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Cytochrome P450 is involved in drug metabolism. Subfamily CYP3 A shows a degree of similarity across different animal species. However, little information is available about its expression and activity in broiler chickens. A RT- PCR method was developed for the quantification of CYP3 A37 expression in the liver and small intestine of broilers. A higher expression in the jejunum was observed compared with that in the ileum. In the liver, a significantly lower expression compared with that in the jejunum was noticed. Thus, the role of the small bowel in drug metabolism cannot be neglected in broilers. CYP3 A activity was studied in vitro using midazolam as a substrate. Two protocols for the preparation of intestinal microsomes were compared. Mincing of the tissues before ultracentrifugation seemed to be more appropriate than a protocol based on ethylenediaminetetra-acetic acid separation. CYP3 A activity revealed to be the highest in the duodenum with a decreasing trend towards the ileum. Activity in liver was comparable to duodenal activity. [ABSTRACT FROM AUTHOR]

Broilers often suffer from subclinical intestinal health problems of ill-defined etiology, which have a negative impact on performance. Macroscopic and microscopic evaluations can be used to monitor intestinal health, but because these are subjective and time-consuming, respectively, objective and easy-to-measure biomarkers are urgently needed. Fecal biomarkers can potentially be used as noninvasive, objective measures to evaluate gut health in broilers. The aim of the current study was to evaluate ovotransferrin (OVT) as a biomarker in fecal/colonic samples derived from broilers from 27 industrial farms by investigating associations between OVT, broiler performance and gut histology parameters. Eight chickens per farm were randomly selected, weighed and euthanized on d 28 of the production round. A duodenal section was collected to measure the intestinal villus structure (villus length, crypt depth) and the inflammatory status of the gut (CD3 + T-lymphocytes area percentage). The coefficient of variation for the OVT (between farms; 83.45%, within farms; 95.13%) was high compared to the villus length (between farms; 10.91%, within farms; 15.48%), crypt depth (between farms; 15.91%, within farms; 14.10%), villus-to-crypt ratio (between farms; 22.08%, within farms; 20.53%), and CD3 + (between farms; 36.38%, within farms; 26.13%). At farm level, colonic OVT was significantly associated with the average slaughter weight (P = 0.005), daily weight gain (P = 0.007) and the European production index (EPI) (P = 0.009). At broiler level, significant associations were found between colonic OVT and the villus length (P = 0.044) and between the colonic OVT and villus-to-crypt ratio (P = 0.050). These results thus show that quantifying OVT in colon can have merit for evaluation of intestinal health in broilers under field conditions., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Galactomannans are abundant nonstarch polysaccharides in broiler feed ingredients. In broilers, diets with high levels of galactomannans have been associated with innate immune response stimulation, poor zootechnical performance, nutrient and lipid absorption, and excessive digesta viscosity. However, data about its effects on the gut microbiome are scarce. β-Mannanases are enzymes that can hydrolyze β-mannans, resulting in better nutrient utilization. In the current study, we have evaluated the effect of guar gum, a source of galactomannans, supplemented to broiler diets, either with or without β-mannanase supplementation, on the microbiota composition, in an attempt to describe the potential role of the intestinal microbiota in β-mannanase-induced gut health and performance improvements. One-day-old broiler chickens (n = 756) were randomly divided into 3 treatments: control diet, guar gum-supplemented diet (1.7%), or guar gum-supplemented diet + β-mannanase (Hemicell 330 g/ton). The zootechnical performance, gut morphometry, ileal and cecal microbiome, and short-chain fatty acid concentrations were evaluated at different time points. The guar gum supplementation decreased the zootechnical performance, and the β-mannanase supplementation restored performance to control levels. The mannan-rich diet-induced dysbiosis, with marked effects on the cecal microbiota composition. The guar gum-supplemented diet increased the cecal abundance of the genera Lactobacillus, Roseburia, Clostridium sensu stricto 1, and Escherichia-Shigella, and decreased Intestinimonas, Alistipes, Butyricicoccus, and Faecalibacterium. In general, dietary β-mannanase supplementation restored the main microbial shifts induced by guar gum to levels of the control group. In addition, the β-mannanase supplementation reduced cecal isobutyric, isovaleric, valeric acid, and branched-chain fatty acid concentrations as compared to the guar gum-supplemented diet group, suggesting improved protein digestion and reduced cecal protein fermentation. In conclusion, a galactomannan-rich diet impairs zootechnical performance in broilers and results in a diet-induced dysbiosis. β-Mannanase supplementation restored the gut microbiota composition and zootechnical performance to control levels., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Maintaining optimal gut health is a key driver for a well-performing broiler flock. Histology of intestinal sections and quantification of villus structure can be used to evaluate gut health. While these measurements have been used in experimental models to evaluate gut health, less is known about the associations of these parameters with performance in commercial broiler farms. The objective of the present study was to evaluate possible associations of intestinal villus structure and the inflammatory condition of the gut with Ross 308 broiler performance in 50 commercial farms. On day 28 of the production round, 20 randomly selected broilers per farm were weighed, euthanized, and a duodenal section was collected to determine villus length, crypt depth and the CD3 + T-lymphocytes area percentage (CD3 + %). We found a relatively low coefficient of variance (CV) for the villus length (between farms; 9.67%, within farms; 15.97%), while the CD3 + (%) had a high CV (between farms; 29.78%, within farms; 25.55%). At flock level, the CD3 + (%) was significantly correlated with the villus length ( r  = -0.334), crypt depth ( r  = 0.523) and the villus-to-crypt ratio ( r  = -0.480). The crypt depth was significantly correlated with the European production index (EPI) ( r  = -0.450) and feed conversion ratio (FCR) ( r  = 0.389). At broiler level, a significant association was found between the individual body weight (day 28), CD3 + (%) and villus-to-crypt ratio. These data thus show that gut villus structure is significantly associated with bird performance under commercial conditions. RESEARCH HIGHLIGHTSGut histology parameters vary between and within farms.Broiler performance is associated with gut morphology.

