BACKGROUND
the gastrointestinal tract contains an immense number of micro-organisms, collectively known as the microbiota.
the major functions of the microbiota include degrading dietary compounds, influencing nutrient partitioning and lipid metabolism, providing essential nutrients generated as a result of microbial metabolism, protecting against invading pathogens and stimulating gut morphology  <cit> .
the gut microbiota also plays an important role in maintaining immune function.
recent work suggests that the commensal microbiota influences processes as complex as pathogen colonization, immune development and homeostasis, t cell differentiation, inflammation, repair and angiogenesis  <cit> .
the impact of the microbiota on host immunity is thought to be critically regulated in early life and inappropriate exposure to bacteria during this developmental window has been linked to the increased incidence of infectious, inflammatory and autoimmune diseases  <cit> .
clearly, the neonatal period is a critical time for gut colonization, and can be affected by numerous factors including gestational age, birth environment, mode of delivery, nutrition and antibiotic use  <cit> .
the increase in immune-mediated disorders, particularly in westernized countries, has led to the so-called hygiene hypothesis, which postulates that the growing incidence of immune-mediated diseases is the consequence of reduced infection and exposure to microbes during early childhood  <cit> .
in this context, the high-hygiene status of western lifestyle, decreased infection rates and reduced bacterial load as a result of widespread use of vaccines and antibiotics are likely to be important contributory factors  <cit> .
animal models have provided some insight into immune-disease aetiology: animals susceptible to autoimmune disease have an increased incidence and severity of disease when bred under germ-free conditions whereas disease is prevented when the animals are exposed to bacteria  <cit> .
this evidence supports the notion that, in addition to naturally-acquired infections, colonization by the normal commensal microbiota is an important factor limiting the incidences of immune-mediated diseases.
consistent with this is the growing awareness of the importance of the commensal microbiota in immune education in early life  <cit> , which appears to involve complex mechanisms of host-bacterial crosstalk  <cit> .
in the current study we have investigated potential interactions between the rearing environment, gut microbiota and immune function in the developing pig gut using molecular methods to evaluate both microbial diversity and host immune gene expression.
microbial diversity in the gastrointestinal tract of these animals was characterized by sequence analysis of 16s rrna gene libraries.
specific responses in transcriptome expression patterns of gut ileal tissue were studied using affymetrix genechip porcine genome microarrays .
biomarkers associated with immune function and altered by rearing environment were identified and investigated more thoroughly by real-time polymerase chain reaction .
RESULTS
mucosal microbial diversity in the ileum of pigs from different environments
we investigated the influence of environmentally-acquired bacteria on the composition of the adult mucosa-adherent ileal microbiota in the pig.
animals were housed in an indoor  or an outdoor facility , as well as in individual isolator units receiving daily doses of antibiotics .
mucosa-adherent bacterial samples from the ileum and fecal samples were collected from all experimental animals at day  <dig>  in addition to this, fecal samples were taken from adult sows from both the indoor  and outdoor  environments to confirm 'environment' as the major factor contributing to the experimental differences.
microbial composition of the ileum was examined by calculation of diversity indices and analysis of the phylogenetic distribution of 16s rrna gene sequences derived from clone libraries of each treatment.
after quality control, a total of  <dig>  validated clones were analyzed.
diversity measures
we first investigated the effects of environment and high-hygiene status on a number of bacterial diversity indices.
estimates of diversity, richness and library coverage for the 16s rrna clone libraries from in, out and ir are shown in table  <dig>  species richness, estimated by chao <dig>  was highest in the ir and in groups but lower in the out group.
good's coverage was  <dig>  to  <dig> % for all three treatment groups, with the lowest coverage in ir libraries.
rarefaction analysis of clone libraries confirmed these findings and suggested that the ir and in groups possessed the most diverse mucosa-adherent bacterial community, whereas the out group showed lower microbial diversity .
calculations were made based on otu definition at 99% sequence identity .
collector's curves of the observed and estimated phylotype richness are shown in figure 2a-c. each curve reflects the series of observed or estimated richness values obtained as more clones were added to the data set.
after an initial steep rise, the curves level out, suggesting that the majority of phylotypes in the treatment groups were adequately sampled.
in the early stages of sampling and clone sequencing, both chao <dig> and abundance-based coverage estimator  showed a sharp increase, together with the observed phylotype number, in the in group .
after the sampling of about  <dig> clones, the gap between the observed and estimated phylotype richness was relatively constant, indicating repeated sampling of same phylotypes within samples.
in the out group, the gap between the observed and estimated phylotype richness was constant after the sampling point of  <dig> clones .
the difference between the estimated and observed phylotype richness was highest in the ir mucosal libraries.
novel phylotypes continued to be identified up to the end of sampling .
phylogenetic affiliation of 16s rrna gene sequences
phylogenetic analysis was performed to establish taxonomic positioning of obtained sequences.
all 16s rrna gene sequences from the mucosa-adherent ileal and fecal samples were subjected to the ribosomal database project  classifier analysis .
based on the classification results, the majority of clones were assigned to four phyla: firmicutes , proteobacteria , bacteroidetes , and actinobacteria  .
the two major phyla, firmicutes and bacteroidetes, were significantly different between the libraries: the firmicutes showed a significant increase in the out group compared to the ir group, while the bacteroidetes were significantly increased in both the ins and outs fecal libraries compared to the mucosa-adherent ileal libraries.
firmicutes
seventy percent of all sequences were affiliated with the firmicutes phylum.
the outdoor environment favoured the expansion of firmicutes compared to the hygienic environment .
at the lower taxa level this difference was even more pronounced.
a large number of sequenced clones fell into the bacilli class.
the most abundant order was lactobacillales, which was dominated by lactobacillaceae, but also contained streptococcaceae, leuconostocaceae, enterococcaceae, carnobacteriaceae and aerococcaceae, although present in lower abundance.
the lactobacillaceae family in the out group  consisted of a small number of operational taxonomic units , including lactobacillus reuteri, l. amylovorous lab <dig>  l. johnsonii, l. delbrueckii subsp.
bulgaricus, l. salivarius and l. mucosae .
in contrast, the in library contained only  <dig> % lactobacillaceae-affiliated clones, although phylotypes were similar to those observed in the out group.
l. reuteri, l. delbrueckii and l. johnsonii were all significantly decreased compared to the out group.
the high-hygiene conditions associated with ir exacerbated these differences.
l. amylovorous lab <dig> and l. brevis were present in very low abundance in the ir libraries  whereas l. reuteri, l. delbrueckii subsp.
bulgaricus, l. johnsonii and l. mucosae were not detected in this treatment group.
the observed differences in lactobacillus levels between the ir and out group were confirmed by enumeration of bacteria in gut contents of both ileum and colon on de man, rogosa and sharpe  agar.
the out group had three to four log <dig> colony forming units lactobacilli/g more than the ir group, thus validating the 16s rrna gene library results .
members of the clostridia class were present in all treatment groups with  <dig> % of all sequenced clones classified as clostridia.
