Effects of removing in-feed antibiotics and zinc oxide on the taxonomy and functionality of the microbiota in post weaning pigs.

BACKGROUND: Post weaning diarrhoea (PWD) causes piglet morbidity and mortality at weaning and is a major driver for antimicrobial use worldwide. New regulations in the EU limit the use of in-feed antibiotics (Ab) and therapeutic zinc oxide (ZnO) to prevent PWD. New approaches to control PWD are needed, and understanding the role of the microbiota in this context is key. In this study, shotgun metagenome sequencing was used to describe the taxonomic and functional evolution of the faecal microbiota of the piglet during the first two weeks post weaning within three experimental groups, Ab, ZnO and no medication, on commercial farms using antimicrobials regularly in the post weaning period. RESULTS: Diversity was affected by day post weaning (dpw), treatment used and diarrhoea but not by the farm. Microbiota composition evolved towards the dominance of groups of species such as Prevotella spp. at day 14dpw. ZnO inhibited E. coli overgrowth, promoted higher abundance of the family Bacteroidaceae and decreased Megasphaera spp. Animals treated with Ab exhibited inconsistent taxonomic changes across time points, with an overall increase of Limosilactobacillus reuteri and Megasphaera elsdenii. Samples from non-medicated pigs showed virulence-related functions at 7dpw, and specific ETEC-related virulence factors were detected in all samples presenting diarrhoea. Differential microbiota functions of pigs treated with ZnO were related to sulphur and DNA metabolism, as well as mechanisms of antimicrobial and heavy metal resistance, whereas Ab treated animals exhibited functions related to antimicrobial resistance and virulence. CONCLUSION: Ab and particularly ZnO maintained a stable microbiota composition and functionality during the two weeks post weaning, by limiting E. coli overgrowth, and ultimately preventing microbiota dysbiosis. Future approaches to support piglet health should be able to reproduce this stable gut microbiota transition during the post weaning period, in order to maintain optimal gut physiological and productive conditions.

Fine-tuning of post-weaning pig microbiome structure and functionality by in-feed zinc oxide and antibiotics use.

Introduction: Post-weaning diarrhoea (PWD) is a multifactorial disease that affects piglets after weaning, contributing to productive and economic losses. Its control includes the use of in-feed prophylactic antibiotics and therapeutic zinc oxide (ZnO), treatments that, since 2022, are no longer permitted in the European Union due to spread of antimicrobial resistance genes and pollution of soil with heavy metals. A dysbiosis in the microbiota has been suggested as a potential risk factor of PWD onset. Understanding pig's microbiota development around weaning and its changes in response to ZnO and antibiotics is crucial to develop feasible alternatives to prophylactic and metaphylactic antimicrobial use. Methods: This study used shotgun metagenomic sequencing to investigate the environmental and faecal microbiota on 10 farms using (Treated) or not using (ZnO-free) in-feed antibiotics and ZnO during the first 14 days post-weaning (dpw). Environmental samples from clean pens were collected at weaning day (0dpw), and faecal samples at 0, 7 and 14dpw. Diarrhoeic faecal samples were collected at 7dpw when available. Results: The analysis of data revealed that the faecal microbiota composition and its functionality was impacted by the sampling time point (microbiota maturation after weaning) but not by the farm environment. Treatment with antibiotics and ZnO showed no effects on diversity indices while the analyses of microbiota taxonomic and functional profiles revealed increased abundance of taxa and metabolic functions associated with Phascolarctobacterium succinatutens or different species of Prevotella spp. on the Treated farms, and with Megasphaera elsdenii and Escherichia coli on the ZnO-free farms. The analysis of diarrhoea samples revealed that the treatment favoured the microbiota transition or maturation from 0dpw to 14dpw in Treated farms, resembling the composition of healthy animals, when compared to diarrhoea from ZnO-free farms, which were linked in composition to 0dpw samples. Discussion: The results provide a comprehensive overview of the beneficial effects of ZnO and antibiotics in PWD in the microbiota transition after weaning, preventing the overgrowth of pathogens such as pathogenic E. coli and revealing the key aspects in microbiota maturation that antibiotics or ZnO alternatives should fulfil.

SARS-CoV-2 accessory proteins involvement in inflammatory and profibrotic processes through IL11 signaling.

