Measuring the gut microbiome in birds: comparison of faecal and cloacal sampling

Abstract The gut microbiomes of birds and other animals are increasingly being studied in ecological and evolutionary contexts. While methods for preserving samples and processing high-throughput sequence data to characterise bacterial communities have received considerable attention, there has been little evaluation of non-invasive sampling methods. Numerous studies on birds and reptiles have made inferences about gut microbiota using cloacal sampling, however, it is not known whether the bacterial community of the cloaca provides an accurate representation of the avian gut microbiome. We examined the accuracy with which cloacal swabs and faecal samples measure the microbiota in three different parts of the gastrointestinal tract (ileum, caecum, and colon) using a case study on juvenile ostriches, Struthio camelus, and high-throughput 16S rRNA sequencing. We found that faeces were significantly better than cloacal swabs in representing the bacterial community of the colon. Cloacal samples had a higher abundance of Gammaproteobacteria and fewer Clostridia relative to the gut and faecal samples. However, both faecal and cloacal samples were poor representatives of the microbial communities in the caecum and ileum. Furthermore, the accuracy of the sampling methods in measuring the abundance of different bacterial taxa was highly variable: Bacteroidetes was the most highly correlated phylum between all three gut sections and both methods, whereas colonic Actinobacteria correlated strongly only with faecal samples. This study demonstrates that sampling methods can have significant effects on the inferred gut microbiome in studies of birds. Based on our results, we recommend sampling faeces, whenever possible, as this provides the most accurate assessment of the colon microbiome. The fact that neither sampling technique portrayed the bacterial community of the ileum or the caecum illustrates the difficulty in non-invasively monitoring gut bacteria located further up in the gastrointestinal tract. These results have important implications for the interpretation of avian gut microbiome studies.