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The ecological interactions within the gut microbial communities are complex and far from being fully understood. Here we report the first study that aims at defining the interaction network of the gut microbiota in pigs and comparing it with the enterotype-like clustering analysis. Fecal microbiota of 518 healthy piglets was characterized by 16S ribosomal RNA gene sequencing. Two networks were constructed at the genus and operational taxonomic unit levels. Within-network interactions mirrored the human gut microbiota relationships, with a strong co-exclusion between Prevotella and Ruminococcus genera, and were consistent with the two enterotype-like clusters identified in the pig microbiota. Remarkably, the cluster classification of the individuals was significantly associated with the body weight at 60 days of age (P=0.005) and average daily gain (P=0.027). To the best of our knowledge, this is the first study to provide an integrated overview of the porcine gut microbiota that suggests a conservation of the ecological community interactions and functional architecture between humans and pig. Moreover, we show that the microbial ecosystems and porcine growth traits are linked, which allows us to foresee that the enterotype concept may have an important role in the animal production industry.The ISME Journal advance online publication, 13 May 2016; doi:10.1038/ismej.2016.77.

Background In pig production systems, weaning is a crucial period characterized by nutritional, environmental, and social stresses. Piglets transition from a milk-based diet to a solid, more complex plant-based diet, and their gut physiology must adapt accordingly. It is well established that piglets weaned later display improved health, better wean-to-finish growth performance, and lower mortality rates. The aim of this study was to evaluate the impact of weaning age on fecal microbiota diversity and composition in piglets. Forty-eight Large White piglets were divided into 4 groups of 12 animals that were weaned at different ages: 14 days (early weaning), 21 days (a common weaning age in intensive pig farming), 28 days ( idem ), and 42 days (late weaning). Microbiota composition was assessed in each group by sequencing the 16S rRNA gene using fecal samples taken on the day of weaning, 7 days later, and at 60 days of age. Results In each group, there were significant differences in fecal microbiota composition before and after weaning ( p  < 0.05), confirming that weaning can drastically change the gut microbiota. Microbiota diversity was positively correlated with weaning age: microbial alpha diversity and richness were higher in piglets weaned at 42 days of age both on the day of weaning and 7 days later. The abundance of Faecalibacterium prausnitzii operational taxonomic units (OTUs) was also higher in piglets weaned at 42 days of age. Conclusions Overall, these results show that late weaning increased gut microbiota diversity and the abundance of F. prausnitzii , a microorganism with positive effects in humans. Piglets might thus derive a competitive advantage from later weaning because they have more time to accumulate a higher diversity of potentially beneficial microbes prior to the stressful and risky weaning period.

and which often tend to be obese. The SLAdd (DD) minipig line was created by the NIH and selected as homozygous at the SLA locus. It was brought to France more than 30 years ago and maintained inbred ever since. In this report, we characterized the physiological status of a herd of French DD pigs by measuring intermediate phenotypes from blood and faeces and by using Large White (LW) pigs as controls. Three datasets were produced, i.e. complete blood counts (CBCs), microarray-based blood transcriptome, and faecal microbiota obtained by 16S rRNA sequencing. CBCs and expression profiles suggested a non-alcoholic fatty liver disease (NAFLD)-related pathology associated to comorbid cardiac diseases. The characterization of 16S sequencing data was less straightforward, suggesting only a potential weak link to obesity. The integration of the datasets identified several fine-scale associations between CBCs, gene expression, and faecal microbiota composition. NAFLD is a common cause of chronic liver disease in Western countries and is linked to obesity, type 2 diabetes mellitus and cardiac pathologies. Here we show that the French DD herd is potentially affected by this syndrome.

We focused on a developmentally regulated growth acceleration in the darkgrown Arabidopsis hypocotyl to study the role of changes in cell wall metabolism in the control of cell elongation. To this end, precise transcriptome analysis on dissected dark-grown hypocotyls, ourier transform infrared (FT-IR) microspectroscopy and kinematic analysis were used. Using a cellulose synthesis inhibitor, we showed that the growth acceleration marks a developmental transition during which growth becomes uncoupled from cellulose synthesis. We next investigated the cellular changes that take place during this transition. FT-IR microspectroscopy revealed significant changes in cell wall composition during, but not after, the growth acceleration. Transcriptome analysis suggested a role for cell wall remodeling, in particular pectin modification, in this growth acceleration. This was confirmed by the overexpression of a pectin methylesterase inhibitor, which caused a delay in the growth acceleration. This study shows that the acceleration of cell elongation marks a developmental transition in dark-grown hypocotyl cells and supports a role for pectin demethylesterification in the timing of this event.

