Effect of dietary supplementation with algae extracts on growth performance and caecal microbiota of broiler chickens.

1. The objective of this study was to test the dose response of dietary supplementation with algae extracts rich in marine-sulphated polysaccharides (MSP1 and MSP2) on the growing performance, body composition at slaughter and caecal microbiota of broiler chickens.2. Male broiler Ross 308 chicks 1-d-old were distributed into eight groups, a control group (unsupplemented), four groups supplemented with increasing doses of algae extract MSP1 (40, 81, 121 and 162 g/ton feed) and three groups supplemented with increasing doses of algae extract MSP2 (40, 81 and 162 g/ton feed). Each group comprised six pens of 56 chickens.3. All chickens were reared under challenging conditions, i.e. high rearing density of 42 kg/m2, fed growing and finishing diets containing, palm oil, rye and high levels of wheat and subjected to short daily fasting periods. The growth performance was recorded during rearing. At 10, 22 and 31 d of age, 12 chickens per group were euthanised to collect the caecal contents and determine microbiota composition and short-chain fatty acid levels. At d 35, the quality of litter and the condition of feathers, footpads and tarsals were scored. At d 36, 7 chickens per pen were slaughtered under commercial conditions to determine carcass composition and breast meat quality (ultimate pH and colour).4. Algal extract MSP1 increased the weight of the caeca and butyrate concentration in the caeca at d 22 (p ≤ 0.05). It increased the ultimate pH of breast fillet measured after slaughter at d 36 (p ≤ 0.05). Moreover, the group receiving 162 g/t MSP1 had a more diverse microbiota at d22. However, algal extract MSP2 had negligible effect on the different measured parameters.

Genetic relationships between efficiency traits and gut microbiota traits in growing pigs being fed with a conventional or a high-fiber diet.

In pigs, the gut microbiota composition plays a major role in the process of digestion, but is influenced by many external factors, especially diet. To be used in breeding applications, genotype by diet interactions on microbiota composition have to be quantified, as well as their impact on genetic covariances with feed efficiency (FE) and digestive efficiency (DE) traits. This study aimed at determining the impact of an alternative diet on variance components of microbiota traits (genera and alpha diversity indices) and estimating genetic correlations between microbiota and efficiency traits for pigs fed a conventional (CO) or a high-fiber (HF) diet. Fecal microbes of 812 full-siblings fed a CO diet and 752 pigs fed the HF diet were characterized at 16 weeks of age by sequencing the V3-V4 region of the 16S rRNA gene. A total of 231 genera were identified. Digestibility coefficients of nitrogen, organic matter, and energy were predicted analyzing the same fecal samples with near infrared spectrometry. Daily feed intake, feed conversion ratio, residual feed intake and average daily gain (ADG) were also recorded. The 71 genera present in more than 20% of individuals were retained for genetic analyses. Heritability (h²) of microbiota traits were similar between diets (from null to 0.38 ±â€…0.12 in the CO diet and to 0.39 ±â€…0.12 in the HF diet). Only three out of the 24 genera and two alpha diversity indices with significant h² in both diets had genetic correlations across diets significantly different from 0.99 (P < 0.05), indicating limited genetic by diet interactions for these traits. When both diets were analyzed jointly, 59 genera had h² significantly different from zero. Based on the genetic correlations between these genera and ADG, FE, and DE traits, three groups of genera could be identified. A group of 29 genera had abundances favorably correlated with DE and FE traits, 14 genera were unfavorably correlated with DE traits, and the last group of 16 genera had abundances with correlations close to zero with production traits. However, genera abundances favorably correlated with DE and FE traits were unfavorably correlated with ADG, and vice versa. Alpha diversity indices had correlation patterns similar to the first group. In the end, genetic by diet interactions on gut microbiota composition of growing pigs were limited in this study. Based on this study, microbiota-based traits could be used as proxies to improve FE and DE in growing pigs.