Intestinal integrity losses have been identified as a main driver for poor performance in broilers. The oral administration of markers such as iohexol is a major asset for measuring intestinal permeability (IP) alterations. The aim of the current study was to evaluate oral iohexol administration and serum levels as a quantitative measure for IP in Ross 308 broilers and to identify possible associations with histologic measurements. A total of 40, day-old broiler chickens were randomly divided into 4 groups of 10 broilers and a coccidiosis model was used to induce IP. Three challenge groups received a mixture of different field strains and concentrations of Eimeria acervulina and Eimeria maxima at d 16, and 1 group operated as an uninfected control group. On d 20, 5 birds per group were orally administered the permeability marker iohexol at a dose of 64.7 mg/kg body weight and blood was taken 60 min after the oral gavage. On d 21 these 5 birds per group were euthanized. On d 21, 5 other birds per group were given iohexol where after blood was taken. These birds were euthanized on d 22. During necropsy, birds were scored for coccidiosis lesions and a duodenal segment was taken for histology. The Eimeria challenge had a significant impact on the villus length, crypt depth, villus-to-crypt ratio and CD3 + T-lymphocytes area percentage. Challenged birds had a significant higher concentration of serum iohexol on both sampling days, as compared to the uninfected controls. A significant correlation could be found between the serum iohexol concentration and the histologic parameters (villus length, crypt depth and villus-to-crypt ratio) on the first sampling day. This suggests that iohexol may be used as a gut permeability marker in broilers under Eimeria challenge., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Intestinal health is critically important for the digestion and absorption of nutrients and thus is a key factor in determining performance. Intestinal health issues are very common in high performing poultry lines due to the high feed intake, which puts pressure on the physiology of the digestive system. Excess nutrients which are not digested and absorbed in the small intestine may trigger dysbiosis, i.e. a shift in the microbiota composition in the intestinal tract. Dysbiosis as well as other stressors elicit an inflammatory response and loss of integrity of the tight junctions between the epithelial cells, leading to gut leakage. In this paper, key factors determining intestinal health and the most important nutritional tools which are available to support intestinal health are reviewed., Competing Interests: We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper., (© 2023 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd.)

