Improving gut function in pigs to prevent pathogen colonization

Intestinal pathogens causing either clinical or sub-clinical infections increase pig morbidity and (or) mortality, resulting in economic losses and wider socio-economic impacts on pig production. An optimally functioning gastrointestinal tract (GIT) is fundamental to combatting intestinal pathogen colonisation at all the stages of life. This requires successful development and maintenance of key gut functions: digestive function; the gastro-intestinal cell line barrier; gut-associated lymphoid tissue (GALT); and gut-associated microbiota. This chapter first discusses research on genes associated with pathogen resistance and porcine immune response. It then reviews risk factors associated with gut mucosa impairment as well as dietary strategies to control risk factors and improve gut functionality in preventing intestinal pathogen colonisation.

Fungal xenosiderophores from mycobiota and diet promote Salmonella colonization of the inflamed gut

The fungal gut microbiota (mycobiota) has been implicated in diseases that disturb gut homeostasis. However, little is known about functional relationships between bacteria and fungi in the gut during infectious colitis. We investigated the role of fungal metabolites during infection with the intestinal pathogen Salmonella enterica serovar Typhimurium. We found that in the gut lumen, both the mycobiota and fungi present in the diet can be a source of siderophores, small molecules that scavenge iron from the host. The ability to use fungal siderophores, such as ferrichrome and coprogen, conferred a competitive growth advantage to Salmonella strains expressing the fungal siderophore receptors FhuA or FhuE in vitro and in a mouse model. Our study highlights the role of inter-kingdom cross-feeding between fungi and Salmonella, and elucidates a new function for the gut mycobiota, revealing the importance of these under-studied members of the gut ecosystem during bacterial infection.

Diet diversity and environment determine the intestinal microbiome and bacterial pathogen load of fire salamanders

Diverse communities of symbiotic microbes inhabit the digestive systems of vertebrates and play a crucial role in animal health, and host diet plays a major role in shaping the composition and diversity of these communities. Here, we characterized diet and gut microbiome of fire salamander populations from three Belgian forests. We carried out DNA metabarcoding on fecal samples, targeting eukaryotic 18S rRNA of potential dietary prey items, and bacterial 16S rRNA of the concomitant gut microbiome. Our results demonstrated an abundance of soft-bodied prey in the diet of fire salamanders, and a significant difference in the diet composition between males and females. This sex-dependent effect on diet was also reflected in the gut microbiome diversity, which is higher in males than female animals. Proximity to human activities was associated with increased intestinal pathogen loads. Collectively, the data supports a relationship between diet, environment and intestinal microbiome in fire salamanders, with potential health implications.

Disease-Alleviating Effects of Peroral Activated Charcoal Treatment in Acute Murine Campylobacteriosis

Foodborne Campylobacter jejuni infections are on the rise and responsible for worldwide serious health issues. Increasing resistance of C. jejuni strains against antimicrobial treatments, necessitates antibiotics-independent treatment options for acute campylobacteriosis. Activated charcoal (AC) constitutes a long-known and safe compound for the treatment of bacterial enteritis. In this preclinical intervention study, we addressed potential anti-pathogenic and immune-modulatory effects of AC during acute experimental campylobacteriosis. Therefore, microbiota-depleted IL-10−/− mice were infected with C. jejuni by gavage and challenged with either AC or placebo via the drinking water starting on day 2 post-infection. On day 6 post-infection, AC as compared to placebo-treated mice did not only harbor lower intestinal pathogen loads but also presented with alleviated C. jejuni-induced clinical signs such as diarrhea and wasting symptoms. The improved clinical outcome of AC-treated mice was accompanied by less colonic epithelial cell apoptosis and reduced pro-inflammatory immune responses in the intestinal tract. Notably, AC treatment did not only alleviate intestinal, but also extra-intestinal and systemic immune responses as indicated by dampened pro-inflammatory mediator secretion. Given the anti-pathogenic and immune-modulatory properties of AC in this study, a short-term application of this non-toxic drug constitutes a promising antibiotics-independent option for the treatment of human campylobacteriosis.