The link between the composition of the gut microbiota, i.e the composition of microorganisms in the gut, in pigs and their feed efficiency, i.e. their ability to utilize nutrients, as well as their ability to digest were studied from a genetic point of view. A family structure of 1,564 pigs were studied and fed with two different diets. One of the full-sib was fed a conventional diet used in breeding farms and the other one an alternative diet containing raw materials, less expensive but with a higher content of dietary fibers more difficult to digest. This study has shown that some microbiota microorganisms were genetically correlated with feed and digestive efficiency performances, positively or negatively, depending on the microorganisms. In addition, the diversity of microorganisms in the animal's gut was favorably correlated with the feed and digestive performances studied. Therefore, there is a genetic link between these performances and the composition of the animal's gut microbiota. Thus, a potential genetic selection on some intestinal microorganisms or diversity of microorganisms would allow to improve these performances, and in particular when pigs are fed with diet more difficult to digest.

Transient effect of single or repeated acute deoxynivalenol and zearalenone dietary challenge on fecal microbiota composition in female finishing pigs.

Mycotoxins are a major contaminant of pig feed and have negative effects on health and performance. The present study investigated the impact of single or repeated acute challenges with a diet naturally contaminated with deoxynivalenol (DON) and zearalenone (ZEN) on growth performances of finishing pigs and their fecal microbiota composition. A total of 160 pigs (castrated males and females) in two successive batches were randomly divided into four experimental groups of 40 pigs each. The control group received a control finisher diet from 99 to 154 days of age. Challenged groups were subjected to a 7-day acute challenge by being fed a DON- and ZEN-contaminated diet (3.02 mg DON/kg feed and 0.76 mg ZEN/kg feed) at 113 days (group DC), 134 days (group CD) or both 113 and 134 days (group DD). Microbiota composition was analyzed via 16S rRNA sequencing from fecal samples collected from the 80 females at 99, 119, 140 and 154 days. Challenged pigs (i.e. groups DC, CD and DD) reduced their average daily feed intake by 25% and 27% (P < 0.001) and feed efficiency by 34% and 28% (P < 0.05) during the first and second mycotoxin exposure, respectively. Microbiota composition was affected by mycotoxin exposure (P = 0.07 during the first exposure and P = 0.01 during the second exposure). At the family level, mycotoxin exposure significantly (P < 0.05) decreased the relative abundances of Ruminococcaceae, Streptococcaceae and Veillonellaceae and increased that of Erysipelotrichaceae at both 119 and 140 days of age. After the 7-day DON/ZEN challenge, the relative abundance of 6 to 148 operational taxonomic units (OTUs) differed among the treatment groups. However, none of these OTUs changed in all treatment groups. Using 27 functional pathways, pigs exposed to DON/ZEN challenges could be distinguished from control pigs using sparse partial least squares discriminant analysis, with a 15% misclassification rate. Regarding the functionality of these predictors, two pathways were involved in detoxifying mycotoxins: drug metabolism and xenobiotic metabolism by cytochrome P450. In challenged pigs, microbiota composition returned to the initial state within 3 weeks after the end of a single or repeated DON/ZEN challenge, highlighting the resilience of the gut microbiome. The feeding and growth performances of the pigs during challenge periods were significantly correlated with biological pathways related to health problems and modifications in host metabolism. To conclude, short-term DON/ZEN challenges resulted in transient modifications in the composition and functions of fecal microbiota.

Impact of feed restriction and housing hygiene conditions on specific and inflammatory immune response, the cecal bacterial community and the survival of young rabbits.

