High-level integration of murine intestinal transcriptomics data highlights the importance of the complement system in mucosal homeostasis.

BACKGROUND: The mammalian intestine is a complex biological system that exhibits functional plasticity in its response to diverse stimuli to maintain homeostasis. To improve our understanding of this plasticity, we performed a high-level data integration of 14 whole-genome transcriptomics datasets from samples of intestinal mouse mucosa. We used the tool Centrality based Pathway Analysis (CePa), along with information from the Reactome database. RESULTS: The results show an integrated response of the mouse intestinal mucosa to challenges with agents introduced orally that were expected to perturb homeostasis. We observed that a common set of pathways respond to different stimuli, of which the most reactive was the Regulation of Complement Cascade pathway. Altered expression of the Regulation of Complement Cascade pathway was verified in mouse organoids challenged with different stimuli in vitro. CONCLUSIONS: Results of the integrated transcriptomics analysis and data driven experiment suggest an important role of epithelial production of complement and host complement defence factors in the maintenance of homeostasis.

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.

The Importance of Endophenotypes to Evaluate the Relationship between Genotype and External Phenotype.

With the exception of a few Mendelian traits, almost all phenotypes (traits) in livestock science are quantitative or complex traits regulated by the expression of many genes. For most of the complex traits, differential expression of genes, rather than genomic variation in the gene coding sequences, is associated with the genotype of a trait. The expression profiles of the animal's transcriptome, proteome and metabolome represent endophenotypes that influence/regulate the externally-observed phenotype. These expression profiles are generated by interactions between the animal's genome and its environment that range from the cellular, up to the husbandry environment. Thus, understanding complex traits requires knowledge about not only genomic variation, but also environmental effects that affect genome expression. Gene products act together in physiological pathways and interaction networks (of pathways). Due to the lack of annotation of the functional genome and ontologies of genes, our knowledge about the various biological systems that contribute to the development of external phenotypes is sparse. Furthermore, interaction with the animals' microbiome, especially in the gut, greatly influences the external phenotype. We conclude that a detailed understanding of complex traits requires not only understanding of variation in the genome, but also its expression at all functional levels.

Major differential gene regulation in Coxiella burnetii between in vivo and in vitro cultivation models.

BACKGROUND: Coxiella burnetii is the causative agent of the zoonotic disease Q fever. As it is an intracellular pathogen, infection by C. burnetii requires adaptation to its eukaryotic host and intracellular environment. The recently developed cell-free medium also allows the bacteria to propagate without host cells, maintaining its infection potential. The adaptation to different hosts or extracellular environments has been assumed to involve genome-wide modulation of C. burnetii gene expression. However, little is currently known about these adaptation events which are critical for understanding the intracellular survival of C. burnetii. RESULTS: We studied C. burnetii genome-wide transcriptional patterns in vivo (mice spleen) and in cell and cell-free in vitro culture models to examine its metabolic pathways and virulence associated gene expression patterns that are required to colonize and persist in different environments. Within each model, the gene expression profiles of the Dutch C. burnetii outbreak strain (602) and NM reference strains were largely similar. In contrast, modulation of gene-expression was strongly influenced by the cultivation method, indicating adaptation of the bacterium to available components. Genome-wide expression profiles of C. burnetii from in vitro cell culture were more similar to those seen for in vivo conditions, while gene expression profiles of cell-free culture were more distant to in vivo. Under in vivo conditions, significant alterations of genes involved in metabolism and virulence were identified. We observed that C. burnetii under in vivo conditions predominantly uses glucose as a carbon source (mostly for biosynthetic processes) and fatty acids for energy generation. C. burnetii experienced nutrient limitation and anaerobiosis as major stressors, while phosphate limitation was identified as an important signal for intracellular growth inside eukaryotic host cells. Finally, the in vivo environment significantly induced expression of several virulence genes, including those implicated in LPS synthesis, colonization, host component modulation and DNA repair mechanisms. CONCLUSION: Our study shows that C. burnetii, with its relative small genome, requires only a subset of core gene functions to survive under in vitro conditions, but requires the induction of full repertoire of genes for successful pathogenesis and thriving in harsh environments in vivo.

