Impact of feed additives and host-related factors on bacterial metabolites, mucosal integrity and immune response in the ileum of broilers

The present study aimed to investigate the effect of age, breed, and sex of broilers, as well as a probiotic or phytobiotic product on mucosal morphology, bacterial metabolites, and immune traits in the ileum of broilers. A total of 2,880 oneday-old male and female broiler chicks from two breeds (Ross308® and Cobb500®) were randomly assigned to 72 pens. Broilers were offered a wheat-soybean diet without (CO), or with either a probiotic (PO; 2.4 ×109 CFU/kg of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (PY; grape extract, 165 ppm procyanidin and 585 ppm polyphenols of the diet) product. The trial was conducted with a 3 ×2 ×2 factorial arrangement of diet, breed, and sex in a completely randomized design (6 replicate-pens per treatment). At day 7, 21, and 35, one chicken per pen was slaughtered for collecting ileal tissue to evaluate of histomorphology and mRNA expression, as well as ileal digesta to measure bacterial metabolites. Data were subjected to ANOVA (the main factors; age, diet, breed, and sex) and Four-Way ANOVA (interactions) using GLM procedure. Overall, the concentration of acetate and total short chain fatty acids reached the peak and lactate decreased to its lowest on day 21, but their concentrations at day 7 and 35 were similar (p >0.05). Spermine, spermidine, and ammonia decreased after day 7, while putrescine and cadaverine increased after day 21 (p 0.05). In conclusion, aging of broilers affected ileal histomorphology, cytokine expression, and barrier integrity, as well as bacterial activity. These observed impacts could be attributed to host-microbiota interaction and the direct effects of bacterial metabolites on intestinal cells and immune system.info:eu-repo/semantics/publishedVersio

Synergistic Effects of Probiotics and Phytobiotics on the Intestinal Microbiota in Young Broiler Chicken

Probiotics and phytobiotics have been studied as in-feed antibiotic alternatives for decades, yet there are no studies on their possible symbiotic effects. In the present study, newly hatched chickens were fed with feeds supplemented either with host-specific Lactobacillus strains (L. agilis and L. salivarius), commercial phytobiotics, or combinations of both. After 13 days of life, crops and caecums were analyzed for bacterial composition (16S rDNA sequencing, qPCR) and activity (bacterial metabolites). Crop and caecum samples were also used to study the ex vivo survival of a broiler-derived extended-spectrum beta-lactamase (ESBL) producing Escherichia coli strain. In the crop, combinations of probiotics and phytobiotics, but not their single application, increased the dominance of lactobacilli. The single application of phytobiotics reduced the metabolite concentrations in the crop, but certain combinations synergistically upregulated the metabolites. Changes in the qualitative and quantitative composition of the caecal microbiota were less pronounced than in the crop. Acetate concentrations were significantly lower for phytobiotics or the L. agilis probiotic strain compared to the control group, but the L. salivarius probiotic showed significantly higher acetate concentrations alone or in combination with one phytobiotic. The synergistic effects on the reduction of the ex vivo survival of an ESBL producing E. coli strain in crop or caecum contents were also observed for most combinations. This study shows the beneficial synergistic effects of probiotics and phytobiotics on the intestinal bacterial composition and their metabolic activity in young broilers. The reduced survival of potentially problematic bacteria, such as ESBL-producing E. coli further indicates that combinations of probiotics and phytobiotics may lead to a more enhanced functionality than their individual supplementation

Impact of feed additives and host-related factors on bacterial metabolites, mucosal integrity and immune response in the ileum of broilers

