Chicken intestinal development in health and disease : transcriptomic and modeling approaches

Intestinal health is an important condition for sustainable animal production. Since it is known that there is significant variation in intestinal health and functionality, there is much to gain in this respect. However, to fully exploit the biological potential of the animal’s gastro-intestinal tract, the mechanism and regulation of major intestinal processes need to be unraveled first. In addition, identification of key components and processes involved in intestinal adaptation mechanisms may help to identify internal and external factors that influence the health and functioning of the gut. Improved knowledge in this area may contribute in defining rational strategies to improve sustainable animal production. Traditionally research used reductionist approaches and focused on specific components or isolated processes related to intestinal functioning. However, the recent developments in the areas of genomics and computational sciences provide tools and methods that allow studying the system of the gut as a whole. In this thesis we have set first steps in the use of such Systems Biology approaches towards the identification of the key components and processes involved in intestinal functioning and health. We investigated molecular processes associated with gut development in chickens under two extreme contrasting conditions. We used an infection with Salmonella immediately after hatch and control animals to create the two contrasting phenotypic conditions. We used microarray-based genome-wide mRNA profiling to identify patterns of gene expression and cellular processes associated with each conditions. Comparisons between the two conditions and the application of modeling approaches revealed genes, groups of genes, molecular pathways, gene networks, and high level regulators of system behavior. We also used a mathematical modeling approach to describe the dynamics of cellular components of the immune system and their corresponding interactions under the same two contrasting conditions. We identified different temporal gene expression profiles associated with morphological, functional and immunological processes. Several of these processes differed between the two contrasting conditions, whereas others were not affected be the experimental treatments. By inferring gene association networks, we observed that an infection with Salmonella considerably changes the behavior of intestinal tissue as well as the regulation of the underlying molecular processes. For each contrasting condition, we identified a specific set of potential high-level regulator genes (hubs). We hypothesize that these hubs are steering systems behavior. Bioinformatic analysis of the hubs suggested that the disturbance with Salmonella is associated with a shift from transcriptional regulation in the non-disturbed tissue to cell-cell communication in the disturbed tissue. Furthermore, the generated mathematical model describes the dynamics of the cellular components of the immune system as well as the dynamics of the invading pathogen well. The model was able to predict the cellular immune response of the host against an invading pathogen. We developed basic knowledge of (molecular) processes that are associated with different physiological conditions of intestinal tissue and we acquired global views on adaptation mechanisms of the intestine, including the regulation thereof. This information can be used to formulate new hypotheses about behavioral aspects of the gut, for the discovery of new biological mechanisms, and ultimately for the development of tools and rational strategies to improve intestinal functionality and health, either via diet and/or the host genotype. Such developments are urgently required to diminish the incidence and impact of intestinal diseases in farm animal species and to reduce the use of antibiotics in animal husbandry. </p

Early-life environmental variation affects intestinal microbiota and immune development in new-born piglets

Background - Early-life environmental variation affects gut microbial colonization and immune competence development; however, the timin Early-life environmental variation affects gut microbial colonization and immune competence development; however, the timing and additional specifics of these processes are unknown. The impact of early-life environmental variations, as experienced under real life circumstances, on gut microbial colonization and immune development has not been studied extensively so far. We designed a study to investigate environmental variation, experienced early after birth, to gut microbial colonization and intestinal immune development. Methodology/Principal Findings - To investigate effects of early-life environmental changes, the piglets of 16 piglet litters were divided into 3 groups per litter and experimentally treated on day 4 after birth. During the course of the experiment, the piglets were kept with their mother sow. Group 1 was not treated, group 2 was treated with an antibiotic, and group 3 was treated with an antibiotic and simultaneously exposed to several routine, but stressful management procedures, including docking, clipping and weighing. Thereafter, treatment effects were measured at day 8 after birth in 16 piglets per treatment group by community-scale analysis of gut microbiota and genome-wide intestinal transcriptome profiling. We observed that the applied antibiotic treatment affected the composition and diversity of gut microbiota and reduced the expression of a large number of immune-related processes. The effect of management procedures on top of the use of an antibiotic was limited. Conclusions/Significance - We provide direct evidence that different early-life conditions, specifically focusing on antibiotic treatment and exposure to stress, affect gut microbial colonization and intestinal immune development. This reinforces the notion that the early phase of life is critical for intestinal immune development, also under regular production circumstances. g and additional specifics of these processes are unknown. The impact of early-life environmental variations, as experienced under real life circumstances, on gut microbial colonization and immune development has not been studied extensively so far. We designed a study to investigate environmental variation, experienced early after birth, to gut microbial colonization and intestinal immune development. Methodology/Principal Findings To investigate effects of early-life environmental changes, the piglets of 16 piglet litters were divided into 3 groups per litter and experimentally treated on day 4 after birth. During the course of the experiment, the piglets were kept with their mother sow. Group 1 was not treated, group 2 was treated with an antibiotic, and group 3 was treated with an antibiotic and simultaneously exposed to several routine, but stressful management procedures, including docking, clipping and weighing. Thereafter, treatment effects were measured at day 8 after birth in 16 piglets per treatment group by community-scale analysis of gut microbiota and genome-wide intestinal transcriptome profiling. We observed that the applied antibiotic treatment affected the composition and diversity of gut microbiota and reduced the expression of a large number of immune-related processes. The effect of management procedures on top of the use of an antibiotic was limited. Conclusions/Significance We provide direct evidence that different early-life conditions, specifically focusing on antibiotic treatment and exposure to stress, affect gut microbial colonization and intestinal immune development. This reinforces the notion that the early phase of life is critical for intestinal immune development, also under regular production circumstances. Figure

