Construction and characterization of a saturated Tn-seq library of Salmonella Typhimurium ATCC 14028.

Salmonella enterica is an important foodborne pathogen. Here, we present the construction and characterization of a high-density transposon sequencing library of the Salmonella Typhimurium ATCC 14028 strain. Essential, advantageous, and disadvantageous genes for growth in rich culture medium were identified on the chromosome and the pSLT plasmid.

In ovo administration of a phage cocktail partially prevents colibacillosis in chicks.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the main bacterial disease in poultry leading to significant economic losses worldwide. Antibiotic treatments favor the emergence of multidrug-resistant bacteria, and preventive measures are insufficient to control the disease. There is increasing interest in using the potential of bacteriophages, not only for phage therapy but also for prevention and biocontrol. This study aimed to evaluate the efficacy of a phage cocktail administered in ovo to prevent avian colibacillosis in chicks. When 4 different phages (REC, ESCO3, ESCO47, and ESCO58), stable under avian physiological conditions, were combined and inoculated at 17 embryogenic days (ED), they were transmitted to the newly hatched chicks. In a second trial, the 4-phage cocktail was inoculated into the allantoic fluid at ED16 and after hatch 1-day-old chicks were challenged with the O2 APEC strain BEN4358 inoculated subcutaneously. Two phages (REC and ESCO3) were still detected in the ceca of surviving chicks at the end of the experiment (7-days postinfection). Chicks that received the phages in ovo did not develop colibacillosis lesions and showed a significant decrease in intestinal BEN4358 load (8.00 × 107 CFU/g) compared to the challenged chicks (4.52 × 108 CFU/g). The majority of the reisolated bacteria from the ceca of surviving chicks had developed full resistance to ESCO3 phage, and only 3 were resistant to REC phage. The partially or complete resistance of REC phage induced a considerable cost to bacterial virulence. Here, we showed that phages inoculated in ovo can partially prevent colibacillosis in 1-wk-old chicks. The reduction in the APEC load in the gut and the decreased virulence of some resistant isolates could also contribute to control the disease.

Differential Salmonella Typhimurium intracellular replication and host cell responses in caecal and ileal organoids derived from chicken.

Chicken infection with Salmonella Typhimurium is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the caecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Here, we established intestinal crypt-derived chicken organoids to better decipher the impact of Salmonella intracellular replication on avian intestinal epithelium. Using a 3D organoid model, we observed a significantly higher replication rate of the intracellular bacteria in caecal organoids than in ileal organoids. Our model thus recreates intracellular environment, allowing Salmonella replication of avian epithelium according to the intestinal segment. Moreover, an inhibition of the cellular proliferation was observed in infected ileal and caecal organoids compared to uninfected organoids. This appears with a higher effect in ileal organoids, as well as a higher cytokine and signaling molecule response in infected ileal organoids at 3 h post-infection (hpi) than in caecal organoids that could explain the lower replication rate of Salmonella observed later at 24 hpi. To conclude, this study demonstrates that the 3D organoid is a model allowing to decipher the intracellular impact of Salmonella on the intestinal epithelium cell response and illustrates the importance of the gut segment used to purify stem cells and derive organoids to specifically study epithelial cell -Salmonella interaction.

Isolation and Characterization of a Novel Phage Collection against Avian-Pathogenic Escherichia coli.