The present study aimed to investigate the effect of age, breed, and sex of broilers, as well as a probiotic or phytobiotic product on mucosal morphology, bacterial metabolites, and immune traits in the ileum of broilers. A total of 2,880 one-day-old male and female broiler chicks from two breeds (Ross308® and Cobb500®) were randomly assigned to 72 pens. Broilers were offered a wheat-soybean diet without (CO), or with either a probiotic (PO; 2.4 × 109 CFU/kg of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (PY; grape extract, 165 ppm procyanidin and 585 ppm polyphenols of the diet) product. The trial was conducted with a 3 × 2 × 2 factorial arrangement of diet, breed, and sex in a completely randomized design (6 replicate-pens per treatment). At day 7, 21, and 35, one chicken per pen was slaughtered for collecting ileal tissue to evaluate of histomorphology and mRNA expression, as well as ileal digesta to measure bacterial metabolites. Data were subjected to ANOVA (the main factors; age, diet, breed, and sex) and Four-Way ANOVA (interactions) using GLM procedure. Overall, the concentration of acetate and total short chain fatty acids reached the peak and lactate decreased to its lowest on day 21, but their concentrations at day 7 and 35 were similar (p > 0.05). Spermine, spermidine, and ammonia decreased after day 7, while putrescine and cadaverine increased after day 21 (p  0.05). In conclusion, aging of broilers affected ileal histomorphology, cytokine expression, and barrier integrity, as well as bacterial activity. These observed impacts could be attributed to host-microbiota interaction and the direct effects of bacterial metabolites on intestinal cells and immune system

Corrigendum: Screening of Host Specific Lactic Acid Bacteria Active Against Escherichia coli From Massive Sample Pools With a Combination of in vitro and ex vivo Methods

A Corrigendum on: Screening of Host Specific Lactic Acid Bacteria Active Against Escherichia Coli From Massive Sample Pools With a Combination of in vitro and ex vivo Methods by Ren, H., Saliu, E.-M., Zentek, J., Goodarzi Boroojeni, F., and Vahjen, W. (2019). Front. Microbiol. 10:2705. doi: 10.3389/fmicb.2019.02705 In the original article, there was a mistake in Figure 3 as published. The authors reversed the order of Figures 3A,B by mistake when uploading the figures. The corrected figure appears below

The Impact of Direct-Fed Microbials and Phytogenic Feed Additives on Prevalence and Transfer of Extended-Spectrum Beta-Lactamase Genes in Broiler Chicken

Poultry frequently account for the highest prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in livestock. To investigate the impact of direct-fed microbials (DFM) and phytobiotic feed additives on prevalence and conjugation of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, an animal trial was conducted. Lactobacillus agilis LA73 and Lactobacillus salivarius LS1 and two commercial phytogenic feed additives (consisting of carvacrol, cinnamaldehyde, and eugenol) were used as feed additives either alone or as a combination of DFM and phytogenic feed additive. An ESBL-producing E. coli donor and a potentially pathogenic Salmonella Typhimurium recipient were inoculated at 5 × 109 cells/mL in cecal contents from 2-week-old broilers. Conjugation frequencies were determined after 4 h aerobic co-incubation at 37 °C and corrected for the impact of the sample matrix on bacterial growth of donor and recipient. Surprisingly, indigenous Enterobacteriaceae acted as recipients instead of the anticipated Salmonella recipient. The observed increase in conjugation frequency was most obvious in the groups fed the combinations of DFM and phytogenic product, but merely up to 0.6 log units. Further, cecal samples were examined for ESBL-producing Enterobacteriaceae on five consecutive days in broilers aged 27–31 days. All samples derived from animals fed the experimental diet showed lower ESBL-prevalence than the control. It is concluded that Lactobacillus spp. and essential oils may help to reduce the prevalence of ESBL-harboring plasmids in broilers, while the effect on horizontal gene transfer is less obvious

Novel strategies to improve chicken performance and welfare by unveiling host-microbiota interactions through hologenomics

