Topical Application of Adult Cecal Contents to Eggs Transplants Spore-Forming Microbiota but Not Other Members of the Microbiota to Chicks

Abstract

The intestinal microbiota plays an essential role in the metabolism and immune competence of chickens from the first day after hatching. In modern production systems, chicks are isolated from adult chickens, instead hatching in a clean environment. As a result, chicks are colonized by environmental bacteria, including potential pathogens. There is a need to investigate methods by which chicks can be exposed to a more appropriate microbial community at hatching. Such methods must be easy to apply in a hatchery and produce consistent results. The development of the intestinal microbiota of chicks hatched from eggs sprayed with dilute adult cecal content during incubation was observed at 0, 3, 7, and 14 days posthatching (dph) across two experiments. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. A topical treatment of dilute adult cecal content was sufficient to transplant spore-forming bacteria such as Lachnospiraceae and Ruminococcaceae However, this treatment was not able to transplant other taxa that are considered to be core elements of the chicken cecal microbiota, such as Bacteroidaceae, Lactobacillaceae, Bifidobacteriaceae, and Burkholderiaceae The topical treatment significantly altered the microbiota of chicks immediately posthatching and accelerated the normal development of the microbiota with earlier colonization by Ruminococcaceae in the cecum and "Candidatus Arthromitus" in the ileum. The effect of the treatment on the cecal microbiota was maximal at 3 dph but diminished over time.IMPORTANCE Over the last 60 years poultry production has intensified in response to increased demand for meat. In modern systems, chicks hatch without contacting chickens and their gut bacteria. Consequently, they are colonized by environmental bacteria that may cause disease. The normal bacteria that live in the gut, or intestinal microbiota, play an important role in the development of the immune system. Therefore, it is essential to find easy ways to expose chicks to the more appropriate bacteria at hatching. This experiment investigated whether spraying eggs with adult cecal contents was sufficient to transfer an adult microbiota to chicks. Our findings show that spore-forming bacteria were transplanted, but other members of the microbiota were not. In this respect, the spray application was partially successful, but the timing of the spray needs to be modified to ensure that more bacteria are transferred.

Keywords: broiler; cecum; chicken; egg; microbiome; transplant.

FIG 1

Sampling regimes for the pilot (A) and repeat (B) experiments, including abbreviations for sample groups used when discussing the results.

FIG 2

Alpha diversity measured with a Shannon index at a sequencing depth of 5,000. Sample groups divided by experiment (P, pilot; R, repeat), treatment (C, control; T, treated), and age. Shannon diversity of transplant material (TRPL) is also shown. The alpha diversity of treated chicks was significantly higher than that of control chicks at 3 dph (both experiments) and 0 dph (repeat experiment). *, P < 0.05.

FIG 3

Principal coordinate analysis (PCoA) plot showing differences in unweighted (A) and weighted (B) UniFrac beta diversity between sample groups and treatment groups in pilot (triangle) and repeat experiments (circle) and transplant samples (square). Each point represents an individual sample, with the distance between points representative of differences in microbiota composition.

FIG 4

A dendrogram heatmap of ASV log abundance in the cecal microbiota of control and treated chicks at 3 and 7 dph in the pilot experiment. The dendrogram represents the organization of ASVs within the system of balances created by hierarchical clustering. Each node on the dendrogram is a balance, with the first node designated balance y0. Each terminal branch represents an ASV present within the analysis. The bar charts visualize which ASVs are denominator (dark red) and which are numerator (light red) ASVs for each balance. The heatmap shows the log abundance of each ASV in the samples organized by group. Low-abundance ASVs are represented by blue, while higher-abundance ASVs are represented by red.

FIG 5

A dendrogram heatmap showing the log abundance of ASVs in the cecal microbiota of control and treated chicks at 0, 3, 7, and 14 dph in the repeat experiment. The dendrogram represents the organization of ASVs within the system of balances created by hierarchical clustering. Each node on the dendrogram is a balance, with the first node designated balance y0. Each terminal branch represents an ASV present within the analysis. The bar charts visualize which ASVs are denominator (dark red) and which are numerator (light red) ASVs for each balance. The heatmap shows the log abundance of each ASV in the samples organized by group. Low-abundance ASVs are represented by blue, while higher-abundance ASVs are represented by red.

FIG 6

Relative abundance of Enterobacteriaceae (A), Clostridium (B), Clostridium cluster IV (C), and Clostridium cluster XIV (a and b) (D) in the ceca of treated and control chicks between 0 and 14 dph. Significant differences between treated and control chicks in the pilot (black lines) and repeat (blue lines) experiments are indicated.

FIG 7

Relative abundance of “Candidatus Arthromitus” (A), Enterobacteriaceae (B), and Clostridium (C) in the ilea of treated and control chicks between 0 and 14 dph. Significant differences between treated and control chicks in the pilot (black lines) and repeat (blue lines) experiments are indicated.