The increase in antibiotic-resistant avian-pathogenic Escherichia coli (APEC), the causative agent of colibacillosis in poultry, warrants urgent research and the development of alternative therapies. This study describes the isolation and characterization of 19 genetically diverse, lytic coliphages, 8 of which were tested in combination for their efficacy in controlling in ovo APEC infections. Genome homology analysis revealed that the phages belong to nine different genera, one of them being a novel genus (Nouzillyvirus). One phage, REC, was derived from a recombination event between two Phapecoctavirus phages (ESCO5 and ESCO37) isolated in this study. Twenty-six of the 30 APEC strains tested were lysed by at least one phage. Phages exhibited varying infectious capacities, with narrow to broad host ranges. The broad host range of some phages could be partially explained by the presence of receptor-binding protein carrying a polysaccharidase domain. To demonstrate their therapeutic potential, a phage cocktail consisting of eight phages belonging to eight different genera was tested against BEN4358, an APEC O2 strain. In vitro, this phage cocktail fully inhibited the growth of BEN4358. In a chicken lethality embryo assay, the phage cocktail enabled 90% of phage-treated embryos to survive infection with BEN4358, compared with 0% of nontreated embryos, indicating that these novel phages are good candidates to successfully treat colibacillosis in poultry. IMPORTANCE Colibacillosis, the most common bacterial disease affecting poultry, is mainly treated by antibiotics. Due to the increased prevalence of multidrug-resistant avian-pathogenic Escherichia coli, there is an urgent need to assess the efficacy of alternatives to antibiotherapy, such as phage therapy. Here, we have isolated and characterized 19 coliphages that belong to nine phage genera. We showed that a combination of 8 of these phages was efficacious in vitro to control the growth of a clinical isolate of E. coli. Used in ovo, this phage combination allowed embryos to survive APEC infection. Thus, this phage combination represents a promising treatment for avian colibacillosis.

Epithelial cell invasion by salmonella typhimurium induces modulation of genes controlled by aryl hydrocarbon receptor signaling and involved in extracellular matrix biogenesis.

Salmonella is the only bacterium able to enter a host cell by the two known mechanisms: trigger and zipper. The trigger mechanism relies on the injection of bacterial effectors into the host cell through the Salmonella type III secretion system 1. In the zipper mechanism, mediated by the invasins Rck and PagN, the bacterium takes advantage of a cellular receptor for invasion. This study describes the transcriptomic reprogramming of the IEC-6 intestinal epithelial cell line to Salmonella Typhimurium strains that invaded cells by a trigger, a zipper, or both mechanisms. Using S. Typhimurium strains invalidated for one or other entry mechanism, we have shown that IEC-6 cells could support both entries. Comparison of the gene expression profiles of exposed cells showed that irrespective of the mechanism used for entry, the transcriptomic reprogramming of the cell was nearly the same. On the other hand, when gene expression was compared between cells unexposed or exposed to the bacterium, the transcriptomic reprogramming of exposed cells was significantly different. It is particularly interesting to note the modulation of expression of numerous target genes of the aryl hydrocarbon receptor showing that this transcription factor was activated by S. Typhimurium infection. Numerous genes associated with the extracellular matrix were also modified. This was confirmed at the protein level by western-blotting showing a dramatic modification in some extracellular matrix proteins. Analysis of a selected set of modulated genes showed that the expression of the majority of these genes was modulated during the intracellular life of S. Typhimurium.

Inflammatory Responses Induced by the Monophasic Variant of Salmonella Typhimurium in Pigs Play a Role in the High Shedder Phenotype and Fecal Microbiota Composition.