Bacterial chondronecrosis with osteomyelitis (BCO) is a common cause of broiler lameness. Bacteria that are found in BCO lesions are intestinal bacteria that are proposed to have translocated through the intestinal epithelium and have spread systemically. One of the specific bacterial species frequently isolated in BCO cases is Enterococcus cecorum. In the current study, caecal isolates were obtained from birds derived from healthy flocks (12 isolates from 6 flocks), while isolates derived from caeca, colon, pericardium, caudal thoracic vertebrae, coxo-femoral joint, knee joint and intertarsal joint (hock) were obtained from broilers derived from BCO outbreaks (111 isolates from 10 flocks). Pulsed field gel electrophoresis was performed to determine similarity. Clonal E. cecorum populations were isolated from different bones/joints and pericardium from animals within the same flock, with intestinal strains carrying the same pulsotype, pointing to the intestinal origin of the systemically present bacteria. Isolates from the intestinal tract of birds from healthy flocks clustered away from the BCO strains. Isolates from the gut, bones/joints and pericardium of affected animals contained a set of genes that were absent in isolates from the gut of healthy animals, such as genes encoding for enterococcal polysaccharide antigens (epa genes), cell wall structural components and nutrient transporters. Isolates derived from the affected birds induced a significant higher mortality in the embryo mortality model as compared to the isolates from the gut of healthy birds, pointing to an increased virulence., (© 2023. The Author(s).)

Muramidases constitute a superfamily of enzymes that hydrolyse peptidoglycan (PGN) from bacterial cell walls. Recently, a fungal muramidase derived from Acremonium alcalophilum has been shown to increase broiler performance when added as a feed additive. However, the underlying mechanisms of action are not yet identified. Here, we investigated the hypothesis that this muramidase can cleave PGN to muramyl dipeptide (MDP), activating nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) receptors in eukaryotic cells, potentially inducing anti-inflammatory host responses. Using Micrococcus luteus as a test bacterium, it was shown that muramidase from A. alcalophilum did not display antimicrobial activity, while it could cleave fluorescently labelled PGN. It was shown that the muramidase could degrade PGN down to its minimal bioactive structure MDP by using UPLC-MS/MS. Using HEK-Blue™-hNOD2 reporter cells, it was shown that the muramidase-treated PGN degradation mixture could activate NOD2. Muramidase supplementation to broiler feed increased the duodenal goblet cell and intraepithelial lymphocyte abundance while reducing duodenal wall CD3+ T lymphocyte levels. Muramidase supplementation to broiler feed only had moderate effects on the duodenal, ileal and caecal microbiome. It was shown that the newly discovered muramidase hydrolysed PGN, resulting in MDP that activates NOD2, potentially steering the host response for improved intestinal health.

Objective: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action., Design: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis., Results: Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum , Holdemanella and Parabacteroides . In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma., Conclusion: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

The grains that form the basis of most commercial chicken diets are rich in cellulose, an unbranched β-1,4-linked D-glucopyranose polymer, used as a structural molecule in plants. Although it is a predominant polysaccharide in cereal hulls, it is considered an inert non-fermentable fiber. The aim of the current study was to analyze the effect of in-feed supplementation of cellulose on the gut microbiota composition of broilers. Administration of cellulose to chickens, on top of a wheat-based diet, changed the caecal microbiota composition, as determined using pyrosequencing of the 16S rRNA gene. At day 26, a significantly (P < 0.01) higher relative abundance of the Alistipes genus was observed in the caeca of broilers fed the cellulose-supplemented diet, compared to animals fed the control diet. An in vitro batch fermentation assay showed a significant (P < 0.01) growth stimulation of Alistipes finegoldii in the presence of cellulose. In conclusion, in-feed supplementation of cellulose alters the microbiota composition at the level of the phylum Bacteroidetes, specifically the Alistipes genus. This suggests that cellulose is not essentially inert but can alter the gut micro-environment., (© 2019 Poultry Science Association Inc.)