SARS-CoV-2, the cause of the COVID-19 pandemic, possesses eleven accessory proteins encoded in its genome. Their roles during infection are still not completely understood. In this study, transcriptomics analysis revealed that both WNT5A and IL11 were significantly up-regulated in A549 cells expressing individual accessory proteins ORF6, ORF8, ORF9b or ORF9c from SARS-CoV-2 (Wuhan-Hu-1 isolate). IL11 is a member of the IL6 family of cytokines. IL11 signaling-related genes were also differentially expressed. Bioinformatics analysis disclosed that both WNT5A and IL11 were involved in pulmonary fibrosis idiopathic disease and functional assays confirmed their association with profibrotic cell responses. Subsequently, data comparison with lung cell lines infected with SARS-CoV-2 or lung biopsies from patients with COVID-19, evidenced altered profibrotic gene expression that matched those obtained in this study. Our results show ORF6, ORF8, ORF9b and ORF9c involvement in inflammatory and profibrotic responses. Thus, these accessory proteins could be targeted by new therapies against COVID-19 disease.

Salmonella Typhimurium induces genome-wide expression and phosphorylation changes that modulate immune response, intracellular survival and vesicle transport in infected neutrophils.

Salmonella Typhimurium is a food-borne pathogen that causes salmonellosis. When in contact with the host, neutrophils are rapidly recruited to act as first line of defense. To better understand the pathogenesis of this infection, we used an in vitro model of neutrophil infection to perform dual RNA-sequencing (both host and pathogen). In addition, and given that many pathogens interfere with kinase-mediated phosphorylation in host signaling, we performed a phosphoproteomic analysis. The immune response was overall diminished in infected neutrophils, mainly JAK/STAT and toll-like receptor signaling pathways. We found decreased expression of proinflammatory cytokine receptor genes and predicted downregulation of the mitogen-activated protein (MAPK) signaling pathway. Also, Salmonella infection inhibited interferons I and II signaling pathways by upregulation of SOCS3 and subsequent downregulation of STAT1 and STAT2. Additionally, phosphorylation of PSMC2 and PSMC4, proteasome regulatory proteins, was decreased in infected neutrophils. Cell viability and survival was increased by p53 signaling, cell cycle arrest and NFkB-proteasome pathways activation. Combined analysis of RNA-seq and phosphoproteomics also revealed inhibited vesicle transport mechanisms mediated by dynein/dynactin and exocyst complexes, involved in ER-to-Golgi transport and centripetal movement of lysosomes and endosomes. Among the overexpressed virulence genes from Salmonella we found potential effectors responsible of these dysregulations, such as spiC, sopD2, sifA or pipB2, all of them involved in intracellular replication. Our results suggest that Salmonella induces (through overexpression of virulence factors) transcriptional and phosphorylation changes that increases neutrophil survival and shuts down immune response to minimize host response, and impairing intracellular vesicle transport likely to keep nutrients for replication and Salmonella-containing vacuole formation and maintenance.

Impairment of antiviral immune response and disruption of cellular functions by SARS-CoV-2 ORF7a and ORF7b.

SARS-CoV-2, the causative agent of the present COVID-19 pandemic, possesses eleven accessory proteins encoded in its genome, and some have been implicated in facilitating infection and pathogenesis through their interaction with cellular components. Among these proteins, accessory protein ORF7a and ORF7b functions are poorly understood. In this study, A549 cells were transduced to express ORF7a and ORF7b, respectively, to explore more in depth the role of each accessory protein in the pathological manifestation leading to COVID-19. Bioinformatic analysis and integration of transcriptome results identified defined canonical pathways and functional groupings revealing that after expression of ORF7a or ORF7b, the lung cells are potentially altered to create conditions more favorable for SARS-CoV-2, by inhibiting the IFN-I response, increasing proinflammatory cytokines release, and altering cell metabolic activity and adhesion. Based on these results, it is plausible to suggest that ORF7a or ORF7b could be used as biomarkers of progression in this pandemic.

Using Shotgun Sequencing to Describe the Changes Induced by In-Feed Zinc Oxide and Apramycin in the Microbiomes of Pigs One Week Postweaning.

