Browsing by Subject "Mikrobiota"
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Publication Comprehensive characterization of microbiota in the gastrointestinal tract of quails and two high yielding laying hen breeds(2023) Roth, Christoph Florian; Camarinha-Silva, AméliaThe 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.Publication Genomic analyses of behavior traits in laying hen lines divergently selected for feather pecking(2021) Iffland, Hanna; Bennewitz, JörnFeather pecking is a longstanding problem in commercial layer flocks. It often causes injured birds and even cannibalism. In the past, hens were beak trimmed to reduce feather pecking. Nevertheless, this procedure is already prohibited in some EU countries. Hence, a solution to this problem is urgently needed. The experimental populations analyzed in this thesis were formed by hens based on a White Leghorn layer strain which were divergently selected for high and low feather pecking since 1995. The first experimental population of this thesis was an F2 cross of about 900 hens which was established of the 10th generation of the pure selection lines. The second population consisted of about 500 hens of the 15th generation of these two lines. The aim of this thesis was to gain further knowledge of the genetic background of feather pecking and its relation to additional behavior traits and the gut microbiome. In chapter one, a novel model to detect extreme feather pecking hens was developed. Therefore, a mixture of two negative binomial distributions was fitted to feather pecking data of the F2 cross. With the estimated parameters, the trait posterior probability of a hen to belong to the extreme feather pecking subgroup (pEFP) was calculated. The fear tests tonic immobility and emerge box were conducted at juvenile and adult age of the hens to relate fearfulness to pEFP. After dichotomization, all traits were analyzed in a multivariate threshold model and subsequent genomewide association studies (GWAS) were performed. The fit revealed that extreme feather peckers made up a proportion of about one third of the hens. The new trait pEFP has a medium heritability of 0.35 and is positively correlated with the fear traits. Breeding for this new trait could be an option to reduce the proportion of extreme feather peckers. An index of fear related traits might serve as a proxy to breed indirectly against pEFP. In chapter two, the model to detect extreme feather pecking hens was applied to the pure selection lines. After calculation of the trait pEFP, GWAS with a subsequent post GWAS analysis were performed. Additionally, to find genomic regions influencing feather pecking, selection signatures were mapped by applying the intra-population iHS and the inter-population FST approach. Mapping of selection signatures revealed no clear regions under selection. GWAS revealed a region on chromosome one, where the existence of a quantitative trait locus (QTL) influencing feather pecking is likely. The candidate genes found in this region are a part of the GABAergic system. Despite the polygenic nature of feather pecking, selection on these candidate genes may reduce the extreme occurrence of it. In chapter three, the relation between agonistic behavior and feather pecking was analyzed. Therefore, the active parts of the traits (delivery of feather pecking, aggressive pecking or threatening) as well as the passive parts (reception of the traits) were considered. These groups of traits were additionally summarized by means of an index formation which led to the two additional traits Activity and Passivity, because all these behaviors are undesired in their excessive manifestations. Moreover, Indices were built by subtracting the passive traits from the respective active traits to obtain the feather pecking index, the aggression index and the threat index. Phenotypic correlations were estimated between all traits which were followed by heritability estimations and GWAS. Feather pecking is significantly positively correlated with the agonistic traits in both lines. The active traits and the feather pecking index show medium heritabilities. Hence, selection on high feather pecking leads to an increase of agonistic behavior whereas the correlation probably depends on the phase of establishing the social hierarchy and might disappear, after a stable ranking is established. GWAS revealed that the heritable traits in this study seem to be typical quantitative traits. Chapter four provides the analyses of the gut microbial composition of the two feather pecking lines, followed by the estimation of microbiabilities for feather pecking and the two agonistic behavior traits, to study the influence of the gut microbiome on behavior. Microbiota samples from digesta and mucosa were taken from ileum and caecum. The microbial communities