Intestinal health problems are a major issue in the poultry industry. Quantifiable easy-to-measure biomarkers for intestinal health would be of great value to monitor subclinical intestinal entities that cause performance problems and to evaluate control methods for intestinal health. The aim of the study was to identify host protein biomarkers for intestinal inflammation and intestinal barrier damage. Proteomic analysis was conducted on ileal and colonic content samples of broilers under an experimental gut damage and inflammation model. Effects of the challenge treatment resulted in a worse gut condition based on macroscopic gut appearance (p < 0.0001). Also microscopic changes such as shortening of the villi and increased crypt depth (p < 0.0001) as well as higher infiltration of T-lymphocytes (p < 0.0001) were seen in the duodenal tissue of challenged animals. Several candidate proteins associated with inflammation, serum leakage and/or tissue damage were identified with an increased abundance in intestinal content of challenged animals (p < 0.05). Conversely, brush border enzymes were less abundant in intestinal content of challenged animals (p < 0.05). These candidate biomarkers have potential to be used in the field for detection of gut barrier failure in broilers.

The impact of cortisol on Flavobacterium columnare biofilm formation was explored. Firstly, the dynamics of biofilm formation by one highly (HV) and one low virulent (LV) F. columnare isolate with and without the stress hormone cortisol under microfluidic flow conditions was characterized. This to confirm that F. columnare cells could form biofilm under cortisol supplementation, and to compare the temporal and structural differences between different treatment groups. One trial revealed that in both isolates cell aggregates resembling biofilms occurred within 7-h post-inoculation. Consequently, cell clusters were sloughed away, followed by a rebuilding of bacterial cell aggregates, suggestive for a high spreading capacity. While the HV isolate revealed cell aggregates formed upstream at all time-points, for the LV isolate this was only seen upon cortisol supplementation. Secondly, the transcriptional effect of genes (gldK, gldL, gldM, gldN, sprA, sprE, sprT, and porV) belonging to the Type IX secretion system involved in gliding motility was investigated in planktonic and biofilm cells of a HV and LV isolate to which no, a low (LD) or high (HD) dose of cortisol was added. Significantly lower expression of gliding genes gldK, gldL, gldM and gldN, and of protein secretion regulator porV was seen in the LV isolate planktonic cells supplemented with a HD-cortisol. The LV isolate biofilm cells treated with the HD-cortisol showed a significant upregulation of sprT, encoding mobile surface adhesion important in bacterial colonization. This is the first evidence for the co-regulatory effect of cortisol on biofilm formation and F. columnare gliding gene expression.

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.

Advances in gut microbiota research have triggered interest in developing colon butyrate producers as niche-specific next-generation probiotics, targeted at increasing colon butyrate production and countering disease-associated microbiota alterations. Crucial steps in the development of next-generation probiotics are the design of formulations with a reasonable shelf life as well as the safety demonstration of an intervention in healthy volunteers. One such potential next-generation butyrate-producing probiotic is Butyricicoccus pullicaecorum 25-3 T , with demonstrated safety in in vitro as well as animal models. Here, we examined the strain's safety, tolerability, and impact on microbiota composition and metabolic activity in healthy volunteers in a randomized, double-blind, placebo-controlled crossover study in 30 healthy volunteers. The study design consisted of two 4-week intervention periods (10 8 CFU B. pullicaecorum [treatment] or maltodextrin [placebo] per day) with a 3-week washout in between. We assessed adverse events, blood parameters (primary endpoints), and fecal microbiota composition and metabolite profiles (secondary endpoints). The number of reported adverse events during the B. pullicaecorum treatment was similar to that of placebo intervention, as were observed changes in blood chemistry parameters, bowel habits, and fecal calprotectin concentrations. Administration of the strain did not induce any disruptive effect in microbiota composition or metabolic activity. In this first human intervention trial with a butyrate-producing Clostridium cluster IV isolate, we demonstrated B. pullicaecorum 25-3 T administration to be both safe and well tolerated by healthy participants. This safety study paves the way for the further development of the strain as a next-generation probiotic. IMPORTANCE This study is the first to determine the safety and tolerance in humans of a butyrate-producing Clostridium cluster IV next-generation probiotic. Advances in gut microbiota research have triggered interest in developing colon butyrate producers as next-generation probiotics. Butyricicoccus pullicaecorum 25-3 T is one such potential probiotic, with demonstrated safety in vitro as well as in animal models. Here, we produced an encapsulated B. pullicaecorum formulation that largely preserved its viability over an 8-month storage period at 4°C. Administration of this formulation to healthy volunteers allowed us to establish the intervention as safe and well tolerated. The probiotic intervention did not cause disruptive alterations in the composition or metabolic activity of health-associated microbiota. The results presented pave the way for the exploration of the impact of the strain on microbiota alterations in a clinical setting.