Network analysis of temporal functionalities of the gut induced by perturbations in new-born piglets.

BACKGROUND: Evidence is accumulating that perturbation of early life microbial colonization of the gut induces long-lasting adverse health effects in individuals. Understanding the mechanisms behind these effects will facilitate modulation of intestinal health. The objective of this study was to identify biological processes involved in these long lasting effects and the (molecular) factors that regulate them. We used an antibiotic and the same antibiotic in combination with stress on piglets as an early life perturbation. Then we used host gene expression data from the gut (jejunum) tissue and community-scale analysis of gut microbiota from the same location of the gut, at three different time-points to gauge the reaction to the perturbation. We analysed the data by a new combination of existing tools. First, we analysed the data in two dimensions, treatment and time, with quadratic regression analysis. Then we applied network-based data integration approaches to find correlations between host gene expression and the resident microbial species. RESULTS: The use of a new combination of data analysis tools allowed us to identify significant long-lasting differences in jejunal gene expression patterns resulting from the early life perturbations. In addition, we were able to identify potential key gene regulators (hubs) for these long-lasting effects. Furthermore, data integration also showed that there are a handful of bacterial groups that were associated with temporal changes in gene expression. CONCLUSION: The applied systems-biology approach allowed us to take the first steps in unravelling biological processes involved in long lasting effects in the gut due to early life perturbations. The observed data are consistent with the hypothesis that these long lasting effects are due to differences in the programming of the gut immune system as induced by the temporary early life changes in the composition and/or diversity of microbiota in the gut.

Early life microbial colonization of the gut and intestinal development differ between genetically divergent broiler lines.

BACKGROUND: Host genetic makeup plays a role in early gut microbial colonization and immune programming. Interactions between gut microbiota and host cells of the mucosal layer are of paramount importance for a proper development of host defence mechanisms. For different livestock species, it has already been shown that particular genotypes have increased susceptibilities towards disease causing pathogens. The objective of this study was to investigate the impact of genotypic variation on both early microbial colonization of the gut and functional development of intestinal tissue. From two genetically diverse chicken lines intestinal content samples were taken for microbiota analyses and intestinal tissue samples were extracted for gene expression analyses, both at three subsequent time-points (days 0, 4, and 16). RESULTS: The microbiota composition was significantly different between lines on each time point. In contrast, no significant differences were observed regarding changes in the microbiota diversity between the two lines throughout this study. We also observed trends in the microbiota data at genus level when comparing lines X and Y. We observed that approximately 2000 genes showed different temporal gene expression patterns when comparing line X to line Y. Immunological related differences seem to be only present at day 0, because at day 4 and 16 similar gene expression is observed for these two lines. However, for genes involved in cell cycle related processes the data show higher expression over the whole course of time in line Y in comparison to line X. CONCLUSIONS: These data suggest the genetic background influences colonization of gut microbiota after hatch in combination with the functional development of intestinal mucosal tissue, including the programming of the immune system. The results indicate that genetically different chicken lines have different coping mechanisms in early life to cope with the outside world.

Association of Egg Mass and Egg Sex: Gene Expression Analysis from Maternal RNA in the Germinal Disc Region of Layer Hens (Gallus gallus).