Postweaning diarrhea (PWD) is a relevant problem associated with early weaning on pig farms. For decades, in-feed antibiotics and therapeutic zinc oxide (ZnO) have been widely used to prevent PWD in piglets. The European Union is banning both strategies in 2022 due to antimicrobial resistance and environmental contamination concerns, respectively. Understanding the effects of these products on the pig microbiome is crucial for correcting potential microbial disbalances that would prompt PWD. Using shotgun sequencing, three trials were carried out to explore the impact of in-feed apramycin and ZnO, combined with different farm hygiene protocols, on the fecal microbiomes of piglets 7 days postweaning. In trial 1, 28-day-old piglets were allocated to one of three groups: control diet (Ct), Ct + ZnO (Zn), and Ct + apramycin (Ab). In trials 2 and 3, piglets were allocated to the same treatments, but the trials also included different cleaning protocols, achieving different hygiene levels. In-feed treatments impacted the richness, diversity, and relative abundance of the piglets' microbiome more than hygiene. Pigs in the Ct group showed higher species richness than pigs in the Ab and Zn groups. A clustering analysis evidenced a link between Enterobacteriaceae in the Ct group; Lactobacillaceae and Veillonellaceae mainly in the Ct group; and Bacteroidaceae, Ruminococcaceae, Oscillospiraceae, Acidaminococcaceae, and Lactobacillaceae in the Ab and Zn groups. Functional data analysis revealed a higher abundance of virulence genes in the Ct group microbiomes and heavy metal and antimicrobial resistance-related functions in the Zn treatment group. The results demonstrate that alternatives to Ab and ZnO should balance the microbial abundance and stimulate the growth of commensals to outcompete potential pathogens. IMPORTANCE Weaning is a critical period for piglets, during which potentially harmful bacteria such as Escherichia coli can increase in abundance in the intestine, creating digestive problems and diarrhea. In-feed antibiotics, the most frequent administration route for antibiotics in livestock, and therapeutic doses of zinc oxide (ZnO) help to control diarrhea but prompt secondary problems such as antimicrobial resistance and soil pollution from heavy metals. Understanding how these strategies impact the gut microbiota is crucial for establishing health biomarkers and designing successful replacement strategies. Using shotgun sequencing, this study compares the microbiota of pigs after early weaning when treated with in-feed antibiotics, ZnO, or treatment-free diets to describe differences that could define the susceptibility to infections, providing the basis for future research on improving intestinal resilience through microbiota-based strategies.

CD9 expression in porcine blood CD4+ T cells delineates two subsets with phenotypic characteristics of central and effector memory cells.

In this report, we describe the characterization of a new monoclonal antibody, named 4H5CR4, against porcine CD9. Its use in combination with antibodies to CD4, CD8α, and 2E3 allows to distinguish at least five main CD4+ T cell subsets. Analysis on these subsets of CD45RA, CD27, CD29, CD95, CCR7, and SLA-DR markers depicts a progressive model of CD4+ T cell development. CD4+ 2E3+ CD8α- CD9- cells are the least differentiated population of naïve cells, whereas the CD4+ 2E3- CD8α+CD9+ and CD4+ 2E3- CD8α+ CD9- cells display phenotypic features of central and effector memory T helper cells, respectively. The latter subsets were able to produce IFN-γ after polyclonal activation with PMA/Ionomycin; however, in vitro virus-specific IFN-γ production of PBMCs collected at 38-44 days after pseudorabies virus vaccination was dominated by cells with a CD9+ phenotype. Therefore, CD9 appears to be a useful marker to investigate CD4+ T cell heterogeneity in swine.

Study of microRNA expression in Salmonella Typhimurium-infected porcine ileum reveals miR-194a-5p as an important regulator of the TLR4-mediated inflammatory response.

Infection with Salmonella Typhimurium (S. Typhimurium) is a common cause of food-borne zoonosis leading to acute gastroenteritis in humans and pigs, causing economic losses to producers and farmers, and generating a food security risk. In a previous study, we demonstrated that S. Typhimurium infection produces a severe transcriptional activation of inflammatory processes in ileum. However, little is known regarding how microRNAs regulate this response during infection. Here, small RNA sequencing was used to identify 28 miRNAs differentially expressed (DE) in ileum of S. Typhimurium-infected pigs, which potentially regulate 14 target genes involved in immune system processes such as regulation of cytokine production, monocyte chemotaxis, or cellular response to interferon gamma. Using in vitro functional and gain/loss of function (mimics/CRISPR-Cas system) approaches, we show that porcine miR-194a-5p (homologous to human miR-194-5p) regulates TLR4 gene expression, an important molecule involved in pathogen virulence, recognition and activation of innate immunity in Salmonella infection.

