Plasticity of intestinal gene expression profile signatures reflected by nutritional interventions in piglets

Background: Immediately after birth, the porcine intestine rapidly develops morphologically, functionally, and immunologically. The jejunum, the second part of the small intestine, is of importance for nutrient uptake and immune surveillance. To study the early postnatal development of the jejunum, a meta-analysis was performed on different transcriptomic datasets. These datasets were acquired from different experimental in-house studies or from experiments described in literature of porcine jejunum mucosa. Gene expression was measured under different experimental interventions, such as nutritional intervention, at various time-points (age). Results: The studies included in the meta-analysis provided gene expression data for various time-points (piglet ages) for piglets that had received a treatment versus control piglets. In separate studies, treatments were administered to the sow (i.e. amoxicillin), or nutritional supplementation directly to the piglets with medium chain fatty acids (MCFAs), and oral administration of fructooligosaccharides (FOS) or a high dose of zinc-oxide, respectively. In the meta-analysis, genes were grouped into 16 clusters according to their temporal gene expression profiles for control piglets, i.e. the changes of gene expression level over time. Functional analysis showed that these temporal profile clusters had different dominant processes, such as immune related processes or barrier function. Transcriptomics data of treatment piglets was subsequently superimposed over the control temporal profiles. In this way we could investigate which temporal profile clusters (and which biological processes) were modulated by the treatments. Interestingly, not all 16 temporal profiles were modulated. Conclusions: We showed that it is possible to re-use (publicly available) transcriptomics data and produce temporal gene expression profiles for control piglets with overexpression of genes representing specific biological processes. Subsequently, by superimposing gene expression data from (nutritional) intervention studies we observed deviations from some of these reference profile(s) and thus the plasticity of the system. By employing this meta-analysis approach we highlighted the importance of birth and weaning and the underlying biological processes.</p

Factors Influencing the Succession of the Fecal Microbiome in Broilers

Health and performance are important aspects in the broiler industry. Underlying complex traits like total mean weight and feed efficiency are polygenic and related to genetic background and an association of the microbiota with these traits has been identified. Whether this association is also reflected in the fecal samples of broilers is not extensively investigated. The objective of this study was to investigate to what extend diet, genetics, and environment influence the fecal microbiome composition during the life time of broilers. Two experiments were performed, in the first experiment the focus was on investigating if a European (EU) or United States (USA) diet effects the fecal microbiota in a commercial line (Cobb500). Whereas in the second diet (EU/USA) and lines with a genetic background (EU/USA) were investigated in relation to the fecal microbiota. In the first experiment we observed a significant effect in commercial broiler line (Cobb500) of the 3-way interaction for age by feed by sex on Total Mean Weight (TMW), and the 2-way interaction of age by feed for Feed Conversion Ratio (FCR). For the microbiota data, we observed differences in alpha-diversity for Age. When comparing the diets on a time-point, this resulted in significant differences for Observed species at day 21 and for Observed species, Shannon index, and Pielou's evenness at day 35. In the beta-diversity, a significant effect of age by feed interaction was observed. Two genera were significantly different in feces of broilers between diets, i.e. Streptococcus on day 7 and Bilophila on day 21. In the second experiment we observed only a significant effect for the main effect age on TMW. Alpha-diversity showed a significant increase for all three measures for age. Furthermore, a significant effect of environment was observed in the Observed species. This effect of environment was also observed in the beta-diversity, where a significant effect for age and environment was observed. This environmental effect was not expected, because here environment represents two different compartments within the same stable, unfortunately it was not possible to perform further down-stream analyses. This research shows the different aspects (feed, sex, genetics, and environment) influence complex traits, like TMW and FCR and are affecting the fecal microbiome. We have shown that interventions, like feed and the effect on microbiome, are reproducible between experiments. Moreover, these results with these two genetic divers chicken lines suggest that the succession of the fecal microbiota was independent of genetic background.</p

Antibiotics in 16-day-old broilers temporarily affect microbial and immune parameters in the gut