Survey of Pathogen-Lowering and Immuno-Modulatory Effects Upon Treatment of Campylobacter coli-Infected Secondary Abiotic IL-10-/- Mice with the Probiotic Formulation Aviguard®

The prevalence of infections with the zoonotic enteritis pathogen Campylobacter coli is increasing. Probiotic formulations constitute promising antibiotic-independent approaches to reduce intestinal pathogen loads and modulate pathogen-induced immune responses in the infected human host, resulting in acute campylobacteriosis and post-infectious sequelae. Here, we address potential antipathogenic and immuno-modulatory effects of the commercial product Aviguard® during experimental campylobacteriosis. Secondary abiotic IL-10-/- mice were infected with a C. coli patient isolate on days 0 and 1, followed by oral Aviguard® treatment on days 2, 3 and 4. Until day 6 post-infection, Aviguard® treatment could lower the pathogen burdens within the proximal but not the distal intestinal tract. In contrast, the probiotic bacteria had sufficiently established in the intestines with lower fecal loads of obligate anaerobic species in C. coli-infected as compared to uninfected mice following Aviguard® treatment. Aviguard® application did not result in alleviated clinical signs, histopathological or apoptotic changes in the colon of infected IL-10-/- mice, whereas, however, Aviguard® treatment could dampen pathogen-induced innate and adaptive immune responses in the colon, accompanied by less distinct intestinal proinflammatory cytokine secretion. In conclusion, Aviguard® constitutes a promising probiotic compound to alleviate enteropathogen-induced proinflammatory immune responses during human campylobacteriosis.

Identification and Characterization of Long Non-coding RNAs in the Intestine of Olive Flounder (Paralichthys olivaceus) During Edwardsiella tarda Infection

Long non-coding RNAs (lncRNAs) play widespread roles in fundamental biological processes, including immune responses. The olive flounder (Paralichthys olivaceus), an important economical flatfish widely cultured in Japan, Korea, and China, is threatened by infectious pathogens, including bacteria, viruses, and parasites. However, the role of lncRNAs in the immune responses of this species against pathogen infections is not well-understood. Therefore, in this study, we aimed to identify lncRNAs in the intestine of olive flounder and evaluate their differential expression profiles during Edwardsiella tarda infection, which is an important zoonotic and intestinal pathogen. A total of 4,445 putative lncRNAs were identified, including 3,975 novel lncRNAs and 470 annotated lncRNAs. These lncRNAs had shorter lengths and fewer exons compared with mRNAs. In total, 115 differentially expressed lncRNAs (DE-lncRNAs) were identified during E. tarda infection. To validate the expression pattern of lncRNAs, six DE-lncRNAs were randomly selected for quantitative real-time PCR. The co-located and co-expressed mRNAs of DE-lncRNAs were predicted, which were used to conduct the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The target genes of DE-lncRNAs enriched numerous immune-related processes and exhibited a strong correlation with immune-related signaling pathways. To better understand the extensive regulatory functions of lncRNAs, the lncRNA–miRNA–mRNA regulatory networks were constructed, and two potential competing endogenous RNA (ceRNA) networks, LNC_001979-novel_171-Potusc2 and LNC_001979-novel_171-Podad1, were preliminarily identified from the intestine of olive flounders for the first time. In conclusion, this study provides an invaluable annotation and expression profile of lncRNAs in the intestine of olive flounder infected with E. tarda; this forms a basis for further studies on the regulatory function of lncRNAs in the intestinal mucosal immune responses of olive flounder.