interestingly, pigs raised in the indoor environment showed the highest abundance of this class.
clostridiaceae-affiliated clones were highly abundant in the in group and mainly identified as uncultured species.
clostridium beijerinckii ncimb  <dig> was significantly elevated in the in libraries compared to the out and ir libraries .
the indoor environment also favoured the expansion of the bacterial clone hh_aai33h <dig>  on the ileal mucosa compared to the outdoor environment.
the peptostreptoccocaceae family was another abundant member of the clostridia class, accounting for  <dig> % of the sequenced clones in all treatment groups.
the indoor environment favoured the expansion of peptostreptoccocaceae.
seven predominant otus, represented by mainly uncultured clones, were identified.
sequences of uncultured clones barb_aaa02d <dig> were significantly higher in the in and ir groups compared to the out group and were not detected in the indoor fecal libraries .
this possibly points to a preferential colonization of the ileal mucosa in the indoor environment.
uncultured bacterium clone barb_aaa01f <dig> was significantly increased in the ileal mucosal libraries of the in group compared to the out and ir groups, indicating a potential antibiotic sensitivity of this bacterium.
bacteroidetes
bacteroidetes were found in all libraries but in different abundance.
the most abundant group within this phylum was represented by members of the prevotellaceae family, followed by porphyromonadaceae, bacteroidaceae and, to a lesser extent, sphingobacteriaceae and flavobacteriaceae.
all 16s rrna gene libraries contained members of prevotellaceae, yet they were most prevalent in the indoor environment, particularly in fecal libraries.
high-hygiene conditions increased the numbers of prevotellaceae on the ileal mucosa.
porphyromonadaceae were mainly obtained from the fecal libraries of both farms.
most clones had only 97% similarity to previously isolated clones, specifically the porphyromonadaceae bacterium sp djf_b <dig>  and uncultured bacterial clones .
bacteroidaceae were exclusively obtained from the indoor environment.
within the in and ir groups, these included bacteroides vulgatus  and uncultured bacterial clones .
in the in fecal libraries two species were related to b. propionifaciens  and uncultured bacterium clone p-240-o <dig> .
proteobacteria
eighteen percent of all clones were placed into the proteobacteria phylum.
γ-proteobacteria and ε-proteobacteria were the most abundant groups, while members of the α- and β-proteobacteria were found only sporadically.
twenty-eight γ-proteobacteria clones were obtained from the out mucosa-adherent libraries.
these included e. coli, actinobacillus minor and a. porcinus.
six otus belonging to actinobacillus spp.
were predominately present in the in group, including actinobacillus minor, a. porcinus strains h1498/h <dig> and a. rossii strain jf <dig>  this clone has been isolated from the intestine and reproductive tract of pigs and is considered an opportunistic pathogen implicated in spontaneous abortion.
high-hygiene status increased the number of γ-proteobacteria on the ileal mucosa.
all 16s rrna gene libraries from the ir group contained members of the γ-proteobacteria class and grouped mainly with enterobacteriaceae, including sequences identified as e. coli spp.
with pathogenic properties which may pose a health risk for the young pig as well as the human population.
members of the ε-proteobacteria were the second most abundant group within the proteobacteria phylum and were represented by two major bacterial families, helicobacteraceae and campylobacteraceae.
most clones were obtained from the in group and included bacteria of recognized pathogenic phenotype .
transcriptomic analysis of gene expression patterns in the ileum of pigs from different environments
while the comprehensive profiling of the mucosa-adherent microbial community revealed large differences in composition attributable to differences in housing environment, a key goal of this study was to determine whether this translated into different host-specific gene responses.
therefore, an affymetrix genechip microarray analysis was conducted on ileum tissue from the same site used for 16s rrna gene library construction.
effects of treatment extremes on gene expression
perhaps not surprisingly, mucosa-adherent microbial diversity in the ileum was most affected by experimental isolator housing, as this constituted a high-hygiene environment.
to ascertain the differences in host-specific transcriptional responses between this treatment and the natural outdoor environment , affymetrix microarray analysis was performed on the comparison ir versus out at day  <dig> , day  <dig>  and day  <dig> .
seventy-four probesets were differentially expressed  at the neonatal stage .
fifty-six of these genes were highly expressed in the ir group, while  <dig> genes were higher in the out group.
interestingly, within the ir gene set, increased expression of genes that are closely linked to type  <dig> interferon  signalling was observed.
these genes included irf <dig>  fam14a, ube2l <dig>  gbp <dig> and usp <dig>  some of the most highly-regulated genes  were viperin, a tightly regulated isgf <dig> target gene  <cit> , and irp <dig>  a pig-specific gene homologous to human viperin.
another group showing increased expression in the ir group included  <dig> genes involved in cholesterol synthesis, such as dhcr <dig>  dhc <dig>  sc5dl, hmgcs <dig>  cyp51a <dig> and erg <dig>  genes of interest showing higher expression in the out group compared to the ir group included tlr <dig> as well as hbb and hba <dig>  both of which code for haemoglobin proteins.
at day  <dig>   <dig> genes were differentially expressed .
twenty-one of the  <dig> transcripts expressed at higher levels in ir were the same as those found at day  <dig>  and included the ifn-induced genes irf <dig> and gbp <dig>  several other type  <dig> ifn-induced genes  were higher in ir animals compared to out animals, indicating a consistent treatment effect on type  <dig> ifn signalling pathways.
also in common with the day  <dig> gene expression set, nine cholesterol synthesis genes were increased in the ir group.
consistent with these findings, microbiota-driven effects on cholesterol metabolism and trafficking have been previously documented  <cit> .
other transcripts expressed higher in the ir group compared to the out group included the chemokines cxcl <dig>  ccl <dig>  ccl <dig>  ccl <dig> and cxcl <dig>  the chemokine receptor ccr <dig> and the chemokine ligand ccl4l.
pmp <dig> was increased in the out group.
this gene is co-expressed with occludin and zona occludens  <dig> at tight junctions in epithelial cells  <cit> .
sixty-six genes were differentially expressed between ir and out at day  <dig> .
some of the genes showing higher expression in the ir group included kai <dig>  cebpb, ltb4dh, col14a <dig> and col1a <dig>  changes in cebpb  expression in ir animals may be functionally important as this gene is involved in the regulation of inflammatory responses  <cit> .
notably, a group of t-cell-related genes was increased in out animals, including tca_human, ly <dig>  cd8a, trgv <dig>  lcp <dig>  lcp <dig>  cxcl <dig> and tec, all of which are involved in t cell signalling, expansion, activation and trafficking.
other highly expressed transcripts in the out group included egr <dig>  sell , pigr  and pik3cg.
consistently, pdk <dig> was higher in the out group compared to the ir group at all three time-points.
pdk <dig> has an important function in glucose metabolism, and its expression is regulated by glucocorticoids, retinoic acid and insulin; however, its potential relevance in host-microbe interactions is currently unknown.
biological pathway analysis revealed that a large number of immune response pathways were affected .
other highly represented pathways included g-protein and congenital, hereditary and neonatal diseases and abnormalities.
consistent with the analysis of individual gene data, the pathway for immune response-ifn alpha/beta signalling was increased at day  <dig> and day  <dig> in the ir group compared to the out group.
immune response-antigen presentation by mhc class l was affected at all three time-points and also higher in ir compared to out.
gene ontology -enrichment analysis  further confirmed these findings.
while a number of go categories were consistently affected by treatment, including immune response , the major biological process affected was antigen processing and presentation .
other affected go-processes were antigen processing and presentation of peptide antigen via mhc class i , antigen processing and presentation of peptide antigen  and antigen processing and presentation of peptide or polysaccharide antigen via mhc class ii .
differentially expressed genes  were imported into genego metacore analytical software to determine the significantly enriched canonical pathways in the treatment comparison ir vs out.
the top ten pathways for each comparison are shown, with the number of genes assigned to each pathway, and the corresponding p-value.