Pigs infected with Salmonella may excrete large amounts of Salmonella, increasing the risk of spread of this pathogen in the food chain. Identifying Salmonella high shedder pigs is therefore required to mitigate this risk. We analyzed immune-associated markers and composition of the gut microbiota in specific-pathogen-free pigs presenting different shedding levels after an oral infection with Salmonella. Immune response was studied through total blood cell counts, production of anti-Salmonella antibodies and cytokines, and gene expression quantification. Total Salmonella shedding for each pig was estimated and hierarchical clustering was used to cluster pigs into high, intermediate, and low shedders. Gut microbiota compositions were assessed using 16S rRNA microbial community profiling. Comparisons were made between control and inoculated pigs, then between high and low shedders pigs. Prior to infection, high shedders had similar immunological profiles compared to low shedders. As soon as 1 day postinoculation (dpi), significant differences on the cytokine production level and on the expression level of several host genes related to a proinflammatory response were observed between high and low shedders. Infection with Salmonella induced an early and profound remodeling of the immune response in all pigs, but the intensity of the response was stronger in high shedders. In contrast, low shedders seroconverted earlier than high shedders. Just after induction of the proinflammatory response (at 2 dpi), some taxa of the fecal microbiota were specific to the shedding phenotypes. This was related to the enrichment of several functional pathways related to anaerobic respiration in high shedders. In conclusion, our data show that the immune response to Salmonella modifies the fecal microbiota and subsequently could be responsible for shedding phenotypes. Influencing the gut microbiota and reducing intestinal inflammation could be a strategy for preventing Salmonella high shedding in livestock. IMPORTANCE Salmonellosis remains the most frequent human foodborne zoonosis after campylobacteriosis and pork meat is considered one of the major sources of human foodborne infections. At the farm, host heterogeneity in pig infection is problematic. High Salmonella shedders contribute more significantly to the spread of this foodborne pathogen in the food chain. The identification of predictive biomarkers for high shedders could help to control Salmonella in pigs. The purpose of the present study was to investigate why some pigs become super shedders and others low shedders. We thus investigated the differences in the fecal microbial composition and the immune response in orally infected pigs presenting different Salmonella shedding patterns. Our data show that the proinflammatory response induced by S. Typhimurium at 1 dpi could be responsible for the modification of the fecal microbiota composition and functions observed mainly at 2 and 3 dpi and to the low and super shedder phenotypes.

Super Shedding in Enteric Pathogens: A Review.

Super shedding occurs when a small number of individuals from a given host population shed high levels of a pathogen. Beyond this general definition, various interpretations of the shedding patterns have been proposed to identify super shedders, leading to the description of the super shedding phenomenon in a wide range of pathogens, in particular enteric pathogens, which are of considerable interest. Several underlying mechanisms may explain this observation, including factors related to the environment, the gut microbiota, the pathogen itself (i.e., genetic polymorphism), and the host (including immune factors). Moreover, data suggest that the interplay of these parameters, in particular at the host-pathogen-gut microbiota interface, is of crucial importance for the determination of the super shedding phenotype in enteric pathogens. As a phenomenon playing an important role in the epidemics of enteric diseases, the evidence of super shedding has highlighted the need to develop various control strategies.

Comparative analysis of the caecal tonsil transcriptome in two chicken lines experimentally infected with Salmonella Enteritidis.

Managing Salmonella enterica Enteritidis (SE) carriage in chicken is necessary to ensure human food safety and enhance the economic, social and environmental sustainability of chicken breeding. Salmonella can contaminate poultry products, causing human foodborne disease and economic losses for farmers. Both genetic selection for a decreased carriage and gut microbiota modulation strategies could reduce Salmonella propagation in farms. Two-hundred and twenty animals from the White Leghorn inbred lines N and 61 were raised together on floor, infected by SE at 7 days of age, transferred into isolators to prevent oro-fecal recontamination and euthanized at 12 days post-infection. Caecal content DNA was used to measure individual Salmonella counts (ISC) by droplet digital PCR. A RNA sequencing approach was used to measure gene expression levels in caecal tonsils after infection of 48 chicks with low or high ISC. The analysis between lines identified 7516 differentially expressed genes (DEGs) corresponding to 62 enriched Gene Ontology (GO) Biological Processes (BP) terms. A comparison between low and high carriers allowed us to identify 97 DEGs and 23 enriched GO BP terms within line 61, and 1034 DEGs and 288 enriched GO BP terms within line N. Among these genes, we identified several candidate genes based on their putative functions, including FUT2 or MUC4, which could be involved in the control of SE infection, maybe through interactions with commensal bacteria. Altogether, we were able to identify several genes and pathways associated with differences in SE carriage level. These results are discussed in relation to individual caecal microbiota compositions, obtained for the same animals in a previous study, which may interact with host gene expression levels for the control of the caecal SE load.