Intestinal health is determined by host (immunity, mucosal barrier), nutritional, microbial and environmental factors. Deficiencies in intestinal health are associated with shifts in the composition of the intestinal microbiome (dysbiosis), leakage of the mucosal barrier and/or inflammation. Since the ban on growth promoting antimicrobials in animal feed, these dysbiosis-related problems have become a major issue, especially in intensive animal farming. The economical and animal welfare consequences are considerable. Consequently, there is a need for continuous monitoring of the intestinal health status, particularly in intensively reared animals, where the intestinal function is often pushed to the limit. In the current review, the recent advances in the field of intestinal health biomarkers, both in human and veterinary medicine are discussed, trying to identify present and future markers of intestinal health in poultry. The most promising new biomarkers will be stable molecules ending up in the feces and litter that can be quantified, preferably using rapid and simple pen-side tests. It is unlikely, however, that a single biomarker will be sufficient to follow up all aspects of intestinal health. Combinations of multiple biomarkers and/or metabarcoding, metagenomic, metatranscriptomic, metaproteomic and metabolomic approaches will be the way to go in the future. Candidate biomarkers currently are being investigated by many research groups, but the validation will be a major challenge, due to the complexity of intestinal health in the field.

Salmonella is an important zoonotic agent, and poultry products remain one of the main sources of infection for humans. Salmonella Infantis is an emerging serotype in poultry worldwide, reflected by an increased prevalence in poultry flocks, on broiler meat and in human foodborne illness cases. In the current study, the efficacy of oral administration of a live monovalent Salmonella Enteritidis and a live bivalent Salmonella Enteritidis/Typhimurium vaccine, against a Salmonella Enteritidis and Infantis infection, was determined. Oral administration of the live vaccines to day-old chickens caused a decrease in caecal colonization by Salmonella Enteritidis, but not Infantis, at day 7, when challenged at day 2. Vaccination with the bivalent vaccine at day 1 resulted in a decreased spleen colonization by both Salmonella Infantis and Enteritidis. Twice (at day 1 and week 6) and thrice vaccination (at day 1, week 6 and 16) of laying hens with the bivalent vaccine resulted in a decreased caecal colonization by Salmonella Enteritidis and Infantis, and significantly lower oviduct colonization levels by Salmonella Enteritidis. These data show cross-protection against Salmonella Infantis by oral administration of live vaccine strains belonging to other serogroups., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: High protein diets shift the faecal microbiota into a more unfavourable composition in obese humans. In lean dogs, higher protein consumption is accompanied with increased production of putrefactive fermentation products, whereas obese dogs have a different gut microbiota compared to lean dogs. Still, the impact of high dietary protein on gut microbiota in obese dogs remains unclear. The aim of this study was to investigate faecal microbial changes in lean and obese dogs in response to two different levels of dietary protein. Six healthy lean and six obese Beagles were fed a high protein diet (HP) and a low protein diet (LP) for 28 days each in a crossover design. Denaturing gradient gel electrophoresis and quantitative PCR were performed on faecal samples for microbial profiling. Plasma acylcarnitine and fermentation metabolites were measured., Results: Dogs fed HP had higher concentrations of protein fermentation metabolites including faecal ammonia, isovalerate, isobutyrate, phenol, indole, serum indoxyl sulphate and plasma 3-OH isovalerylcarnitine compared to dogs fed LP, whereas no changes in faecal concentrations of acetate and butyrate were observed. The abundances of clostridial clusters IV and XIVa, covering the majority of butyrate-producing bacteria, and of the butyrate kinase gene, one of the terminal genes of the butyrate synthesis pathway were higher in dogs on HP compared to LP. Significant interactions between diet and body condition were found for the abundance of Firmicutes, Lactobacillus and clostridial cluster I. The similarity coefficient of faecal microbiota between the two diets was smaller in obese dogs than in lean dogs., Conclusions: High protein diet increased the abundance and activity of butyrate-producing bacteria in Beagles independent of the body condition. In addition, increasing dietary protein content had a greater overall impact on faecal microbiota in obese compared to lean dogs.

