Galacto-oligosaccharides modulate the juvenile gut microbiome and innate immunity to improve broiler chicken performance

Abstract

Improvements in growth performance and health are key goals in broiler chicken production. Inclusion of prebiotic galacto-oligosaccharides in broiler feed enhanced the growth rate and feed conversion of chickens relative to a calorie-matched control diet. Comparison of the cecal microbiota identified key differences in abundance of Lactobacillus spp. Increased levels of L. johnsonii in GOS-fed juvenile birds at the expense of L. crispatus was linked to improved performance (growth rate and market weight). Investigation of the innate immune responses highlighted increases of ileal and cecal IL-17A gene expression counterposed to a decrease in IL-10 and IL-17F. Quantification of the autochthonous Lactobacillus ssp. revealed a correlation between bird performance and L. johnsonii abundance. Shifts in the cecal populations of key Lactobacillus spp. of juvenile birds primed intestinal innate immunity without harmful pathogen challenge. IMPORTANCEImprovements in the growth rate of broiler chickens can be achieved through dietary manipulation of the naturally occurring bacterial populations whilst mitigating the withdrawal of antibiotic growth promoters. Prebiotic galacto-oligosaccharides (GOS) are manufactured as a by-product of dairy cheese production, which can be incorporated in the diets of juvenile chickens to improve their health and performance. This study investigates the key mechanisms behind this progression and pin points L. johnsonii as a key species that facilitates the enhancements in growth rate and gut health. It also relates the role of the innate immune system in the response to the GOS diet.in the PCR reaction and converted into genome copy number per gram of cecal content based on the mass of cecal material and elution volume applied for DNA extraction.Data and Statistical Analysis. For the microbiota -diversity analysis Bray Curtis distances were tested for significance using AMOVA implemented within Mothur (79). Linear discriminant analysis effect size (LEfSe) was used to identify differentially abundant OTUs (82) and implemented within Mothur (79). Data processing and ordination were performed using R (R Development Core Team, 2013. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0; URL http://www.R-project.org ; 83). With the exception of Figure S2 all figures were drawn using R version 3.5.3 (2019-03-11) in Rstudio 1.1 (84,85). R scripts used to draw figures are available at: https://github.com/PJRichards/Richards_GOS_broiler. Significance tests of heterophil counts, villus and crypt measurements were performed using single-factor ANOVA with p < 0.05 used as the level significance. Non-normally distributed gene expression data were compared using the non-parametric Kruskal-Wallis test. Data Availability. The genome DNA sequences of L. crispatus DC21.1 appear in the NCBI database under the accession numbers CP039266-CP039267.The genome DNA sequences L. johnsonii DC22.2 appear in the NCBI database under the access...

Prebiotic Driven Increases in IL-17A Do Not Prevent Campylobacter jejuni Colonization of Chickens

“…Consistent with previous reports we observed GOS-driven changes in the cecal microbiota of chickens featuring specific operational taxonomic units identifiable as lactobacilli (Hughes et al, 2017;Azcarate-Peril et al, 2018). Colonization by C. jejuni did not prevent the differential increase in abundance of Otu0017 (L. johnsoniii) associated previously with dietary GOS (Richards et al, 2019b). At 2dpi for the 6-dc birds Otu0002 (L. crispatus) and Otu0017 (L. johnsoniii) showed increases in abundance in association with the GOS-diet.…”

“…OTU0001 did not discriminate the microbiota of the Campylobacter-challenged birds on the control diet (Campylobacter treatment) from the microbiota of mockchallenged birds on the GOS diet (Supplementary Figure S2C). In addition, previous analysis of the mock-challenged controls by our laboratory indicated that Lactobacillus johnsonii outcompetes L. crispatus in GOS-fed birds (Richards et al, 2019b). In the analysis presented here OTU0002 has strong sequence similarity with the L. crispatus strain (97.21%) and OTU0017 has a strong sequence similarity with L. johnsoniii (98.03%).…”

“…Here, we demonstrate dietary GOS inclusion can maintain transcript levels of IL-17A and suppress IL-17F post colonization with C. jejuni. Previously we have shown C. jejuni challenge at 6 days triggered a transient increases in IL-17A and IL-17F in broiler chickens at 15 days (9 dpi) compared with non-colonized birds (Connerton et al, 2018), and more recently that dietary GOS increases the expression of IL-17A in juvenile birds up to 15 days (Richards et al, 2019b). In mice IL-17A expression has been proposed to benefit intestinal barrier function and IL-17F to weaken intestinal integrity, since IL-17A inhibition exacerbates induced colitis (Maxwell et al, 2015) and IL-17F suppression is protective (Tang et al, 2018).…”

“…Under increasing economic pressure, the poultry industry is pursuing effective alternative methods to promote bird growth whilst controlling farm sources of antimicrobial resistance and zoonotic disease. Prebiotic feed additives such as GOS, a byproduct of the dairy industry, have revealed potential growthpromoting effects in piglets (Alizadeh et al, 2016) and chickens (Richards et al, 2019b), whilst fructo-ologosaccharide (FOS) have been reported to reduce Salmonella colonization of chicks (Fukata et al, 1999). Here, we assess the impact of a GOS prebiotic on chickens colonized by C. jejuni at either 6 or 20 days with respect to their zootechnical performance, gut architecture and differences in intestinal cytokine and chemokine transcription (the experimental designs and corresponding data are summarized in Figure 4).…”

“…We have recently reported that the inclusion of the prebiotic GOS in juvenile broiler feed enhances the growth and feed conversion rates of broiler chickens, increases ileal and cecal IL-17A gene expression and brings about changes in the cecal populations of key Lactobacillus spp. (Richards et al, 2019b). GOS represent host-indigestible carbohydrates that have been identified as promoting beneficial bacteria in humans and animals, which include Bifidobacteria, Bacteroides, and Lactobacillaceae (Jung et al, 2008;Hughes et al, 2017;Van Bueren et al, 2017;Tian et al, 2019).…”