Female birds have been shown to manipulate offspring sex ratio. However, mechanisms of sex ratio bias are not well understood. Reduced feed availability and change in body condition can affect the mass of eggs in birds that could lead to a skew in sex ratio. We employed feed restriction in laying chickens (Gallus gallus) to induce a decrease in body condition and egg mass using 45 chicken hens in treatment and control groups. Feed restriction led to an overall decline of egg mass. In the second period of treatment (Days 9-18) with more severe feed restriction and a steeper decline of egg mass, the sex ratio per hen (proportion of male eggs) had a significant negative association with mean egg mass per hen. Based on this association, two groups of hens were selected from feed restriction group, that is, hens producing male bias with low egg mass and hens producing female bias with high egg mass with overall sex ratios of 0.71 and 0.44 respectively. Genomewide transcriptome analysis on the germinal disks of F1 preovulatory follicles collected at the time of occurrence of meiosis-I was performed. We did not find significantly differentially expressed genes in these two groups of hens. However, gene set enrichment analysis showed that a number of cellular processes related to cell cycle progression, mitotic/meiotic apparatus, and chromosomal movement were enriched in female-biased hens or high mean egg mass as compared with male-biased hens or low mean egg mass. The differentially expressed gene sets may be involved in meiotic drive regulating sex ratio in the chicken.

Effect of corticosterone and hen body mass on primary sex ratio in laying hen (Gallus gallus), using unincubated eggs.

In various studies, chronic elevation of corticosterone levels in female birds under natural or experimental conditions resulted in female biased offspring sex ratios. In chicken, one study with injected corticosterone resulted in a male sex ratio bias. In the current study, we chronically elevated blood plasma corticosterone levels through corticosterone feeding (20 mg/kg feed) for 14 days using 30 chicken hens in each of treatment and control groups and studied the primary offspring sex ratio (here defined as the proportion of male fertile eggs determined in freshly laid eggs, i.e., without egg incubation). Mean plasma corticosterone concentrations were significantly higher in the treatment group but were not associated with sex ratio, laying rate, and fertility rate. Corticosterone treatment by itself did not affect egg sex but affected sex ratio as well as laying rate and fertility rate in interaction with hen body mass. Body mass had a negative association with sex ratio, laying rate, and fertility rate per hen in the corticosterone group, but a positive association with sex ratio in untreated hens. These interactions were already seen when taking the body mass at the beginning of the experiment, indicating intrinsic differences between light and heavy hens with regard to their reaction to corticosterone treatment. The effects on laying rate, fertility rate, and sex ratio suggest that some factor related to body mass act together with corticosterone to modulate ovarian functions. We propose that corticosterone treatment in conjunction with hen body mass can interfere with meiosis, which can lead to meiotic drive and to chromosomal aberrations resulting in postponed ovulation or infertile ova.

Yolk concentrations of hormones and glucose and egg weight and egg dimensions in unincubated chicken eggs, in relation to egg sex and hen body weight.

Birds can manipulate offspring sex ratio under natural and experimental conditions and maternal hormones have been shown to be involved in this process. Studies also provided evidence for the presence of sex specific concentrations of yolk hormones in avian eggs. These findings led to the suggestion that yolk hormones could influence genetic sex determination in birds. However, in previous studies, yolk hormone concentrations and egg sex were studied in incubated eggs, although incubation of the eggs and embryonic development can alter yolk hormone concentrations and measured sex ratio. This study is the first to determine a wide array of egg components and hen body weight in relation to the sex of the egg in unincubated eggs. Egg parameters studied were yolk concentrations of testosterone, estradiol, androstenedione, progesterone, dihydrotestosterone, and glucose, and egg weight and dimensions. In addition, we studied the associations among all measured parameters. Associations were found between a number of yolk hormones (progesterone associated with testosterone, estradiol and androstenedione; androstenedione with testosterone; dihydrotestosterone with estradiol and androstenedione) as well as between yolk testosterone and egg length and egg weight. There were no significant overall differences between male and female chicken eggs in any of the measured egg parameters. However, there were a few interactions such as the interaction of egg sex with dihydrotestosterone and with hen body weight which predicted estradiol levels and an interaction of estradiol levels with egg width for predicting sex of egg. Their biological relevance need, however, further study.

Meta-analysis of chicken--salmonella infection experiments.

