Characterization of intestinal microbiota and response to dietary virginiamycin supplementation in the broiler chicken

Abstract

The inclusion of antibiotic growth promoters, such as virginiamycin, at subtherapeutic levels in poultry feeds has a positive effect on health and growth characteristics, possibly due to beneficial effects on the host gastrointestinal microbiota. To improve our understanding of the chicken gastrointestinal microbiota and the effect of virginiamycin on its composition, we characterized the bacteria found in five different gastrointestinal tract locations (duodenal loop, mid-jejunum, proximal ileum, ileocecal junction, and cecum) in 47-day-old chickens that were fed diets excluding or including virginiamycin throughout the production cycle. Ten libraries (five gastrointestinal tract locations from two groups of birds) of approximately 555-bp chaperonin 60 PCR products were prepared, and 10,932 cloned sequences were analyzed. A total of 370 distinct cpn60 sequences were identified, which ranged in frequency of recovery from 1 to 2,872. The small intestinal libraries were dominated by sequences from the Lactobacillales (90% of sequences), while the cecum libraries were more diverse and included members of the Clostridiales (68%), Lactobacillales (25%), and Bacteroidetes (6%). To assess the effects of virginiamycin on the gastrointestinal microbiota, 15 bacterial targets were enumerated using quantitative, real-time PCR. Virginiamycin was associated with increased abundance of many of the targets in the proximal gastrointestinal tract (duodenal loop to proximal ileum), with fewer targets affected in the distal regions (ileocecal junction and cecum). These findings provide improved profiling of the composition of the chicken intestinal microbiota and indicate that microbial responses to virginiamycin are most significant in the proximal small intestine.

FIG. 1.

Taxonomic group assignments and clone frequencies of cpn60 sequences isolated from five gastrointestinal tract locations in control birds (no virginiamycin).

FIG.2.

Abundance of 15 bacterial targets in each gastrointestinal tract location in the presence and absence of virginiamycin as measured by qPCR. The dotted lines indicate a 10-fold increase (upper line), a 10-fold decrease (lower line), or no change (middle line). For all graphs, the data points are labeled as follows (see Table S1 in the supplemental material for library group identifications): 1, library group D; 2, L. crispatus; 3, L. gasseri; 4, library group V; 5, library group X; 6, library group W; 7, E. faecalis; 8, E. coli; 9, library group M; 10, library group E; 11, library group N; 12, library group K; 13, library group L; 14, S. lentus; and 15, L. johnsonii. Note that all primer sets were assayed in all gastrointestinal tract locations, and the absence of a target on a graph indicates that it was not detected in one or both treatment groups. (A) Duodenal loop. (B) Mid-jejunum. (C) Proximal ileum. (D) Ileocecal junction. (E) Cecum.

FIG. 3.

Abundance of each of 15 bacterial targets in the presence and absence of dietary virginiamycin grouped according to gastrointestinal tract location. Each gastrointestinal tract location is plotted separately: duodenal loop (□), mid-jejunum (▵), proximal ileum (○), ileocecal junction (♦), and cecum (▪). Note that the proximal locations are represented by open symbols while the distal locations are represented by closed symbols.