* the number of genes on each map that are differentially expressed in the specific treatment comparison
** the total number of genes on each map
differentially expressed transcripts  are shown assigned to the go-category 'biological process'.
the top ten go-categories are shown.
the number of transcripts for each function is shown, with the corresponding p-value.
*genes involved in the specific go-category
effects of housing environment on gene expression
differences in ileal mucosa-adherent microbial composition between the ir group and the out group were associated with large host-specific transcriptional differences in the ileum.
we next set out to assess whether the microbial differences associated with the in and out environments had a similar impact on the gut transcriptome of the pig.
while the number of differentially expressed genes between in and out housed animals was smaller than between the treatment extremes , similar trends could be discerned.
in the neonatal pig, the expression levels of  <dig> probesets were differentially expressed between the in and out animals .
nine genes were higher in in animals, and this included cxcl <dig>  which is involved in t cell trafficking.
four genes showed higher expression in out animals, including tfrc.
in weaning animals,  <dig> genes were differentially expressed between the two rearing environments .
twelve transcripts were higher in in animals, including tafa <dig> , ccr <dig> and cxcr <dig>  of the  <dig> genes that were higher in the out group, genes of interest included pmp <dig>  cnksr <dig>  tjp <dig> and ltbr .
the largest differences in gene expression were observed at day  <dig>  when  <dig> genes were differentially expressed between the treatments .
transcripts increased in in animals  included three type  <dig> ifn-inducible genes .
the antibacterial peptide genes lyz , pi <dig>  and bpi  were increased  <dig> -,  <dig> - and  <dig> -fold, respectively, in in animals compared to out animals and may contribute to the observed differences in microbiota composition between these groups.
furthermore, these peptides appear to maintain gut homeostasis as evidenced by their aberrant expression in crohn's disease  <cit>  and ulcerative colitis  <cit> .
ccl <dig>  was also higher in the in group.
some of the  <dig> genes increased in out animals were pmp <dig> and sell, in accordance with the observations from the ir and out comparison.
the most affected pathways belonged to immune response, g-protein and congenital, hereditary, and neonatal diseases and abnormalities , as observed previously in the treatment extremes comparison.
differentially expressed genes  were imported into genego metacore analytical software to determine the significantly enriched canonical pathways in the treatment comparison in vs out.
the top ten pathways for each comparison are shown, with the number of genes assigned to each pathway, and the corresponding p-value.
* the number of genes on each map that are differentially expressed in the specific treatment comparison
** the total number of genes on each map
real-time quantitative pcr to analyze differentially expressed genes
real-time pcr was performed for ccl <dig>  ccl <dig>  cxcl <dig>  cxcl <dig>  cxcr <dig>  ifit <dig>  fkbp <dig>  irf <dig>  irp <dig>  mt1j, mx, pdk <dig>  pi <dig>  sell, sqle and tfrc.
these genes were selected from the gene expression data set both because they showed significant changes and because of their involvement in key immune system pathways.
verification of the true differential expression between treatment groups of these genes by real-time pcr was therefore considered essential for further biological interpretation.
the subsequent correlation between affymetrix microarray and real-time pcr data  was positive, further substantiating the biological importance of the selected genes and identified pathways.
real-time pcr verification for the comparison ir versus out showed that direction and magnitude of fold change correlated well with the affymetrix microarray results .
in some cases the fold changes detected by real-time pcr were lower than those observed by microarray analysis.
real-time pcr results at each time-point for the comparisons ir vs out and in vs out.
*pcr values are expressed as mean Δct ± sd, n =  <dig>
**standard error of difference
the differentially expressed genes from the in versus out comparison were examined by real-time pcr and again correlated well with the affymetrix microarray results .
only cxcr <dig> expression in the in group showed disagreement between the two platforms, as it was increased using microarray analysis, and decreased using real-time pcr.
discussion
the current study sought to investigate the effects of environmental hygiene on microbial colonization and composition of the gut microbiota.
additionally, transcriptomic profiling was performed to assess the impact of environmental hygiene on gene expression, in particular those genes and pathways associated with immune function.
both indoor and isolator , and outdoor  conditions were compared using pigs as an experimental model.
using extensive analysis of 16s rrna gene libraries our study categorically revealed that early-life environment has a major impact on microbial diversity and that these differences are sustainable throughout adult life.
many of the bacterial phylotypes identified in our study are commonly found in the human and animal gastrointestinal tract  <cit> .
our results also identified that only  <dig> % of the clones had less than 97% sequence similarity to existing database entries.
a major finding of the current study was the significant increase in the firmicutes phylum in sow-reared pigs housed in outdoor environments compared to littermates housed in isolators with daily antibiotic treatment.
within the firmicutes phylum, the most compelling observation was the abundance of lactobacilli in animals reared in the outdoor environment.
lactobacilli are often associated with the suckling pig and early stages of colonization in the gastrointestinal tract.
in this study, the high abundance of lactobacilli in the fecal samples obtained from truly adult sows identified lactobacilli as normal colonizers of the adult pig microbiota in the outdoor environment.
leser et al.
