Effect of in feed administration of different butyrate formulations on Salmonella Enteritidis colonization and cecal microbiota in broilers.

Butyrate has been used extensively as a feed additive to improve gut health and to decrease Salmonella colonization in poultry. Salmonella mainly colonizes the ceca so butyrate concentrations should be increased in this gut segment. Discrepancies on the effects of butyrate on Salmonella colonization, described in the scientific literature, could thus be due to butyrate release location effects. In this study, newly developed butyrate formulations were evaluated for their effect on cecal butyrate concentrations and on colonization by Salmonella Enteritidis. In a first trial, broilers were randomly allocated to 7 dietary treatment groups with formulations based on different approaches to modify the butyrate release profile: release from wax matrices based on diffusion/erosion; micropellets supposedly release butyrate around pH 7 in the colon; tributyrin is based on the hydrolysis of esters in the small intestine. Fat-protected butyrate was included as a reference, because of its known effect on reduction of Salmonella colonization. Four days after infection, the number of cfu Salmonella per g cecal content and spleen were determined. Butyrate formulations in a wax matrix significantly reduced the Salmonella colonization in cecal content. In a second trial, wax and fat-protected butyrate treatments were replicated and results from the first trial were confirmed. Compared to the control group a higher proportion of butyrate concentration was observed in ceca for those groups with reduced Salmonella colonization. This was associated with a beneficial shift in the cecal microbiota. In conclusion, formulations that increase cecal butyrate concentrations are superior in protecting against Salmonella Enteritidis colonization.

Valid publication of the names Caecibacterium and Caecibacterium sporoformans.

Descriptions of the genus Caecibacterium and its proposed type species Caecibacterium sporoformans were published in the IJSEM by Onrust et al. (Int J Syst Evol Microbiol 2017;67:4589-4594). The type strain was deposited as LMG 27730 and DSM 26959. DSM 26959 is a patent strain, and therefore the names were effectively, but not validly, published based on Rule 30(4) of the International Code of Nomenclature of Prokaryotes. The type strain of C. sporoformans is now available from the Deutsche Sammlung von Mikroorganismen und Zellkulturen as DSM 103070 and no restrictions have been placed on its distribution. We here present new descriptions of the genus and its type species so that the names can be validly published.

Caecibacterium sporoformans gen. nov., sp. nov., an anaerobic, butyrate-producing, spore-forming bacterium isolated from chicken caecum.

Strains of a Gram-stain-negative, rod-shaped and immotile bacterium were isolated from broiler chicken caecal content. The isolates required strict anaerobic conditions for growth, formed spores, were catalase-positive and oxidase-negative. They produced butyrate as the major metabolic end product in reinforced clostridial medium broth. The genomic DNA G+C content of the isolated strains was 32.5-34.6 mol%. The major cellular fatty acids were C16 : 0 FAME, C14 : 0 FAME, C19 : 0CYC 9,10DMA and C16 : 0DMA. The fatty acid composition of the cell wall showed no similarity to any strain in the midi database. 16S rRNA gene sequence analysis showed that the nearest phylogenetic neighbours were Anaerostipes hadrus and Clostridium populeti (92 % sequence similarity) within Clostridium cluster XIVa of the phylum Firmicutes. Therefore, a novel genus is proposed, with the name Caecibacterium sporoformans gen. nov., sp. nov. The type strain of Caecibacterium sporoformans is LMG 27730T=DSM 26959T.

Microbial shifts associated with necrotic enteritis.

