Browsing by Subject "Darmflora"

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Analyse komplexer Merkmale beim Schwein mittels SNP-Chip Genotypen, Darmmikrobiota- und Genexpressionsdaten
(2017) Maushammer, Maria; Bennewitz, Jörn
In the present scientific research, SNP chip genotypes, gut microbiota and gene expression data were used for analysing complex traits in a Piétrain population. These data were collected from around 200 performance tested sows and were used for genetic and microbial analyses of complex trait as well as for structural and functional meat quality traits. The gut microbiome plays a major role in the immune system development, state of health and energy supply of the host. Quantitative-genetic methods were applied to analyse the interrelationship between pig gut microbiota compositions, complex traits (daily gain, feed conversion and feed intake) and pig genomes. The specific aims were to characterize the gut microbiota of the pigs, to analyse the effects of host genetics on gut microbial composition, and to investigate the role of gut microbial composition on the host’s complex traits. The pigs were genotyped with a standard 60K SNP chip. Microbial composition was characterized by 16S rRNA gene amplicon sequencing technology. Ten out of 51 investigated bacterial genera showed a significant host heritability, ranging from 0.32 to 0.57. Conducting genome wide association analysis showed associations of 22 SNPs and six bacterial genera. The potential candidate genes identified are involved in the immune system, mucosa structure and secretion of digestive juice. These results show, that parts of the gut microbiota are heritable and that the gut microbiome can be seen as quantitative trait. Microbial mixed linear models were applied to estimate the microbiota variance for each of the investigated traits. The fraction of phenotypic variance explained by the microbial variance was 0.28, 0.21, and 0.16 for daily gain, feed conversion, and feed intake, respectively. The SNP data and the microbiota data were used to predict the phenotypes of the traits using both, genomic best linear unbiased prediction (G-BLUP) and microbial best linear unbiased prediction (M-BLUP) methods. The prediction accuracies of G-BLUP were 0.35, 0.23, and 0.20 for daily gain, feed conversion, and feed intake, respectively. The corresponding prediction accuracies of M-BLUP were 0.41, 0.33, and 0.33. Thus, the gut microbiota can be seen as an explaining variable for complex traits like daily gain, feed conversion and feed intake. In addition, in combination with meat quality traits, transcript levels of muscle tissue were analysed at time of slaughtering. This study should give an insight into the biological processes involved in meat quality characteristics. The aims were to functionally characterise differentially expressed genes, to link the functional information with structural information obtained from GWAS, and to identify potential candidate genes based on these results. An important meat quality trait is the intramuscular fat content, since it affects the juiciness, the taste and the tenderness of the meat. Another important trait is drip loss which causes not only a loss of weight but also a loss of important proteins. Both traits have an impact on the consumer acceptance of fresh meat products. For each of the two traits, eight discordant sibling pairs were selected out of the Piétrain sample and were used for genome-wide gene expression analyses. Thirty five and 114 genes were identified as differentially expressed and trait correlated genes for intramuscular fat content and drip loss, respectively. On the basis of functional annotation, gene groups belonging to the energy metabolism of the mitochondria, the immune response and the metabolism of fat, were associated with intramuscular fat content. Gene groups associated with protein ubiquitination, mitochondrial metabolism, and muscle structural proteins were associated with drip loss. Furthermore, genome-wide association analyses were carried out for these traits and their results were linked to the genome-wide expression analysis by functional annotation. In this context, intramuscular fat was related to muscle contraction, transmembrane transport and nucleotide binding. Drip loss was characterized by the endomembrane system, the energy generation of cells, and phosphorus metabolic processes. Three and four potential candidate genes were identified for intramuscular fat content and drip loss, respectively.
Antimikrobielle Aktivität der Histone bei chronisch entzündlichen Darmerkrankungen
(2017) Kunkel, Yasmin; Stange, Eduard F.