Limiting the post-weaning intake of the young rabbit is known to improve its resistance to digestive disorders, whereas a degradation of its housing hygiene is assumed to have a negative impact on its health. This study aims at providing insights into the mechanism of digestive health preservation regarding both host (growth and immune response) and its symbiotic digestive microbiota. A 2×2 factorial design from weaning (day 28) to day 64 was set up: ad libitum intake or restricted intake at 70% of ad libitum, and high v. low hygiene of housing (n=105 per group). At day 36 and day 45, 15 animals/group were subcutaneously immunized with ovalbumin (OVA) to assess their specific immune response. Blood was sampled at 36, 45, 57 and 64 days of age to determine total and anti-OVA immunoglobulin type G (IgG) and haptoglobin levels. The cecal bacterial community was explored (18 per group) by 454 pyrosequencing of genes coding for the 16S ribosomal RNA, whereas cecal pH, NH3 and volatile fatty acid (VFA) concentrations were measured to characterize fermentative activity. A 30% reduction in feed intake reduced the growth by only 17% (P<0.001), and improved the feed conversion ratio by 15% (P<0.001), whereas the degradation of hygiene conditions slightly decreased the feed intake in ad libitum fed rabbits (-3.5%, P<0.02). As poor hygiene conditions did not affect weight gain, feed conversion was improved from day 42 (P<0.05). Restricted feeding led to a lower mortality between day 28 and day 40 (P=0.047), whereas degraded hygiene conditions decreased overall morbidity (7.8% v. 16.6%; P<0.01). Both a reduced intake and low hygiene conditions of housing affected microbiota composition and especially dominant genera belonging to the Ruminococcaceae family (P<0.01). Moreover, low hygiene was associated with a higher Ruminococcaceae/Lachnospiraceae ratio (3.7 v. 2.4; P<0.05). Cecal total VFA and pH were increased (+19%; P<0.001) and decreased (-0.1 pH unit; P<0.05), respectively, in feed-restricted rabbits. Neither specific anti-OVA IgG nor haptoglobin was affected by treatments. Total IgG concentrations were the highest in animals raised in poor hygiene conditions after 8 days of restriction, but decreased after 19 days of restriction in high hygiene conditions (-2.15%; P<0.05). In conclusion, the degradation of hygiene conditions failed to induce a systematic specific and inflammatory response in rabbit, but reduced morbidity instead. Our results suggest that the microbiota composition would be a helpful source of biomarkers of digestive health.

Direct and correlated responses to selection in two lines of rabbits selected for feed efficiency under ad libitum and restricted feeding: I. Production traits and gut microbiota characteristics.

To get insights into selection criteria for feed efficiency, 2 rabbit lines have been created: the ConsoResidual line was selected for residual feed intake (RFI) with ad libitum feeding and the ADGrestrict line was selected for ADG under restricted feeding (-20% of voluntary intake). The first objective of this study was to evaluate, after 9 generations of selection, the direct and correlated responses to selection on production traits in the 2 lines for traits recorded during growth. Second, applying the 2 feeding conditions used for selection to both selected lines plus the control unselected line (generation 0, G0) in a 2 × 3 factorial trial, the line performances were compared and the gut microbiota of the lines was characterized. The correlated responses in feed conversion ratio (FCR) were remarkably equivalent in both selected lines (-2.74 genetic σ) but correlated responses in other traits were notably different. In the ConsoResidual line, selection for decreased RFI resulted in a small negative correlated response in BW at 63 d old (BW63) and in a null response in ADG. In the ADGrestrict line, on the contrary, the correlated response in BW63 was substantial (+1.59 σ). The 2 selected lines had a FCR reduced by 0.2 point compared with the G0 line, and the same difference was found in both feeding regimens ( < 0.001). Indeed, selection on ADG would lead to heavier animals with no significant reduction of feed costs, whereas selection on RFI leads to lower feed costs and no increase of animal BW under ad libitum feeding. Altogether, our results do not suggest any genotype × environment interaction in the response to feeding regimens. The intestinal microbial communities from efficient rabbits differed from their unselected counterparts in terms of fermentation end products and microbial phylotypes, suggesting a central role of these microbes in the better feed efficiency of the rabbits.

Impact of feed restriction on health, digestion and faecal microbiota of growing pigs housed in good or poor hygiene conditions.

