Centre Île-de-France - Jouy-en-Josas - Antony

BACKGROUND AND AIMS: The colonic microflora is involved in the pathogenesis of Crohn's disease (CD) but less than 30% of the microflora can be cultured. We investigated potential differences in the faecal microflora between patients with colonic CD in remission (n=9), patients with active colonic CD (n=8), and healthy volunteers (n=16) using culture independent techniques. METHODS: Quantitative dot blot hybridisation with six radiolabelled 16S ribosomal ribonucleic acid (rRNA) targeting oligonucleotide probes was used to measure the proportions of rRNA corresponding to each phylogenetic group. Temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA was used to evaluate dominant species diversity. RESULTS: Enterobacteria were significantly increased in active and quiescent CD. Probe additivity was significantly lower in patients (65 (11)% and 69 (6)% in active CD and quiescent CD) than in healthy controls (99 (7)%). TTGE profiles varied markedly between active and quiescent CD but were stable in healthy conditions. CONCLUSION: The biodiversity of the microflora remains high in patients with CD. Enterobacteria were observed significantly more frequently in CD than in health, and more than 30% of the dominant flora belonged to yet undefined phylogenetic groups.

Pollution of the environment by human and animal faecal pollution affects the safety of shellfish, drinking water and recreational beaches. To pinpoint the origin of contaminations, it is essential to define the differences between human microbiota and that of farm animals. A strategy based on real-time quantitative PCR (qPCR) assays was therefore developed and applied to compare the composition of intestinal microbiota of these two groups. Primers were designed to quantify the 16S rRNA gene from dominant and subdominant bacterial groups. TaqMan probes were defined for the qPCR technique used for dominant microbiota. Human faecal microbiota was compared with that of farm animals using faecal samples collected from rabbits, goats, horses, pigs, sheep and cows. Three dominant bacterial groups (Bacteroides/Prevotella, Clostridium coccoides and Bifidobacterium) of the human microbiota showed differential population levels in animal species. The Clostridium leptum group showed the lowest differences among human and farm animal species. Human subdominant bacterial groups were highly variable in animal species. Partial least squares regression indicated that the human microbiota could be distinguished from all farm animals studied. This culture-independent comparative assessment of the faecal microbiota between humans and farm animals will prove useful in identifying biomarkers of human and animal faecal contaminations that can be applied to microbial source tracking methods.

Abstract CBA x DBA/2 placentae are quantitatively or qualitatively deficient in their production of the anti-inflammatory Th2-type cytokines IL-4 and IL-10 compared with the nonresorption-prone CBA x BALB/c mating combination. Wastage in this mating combination is accompanied by increased levels of local inflammatory cytokines. In addition, alloimmunization enhances the placental production of IL-4 and IL-10 in CBA x DBA/2 matings. Furthermore, rIL-10 by itself completely reverses the high incidence of fetal resorption after i.p. injection. Conversely, anti-IL-10 increases the resorption rate, but only in CBA x DBA/2 matings. On the other hand, injecting either anti-IFN-gamma or pentoxifillin (an anti-TNF agent) partially reduces the resorption. When given together, they produce a synergistic remission of fetal loss. Finally, we report that recombinant ovine trophoblast protein, an IFN-tau which is known to influence reproductive outcome in ruminants, can also counteract increased CBA x DBA/2 fetal resorption. It simultaneously induces increased placental IL-4 and IL-10 production in this mating combination. These results indicate that the placentally produced anti-inflammatory cytokines can play a vital role in the survival to term of the fetal allograft, by counteracting deleterious inflammatory cytokines.

The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and Bacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides, and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.