Fast optimisation of farming practices is essential to meet environmental sustainability challenges. Hologenomics, the joint study of the genomic features of animals and the microbial communities associated with them, opens new avenues to obtain in-depth knowledge on how host-microbiota interactions affect animal performance and welfare, and in doing so, improve the quality and sustainability of animal production. Here, we introduce the animal trials conducted with broiler chickens in the H2020 project HoloFood, and our strategy to implement hologenomic analyses in light of the initial results, which despite yielding negligible effects of tested feed additives, provide relevant information to understand how host genomic features, microbiota development dynamics and host-microbiota interactions shape animal welfare and performance. We report the most relevant results, propose hypotheses to explain the observed patterns, and outline how these questions will be addressed through the generation and analysis of animal-microbiota multi-omic data during the HoloFood project

Novel strategies to improve chicken performance and welfare by unveiling host-microbiota interactions through hologenomics

Fast optimisation of farming practices is essential to meet environmental sustainability challenges. Hologenomics, the joint study of the genomic features of animals and the microbial communities associated with them, opens new avenues to obtain in-depth knowledge on how host-microbiota interactions affect animal performance and welfare, and in doing so, improve the quality and sustainability of animal production. Here, we introduce the animal trials conducted with broiler chickens in the H2020 project HoloFood, and our strategy to implement hologenomic analyses in light of the initial results, which despite yielding negligible effects of tested feed additives, provide relevant information to understand how host genomic features, microbiota development dynamics and host-microbiota interactions shape animal welfare and performance. We report the most relevant results, propose hypotheses to explain the observed patterns, and outline how these questions will be addressed through the generation and analysis of animal-microbiota multi-omic data during the HoloFood project

Der Einfluss verschiedener Wärmebehandlungen und Zulagen organischer Säuren im Futter auf die Nährstoffverdaulichkeit und die Mikrobiota des Darms von Broilern

