Validation of method for faecal sampling in cats and dogs for faecal microbiome analysis

Abstract

Background: Reproducible and reliable studies of cat and dog faecal microbiomes are dependent on many methodology-based variables including how the faecal stools are sampled and stored prior to processing. The current study aimed to establish an appropriate method for sampling and storing faecal stools from cats and dogs which may also be applied to privately-owned pets. The approach investigated the effects of storing faeces for up to 12 h at room temperature and sampling from various locations within the stool in terms of microbial diversity, relative taxa abundances and DNA yield. Faeces were collected from 10 healthy cats and 10 healthy dogs and stored at room temperature (20 °C). Samples were taken from various locations within the stool (the first emitted part (i), the middle (ii) and the last emitted end (iii), at either surface or core) at 0, 0.5, 1, 2, 3, 6 and 12 h, stabilised and stored at -80 °C. DNA was extracted from all samples, using Illumina NovaSeq.

Results: Faecal bacterial composition of dogs and cats shown no statistically significant differences in alpha diversity. Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria were the most prevalent phyla. Cat and dog samples were characterized by a dominance of Prevotella, and a lack of Fusobacterium in feline stools. Room temperature storage of cat and dog faecal samples generally had no significant effect on alpha diversity, relative taxa abundance or DNA yield for up to 12 h. Sampling from regions i, ii or iii of the stool at the surface or core did not significantly influence the outcome. However, surface cat faecal samples stored at room temperature for 12 h showed a significant increase in two measures of alpha diversity and there was a tendency for a similar effect in dogs. When comparing samples with beta diversity measures, it appeared that for dog and cat samples, individual effect has the strongest impact on the observed microbial diversity (R2 0.64 and 0.88), whereas sampling time, depth and horizontal locations significantly affected the microbial diversity but with less impact.

Conclusion: Cat and dog faeces were stable at room temperature for up to 12 h, with no significant changes in alpha diversity, relative taxa abundance and DNA concentration. Beta diversity analysis demonstrated that despite an impact of the sampling storing time and the surface of the sampling, we preserved the identity of the microbial structure linked to the individual. Finally, the data suggest that faecal stools stored for > 6 h at room temperature should be sampled at the core, not the surface.

Keywords: Cat; Dog; Faeces; Gastrointestinal; Microbiome; Storage; Temperature.

Fig. 1

Relative abundance of predominant phyla in faecal samples from 10 healthy cats and 10 healthy dogs

Fig. 2

Relative abundance of predominant genera in faecal samples from 10 healthy cats and 10 healthy dogs

Fig. 3

Relative abundance of predominant genera in faecal samples from 10 healthy cats (C) and 10 healthy dogs (D), by individual

Fig. 4

Cat and dog faecal microbial diversity according to three methods. Samples were taken at T0 from region ii of faeces from 10 healthy cats and 10 healthy dogs

Fig. 5

Comparisons of microbial diversity between regions i, ii and iii in healthy cat (A-C) and healthy dog (D-F) stool samples at T0

Fig. 6

Comparisons of microbial diversity between core and surface samples from healthy cat (A-C) and healthy dog (D-F) faecal stools stored at room temperature for 12 h

Fig. 7

Beta diversity representation with Principal Component Analysis (PCA). A: PCA representation with all samples from the study; B: PERMANOVA results for the cat samples; C: PERMANOVA results for the dog samples; D: PCA representation with dog samples; E PCA representation with cat samples

Fig. 8

DNA concentration in T0 samples for 10 individual healthy cats (C) and 10 individual healthy dogs [10]

Fig. 9

DNA concentration in core faecal samples from region ii at T0 in 10 healthy cats and 10 healthy dogs