Recent studies have shown that the human fecal microbiota is composed of a consortium of species specific to the host and resistant to modifications over time. Antibiotics are known to affect the intestinal microflora, and ensuing changes may result in antibiotic-associated diarrhea. It is therefore important to characterize the nature and amplitude of these modifications and the ability of this ecosystem to return to its original profile-i.e., its resilience. Six healthy volunteers received oral amoxicillin (1.5 g/day) for 5 days. Fecal samples were collected at day 0 (D0) before antibiotic treatment and at set intervals until 60 days thereafter. Fecal DNA was isolated, and V6-to-V8 regions of the 16S rRNA genes were amplified by PCR with general primers and analyzed by temporal temperature gradient gel electrophoresis. Dominant species profiles were compared on the basis of similarity (Pearson correlation coefficient). Dominant species profiles at D0 were used as a reference. The fecal microbiota showed a major shift in dominant species upon antibiotic treatment, starting 24 h after treatment initiation and reaching an average similarity of only 74% after 4 days. Within 30 days following antibiotic treatment, the fecal microbiota tended to reach an average similarity of 88% to the D0 value; within 60 days, the average similarity to the D0 value was 89%. However, in one subject, important modifications persisted for at least 2 months, with similarity to the D0 value remaining below 70%. We demonstrated the resilience of the dominant human fecal microbiota upon short-course antibiotic challenge. Yet the persistence of long-term alterations in some subjects may explain susceptibilities to antibiotic-associated diarrhea. Furthermore, these findings suggest that strategies reinforcing the ability of the fecal microbiota to resist modifications would be of clinical relevance.

Mucosal dendritic cells are at the heart of decision-making processes that dictate immune reactivity to intestinal microbes. They ensure tolerance to commensal bacteria and a vigorous immune response to pathogens. It has recently been demonstrated that the former involves a limited migration of bacterially loaded dendritic cells from the Peyer's patches to the mesenteric lymph nodes. During lactation, cells from gut-associated lymphoid tissue travel to the breast via the lymphatics and peripheral blood. Here, we show that human peripheral blood mononuclear cells and breast milk cells contain bacteria and their genetic material during lactation. Furthermore, we show an increased bacterial translocation from the mouse gut during pregnancy and lactation and the presence of bacterially loaded dendritic cells in lactating breast tissue. Our observations show bacterial translocation as a unique physiological event, which is increased during pregnancy and lactation. They suggest endogenous transport of intestinally derived bacterial components within dendritic cells destined for the lactating mammary gland. They also suggest neonatal immune imprinting by milk cells containing commensal-associated molecular patterns.

Current knowledge on chicken domestication is reviewed on the basis of archaeological, historical and molecular data. Several domestication centres have been identified in South and South-East Asia. Gallus gallus is the major ancestor species, but Gallus sonneratii has also contributed to the genetic make-up of the domestic chicken. Genetic diversity is now distributed among traditional populations, standardized breeds and highly selected lines. Knowing the genome sequence has accelerated the identification of causal mutations determining major morphological differences between wild Gallus and domestic breeds. Comparative genome resequencing between Gallus and domestic chickens has identified 21 selective sweeps, one involving a non-synonymous mutation in the TSHR gene, which functional consequences remain to be explored. The resequencing approach could also identify candidate genes responsible of quantitative traits loci (QTL) effects in selected lines. Genomics is opening new ways to understand major switches that took place during domestication and subsequent selection.

Regulatory ncRNAs (non-coding RNAs) adjust bacterial physiology in response to environmental cues. ncRNAs can base-pair to mRNAs and change their translation efficiency and/or their stability, or they can bind to proteins and modulate their activity. ncRNAs have been discovered in several species throughout the bacterial kingdom. This review illustrates the diversity of physiological processes and molecular mechanisms where ncRNAs are key regulators.

We review the evidence that strongly suggests a role of the intestinal microbiota in the onset and perpetuation of inflammatory bowel disease (IBD). Experimental studies consisted of suppressing micro-organisms from the microbiota (using germ-free or gnotoxenic animals or antibiotics), introducing new micro-organisms or microbial components (e.g. probiotics, CpG-DNA) or selectively increasing some endogenous bacteria (e.g. using prebiotics). Intervention studies were performed in patients or animal models of spontaneous or chemically-induced colitis. Information was also obtained from observational studies that described the composition of the faecal and mucosal microbiota at various stages of the disease process and in controls. Many have used culture-independent techniques that identify bacteria based on the nucleic acid sequence of ribosomal RNA molecules. Microbiota in patients with IBD seem to be characterized by high concentrations of bacteria in contact with the mucosa, instability, the presence of high numbers of unusual bacteria and sometimes a reduction in the biodiversity. Studies searching for a generalized or localized dysbiosis in IBD are discussed, as well as those trying to identify bacterial molecules and receptors, which may be implicated in triggering the inflammatory process.