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 27730 T =DSM 26959 T .

More sustainable broiler meat production can be facilitated by the increased use of cheap by-products and local crops as feed ingredients, while not affecting animal performance and intestinal health, or even improving intestinal health, so that antibiotic usage is further reduced. To achieve this, knowledge of the relationship between the taxonomic and functional microbiota composition and intestinal health is required. In addition, the relationship between the novel feed sources, the substrates present in these feed sources, and the breakdown by enzymes and microbial networks can be crucial, because this can form the basis for development of tailored feed-type specific solutions for optimal digestion and animal performance., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Background and Aims: Butyricicoccus is a butyrate-producing clostridial cluster IV genus whose numbers are reduced in the stool of ulcerative colitis [UC] patients. Conditioned medium of Butyricicoccus [B.] pullicaecorum prevents tumour necrosis factor alpha [TNFα]-induced increase in epithelial permeability in vitro. Since butyrate influences intestinal barrier integrity, we further investigated the relationship between the abundance of mucosa-associated Butyricicoccus and the expression of butyrate-regulated tight junction [TJ] genes., Methods: Tight junction protein 1 [TJP1], occludin [OCLN], claudin-1 [CLDN1], and Butyricicoccus 16S rRNA expression was analysed in a collection of colonic biopsies of healthy controls and UC patients with active disease. The effect of butyrate and B. pullicaecorum conditioned medium on TJ gene expression was investigated in TNFα-stimulated Caco-2 monolayers and inflamed mucosal biopsies of UC patients., Results: TJP1 expression was significantly decreased in inflamed UC mucosa, whereas CLDN1 mRNA levels were increased. OCLN did not differ significantly between the groups. Mucosa-associated Butyricicoccus 16S rRNA transcripts were reduced in active UC patients compared with healthy controls. Interestingly, Butyricicoccus activity negatively correlated with CLDN1 expression. Butyrate reversed the inflammation-induced increase of CLDN1 protein levels, and stimulation of inflamed UC biopsies with B. pullicaecorum conditioned medium normalized CLDN1 mRNA levels., Conclusions: Butyricicoccus is a mucosa-associated bacterial genus under-represented in colonic mucosa of patients with active UC, whose activity inversely correlates with CLDN1 expression. Butyrate and B. pullicaecorum conditioned medium reduce CLDN1 expression, supporting its use as a pharmabiotic preserving epithelial TJ integrity., (Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: Pouchitis is the most common complication after colectomy with ileal pouch-anal anastomosis (IPAA) for UC and the risk is the highest within the 1st year after surgery. The pathogenesis is not completely understood but clinical response to antibiotics suggests a role for gut microbiota. We hypothesised that the risk for pouchitis can be predicted based on the faecal microbial composition before colectomy., Design: Faecal samples from 21 patients with UC undergoing IPAA were prospectively collected before colectomy and at predefined clinical visits at 1 month, 3 months, 6 months and 12 months after IPAA. The predominant microbiota was analysed using community profiling with denaturing gradient gel electrophoresis followed by quantitative real-time PCR validation., Results: Cluster analysis before colectomy distinguished patients with pouchitis from those with normal pouch during the 1st year of follow-up. In patients developing pouchitis, an increase of Ruminococcus gnavus (p<0.001), Bacteroides vulgatus (p=0.043), Clostridium perfringens (p=0.011) and a reduction of two Lachnospiraceae genera (Blautia (p=0.04), Roseburia (p=0.008)) was observed. A score combining these five bacterial risk factors was calculated and presence of at least two risk factors showed a sensitivity and specificity of 100% and 63.6%, respectively., Conclusions: Presence of R. gnavus, B. vulgatus and C. perfringens and absence of Blautia and Roseburia in faecal samples of patients with UC before surgery is associated with a higher risk of pouchitis after IPAA. Our findings suggest new predictive and therapeutic strategies in patients undergoing colectomy with IPAA., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Probiotics which do not result in the development and spread of microbial resistance are among the candidate replacements for antibiotics previously used as growth promotors. In this study the effect of in-feed supplementation of the butyrate producing Butyricicoccus pullicaecorum strain 25-3 T on performance, intestinal microbiota and prevention of necrotic enteritis (NE), a disease caused by Clostridium perfringens was evaluated in broilers. For the performance study, day old Ross 308 chicks were randomly allocated into two treatment groups and fed either a non-supplemented diet or a diet supplemented with 10 9 cfu lyophilized B. pullicaecorum per kg feed for 40 days. On day 40 broilers administered B. pullicaecorum had a significant lower bodyweight (2675 g vs. 2762 g; p = 0.0025) but supplementation of B. pullicaecorum decreased the feed conversion ratio significantly (1.518 vs. 1.632; p < 0.0001). Additionally, ingestion of the Butyricicoccus strain significantly lowered the abundance of Campylobacter spp. in the caecum and Enterococcus and Escherichia / Shigella spp. in the ileum at day 40. In feed supplementation of B. pullicaecorum in the NE trials resulted in a significant decrease in the number of birds with necrotic lesions compared with the untreated control group. These studies show that supplementation of B. pullicaecorum is able to improve feed conversion, to reduce the abundance of some potentially important pathogens in the caeca and ileum and to contribute to the prevention of NE in broilers, making the strain a potential valuable probiotic.