Saccharomyces Cerevisiae Var Boulardii CNCM I-1079 Reduces Expression of Genes Involved in Inflammatory Response in Porcine Cells Challenged by Enterotoxigenic E. Coli and Influences Bacterial Communities in an In Vitro Model of the Weaning Piglet Colon.

Enterotoxigenic Escherichia coli (ETEC) is the main infectious agent responsible for piglet post-weaning diarrhea with high mortality rates. Antimicrobials represent the current principal strategy for treating ETEC infections in pig farms, but the occurrence of multi-resistant bacterial strains has considerably increased in the last decades. Thus, finding non-antibiotic alternatives becomes a real emergency. In this context, we investigated the effect of a live yeast strain, Saccharomyces cerevisiae var boulardii CNCM I-1079 (SB) in an in vitro model of the weaning piglet colon implemented with a mucus phase (MPigut-IVM) inoculated with ETEC and coupled with an intestinal porcine cell line IPI-2I. We showed that SB was able to modulate the in vitro microbiota through an increase in Bacteroidiaceae and a decrease in Prevotellaceae families. Effluents collected from the SB treated bioreactors were able to mitigate the expression level of genes encoding non-gel forming mucins, tight junction proteins, innate immune pathway, and pro-inflammatory response in IPI-2I cells. Furthermore, SB exerted a significant protective effect against ETEC adhesion on porcine IPEC-J2 intestinal cells in a dose-dependent manner and showed a positive effect on ETEC-challenged IPEC-J2 by lowering expression of genes involved in pro-inflammatory immune responses. Our results showed that the strain SB CNCM I-1079 could prevent microbiota dysbiosis associated with weaning and protect porcine enterocytes from ETEC infections by reducing bacterial adhesion and modulating the inflammatory response.

CPEB alteration and aberrant transcriptome-polyadenylation lead to a treatable SLC19A3 deficiency in Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder of the basal ganglia for which disease-modifying treatments are not yet available. Although gene-silencing therapies are currently being tested, further molecular mechanisms must be explored to identify druggable targets for HD. Cytoplasmic polyadenylation element binding proteins 1 to 4 (CPEB1 to CPEB4) are RNA binding proteins that repress or activate translation of CPE-containing transcripts by shortening or elongating their poly(A) tail. Here, we found increased CPEB1 and decreased CPEB4 protein in the striatum of patients and mouse models with HD. This correlated with a reprogramming of polyadenylation in 17.3% of the transcriptome, markedly affecting neurodegeneration-associated genes including PSEN1, MAPT, SNCA, LRRK2, PINK1, DJ1, SOD1, TARDBP, FUS, and HTT and suggesting a new molecular mechanism in neurodegenerative disease etiology. We found decreased protein content of top deadenylated transcripts, including striatal atrophy­linked genes not previously related to HD, such as KTN1 and the easily druggable SLC19A3 (the ThTr2 thiamine transporter). Mutations in SLC19A3 cause biotin-thiamine­responsive basal ganglia disease (BTBGD), a striatal disorder that can be treated with a combination of biotin and thiamine. Similar to patients with BTBGD, patients with HD demonstrated decreased thiamine in the cerebrospinal fluid. Furthermore, patients and mice with HD showed decreased striatal concentrations of thiamine pyrophosphate (TPP), the metabolically active form of thiamine. High-dose biotin and thiamine treatment prevented TPP deficiency in HD mice and attenuated the radiological, neuropathological, and motor HD-like phenotypes, revealing an easily implementable therapy that might benefit patients with HD.

Pathogen Challenge and Dietary Shift Alter Microbiota Composition and Activity in a Mucin-Associated in vitro Model of the Piglet Colon (MPigut-IVM) Simulating Weaning Transition.