The development of pancreatic tissue (fresh weight, total proteins, RNA and DNA) and of the level of pancreatic enzymes (trypsin, chymotrypsin, lipase and amylase) of the piglet has been followed from birth to the age of 8 weeks in 10 animals at each of 7 stages. There was an increase with age and body weight of the total fresh weight of the exocrine pancreas. From birth until 4 weeks the development of the pancreatic gland was due to hyperplasia; from the 4th week till the 8th week of age, it was due both to hyperplasia and hypertrophy. There was a specific period, at the age of 3--4 weeks, from which total enzymatic activities markedly increased. Furthermore, from the 4th week of age there was a rise in the intake of total dietary proteins, fat and carbohydrates, due to the intake of solid food.

BACKGROUND: The advent and democratization of next generation sequencing and genotyping technologies lead to a huge amount of data for the characterization of population genetic diversity in model and non model-species. However, efficient storage, management, cross-analyzing and exploration of such dense genotyping datasets remain challenging. This is particularly true for the bovine species where many SNP datasets have been generated in various cattle populations with different genotyping tools. DESCRIPTION: We developed WIDDE, a Web-Interfaced Next Generation Database that stands as a generic tool applicable to a wide range of species and marker types ( http://widde.toulouse.inra.fr). As a first illustration, we hereby describe its first version dedicated to cattle biodiversity, which includes a large and evolving cattle genotyping dataset for over 750,000 SNPs available on 129 (89 public) different cattle populations representative of the world-wide bovine genetic diversity and on 7 outgroup bovid species. This version proposes an optional marker and individual filtering step, an export of genotyping data in different popular formats, and an exploration of genetic diversity through a principal component analysis. Users can also explore their own genotyping data together with data from WIDDE, assign their samples to WIDDE populations based on distance assignment method and supervised clustering, and estimate their ancestry composition relative to the populations represented in the database. CONCLUSION: The cattle version of WIDDE represents to our knowledge the first database dedicated to cattle biodiversity and SNP genotyping data that will be very useful for researchers interested in this field. As a generic tool applicable to a wide range of marker types, WIDDE is overall intended to the genetic diversity exploration of any species and will be extended to other species shortly. The structure makes it easy to include additional output formats and new tools dedicated to genetic diversity exploration.

Gibbs random fields (GRF) are polymorphous statistical models that can be used to analyse different types of dependence, in particular for spatially correlated data. However, when those models are faced with the challenge of selecting a dependence structure from many, the use of standard model choice methods is hampered by the unavailability of the normalising constant in the Gibbs likelihood. In particular, from a Bayesian perspective, the computation of the posterior probabilities of the models under competition requires special likelihood-free simulation techniques like the Approximate Bayesian Computation (ABC) algorithm that is intensively used in population genetics. We show in this paper how to implement an ABC algorithm geared towards model choice in the general setting of Gibbs random fields, demonstrating in particular that there exists a sufficient statistic across models. The accuracy of the approximation to the posterior probabilities can be further improved by importance sampling on the distribution of the models. The practical aspects of the method are detailed through two applications, the test of an iid Bernoulli model versus a first-order Markov chain, and the choice of a folding structure for two proteins.

The interplay between dietary nutrients, gut microbiota and mammalian host tissues of the gastrointestinal tract is recognised as highly relevant for host health. Combined transcriptome, metabonome and microbial profiling tools were employed to analyse the dynamic responses of germfree mouse colonic mucosa to colonisation by normal mouse microbiota (conventionalisation) at different time-points during 16 days. The colonising microbiota showed a shift from early (days 1 and 2) to later colonisers (days 8 and 16). The dynamic changes in the microbial community were rapidly reflected by the urine metabolic profiles (day 1) and at later stages (day 4 onward) by the colon mucosa transcriptome and metabolic profiles. Correlations of host transcriptomes, metabolite patterns and microbiota composition revealed associations between Bacilli and Proteobacteria, and differential expression of host genes involved in energy and anabolic metabolism. Differential gene expression correlated with scyllo- and myo-inositol, glutamine, glycine and alanine levels in colonic tissues during the time span of conventionalisation. Our combined time-resolved analyses may help to expand the understanding of host-microbe molecular interactions during the microbial establishment.