The human intestine harbours a multitude of microorganisms. In addition to its func-tion as a protective layer against pathogens, it has to prevent an excessive immune answer against commensales simultaneously. Antimicrobial Peptides (AMPs) with their cationic character are playing an essential role in protection, because they are able to form voltage dependent channels on the surface of microorganism, which kill pathogens. In addition to the classical AMPs more antimicrobial active polypeptides, such as members of the histone family, were isolated. Histones are alkaline proteins, which are components of the chromatine. They are foremost responsible for packaging the DNA and for posttranslational modifications. Five different families can be differentiated: the core histones H2A, H2B, H3 and H4, as well as the linker histone H1. While extracellular histones show strong antimicrobial activity against a broad spectrum of microorganisms, the mechanism of their toxicity has not yet been sufficiently determined. If the antimicrobial protection layer of the intestine is weakened, due to a diminished expression of AMPs for instance, microorganisms can penetrate the mucosa and trigger inflammations. These findings have been confirmed in different tissues of patients with inflammatory bowel diseases (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC). The aim of this work is to determine whether histones play a role in IBD. In the first part a systematic analysis of the transcriptome (Q-PCR) and the transla-tome (Western Blotting) of the core histones of colonic tissue was performed. In tis-sues of patients with CD gene expression data showed generally an increase of his-tones. In the cases of H2A and H2B the increase was significant. The quantification on the protein level offered an extreme variance of the expression of all core histones, irrespective of the analysed group. Significant differences were not detected. However, in trend H2B was lower in inflammation. After the systematic analysis, histones were then isolated of human colonic tissue. Before the extracted histones were fractionated via RP-HPLC and screened via MALDI-TOF-MS, different methods for the isolation of histones had been compared. The antimicrobial activity of the isolated histones of different intestinal tissues and mucus showed no differences between healthy controls and patients with IBD in flow cytometric tests. A significant increase of the histone activity in inflamed tissues of UC was only detected against S. aureus. The impact of the extracted histones seems to be strain-specific and higher against gram-positive species. As expected, extracel-lular histones could be detected in the mucus by immunehistological staining. Through ELISAs, protein concentrations of H2A and H2B were determined in the mucus and thus a slight increase of the histone proteins in UC was observed. In the last part of this work, the interactions of recombinant histones among them-selves and with other AMPs were analysed by flow cytometric viability assays. A strain-specific increase of the antimicrobial activity of histones among themselves and with AMPs was found. Thereby synergistic effects occurred frequently. The in-teractions of histones against several bacteria were visualised by electron microscope images and furthermore an agglutination of the microorganisms as well as a massive loss of cell integrity were detected. Variantions of the histones’ transcriptome and the translatome, as well as variations of the antimicrobial activity of histones in CED would have been evaluated as patho-logic defects. However, in this work such effects could not been confirmed. Because of their enormous antimicrobial activity histones still play an important role in the protection against microorganisms in the colon. Further studies have to show, if his-tones possess a therapeutic potential, and if they can be used as new antibiotics. This work was able to verify the strong potential of histones against different pathogens, which is absolutely comparable with the potential of classic AMPs, and could pro-mote inspiration for subsequent studies.
Comprehensive characterization of microbiota in the gastrointestinal tract of quails and two high yielding laying hen breeds
(2023) Roth, Christoph Florian; Camarinha-Silva, Amélia
The microbiomes composition in the gastrointestinal tract (GIT) is subject to several changes and influences. In addition to breed, sex, or diet, age affects the GIT microbiome dynamics of laying hens and quails. From the first day, the microbiome develops and increases its bacterial load to thousands of species. Then, depending on the diet fed, the animals microbiome and associated active bacteria vary and directly influence the animals nutrient uptake and efficiency. Omics technologies give insights into changes in microbes in the GIT (crop, gizzard, duodenum, ileum, caeca). In addition, they can reveal how feed supplements such as calcium (Ca) or phosphorus (P) can affect host health and performance through alterations in the microbiome. The Japanese quail has been an established animal model for nutritional and biological studies in poultry for the last 60 years. In particular, its short development time makes it a convenient model for microbiome research. However, compared to broiler microbiome research, the quail microbiome is still poorly understood. Animals of the breed Coturnix japonica were housed under the same conditions, fed a diet with P below recommendation, and the ileum microbiota characterized. Microbiota relations with gender and higher or lower predisposition of the birds for PU, CaU, FI, BWG, and FC were described (Chapter II). In addition, these performance parameters influenced the relative average abundance of bacteria like Candidatus Arthromitus, Bacillus, and Leuconostoc. Gender affects specific bacterial groups