BACKGROUND: Chicken meat and eggs can be a source of human zoonotic pathogens, especially Salmonella species. These food items contain a potential hazard for humans. Chickens lines differ in susceptibility for Salmonella and can harbor Salmonella pathogens without showing clinical signs of illness. Many investigations including genomic studies have examined the mechanisms how chickens react to infection. Apart from the innate immune response, many physiological mechanisms and pathways are reported to be involved in the chicken host response to Salmonella infection. The objective of this study was to perform a meta-analysis of diverse experiments to identify general and host specific mechanisms to the Salmonella challenge. RESULTS: Diverse chicken lines differing in susceptibility to Salmonella infection were challenged with different Salmonella serovars at several time points. Various tissues were sampled at different time points post-infection, and resulting host transcriptional differences investigated using different microarray platforms. The meta-analysis was performed with the R-package metaMA to create lists of differentially regulated genes. These gene lists showed many similarities for different chicken breeds and tissues, and also for different Salmonella serovars measured at different times post infection. Functional biological analysis of these differentially expressed gene lists revealed several common mechanisms for the chicken host response to Salmonella infection. The meta-analysis-specific genes (i.e. genes found differentially expressed only in the meta-analysis) confirmed and expanded the biological functional mechanisms. CONCLUSIONS: The meta-analysis combination of heterogeneous expression profiling data provided useful insights into the common metabolic pathways and functions of different chicken lines infected with different Salmonella serovars.

Serum protein profiles as potential biomarkers for infectious disease status in pigs.

BACKGROUND: In veterinary medicine and animal husbandry, there is a need for tools allowing the early warning of diseases. Preferably, tests should be available that warn farmers and veterinarians during the incubation periods of disease and before the onset of clinical signs. The objective of this study was to explore the potential of serum protein profiles as an early biomarker for infectious disease status. Serum samples were obtained from an experimental pig model for porcine circovirus-associated disease (PCVAD), consisting of Porcine Circovirus type 2 (PCV2) infection in combination with either Porcine Parvovirus (PPV) or Porcine Reproductive and Respiratory Syndrome virus (PRRSV). Sera were collected before and after onset of clinical signs at day 0, 5 and 19 post infection. Serum protein profiles were evaluated against sera from non-infected control animals. RESULTS: Protein profiles were generated by SELDI-TOF mass spectrometry in combination with the Proteominer™ technology to enrich for low-abundance proteins. Based on these protein profiles, the experimentally infected pigs could be classified according to their infectious disease status. Before the onset of clinical signs 88% of the infected animals could be classified correctly, after the onset of clinical sigs 93%. The sensitivity of the classification appeared to be high. The protein profiles could distinguish between separate infection models, although specificity was moderate to low. Classification of PCV2/PRRSV infected animals was superior compared to PCV2/PPV infected animals. Limiting the number of proteins in the profiles (ranging from 568 to 10) had only minor effects on the classification performance. CONCLUSIONS: This study shows that serum protein profiles have potential for detection and identification of viral infections in pigs before clinical signs of the disease become visible.

TroA of Streptococcus suis is required for manganese acquisition and full virulence.

Streptococcus suis causes infections in pigs and occasionally in humans, resulting in manifestations as meningitis, sepsis, arthritis, and septic shock. For survival within the host, S. suis requires numerous nutrients including trace metals. Little is known about the specific proteins involved in metal scavenging in S. suis. In this study we evaluated the role of the putative high-affinity metal binding lipoprotein TroA in metal acquisition and virulence. A mutant strain deficient in the expression of TroA (ΔtroA mutant) was constructed. Growth of the ΔtroA mutant in Todd-Hewitt broth was similar to wild-type growth; however, growth of the ΔtroA mutant in cation-deprived Todd-Hewitt broth and in porcine serum was strongly reduced compared to growth of wild-type bacteria. Supplementing the medium with extra manganese but not with magnesium, zinc, copper, nickel, or iron restored growth to wild-type levels, indicating that TroA is specifically required for growth in environments low in manganese. The ΔtroA mutant also showed increased susceptibility to H2O2, suggesting that TroA is involved in counteracting oxidative stress. Furthermore, the expression of the troA gene was subject to environmental regulation at the transcript level. In a murine S. suis infection model, the ΔtroA mutant displayed a nonvirulent phenotype. These data indicate that S. suis TroA is involved in manganese acquisition and is required for full virulence in mice.

