16S rRNA gene-based assessment of common broiler chicken sampling methods: evaluating intra-flock sample size, cecal pair similarity, and cloacal swab similarity to other alimentary tract locations

Authors: Weinroth, Margaret - Maggie; OAKLEY, BRIAN - Western University Of Health Sciences; RAMIREZ, GUSTAVO - Western University Of Health Sciences; REYES, ARQUIMIDES - University Of Wisconsin; Harris, Caitlin; Buhr, Richard - Jeff

Submitted to: Frontiers in Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/3/2022
Publication Date: 10/19/2022
Citation: Weinroth, M.D., Oakley, B.B., Ramirez, G.A., Reyes, A., Harris, C.E., Buhr, R.J. 2022. 16S rRNA gene-based assessment of common broiler chicken sampling methods: evaluating intra-flock sample size, cecal pair similarity, and cloacal swab similarity to other alimentary tract locations. Frontiers in Physiology. https://doi.org/10.3389/fphys.2022.996654.
DOI: https://doi.org/10.3389/fphys.2022.996654

Interpretive Summary: 16S rRNA sequencing is a tool that is being used more widely for microbiome studies as costs fall and computational resources become more accessible. With increased use of this technology, 16S rRNA sequencing can help guide most appropriate sampling techniques. The objective of this study was to use 16S rRNA sequencing to examine common sampling practices in broiler chicken studies such as: the number of birds selected from a flock, the differences between cecal pairs within the same bird, and using cloacal swabs as a representation of other alimentary tract (AT) locations. Nine market age broilers were euthanized and immediately sampled in ten AT locations: (1) crop, (2) gizzard, (3) proventriculus, (4) duodenum, (5) jejunum, (6) ileum, (7-8) cecal samples from pouch, (9) colon, and (10) cloacal swab. DNA was extracted and subjected to 16S rRNA sequencing. Each location in the AT was had difference levels of bacteria present. Across locations in the AT, when 3 birds resulted in less than a 10% increase in new bacteria from the next sample and after the 8th bird less than 1%. Additionally, when cecal pairs from the same bird were evaluated, there was high similarity between pairs (95.3% of the bacteria captured by both cecal samples). Finally, when cloacal swabs were compared to other AT locations, the samples closer to the swab in the AT tract were more like the swab. These data provide new analysis on appropriate sample size selection within flocks as well as add to the consensus data regarding cecal pair similarity and destructive versus non-destructive sampling method agreement.

Technical Abstract: 16S rRNA sequencing for characterization of poultry associated microbiomes has been increased in use in poultry research as sequencing costs fall and computational resources become more accessible. As these tools are put into wider use, they can not only be used to answer specific research questions but also help determine the most appropriate sampling techniques. The objective of this study was to use 16S rRNA sequencing to examine common sampling practices in broiler chicken studies such as: the number of birds selected from a flock to capture microbiome diversity, the differences between cecal pairs within the same bird, and using cloacal swabs as a representation of other alimentary tract (AT) locations. Nine market age broilers were euthanized and immediately sampled in ten AT locations: (1) crop, (2) gizzard, (3) proventriculus, (4) duodenum, (5) jejunum, (6) ileum, (7-8) cecal samples from pouch, (9) colon, and (10) cloacal swab. DNA was extracted and subjected to 16S rRNA sequencing. Each location within the broiler AT was distinct and generally samples more aboral had a higher alpha diversity. When each sampling location was considered, it was found that at 2.8 birds sampled (range 2 to 4) subsequent additional of birds resulted in less than 10% new amplicon sequencing variants (ASV) being added while sampling after 7.6 birds (range 6 to 10) resulted to further samples increasing by less than 1% new ASVs. Additionally, when cecal pairs from the same bird were evaluated, there was high similarity between pairs. An average of 95.3% of the relative abundance of reads was captured by both cecal samples; although when unique ASV were compared this number fell to 56.8% present in both samples. Meaning, cecal pair mates are an adequate replication if interested in the total microbiome but may be less useful if a rare bacterium is of interest. Finally, when cloacal swabs were compared to other AT locations, the more aboral samples were the more similar they were to the swab (although all samples were distinct from one another). On the other hand, when the number of reads assigned to Enterobacteriaceae were specifically compared between cloacal swabs and other AT locations, only the colon samples were correlated (P = 0.035, R = 0.70). These findings indicate that while cloacal swabs can approximate overall changes in microbiome composition, they are not adequate for inferring changes to specific phyla—even those that are highly abundant within the microbial community. These data provide new analysis on appropriate sample size selection within flocks as well as add to the consensus data regarding cecal pair similarity and destructive versus non-destructive sampling method agreement.