Two In Vivo Models to Study Salmonella Asymptomatic Carrier State in Chicks.

In chicken, Salmonella Enteritidis and Salmonella Typhimurium, the two main serotypes isolated in human infections, can persist in the host organism for many weeks and up to many years without causing any symptoms. This persistence generally occurs after a short systemic infection that may either lead to death of very young birds or develop into cecal asymptomatic persistence, which is often accompanied by a high level of bacterial excretion, facilitating Salmonella transmission to counterparts. Here we describe two models of chick infection. The first model reproduces well the poultry infection in farm flocks. Numerous reinfections and animal-animal recontaminations occur leading to a high level of cecal colonization and fecal excretion in all chicks in the flock, over several weeks. In the second model, these animal reinfections and recontaminations are hampered leading to heterogeneity of infection characterized by the presence of low and super-shedders. This model allows for more mechanistic studies of Salmonella/chicks interactions as animal recontaminations are lowered.

Differences in caecal microbiota composition and Salmonella carriage between experimentally infected inbred lines of chickens.

BACKGROUND: Salmonella Enteritidis (SE) is one of the major causes of human foodborne intoxication resulting from consumption of contaminated poultry products. Genetic selection of animals that are more resistant to Salmonella carriage and modulation of the gut microbiota are two promising ways to decrease individual Salmonella carriage. The aims of this study were to identify the main genetic and microbial factors that control the level of Salmonella carriage in chickens (Gallus gallus) under controlled experimental conditions. Two-hundred and forty animals from the White Leghorn inbred lines N and 61 were infected by SE at 7 days of age. After infection, animals were kept in isolators to reduce recontamination of birds by Salmonella. Caecal contents were sampled at 12 days post-infection and used for DNA extraction. Microbiota DNA was used to measure individual counts of SE by digital PCR and to determine the bacterial taxonomic composition, using a 16S rRNA gene high-throughput sequencing approach. RESULTS: Our results confirmed that the N line is more resistant to Salmonella carriage than the 61 line, and that intra-line variability is higher for the 61 line. Furthermore, the 16S analysis showed strong significant differences in microbiota taxonomic composition between the two lines. Among the 617 operational taxonomic units (OTU) observed, more than 390 were differentially abundant between the two lines. Furthermore, within the 61 line, we found a difference in the microbiota taxonomic composition between the high and low Salmonella carriers, with 39 differentially abundant OTU. Using metagenome functional prediction based on 16S data, several metabolic pathways that are potentially associated to microbiota taxonomic differences (e.g. short chain fatty acids pathways) were identified between high and low carriers. CONCLUSIONS: Overall, our findings demonstrate that the caecal microbiota composition differs between genetic lines of chickens. This could be one of the reasons why the investigated lines differed in Salmonella carriage levels under experimental infection conditions.

Microbiota and stress: a loop that impacts memory.

Chronic stress and the gut microbiota appear to comprise a feed-forward loop, which contributes to the development of depressive disorders. Evidence suggests that memory can also be impaired by either chronic stress or microbiota imbalance. However, it remains to be established whether these could be a part of an integrated loop model and be responsible for memory impairments. To shed light on this, we used a two-pronged approach in Japanese quail: first stress-induced alterations in gut microbiota were characterized, then we tested whether this altered microbiota could affect brain and memory function when transferred to a germ-free host. The cecal microbiota of chronically stressed quails was found to be significantly different from that of unstressed individuals with lower α and ß diversities and increased Bacteroidetes abundance largely represented by the Alistipes genus, a well-known stress target in rodents and humans. The transfer of this altered microbiota into germ-free quails decreased their spatial and cue-based memory abilities as previously demonstrated in the stressed donors. The recipients also displayed increased anxiety-like behavior, reduced basal plasma corticosterone levels and differential gene expression in the brain. Furthermore, cecal microbiota transfer from a chronically stressed individual was sufficient to mimic the adverse impact of chronic stress on memory in recipient hosts and this action may be related to the Alistipes genus. Our results provide evidence of a feed-forward loop system linking the microbiota-gut-brain axis to stress and memory function and suggest that maintaining a healthy microbiota could help alleviate memory impairments linked to chronic stress.