Peroral Clove Essential Oil Treatment Ameliorates Acute Campylobacteriosis—Results from a Preclinical Murine Intervention Study

Campylobacter (C.) jejuni infections pose progressively emerging threats to human health worldwide. Given the rise in antibiotic resistance, antibiotics-independent options are required to fight campylobacteriosis. Since the health-beneficial effects of clove have been known for long, we here analyzed the antimicrobial and immune-modulatory effects of clove essential oil (EO) during acute experimental campylobacteriosis. Therefore, microbiota-depleted interleukin-10 deficient (IL-10−/−) mice were perorally infected with C. jejuni and treated with clove EO via drinking water starting on day 2 post-infection. On day 6 post-infection, lower small- and large-intestinal pathogen loads could be assessed in clove EO as compared to placebo treated mice. Although placebo mice suffered from severe campylobacteriosis as indicated by wasting and bloody diarrhea, clove EO treatment resulted in a better clinical outcome and in less severe colonic histopathological and apoptotic cell responses in C. jejuni infected mice. Furthermore, lower colonic numbers of macrophages, monocytes, and T lymphocytes were detected in mice from the verum versus the placebo cohort that were accompanied by lower intestinal, extra-intestinal, and even systemic proinflammatory cytokine concentrations. In conclusion, our preclinical intervention study provides first evidence that the natural compound clove EO constitutes a promising antibiotics-independent treatment option of acute campylobacteriosis in humans.

HilE is required for synergistic activation of SPI-1 gene expression in Salmonella enterica serovar Typhimurium

Background Salmonella enterica serovar Typhimurium is an intestinal pathogen capable of infecting a wide range of animals. It initiates infection by invading intestinal epithelial cells using a type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1). The SPI-1 genes are regulated by multiple interacting transcription factors. The master regulator is HilD. HilE represses SPI-1 gene expression by binding HilD and preventing it from activating its target promoters. Previous work found that acetate and nutrients synergistically induce SPI-1 gene expression. In the present study, we investigated the role of HilE, nominally a repressor of SPI-1 gene expression, in mediating this response to acetate and nutrients. Results HilE is necessary for activation of SPI-1 gene expression by acetate and nutrients. In mutants lacking hilE, acetate and nutrients no longer increase SPI-1 gene expression but rather repress it. This puzzling response is not due to the BarA/SirA two component system, which governs the response to acetate. To identify the mechanism, we profiled gene expression using RNAseq in the wild type and a ΔhilE mutant under different growth conditions. Analysis of these data suggested that the Rcs system, which regulates gene expression in response to envelope stress, is involved. Consistent with this hypothesis, acetate and nutrients were able to induce SPI-1 gene expression in mutants lacking hilE and the Rcs system. Conclusions While the exact mechanism is unknown, these results demonstrate the HilE, nominally a repressor of SPI-1 gene expression, can also function as an activator under the growth conditions investigated. Collectively, these results provide new insights regarding SPI-1 gene regulation and demonstrate that HilE is more complex than initially envisioned.

The Mastigamoeba balamuthi Genome and the Nature of the Free-Living Ancestor of Entamoeba

The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living Mastigamoeba balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host–parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).

Anti-Pathogenic and Immune-Modulatory Effects of Peroral Treatment with Cardamom Essential Oil in Acute Murine Campylobacteriosis

Human infections with enteropathogenic Campylobacter jejuni (C. jejuni) including multi-drug resistant isolates are emerging worldwide. Antibiotics-independent approaches in the combat of campylobacteriosis are therefore highly desirable. Since the health-beneficial including anti-inflammatory and anti-infectious properties of cardamom have been acknowledged for long, we here addressed potential anti-pathogenic and immune-modulatory effects of this natural compound during acute campylobacteriosis. For this purpose, microbiota-depleted IL-10−/− mice were orally infected with C. jejuni strain 81–176 and subjected to cardamom essential oil (EO) via the drinking water starting on day 2 post-infection. Cardamom EO treatment resulted in lower intestinal pathogen loads and improved clinical outcome of mice as early as day 3 post-infection. Furthermore, when compared to mock controls, cardamom EO treated mice displayed less distinct macroscopic and microscopic inflammatory sequelae on day 6 post-infection that were paralleled by lower colonic numbers of macrophages, monocytes, and T cells and diminished pro-inflammatory mediator secretion not only in the intestinal tract, but also in extra-intestinal and, remarkably, systemic organs. In conclusion, our preclinical intervention study provides the first evidence that cardamom EO comprises a promising compound for the combat of acute campylobacteriosis and presumably prevention of post-infectious morbidities.