were determined by using 16S RNA gene sequencing techniques. Although both lines differ significantly in some fractions of their gut microbial composition, the microbial animal effects were mostly negligibly small. Thus, the calculated microbiabilities were close to zero and not significant in both lines and for all traits investigated. Hence, trait variations were not affected by the gut microbial composition in both feather pecking lines. The thesis ends with a general discussion where additional results of a meta-analysis of pEFP and breeding strategies against feather pecking are considered.Publication The effect of aging in the murine gut microbiome(2020) Hernández Arriaga, Angélica; Camarinha-Silva, AméliaAging 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.Publication The porcine intestinal microbiota : studies on diversity and dietary impact(2018) Burbach, Katharina; Seifert, JanaThe entirety of microbial communities within the gastrointestinal tract is referred to as intestinal microbiota and is predominantly composed of bacteria. Interactions between the microbiota, the host and the diet are essential for maintaining a healthy and functional intestinal ecosystem. The overarching aim of this thesis was the characterization of the porcine intestinal microbiota and further to enhance knowledge about the effects of varying diets. High-throughput sequencing of the 16S rRNA gene facilitates exploration of the taxonomic composition of the microbiota. However, the respective findings may be impaired by methodological variations. Thus, within this thesis, commercial DNA extraction kits are evaluated for their suitability in porcine microbiota analysis. The tested extractions yield into variations of quantity and quality of DNA. The DNA extracts are further used to elucidate the structure of the microbiota by a rapid fingerprinting (Terminal Restriction Fragment Length Polymorphism) and high-resolution sequencing (Illumina amplicon sequencing). While different variable regions of the 16S rRNA gene vary in the taxonomical resolution, sequencing analyses exhibit a good comparability of the two regions V1-V2 and the V5-V6. Furthermore, the microbiota profiles reveal a high consistency by the fingerprinting and sequencing approach but are distinguished by the different DNA extraction kits. Based on criteria of DNA extraction and the depicted microbiota composition, it is recommended to use the FastDNA SPIN Kit for Soil for further analysis of porcine intestinal microbiota. Subsequently, these methodological findings are applied to investigate the impact of varying diets. Illumina amplicon sequencing of the V1-V2 region of the 16S rRNA gene reveals different microbiota structures when diets are solely composed of rye or triticale. Besides the taxonomic analyses of ileal digesta and fecal samples, the concentrations of bacterial metabolites in feces are determined. In summary, rye promotes an increased abundance of saccharolytic bacteria like Lactobacillus, Bifidobacterium, and Prevotella and results in higher concentrations of bacterial metabolites in fecal samples. In contrast, a diet based on triticale is associated with an increased abundance of Clostridium sensu stricto, which may indicate an enhanced cellulolytic potential of the microbiota. When the crude protein content is increased (18%), compared to a lower content (14%), an increased abundance of Lactobacillus is demonstrated in microbiota of ileal digesta samples. However, the content of crude protein did not affect the overall microbiota significantly. In addition, dietary supplementation with probiotic Bacillus spp. shows no effect. In conclusion, these dietary effects on microbiota are considered together with results of a protein digestibility analysis. Moreover, an impact of dietary calcium and phosphorus in combination with different sources of dietary protein is analyzed by fingerprinting approach of digesta samples. Here, the content of calcium-phosphorus shows significant effects on the microbiota of caecal digesta and the putative identities of discriminative variables are determined by a cloning-sequencing approach. Similar, 16S rRNA gene sequencing reveals a significant impact of dietary calcium-phosphorus on the overall fecal microbiota without indicating specific discriminating variables. In combination with the results of a meta-proteomic approach, a gradual adaptation on dietary changes is indicated and consequently, a prolonged adaptation time of three to four weeks is recommended for diet-microbiota studies. This thesis includes a comprehensive analysis of the microbiota across and along the gastrointestinal tract of piglets and explores the dietary inclusion of four levels of insect larvae meal. Feeding insects represent an alternative source of dietary protein, whereby the increased