The microbiota of the gastro-intestinal tract: a biological compartment to explore in livestock species

In a perspective of safe, competitive and sustainable animal breeding systems, deepening animal phenotyping and defining new breeding goals are identified as major concerns. The intestine hosts a microbial ecosystem referred to as the gut microbiota. The gut epithelium, at the interface between the host and the microbiota, plays fundamental roles in nutrient absorption, local and systemic immune functions, as well as adaptive functions. The onset of high throughput sequencing methods have paved the way toward an unprecedented characterization of the microbiota by producing data on the non cultivable bacteria set. In livestock species, the scientific and technological context is in place to study the gut microbiota at both phylogenetic (bacteria diversity) and functional levels, and analyze the impact on shaping the host phenotypeL’élaboration de systèmes productifs et durables en élevage est une priorité. Dans ce contexte, il convient d’élargir le champ du phénotypage et d’identifier des leviers d’action. L’intestin héberge une communauté microbienne appelée microbiote intestinal. La muqueuse intestinale, à l’interface entre l’hôte et son microbiote, assure des fonctions vitales dans l’absorption des nutriments, dans l’équilibre immunitaire à la fois local et systémique, et dans la dynamique adaptative au cours de la vie. L’avènement de méthodes de séquençage à haut débit permet aujourd’hui de caractériser le microbiote intestinal avec une précision encore jamais atteinte, fournissant des données sur la portion bactérienne non cultivable. Chez les animaux d’élevage, le contexte scientifique et technologique est favorable pour mettre en place des projets qui croisent des informations individuelles liées au microbiote intestinal (diversité, écologie fonctionnelle) et à l’hôte (génétique, caractères zootechniques et cliniques, statut immunitaire), afin d’aborder de manière intégrée les nouveaux caractères à mesurer et améliore

The porcine Major Histocompatibility Complex and related paralogous regions: a review

The physical alignment of the entire region of the pig major histocompatibility complex (MHC) has been almost completed. In swine, the MHC is called the SLA (swine leukocyte antigen) and most of its class I region has been sequenced. Over one hundred genes have been characterised, including the classical class I and class I-related genes, as well as the class II gene families. These results in swine provide new evidence for the striking conservation during the evolution of a general MHC framework, and are consistent with the location of the class I genes on segments referred to as permissive places within the MHC class I region. Recent results confirm the involvement of the SLA region in numerous quantitative traits

Transcription variants of SLA-7, a swine non classical MHC class I gene

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

Demonstration of microchimerism in pregnant sows and effects of congenital PRRSV infection

The presence of foreign cells within the tissue/circulation of an individual is described as microchimerism. The main purpose of the present investigation was to study if microchimerism occurs in healthy sows/fetuses and if porcine reproductive and respiratory syndrome virus (PRRSV) infection influences this phenomenon. Six dams were inoculated intranasally with PRRSV and three non-inoculated dams served as controls. Male DNA was detected in female fetal sera of all dams via PCR. Male DNA was also detected in the maternal circulation. Sex-typing FISH showed the presence of male cells in the female fetal organs and vice versa. PRRSV infection did not influence microchimerism, but might misuse maternal and sibling microchimeric cells to enter fetuses

Transcriptomic analysis of the dialogue between Pseudorabies virus and porcine epithelial cells during infection