Gene expression patterns in four brain areas associate with quantitative measure of estrous behavior in dairy cows.

BACKGROUND: The decline noticed in several fertility traits of dairy cattle over the past few decades is of major concern. Understanding of the genomic factors underlying fertility, which could have potential applications to improve fertility, is very limited. Here, we aimed to identify and study those genes that associated with a key fertility trait namely estrous behavior, among genes expressed in four bovine brain areas (hippocampus, amygdala, dorsal hypothalamus and ventral hypothalamus), either at the start of estrous cycle, or at mid cycle, or regardless of the phase of cycle. RESULTS: An average heat score was calculated for each of 28 primiparous cows in which estrous behavior was recorded for at least two consecutive estrous cycles starting from 30 days post-partum. Gene expression was then measured in brain tissue samples collected from these cows, 14 of which were sacrificed at the start of estrus and 14 around mid cycle. For each brain area, gene expression was modeled as a function of the orthogonally transformed average heat score values using a Bayesian hierarchical mixed model. Genes whose expression patterns showed significant linear or quadratic relationships with heat scores were identified. These included genes expected to be related to estrous behavior as they influence states like socio-sexual behavior, anxiety, stress and feeding motivation (OXT, AVP, POMC, MCHR1), but also genes whose association with estrous behavior is novel and warrants further investigation. CONCLUSIONS: Several genes were identified whose expression levels in the bovine brain associated with the level of expression of estrous behavior. The genes OXT and AVP play major roles in regulating estrous behavior in dairy cows. Genes related to neurotransmission and neuronal plasticity are also involved in estrous regulation, with several genes and processes expressed in mid-cycle probably contributing to proper expression of estrous behavior in the next estrus. Studying these genes and the processes they control improves our understanding of the genomic regulation of estrous behavior expression.

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources.

BACKGROUND: Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. RESULTS: Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1.The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. CONCLUSIONS: This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

Local intestinal microbiota response and systemic effects of feeding black soldier fly larvae to replace soybean meal in growing pigs.

Black soldier fly (Hermetia illucens; BSF) larvae as dietary protein source have the ability to deliver nutrients and could possess functional properties that positively support animal productivity and health. More knowledge, however, is needed to assess the impact of feeding a BSF based diet on gut and animal health. Sixteen post-weaned male pigs were randomly assigned to two groups and fed for three weeks with iso-caloric and iso-proteinaceous experimental diets prepared with either soybean meal (SBM) as reference protein source or with BSF as single source of dietary protein. At the end of the trial, the pigs were sacrificed to collect relevant digesta, gut tissue and blood samples to study changes induced by the dietary treatments using ~ omics based analyses. Inclusion of BSF in the diet supports the development of the intestinal microbiome that could positively influence intestinal health. By amine metabolite analysis, we identified two metabolites i.e. sarcosine and methionine sulfoxide, in plasma that serve as markers for the ingestion of insect based ingredients. BSF seems to possess functional properties indicated by the appearance of alpha-aminobutyric acid and taurine in blood plasma of pigs that are known to induce health beneficial effects.

The effect of maternal antibiotic use in sows on intestinal development in offspring.