of the GIT, such as Lactobacillus, Streptococcus, Escherichia, and Clostridium, which differ in average abundance between male and female quails. Despite the comprehensive microbiota analysis, the interplay between animal genetics, diet, sex, and microbiome functionality is not yet understood. The laying hen breeds Lohmann LSL-Classic and Lohmann Brown-Classic are used worldwide. Little is known about the interaction with microbiome composition, performance, dietary effects, and changes during the productive life that might help develop feeding strategies and microbiome responses on a large scale. Because of the importance of P and Ca in poultry diet, the research in Chapter III was conducted to challenge laying hens with reduced dietary P and Ca and describe the effect on GIT active microbiota. The breed was the primary driver of microbial differences. A core microbiome of active bacteria, present along the complete GIT, was revealed for the first time and consisted of five bacteria detected in 97% of all samples, including digesta and mucosa samples (uncl. Lactobacillus, Megamonas funiformis, Ligilactobacillus salivarius, Lactobacillus helveticus, uncl. Fuscatenibacter). Furthermore, significant microbial differences between the GIT sections and between the breeds were described. Minor dietary effects of the P and Ca reduction on the microbiota showed that a further decrease in Ca and P supplementation might be possible without affecting the gut microbial composition and bird performance. Furthermore, the microbiome of laying hens was characterized at five productive stages (weeks 10, 16, 24, 30, and 60) to analyze the age effect on the GIT microbiome (Chapter IV). Although the two breeds of laying hens were offered the same diet and housed under similar conditions, the active microbiota composition changed between the analyzed productive stages, the breed and the GIT sections. The major shift occurred between weeks 16 and 24 and supported the hypothesis of bacterial fluctuations due to the onset of the laying period. Those changes occurred mainly in the abundance of the genera Lactobacillus and Ligilactobacillus. However, it remains unclear whether the dietary changes, due to the development of the birds, influenced the microbiota shifts or if the anatomical and physiological modifications influenced the GIT microbiota. Furthermore, the shotgun metagenomic analysis revealed differences in regulatory functions and pathways between breeds, sections, and the two production stages. Different relative abundance levels of the microbial composition were observed between the RNA-based targeted sequencing and the DNA-based shotgun metagenomics. In conclusion, the comprehensive characterization of the microbiota in the GIT of quails and two high-yielding breeds of laying hens contributes to a broader knowledge of the microbiome dynamics within the fowl GIT. Age and breed play a more important role than diet in influencing the dynamics of microbial composition in laying hens, and individual performance and sex in quails. Research characterizing the microbiome in poultry and its effect on diet and host genetics will help improve feeding and breeding strategies in the future and reduce excretion of nutrients into the environment while ensuring overall animal health.
High-throughput sequencing techniques to analyze microbial communities in the gastrointestinal tract of broiler chickens
(2018) Borda Molina, Daniel Enrique; Camarinha-Silva, Amélia
Broiler chicken represents an excellent case-study to elucidate the inter-communication between the host and its microbial communities. The general aim of this thesis was to describe the changes in bacterial community structure that occurred in chickens, in response to different experimental diets. An update of the state of the art of the chicken gastrointestinal microbiota was done in chapter 2. The composition and functionality are described through the most recent technologies that provide taxonomic information at DNA level using 16S rRNA genes. Gene catalogs and their abundance are deciphered through shotgun metagenome sequencing, which is still at its infancy and only eight publications have been published so far. At the protein level, only two studies were found that contribute metaproteomic information. Thanks to these technologies many studies were able to focus on answering how feed supplementations altered the microbes in GIT sections. The second part presented in chapter 3 comprises an extensive investigation of the broiler chicken microbiota composition in digesta and mucosa of individual samples under varying supplementation of calcium, phosphorus, and phytase. The dietary impact on the distribution of the microbial communities was studied in the crop, ileum, and caecum through illumina sequencing of the 16S rRNA gene. One important outcome was the high variability in the microbial composition between individual samples. Significant differences were observed between the digesta and mucosa samples, supporting the hypothesis that being close to the host, mucosa-associated communities show a different composition. A calcium effect on the performance was observed, where values for body weight gain and feed conversion were lower in comparison to the other treatments. Microbial communities in the crop mucosa revealed a dietary effect, while in the digesta samples no significant changes were seen. Regarding the ileum mucosa, there was an effect of P addition on the microbial distribution. As expected, caeca-derived samples showed an increase in the diversity indexes when compared to the ileum and crop and butyrate producers were detected in higher abundance. A lower microbial diversity in the crop was linked to lower growth performance regarding the supplementation of Ca. Hence, each dietary treatment affected the microbial communities; nevertheless, none