Prevotella in Pigs: The Positive and Negative Associations with Production and Health.

A diverse and dynamic microbial community (known as microbiota) resides within the pig gastrointestinal tract (GIT). The microbiota contributes to host health and performance by mediating nutrient metabolism, stimulating the immune system, and providing colonization resistance against pathogens. Manipulation of gut microbiota to enhance growth performance and disease resilience in pigs has recently become an active area of research in an era defined by increasing scrutiny of antimicrobial use in swine production. In order to develop microbiota-targeted strategies, or to identify potential next-generation probiotic strains originating from the endogenous members of GIT microbiota in pigs, it is necessary to understand the role of key commensal members in host health. Many, though not all, correlative studies have associated members of the genus Prevotella with positive outcomes in pig production, including growth performance and immune response; therefore, a comprehensive review of the genus in the context of pig production is needed. In the present review, we summarize the current state of knowledge about the genus Prevotella in the intestinal microbial community of pigs, including relevant information from other animal species that provide mechanistic insights, and identify gaps in knowledge that must be addressed before development of Prevotella species as next-generation probiotics can be supported.

Discovery of Predictors of Mycoplasma hyopneumoniae Vaccine Response Efficiency in Pigs: 16S rRNA Gene Fecal Microbiota Analysis.

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.

Denosumab Regulates Gut Microbiota Composition and Cytokines in Dinitrobenzene Sulfonic Acid (DNBS)-Experimental Colitis.

The pro-inflammatory mediator receptor activator of nuclear factor-kappa B ligand (RANKL) plays a significant role in the development of rheumatoid arthritis; however, its role in inflammatory bowel disease is unknown. Genome-wide association meta-analysis for Crohn's disease (CD) identified a variant near the TNFSF11 gene that encodes RANKL and CD risk allele increased expression of RANKL in specific cell lines. This study aims to elucidate if the RANKL inhibitor denosumab can reduce the severity of experimental colitis and modify the gut microbiota composition using murine dinitrobenzenesulfonic acid (DNBS)-experimental model of colitis mimicking CD. In colitic conditions, denosumab treatment significantly decreased the pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α within the colonic mucosa. Moreover, colitis was accompanied by disruption of gut microbiota, and preventative treatment with denosumab modulated this disruption. Denosumab treatment also modified the alpha- and beta diversity of colonic mucosa and fecal microbiota. These results provide a rationale for considering denosumab as a future potential therapy in CD; however, more detailed experimental and clinical studies are warranted.

Characterization of whole blood transcriptome and early-life fecal microbiota in high and low responder pigs before, and after vaccination for Mycoplasma hyopneumoniae.

We investigated gene expression patterns in whole blood and fecal microbiota profile as potential predictors of immune response to vaccination, using healthy M. hyopneumoniae infection free piglets (n = 120). Eighty piglets received a dose of prophylactic antibiotics during the first two days of life, whereas the remaining 40 did not. Blood samples for RNA-Seq analysis were collected on experimental Day 0 (D0; 28 days of age) just prior to vaccination, D2, and D6 post-vaccination. A booster vaccine was given at D24. Fecal samples for microbial 16SrRNA sequencing were collected at 7 days of age, and at D0 and D35 post-vaccination. Pigs were ranked based on the levels of M. hyopneumoniae-specific antibodies in serum samples collected at D35, and groups of 'high' (HR) and 'low' (LR) responder pigs (n = 15 each) were selected. Prophylactic antibiotics did not influence antibody titer levels and differential expression analysis did not reveal differences between HR and LR at any time-point (FDR > 0.05); however, based on functional annotation with Ingenuity Pathway Analysis, D2 post-vaccination, HR pigs were enriched for biological terms relating to increased activation of immune cells. In contrast, the immune activation decreased in HR, 6 days post-vaccination. No significant differences were observed prior to vaccination (D0). Two days post-vaccination, multivariate analysis revealed that ADAM8, PROSER3, B4GALNT1, MAP7D1, SPP1, HTRA4, and ENO3 genes were the most promising potential biomarkers. At D0, OTUs annotated to Prevotella, CF21, Bacteroidales and S24-7 were more abundant in HR, whereas Fibrobacter, Paraprevotella, Anaerovibrio, [Prevotella], YRC22, and Helicobacter positively correlated with the antibody titer as well as MYL1, SPP1, and ENO3 genes. Our study integrates gene differential expression and gut microbiota to predict vaccine response in pigs. The results indicate that post-vaccination gene-expression and early-life gut microbiota profile could potentially predict vaccine response in pigs, and inform a direction for future research.

Reactivation of Intestinal Inflammation Is Suppressed by Catestatin in a Murine Model of Colitis via M1 Macrophages and Not the Gut Microbiota.

