Table_1_Temporal changes in ewe vaginal microbiota throughout gestation.XLSX

IntroductionNumerous factors are known to influence reproductive efficiency in ewes, but few studies have investigated the potential role of vaginal microbiota in sheep reproductive success. The objective of this study was to thoroughly characterize the ewe vaginal microbiota throughout the course of pregnancy.MethodsVaginal samples were collected from 31 pregnant Hampshire and Hampshire X Suffolk crossbred ewes on a weekly basis from pre-breeding to pregnancy testing and then biweekly until just after lambing. To characterize the vaginal microbial communities, DNA was extracted and 16S rRNA gene Illumina MiSeq amplicon sequencing was performed.Results and DiscussionAlpha diversity metrics indicated an increase in species richness, evenness, and overall diversity throughout gestation. Distinct shifts in the bacterial communities were observed during gestation and were segregated into three periods: early gestation, a transitional period and mid/late gestation. During early gestation, Actinobacillus, Histophilus, and unclassified Leptotrichiaceae were found in greater relative abundance. During the transitional period, a population shift occurred characterized by increasing relative abundance of Streptococcus and Staphylococcus. During mid/late gestation, Staphylococcus, Streptococcus, and Ureaplasma had the greatest relative abundance. These shifts in the microbial population throughout the ewe’s gestation are likely related to hormonal changes triggered by the growing conceptus, specifically increasing blood concentration of progesterone. The transitional period shift in vaginal microbial communities potentially aligns with the placental take-over of progesterone production from the corpus luteum at approximately day 50 after conception (gestational week 7). Understanding the observed variability of the vaginal microbiota throughout pregnancy will allow for future comparison of ewes that did not become pregnant or had abnormal pregnancies, which could lead to the discovery of potential bacterial biomarkers for pregnancy outcome; this understanding could also lead to development of probiotics to improve sheep reproductive success.</p

Table_2_Temporal changes in ewe vaginal microbiota throughout gestation.XLSX

IntroductionNumerous factors are known to influence reproductive efficiency in ewes, but few studies have investigated the potential role of vaginal microbiota in sheep reproductive success. The objective of this study was to thoroughly characterize the ewe vaginal microbiota throughout the course of pregnancy.MethodsVaginal samples were collected from 31 pregnant Hampshire and Hampshire X Suffolk crossbred ewes on a weekly basis from pre-breeding to pregnancy testing and then biweekly until just after lambing. To characterize the vaginal microbial communities, DNA was extracted and 16S rRNA gene Illumina MiSeq amplicon sequencing was performed.Results and DiscussionAlpha diversity metrics indicated an increase in species richness, evenness, and overall diversity throughout gestation. Distinct shifts in the bacterial communities were observed during gestation and were segregated into three periods: early gestation, a transitional period and mid/late gestation. During early gestation, Actinobacillus, Histophilus, and unclassified Leptotrichiaceae were found in greater relative abundance. During the transitional period, a population shift occurred characterized by increasing relative abundance of Streptococcus and Staphylococcus. During mid/late gestation, Staphylococcus, Streptococcus, and Ureaplasma had the greatest relative abundance. These shifts in the microbial population throughout the ewe’s gestation are likely related to hormonal changes triggered by the growing conceptus, specifically increasing blood concentration of progesterone. The transitional period shift in vaginal microbial communities potentially aligns with the placental take-over of progesterone production from the corpus luteum at approximately day 50 after conception (gestational week 7). Understanding the observed variability of the vaginal microbiota throughout pregnancy will allow for future comparison of ewes that did not become pregnant or had abnormal pregnancies, which could lead to the discovery of potential bacterial biomarkers for pregnancy outcome; this understanding could also lead to development of probiotics to improve sheep reproductive success.</p

Mucosa-Associated Bacterial Microbiome of the Gastrointestinal Tract of Weaned Pigs and Dynamics Linked to Dietary Calcium-Phosphorus