<cit>  found similar high-abundance phylotypes associated with the ileum, including l. amylovorous, l. johnsonii and l. reuteri, in pigs from different rearing environments.
our study further revealed that an increase in hygiene status in pigs housed both indoor and in isolators with antibiotic administration was associated with a significant decrease in mucosa-adherent lactobacilli.
affected species included l. reuteri, l. delbrueckii, l. amylovorous, l. johnsonii and l. mucosae.
the reduced microbial diversity in outdoor animals compared to indoor and isolator housed groups was a somewhat surprising outcome.
these outdoor animals were exposed to a huge variety of different bacterial species, as well as fungi, archaea and viruses, originating from both maternal and environmental sources.
the soil especially is hugely abundant in micro-organisms, and estimates of soil diversity show the presence of at least  <dig> phyla, the dominant members of which are proteobacteria, bacteroidetes and firmicutes  <cit> .
soil ecosystems potentially provide an important source of microbes for gut colonization of outdoor animals.
however, only a selective subset of environmental bacteria colonize the intestine, since we noted that the pig gut microbiota was comprised of a restricted number of phyla, dominated by bacteroidetes and firmicutes, consistent with published findings on the diversity of the adult human gut  <cit> .
current thinking has focussed on the benefits of a highly diverse gut microbiota, as it has long been considered that this confers greater plasticity of the bacterial community to respond to perturbations within the gut ecosystem  <cit> .
paradoxically, we found that exposure to a large variety of environmental microbes in early life does not generate greater diversity in the adult gut but rather leads to a microbiota that is dominated by a limited number of phyla composed of bacteria with proven health-promoting properties.
lactobacilli have long been known for their health-promoting effects and they directly limit the prevalence of several intestinal pathogens including e. coli and salmonella  <cit> .
in this study, l. reuteri was one of the most abundant members of the mucosa-adherent microbiota of the outdoor group.
reuterin, a broad-spectrum antimicrobial substance, is produced by l. reuteri  <cit>  and inhibits most intestinal bacteria with the exception of lactobacillus strains  <cit> .
importantly, the greater abundance of l. reuteri in the outdoor animals may contribute to the enhanced presence of other lactobacillus species as well as the decreased microbial diversity observed in these animals.
a further point meriting comment is the reduced presence of potentially pathogenic phylotypes in outdoor-housed pigs.
these phylotypes were clearly present in both indoor and isolator housed animals, although animals showed no overt signs of infection.
the specific reduction in firmicutes, in particular lactobacilli, in these pigs may affect the normal mechanisms of colonization resistance that control potentially pathogenic populations within the gut ecosystem.
although there has been a major focus on health-promoting probiotic actions of lactobacilli following their introduction as oral supplements, significantly less attention has been paid to the effects of naturally-acquired, gut-colonizing  lactobacilli.
given that immune modulation is dependent on gut colonization, close proximity to the mucosa and host adaptation, naturally-acquired lactobacilli clearly deserve greater attention.
of those species studied, l. casei, l. johnsonii and l. plantarum are strong inducers of il- <dig> and/or inf-γ, thereby favouring a th <dig> cytokine profile  <cit> .
conversely, l. reuteri inhibits the induction of il- <dig> and tnf-α and also attenuates l. casei-induced il- <dig>  <cit> .
a fine balance between th1-polarising lactobacilli strains and those which counterbalance such responses may be an important factor in maintaining mucosal immune homeostasis and explain the lack of overt th <dig> or th <dig> responses in outdoor-housed pigs in the current study.
while there was no evidence of th1/th <dig> pathways being affected, we found significant effects of environment on the type  <dig> interferon  signalling pathways.
isolator-reared pigs exhibited increased gene expression levels of the ifnα/β transcription/signalling factors irf <dig> and usp <dig>  type  <dig> ifn signalling induces the expression of a large number of target genes, which in the current study included mx <dig>  g1p <dig>  isg <dig>  fam14a, ifit <dig> and ifit <dig>  three type  <dig> ifn-inducible genes  were increased in indoor-housed animals compared to outdoor-housed animals, indicating that the ifnα/β pathway is directly affected by the housing environment.
a number of recent studies further support our data describing the influence of the gut microbiota on the type  <dig> ifn pathway.
for example, conventionalized pigs exhibited increased expression of irf <dig>  stat <dig> and stat <dig> when compared with their germ-free counterparts  <cit> .
conversely, bacterial colonization of germ-free mice led to a decreased expression of the ifn-related genes irf <dig>  isgf3g, ifit <dig> and stat <dig> <cit> .
our study further qualifies these findings by establishing that specific microbial composition, rather than the microbiota as such, influences type  <dig> ifn signalling during early colonization and development.
type  <dig> ifns have many biological properties, including innate, cellular and humoral adaptive immune responses  <cit> .
much evidence has focussed on their central role in pathogen resistance, especially viral immunity through recognition of dsrna.
the significance of type  <dig> ifns in response to bacterial colonization and infection is receiving increasingly more attention  <cit> .
ifn expression is induced in numerous cell lineages, including macrophages and plasmacytoid dendritic cells, by bacterial components such as lps and cpg dna  <cit> .
it is worth noting that the transcriptome analysis was performed on whole ileal tissue samples, rather than on a specific cell subset.
in this study we elected to study interactions and contributions of all cell lineages present in the gut to comprehensively characterize the transcriptomic changes induced by different microbiota compositions.
however, the contribution of individual lineages such as plasmacytoid dendritic cells , which naturally produce type  <dig> ifn, will be addressed in subsequent studies.
ifn-α/β has profound effects on immune cell development  <cit>  by regulating the differentiation of b and t cells, myeloid dcs and natural killer cells.
activation of immature dcs by ifn-α/β upregulates major histocompatibility complex  class i. consistent with this, we found that antigen presentation by mhc class i was also affected by the microbiota and was upregulated in indoor reared animals which also displayed increased type  <dig> ifn levels.
mhc class i molecules are type  <dig> ifn-inducible genes whose promoter regions contain typical ifn-stimulated response elements .
mhc class i molecules are specialized for presentation of endogenously synthesized proteins, including self-proteins, to the tcr of cd8+ t-cells  <cit> .
the cross-presentation of antigens on mhc class i molecules, the induction of ctl responses and the subsequent memory cd8+ t cell survival are also dependent on ifn-α/β.
increased expression of mhc class i in the indoor environment was accompanied by the upregulation of a plethora of chemokines, including ccl <dig>  ccl <dig>  ccl <dig>  ccr <dig>  cxcr <dig> and cxcl <dig>  chemokines are chemotactic cytokines that function during immune responses to recruit effector cells to sites of inflammation and infection.
they are involved in the pathophysiology of many diseases.
numerous chemokines have been implicated in the pathology and perpetuation of tissue destructive inflammatory processes in patients with ibd, including ccl <dig>  <cit>  and ccl <dig>  <cit> .
increased expression of these chemokines in the indoor-housed animals indicates the presence of an immune-activated gut microenvironment.
this contrasts with the lack of innate and pro-inflammatory gene expression in the outdoor-housed animals, which may be indicative of a more immune-tolerant and homeostatic mucosal immune system in these animals.
further studies are required to assess the impact of the microbiota, immune gene transcription and immune cell lineages on specific tolerance towards food and environmental antigens and long-term predisposition to infection, food intolerance and allergy.
CONCLUSIONS
environmental exposure in early life has a significant impact on microbiota composition of the adult gut and the immune transcriptome during development.
rural, outdoor environments support the establishment of a natural microbiota dominated by lactobacilli and containing low numbers of potentially pathogenic bacteria and this may be an important factor in maintaining mucosal immune homeostasis and limiting excessive inflammatory responses in the gut.
the significance of the microbiome and transcriptome data presented herein in relation to immune events such as oral tolerance and host defence against enteric pathogens is a major focus of our future studies.

