Porcine Colostrum Protects the IPEC-J2 Cells and Piglet Colon Epithelium against Clostridioides (syn. Clostridium) difficile Toxin-Induced Effects

Clostridioides difficile toxins are one of the main causative agents for the clinical symptoms observed during C. difficile infection in piglets. Porcine milk has been shown to strengthen the epithelial barrier function in the piglet’s intestine and may have the potential to neutralise clostridial toxins. We hypothesised that porcine colostrum exerts protective effects against those toxins in the IPEC-J2 cells and in the colon epithelium of healthy piglets. The IPEC-J2 cells were treated with either the toxins or porcine colostrum or their combination. Analyses included measurement of trans-epithelial electrical resistance (TEER), cell viability using propidium iodide by flow cytometry, gene expression of tight junction (TJ) proteins and immune markers, immunofluorescence (IF) histology of the cytoskeleton and a TJ protein assessment. Colon tissue explants from one- and two-week-old suckling piglets and from five-week-old weaned piglets were treated with C. difficile toxins in Ussing chamber assays to assess the permeability to macromolecules (FITC-dextran, HRP), followed by analysis of gene expression of TJ proteins and immune markers. Toxins decreased viability and integrity of IPEC-J2 cells in a time-dependent manner. Porcine colostrum exerted a protective effect against toxins as indicated by TEER and IF in IPEC-J2 cells. Toxins tended to increase paracellular permeability to macromolecules in colon tissues of two-week-old piglets and downregulated gene expression of occludin in colon tissues of five-week-old piglets (p = 0.05). Porcine milk including colostrum, besides other maternal factors, may be one of the important determinants of early immune programming towards protection from C. difficile infections in the offspring

Influence of fermentable carbohydrates or protein on large intestinal and urinary metabolomic profiles in piglets

It was recently shown that variations in the ratio of dietary fermentable carbohydrates (fCHO) and fermentable protein (fCP) differentially affect large intestinal microbial ecology and the mucosal response. Here we investigated the use of mass spectrometry to profile changes in metabolite composition in colon and urine associated with variation in dietary fCHO and fCP composition and mucosal physiology. Thirty-two weaned pigletswere fed 4 diets in a 2 × 2 factorial design with low fCP and low fCHO, low fCP and high fCHO, high fCP and low fCHO, and high fCP and high fCHO. After 21 to 23 d, all pigs were euthanized and colon digesta and urine metabolite profiles were obtained by mass spectrometry. Analysis of mass spectra by partial least squares approach indicated a clustering of both colonic and urinary profiles for each pig by feeding group. Metabolite identification and annotation using the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways revealed increased abundance of metabolites associated with arachidonic acid metabolism in colon of pigs fed a high concentration of fCP irrespective of dietary fCHO. Urinary metabolites did not show as clear patterns. Mass spectrometry can effectively differentiate metabolite profiles in colon contents and urine associated with changes in dietary composition. Whether metabolite profiling is an effective tool to identify specific metabolites (biomarkers) or metabolite profiles associated with gut function and integrity needs further elucidation

Carbon on the Northwest European Shelf: Contemporary Budget and Future Influences