<div><p>Dietary composition largely influences pig’s gastrointestinal microbiota and represents a useful prophylactic tool against enteric disturbances in young pigs. Despite the importance for host-microbe interactions and bacterial colonization, dietary responses of the mucosa-associated bacterial communities are less well investigated. In the present study, we characterized the mucosa-associated bacterial communities at the <i>Pars non-glandularis</i> of the stomach, ileum and colon, and identified shifts in these communities in response to different dietary calcium-phosphorus (Ca-P) contents (100% versus 190% of the Ca and P requirements) in combination with two basal diets (wheat-barley- or corn-based) in weaned pigs. Pyrosequencing of 16S rRNA genes from 93 mucosal samples yielded 447,849 sequences, clustering into 997 operational taxonomic units (OTUs) at 97% similarity level. OTUs were assigned to 198 genera belonging to 14 different phyla. Correlation-based networks revealed strong interactions among OTUs at the various gastrointestinal sites. Our data describe a previously not reported high diversity and species richness at the <i>Pars non-glandularis</i> of the stomach in weaned pigs. Moreover, high versus adequate Ca-P content significantly promoted <i>Lactobacillus</i> by 14.9% units (1.4 fold change) at the gastric <i>Pars non-glandularis</i> (<i>P</i> = 0.035). Discriminant analysis revealed dynamic changes in OTU composition in response to dietary cereals and Ca-P contents at all gastrointestinal sites which were less distinguishable at higher taxonomic levels. Overall, this study revealed a distinct mucosa-associated bacterial community at the different gut sites, and a strong effect of high Ca-P diets on the gastric community, thereby markedly expanding our comprehension on mucosa-associated microbiota and their diet-related dynamics in weaned pigs.</p></div

The 50

<p>All OTUs were blasted against NCBI GenBank nr. Closest reference strains, accession numbers and similarity values are listed.</p>1<p>BlastN against the NCBI nr excluding uncultured/environmental sample sequences.</p

Correlation networks for the microbial communities at the mucosa of (A) the <i>Pars non-glandularis</i> of the stomach, (B) the ileum and (C) the colon.

<p>The network depicts correlations between the top 10% of all OTUs per GIT site (r >0.5, <i>P</i><0.001). OTUs belonging to the 10 most abundant genera are shown in the same color. Correlation networks were calculated in MENAP (<a href="http://ieg2.ou.edu/MENA" target="_blank">http://ieg2.ou.edu/MENA</a>; molecular ecological network analysis pipeline) and visualized with Cytoscape.</p

Relative abundances of bacterial phyla attached to the gastrointestinal mucosa independent of diet.

<p>(A) Phyla detected in stomach, ileum and colon mucosa samples with >0.5% mean abundance were shown for the total V1–V2 region and for each gastrointestinal site separately. (B) Relative abundances of rare phyla. Error bars represent standard deviation from the mean.</p

Survival of <i>L. monocytogenes</i> QOC1 and QOC2 in minimal media adjusted to pH 2, pH 3, pH 11 and pH 12; and in gastric fluid.

<p>Values, given as percentage of survival, represent mean values ± SD of four biological replicates performed in triplicate. *indicates statistical significant differences (P<0.05) between QOC1 and QOC2. n.d.: not detectable.</p

Diversity of mucosa-associated bacteria.

<p>(A) Rarefaction and rank abundance curves based on an OTU definition threshold of 0.03 16S rRNA distance are shown. Rarefaction and rank abundance curves were calculated for each sample and depicted as mean per gastrointestinal site (<i>Pars non-glandularis</i> of the stomach, ileum and colon mucosa). Sequence numbers of samples were normalized by random selection before calculation. (B) Species richness and diversity estimates for bacteria at gastrointestinal mucosa. Significant differences between GIT sites, calculated with PROC MIXED of SAS, were listed. Significance was declared at <i>P≤</i>0.05. (C) Number of OTUs detected per gastrointestinal site and Venn diagram showing the number of shared OTUs between GIT sites. The size of the circles is in proportion to the number of OTUs detected in each gastrointestinal site.</p

Invasion efficiency and intracellular proliferation of <i>L. monocytogenes</i> strains.

<p>Invasion efficiency (panel A and B) and intracellular growth coefficient (IGC, panel C) of type strain EGDe, QOC1 and QOC2 using four different human cell lines (intestinal epithelial Caco2, hepatocytic HepG2 and macrophage-like U937 and THP1 cells) and primary mouse bone-marrow derived macrophages (mBMDM). Values represent mean values ± SD of four biological replicates performed in duplicate. Different letters indicate statistically significant differences (P<0.05).</p

General features of the <i>L. monocytogenes</i> QOC1 and QOC2 genomes.

<p>*genomes are not closed.</p>#<p>excluding the monocin regions and contig 7 of strain QOC1.</p