Feed restriction could be a relevant strategy to preserve gut health, reduce systemic inflammatory response and finally limit antibiotic use. This study assessed the effect of feed restriction on growing pigs submitted to a moderate inflammatory challenge induced by the degradation of the environmental hygiene that is known to alter growth rate. The experiment was run on 80 pigs selected at 7 weeks of age according to a 2×2 factorial design: two feeding levels, ad libitum (AL) and feed restricted (FR) at 60% of AL, and two conditions of environmental hygiene, clean and dirty. Pigs were housed individually throughout the experiment. From 61 to 68 days of age (day 0 to 7), pigs were housed in a post weaning unit and feed restriction was applied to half of the pigs from day 0 to day 29. At 68 days of age (day 7 of the experiment), pigs were transferred in a growing unit where half of FR and half of AL pigs were housed in a dirty environment (poor hygiene) and the other half in a clean environment (good hygiene) until day 42. Growth performance was recorded weekly. Blood and faeces samples were collected to measure indicators of inflammation, nutrient digestibility and microbiota composition. Faecal consistency was monitored daily to detect diarrhoeas. Feed restriction decreased daily weight gain (-35% to -50%, P<0.001), increased the feed conversion ratio (+15%, P<0.001) and CP digestibility (+3%, P<0.05) and reduced the occurrence of diarrhoeas irrespective of hygiene conditions. Poor hygiene conditions decreased growth performance (-20%, P<0.05) and total tract digestibility of all nutrients (P<0.001). Haptoglobin (+50%) concentrations and lymphocyte (+10%) and granulocyte (+40%) numbers were higher in poor hygiene conditions (P<0.05), confirming that the model was effective to induce a systemic inflammatory response. Both feed restriction and hygiene modified the profile of the faecal microbiota. In this study, feed restriction did not reduce the systemic inflammatory response caused by poor hygiene conditions despite the limitation of the occurrence of digestive disorders. However, our study opens discussions regarding the impact of hygiene and feed restriction on gut microbial communities and digestive health.

Phage adsorption to bacteria in the light of the electrostatics: a case study using E. coli, T2 and flow cytometry.

The addition of sodium chloride to freshwater or diluted minimal salt medium increases the adsorption of T2 phages on Escherichia coli. For the first time the adsorption in diluted minimal salt medium was measured by counting unadsorbed phages (i.e. free particles) using flow cytometry, allowing a gentle separation between adsorbed and unadsorbed phages. Flow cytometry was able to detect weakly adsorbed phage that remained undetected using classical centrifugation-based methods and this allowed us to show that increasing ionic strength enhances the phage adsorption to its bacterial host with an extremely low detection limit. A key result was that the adsorption in high ionic strength (i.e. 100 mM) reached 4.5±0.1×10⁻5 mL/min which is 1400 fold higher than previously reported values. In order to understand the mechanism underpinning such a weak phage adsorption, the zeta potentials and the diffusion coefficient of the particles were measured by dynamic light scattering. The bacterial cells and the phages had zeta potentials between -60 mV and -10 mV and -30 mV and -10 mV, respectively. The diffusion coefficient of the phage was 2.8±0.4×10⁻¹² m² s⁻¹ corresponding to a hydrodynamic radius of 104±15 nm. However significant adsorption occurs in conditions where the DLVO theory predicts that minimal encounter, suggesting that forces other that electrostatic repulsion and Van der Waals interaction (e.g. potential impurities, particle shape and other biological characteristics) are likely to interplay.

Bacterial and fungal community composition over time in chicken litter with high or low moisture content.

1. Changes in bacterial and fungal communities in chicken litter with high and low moisture content over a five week period during a single chicken grow out cycle in a poultry shed in subtropical Australia were investigated to study the association between specific microbes and odour production. 2. Microbial biomass, as indicated by DNA yields, was higher and community composition was more dynamic over time in moist compared with dry chicken litter. 3. Bacillus, Atopostipes and Aspergillus species increased in relative abundance in moist chicken litter samples over time reflecting the relatively high fitness and hence activity of these specific bacteria and this specific fungus in this environment.

Improvement of RNA-SIP by pyrosequencing to identify putative 4-n-nonylphenol degraders in activated sludge.