Animal health benefits from a stable intestinal homeostasis, for which proper development and functioning of the intestinal microbiota and immune system are essential. It has been established that changes in microbial colonization in early life (the first 2 wk post hatch) impacts the functioning of the adult gut and the associated crosstalk between microbiota and intestinal mucosal cells. The aim of the present study was to study the effect of the administration of antibiotics later in life (d 15 to 20 post hatch) on microbiota and immune parameters. For this purpose, chickens received from 15 d post hatch during 5 d amoxicillin or enrofloxacin through their drinking water. Before and at 6, 16, and 27 d after start of the administration of antibiotics, the composition of the microbiota in the jejunum was determined using a 16S ribosomal RNA gene-targeted DNA microarray, the CHICKChip. At 6 d after the start of the administration of the antibiotics, the composition and diversity of the microbiota were affected significantly (P < 0.05), but this change was small and observed only temporarily since differences disappeared at 16 d after initiating treatment with amoxillin and at 27 d after starting treatment with enrofloxacin. Intestinal morphology and development were not visibly affected since there were no differences between villus/crypt ratios and numbers of PAS+ and PCNA+ cells in the duodenum and jejunum at any time point. At 16 d after the start of antibiotic administration, the number of CD4+ T-cells and CD8+ T-cells in the duodenum was lower compared to the control animals; however, this difference was not significant. At some time points, significant differences (P < 0.05) were observed among the groups to locally expressed IL-8, IL-1β, IFN-γ, IL-2, and IL-4 mRNA. However, this effect was not long lasting, as differences that were observed at 16 d after starting the treatment had disappeared at 27 d after treatment was started. The results of this study indicate that later in the broiler's life, antibiotics only temporarily affect intestinal microbial and immune parameters

Successional Dynamics in the Gut Microbiome Determine the Success of Clostridium difficile Infection in Adult Pig Models

Clostridium difficile infections (CDI) are a major cause of antibiotic-associated diarrhea. It is hypothesized that CDI develops due to the antibiotic-induced disruption of the intestinal microbial community structure, which allows C. difficile to flourish. Here, we pre-treated weaned pigs with the antibiotics Clindamycin or Ciprofloxacin for 1 day, and subsequently inoculated them with a human and pig enteropathogenic C. difficile strain 078 spores. Body temperature, clinical signs of disease, and the fecal microbiome were monitored daily for 15 days. Clindamycin had a stronger effect on the pigs than Ciprofloxacin, resulting in drastic shifts in the fecal microbiome, decreases in microbial diversity and significant increases in body temperature, even in the absence of C. difficile. Fecal shedding of C. difficile was detectable for 3 and 9 days in Ciprofloxacin and Clindamycin treated pigs inoculated with C. difficile, respectively, and in both cases decreased cell proliferation rates were detected in colon tissue. The timing of C. difficile shedding coincided with the decrease in a large cluster of Firmicutes following Clindamycin treatment, a pattern which was also independent of C. difficile inoculation. The observed community patterns suggest that successional dynamics following antibiotic treatment facilitate C. difficile establishment. The similarities between the microbiome responses observed in our study and those previously reported in CDI-infected humans further support the utility of adult pigs as models for the study of CDI.</p

Validation of non-invasive sensor technologies to measure interaction with enrichment material in weaned fattening pigs

Measuring animal behaviour is important in the assessment of animal welfare. When interaction with the enrichment material (EM) can be measured, it can be used for detecting an increasing/decreasing interest in a certain EM. In this study, non-invasive sensor technologies were validated for measuring interaction with EM in pens with weaned fattening pigs. The experiment was carried out in four pens with six weaned pigs per pen (until a body weight of ± 25 kg) at a semi-commercial farm. Pens were provided with EM (ball and piece of wood (and a rope in two of the four pens) connected to a chain). Different sensor technologies were tested: passive infra-red detectors (PIDs), tri-axial accelerometers (TAA) and neural network model algorithms (NNMA 1 and NNMA 2) based on video images. Per pen, a PID was placed above the EM which measured the movement of body heat around the chain (⌀20 cm) in volts per second. A TAA was attached to the EM (at the top of the chain) and measured acceleration based on X-, Y- and Z-axis co-ordinates every second. A video camera was placed above each pen to record video images that were used to feed the NNMAs and for behavioural observations. Interaction with EM (shake, carry, nose, bite, chew or root) was manually scored per second per pig (pooled per pen afterwards) for 30 min of video footage per pen per week and was compared with data from PIDs, TAAs and NNMAs. F1 score (F1) and Matthews Correlation Coefficient (MCC) were calculated to measure the performance of the sensor technologies. PIDs (F1 = 0.380, MCC = 0.192), as well as TAAs (X-axis: F1 = 0.482, MCC = 0.345; Y-axis: F1 = 0.524, MCC = 0.401; Z-axis: F1 = 0.465, MCC = 0.320; XYZ-axis: F1 = 0.474, MCC = 0.333), overestimated interaction with the EM which might be due to the relatively small pen size, resulting in piglets touching the EM without intentional interaction with the EM. NNMAs achieved the highest performance parameters (NNMA 1: F1 = 0.554, MCC = 0.466; NNMA 2: F1 = 0.540, MCC = 0.445). Overall, only moderate F1s and MCCs were reached. The results indicated that the individual sensor technologies are not yet appropriate to measure interaction with the EM. However, there is potential to measure interaction with EM by applying a multi-sensor approach (combination of PID, TAA and NNMA), but this merits further study