The objective of this study is to investigate the effect of a maternal antibiotic administration during the last week of gestation on the early life intestinal development in neonatal piglets. Colonization of the gut with bacteria starts during birth and plays a major role in the intestinal and immunological development of the intestine. We demonstrate that maternal interventions induced changes in the sows (n = 6 to 8 per treatment) fecal microbiota diversity around birth (P < 0.001, day 1). Whole-genome microarray analysis in small intestinal samples of 1-d old piglets (n = 6 to 8 per treatment) showed significantly expressed genes (Padj < 0.05) which were involved in processes of tight junction formation and immunoglobulin production. Furthermore, when performing morphometry analysis, the number of goblet cells in jejunum was significantly (P < 0.001) lower in piglets from amoxicillin administered sows compared with the respective control piglets. Both significantly expressed genes (Padj < 0.05) and significant morphometry data (jejunum P < 0.05 and ileum P < 0.01) indicate that the crypts of piglets from amoxicillin administered sows deepen around weaning (day 26) as an effect of the amoxicillin administration in sows. The latter might imply that the intestinal development of piglets was delayed by maternal antibiotic administration. Taken together, these results show that maternally oral antibiotic administration changes in early life can affect intestinal development of the offspring piglets for a period of at least 5 wk after the maternal antibiotic administration was finished. These results show that modulation of the neonatal intestine is possible by maternal interventions.

Effects of undigested protein-rich ingredients on polarised small intestinal organoid monolayers.

ABSTRACT: Here, we describe the use of monolayers of intestinal epithelial cells derived from intestinal organoids and transcriptomics to investigate the direct effects of dietary protein sources on epithelial function. Mechanically dissociated 3D organoids of mouse duodenum were used to generate a polarized epithelium containing all cell types found in the tissue of origin. The organoid-derived cell monolayers were exposed to 4% (w/v) of 'undigested (non-hydrolysed)-soluble' fraction of protein sources used as feed ingredients [soybean meal (SBM) and casein], or alternative protein sources (spray dried plasma protein, and yellow meal worm), or controls for 6 h prior to RNA isolation and transcriptomics. All protein sources altered expression of unique biological processes in the epithelial cells. Exposure of intestinal organoids to SBM downregulated expression of retinol and retinoid metabolic processes as well as cholesterol and lipid biosynthetic pathways, consistent with the reported hypotriglyceridaemic effect of soy protein in vivo. These findings support the use of intestinal organoids as models to evaluate complex interactions between dietary ingredients and the intestinal epithelium and highlights some unique host effects of alternative protein sources in animal feed and potentially human food. GRAPHICAL ABSTRACT: Schematic representation of the study. 3-dimensional organoids were generated from mouse duodenum (1). The organoids were subsequently dissociated into single cells (2) and grown as 2-dimensional polarised monolayers (3). Polarized monolayers of organoid cells were exposed to different protein sources [CAS, SBM, SDPP, YMW, or medium control (MC)] for 6 h (4) and further processed for imaging (5) gene expression (6), and biochemical assays (7), to investigate the effects of undigested protein sources on the duodenal epithelium.

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.

Mannose-specific interaction of Lactobacillus plantarum with porcine jejunal epithelium.

Host-microorganism interactions in the intestinal tract are complex, and little is known about specific nonpathogenic microbial factors triggering host responses in the gut. In this study, mannose-specific interactions of Lactobacillus plantarum 299v with jejunal epithelium were investigated using an in situ pig Small Intestinal Segment Perfusion model. The effects of L. plantarum 299v wild-type strain were compared with those of two corresponding mutant strains either lacking the gene encoding for the mannose-specific adhesin (msa) or sortase (srtA; responsible for anchoring of cell surface proteins like Msa to the cell wall). A slight enrichment of the wild-type strain associated with the intestinal surface could be observed after 8 h of perfusion when a mixture of wild-type and msa-mutant strain had been applied. In contrast to the mutant strains, the L. plantarum wild-type strain tended to induce a decrease in jejunal net fluid absorption compared with control conditions. Furthermore, after 8 h of perfusion expression of the host gene encoding pancreatitis-associated protein, a protein with proposed bactericidal properties, was found to be upregulated by the wild-type strain only. These observations suggest a role of Msa in the induction of host responses in the pig intestine.