Isolates of Flavobacterium psychrophiluln (formerly Cytophaga psychrophila and Flexlbacter psychrophilus) mainly originating from clinical outbreaks of either coldwater disease (CWD) or rainbow trout fry syndrome (RTFS) were studied for selected biochemical, physiological, rnorphological and genomic characteristics, and compared with previously characterized French and American strains. DNA hybridization studies showed that the Danish isolates were highly related to the type strain, F. psychrophilum NCIMB 1 9 4 7 ~ Plasmid profiling of Danish isolates and those from other European countries revealed differences, which might be related to differences in pathogenicity. European ~solates originating from clinical outbreaks of either RTFS or CWD usually harboured one plasmid of 3.2 kb, whereas isolates originating from fish with different or no disease slgns had other profiles. Phenotypically, the Danish isolates appeared very homogeneous and shared most characteristics with the type strain, and with French and American strains studied by other authors. Further studies on the importance of the plasmids and the proteolytic activities of the bacterium might help in elucidating possible virulence factors.

OBJECTIVE: Yersinia pseudotuberculosis causes ileitis and mesenteric lymphadenitis by mainly invading the Peyer's patches that are positioned in the terminal ileum. Whereas toll-like-receptor 2 (TLR2) controls mucosal inflammation by detecting certain microbiota-derived signals, its exact role in protecting Peyer's patches against bacterial invasion has not been defined. DESIGN: Wild-type, Tlr2-, Nod2- and MyD88-deficient animals were challenged by Y pseudotuberculosis via the oral or systemic route. The role of microbiota in conditioning Peyer's patches against Yersinia through TLR2 was assessed by delivering, ad libitum, exogenous TLR2 agonists in drinking water to germ-free and streptomycin-treated animals. Bacterial eradication from Peyer's patches was measured by using a colony-forming unit assay. Expression of cryptdins and the c-type lectin Reg3 beta was quantified by quantitative reverse transcriptase polymerase chain reaction analysis. RESULTS: Our data demonstrated that Tlr2-deficient mice failed to limit Yersinia dissemination from the Peyer's patches and succumbed to sepsis independently of nucleotide-binding and oligomerisation domain 2 (NOD2). Recognition of both microbiota-derived and myeloid differentiation factor 88 (MyD88)-mediated elicitors was found to be critically involved in gut protection against Yersinia-induced lethality, while TLR2 was dispensable to systemic Yersinia infection. Gene expression analyses revealed that optimal epithelial transcript level of the anti-infective Reg3 beta requires TLR2 activation. Consistently, Yersinia infection triggered TLR2-dependent Reg3 beta expression in Peyer's patches. Importantly, oral treatment with exogenous TLR2 agonists in germ-free animals was able to further enhance Yersinia-induced expression of Reg3 beta and to restore intestinal resistance to Yersinia. Lastly, genetic ablation of Reg3 beta resulted in impaired clearance of the bacterial load in Peyer's patches. CONCLUSIONS: TLR2/REG3 beta is thus an essential component in conditioning epithelial defence signalling pathways against bacterial invasion.

The Swine Genome Sequencing Consortium (SGSC) was formed in September 2003 by academic, government and industry representatives to provide international coordination for sequencing the pig genome. The SGSC's mission is to advance biomedical research for animal production and health by the development of DNAbased tools and products resulting from the sequencing of the swine genome. During the past 2 years, the SGSC has met bi-annually to develop a strategic roadmap for creating the required scientific resources, to integrate existing physical maps, and to create a sequencing strategy that captured international participation and a broad funding base. During the past year, SGSC members have integrated their respective physical mapping data with the goal of creating a minimal tiling path (MTP) that will be used as the sequencing template. During the recent Plant and Animal Genome meeting (January 16, 2005 San Diego, CA), presentations demonstrated that a human-pig comparative map has been completed, BAC fingerprint contigs (FPC) for each of the autosomes and X chromosome have been constructed and that BAC end-sequencing has permitted, through BLAST analysis and RH-mapping, anchoring of the contigs. Thus, significant progress has been made towards the creation of a MTP. In addition, whole-genome (WG) shotgun libraries have been constructed and are currently being sequenced in various laboratories around the globe. Thus, a hybrid sequencing approach in which 3x coverage of BACs comprising the MTP and 3x of the WG-shotgun libraries will be used to develop a draft 6x coverage of the pig genome.