Background: Inflammatory bowel disease (IBD) refers to a diverse group of chronic gastrointestinal diseases, and gut microbial dysbiosis has been proposed as a modulating factor in its pathogenesis. Several studies have investigated the gut microbial ecology of dogs with IBD but it is yet unclear if this microbial profile can alter the nutrient metabolism of the host. The aim of the present study was to characterize the faecal bacterial profile and functionality as well as to determine host metabolic changes in IBD dogs. Twenty-three dogs diagnosed with IBD and ten healthy control dogs were included. Dogs with IBD were given a clinical score using the canine chronic enteropathy clinical activity index (CCECAI). Faecal short-chain fatty acids (SCFA) and ammonia concentrations were measured and quantitative PCR was performed. The concentration of plasma amino acids, acylcarnitines, serum folate, cobalamin, and indoxyl sulfate was determined., Results: No significant differences in the abundance of a selection of bacterial groups and fermentation metabolites were observed between the IBD and control groups. However, significant negative correlations were found between CCECAI and the faecal proportion of Lactobacillus as well as between CCECAI and total SCFA concentration. Serum folate and plasma citrulline were decreased and plasma valine was increased in IBD compared to control dogs. Increased plasma free carnitine and total acylcarnitines were observed in IBD compared with control dogs, whereas short-chain acylcarnitines (butyrylcarnitine + isobutyrylcarnitine and, methylmalonylcarnitine) to free carnitine ratios decreased. Dogs with IBD had a higher 3-hydroxyisovalerylcarnitine + isovalerylcarnitine to leucine ratio compared to control dogs., Conclusions: Canine IBD induced a wide range of changes in metabolic profile, especially for the plasma concentrations of short-chain acylcarnitines and amino acids, which could have evolved from tissue damage and alteration in host metabolism. In addition, dogs with more severe IBD were characterised by a decrease in faecal proportion of Lactobacillus.

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.

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.