 the gastrointestinal tract contains an immense number of micro-organisms, collectively known as the microbiota. the major functions of the microbiota include degrading dietary compounds, influencing nutrient partitioning and lipid metabolism, providing essential nutrients generated as a result of microbial metabolism, protecting against invading pathogens and stimulating gut morphology. the gut microbiota also plays an important role in maintaining immune function. recent work suggests that the commensal microbiota influences processes as complex as pathogen colonization, immune development and homeostasis, t cell differentiation, inflammation, repair and angiogenesis. the impact of the microbiota on host immunity is thought to be critically regulated in early life and inappropriate exposure to bacteria during this developmental window has been linked to the increased incidence of infectious, inflammatory and autoimmune diseases. clearly, the neonatal period is a critical time for gut colonization, and can be affected by numerous factors including gestational age, birth environment, mode of delivery, nutrition and antibiotic use. the increase in immune-mediated disorders, particularly in westernized countries, has led to the so-called hygiene hypothesis, which postulates that the growing incidence of immune-mediated diseases is the consequence of reduced infection and exposure to microbes during early childhood. in this context, the high-hygiene status of western lifestyle, decreased infection rates and reduced bacterial load as a result of widespread use of vaccines and antibiotics are likely to be important contributory factors. animal models have provided some insight into immune-disease aetiology: animals susceptible to autoimmune disease have an increased incidence and severity of disease when bred under germ-free conditions whereas disease is prevented when the animals are exposed to bacteria. this evidence supports the notion that, in addition to naturally-acquired infections, colonization by the normal commensal microbiota is an important factor limiting the incidences of immune-mediated diseases. consistent with this is the growing awareness of the importance of the commensal microbiota in immune education in early life, which appears to involve complex mechanisms of host-bacterial crosstalk. in the current study we have investigated potential interactions between the rearing environment, gut microbiota and immune function in the developing pig gut using molecular methods to evaluate both microbial diversity and host immune gene expression. microbial diversity in the gastrointestinal tract of these animals was characterized by sequence analysis of 16s rrna gene libraries. specific responses in transcriptome expression patterns of gut ileal tissue were studied using affymetrix genechip porcine genome microarrays. biomarkers associated with immune function and altered by rearing environment were identified and investigated more thoroughly by real-time polymerase chain reaction.  mucosal microbial diversity in the ileum of pigs from different environments we investigated the influence of environmentally-acquired bacteria on the composition of the adult mucosa-adherent ileal microbiota in the pig. animals were housed in an indoor or an outdoor facility, as well as in individual isolator units receiving daily doses of antibiotics. mucosa-adherent bacterial samples from the ileum and fecal samples were collected from all experimental animals at day in addition to this, fecal samples were taken from adult sows from both the indoor and outdoor environments to confirm'environment' as the major factor contributing to the experimental differences. microbial composition of the ileum was examined by calculation of diversity indices and analysis of the phylogenetic distribution of 16s rrna gene sequences derived from clone libraries of each treatment. after quality control, a total of validated clones were analyzed. diversity measures we first investigated the effects of environment and high-hygiene status on a number of bacterial diversity indices. estimates of diversity, richness and library coverage for the 16s rrna clone libraries from in, out and ir are shown in table species richness, estimated by chao was highest in the ir and in groups but lower in the out group. good's coverage was to % for all three treatment groups, with the lowest coverage in ir libraries. rarefaction analysis of clone libraries confirmed these findings and suggested that the ir and in groups possessed the most diverse mucosa-adherent bacterial community, whereas the out group showed lower microbial diversity. calculations were made based on otu definition at 99% sequence identity. collector's curves of the observed and estimated phylotype richness are shown in figure 2a-c. each curve reflects the series of observed or estimated richness values obtained as more clones were added to the data set. after an initial steep rise, the curves level out, suggesting that the majority of phylotypes in the treatment groups were adequately sampled. in the early stages of sampling and clone sequencing, both chao and abundance-based coverage estimator showed a sharp increase, together with the observed phylotype number, in the in group. after the sampling of about clones, the gap between the observed and estimated phylotype richness was relatively constant, indicating repeated sampling of same phylotypes within samples. in the out group, the gap between the observed and estimated phylotype richness was constant after the sampling point of clones. the difference between the estimated and observed phylotype richness was highest in the ir mucosal libraries. novel phylotypes continued to be identified up to the end of sampling. phylogenetic affiliation of 16s rrna gene sequences phylogenetic analysis was performed to establish taxonomic positioning of obtained sequences. all 16s rrna gene sequences from the mucosa-adherent ileal and fecal samples were subjected to the ribosomal database project classifier analysis. based on the classification results, the majority of clones were assigned to four phyla: firmicutes, proteobacteria, bacteroidetes, and actinobacteria. the two major phyla, firmicutes and bacteroidetes, were significantly different between the libraries: the firmicutes showed a significant increase in the out group compared to the ir group, while the bacteroidetes were significantly increased in both the ins and outs fecal libraries compared to the mucosa-adherent ileal libraries. firmicutes seventy percent of all sequences were affiliated with the firmicutes phylum. the outdoor environment favoured the expansion of firmicutes compared to the hygienic environment. at the lower taxa level this difference was even more pronounced. a large number of sequenced clones fell into the bacilli class. the most abundant order was lactobacillales, which was dominated by lactobacillaceae, but also contained streptococcaceae, leuconostocaceae, enterococcaceae, carnobacteriaceae and aerococcaceae, although present in lower abundance. the lactobacillaceae family in the out group consisted of a small number of operational taxonomic units, including lactobacillus reuteri, l. amylovorous lab l. johnsonii, l. delbrueckii subsp. bulgaricus, l. salivarius and l. mucosae. in contrast, the in library contained only % lactobacillaceae-affiliated clones, although phylotypes were similar to those observed in the out group. l. reuteri, l. delbrueckii and l. johnsonii were all significantly decreased compared to the out group. the high-hygiene conditions associated with ir exacerbated these differences. l. amylovorous lab and l. brevis were present in very low abundance in the ir libraries whereas l. reuteri, l. delbrueckii subsp. bulgaricus, l. johnsonii and l. mucosae were not detected in this treatment group. the observed differences in lactobacillus levels between the ir and out group were confirmed by enumeration of bacteria in gut contents of both ileum and colon on de man, rogosa and sharpe agar. the out group had three to four log colony forming units lactobacilli/g more than the ir group, thus validating the 16s rrna gene library results. members of the clostridia class were present in all treatment groups with % of all sequenced clones classified as clostridia. interestingly, pigs raised in the indoor environment showed the highest abundance of this class. clostridiaceae-affiliated clones were highly abundant in the in group and mainly identified as uncultured species. clostridium beijerinckii ncimb was significantly elevated in the in libraries compared to the out and ir libraries. the indoor environment also favoured the expansion of the bacterial clone hh_aai33h on the ileal mucosa compared to the outdoor environment. the peptostreptoccocaceae family was another abundant member of the clostridia class, accounting for % of the sequenced clones in all treatment groups. the indoor environment favoured