of the dietary treatments displayed a consistent effect across the studied gut sections. Additionally, the effects of supplementing different proteases and one phytase on the microbial community of the ileum of broiler chickens was assessed. Thus, the specific aim of chapter 4 was to determine how enzyme supplementation affects the microbiota composition in the ileum of broilers and whether these effects were related to differences in pre-caecal AA digestibility. Three different protease sources at a low and high level were included. The microbial taxonomy was assessed through 16S rRNA gene Illumina amplicon sequencing. Performance results revealed a significant increase in growth and feed efficiency in broilers fed with phytase only and the high dosage of protease C, in comparison to the control. Most of the AA showed a significant difference between the control diet and protease C at high dosage and phytase diets. Effects on microbiota composition were observed at the genus level for some protease and phytase supplementations. The genera Streptococcus, Lactobacillus, and uncultured Clostridiaceae were responsible for these differences. This study demonstrates that effects of enzyme supplementation were evident in the terminal small intestine microbiota composition, and, to a lesser extent, in pc AA digestibility. However, the changes in microbiota composition and pc AA digestibility could not be correlated which may indicate the absence of a causal relationship. Finally, an outlook with metagenome sequencing is presented in chapter 5, to further characterize the result of feeding strategies. Metabolism information, essential to microbial activities registered 50% of abundant genes in the supplemented diets while being reduced to 40% in the control samples Phosphatases pathways and butyrate production increased in the supplemented diets while calcium signaling pathway was higher in the control. In conclusion, within this project a method of standardization to study the microbiota along the gastrointestinal tract of broiler chickens was successfully established. The obtained results revealed a significant impact of both, enzyme and mineral supplementation in the individual sections of the GIT. Also, it was proved that even if the GIT works as an interconnected system, its compartmentalization creates different environmental conditions which influence the microbiota. This study provides insights into the responses of the bacteria and their functionality which were stimulated by the feed supplementations.
Mechanismen der Mastzellaktivierung durch gram-negative Bakterien und Bakterienprodukte aus der Darmflora.
(2006) Krämer, Sigrid; Bischoff, Stephan C.
The role of mast cells (MC) as effector cells in IgE dependent processes like the type 1 allergy has been known for a long time. During the decade, it has been shown that MC are also involved in other pathophysiological processes such as mucosal polyposis, rheumatoid arthritis, inflammatory bowl disease, tissue fibrosis, and atherosclerosis. Furthermore, MC play an important role in the regulation of host defense against microbes, tissue remodeling processes, and neuro-immunology-interaction. The first aim of the present study was to clarify the question whether human intestinal MC express toll-like receptors (TLR), which recognize conserved bacterial and viral components, and can MC be activated through TLR-ligands. The second major focus of the present study was to investigate if the stimulation of human intestinal MC with different E. coli and Shigella strains, respectively, results in an activation of MC and to identify the underlying mechanism(s). Accordingly, human intestinal MC were isolated from surgery tissue with a mechanical and enzymatical protocol. The purity of the MC cultures used in all experiments was between 98 and 100% which was achieved by positive selection (MACS). We could show, that human intestinal MC express mRNA for TLR 1, 2, 3, 4, 5, 6, 8, and 9. However, neither the stimulation with LPS (lipopolysaccharide, TLR 4 ligand), LTA (lipoteichoic acid, TLR 2 ligand), Zymosan (TLR 2 ligand), poly I:C (polyinosinic-polycytidylic acid, TLR 3 ligand), R848 (TLR 7/8 ligand), CpG (C poly G oligo-desoxy-nucleotide, TLR 9 ligand) and non CpG, respectively resulted in a release of histamine, leucotriens, TNF-alpha, or IL-8. Furthermore, mRNA expression levels of TNF-alpha and IL-8 were not induced by any of the treatments. Similar results where found when human intestinal MC were stimulated with E. coli (O101:H-) isolated from human faeces or the probiotic strain E. coli Nissle 1917. Even after stimulation with pathogenic bacteria strains such as the invasive S. flexneri M90T and the fimbriated E. coli, respectively, no induction of any of the parameters mentioned above was found. However, E. coli strains activate the intracellular signal molecule and transcription factors ERK1/2, c-Fos, and AP1, but this activation failed to induce a complete immune answer. In contrast, the hemolytic E. coli stains ATCC 25922 and ATCC 35218 provoked strong activation of intestinal MC. Using the isogenic hemolysin negative E. coli mutants and the hemolysin positive transformants of the probiotic E. coli Nissle 1917 it was shown, that human intestinal MC are sensitive target cells for E. coli alpha hemolysin. Stimulation of MC with sublytic concentration of hemolysin resulted in an induction of TNF-alpha, IL-3, IL-5, IL-6, IL-8 mRNA expression, the release of histamine as well as leucotrien. This activation was found to be regulated by calcium dependent signal cascades. Inhibition of intracellular signal molecules showed that the activation depends on L-typ calcium channels, calcineurin, NFAT and NFkappaB. Prolonged infection with hemolytic E. coli strains resulted in lysis of intestinal MC indicating a biphasic activation of hemolysin.