While there is growing awareness of a relationship between chromogranin-A (CHGA) and susceptibility to inflammatory conditions, the role of human catestatin [(hCTS); CHGA352-67] in the natural history of established inflammatory bowel disease is not known. Recently, using two different experimental models, we demonstrated that hCTS-treated mice develop less severe acute colitis. We have also shown the implication of the macrophages in this effect. The aims of this study were to determine (1) whether hCTS treatment could attenuate the reactivation of inflammation in adult mice with previously established chronic colitis; (2) whether this effect is mediated through macrophages or the gut microbiota. Quiescent colitis was induced in 7-8-week-old C57BL6 mice using four cycles (2-4%) of dextran sulfate sodium. hCTS (1.5 mg/kg/day) treatment or vehicle started 2 days before the last induction of colitis and continuing for 7 days. At sacrifice, macro- and microscopic scores were determined. Colonic pro-inflammatory cytokines [interleukin (IL)-6, IL-1ß, and TNF- α], anti-inflammatory cytokines (IL-10, TGF- ß), classically activated (M1) (iNOS, Mcp1), and alternatively activated (M2) (Ym1, Arg1) macrophages markers were studied using ELISA and/or RT-qPCR. In vitro, peritoneal macrophages isolated from naïve mice and treated with hCTS (10-5 M, 12 h) were exposed to either lipopolysaccharide (100 ng/ml, 12 h) to polarize M1 macrophages or to IL-4/IL-13 (20 ng/ml) to polarize M2 macrophages. M1/M2 macrophage markers along with cytokine gene expression were determined using RT-qPCR. Feces and mucosa-associated microbiota (MAM) samples were collected, and the V4 region of 16 s rRNA was sequenced. Micro- and macroscopic scores, colonic IL-6, IL-1ß, TNF- α, and M1 macrophages markers were significantly decreased in the hCTS-treated group. Treatment did not have any effect on colonic IL-10, TGF-ß, and M2 markers nor modified the bacterial richness, diversity, or the major phyla in colitic fecal and MAM samples. In vitro, pro-inflammatory cytokines levels, as well as their gene expression, were significantly reduced in hCTS-treated M1 macrophages. hCTS treatment did not affect M2 macrophage markers. These findings suggest that hCTS treatment attenuates the severity of inflammatory relapse through the modulation of the M1 macrophages and the release of pro-inflammatory cytokines.

Carrageenan Gum and Adherent Invasive Escherichia coli in a Piglet Model of Inflammatory Bowel Disease: Impact on Intestinal Mucosa-associated Microbiota.

Inflammatory bowel diseases (IBD) including Crohn's disease (CD), and ulcerative colitis (UC), are chronic conditions characterized by chronic intestinal inflammation. Adherent invasive Escherichia coli (AIEC) pathotype has been increasingly implicated in the etiopathogenesis of IBD. In a 21-day study, we investigated the effects of AIEC strain UM146 inoculation on microbiota profile of the ileal, cecal, ascending and descending colon in a pig model of experimental colitis. Carrageenan gum (CG) was used to induce colitis in weaner piglets whereas AIEC strain UM146 previously isolated from a CD patient was included to investigate a cause or consequence effect in IBD. Treatments were: (1) control; (2) CG; (3) AIEC strain UM146; and (4) CG+UM146. Pigs in groups 2 and 4 received 1% CG in drinking water from day 1 of the study while pigs in groups 3 and 4 were inoculated with UM146 on day 8. Following euthanization on day 21, tissue mucosal scrapings were collected and used for DNA extraction. The V4 region of bacterial 16S rRNA gene was then subjected to Illumina sequencing. Microbial diversity, composition, and the predicted functional metagenome were determined in addition to short chain fatty acids profiles in the digesta and inflammatory cytokines in the intestinal tissue. CG-induced colitis decreased bacterial species richness and shifted community composition. At the phylum level, an increase in Proteobacteria and Deferribacteres and a decrease in Firmicutes, Actinobacteria, and Bacteroidetes were observed in CG and CGUM146 compared to control and UM146. The metabolic capacity of the microbiome was also altered in CG and CGUM146 compared to UM146 and control in the colon. We demonstrated that CG resulted in bacterial dysbiosis and shifted community composition similar to what has been previously observed in IBD patients. However, AIEC strain UM146 alone did not cause any clear changes compared to CG or control in our experimental IBD pig model.

Human Catestatin Alters Gut Microbiota Composition in Mice.

The mammalian intestinal tract is heavily colonized with a dense, complex, and diversified microbial populations. In healthy individuals, an array of epithelial antimicrobial agents is secreted in the gut to aid intestinal homeostasis. Enterochromaffin cells (EC) in the intestinal epithelium are a major source of chromogranin A (CgA), which is a pro-hormone and can be cleaved into many bioactive peptides that include catestatin (CST). This study was carried out to evaluate the possible impact of CST on gut microbiota in vivo using a mouse model. The CST (Human CgA352-372) or normal saline was intrarectally administered in C57BL/6 male mice for 6 days and then sacrificed. Feces and colonic mucosa tissue samples were collected, DNA was extracted, the V4 region of bacterial 16S rRNA gene was amplified and subjected to MiSeq Illumina sequencing. The α-diversity was calculated using Chao 1 and ß-diversity was determined using QIIME. Differences at the genus level were determined using partial least square discriminant analysis (PLS-DA). Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was used to predict functional capacity of bacterial community. CST treatment did not modify bacterial richness in fecal and colonic mucosa-associated microbiota; however, treatment significantly modified bacterial community composition between the groups. Also, CST-treated mice had a significantly lower relative abundance of Firmicutes and higher abundance of Bacteroidetes, observed only in fecal samples. However, at lower phylogenetic levels, PLS-DA analysis revealed that some bacterial taxa were significantly associated with the CST-treated mice in both fecal and colonic mucosa samples. In addition, differences in predicted microbial functional pathways in both fecal and colonic mucosa samples were detected. The results support the hypothesis that CST treatment modulates gut microbiota composition under non-pathophysiological conditions, however, the result of this study needs to be further validated in a larger experiment. The data may open new avenues for the development of a potential new line of antimicrobial peptides and their use as therapeutic agents to treat several inflammatory conditions of the gastrointestinal tract, such as inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS), or other health conditions.