the expansion of peptostreptoccocaceae. seven predominant otus, represented by mainly uncultured clones, were identified. sequences of uncultured clones barb_aaa02d were significantly higher in the in and ir groups compared to the out group and were not detected in the indoor fecal libraries. this possibly points to a preferential colonization of the ileal mucosa in the indoor environment. uncultured bacterium clone barb_aaa01f was significantly increased in the ileal mucosal libraries of the in group compared to the out and ir groups, indicating a potential antibiotic sensitivity of this bacterium. bacteroidetes bacteroidetes were found in all libraries but in different abundance. the most abundant group within this phylum was represented by members of the prevotellaceae family, followed by porphyromonadaceae, bacteroidaceae and, to a lesser extent, sphingobacteriaceae and flavobacteriaceae. all 16s rrna gene libraries contained members of prevotellaceae, yet they were most prevalent in the indoor environment, particularly in fecal libraries. high-hygiene conditions increased the numbers of prevotellaceae on the ileal mucosa. porphyromonadaceae were mainly obtained from the fecal libraries of both farms. most clones had only 97% similarity to previously isolated clones, specifically the porphyromonadaceae bacterium sp djf_b and uncultured bacterial clones. bacteroidaceae were exclusively obtained from the indoor environment. within the in and ir groups, these included bacteroides vulgatus and uncultured bacterial clones. in the in fecal libraries two species were related to b. propionifaciens and uncultured bacterium clone p-240-o. proteobacteria eighteen percent of all clones were placed into the proteobacteria phylum. γ-proteobacteria and ε-proteobacteria were the most abundant groups, while members of the α- and β-proteobacteria were found only sporadically. twenty-eight γ-proteobacteria clones were obtained from the out mucosa-adherent libraries. these included e. coli, actinobacillus minor and a. porcinus. six otus belonging to actinobacillus spp. were predominately present in the in group, including actinobacillus minor, a. porcinus strains h1498/h and a. rossii strain jf this clone has been isolated from the intestine and reproductive tract of pigs and is considered an opportunistic pathogen implicated in spontaneous abortion. high-hygiene status increased the number of γ-proteobacteria on the ileal mucosa. all 16s rrna gene libraries from the ir group contained members of the γ-proteobacteria class and grouped mainly with enterobacteriaceae, including sequences identified as e. coli spp. with pathogenic properties which may pose a health risk for the young pig as well as the human population. members of the ε-proteobacteria were the second most abundant group within the proteobacteria phylum and were represented by two major bacterial families, helicobacteraceae and campylobacteraceae. most clones were obtained from the in group and included bacteria of recognized pathogenic phenotype. transcriptomic analysis of gene expression patterns in the ileum of pigs from different environments while the comprehensive profiling of the mucosa-adherent microbial community revealed large differences in composition attributable to differences in housing environment, a key goal of this study was to determine whether this translated into different host-specific gene responses. therefore, an affymetrix genechip microarray analysis was conducted on ileum tissue from the same site used for 16s rrna gene library construction. effects of treatment extremes on gene expression perhaps not surprisingly, mucosa-adherent microbial diversity in the ileum was most affected by experimental isolator housing, as this constituted a high-hygiene environment. to ascertain the differences in host-specific transcriptional responses between this treatment and the natural outdoor environment, affymetrix microarray analysis was performed on the comparison ir versus out at day, day and day. seventy-four probesets were differentially expressed at the neonatal stage. fifty-six of these genes were highly expressed in the ir group, while genes were higher in the out group. interestingly, within the ir gene set, increased expression of genes that are closely linked to type interferon signalling was observed. these genes included irf fam14a, ube2l gbp and usp some of the most highly-regulated genes were viperin, a tightly regulated isgf target gene, and irp a pig-specific gene homologous to human viperin. another group showing increased expression in the ir group included genes involved in cholesterol synthesis, such as dhcr dhc sc5dl, hmgcs cyp51a and erg genes of interest showing higher expression in the out group compared to the ir group included tlr as well as hbb and hba both of which code for haemoglobin proteins. at day genes were differentially expressed. twenty-one of the transcripts expressed at higher levels in ir were the same as those found at day and included the ifn-induced genes irf and gbp several other type ifn-induced genes were higher in ir animals compared to out animals, indicating a consistent treatment effect on type ifn signalling pathways. also in common with the day gene expression set, nine cholesterol synthesis genes were increased in the ir group. consistent with these findings, microbiota-driven effects on cholesterol metabolism and trafficking have been previously documented. other transcripts expressed higher in the ir group compared to the out group included the chemokines cxcl ccl ccl ccl and cxcl the chemokine receptor ccr and the chemokine ligand ccl4l. pmp was increased in the out group. this gene is co-expressed with occludin and zona occludens at tight junctions in epithelial cells. sixty-six genes were differentially expressed between ir and out at day. some of the genes showing higher expression in the ir group included kai cebpb, ltb4dh, col14a and col1a changes in cebpb expression in ir animals may be functionally important as this gene is involved in the regulation of inflammatory responses. notably, a group of t-cell-related genes was increased in out animals, including tca_human, ly cd8a, trgv lcp lcp cxcl and tec, all of which are involved in t cell signalling, expansion, activation and trafficking. other highly expressed transcripts in the out group included egr sell, pigr and pik3cg. consistently, pdk was higher in the out group compared to the ir group at all three time-points. pdk has an important function in glucose metabolism, and its expression is regulated by glucocorticoids, retinoic acid and insulin; however, its potential relevance in host-microbe interactions is currently unknown. biological pathway analysis revealed that a large number of immune response pathways were affected. other highly represented pathways included g-protein and congenital, hereditary and neonatal diseases and abnormalities. consistent with the analysis of individual gene data, the pathway for immune response-ifn alpha/beta signalling was increased at day and day in the ir group compared to the out group. immune response-antigen presentation by mhc class l was affected at all three time-points and also higher in ir compared to out. gene ontology -enrichment analysis further confirmed these findings. while a number of go categories were consistently affected by treatment, including immune response, the major biological process affected was antigen processing and presentation. other affected go-processes were antigen processing and presentation of peptide antigen via mhc class i, antigen processing and presentation of peptide antigen and antigen processing and presentation of peptide or polysaccharide antigen via mhc class ii. differentially expressed genes were imported into genego metacore analytical software to determine the significantly enriched canonical pathways in the treatment comparison ir vs out. the top ten pathways for each comparison are shown, with the number of genes assigned to each pathway, and the corresponding p-value. * the number of genes on each map that are differentially expressed in the specific treatment comparison ** the total number of genes on each map differentially expressed transcripts are shown assigned to the go-category'biological process'. the top ten go-categories are shown. the number of transcripts for each function is shown, with the corresponding p-value. *genes involved in the specific go-category effects of housing environment on gene expression differences in ileal mucosa-adherent microbial composition between the ir group and the out group were associated with large host-specific transcriptional differences in the ileum. we next set out to assess whether the microbial differences associated with the in and out environments had a similar impact on the gut transcriptome of the pig. while the number of differentially expressed genes between in and out housed animals was smaller than between the treatment extremes, similar