Poultry feed could be a potential vector in the transmission of pathogens to the poultry. The processing facilities of poultry products can be contaminated by carrier or infected carcasses which lead to transmit of pathogens through the food chain to human. There is a correlation between the presence of Salmonella in poultry feed and their isolation on poultry meet. On the other hand, over 50% of all traceable human cases of salmonellosis have been associated with the consumption of eggs and other poultry products. The need for biological safety of the poultry products initiated a worldwide search to find an economically efficacious feed management strategy, which decontaminates feed, and meanwhile, would be putatively beneficial for the gut microbiota and have no negative impact on the animal welfare, physiology, performance, nutrient digestibility and consumer health (Chapter 1). In Chapter 2, a summary of the literature on different types of feed management strategies is given. The main objective of the present thesis was to investigate the effects of different thermal treatments including pelleting, long-term conditioning at 85°C for 3 minutes, or expanding at 110°C and 130°C for 3-5 seconds without or with 0.75 and 1.5% organic acid supplementation (63.75% formic acid, 25.00% propionic acid and 11.25% water) and their interactions on hygienic status of broiler feed, nutrient digestibility and bacterial composition and activity in gastrointestinal tract of broiler chicks. The detailed information on the main aims and hypotheses of the present study can be found in Chapter 3. In Chapter 4 and 5, the effects of different thermal treatments including pelleting (P), long-term conditioning at 85°C for 3 minutes (L), or expanding at 110°C (E110) and 130°C for 3-5 seconds (E130) without or with 0.75 and 1.5% organic acids supplementation on hygienic status of feed, performance, nutrient digestibility, gastrointestinal microbiota and organ weights of broilers are investigated an disccused. In total, 960 one-day-old broiler chicks were randomly assigned to 8 replicates using a 3 × 4 factorial arrangement. Performance variables were determined, and the relative organ weights, ileal and total amino acid (AA) digestibilities were measured at d 35. Also, at d 35, bacterial cell numbers in the crop, ileum and caecum, and bacterial metabolites in the crop, gizzard, ileum and caecum were determined. The organic acids inclusion linearly improved feed efficiency in the first week (P ≤ 0.05). The acid inclusion levels and thermal treatments had no significant effect on the performance variables at later intervals of the growing period. The inclusion of organic acids had a quadratic effect on total and ileal digestibility of isoleucine (P ≤ 0.05); while it had no significant effect on the ileal and total digestibility of other AA and nutrients. The inclusion of 1.5% organic acids increased cell numbers of all clostridial clusters in the crop. The organic acids supplementation increased the propionic acid concentration in the crop and gizzard whilst there was a decrease in lactic acid concentration. In the ileum, the 0% organic acids group had the highest numbers of Lactobacillus spp. and enterobacteria. Inclusion of 1.5% organic acids increased ileal acetate concentration. The L group showed the lowest ileal AA and crude protein digestibility. The relative weights of jejunum and small intestine were significantly higher in the E130 group compared to P and L (P ≤ 0.05). Increasing the feed processing temperature led to an increase of lactobacilli in the crop and ileum, while clostridia and enterobacteria seemed unaffected. Similarly, lactate concentrations increased in the ileum, but short chain fatty acids remained identical. In the crop, an increase for acetate was found for the E130 group compared to all other thermal treatments. In conclusion, the results of the present thesis demonstrated that the applied thermal and organic acids treatments were efficient strategies in order to hygienize broiler feed. The long-term thermal conditioning might decrease the ileal nutrient digestibility, while pelleting and expansion, without or/and with organic acids supplementation, seemed to be less critical. While expansion increased lactobacilli and lactate in the crop and ileum, organic acid addition remarkably influenced bacterial composition and activity in the crop. Taking into account all the observed effects of applied thermal and organic acids treatments on the microbiota of gastrointestinal tract, the evaluated treatments in the present study might have positive effects on the bacterial composition and activity of the upper gastrointestinal tract in broiler chicks that might offer interesting perspectives to achieve a better control of intestinal