An outbreak of necrotic enteritis (NE) is a complex process requiring one or a number of predisposing factors rather than just the presence of pathogenic Clostridium perfringens. Examples are dietary influences, such as high levels of non-starch polysaccharides and fishmeal, and factors that evoke epithelial cell damage, such as Fusarium mycotoxins in feed and Eimeria infections. Recent studies have shown that different predisposing factors induce similar shifts in the intestinal microbiota composition. Butyrate-producing-strains of the Ruminococcaceae family are decreased in abundance by both fishmeal and Eimeria. Similarly, a decreased abundance of butyrate-producing-strains belonging to the Lachnospiraceae family has been induced by fishmeal. Also shifts are observed in the lactic acid-producing bacteria, such as decreased abundance of Lactobacillus johnsonii or Weissella confusa, when broilers were fed a fishmeal-based diet or a Fusarium mycotoxin contaminated diet. Finally, the abundance of Candidatus Savagella was decreased in broilers following Eimeria challenge or feeding a fumonisins contaminated diet. The nature of the microbiota shifts indicate that immune modulatory actions of the intestinal microbiota may play a critical role in the effect on the necrosis inducing activity of C. perfringens. Indeed, colonization with butyrate-producing bacteria plays a key role in counteracting inflammation in the gut and preserving intestinal integrity, while Candidatus Savagella is involved in stimulating Th17 and immunoglobulin A responses. Lactic acid bacteria stimulate colonization of lactate-utilizing and butyrate-producing Lachnospiraceae. Future research needs to clarify the role of the microbiota changes in the pathogenesis of NE.

Valeric acid glyceride esters in feed promote broiler performance and reduce the incidence of necrotic enteritis.

Valeric acid is a C5 fatty acid, naturally produced in low concentrations by specific members of the microbiota of the lower intestinal tract. Effects of valeric acid on intestinal health have been poorly investigated. Valeric acid derivatives can be produced as glyceride esters and added to broiler feed. In the current study, experiments were carried out to evaluate the effect of valeric acid glycerides (GVA) on growth performance, on the morphology of the small intestinal mucosa and on protection against necrotic enteritis. In a first feeding trial, Ross-308 chicks were randomly divided into 2 dietary treatment groups and fed either a non-supplemented diet or a diet supplemented with GVA (1.5 g/kg). In the GVA supplemented group, the feed conversion ratio was significantly decreased during the entire trial period (D1-37). In a second trial, gut wall morphology was evaluated. In broilers fed a GVA-containing diet at 5 g/kg, the villus height/crypt depth ratio in the jejunum was significantly increased (P ≤ 0.05), and the crypt depth was significantly decreased at 28 d. In a third trial, immunohistochemistry showed that the density of glucagon-like peptide-2 immunoreactive cells in jejunal and ileal villi from broilers supplemented with GVA (5 g/kg) was significantly increased (P ≤ 0.05) on d 10. In a necrotic enteritis challenge model, a significant reduction of the number of birds with necrotic lesions was found at d 21, using in-feed supplementation of low and high regimen of GVA. These data show that GVA supplementation to broiler feed can decrease the feed conversion, positively affect the morphology of the small intestinal mucosa, increase the density of glucagon-like peptide-2 producing enteroendocrine cells, and reduce the incidence of necrotic enteritis, making GVA a valuable candidate feed additive for broilers.

Steering Endogenous Butyrate Production in the Intestinal Tract of Broilers as a Tool to Improve Gut Health.

The ban on antimicrobial growth promoters and efforts to reduce therapeutic antibiotic usage has led to major problems of gastrointestinal dysbiosis in livestock production in Europe. Control of dysbiosis without the use of antibiotics requires a thorough understanding of the interaction between the microbiota and the host mucosa. The gut microbiota of the healthy chicken is highly diverse, producing various metabolic end products, including gases and fermentation acids. The distal gut knows an abundance of bacteria from within the Firmicutes Clostridium clusters IV and XIVa that produce butyric acid, which is one of the metabolites that are sensed by the host as a signal. The host responds by strengthening the epithelial barrier, reducing inflammation, and increasing the production of mucins and antimicrobial peptides. Stimulating the colonization and growth of butyrate-producing bacteria thus may help optimizing gut health. Various strategies are available to stimulate butyrate production in the distal gut. These include delivery of prebiotic substrates that are broken down by bacteria into smaller molecules which are then used by butyrate producers, a concept called cross-feeding. Xylo-oligosaccharides (XOS) are such compounds as they can be converted to lactate, which is further metabolized to butyrate. Probiotic lactic acid producers can be supplied to support the cross-feeding reactions. Direct feeding of butyrate-producing Clostridium cluster IV and XIVa strains are a future tool provided that large scale production of strictly anaerobic bacteria can be optimized. Current results of strategies that promote butyrate production in the gut are promising. Nevertheless, our current understanding of the intestinal ecosystem is still insufficient, and further research efforts are needed to fully exploit the capacity of these strategies.