The influences of microbial colonisation and germ-free status on the chicken TCRß repertoire.

Microbial colonisation is paramount to the normal development of the immune system, particularly at mucosal sites. However, the relationships between the microbiome and the adaptive immune repertoire have mostly been explored in rodents and humans. Here, we report a high-throughput sequencing analysis of the chicken TCRß repertoire and the influences of microbial colonisation on tissue-resident TCRß+ cells. The results reveal that the microbiome is an important driver of TCRß diversity in both intestinal tissues and the bursa of Fabricius, but not in the spleen. Of note, public TCRß sequences (shared across individuals) make a substantial contribution to the repertoire. Additionally, different tissues exhibit biases in terms of their V family and J gene usage, and these effects were influenced by the gut-associated microbiome. TCRß clonal expansions were identified in both colonised and germ-free birds, but differences between the groups were indicative of an influence of the microbiota. Together, these findings provide an insight into the avian adaptive immune system and the influence of the microbiota on the TCRß repertoire.

Investigation of the invasion mechanism mediated by the outer membrane protein PagN of Salmonella Typhimurium.

BACKGROUND: Salmonella can invade host cells via a type three secretion system called T3SS-1 and its outer membrane proteins, PagN and Rck. However, the mechanism of PagN-dependent invasion pathway used by Salmonella enterica, subspecies enterica serovar Typhimurium remains unclear. RESULTS: Here, we report that PagN is well conserved and widely distributed among the different species and subspecies of Salmonella. We showed that PagN of S. Typhimurium was sufficient and necessary to enable non-invasive E. coli over-expressing PagN and PagN-coated beads to bind to and invade different non-phagocytic cells. According to the literature, PagN is likely to interact with heparan sulfate proteoglycan (HSPG) as PagN-mediated invasion could be inhibited by heparin treatment in a dose-dependent manner. This report shows that this interaction is not sufficient to allow the internalization mechanism. Investigation of the role of ß1 integrin as co-receptor showed that mouse embryo fibroblasts genetically deficient in ß1 integrin were less permissive to PagN-mediated internalization. Moreover, PagN-mediated internalization was fully inhibited in glycosylation-deficient pgsA-745 cells treated with anti-ß1 integrin antibody, supporting the hypothesis that ß1 integrin and HSPG cooperate to induce the PagN-mediated internalization mechanism. In addition, use of specific inhibitors and expression of dominant-negative derivatives demonstrated that tyrosine phosphorylation and class I phosphatidylinositol 3-kinase were crucial to trigger PagN-dependent internalization, as for the Rck internalization mechanism. Finally, scanning electron microscopy with infected cells showed microvillus-like extensions characteristic of Zipper-like structure, engulfing PagN-coated beads and E. coli expressing PagN, as observed during Rck-mediated internalization. CONCLUSIONS: Our results supply new comprehensions into T3SS-1-independent invasion mechanisms of S. Typhimurium and highly indicate that PagN induces a phosphatidylinositol 3-kinase signaling pathway, leading to a Zipper-like entry mechanism as the Salmonella outer membrane protein Rck.

Early-Life Immune System Maturation in Chickens Using a Synthetic Community of Cultured Gut Bacteria.