Immunological basis of differences in disease resistance in the chicken

Genetic resistance to diseases is a multigenic trait governed mainly by the immune system and its interactions with many physiologic and environmental factors. In the adaptive immunity, T cell and B cell responses, the specific recognition of antigens and interactions between antigen presenting cells, T cells and B cells are crucial. It occurs through a network of mediator proteins such as the molecules of the major histocompatibility complex (MHC), T cell receptors, immunoglobulins and secreted proteins such as the cytokines and antibodies. The diversity of these proteins that mainly is due to an intrinsic polymorphism of the genes causes phenotypic variation in disease resistance. The well-known linkage of MHC polymorphism and Marek's disease resistance difference represents a classic model revealing immunological factors in resistance differences and diversity of mediator molecules. The molecular bases in any resistance variation to infectious pathogens are vaguely understood. This paper presents a review of the major immune mediators involved in resistance and susceptibility to infectious diseases and their functional mechanisms in the chicken. The genetic interaction of disease resistance with production traits and the environment is mentioned.Bases immunologiques des différences dans la résistance aux maladies chez le poulet. La résistance génétique aux maladies est un trait multigénique qui est surtout gouverné par le système immunitaire et ses interactions avec de nombreux facteurs physiologiques et environnementaux. Dans l'immunité acquise, les réponses des cellules T et B, la reconnaissance spécifique des antigènes et les interactions entre les cellules présentant des antigènes, les cellules T et B, sont cruciales. Cela se produit à travers un réseau de protéines médiatrices telles que les molécules du complexe majeur d'histocompatibilité (CMH), les récepteurs des cellules T, les immunoglobulines et les protéines sécrétées telles que les cytokines et les anticorps. La diversité de ces protéines, qui est surtout due au polymorphisme intrinsèque des gènes, entraîne une variation phénotypique dans la résistance aux maladies. Le lien bien connu entre le polymorphisme du CMH et les différences dans la résistance à la maladie de Marek représente un modèle classique révélant les facteurs immunologiques dans les différences de résistance et la diversité dans les molécules médiatrices. Les bases moléculaires impliquées dans toute variation de résistance aux pathogènes infectieux sont à peu près comprises. Cet article présente une synthèse sur les principaux médiateurs de l'immunité impliqués dans la résistance et la sensibilité aux maladies infectieuses chez le poulet et leurs mécanismes fonctionnels. L'interaction génétique entre la résistance aux maladies et les traits de production et l'environnement est mentionnée

Environmentally enriched housing conditions affect pig welfare, immune system and gut microbiota in early life

BackgroundConventional pig housing and management conditions are associated with gastrointestinal pathophysiology and disease susceptibility in early life. Developing new strategies to reduce both therapeutic and prophylactic antibiotic use is urgent for the sustainable swine production globally. To this end, housing methodology providing effective environmental enrichment could be a promising alternative approach to reduce antibiotic usage, as it has been proven to positively influence pig welfare and immune status and reduce susceptibility to infections. It is, however, poorly understood how this enriched housing affects systemic and local pulmonary immune status and gut microbiota colonization during early life. In the present study, we compared the effects of two housing conditions, i.e., conventional housing: (CH) versus enriched housing (EH), on immune status and gut microbiota from birth until 61 days of age.ResultsThe expected benefits of enrichment on pig welfare were confirmed as EH pigs showed more positive behaviour, less aggression behaviour during the weaning transition and better human animal relation during the post weaning phase. Regarding the pigs’ immune status, EH pigs had higher values of haemoglobin and mean corpuscular volume in haematological profiles and higher percentages of T cells and cytotoxic T cells in peripheral blood. Furthermore, EH pigs showed higher ex vivo secretion of IL1ß and TNF-α after lipopolysaccharide stimulation of whole blood than CH pigs. The structure of the developing faecal microbiota of CH and EH pigs significantly differed as early as day 12 with an increase in the relative abundance of several bacterial groups known to be involved in the production of short chain fatty acids, such as Prevotella_2, Christensenellaceae_R_7_group and Ruminococcus gauvreauii group. Furthermore, the main difference between both housing conditions post weaning was that on day 61, CH pigs had significantly larger inter-individual variation of ileal and colonic microbiota than EH pigs. In addition to housing, other intrinsic factors (e.g., sex) were associated with gut microbiota development and immune competence.ConclusionsIn addition to the known welfare benefits for pigs, environmentally enriched housing also positively drives important aspects of the development of the immune system and the establishment of gut microbiota in early life. Consequently, EH may contribute to increasing productivity of pigs and reducing antibiotic use