Shift in chicken intestinal gene association networks after infection with Salmonella

A primary infection of Salmonella enteritidis causes a spatial-temporal dependent change in the gene expression patterns in the intestine of chickens (Gallus gallus). This is the result of a dynamic intestinal response to adapt to the altered environment and to optimize its ‘health’ and functionality under the new circumstances. By inferring gene association networks (GANs), the complexities of and changes in biological networks can be uncovered. Within such GANs highly interacting (hub) genes can be identified, which are supposed to be high-level regulators connected to multiple processes. By exploring the intestinal expression of genes differing between control and Salmonella infected chicken in a time-dependent manner differences in GANs were found. In control chickens more developmental processes were observed, whereas in infected chickens relatively more processes were associated to ‘defense/pathogen response’. Moreover the conserved protein domains of the identified hub genes in controls were nuclear-associated, whereas hub genes in infected chickens were involved in ‘cellular communication’. The shift in topology and functionality of the intestinal GANs in control and Salmonella infected animals and the identification of GAN-specific hubs is a first step to understand the complexity of biological networks and processes regulating intestinal health and functionality under normal and disturbed conditions

Gene expression patterns associated with chicken jejunal development

Jejunal development occurs in a spatio-temporal pattern and is characterized by morphological and functional changes. To investigate jejunal development at the transcriptomic level, we performed microarray studies in 1–21-day-old chickens. Nine gene clusters were identified, each with a specific gene expression pattern. Subsequently, groups of genes with similar functions could be identified. Genes involved in morphological and functional development were highly expressed immediately after hatch with declining expression patterns afterwards. Immunological development can be roughly divided based on expression patterns into three processes over time; first innate response and immigration of immune cells, secondly differentiation and specialization, and thirdly maturation and immune regulation. We conclude that specific gene expression patterns coincide with the immunological, morphological, and functional development as measured by other methods. Our data show that transcriptomic approaches provide more detailed information on the biological processes underlying jejunal developmen

Effects of Salmonella on spatial-temporal processes of jejunal development in chickens

To study effects of Salmonella enteritidis on morphological and functional changes in chicken jejunal development, we analysed gene expression profiles at seven points post-infection in 1-21 day-old broiler chickens. Nine clusters with different gene expression patterns were identified, and the genes in each cluster were further analyzed by a functional annotation clustering method (DAVID). Functional and morphological developmental processes dominated in all the nine clusters. Salmonella infection caused delays in several intestinal-morphological processes, whereas functional metabolic processes occurred in a similar spatial-temporal frame compared to normal jejunum development. A clear difference between normal developing- and Salmonella disturbed jejunum was the higher expression of genes involved in cell turn-over at early stages in the infected jejunum. Surprisingly, we found no clustered immune related processes in the infected birds. To compare the immunological processes between control and Salmonella infected chickens, the gene expression data was superimposed on known immunological KEGG pathways. Furthermore an in-depth analysis on the immune gene level was performed. As expected, we did find immunological processes in the Salmonella infected jejunum. Several of these processes could be verified by immunohistochemistry measurements of different immunological cell types. However, the well-ordered spatial-temporal development of the immune system, as observed in control non-infected animals, was completely abolished in the infected animals. Several immunological processes started much earlier in time, whereas other processes are disorganised. These data indicate that normal morphological and immunological development of jejunum is changed dramatically by a disturbance due to Salmonella infection. Due to the disturbance, the well-organized spatial-temporal development of morphological processes are delayed, those of the immunological development are scattered, whereas metabolic functional processes are almost not affected. This demonstrates the flexibility of developmental processes in the broiler chicken intestine. (C) 2010 Elsevier Ltd. All rights reserved