This study describes the associations between fecal microbiota and vaccine response variability in pigs, using 98 piglets vaccinated against the influenza A virus at 28 days of age (D28) with a booster at D49. Immune response to the vaccine is measured at D49, D56, D63, and D146 by serum levels of IAV-specific IgG and assays of hemagglutination inhibition (HAI). Analysis of the pre-vaccination microbiota characterized by 16S rRNA gene sequencing of fecal DNA reveals a higher vaccine response in piglets with a richer microbiota, and shows that 23 operational taxonomic units (OTUs) are differentially abundant between high and low IAV-specific IgG producers at D63. A stronger immune response is linked with OTUs assigned to the genus Prevotella and family Muribaculaceae, and a weaker response is linked with OTUs assigned to the genera Helicobacter and Escherichia-Shigella . A set of 81 OTUs accurately predicts IAV-specific IgG and HAI titer levels at all time points, highlighting early and late associations between pre-vaccination fecal microbiota composition and immune response to the vaccine.

Background The impact of individual genetic and genomic variations on immune responses is an emerging lever investigated in vaccination strategies. In our study, we used genetic and pre-vaccination blood transcriptomic data to study vaccine effectiveness in pigs. Results A cohort of 182 Large White pigs was vaccinated against Mycoplasma hyopneumoniae ( M. hyo ) at weaning (28 days of age), with a booster 21 days later. Vaccine response was assessed by measuring seric M. hyo antibodies (Ab) at 0 (vaccination day), 21 (booster day), 28, 35, and 118 days post-vaccination (dpv). Inter-individual variability of M. hyo Ab levels was observed at all time points and the corresponding heritabilities ranged from 0.46 to 0.57. Ab persistence was higher in females than in males. Genome-wide association studies with a 658 K SNP panel revealed two genomic regions associated with variations of M. hyo Ab levels at 21 dpv at positions where immunity-related genes have been mapped, DAB2IP on chromosome 1, and ASAP1 , CYRIB and GSDMC on chromosome 4. We studied covariations of Ab responses with the pre-vaccination blood transcriptome obtained by RNA-Seq for a subset of 82 pigs. Weighted gene correlation network and differential expression analyses between pigs that differed in Ab responses highlighted biological functions that were enriched in heme biosynthesis and platelet activation for low response at 21 dpv, innate antiviral immunity and dendritic cells for high response at 28 and 35 dpv, and cell adhesion and extracellular matrix for high response at 118 dpv. Sparse partial least squares discriminant analysis identified 101 genes that efficiently predicted divergent responders at all time points. We found weak negative correlations of M. hyo Ab levels with body weight traits, which revealed a trade-off that needs to be further explored. Conclusions We confirmed the influence of the host genetics on vaccine effectiveness to M. hyo and provided evidence that the pre-vaccination blood transcriptome co-varies with the Ab response. Our results highlight that both genetic markers and blood biomarkers could be used as potential predictors of vaccine response levels and more studies are required to assess whether they can be exploited in breeding programs.

Background Designing sustainable animal production systems that better balance productivity and resistance to disease is a major concern. In order to address questions related to immunity and resistance to disease in pig, it is necessary to increase knowledge on its immune system and to produce efficient tools dedicated to this species. Results A long-oligonucleotide-based chip referred to as SLA-RI/NRSP8-13K was produced by combining a generic set with a newly designed SLA-RI set that targets all annotated loci of the pig major histocompatibility complex (MHC) region (SLA complex) in both orientations as well as immunity genes outside the SLA complex. The chip was used to study the immune response of pigs following stimulation of porcine peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) or a mixture of phorbol myristate acetate (PMA) and ionomycin for 24 hours. Transcriptome analysis revealed that ten times more genes were differentially expressed after PMA/ionomycin stimulation than after LPS stimulation. LPS stimulation induced a general inflammation response with over-expression of SAA1, pro-inflammatory chemokines IL8, CCL2, CXCL5, CXCL3, CXCL2 and CCL8 as well as genes related to oxidative processes (SOD2) and calcium pathways (S100A9 and S100A12). PMA/ionomycin stimulation induced a stronger up-regulation of T cell activation than of B cell activation with dominance toward a Th1 response, including IL2, CD69 and TNFRSF9 (tumor necrosis factor receptor superfamily, member 9) genes. In addition, a very intense repression of THBS1 (thrombospondin 1) was observed. Repression of MHC class I genes was observed after PMA/ionomycin stimulation despite an up-regulation of the gene cascade involved in peptide processing. Repression of MHC class II genes was observed after both stimulations. Our results provide preliminary data suggesting that antisense transcripts mapping to the SLA complex may have a role during immune response. Conclusion The SLA-RI/NRSP8-13K chip was found to accurately decipher two distinct immune response activations of PBMCs indicating that it constitutes a valuable tool to further study immunity and resistance to disease in pig. The transcriptome analysis revealed specific and common features of the immune responses depending on the stimulation agent that increase knowledge on pig immunity.