bacterial colonization in poultry. These indicate the potential of expansion process and organic acids addition for the hygienization of broiler feed (Chapter 6).Geflügelfutter stellt eine potenzielle Übertragungsmöglichkeit von pathogenen Erregern auf das Geflügel dar. Zur Verbesserung der Sicherheit bei Geflügelprodukten sind ökonomische Fütterungsstrategien erforderlich, die das Futter dekontaminieren, vorteilhaft für die Mikrobiota des Darms sind und die keine negativen Auswirkungen auf die Tiergesundheit, die Physiologie und Leistung der Tiere, die Nährstoffverdaulichkeit und die Sicherheit der Lebensmittel haben (Kapitel 1). In Kapitel 2 sind verschiedene Fütterungsstrategien aus der Literatur zusammengefasst. Ziel dieser Arbeit war es den Einfluss verschiedener Wärmebehandlungen, darunter das Pelletieren, die Langzeit-Aufbereitung bei 85 °C für 3 Minuten, oder die Expansion bei 110 °C und 130 °C für 3-5 Sekunden mit oder ohne den Zusatz von 0,75 und 1,5 % organischer Säure (63,75 % Ameisensäure, 25,00 % Propansäure und 11,25 % Wasser) und deren Wechselwirkungen auf den Hygienestatus von Broilerfutter, die Nährstoffverdaulichkeit und bakterielle Zusammensetzung und die Aktivität des Magen-Darm-Trakts von Broilerküken zu untersuchen. Detaillierte Informationen zu den Hauptzielen und Hypothesen dieser Arbeit sind in Kapitel 3 aufgeführt. In Kapitel 4 und 5 wurden die Einflüsse verschiedener Wärmebehandlungen, darunter das Pelletieren (P), Langzeit-Aufbereitung bei 85 °C für 3 Minuten (L), oder Expansion bei 110 °C (E110) und 130 °C für 3-5 Sekunden (E130) mit oder ohne den Zusatz von 0,75 und 1,5 % organischer Säure auf den Hygienestatus des Futters, die Leistung, Nährstoffverdaulichkeit, die Mikrobiota des Magen-Darm-Trakts und die Organgewichte von Broilern untersucht und diskutiert. Insgesamt wurden 960 Eintagsküken randomisiert in einem 3 × 4 faktoriellen Versuchsplan acht Wiederholungen zugeordnet. Die Leistungsvariablen wurden ermittelt und die relativen Organgewichte sowie die ileale und die gesamte Verdaulichkeit der Aminosäuren an Tag 35 gemessen. An Tag 35 wurden zudem die bakteriellen Zellzahlen in Kropf, Ileum und Caecum sowie die bakteriellen Metabolite in Kropf, Muskelmagen, Ileum und Caecum bestimmt. Der Zusatz von organischen Säuren führte zu einer Verbesserung der Futtereffizienz in der ersten Woche (P ≤ 0.05). Die verschiedenen Mengen an zugeführten organischen Säuren und die Wärmebehandlungen zeigten zu späteren Zeitpunkten der Wachstumsphase keine signifikanten Auswirkungen auf die Leistungsvariablen. Der Säurenzusatz zeigte einen quadratischen Effekt auf die ileale und die Gesamtverdaulichkeit von Isoleucin (P ≤ 0.05), hatte jedoch keinen signifikanten Einfluss auf die anderen Aminosäuren und Nährstoffe. Der Zusatz von 1,5% eines Produkts mit organischen Säuren erhöhte die Zellzahl aller Clostridiengruppen im Kropf. Durch die Zugabe erhöhte sich die Propionsäurekonzentration in Kropf und Muskelmagen bei einem gleichzeitigen Absinken der Milchsäurekonzentration. Im Ileum zeigte die Gruppe ohne Säurenzusatz die höchsten Zellzahlen an Lactobacillus spp. und Enterobakterien. Der Zusatz organischer Säuren erhöhte die ileale Acetatkonzentration. Die L-Gruppe zeigte die geringste ileale Aminosäuren- und Rohproteinverdaulichkeit. Die relativen Gewichte des Jejunums und des gesamten Dünndarms waren in der E130 Gruppe signifikant höher als in den Gruppen P und L (P ≤ 0.05). Eine Erhöhung der Verarbeitungstemperatur des Futters führte zu einer erhöhten Anzahl an Milchsäurebakterien in Kropf und Ileum, wobei Clostridien und Enterobakterien unbeeinflusst schienen. Zudem erhöhten sich die Laktatkonzentrationen im Ileum, die kurzkettigen Fettsäuren blieben jedoch unverändert. Im Kropf konnte ein erhöhter Acetatgehalt in der E130 Gruppe im Vergleich zu allen anderen Wärmebehandlungen gezeigt werden. Schlussfolgernd konnten die Ergebnisse dieser Arbeit zeigen, dass die angewandten Wärmebehandlungen und der Einsatz organischer Säuren effiziente Strategien einer Hygienisierung von Broilerfutter darstellen. Die Langzeiterhitzung verringert möglicherweise die ileale Nährstoffverdaulichkeit, während Pelletierung und Expansion mit oder ohne Zugabe organischer Säure weniger bedenklich erscheinen. Während die Expansion Milchsäurebakterien und Laktat in Kropf und Ileum erhöhte, beeinflusste die Zugabe organischer Säuren die bakterielle Zusammensetzung und Aktivität im Kropf. Aufgrund der beobachteten Effekte von Wärmebehandlung und organischen Säuren auf die Mikrobiota des Magen-Darm-Trakts führen diese beiden Ansätze möglicherweise zur Veränderung der bakteriellen Zusammensetzung des oberen Magen-Darm-Trakts bei Broilerküken, wodurch sich interessante Aspekte für eine Kontrolle der bakteriellen Besiedlung beim Geflügel ergeben