trends could be discerned. in the neonatal pig, the expression levels of probesets were differentially expressed between the in and out animals. nine genes were higher in in animals, and this included cxcl which is involved in t cell trafficking. four genes showed higher expression in out animals, including tfrc. in weaning animals, genes were differentially expressed between the two rearing environments. twelve transcripts were higher in in animals, including tafa, ccr and cxcr of the genes that were higher in the out group, genes of interest included pmp cnksr tjp and ltbr. the largest differences in gene expression were observed at day when genes were differentially expressed between the treatments. transcripts increased in in animals included three type ifn-inducible genes. the antibacterial peptide genes lyz, pi and bpi were increased -, - and -fold, respectively, in in animals compared to out animals and may contribute to the observed differences in microbiota composition between these groups. furthermore, these peptides appear to maintain gut homeostasis as evidenced by their aberrant expression in crohn's disease and ulcerative colitis. ccl was also higher in the in group. some of the genes increased in out animals were pmp and sell, in accordance with the observations from the ir and out comparison. the most affected pathways belonged to immune response, g-protein and congenital, hereditary, and neonatal diseases and abnormalities, as observed previously in the treatment extremes comparison. differentially expressed genes were imported into genego metacore analytical software to determine the significantly enriched canonical pathways in the treatment comparison in vs out. the top ten pathways for each comparison are shown, with the number of genes assigned to each pathway, and the corresponding p-value. * the number of genes on each map that are differentially expressed in the specific treatment comparison ** the total number of genes on each map real-time quantitative pcr to analyze differentially expressed genes real-time pcr was performed for ccl ccl cxcl cxcl cxcr ifit fkbp irf irp mt1j, mx, pdk pi sell, sqle and tfrc. these genes were selected from the gene expression data set both because they showed significant changes and because of their involvement in key immune system pathways. verification of the true differential expression between treatment groups of these genes by real-time pcr was therefore considered essential for further biological interpretation. the subsequent correlation between affymetrix microarray and real-time pcr data was positive, further substantiating the biological importance of the selected genes and identified pathways. real-time pcr verification for the comparison ir versus out showed that direction and magnitude of fold change correlated well with the affymetrix microarray results. in some cases the fold changes detected by real-time pcr were lower than those observed by microarray analysis. real-time pcr results at each time-point for the comparisons ir vs out and in vs out. *pcr values are expressed as mean Δct ± sd, n = **standard error of difference the differentially expressed genes from the in versus out comparison were examined by real-time pcr and again correlated well with the affymetrix microarray results. only cxcr expression in the in group showed disagreement between the two platforms, as it was increased using microarray analysis, and decreased using real-time pcr. discussion the current study sought to investigate the effects of environmental hygiene on microbial colonization and composition of the gut microbiota. additionally, transcriptomic profiling was performed to assess the impact of environmental hygiene on gene expression, in particular those genes and pathways associated with immune function. both indoor and isolator, and outdoor conditions were compared using pigs as an experimental model. using extensive analysis of 16s rrna gene libraries our study categorically revealed that early-life environment has a major impact on microbial diversity and that these differences are sustainable throughout adult life. many of the bacterial phylotypes identified in our study are commonly found in the human and animal gastrointestinal tract. our results also identified that only % of the clones had less than 97% sequence similarity to existing database entries. a major finding of the current study was the significant increase in the firmicutes phylum in sow-reared pigs housed in outdoor environments compared to littermates housed in isolators with daily antibiotic treatment. within the firmicutes phylum, the most compelling observation was the abundance of lactobacilli in animals reared in the outdoor environment. lactobacilli are often associated with the suckling pig and early stages of colonization in the gastrointestinal tract. in this study, the high abundance of lactobacilli in the fecal samples obtained from truly adult sows identified lactobacilli as normal colonizers of the adult pig microbiota in the outdoor environment. leser et al. found similar high-abundance phylotypes associated with the ileum, including l. amylovorous, l. johnsonii and l. reuteri, in pigs from different rearing environments. our study further revealed that an increase in hygiene status in pigs housed both indoor and in isolators with antibiotic administration was associated with a significant decrease in mucosa-adherent lactobacilli. affected species included l. reuteri, l. delbrueckii, l. amylovorous, l. johnsonii and l. mucosae. the reduced microbial diversity in outdoor animals compared to indoor and isolator housed groups was a somewhat surprising outcome. these outdoor animals were exposed to a huge variety of different bacterial species, as well as fungi, archaea and viruses, originating from both maternal and environmental sources. the soil especially is hugely abundant in micro-organisms, and estimates of soil diversity show the presence of at least phyla, the dominant members of which are proteobacteria, bacteroidetes and firmicutes. soil ecosystems potentially provide an important source of microbes for gut colonization of outdoor animals. however, only a selective subset of environmental bacteria colonize the intestine, since we noted that the pig gut microbiota was comprised of a restricted number of phyla, dominated by bacteroidetes and firmicutes, consistent with published findings on the diversity of the adult human gut. current thinking has focussed on the benefits of a highly diverse gut microbiota, as it has long been considered that this confers greater plasticity of the bacterial community to respond to perturbations within the gut ecosystem. paradoxically, we found that exposure to a large variety of environmental microbes in early life does not generate greater diversity in the adult gut but rather leads to a microbiota that is dominated by a limited number of phyla composed of bacteria with proven health-promoting properties. lactobacilli have long been known for their health-promoting effects and they directly limit the prevalence of several intestinal pathogens including e. coli and salmonella. in this study, l. reuteri was one of the most abundant members of the mucosa-adherent microbiota of the outdoor group. reuterin, a broad-spectrum antimicrobial substance, is produced by l. reuteri and inhibits most intestinal bacteria with the exception of lactobacillus strains. importantly, the greater abundance of l. reuteri in the outdoor animals may contribute to the enhanced presence of other lactobacillus species as well as the decreased microbial diversity observed in these animals. a further point meriting comment is the reduced presence of potentially pathogenic phylotypes in outdoor-housed pigs. these phylotypes were clearly present in both indoor and isolator housed animals, although animals showed no overt signs of infection. the specific reduction in firmicutes, in particular lactobacilli, in these pigs may affect the normal mechanisms of colonization resistance that control potentially pathogenic populations within the gut ecosystem. although there has been a major focus on health-promoting probiotic actions of lactobacilli following their introduction as oral supplements, significantly less attention has been paid to the effects of naturally-acquired, gut-colonizing lactobacilli. given that immune modulation is dependent on gut colonization, close proximity to the mucosa and host adaptation, naturally-acquired lactobacilli clearly deserve greater attention. of those species studied, l. casei, l. johnsonii and l. plantarum are strong inducers of il- and/or inf-γ, thereby favouring a th cytokine profile. conversely, l. reuteri inhibits the induction of il- and tnf-α and also attenuates l. casei-induced il-. a fine balance between th1-polarising lactobacilli strains and those which counterbalance such responses may be an important factor in maintaining mucosal immune homeostasis and explain