The gut microbiota comprises a large and diverse community of bacteria that play a significant role in swine health. Indeed, there is a tight association between the enteric immune system and the overall composition and richness of the microbiota, which is key in the induction, training and function of the host immunity, and may therefore, influence the immune response to vaccination. Using vaccination against Mycoplasma hyopneumoniae (M. hyo) as a model, we investigated the potential of early-life gut microbiota in predicting vaccine response and explored the post-vaccination dynamics of fecal microbiota at later time points. At 28 days of age (0 days post-vaccination; dpv), healthy piglets were vaccinated, and a booster vaccine was administered at 21 dpv. Blood samples were collected at 0, 21, 28, 35, and 118 dpv to measure M. hyo-specific IgG levels. Fecal samples for 16S rRNA gene amplicon sequencing were collected at 0, 21, 35, and 118 dpv. The results showed variability in antibody response among individual pigs, whilst pre-vaccination operational taxonomic units (OTUs) primarily belonging to Prevotella, [Prevotella], Anaerovibrio, and Sutterella appeared to best-predict vaccine response. Microbiota composition did not differ between the vaccinated and non-vaccinated pigs at post-vaccination time points, but the time effect was significant irrespective of the animals’ vaccination status. Our study provides insight into the role of pre-vaccination gut microbiota composition in vaccine response and emphasizes the importance of studies on full metagenomes and microbial metabolites aimed at deciphering the role of specific bacteria and bacterial genes in the modulation of vaccine response.

Background: Increasing robustness via improvement of resistance to pathogens is a major selection objective in livestock breeding. As resistance traits are difficult or impossible to measure directly, potential indirect criteria are measures of immune traits (ITs). Our underlying hypothesis is that levels of ITs with no focus on specific pathogens define an individual's immunocompetence and thus predict response to pathogens in general. Since variation in ITs depends on genetic, environmental and probably epigenetic factors, our aim was to estimate the relative importance of genetics. In this report, we present a large genetic survey of innate and adaptive ITs in pig families bred in the same environment.[br/] Methodology/Principal Findings: Fifty four ITs were studied on 443 Large White pigs vaccinated against Mycoplasma hyopneumoniae and analyzed by combining a principal component analysis (PCA) and genetic parameter estimation. ITs include specific and non specific antibodies, seric inflammatory proteins, cell subsets by hemogram and flow cytometry, ex vivo production of cytokines (IFNa, TNFa, IL6, IL8, IL12, IFNc, IL2, IL4, IL10), phagocytosis and lymphocyte proliferation. While six ITs had heritabilities that were weak or not significantly different from zero, 18 and 30 ITs had moderate (0.1,h2#0.4) or high (h2.0.4) heritability values, respectively. Phenotypic and genetic correlations between ITs were weak except for a few traits that mostly include cell subsets. PCA revealed no cluster of innate or adaptive ITs. [br/] Conclusions/Significance: Our results demonstrate that variation in many innate and adaptive ITs is genetically controlled in swine, as already reported for a smaller number of traits by other laboratories. A limited redundancy of the traits was also observed confirming the high degree of complementarity between innate and adaptive ITs. Our data provide a genetic framework for choosing ITs to be included as selection criteria in multitrait selection programmes that aim to improve both production and health traits.

In pig, very little information is available on the non classical class I (Ib) genes of the Major Histocompatibility Complex (MHC) i.e. SLA-6 , -7 and -8 . Our aim was to focus on the transcription pattern of the SLA-7 gene. RT-PCR experiments were carried out with SLA-7 specific primers targeting either the full coding sequence (CDS) from exon 1 to the 3 prime untranslated region (3UTR) or a partial CDS from exon 4 to the 3UTR. We show that the SLA-7 gene expresses a full length transcript not yet identified that refines annotation of the gene with eight exons instead of seven as initially described from the existing RefSeq RNA. These two RNAs encode molecules that differ in cytoplasmic tail length. In this study, another SLA-7 transcript variant was characterized, which encodes a protein with a shorter alpha 3 domain, as a consequence of a splicing site within exon 4. Surprisingly, a cryptic non canonical GA-AG splicing site is used to generate this transcript variant. An additional SLA-7 variant was also identified in the 3UTR with a splicing site occurring 31 nucleotides downstream to the stop codon. In conclusion, the pig SLA-7 MHC class Ib gene presents a complex transcription pattern with two transcripts encoding various molecules and transcripts that do not alter the CDS and may be subject to post-transcriptional regulation.