Enterotoxigenic Escherichia coli (ETEC) is the principal pathogen responsible for post-weaning diarrhea in newly weaned piglets. Expansion of ETEC at weaning is thought to be the consequence of various stress factors such as transient anorexia, dietary change or increase in intestinal inflammation and permeability, but the exact mechanisms remain to be elucidated. As the use of animal experiments raise more and more ethical concerns, we used a recently developed in vitro model of piglet colonic microbiome and mucobiome, the MPigut-IVM, to evaluate the effects of a simulated weaning transition and pathogen challenge at weaning. Our data suggested that the tested factors impacted the composition and functionality of the MPigut-IVM microbiota. The simulation of weaning transition led to an increase in relative abundance of the Prevotellaceae family which was further promoted by the presence of the ETEC strain. In contrast, several beneficial families such as Bacteroidiaceae or Ruminococcaceae and gut health related short chain fatty acids like butyrate or acetate were reduced upon simulated weaning. Moreover, the incubation of MPigut-IVM filtrated effluents with porcine intestinal cell cultures showed that ETEC challenge in the in vitro model led to an increased expression of pro-inflammatory genes by the porcine cells. This study provides insights about the etiology of a dysbiotic microbiota in post-weaning piglets.

SARS-CoV-2 Accessory Proteins in Viral Pathogenesis: Knowns and Unknowns.

There are still many unanswered questions concerning viral SARS-CoV-2 pathogenesis in COVID-19. Accessory proteins in SARS-CoV-2 consist of eleven viral proteins whose roles during infection are still not completely understood. Here, a review on the current knowledge of SARS-CoV-2 accessory proteins is summarized updating new research that could be critical in understanding SARS-CoV-2 interaction with the host. Some accessory proteins such as ORF3b, ORF6, ORF7a and ORF8 have been shown to be important IFN-I antagonists inducing an impairment in the host immune response. In addition, ORF3a is involved in apoptosis whereas others like ORF9b and ORF9c interact with cellular organelles leading to suppression of the antiviral response in infected cells. However, possible roles of ORF7b and ORF10 are still awaiting to be described. Also, ORF3d has been reassigned. Relevant information on the knowns and the unknowns in these proteins is analyzed, which could be crucial for further understanding of SARS-CoV-2 pathogenesis and to design strategies counteracting their actions evading immune responses in COVID-19.

Reprogramming of microRNA expression via E2F1 downregulation promotes Salmonella infection both in infected and bystander cells.

Cells infected with pathogens can contribute to clearing infections by releasing signals that instruct neighbouring cells to mount a pro-inflammatory cytokine response, or by other mechanisms that reduce bystander cells' susceptibility to infection. Here, we show the opposite effect: epithelial cells infected with Salmonella Typhimurium secrete host factors that facilitate the infection of bystander cells. We find that the endoplasmic reticulum stress response is activated in both infected and bystander cells, and this leads to activation of JNK pathway, downregulation of transcription factor E2F1, and consequent reprogramming of microRNA expression in a time-dependent manner. These changes are not elicited by infection with other bacterial pathogens, such as Shigella flexneri or Listeria monocytogenes. Remarkably, the protein HMGB1 present in the secretome of Salmonella-infected cells is responsible for the activation of the IRE1 branch of the endoplasmic reticulum stress response in non-infected, neighbouring cells. Furthermore, E2F1 downregulation and the associated microRNA alterations promote Salmonella replication within infected cells and prime bystander cells for more efficient infection.

Impact of Varroa destructor and associated pathologies on the colony collapse disorder affecting honey bees.

Varroa mite is the major threat to the western honey bee, Apis mellifera, and the cause of significant economic losses in the apiculture industry. Varroa destructor feeds on brood and adult bees being responsible for vectoring virus infections and other diseases. This study analyses the role of Varroa and other associated pathogens, such as viruses or the fungus Nosema ceranae, and their relationships regarding the viability of the bee colony. It has been carried out during one beekeeping season, with the subspecies A. m. iberiensis, commonly used in the apiculture industry of Spain. Our study shows a significant relationship between the presence of Varroa destructor and viral infection by deformed wing virus and acute bee paralysis virus. Nosema ceranae behaved as an opportunistic pathogen. In addition, this study explored a potential naturally occurring subset of peptides, responsible for the humoral immunity of the bees. The expression of the antimicrobial peptides abaecin and melittin showed a significant relationship with the levels of Varroa mite and the deformed wing virus.