The effect of aging in the murine gut microbiome
(2020) Hernández Arriaga, Angélica; Camarinha-Silva, Amélia
Aging is characterized by several physiological changes. During the lifespan, the biological systems from the body of humans and other animals remain dynamic. Throughout the early stages of life, the microbiome develops into a complex ecosystem with thousands of species. Variations related to diet, environmental changes, medications affect the diversity and composition of the microbiota through the lifespan. Some old individuals with higher incidence of chronic diseases have a loss of the stability of the microbiome and an imbalance occurs between the different colonizers of the gut, also named dysbiosis. One of the most distinctive changes occurring with age is the prevalent low grade inflammation, which is named inflamm-aging. This not only changes the microbial composition of the GIT but also affects the permeability. Murine models are well established and help us to understand the complex dynamics between the host and the microbial communities inhabiting the gastrointestinal tract. These models allow us to analyze microbial communities from tissue and mucosa, from all sections of the gut, which is limited in humans. Methods standardization is an important topic in microbiome research. In chapter 2 it was compared the efficiency of two sample methods, cotton swab and tissue biopsy, in characterizing the mouth microbiota. In recent years, the mouth microbiome is being seen as a diagnostic tool for not only oral diseases but also systemic diseases. As physiological changes occur with aging, the microbiome from the mouth is affected and there is an increase of pathogens present in the oral surfaces. In murine models, cotton swab is a common tool used for sampling the microbiome of the oral cavity. In our study, we observed similar microbial community structure using both methodologies. However, the species Streptococcus danieliae, Moraxella osloensis, and some unclassified members of Streptococcus were affected by the different sampling procedures. In this trial, we included mice at two different ages, 2 months old being considered young and 15 months old considered middle aged mice. We observed changes in the genera Actinobacillus, Neisseria, Staphylococcus, and Streptococcus related to the age of the animal and the sampling type. These results showed the importance of sampling standardization in microbiome research and that age has a strong effect on the microbial ecology of the oral cavity. In chapter 3, it was studied the bacterial communities from duodenum and colon of mice at 2, 15, 24 and 30 months of age in combination with the results of the expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function. Besides, in this chapter were also assessed the indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissues. At 24 and 30 months of age there was an increase in inflammation, they developed fibrosis and the levels of endotoxin in plasma were higher. Regarding changes in the microbiome, the duodenum had more changes than the colon related to age. Allobacullum, Bifidobacterium, Olsenella, Corynebacterium were the genera that differed statistically in the duodenum through the murine lifespan. Fewer changes were observed in the colon, as Allobaculum was the only genus that showed differences between young and old mice. Additionaly, it was analyzed the impact of aging in the active microbial communities of mouth, duodenum and colon at 2, 9, 15, 24 and 30 months of age (chapter 4). Changes were observed at every age and different taxonomical levels, with a greater shift at 15 months of age. This is related to the age of the mice, as at middle age systemic changes related to the aging process start to occur. At old ages, there was an increment of the pathobiontic species Helicobacter hepaticus and Helicobacter ganmani in the duodenum and colon. The oral, duodenal and colonic microbial communities are important pieces of information that can be related to the health status of the host. Research that focuses on assessing the changes in the different niches and not only in the feces, gives a broader overview of the microbial community of the host.