Fumonisins (FBs) are mycotoxins produced by Fusarium fungi. This study aimed to investigate the effect of these feed contaminants on the intestinal morphology and microbiota composition, and to evaluate whether FBs predispose broilers to necrotic enteritis. One-day-old broiler chicks were divided into a group fed a control diet, and a group fed a FBs contaminated diet (18.6 mg FB1+FB2/kg feed). A significant increase in the plasma sphinganine/sphingosine ratio in the FBs-treated group (0.21 ± 0.016) compared to the control (0.14 ± 0.014) indicated disturbance of the sphingolipid biosynthesis. Furthermore, villus height and crypt depth of the ileum was significantly reduced by FBs. Denaturing gradient gel electrophoresis showed a shift in the microbiota composition in the ileum in the FBs group compared to the control. A reduced presence of low-GC containing operational taxonomic units in ileal digesta of birds exposed to FBs was demonstrated, and identified as a reduced abundance of Candidatus Savagella and Lactobaccilus spp. Quantification of total Clostridium perfringens in these ileal samples, previous to experimental infection, using cpa gene (alpha toxin) quantification by qPCR showed an increase in C. perfringens in chickens fed a FBs contaminated diet compared to control (7.5 ± 0.30 versus 6.3 ± 0.24 log10 copies/g intestinal content). After C. perfringens challenge, a higher percentage of birds developed subclinical necrotic enteritis in the group fed a FBs contaminated diet as compared to the control (44.9 ± 2.22% versus 29.8 ± 5.46%).

Butyrate-producing bacteria are promising probiotic candidates to target microbial dysbiosis in gastrointestinal disorders like inflammatory bowel diseases. Butyricicoccus pullicaecorum 25-3(T), a butyrate-producing clostridial cluster IV strain, is such a candidate. Little is known about its abundance in the colon microbiota and its butyrogenic properties. We used the M-SHIME(®), an in vitro simulator for the human intestinal microbial ecosystem, to study the effect of supplementing a single dose of B. pullicaecorum 25-3(T) on lumen- and mucus-associated microbiota of eight individuals. Butyricicoccus pullicaecorum was more abundant in mucus-associated microbiota compared with lumen microbiota. Supplementation with a single dose of B. pullicaecorum 25-3(T) resulted in a temporary increase in B. pullicaecorum bacteria in lumen compartment of all individuals. In two cases, the responders, an increased butyrate production was observed as compared with the control. 16S rRNA gene amplicon sequencing revealed the microbiota of responders to be different as compared to non-responder microbiota. We can conclude that B. pullicaecorum 25-3(T) is a mucus-associated bacterium whose potency to stimulate butyrate production is characterized by a large interindividual variability in terms of composition of the receiving microbial community., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The interactions of Flavobacterium columnare isolates of different virulence with the gills of carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum) were investigated. Both fish species were exposed to different high (HV) or low virulence (LV) isolates and sacrificed at seven predetermined times post-challenge. Histopathological and ultrastructural examination of carp and rainbow trout inoculated with the HV-isolate disclosed bacterial invasion and concomitant destruction of the gill tissue, gradually spreading from the filament tips towards the base, with outer membrane vesicles surrounding most bacterial cells. In carp, 5-10% of the fish inoculated with the LV-isolate became moribund and their gill tissue displayed the same features as described for the HV-isolate, albeit to a lesser degree. The bacterial numbers retrieved from the gill tissue were significantly higher for HV- compared to LV-isolate challenged carp and rainbow trout. TUNEL-stained and caspase-3-immunostained gill sections demonstrated significantly higher apoptotic cell counts in carp and rainbow trout challenged with the HV-isolate compared to control animals. Periodic acid-Schiff/alcian blue staining demonstrated a significantly higher total gill goblet cell count for HV- and LV-isolate challenged compared to control carp. Moreover, bacterial clusters were embedded in a neutral matrix while being encased by acid mucins, resembling biofilm formation. Eosinophilic granular cell counts were significantly higher in the HV-isolate compared to LV-isolate inoculated and control carp. The present data indicate a high colonization capacity, and the destructive and apoptotic-promoting features of the HV-isolate, and point towards important dynamic host mucin-F. columnare interactions warranting further research.