimilarity by management strategy was plotted for 1-week-old foals, 5 or 6-week- old foals, and dams using multidimensional scaling (Figure 7). As foals age, their microbiomes become more similar to that of their dams, however the domestic dams and their 5/6 weeks old foals (Figure 7A) clustered more tightly than the semi-feral dams and their 6-week-old foals (Figure 7B) with higher amount of overlap in the ellipsoids of the domestic dams and their 5/6-week-old foals indicating differences between the two groups in the progress of microbiome development. PCoA plots of weighted and unweighted Unifrac distances demonstrates similar trends for Age and Management [Additional file 6]
Community similarities between and within DCM and SFM foal groups, compared using multivariate ANOSIM and PERMANOVA indicated significant differences between and within DCM and SFM foals based on age, grazing access and housing as well as within each domestication group between age groups [Additional file 7]. These findings show that between study groups, both age and management type affected the foals’ hindgut microbiomes. Significant differences were also found between dams and foals and between SFM and DCM when comparing all dam and foal samples. When analyzing dams only, significant differences were found between SFM and DCM dams indicating that management affects adult horse microbiomes as well as foals.
Pairwise comparisons by age for SFM and DCM foals found significant differences for DCM foals between all ages except for week 2 vs. 3, 3 vs. 4, 3 vs. 5, 3 vs. 6, 4 vs. 5, 4 vs. 6 and 5 vs. 6 foals [Additional file 7]. When comparing all ages in the SFM foals, significant differences were found between all ages except for week 3 vs. 4, 4 vs. 5, 4 vs. 6 and 5 vs. 6 foals. There was more variance between ages in DCM foals, which may indicate that the SFM foals had a more consistent microbiome throughout the study period than DCM foals. Significant differences were found between 6-week-old SFM foals and SFM dams as well as between 6-week-old DCM foals and DCM dams. Therefore, it is clear that these foals’ gut microbiomes had not yet stabilized to that of an adult at 6 weeks of age.
Differences in Community Composition
Significantly different OTUs between SFM and DCM foals at different ages as well as SFM and DCM dams are shown on Tables 1. The most highly significant (Kruskal-Wallis, p<0.01) taxa belonging to the Firmicutes and Bacteroidetes phyla are shown on Table 2. Genera from the family Lactobacillaceae were found to be significantly more abundant in DCM foals than in SFM foals and semi-feral and domestic dams (Table 2 and Figure 2C and D). This is interesting because it is a family that contains many lactic acid producing bacteria which have been associated with the onset of starch-induced laminitis [27].
Enriched taxa were also analyzed using LEfSe (Linear Discriminant Analysis Effect Size) [38]. DCM and SFM foals were analyzed separately for each of their 6 age groups (Tables 3 and 4). 182 taxa were found to be significantly enriched in the different age groups in DCM foals and 151 taxa were found to be significantly enriched in the different ages in SFM foals (p<0.05, Kruskal-Wallis, LDA score>2.0). Week 5 SFM foals and week 4 DCM foals were found to have Methanobrevibacter spp. and Methanobacteriaceae gen. enriched in their microbiomes, which are archaea taxa associated with the digestion of complex carbohydrates and methane production. Fibrobacter spp. and Fibrobacteraceae gen. are also associated with complex plant carbohydrate digestion and were found to be enriched in week 4 SFM foals. Lactobacillus spp. and Lactobacillaceae gen. were found to be enriched in DCM foals aged 2 and 3 weeks, which reinforces this same finding using a Kruskal-Wallis test stated previously.
Predicted Functional Analysis of Foal and Dam Hindgut Microbiome
Functional potential of communities was inferred using PICRUSt [28] to generate predictions based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways at Level 3 [29]. These predictions were then sorted into 6 different digestion related categories [Additional file 8].
Significant differences were found between the 6 different age groups and DCM and SFM for all types of digestion: general carbohydrate, lipid, protein, complex carbohydrate, starch and simple carbohydrate (p<0.05, Kruskal-Wallis). Week 1 DCM and SFM foals had the greatest amount of general carbohydrate-, lipid-, protein-, complex carbohydrate-, starch- and simple carbohydrate-digesting bacteria when compared to the rest of the age groups, including dams. This finding is most likely due to nutrient-rich colostrum and mare’s milk during the foal’s first week of life and the gradual decrease in nutrient content as time progressed. As the foals aged, it was apparent that the abundance of the OTUs contributing to each digestion type gradually decreased to reach levels similar to those of their dams (Figure 8). Both SFM and DCM foals at every age group were found to have significantly higher levels in all types of digestion than SFM and DCM dams (p<0.05, Kruskal-Wallis). Significant differences were also found between SFM and DCM foals with SFM foals having a significantly higher mean sequence count in the OTUs contributing to each type of digestion (p<0.001, Kruskal-Wallis). No significant differences were found in the digestion types between SFM and DCM dams, which may indicate that SFM and DCM adult microbiomes are functionally similar.