A carbon budget for the northwest European continental shelf seas (NWES) was synthesized using available estimates for coastal, pelagic and benthic carbon stocks and flows. Key uncertainties were identified and the effect of future impacts on the carbon budget were assessed. The water of the shelf seas contains between 210 and 230 Tmol of carbon and absorbs between 1.3 and 3.3 Tmol from the atmosphere annually. Off-shelf transport and burial in the sediments account for 60–100 and 0–40% of carbon outputs from the NWES, respectively. Both of these fluxes remain poorly constrained by observations and resolving their magnitudes and relative importance is a key research priority. Pelagic and benthic carbon stocks are dominated by inorganic carbon. Shelf sediments contain the largest stock of carbon, with between 520 and 1600 Tmol stored in the top 0.1 m of the sea bed. Coastal habitats such as salt marshes and mud flats contain large amounts of carbon per unit area but their total carbon stocks are small compared to pelagic and benthic stocks due to their smaller spatial extent. The large pelagic stock of carbon will continue to increase due to the rising concentration of atmospheric CO2, with associated pH decrease. Pelagic carbon stocks and flows are also likely to be significantly affected by increasing acidity and temperature, and circulation changes but the net impact is uncertain. Benthic carbon stocks will be affected by increasing temperature and acidity, and decreasing oxygen concentrations, although the net impact of these interrelated changes on carbon stocks is uncertain and a major knowledge gap. The impact of bottom trawling on benthic carbon stocks is unique amongst the impacts we consider in that it is widespread and also directly manageable, although its net effect on the carbon budget is uncertain. Coastal habitats are vulnerable to sea level rise and are strongly impacted by management decisions. Local, national and regional actions have the potential to protect or enhance carbon storage, but ultimately global governance, via controls on emissions, has the greatest potential to influence the long-term fate of carbon stocks in the northwestern European continental shelf

Carbon on the Northwest European Shelf: Contemporary Budget and Future Influences

© Copyright © 2020 Legge, Johnson, Hicks, Jickells, Diesing, Aldridge, Andrews, Artioli, Bakker, Burrows, Carr, Cripps, Felgate, Fernand, Greenwood, Hartman, Kröger, Lessin, Mahaffey, Mayor, Parker, Queirós, Shutler, Silva, Stahl, Tinker, Underwood, Van Der Molen, Wakelin, Weston and Williamson. A carbon budget for the northwest European continental shelf seas (NWES) was synthesized using available estimates for coastal, pelagic and benthic carbon stocks and flows. Key uncertainties were identified and the effect of future impacts on the carbon budget were assessed. The water of the shelf seas contains between 210 and 230 Tmol of carbon and absorbs between 1.3 and 3.3 Tmol from the atmosphere annually. Off-shelf transport and burial in the sediments account for 60–100 and 0–40% of carbon outputs from the NWES, respectively. Both of these fluxes remain poorly constrained by observations and resolving their magnitudes and relative importance is a key research priority. Pelagic and benthic carbon stocks are dominated by inorganic carbon. Shelf sediments contain the largest stock of carbon, with between 520 and 1600 Tmol stored in the top 0.1 m of the sea bed. Coastal habitats such as salt marshes and mud flats contain large amounts of carbon per unit area but their total carbon stocks are small compared to pelagic and benthic stocks due to their smaller spatial extent. The large pelagic stock of carbon will continue to increase due to the rising concentration of atmospheric CO2, with associated pH decrease. Pelagic carbon stocks and flows are also likely to be significantly affected by increasing acidity and temperature, and circulation changes but the net impact is uncertain. Benthic carbon stocks will be affected by increasing temperature and acidity, and decreasing oxygen concentrations, although the net impact of these interrelated changes on carbon stocks is uncertain and a major knowledge gap. The impact of bottom trawling on benthic carbon stocks is unique amongst the impacts we consider in that it is widespread and also directly manageable, although its net effect on the carbon budget is uncertain. Coastal habitats are vulnerable to sea level rise and are strongly impacted by management decisions. Local, national and regional actions have the potential to protect or enhance carbon storage, but ultimately global governance, via controls on emissions, has the greatest potential to influence the long-term fate of carbon stocks in the northwestern European continental shelf

Investigations on the formation of vitamin K2 by the canine intestinal microbiota