International audienceBACKGROUND: Transcriptomic approaches are relevant for studying virus-host cell dialogues to better understand the physiopathology of infection and the immune response at the cellular level. Pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for such studies in pig. Since PrV displays a strong tropism for mucous epithelial cells, we developed a kinetics study of PrV infection in the porcine PK15 epithelial cell line. To identify as completely as possible, viral and cellular genes regulated during infection, we simultaneously analyzed PrV and cellular transcriptome modifications using two microarrays i.e. a laboratory-made combined SLA/PrV microarray, consisting of probes for all PrV genes and for porcine genes contained in the Swine Leukocyte Antigen (SLA) complex, and the porcine generic Qiagen-NRSP8 oligonucleotide microarray. We confirmed the differential expression of a selected set of genes by qRT-PCR and flow cytometry. RESULTS: An increase in the number of differentially expressed cellular genes and PrV genes especially from 4 h post-infection (pi) was observed concomitantly with the onset of viral progeny while no early global cellular shutoff was recorded. Many cellular genes were down-regulated from 4 h pi and their number increased until 12 h pi. UL41 transcripts encoding the virion host shutoff protein were first detected as differentially expressed at 8 h pi. The viral gene UL49.5 encoding a TAP inhibitor protein was differentially expressed as soon as 2 h pi, indicating that viral evasion via TAP inhibition may start earlier than the cellular gene shutoff. We found that many biological processes are altered during PrV infection. Indeed, several genes involved in the SLA class I antigenic presentation pathway (SLA-Ia, TAP1, TAP2, PSMB8 and PSMB9), were down-regulated, thus contributing to viral immune escape from this pathway and other genes involved in apoptosis, nucleic acid metabolism, cytoskeleton signaling as well as interferon-mediated antiviral response were also modulated during PrV infection. CONCLUSION: Our results show that the gene expression of both PrV and porcine cells can be analyzed simultaneously with microarrays, providing a chronology of PrV gene transcription, which has never been described before, and a global picture of transcription with a direct temporal link between viral and host gene expression

Deciphering the genetic control of innate and adaptive immune responses in pig: a combined genetic and genomic study

Improving animal robustness and resistance to pathogens by adding health criteria in selection schemes is one of the challenging objectives of the next decade. In order to better understand the genetic control of immunity in French Large White pigs, we have launched a program combining genetic and genomic studies not focussing on any particular pathogen. Animals recorded for production traits were scored for a wide range of immunity parameters three weeks after vaccination against Mycoplasma hyopneumoniae: i) total white blood cells and lymphocyte counts and proportions of various leucocyte subsets including cells harbouring IgM, γδTCR, CD4/CD8, CD16/CD2 and CD16/CD172a/MHCII, ii) innate immune response parameters (phagocytosis and in vitro production of IL1B, IL6, IL8, TNF, IL12 and IFNαafter blood stimulation), iii) adaptive immune response parameters (lymphocyte proliferation, in vitro production of IL2, IL4, IL10 and IFNγ after blood stimulation, total IgG, IgA, IgM and specific IgG levels) and iv) two acute phase proteins (C-reactive protein and haploglobin). Across traits, heritability estimates reached 0.4 on average (se=0.1) and 42 of the 54 measured parameters showed moderate to high heritabilities (≥0.2), confirming that many parameters are under genetic control and could be included in selection protocols. Functional analyses revealed that the blood transcriptome is informative for part of the immunity traits and should provide relevant phenotypic information to better characterize some immunity traits

Transcriptome analysis of porcine PBMCs after in vitro stimulation by LPS or PMA/ionomycin using an expression array targeting the pig immune response

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Promoter analysis of the rabbit POU5F1 gene and its expression in preimplantation stage embryos

Background: The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos. [br/] Results: The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1. The results of quantitative real-time PCR experiments showed that POU5F1 mRNA was abundantly present in oocytes and zygotes, but it was gradually reduced until the activation of the embryonic genome, thereafter a continuous increase in POU5F1 mRNA level was observed until blastocyst stage. By using the XYClone laser system the inner cell mass (ICM) and trophoblast portions of embryos were microdissected and examined separately and POU5F1 mRNA was detected in both cell types. [br/] Conclusion: In this study we provide a comparative sequence analysis of the regulatory region of rabbit POU5F1 gene. Our data suggest that the POU5F1 gene is strictly regulated during early mammalian development. We proposed that the well conserved CR4 region containing the DE-2A enhancer is responsible for the highly conserved ESC specific gene expression. Notably, we are the first to report that the rabbit POU5F1 is not restricted to ICM cells only, but it is expressed in trophoblast cells as well. This information may be well applicable to investigate further the possible phylogenetic role and the regulation of POU5F1 gene