The gut microbiome is crucial for both maturation of the immune system and colonization resistance against enteric pathogens. Although chicken are important domesticated animals, the impact of their gut microbiome on the immune system is understudied. Therefore, we investigated the effect of microbiome-based interventions on host mucosal immune responses. Increased levels of IgA and IgY were observed in chickens exposed to maternal feces after hatching compared with strict hygienic conditions. This was accompanied by increased gut bacterial diversity as assessed by 16S rRNA gene amplicon sequencing. Cultivation work allowed the establishment of a collection of 43 bacterial species spanning 4 phyla and 19 families, including the first cultured members of 3 novel genera and 4 novel species that were taxonomically described. This resource is available at www.dsmz.de/chibac A synthetic community consisting of nine phylogenetically diverse and dominant species from this collection was designed and found to be moderately efficient in boosting immunoglobulin levels when provided to chickens early in life.IMPORTANCE The immune system plays a crucial role in sustaining animal health. Its development is markedly influenced by early microbial colonization of the gastrointestinal tract. As chicken are fully dependent on environmental microbes after hatching, extensive hygienic measures in production facilities are detrimental to the microbiota, resulting in low colonization resistance against pathogens. To combat enteric infections, antibiotics are frequently used, which aggravates the issue by altering gut microbiota colonization. Intervention strategies based on cultured gut bacteria are proposed to influence immune responses in chicken.

Rck of Salmonella Typhimurium Delays the Host Cell Cycle to Facilitate Bacterial Invasion.

Salmonella Typhimurium expresses on its outer membrane the protein Rck which interacts with the epidermal growth factor receptor (EGFR) of the plasma membrane of the targeted host cells. This interaction activates signaling pathways, leading to the internalization of Salmonella. Since EGFR plays a key role in cell proliferation, we sought to determine the influence of Rck mediated infection on the host cell cycle. By analyzing the DNA content of uninfected and infected cells using flow cytometry, we showed that the Rck-mediated infection induced a delay in the S-phase (DNA replication phase) of the host cell cycle, independently of bacterial internalization. We also established that this Rck-dependent delay in cell cycle progression was accompanied by an increased level of host DNA double strand breaks and activation of the DNA damage response. Finally, we demonstrated that the S-phase environment facilitated Rck-mediated bacterial internalization. Consequently, our results suggest that Rck can be considered as a cyclomodulin with a genotoxic activity.

Gut microbiota composition before infection determines the Salmonella super- and low-shedder phenotypes in chicken.

Heterogeneity of infection and extreme shedding patterns are common features of animal infectious diseases. Individual hosts that are super-shedders are key targets for control strategies. Nevertheless, the mechanisms associated with the emergence of super-shedders remain largely unknown. During chicken salmonellosis, a high heterogeneity of infection is observed when animal-to-animal cross-contaminations and reinfections are reduced. We hypothesized that unlike super-shedders, low-shedders would be able to block the first Salmonella colonization thanks to a different gut microbiota. The present study demonstrates that (i) axenic and antibiotic-treated chicks are more prone to become super-shedders; (ii) super or low-shedder phenotypes can be acquired through microbiota transfer; (iii) specific gut microbiota taxonomic features determine whether the chicks develop a low- and super-shedder phenotype after Salmonella infection in isolator; (iv) partial protection can be conferred by inoculation of four commensal bacteria prior to Salmonella infection. This study demonstrates the key role plays by gut microbiota composition in the heterogeneity of infection and pave the way for developing predictive biomarkers and protective probiotics.

IPEC-1 variable immune response to different serovars of Salmonella enterica subsp. enterica.

Salmonella is a genus of Gram-negative bacteria in the Enterobacteriaceae family causing various illnesses. The ability of the different serovars of Salmonella enterica subsp. enterica to infect a host and to induce pathology relies in part on their cellular and molecular interactions with the intestinal epithelium. In the current study, an in vitro approach using non-polarized or polarized IPEC-1 porcine intestinal epithelial cells were used in order to assess the relation between adhesion, invasion, and induction of the immune response as a function of the serotype of Salmonella. Five serovars, Choleraesuis (host-adapted), Typhimurium (ubiquitous), Typhisuis (host-restricted), which are relevant for pig infection, and Dublin and Gallinarum, which are host-restricted or host-adapted, were studied. A strong variation was observed in the percentages of adhesion and invasion amongst the S. enterica serovars used to interact with the non-polarized and polarized cells. Subsequently, differences were identified between serovars in terms of immune response induced. Serovars Typhimurium and Typhisuis induced a strong innate immune response four and half hours after the beginning of cell stimulation while Choleraesuis, Gallinarum, and Dublin did not. A strong inflammatory response could limit the spread of the porcine serovars to the gut while, with a weak response, bacteria may not be constrained by the immune response enabling severe systemic diseases. Different repertoires of adhesion factors and of secreted protein effectors between Salmonella serovars interacting with IPEC-1 cells probably explains the differences in their early pathogenic behaviours.