the lack of overt th or th responses in outdoor-housed pigs in the current study. while there was no evidence of th1/th pathways being affected, we found significant effects of environment on the type interferon signalling pathways. isolator-reared pigs exhibited increased gene expression levels of the ifnα/β transcription/signalling factors irf and usp type ifn signalling induces the expression of a large number of target genes, which in the current study included mx g1p isg fam14a, ifit and ifit three type ifn-inducible genes were increased in indoor-housed animals compared to outdoor-housed animals, indicating that the ifnα/β pathway is directly affected by the housing environment. a number of recent studies further support our data describing the influence of the gut microbiota on the type ifn pathway. for example, conventionalized pigs exhibited increased expression of irf stat and stat when compared with their germ-free counterparts. conversely, bacterial colonization of germ-free mice led to a decreased expression of the ifn-related genes irf isgf3g, ifit and stat. our study further qualifies these findings by establishing that specific microbial composition, rather than the microbiota as such, influences type ifn signalling during early colonization and development. type ifns have many biological properties, including innate, cellular and humoral adaptive immune responses. much evidence has focussed on their central role in pathogen resistance, especially viral immunity through recognition of dsrna. the significance of type ifns in response to bacterial colonization and infection is receiving increasingly more attention. ifn expression is induced in numerous cell lineages, including macrophages and plasmacytoid dendritic cells, by bacterial components such as lps and cpg dna. it is worth noting that the transcriptome analysis was performed on whole ileal tissue samples, rather than on a specific cell subset. in this study we elected to study interactions and contributions of all cell lineages present in the gut to comprehensively characterize the transcriptomic changes induced by different microbiota compositions. however, the contribution of individual lineages such as plasmacytoid dendritic cells, which naturally produce type ifn, will be addressed in subsequent studies. ifn-α/β has profound effects on immune cell development by regulating the differentiation of b and t cells, myeloid dcs and natural killer cells. activation of immature dcs by ifn-α/β upregulates major histocompatibility complex class i. consistent with this, we found that antigen presentation by mhc class i was also affected by the microbiota and was upregulated in indoor reared animals which also displayed increased type ifn levels. mhc class i molecules are type ifn-inducible genes whose promoter regions contain typical ifn-stimulated response elements. mhc class i molecules are specialized for presentation of endogenously synthesized proteins, including self-proteins, to the tcr of cd8+ t-cells. the cross-presentation of antigens on mhc class i molecules, the induction of ctl responses and the subsequent memory cd8+ t cell survival are also dependent on ifn-α/β. increased expression of mhc class i in the indoor environment was accompanied by the upregulation of a plethora of chemokines, including ccl ccl ccl ccr cxcr and cxcl chemokines are chemotactic cytokines that function during immune responses to recruit effector cells to sites of inflammation and infection. they are involved in the pathophysiology of many diseases. numerous chemokines have been implicated in the pathology and perpetuation of tissue destructive inflammatory processes in patients with ibd, including ccl and ccl. increased expression of these chemokines in the indoor-housed animals indicates the presence of an immune-activated gut microenvironment. this contrasts with the lack of innate and pro-inflammatory gene expression in the outdoor-housed animals, which may be indicative of a more immune-tolerant and homeostatic mucosal immune system in these animals. further studies are required to assess the impact of the microbiota, immune gene transcription and immune cell lineages on specific tolerance towards food and environmental antigens and long-term predisposition to infection, food intolerance and allergy.  environmental exposure in early life has a significant impact on microbiota composition of the adult gut and the immune transcriptome during development. rural, outdoor environments support the establishment of a natural microbiota dominated by lactobacilli and containing low numbers of potentially pathogenic bacteria and this may be an important factor in maintaining mucosal immune homeostasis and limiting excessive inflammatory responses in the gut. the significance of the microbiome and transcriptome data presented herein in relation to immune events such as oral tolerance and host defence against enteric pathogens is a major focus of our future studies. 
pigs housed in a natural outdoor environment showed a dominance of firmicutes, in particular lactobacillus, whereas animals housed in a hygienic indoor environment had reduced lactobacillus and higher numbers of potentially pathogenic phylotypes. the extent to which early-life environment impacts on microbial diversity of the adult gut and subsequent immune processes has not been comprehensively investigated thus far. analysis of over 16s rrna sequences revealed major differences in mucosa-adherent microbial diversity in the ileum of adult pigs attributable to differences in early-life environment. we observed that a microbiota dominated by lactobacilli may function to maintain mucosal immune homeostasis and limit pathogen colonization. we addressed this important question using the pig as a model to evaluate the impact of early-life environment on microbe/host gut interactions during development. affymetrix microarray technology and real-time polymerase chain reaction revealed significant gut-specific gene responses also related to early-life environment. early-life environment significantly affects both microbial composition of the adult gut and mucosal innate immune function. gene ontology and pathway analysis further confirmed these results. early microbial colonization of the gut reduces the incidence of infectious, inflammatory and autoimmune diseases. recent population studies reveal that childhood hygiene is a significant risk factor for development of inflammatory bowel disease, thereby reinforcing the hygiene hypothesis and the potential importance of microbial colonization during early life. genetically-related piglets were housed in either indoor or outdoor environments or in experimental isolators. significantly, indoor-housed pigs displayed increased expression of type interferon genes, major histocompatibility complex class i and several chemokines.
 early microbial colonization of the gut reduces the incidence of infectious, inflammatory and autoimmune diseases. recent population studies reveal that childhood hygiene is a significant risk factor for development of inflammatory bowel disease, thereby reinforcing the hygiene hypothesis and the potential importance of microbial colonization during early life. the extent to which early-life environment impacts on microbial diversity of the adult gut and subsequent immune processes has not been comprehensively investigated thus far. we addressed this important question using the pig as a model to evaluate the impact of early-life environment on microbe/host gut interactions during development.  genetically-related piglets were housed in either indoor or outdoor environments or in experimental isolators. analysis of over 16s rrna sequences revealed major differences in mucosa-adherent microbial diversity in the ileum of adult pigs attributable to differences in early-life environment. pigs housed in a natural outdoor environment showed a dominance of firmicutes, in particular lactobacillus, whereas animals housed in a hygienic indoor environment had reduced lactobacillus and higher numbers of potentially pathogenic phylotypes. our analysis revealed a strong negative correlation between the abundance of firmicutes and pathogenic bacterial populations in the gut. these differences were exaggerated in animals housed in experimental isolators. affymetrix microarray technology and real-time polymerase chain reaction revealed significant gut-specific gene responses also related to early-life environment. significantly, indoor-housed pigs displayed increased expression of type interferon genes, major histocompatibility complex class i and several chemokines. gene ontology and pathway analysis further confirmed these results.  early-life environment significantly affects both microbial composition of the adult gut and mucosal innate immune function. we observed that a microbiota dominated by lactobacilli may function to maintain mucosal immune homeostasis and limit pathogen colonization. 