Differences in the early response of hatchlings of different chicken breeding lines to Salmonella enterica serovar Enteritidis infection

Poultry products are the major source of food-borne Salmonella infection in humans. Broiler lines selected to be more resistant to Salmonella could reduce the transfer of Salmonella to humans. To investigate differences in the susceptibility of newly hatched chicks to oral infection with Salmonella enterica serovar Enteritidis, 3 commercial broiler lines (A, B, and C) were infected immediately after hatch and compared to healthy controls at 0.33, 1, and 2 d postinfection. Weight, bacteriological examination, and the jejunal influx of CD4, CD8, TCRaß, TCR¿d, and KUL01 (macrophages and dendritic cells) cells that are positive was investigated. In addition, the jejunal transcriptional response was analyzed using whole-genome chicken cDNA arrays. Salmonella colony-forming unit counts from cecal content and liver revealed that Salmonella enterica entered the body at 0.33 d postinfection. Broiler line A appeared most susceptible to intestinal colonization and the systemic spread of Salmonella. In addition, the Salmonella-induced jejunal influx of macrophages in this line showed a clear increase in time, which is in contrast to lines B and C. On the other hand, all lines showed a peak of CD4+ cells at 1 d postinfection when infected chicks were compared to control chicks. The transcriptional response of line A clearly differed from the responses in lines B and C. Functional analysis indicated that the majority of the differentially expressed genes at 0.33 d postinfection in line A were involved in cell-cycle functions, whereas at 2 d postinfection the majority of the differentially expressed genes could be assigned to inflammatory disorder, differentiation and proliferation of (T) lymphocytes. These data indicate that hatchlings of different broiler lines differ in their systemic spread of Salmonella and suggest that intestinal barrier functions, as well as immunological responses, may be the underlying factors. We hypothesize that the differences between genetic chicken lines divergent in their response to Salmonella infection at a young age include developmental differences of the gut

Middle Palaeolithic artefact migration due to periglacial processes; a geological investigation into near-surface occurrence of Palaeolithic artefacts (Limburg-Eastern Brabant coversand region, the Netherlands)

The original distribution pattern of Middle-Palaeolithic artefacts may be affected by tectonic movement, sedimentation and periglacial processes. This is e.g. the case in the coversand area of Limburg and Eastern Brabant (NL), where the occurrence of numerous finds in a SW-NE trending zone across the Roer Valley Graben is considered enigmatic. In order to elucidate the processes affecting the spatial distribution and the chance of recovery of such artefacts, we investigated a site in Nederweert. At this site, several Middle-Palaeolithic artefacts had been recovered earlier from unexpectedly shallow depths. A test pit profile and grain size analyses revealed that the shallow sediments at this site have been affected by intense, multi-phase cryoturbation, which has deformed the sand and loam layers and partially mixed them thoroughly. As a result, optically stimulated luminescence dating of these sediments yielded widely scattered single-aliquot equivalent dose distributions. Using a Finite Mixture Model (FMM), it was estimated that cryoturbation caused mixing of sediments deposited between 12 and 50 ka with sediment grains deposited between 60-150 ka. The latter material is probably the original context of the Middle-Paleolithic artefacts. Apparently, cryoturbation and potentially other periglacial processes have transported artefacts closer to the surface. Based on these results, we suggest that the occurrence of MiddlePalaeolithic artefacts is caused by (1) the tectonically-induced spatial distribution of layers of this age and (2) periglacial processes having caused migration of artefacts towards the surface. Although periglacial processes may facilitate finding Middle Palaeolithic artefacts, they may severely disturb the original context to such an extent that Middle Palaeolithic sites can no longer be identified. The results of this study form a basis for improving the Indicative Map of Archaeological Values that is used to predict the presence of archaeological sites. The insights gained are also relevant to other areas where Middle-Palaeolithic sites are affected by periglacial processes.</p