Vitamin K kommt in zwei unterschiedlichen Formen vor. Vitamin K1, Phyllochinon (PK), ist in grünblättrigen Pflanzen und verschiedenen Pflanzenölen vorzufinden. Vitamin K2, Menachinon (MK), wird im Intestinaltrakt von Säugetieren und Vögeln von der dort ansässigen Darmflora gebildet. Die Menachinone unterscheiden sich in der Länge ihrer Seitenkette und werden je nach Anzahl der Isopreneinheiten numerisch gekennzeichnet (MK-n). Ziel dieser Arbeit war es, die Bildung von MK durch die Intestinalflora von Hunden und den Einfluss von Substraten (Protein und Stärke) anhand von zwei In-vitro-Methoden zu untersuchen. Ergänzend wurde bei Hunden die MK-Konzentration im Chymus verschiedener Darmabschnitte (Duodenum, Jejunum, Ileum, Zäkum und Kolon) bestimmt (Analyse von Ex-vivo-Proben). Zunächst wurden zwei In-vitro-Systeme, eine als semikontinuierliche Langzeitinkubation und eine 24stündige Kurzzeitinkubation auf ihre Eignung als methodischer Ansatz für die Fragestellung getestet. Aufgrund der Ergebnisse der Vorversuche wurde die 24stündige Kurzzeitinkubation als geeignete Methode ausgewählt. Es wurden drei Versuchdurchgänge mit jeweils zehn gasdicht verschlossenen Inkubationsgefäßen durchgeführt. Als Inkubationsansatz diente Fäzes von Hunden, welche mit einem anaeroben vorreduzierten Puffer verdünnt (1:100) wurde. Um den Einfluss von Proteinen und Kohlenhydraten auf die Bildung von MK zu überprüfen, wurde pro Versuchsdurchgang jeweils verschiedene Konzentrationen (0,1, 0,5 und 1,0 g) von Maisstärke (Ms), Fleischpepton (Pep) bzw. Maisstärke/Fleischpepton (Ms/Pep; 50:50) hinzugefügt. Als Kontrolle diente eine Inkubationseinheit ohne Substrat. Alle Gefäße wurden für 24 Stunden, bei 37 °C und bei leichter Rotation unter Lichtschutz inkubiert. Nach Ende der Inkubationszeit wurde der pH-Wert gemessen und Proben für die Bestimmung von flüchtigen Fettsäuren, Ammonium und Laktat als Fermentationsparameter sowie für die Vitamin K Analyse per Hochleistungsflüssigkeitschromatographie (HPLC) entnommen. Die Bestimmungen von MK-4 bis -10 ergaben, dass MK-10 das Verteilungsmuster dominierte, gefolgt von MK-9 und MK-4. Am geringsten war die Bildung von MK-7. Ein Einfluss der Substrate war sowohl bei der Gesamtkonzentration als auch bei der Bildung der einzelnen MK zu erkennen. Es zeigte sich, dass die höchsten Konzentrationen von MK-7 bis -10 nach der Zugabe von MS bzw. Ms/Pep gemessen werden konnten und MK-5 und -6 nach dem Zusatz von Ms/Pep. Nach Zugabe von Pep waren die höchsten Konzentrationen von MK-4 zu beobachten. Die Analyse der Digestaproben ergab ein ähnliches Verteilungsmuster. Im Zäkum und Kolon dominierte MK-10, gefolgt von MK-6, -9 und -4. In den Dünndarmproben wurde mehr MK-9 als MK-10 gemessen. Die höchsten Gesamtkonzentrationen wurden im Zäkum (635 ng/g) und Kolon (1045 ng/g) analysiert. Im Dünndarm nahmen die Konzentrationen vom Duodenum zum Ileum ab. Die durchschnittliche Gesamtkonzentrationen der Kurzzeitinkubation und im Koloninhalt ergaben vergleichbare Werte, allerdings muss beachtet werden, dass bei den In-vitro- Versuchen keine Resorption der gebildeten MK stattfinden konnte. Die Ergebnisse zeigen, dass eine mikrobielle MK-Bildung im Intestinaltrakt von Hunden stattfindet und somit ein Beitrag zur Vitamin K-Versorgung durch diese Synthese geleistet werden dürfte. Fragen zur Resorption und biologischen Wirkung der verschiedenen MK bedürfen weiterer Untersuchungen, um die physiologische Bedeutung genauer schätzen zu können.The term Vitamin K refers to at least two different types of quinones. Vitamin K1, phylloquinone (PK), is found in green leafy plants and several vegetable oils. Vitamin K2, menaquinone (MK), is produced by the intestinal microbiota of mammals and birds. MK differ in the length of their side chain and the common nomenclature is MK-n, “n” representing the number of isoprenoids. The objective of this study was to investigate the production of MK by the canine intestinal microbiota and the influence of substrate (protein and starch) in two different in-vitro-fermentation systems. Additionally, MK concentrations were determined in the chyme of different parts of the intestinal tract of dogs (duodenum, jejunum, ileum, caecum and colon). For the in-vitro-study, a semicontinuous long-term- and a 24h short-term incubation system were tested for their suitability. Based on the results of these tests, the 24h short-term incubation system was chosen as method. The trial was performed with three repetitions, each with ten airtight fermentation flasks. Faeces was obtained from dogs and diluted 1:100 in an anaerobic pre-reduced buffer with different amounts (0.1, 0.5 and 1.0 g) of maize starch (Ms), peptone (Pep) and a mixture of both (Ms/Pep; 50:50) including a control. The bottles were incubated with slow rotation for 24 hours at 37°C under subdued light. The pH was measured after incubation and samples were taken for the determination of volatile fatty acids, ammonium and lactate as fermentation parameters, as well as for the detection of MK by high-performance liquid chromatography (HPLC). The pattern of MK-4 to -10 was dominated by MK-10 followed by MK-9 and MK-4. Lowest concentrations were found for MK-7. An influence of the substrates could be observed regarding the total MK-concentration and the production of the different MK. The concentration of MK-7 to -10 increased after the addition of Ms and Ms/Pep and for MK-5 and -6 after Ms/Pep. Highest concentrations of MK-4 were found after the addition of Pep. The analysis of the chyme samples revealed similar MK-pattern. MK-10 was predominant in the caecal and colonal chyme followed by MK-6, -9 and -4. In the small intestine contents the formation of Vitamin K2 was dominated by MK-9. Highest total MK concentrations were measured in caecum and colon with an average of 635 ng/g and 1045 ng/g, respectively. The concentration in the small intestine decreased from duodenum to the ileum. The MK pattern of the short-term incubation and in the colon digesta was similar. In conclusion, the results pointed out that a production of MK takes place in the intestinal tract of dogs and might therefore contribute to cover the requirements of vitamin K. Further investigations about the mechanism of absorption and the biological activity are needed to obtain a better understanding about the physiological importance of MK