Nonylphenols (NP) have estrogenic potential because of their phenolic ring, but the organisms involved in the degradation of this alkylated phenol remain unidentified. Using 16S ribosomal RNA (rRNA)-based stable isotope probing (SIP) and a new method based on pyrosequencing, we identified the bacteria involved in the degradation of the aromatic ring of [U-ring-(13)C] 4-n-NP in aerobic sludge. The first order degradation rate of 4-n-NP was 5.5d(-1). Single strand conformation polymorphism of density-separated labeled and unlabeled 16S rRNA showed significant differences and enabled selection of four representative fractions for pyrosequencing. Nineteen phylotypes showed a significant enrichment in the heavy fraction in the labeled pulse. The relative abundances of these phylotypes were combined with the RNA concentration of each fraction to yield a simple model of the distribution of each phylotype across the gradient. This model was used to estimate the percentage of labeling for each phylotype. The sequences showing the highest labeling (11%) were closely related to Afipia sp. but represented only 2 % of the RNA in the heavy fraction of the labeled pulse. The sequences representing the largest proportion of the RNA in the heavy fraction were related to Propionibacterium acnes and Frateuria aurantia, which are known to possess enzymes for phenol degradation. The model shows that despite Afipia having the highest (13)C enrichment, other species encoding phenol degradation pathways are responsible for more (13)C incorporation. Last, we showed that some species represent 12% of the total RNA but contain only 1% (13)C above natural abundance.

Effect of a transient perturbation on marine bacterial communities with contrasting history.

AIMS: To evaluate the importance of the bacterial composition on the resilience of the organic matter assimilation in the sea. METHODS AND RESULTS: Chemostats were inoculated with coastal and offshore bacterial communities. Bacterial density and protein synthesis increased before stabilizing, and this response to confinement was more marked in the offshore chemostats. Before the toluene perturbation the community structure in the coastal chemostats remained complex whereas the offshore chemostats became dominated by Alteromonas sp. After the perturbation, bacterial protein synthesis was inhibited before peaking briefly at a level fivefold to that observed before the perturbation and then stabilizing at a level comparable to that before the perturbation. Alteromonas dominated both the coastal and the offshore communities immediately after the perturbation and the coastal communities did not recover their initial complexity. CONCLUSIONS: Cell lysis induced by the toluene perturbation favoured the growth of Alteromonas which could initiate growth rapidly in response to the nutrient pulse. Despite their different community structure in situ, the resilience of protein synthesis of coastal and offshore bacterial communities was dependent on Alteromonas, which dominated in the chemostats. SIGNIFICANCE AND IMPACT OF THE STUDY: Here we show that although Alteromonas sp. dominated in artificial offshore and coastal communities in chemostats, their response time to the shock was different. This suggests that future perturbation studies on resilience in the marine environment should take account of ecosystem history.

Efficient method to isolate and purify viruses of bacteria from marine environments.

AIM: To isolate viruses of specific heterotrophic bacterial strains from marine environments using a host addition/virus amplification protocol (HAVAP) for use in phage/host systems. METHODS AND RESULTS: Bacteria-free seawater samples containing natural viruses assemblages were inoculated with a single laboratory grown bacterial host of interest in a nutrient-enriched [peptone, Fe(III) and yeast extract] seawater suspension. These conditions enhanced the replication of only those virus(s) capable of infecting the host bacterium. After incubation, free viruses were recovered at concentrations ranging 10(5)-10(10) infectious virus particles per ml of seawater. Using this approach, 15 viruses were isolated and represented 12 unique phage/host systems. Two of the hosts tested were infected by more than one virus. CONCLUSIONS: Isolation of high concentrations of specific viruses is possible even if their initial concentrations in native waters are low. This approach allows the recovery of phage/host systems that may not be numerically dominant. SIGNIFICANCE AND IMPACT OF THE STUDY: This host enrichment protocol for virus detection and isolation is well-suited for aquatic viral ecology studies that require phage/host systems.