Coxiella burnetii isolates originating from infected cattle induce a more pronounced proinflammatory cytokine response compared to isolates from infected goats and sheep

Coxiella burnetii is the causative agent of Q fever. Although the prevalence of C. burnetii in cattle is much higher than in goats and sheep, infected cattle are rarely associated with human outbreaks. We investigated whether the immune response of humans differs after contact with C. burnetii isolates from different host origins or with different multilocus variable number of tandem repeat analysis (MLVA) genotypes. Cytokine responses were measured in human peripheral blood mononuclear cells (PBMCs) stimulated with 16 C. burnetii isolates with known MLVA genotype from goats, sheep, cattle, acute and chronic Q fever patients. Coxiella burnetii isolates originating from cattle induce significantly more IL-1β, TNF-α and IL-22 than the isolates from goats, sheep or chronic Q fever patients. Comparing the cytokine induction of the isolates based on their MVLA genotype did not reveal differences in response between the MLVA genotypes. The proinflammatory cytokine response induced in human PBMCs by C. burnetii isolates from cattle may explain the low incidence of human Q fever outbreaks caused by cattle. The cytokine profile of PBMCs stimulated with C. burnetii isolates from chronic Q fever patients resembles isolates from goats. Furthermore, cytokine responses seem to be depending on host origin than on MLVA genotype

Respiratory health of broilers following chronic exposure to airborne endotoxin

In and around poultry farms, high concentrations of endotoxins are found that have a negative impact on the health of farmers and local residents. However, little is known about the effects of chronic exposure to endotoxins on the health of poultry. The aim of this study was to identify effects of chronic exposure to airborne endotoxins (E. coli LPS) on the immune system, respiratory tract, disease susceptibility and welfare of broilers. Effects of high (HE) and low endotoxin (LE) concentrations on natural antibody titers (NAb), performance and behavior of broilers were determined. After treatment with a respiratory virus infection, infectious bronchitis virus (IBV), mRNA expression of cytokines and Toll-like receptor (TLR) 4 in the lung, tracheal ciliary activity and lesions in the respiratory tract were determined. Endotoxin affected the immune system and respiratory tract, where HE broilers tended to have lower IgM NAb binding Phosphorylcholine-conjugated to Bovine Serum Albumin, and higher interferon (IFN)-α mRNA expression and more lesions in the nasal tissue compared to LE broilers. Furthermore, HE broilers had higher TLR4 mRNA expression compared to LE broilers. However, endotoxin did not affect NAb levels binding Keyhole Limpet Hemocyanin, IFN-β and interleukin-10 mRNA expression, IBV replication or lesions in the lung and trachea. HE and LE broilers further had similar body weight, but HE broilers showed numerically more passive behavior compared to LE broilers. In conclusion, chronic exposure to high airborne endotoxin concentrations affects components of the immune system and respiratory tract in broilers and could therefore influence disease susceptibility

Perturbation of microbiota in one-day old broiler chickens with antibiotic for 24 hours negatively affects intestinal immune development

Background: Gut microbial colonization and development of immune competence are intertwined and are influenced by early-life nutritional, environmental, and management factors. Perturbation of the gut microbiome at young age affects the crosstalk between intestinal bacteria and host cells of the intestinal mucosa.Results: We investigated the effect of a perturbation of the normal early life microbial colonization of the jejunum in 1-day old chickens. Perturbation was induced by administering 0.8 mg amoxicillin per bird per day) via the drinking water for a period of 24 h. Effects of the perturbation were measured by 16S rRNA profiling of the microbiome and whole genome gene expression analysis. In parallel to what has been observed for other animal species, we hypothesized that such an intervention may have negative impact on immune development.Trends were observed in changes of the composition and diversity of the microbiome when comparing antibiotic treated birds with their controls. in the jejunum, the expression of numerous genes changed, which potentially leads to changes in biological activities of the small intestinal mucosa. Validation of the predicted functional changes was performed by staining immune cells in the small intestinal mucosa and a reduction in the number of macrophage-like (KUL01+) cells was observed due to a direct or indirect effect of the antibiotic treatment. We provide evidence that a short, early life antibiotic treatment affects both the intestinal microbiota (temporarily) and mucosal gene expression over a period of 2 weeks.Conclusion: These results underscore the importance of early life microbial colonization of the gut in relation to immune development and the necessity to explore the capabilities of a variety of early life dietary and/or environmental factors to modulate the programming for immune competence in broilers.<br/