Porcine Colostrum Protects the IPEC-J2 Cells and Piglet Colon Epithelium against Clostridioides (syn. Clostridium) difficile Toxin-Induced Effects

Clostridioides difficile toxins are one of the main causative agents for the clinical symptoms observed during C. difficile infection in piglets. Porcine milk has been shown to strengthen the epithelial barrier function in the piglet’s intestine and may have the potential to neutralise clostridial toxins. We hypothesised that porcine colostrum exerts protective effects against those toxins in the IPEC-J2 cells and in the colon epithelium of healthy piglets. The IPEC-J2 cells were treated with either the toxins or porcine colostrum or their combination. Analyses included measurement of trans-epithelial electrical resistance (TEER), cell viability using propidium iodide by flow cytometry, gene expression of tight junction (TJ) proteins and immune markers, immunofluorescence (IF) histology of the cytoskeleton and a TJ protein assessment. Colon tissue explants from one- and two-week-old suckling piglets and from five-week-old weaned piglets were treated with C. difficile toxins in Ussing chamber assays to assess the permeability to macromolecules (FITC-dextran, HRP), followed by analysis of gene expression of TJ proteins and immune markers. Toxins decreased viability and integrity of IPEC-J2 cells in a time-dependent manner. Porcine colostrum exerted a protective effect against toxins as indicated by TEER and IF in IPEC-J2 cells. Toxins tended to increase paracellular permeability to macromolecules in colon tissues of two-week-old piglets and downregulated gene expression of occludin in colon tissues of five-week-old piglets (p = 0.05). Porcine milk including colostrum, besides other maternal factors, may be one of the important determinants of early immune programming towards protection from C. difficile infections in the offspring