Salmonella Typhimurium Infection Along the Porcine Gastrointestinal Tract and Associated Lymphoid Tissues.

Salmonella is a major foodborne pathogen and pork is one of the main sources of human salmonellosis. Understanding the pathogenesis and progression of the infection within the host is of interest to establish potential approaches to control the disease in pigs. The present study evaluates factors such as intestinal colonization, fecal shedding, and pathogen persistence by 2 studies using experimental challenge with Salmonella Typhimurium in weaned pigs and euthanasia at different time points (1, 2, and 6 and 2, 14, and 30 days postinfection [dpi], respectively). Histopathology of intestine at early time points (1 dpi and 2 dpi) showed severe damage to the epithelium together with an increase in polymorphonuclear cells and macrophages (P < .001), particularly in jejunum and ileum. Large quantities of Salmonella were detected within the contents of the ileum, cecum, and colon in early infection. Salmonella could also be observed in the medulla of tonsils and mesenteric lymph nodes. From 6 dpi onward, signs of recovery were observed, with progressive restoration of the epithelium, reduction of the inflammatory infiltrate, and elimination of Salmonella from the mucosa. Concentration of Salmonella in feces and ileum content decreased, but shedding did not cease even at 4 weeks after infection. Persistence of the bacteria in mesenteric lymph nodes was identified within the connective tissue at 14 and 30 dpi. Our results demonstrate a recovery of the disease after an initial acute phase but also show persistence within the lumen and surrounding lymphoid tissue. These findings are relevant to developing effective control strategies.

Identification and functional characterization of polymorphisms in promoter sequences of porcine NOD1 and NOD2 genes.

NOD-like receptors (NLRs) play a key role in the innate immune system, acting as a second line of surveillance against pathogens. NLRs detect particular bacteria that have gained access to the cytoplasm, evading recognition by other pattern recognition receptors, such as Toll-like receptors. It has been demonstrated that coding sequence-single nucleotide polymorphisms may alter the ligand recognition ability of NLRs, affecting their pathogen-sensing function. However, there have been no data relating to the identification and functional analysis of SNPs in porcine NLR promoters. We examined the promoter sequences of the porcine NOD1 and NOD2 genes with the aim to identify and to evaluate the effect of genetic variations on promoter activity. Six SNPs in NOD1 and three SNPs in NOD2 were identified. Luciferase reporter gene assays showed significant differences in promoter activity between allele variants of NOD1 -920G>A (NC_010460.4:g.42431413G>A) and NOD2 -1670G>A (NC_010448.4:g.34169122T>C) SNPs. The results suggest that promoter polymorphisms could modify the expression levels of porcine NOD1 and NOD2 genes.

Comparative proteomic analysis reveals different responses in porcine lymph nodes to virulent and attenuated homologous African swine fever virus strains.

African swine fever (ASF) is a pathology of pigs against which there is no treatment or vaccine. Understanding the equilibrium between innate and adaptive protective responses and immune pathology might contribute to the development of strategies against ASFV. Here we compare, using a proteomic approach, the course of the in vivo infection caused by two homologous strains: the virulent E75 and the attenuated E75CV1. Our results show a progressive loss of proteins by day 7 post-infection (pi) with E75, reflecting tissue destruction. Many signal pathways were affected by both infections but in different ways and extensions. Cytoskeletal remodelling and clathrin-endocytosis were affected by both isolates, while a greater number of proteins involved on inflammatory and immunological pathways were altered by E75CV1. 14-3-3 mediated signalling, related to immunity and apoptosis, was inhibited by both isolates. The implication of the Rho GTPases by E75CV1 throughout infection is also evident. Early events reflected the lack of E75 recognition by the immune system, an evasion strategy acquired by the virulent strains, and significant changes at 7 days post-infection (dpi), coinciding with the peak of infection and the time of death. The protein signature at day 31 pi with E75CV1 seems to reflect events observed at 1 dpi, including the upregulation of proteosomal subunits and molecules described as autoantigens (vimentin, HSPB1, enolase and lymphocyte cytosolic protein 1), which allow the speculation that auto-antibodies could contribute to chronic ASFV infections. Therefore, the use of proteomics could help understand ASFV pathogenesis and immune protection, opening new avenues for future research.