H-NS is the major repressor of Salmonella Typhimurium Pef fimbriae expression.

Fimbriae play an important role in adhesion and are therefore essential for the interaction of bacteria with the environments they encounter. Most of them are expressed in vivo but not in vitro, thus making difficult the full characterization of these fimbriae. Here, we characterized the silencing of plasmid-encoded fimbriae (Pef) expression, encoded by the pef operon, in the worldwide pathogen Salmonella Typhimurium. We demonstrated that the nucleoid-associated proteins H-NS and Hha, and their respective paralogs StpA and YdgT, negatively regulate at pH 5.1 and pH 7.1 the transcription of the pef operon. Two promoters, PpefB and PpefA, direct the transcription of this operon. All the nucleoid-associated proteins silence the PpefB promoter and H-NS also targets the PpefA promoter. While Hha and YdgT are mainly considered as acting primarily through H-NS to modulate gene transcription, our results strongly suggest that Hha and YdgT silence pef transcription at acidic pH either by interacting with StpA or independently of H-NS and StpA. We also confirmed the previously described post-transcriptional repression of Pef fimbriae by CsrA titration via the fim mRNA and CsrB and CsrC sRNA. Finally, among all these regulators, H-NS clearly appeared as the major repressor of Pef expression. These results open new avenues of research to better characterize the regulation of these bacterial adhesive proteins and to clarify their role in the virulence of pathogens.

Influence of the microbiota-gut-brain axis on behavior and welfare in farm animals: A review.

There is increasing evidence of a pivotal role of the gut microbiota (GUT-M) in key physiological functions in vertebrates. Many studies discuss functional implications of the GUT-M not only on immunity, growth, metabolism, but also on brain development and behavior. However, while the influence of the microbiota-gut-brain axis (MGBA) on behavior is documented in rodents and humans, data on farm animals are scarce. This review will first report the well-known influence of the MGBA on behavior in rodent and human and then describe its influence on emotion, memory, social and feeding behaviors in farm animals. This corpus of experiments suggests that a better understanding of the effects of the MGBA on behavior could have large implications in various fields of animal production. Specifically, animal welfare and health could be improved by selection, nutrition and management processes that take into account the role of the GUT-M in behavior.

Effects of gut microbiota transfer on emotional reactivity in Japanese quails (Coturnix japonica).

The interaction between the gut microbiota (GM) and the brain has led to the concept of the microbiota-gut-brain axis but data for birds remain scarce. We tested the hypothesis that colonization of germ-free chicks from a quail line selected for a high emotional reactivity (E+) with GM from a line with low emotional reactivity (E-) would reduce their emotional behaviour in comparison with germ-free chicks from an E+ line colonized with GM from the same E+ line. The GM composition analysis of both groups revealed a shift in terms of microbial diversity and richness between day 21 and day 35 and the GM of the two groups of quails were closer to each other at day 35 than at day 21 at a phylum level. Quails that received GM from the E- line expressed a lower emotional reactivity than quails colonized by GM from the E+ line in tonic immobility and novel environment tests carried out during the second week of age. This result was reversed in a second tonic immobility test and an open-field run 2 weeks later. These behavioural and GM modifications over time could be the consequence of the resilience of the GM to recover the equilibrium present in the E+ host, which is in part driven by the host genotype. This study shows for the first time that a GM transfer can influence emotional reactivity in Japanese quails, supporting the existence of a microbiota-gut-brain axis in this species of bird.