De midden-paleolithische vuurstenen artefacten van het Rosveld te Nederweert: Rapportage Archeologische Monumentenzorg 171

Tussen 2000 en 2003 heeft het Archeologisch Centrum van de Vrije Universiteit in Amsterdam (ACVU) grootschalig archeologisch onderzoek uitgevoerd op het Rosveld ten zuiden van Nederweert in de provincie Limburg. Het onderzoek was gericht op sporen van bewoning en begravingen uit het tijdvak Late Bronstijd-Volle Middeleeuwen (1100 v.Chr. tot 1300 n.Chr.) en niet op het opsporen en onderzoeken van vindplaatsen met (vuur)stenen artefacten. Desondanks is een veertigtal artefacten verzameld uit het Midden-Paleolithicum (300 000-35 000 jaar voor heden), het Laat-Paleolithicum (12 500-10 000 jaar voor heden) en het Midden-Neolithicum (4200-3400 v.Chr.). Intrigerend zijn vooral de midden-paleolithische vondsten, omdat het opgegraven gebied in de Roerdalslenk ligt waar vondsten van die ouderdom niet aan of nabij het oppervlak verwacht worden. De opgraving lag in een zone waar nat-eolisch sediment (‘Oud Dekzand’) nabij de oppervlakte voorkomt. Het Oud Dekzand is echter vanaf ca. 28 000 jaar geleden afgezet, terwijl de middenpaleolithische artefacten dateren van vóór ca. 35 000 jaar geleden. Er is dus sprake van een discrepantie tussen de veronderstelde datering van het dekzand ter plaatse en de typo-technologische datering van de artefacten. Hiervoor zijn drie verklaringen denkbaar. De eerste is dat de midden-paleolithische artefacten afk omstig zijn uit dieper gelegen lagen en naar boven zijn gehaald door bijvoorbeeld het graven van diepe sporen zoals waterputt en en (paal)kuilen (de zogenoemde opspit). De tweede mogelijke verklaring is dat de artefacten uit diepere lagen afkomstig zijn en door processen als cryoturbatie en opvriezing naar boven zijn getransporteerd. Een derde verklaring of hypothese is dat het dekzand ter plaatse niet dateert uit de periode 28 000-13 000 jaar voor heden, maar veel ouder is. Met behulp van verder ontwikkelde dateringstechnieken is het mogelijk om de afzett ingen absoluut te dateren. Hiervoor is in 2006 een profielput gegraven en zijn van verschillende niveaus OSL-monsters genomen. Daarnaast zijn van verschillende niveaus korrelgrootteanalyses uitgevoerd. Op grond van de onderzoeksresultaten en de verwachte ouderdom van de artefacten (ouder dan 35 000 jaar) concluderen we dat de artefacten uit de diepere lagen (meer dan 1,8 m onder het maaiveld) afk omstig zijn. Het is zeer waarschijnlijk dat de cryoturbate processen die hebben geleid tot menging van sediment uit deze diepere laag tot dicht bij het huidige maaiveld ook de middenpaleolithische artefacten mee omhoog hebben gewerkt. Deze studie toont aan dat het mogelijk is om op basis van korrelgrootteverdelingen de mate van menging tussen verschillende sedimentaire lagen te reconstrueren. Om in de toekomst betrouwbaardere uitspraken te doen over de ouderdom van midden-paleolithische (vuur)stenen artefacten zal het nodig zijn om deze gegevens beter te integreren met OSL-metingen. Voor vervolgonderzoek zou het bijvoorbeeld aan te raden zijn om: 1 diepere profielen te graven (voor bepalen potentiële herkomst zand); 2 grotere aantallen korrelgrootteanalyses te doen, te bestuderen hoe de korrelgrootteverdeling kan zijn ontstaan uit verschillende pakketten die qua korrelgrootte elk min of meer homogeen waren en de onderlinge verhoudingen terug te rekenen; 3 met behulp van micromorfologie de menging van verschillende pakkett en in detail te bestuderen; 4 OSL-dateringen op verschillende korrelgroottefractie

De midden-paleolitische vuurstenen artefacten van het Rosveld te Nederweert

Met lit. opg