Development and Functional Properties of Intestinal Mucus Layer in Poultry

Intestinal mucus plays important roles in protecting the epithelial surfaces against pathogens, supporting the colonization with commensal bacteria, maintaining an appropriate environment for digestion, as well as facilitating nutrient transport from the lumen to the underlying epithelium. The mucus layer in the poultry gut is produced and preserved by mucin-secreting goblet cells that rapidly develop and mature after hatch as a response to external stimuli including environmental factors, intestinal microbiota as well as dietary factors. The ontogenetic development of goblet cells affects the mucin composition and secretion, causing an alteration in the physicochemical properties of the mucus layer. The intestinal mucus prevents the invasion of pathogens to the epithelium by its antibacterial properties (e.g. β-defensin, lysozyme, avidin and IgA) and creates a physical barrier with the ability to protect the epithelium from pathogens. Mucosal barrier is the first line of innate defense in the gastrointestinal tract. This barrier has a selective permeability that allows small particles and nutrients passing through. The structural components and functional properties of mucins have been reviewed extensively in humans and rodents, but it seems to be neglected in poultry. This review discusses the impact of age on development of goblet cells and their mucus production with relevance for the functional characteristics of mucus layer and its protective mechanism in the chicken’s intestine. Dietary factors directly and indirectly (through modification of the gut bacteria and their metabolic activities) affect goblet cell proliferation and differentiation and can be used to manipulate mucosal integrity and dynamic. However, the mode of action and mechanisms behind these effects need to be studied further. As mucins resist to digestion processes, the sloughed mucins can be utilized by bacteria in the lower part of the gut and are considered as endogenous loss of protein and energy to animal. Hydrothermal processing of poultry feed may reduce this loss by reduction in mucus shedding into the lumen. Given the significance of this loss and the lack of precise data, this matter needs to be carefully investigated in the future and the nutritional strategies reducing this loss have to be defined better

Alterations in bacterial metabolites, cytokines, and mucosal integrity in the caecum of broilers caused by feed additives and host-related factors

A total of 2,880 one-day-old male and female broiler chicks from two breeds, Ross308 and Cobb500 were randomly assigned to 72 pens. Broilers were offered three diets: a wheat-soybean diet without (CO), or with either a probiotic (probiotic; 2.4 x 109 CFU/kg diet of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (phytobiotic; grape extract with 165 ppm procyanidin and 585 ppm polyphenol) product. The trial was conducted with a 3 × 2 × 2 factorial arrangement of diet, breed and sex in a completely randomized design and consisted of 6 replicate-pens per treatment (40 birds per pen). At day 7, 21, and 35, one chicken per pen was slaughtered for caecal sampling to quantify bacterial metabolites (digesta) as well as evaluate mRNA abundance and histomorphology (tissue). Data were subjected to ANOVA using GLM procedure to evaluate age, diet, breed and sex and their interactions. Spearman’s correlation (r) was analyzed between metabolite concentration and mRNA abundance. Overall, the concentration of short chain fatty acids increased with age, while lactate decreased from day 7 to 21 (p < 0.05). The mRNA abundance of IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17α, IL-18, IFN-γ and TGF-β2 increased with age but IL-1β and TNF-α increased in abundance from day 7 to 21 and then decreased (p < 0.05). Abundance of MUC2 and CLDN5 increased after day 21 (p < 0.05). Caecal crypt depth increased with age (p < 0.05). Acidic goblet cell (GC) number peaked at day 21 (p < 0.05), while mixed GC number was not affected by age. A few impacts of breed, diet and interactions on the investigated variables showed no meaningful biological pattern. Propionate positively correlated with all cytokines investigated (r = 0.150–0.548), except TNF-α. Lactate negatively correlated with pro-inflammatory cytokines like IL-1β (r = −0.324). Aging affected caecal histomorphology, bacterial activity and genes responsible for barrier integrity and inflammatory response. This effect could be attributed to the interaction between gut microbiota and immune system as well as the direct effect of metabolites on gut histomorphology and cytokine mRNA abundance.info:eu-repo/semantics/publishedVersio