Early Salmonella Typhimurium infection in pigs disrupts Microbiome composition and functionality principally at the ileum mucosa.

Salmonella is a major foodborne pathogen which successfully infects animal species for human consumption such as swine. The pathogen has a battery of virulence factors which it uses to colonise and persist within the host. The host microbiota may play a role in resistance to, and may also be indirectly responsible from some of the consequences of, Salmonella infection. To investigate this, we used 16S rRNA metagenomic sequencing to determine the changes in the gut microbiota of pigs in response to infection by Salmonella Typhimurium at three locations: ileum mucosa, ileum content and faeces. Early infection (2 days post-infection) impacted on the microbiome diversity at the mucosa, reflected in a decrease in representatives of the generally regarded as desirable genera (i.e., Bifidobacterium and Lactobacillus). Severe damage in the epithelium of the ileum mucosa correlated with an increase in synergistic (with respect to Salmonella infection; Akkermansia) or opportunistically pathogenic bacteria (Citrobacter) and a depletion in anaerobic bacteria (Clostridium spp., Ruminococcus, or Dialliser). Predictive functional analysis, together with metabolomic analysis revealed changes in glucose and lipid metabolism in infected pigs. The observed changes in commensal healthy microbiota, including the growth of synergistic or potentially pathogenic bacteria and depletion of beneficial or competing bacteria, could contribute to the pathogen's ability to colonize the gut successfully. The findings from this study could be used to form the basis for further research aimed at creating intervention strategies to mitigate the effects of Salmonella infection.

Regulatory role of microRNA in mesenteric lymph nodes after Salmonella Typhimurium infection.

Salmonellosis is a gastrointestinal disease caused by non-typhoidal Salmonella serovars such as Salmonella Typhimurium. This pathology is a zoonosis, and food animals with subclinical infection constitute a vast reservoir for disease. After intestinal colonization, Salmonella Typhimurium reaches mesenteric lymph nodes (MLN), where infection is controlled avoiding systemic spread. Although the molecular basis of this infection has been extensively studied, little is known about how microRNA (miRNA) regulate the expression of proteins involved in the Salmonella-host interaction. Using small RNA-seq, we examined expression profiles of MLN 2 days after infection with Salmonella Typhimurium, and we found 110 dysregulated miRNA. Among them, we found upregulated miR-21, miR-155, miR-150, and miR-221, as well as downregulated miR-143 and miR-125, all of them previously linked to other bacterial infections. Integration with proteomic data revealed 30 miRNA potentially regulating the expression of 15 proteins involved in biological functions such as cell death and survival, inflammatory response and antigenic presentation. The inflammatory response was found increased via upregulation of miRNA such as miR-21 and miR-155. Downregulation of miR-125a/b, miR-148 and miR-1 were identified as potential regulators of MHC-class I components PSMB8, HSP90B1 and PDIA3, respectively. Furthermore, we confirmed that miR-125a is a direct target of immunoproteasome component PSMB8. Since we also found miR-130 downregulation, which is associated with upregulation of HSPA8, we suggest induction of both MHC-I and MHC-II antigen presentation pathways. In conclusion, our study identifies miRNA that could regulate critical networks for antigenic presentation, inflammatory response and cytoskeletal rearrangements.

Phenotypic and functional characterization of porcine bone marrow monocyte subsets.

Monocytes comprise several subsets with distinct phenotypes and functional capacities. Based on CD163 expression, two major monocyte subsets can be discriminated in the porcine bone marrow. The CD163+ cells expressed higher levels of SLA-DR, Siglec-1, CD11R1 and CD16 when compared to CD163- monocytes, whereas no remarkable differences were observed in the expression of other markers analyzed. Gene expression analysis showed differential expression of several chemokine receptor and TLR genes. Both subsets phagocytosed microspheres with similar efficiency. However, CD163- cells tended to produce higher levels of ROS in response to PMA, whereas CD163+ cells were more efficient in endocytosing and processing antigens (DQ-OVA). CD163- monocytes produced higher levels of TNF-α and IL-10 than CD163+ cells when stimulated with LPS or Imiquimod. Both subsets produced similar amounts of IL-8 in response to LPS; however, CD163+ cells produced more IL-8 after Imiquimod stimulation. Whether these subsets represent different developmental stages, and how are they related remain to be investigated.