Rapid regrowth and detection of microbial contaminants in equine fecal microbiome samples

Advances have been made to standardize 16S rRNA gene amplicon based studies for inter-study comparisons, yet there are many opportunities for systematic error that may render these comparisons improper and misleading. The fecal microbiome of horses has been examined previously, however, no universal horse fecal collection method and sample processing procedure has been established. This study was initialized in large part to ensure that samples collected by different individuals from different geographical areas (i.e., crowdsourced) were not contaminated due to less than optimal sampling or holding conditions. In this study, we examined the effect of sampling the surface of fecal pellets compared to homogenized fecal pellets, and also the effect of time of sampling after defecation on ‘bloom’ taxa (bloom taxa refers to microbial taxa that can grow rapidly in horse feces post-defecation) using v4 16S rRNA amplicon libraries. A total of 1,440,171 sequences were recovered from 65 horse fecal samples yielding a total of 3,422 OTUs at 97% similarity. Sampling from either surface or homogenized feces had no effect on diversity and little effect on microbial composition. Sampling at various time points (0, 2, 4, 6, 12 h) had a significant effect on both diversity and community composition of fecal samples. Alpha diversity (Shannon index) initially increased with time as regrowth taxa were detected in the amplicon libraries, but by 12 h the diversity sharply decreased as the community composition became dominated by a few bloom families, including Bacillaceae, Planococcaeae, and Enterococcaceae, and other families to a lesser extent. The results show that immediate sampling of horse feces must be done in order to ensure accurate representation of horse fecal samples. Also, several of the bloom taxa found in this study are known to occur in human and cattle feces post defecation. The dominance of these taxa in feces shortly after defecation suggests that the feces is an important habitat for these organisms, and horse fecal samples that were improperly stored can be identified by presence of bloom taxa.

Detailed Sampling methods: Flow-charts for each sampling procedure (i -iii) are provided below. For objective (i) three freshly deposited manure piles from three individual horses were visually observed at the time of deposition at the Loranger Farm, and sampling was performed immediately afterwards by taking small (approx 2g) scrapings from the exterior of a single pellet for 'surface' and placing into separate sterile 36oz whirl-paks (Nasco, Inc. Fort Atkinson, WI, USA). Following surface sampling, the remaining fecal pellet was placed into a whirl-pak for 'homogenized' sampling, and all samples were placed on ice and transported directly to a -20°C freezer until DNA extraction, for a total of 18 samples. After samples were thawed immediately before DNA extraction, 'homogenized' samples were homogenized by kneading the pellet inside the whirl-pak by hand for 1min, then DNA extraction proceeded as described in manuscript. For objective (ii) three fresh manure piles were marked off at time of deposition from three individual horses at the Loranger Farm and five samples were taken at timed intervals (14 samples, one lost in processing) from each manure pile. All samples were in shaded areas (barn). The average ambient temperature for the 12 hr period was 32ºC (stdev ± 3.6). At time of deposition (T0) an individual pellet from the manure pile was collected, immediately frozen, then processed using the homogenized sampling method. Additional samples were collected at 2, 4, 6, and 12 hrs (T2, T4, T6, and T12). To address objective (iii) 24 samples were collected, six samples from the Loranger Farm, five samples from the Hammond Farm, and 13 samples from the Folsom Farm in January of 2015. All samples from the Hammond and Loranger Farms were collected using the wait-for-thedrop-method followed by the homogenized technique. Samples collected from the Folsom Farm were collected from individual stalls that housed a single horse with a clay surface with wood shaving. The stalls had been cleaned within the previous six hrs by removal of horse and wood shavings, and addition of new wood shavings. Samples were processed using the homogenized sampling procedure. Objective (i): Difference between surface and homogenized sampling x 3 horses (i.e., 3 sets of samples) 1.) Individual Horses were observed until a defecation event.
2.) Immediately after defecation, three samples were collected from the surface (yellow = pellet surface ~ 1cm) of a single fecal pellet by scraping ~ 2g using a sterile spatula each time placing into separate sterile 36oz whirl-paks, then on ice for transport to freezer.
3.) The remainder of the pellet sampled was placed in it's own 36oz whirl-pak, then on ice for transport to freezer.
5a.) Samples were defrosted, and processing began while still cold.

DNA extraction 6.
Objective (ii): Time series sampling for 'bloom' taxa identification 1.) Individual Horses were observed until a defecation event, and fecal pellet was roped off to prevent horses from disturbing manure pile during 12 hr time-series collection.
2.) Immediately after defecation, a single fecal pellet was removed from the manure pile and placed into a sterile 36oz whirl-paks, then on ice for transport to freezer. This pellet was labeled as Time 0 (T0).
3.) The manure pile was sampled again at 2 hrs, 4hrs, 6 hrs, and 12 hrs, removing an individual pellet at each time point. Pellets that were on the bottom were avoided to avoid direct ground contamination. 4.) Samples stored at -20°C until DNA extraction.
5a.) Samples were defrosted, and processing began while still cold. 5b.) The single pellet collected was homogenized by kneading within whirl-pak for 1min.
6.) DNA extraction was performed using MoBio Powersoil DNA Isolation kit (Mo Bio Laboratories Inc., Carlsbad, CA) according to manufacturers protocol.

5a.
Objective (iii): Feasibility of sampling stalled horses without witnessing actual defecation event.
x 13 stalled horses, samples collected between 0 and 6hrs from time of defecation.
x 11 horses, samples collected immediately after defecation 1.) As part of routine farm maintenance, horses as removed from stall and all manure is removed from stall (cleaning), including soiled wood shavings. New shavings are added.
2.) Horses are brought back into stall, as part of their normal routine.
3.) The stall is re-visited 6 hrs later to collect fecal pellets (a few fecal pellets from top of manure pile are placed into sterile whirl-paks, and placed on ice). Exact time of defecation (i.e., feces age) is unknown, but must be between 0 -6 hrs of age at max. 4.) Samples stored at -20°C until DNA extraction.
5a.) Samples were defrosted, and processing began while still cold.
2.) Immediately after defecation, several pellets were collected from the surface and placed into a sterile 36oz whirlpak, then on ice for transport to freezer.
4a.) Samples were defrosted, and processing began while still cold.
4b.) The pellets collected were homogenized by kneading within whirl-pak for 1min.