Three experiments involving 34 individually fed pigs were conducted in Guadeloupe (16 degrees Lat. N., 61 degrees Long. W.) to determine the effects of environmental temperature (tropical, 22 to 32 degrees C, vs thermoneutral, 17 to 21 degrees C) and feeding method (restricted vs ad libitum) on performance, carcass characteristics and physiological and metabolic responses of pigs at three weight ranges (8 to 25, 29 to 50 and 54 to 79 kg live weight). Compared with the control environment, the tropical climate increased rectal temperature and respiratory rate but depressed growth rate and efficiency of feed utilization. In addition, in the heaviest weight group, feed intake was reduced and body fat increased. Changes in metabolic status, such as increased concentrations of plasma free fatty acids, triglycerides, cholesterol and adipose tissue lipoprotein lipase activity were observed in pigs housed in the tropical environment. Moreover, in these pigs, there was a decreased plasma concentration of thyroid hormones (triiodothyronine and thyroxine). These results indicate that tropical ambient temperature markedly affects the metabolism of pigs and, therefore, probably influences their nutritional requirements.

Egg activation in mammals is caused by cytosolic Ca(2+) oscillations that are essential for development. However, despite increasing knowledge about signal transduction mechanisms, the functional linkage between frequency number, amplitude and duration of the Ca(2+) signal and the kinetics of pronucleus formation has not yet been defined. While a wide range of Ca(2+) signal parameters are efficient in causing egg activation, the basic rules governing how the egg integrates these signalling events are not yet clear. Thus, in the perspective of better understanding how the egg processes Ca(2+) signalling events, the objective of this study was to determine experimentally whether the efficiency of egg activation and the subsequent early developmental stages rely on Ca(2+) signalling summation. Non-fertilized, but freshly ovulated mouse eggs, were subjected to a series of repetitive Ca(2+) influxes of various patterns modulated by a non-invasive membrane electropermeabilization method. Using a combination of two suboptimal treatments we have shown that mouse eggs can sum up the effects caused by various patterns of intracellular Ca(2+) concentrations transient during the period of egg activation. In addition, overloading the intracellular milieu by repetitive Ca(2+) influxes did not seem to inhibit the process of activation. The kinetics of pronuclear formation among a population of eggs treated in the same conditions became accelerated when the total dose of Ca(2+) signal 'experienced' by the eggs was increased. The results suggested that summation of the biological effects of all Ca(2+) signals constitutes an important mode of Ca(2+) signal integration.

Four Cytophaga psychrophila-like strains were isolated from one eel and 3 species of cyprinids with skin lesions and signs of acute septicaemia in Nordrhein-Westfalen (Germany). The strains proved identical in their phenotypic charactenst~cs to each other and to 3 typical strains of C psychrophila, one of which was the type strain. The identification of the new isolates as C. psl/chrophila was confirmed by rapid s l ~d e agglutination tests uslng a specific rabbit anti-C. psychrophila serum. This is the first report of C. psychrophila being isolated from non-salmonid fishes. This pathogen, which has provoked heavy losses in rainbow trout hatcheries in France and Germany since 1984-1985, may also have the potential to become a problem for non-salmonids. As with most other bacteria pathogenic for fish, C. psychrophila shows a lack of strict host specificity.

1. Six non-anaesthetized pigs (mean body-weight 57.0 kg) were used to study the intestinal absorption of amino acids (AA) from either an enzymic hydrolysate of milk (PEP) containing a large percentage of small peptides (about 50% with less than five AA residues) and very few free AA (8%), or from a mixture of free AA with an identical pattern (AAL) infused intraduodenally in one of two amounts (55 or 110 g). Concomitant insulin and glucagon production rates were estimated. 2. Each pig was previously fitted, under anaesthesia, with an electromagnetic flow probe around the portal vein, with permanent catheters in the portal vein, the carotid artery and the duodenum. Each infusion was performed after an 18 h fasting period and each pig received each infusion. The observation period lasted for 5 h. 3. The absorption of AA was greater, more rapid and more homogeneous after PEP infusion than after AAL infusion, independent of the amount infused. 4. For the majority of AA considered individually, the absorption coefficient was higher after infusion of PEP than after that of AAL. The exceptions were methionine with a higher absorption coefficient after AAL infusion, and isoleucine, aspartic acid + asparagine and glutamic acid + glutamine with identical coefficients for both infusions. 5. Some AA, such as asparagine, ornithine, citrulline and taurine, while absent in the infusates, appeared in the portal vein in appreciable amounts after the infusion of both solutions. While a small proportion of taurine may arise from recycling of taurine-containing bile salts, it seems that the gut wall is able to synthesize all four AA. 6. Insulin production did not differ according to the nature or amount of solutions infused. Glucagon production was greater after PEP infusion.