content of chitin indicates a potential shift in microbiota composition compared to a control diet. However, in this case, the structural analysis demonstrates no effects on the overall microbiota’s structure. However, a pairwise comparison between diets reveals significant effects on the microbiota of digesta samples of the small intestine. Dietary inclusion of 5% insect meal increases the abundance of Lactobacillus, whereas the control treatment promotes Bifidobacterium. In conclusion, the results of the present thesis emphasize the importance of standardization within 16S rRNA gene based studies of the porcine intestinal microbiota. Furthermore, the necessity of studying various sampling sites combined with multidisciplinary approaches is demonstrated.Publication The production of melezitose in honeydew and its impact on honey bees (Apis mellifera L.)(2021) Seeburger, Victoria; Hasselmann, MartinHoneydew honey is a honey type which is of high economic importance in Europe. Phloem sap feeding insects of the order Hemiptera (true bugs) excrete honeydew, the key component of honeydew honey. Beekeepers move their hives between forest regions so that their bees can process the honeydew into honey. In case of high osmolality in the phloem sap of the hemipterans’ host trees, they counteract osmotic pressure by osmoregulation and produce oligosaccharides such as melezitose. Melezitose-rich honeydew honey is a major issue for beekeepers; it crystallises and obstructs the combs, leading to an economical loss. Nevertheless, precise analyses of the conditions of the occurrence of melezitose have not been realised. Furthermore, it is not known which impacts the trisaccharide has on honey bee health and the honeydew flow disease documented in beekeepers’ journals can have one explanation in the nutrition on melezitose. In order to determine influence factors for the emergence of melezitose, more than 600 honeydew droplets from defined honeydew producer species were collected under different environmental conditions (hemipteran species (host tree specific), natural area, air temperature, relative humidity, altitude, time of the year and of the day) between 2016 and 2019. The sugar spectra were analysed by high performance anion exchange chromatography with pulsed amperometric detection. To obtain the impact of melezitose on honey bee health, additional feeding experiments with daily evaluation of food uptake, gut-body weight ratio and mortality have been realised between 2017 and 2019. Additionally, comprehensive 16S rRNA Illumina sequencing of the gut microbial community has been performed. Remarkable differences could be found in the amount of melezitose between honeydew samples collected from different honeydew producer species and according to different environmental conditions. Air temperature increases and decreases in relative humidity increased the melezitose production in honeydew by the observed seven hemipteran species. Both, scale insect species on Picea abies and aphid species on Abies alba produced significantly less honeydew containing melezitose than aphid species on Picea abies. Additionally, honeydew with increased melezitose content was significantly more frequent collected in natural areas with limited water reservoir capacities, at higher altitudes and years with low precipitation. All results lead to the conclusion that hemipteran species produce more melezitose when the host trees have less access to water, increasing the osmolality of the phloem sap and indirectly enhancing the osmoregulation with producing melezitose by hemipteran species. Bees fed with melezitose showed increased food uptake and higher gut-body weight ratio than the control groups. Furthermore, melezitose feeding caused disease symptoms such as swollen abdomen, abdomen tipping and impaired movement and a significantly higher mortality than in control groups. Gut microbiota analyses indicated a shift of the bacterial species Lactobacillus Firm-4 and Lactobacillus kunkeei in favour of Lactobacillus Firm-5 in melezitose fed bees. This PhD project provides the important knowledge about the indicators that point out an enhanced melezitose production. This is a valuable contribution to design a warning system for beekeepers that will help to prevent harmful nutrition for honey bees or crystallised honey in the future by timely removal of bee colonies from local regions at risk. Additionally, feeding experiments point out the high effort that is required for the degradation process of the large-molecule melezitose. This effort might lead to a higher uptake of food, heavier guts, shorter lifespan and a higher susceptibility to intestinal diseases. Finally, an evidence was presented that the lactic acid bacteria of the gut microbiota are significantly involved in the digestion of melezitose.