An ambient temperature collection and stabilization strategy for canine microbiota studies

Abstract

Similar to humans, the fecal microbiome of dogs may be useful in diagnosing diseases or assessing dietary interventions. The accuracy and reproducibility of microbiome data depend on sample integrity, which can be affected by storage methods. Here, we evaluated the ability of a stabilization device to preserve canine fecal samples under various storage conditions simulating shipping in hot or cold climates. Microbiota data from unstabilized samples stored at room temperature (RT) and samples placed in PERFORMAbiome·GUT collection devices (PB-200) (DNA Genotek, Inc. Ottawa, Canada) and stored at RT, 37 °C, 50 °C, or undergoing repeated freeze-thaw cycles, were compared with freshly extracted samples. Alpha- and beta diversity indices were not affected in stabilized samples, regardless of storage temperature. Unstabilized samples stored at RT, however, had higher alpha diversity. Moreover, the relative abundance of dominant bacterial phyla (Firmicutes, Fusobacteria, Bacteriodetes, and Actinobacteria) and 24 genera were altered in unstabilized samples stored at RT, while microbiota abundance was not significantly changed in stabilized samples stored at RT. Our results suggest that storage method is important in microbiota studies and that the stabilization device may be useful in maintaining microbial profile integrity, especially for samples collected off-site and/or those undergoing temperature changes during shipment or storage.

Figure 1

Experimental design. Fresh fecal samples were collected from 30 healthy beagles. Each sample was aliquoted into three stabilization devices and one unstabilized aliquot, and incubated under different conditions. One stabilized sample was aliquoted and incubated at 37 °C and 50 °C after baseline microbiota data were obtained. Microbiota data were obtained at different time points as marked.

Figure 2

Fecal microbiota communities of baseline stabilized samples (D0-37/50, D0-FT, D0-RT) and unstabilized sample (D0-UNST). (A) Alpha diversity measures, including phylogenetic diversity (PD) whole tree (shown), suggested that species richness and diversity were not affected by collection method at baseline (ANOVA, p = 0.506). Principal coordinates analysis plots of unweighted (B) and weighted (C) UniFrac distances of fecal microbial communities revealed that beta diversity was not altered by collection method at baseline.

Figure 3

Fecal microbiota communities of stabilized samples at baseline (D0-37/50) and stored at 37 °C and 57 °C for 1 (D1-37, D1-50) and 3 days (D3-37, D3-50). (A) Alpha diversity measures, including phylogenetic diversity (PD) whole tree (shown), suggested that species richness and diversity were not affected by storage condition (ANOVA, p = 0.944). Principal coordinates analysis plots of unweighted (B) and weighted (C) UniFrac distances of fecal microbial communities as well as unweighted (D) and weighted (E) UniFrac distances from baseline revealed that beta diversity was not altered by storage condition. Scatterplots show the relative abundance of each genus in baseline samples against samples stored at 37 °C for 1 (F) and 3 days (G), and samples stored at 50 °C for 1 (H) and 3 days (I).

Figure 4

Fecal microbiota communities of stabilized samples at baseline (D0-FT) and stored at freeze–thaw cycles (D14-FT). (A) Alpha diversity measures, including phylogenetic diversity (PD) whole tree (shown), suggested that species richness and diversity were not affected by storage condition (ANOVA, p = 0.630). Principal coordinates analysis plots of unweighted (B) and weighted (C) UniFrac distances of fecal microbial communities revealed that beta diversity was not altered by storage condition. The scatterplot (D) shows the relative abundance of each genus in baseline samples against samples after freeze–thaw cycles.

Figure 5

Fecal microbiota communities of stabilized and unstabilized samples at baseline (D0-RT, D0-UNST) and stored at room temperature (RT) for 14 (D14-RT, D14-UNST) and 60 days (D60-RT). (A) Alpha diversity measures, including phylogenetic diversity (PD) whole tree, Chao1, and observed operational taxonomic units (OTUs), suggested that species richness and diversity were greater in unstabilized samples stored at RT for 14 days (Tukey’s HSD, p < 0.001). The Chao1 metric showed a lower diversity in D60-RT than those of D0-RT (Tukey’s HSD, p = 0.0193), D14-RT (Tukey’s HSD, p = 0.0431), and D0-UNST (Tukey’s HSD, p = 0.013). Principal coordinates analysis (PCoA) plots of unweighted (B) UniFrac distances of fecal microbial communities revealed that beta diversity was not altered by storage condition. PCoA plots of weighted (C) UniFrac distance showed that D14-UNST samples clustered together (circled area) and away from other samples. Unweighted UniFrac distance from baseline (D) revealed that D14-UNST and D60-RT had greater distance than 14-RT. Weighted UniFrac distance (E) showed that D14-UNST had a greater distance than stabilized samples. ^a,b mean values with unlike letters were significantly different (Tukey’s HSD, p < 0.05). Scatterplots show the relative abundance of each genus in baseline samples against stabilized samples stored at RT for 14 (F) and 60 days (G), and unstabilized samples stored at RT for 14 days (H).