Super- and low-shedding phenomena have been observed in genetically homogeneous hosts infected by a single bacterial strain. To decipher the mechanisms underlying these phenotypes, we conducted an experiment with chicks infected with Salmonella Enteritidis in a non-sterile isolator, which prevents bacterial transmission between animals while allowing the development of the gut microbiota. We investigated the impact of four commensal bacteria called Mix4, inoculated at hatching, on chicken systemic immune response and intestinal microbiota composition and functions, before and after Salmonella infection. Our results revealed that these phenotypes were not linked to changes in cell invasion capacity of bacteria during infection. Mix4 inoculation had both short- and long-term effects on immune response and microbiota and promoted the low-shedder phenotype. Kinetic analysis revealed that Mix4 activated immune response from day 4, which modified the microbiota on day 6. This change promotes a more fermentative microbiota, using the aromatic compounds degradation pathway, which inhibited Salmonella colonization by day 11 and beyond. In contrast, control animals exhibited a delayed TNF-driven pro-inflammatory response and developed a microbiota using anaerobic respiration, which facilitates Salmonella colonization and growth. This strategy offers promising opportunities to strengthen the barrier effect against Salmonella and possibly other pathogens.

• Keywords
• Salmonella, carrier-state, chicken, excretion, immune response, microbiota, super-shedder, virulence,
• MeSH
• Bacteria * immunology   classification   genetics   MeSH
• Chickens immunology   microbiology   MeSH
• Poultry Diseases * microbiology   immunology   prevention & control   MeSH
• Salmonella enteritidis * immunology   growth & development   physiology   MeSH
• Salmonella Infections, Animal * immunology   microbiology   prevention & control   MeSH
• Gastrointestinal Microbiome * immunology   MeSH
• Symbiosis MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Chickens represent one of the most important sources of animal protein for the human population. However, chickens also represent one of the most important reservoirs of Salmonella for humans. Measures to decrease the Salmonella incidence in chickens are therefore continuously sought. In this study, we tested feed supplementation with a mixture of C1 to C12 monoacylglycerides. At 0.7 and 1.5 kg per ton of feed, such supplementation significantly decreased Salmonella counts in the caecum but not in the liver. The chickens were infected on day 4 and the protective effect in the caecum was recorded on day 22 and 23 of life. Supplementation also decreased the inflammatory response of chickens to Salmonella infection determined by avidin, SAA, ExFABP, MMP7, IL1β, IL4I and MRP126 gene expression but did not affect immunoglobulin expression in the caecum. C1 to C12 monoacylglycerides can be used as a feed supplement which, if continuously provided in feed, decrease Salmonella counts in chickens just prior slaughter.

• Keywords
• Caecum, Chicken, Monoacylglyceride, Salmonella,
• MeSH
• Cecum microbiology   MeSH
• Diet veterinary   MeSH
• Animal Feed analysis   MeSH
• Chickens * MeSH
• Monoglycerides * administration & dosage   metabolism   MeSH
• Random Allocation MeSH
• Poultry Diseases * immunology   microbiology   prevention & control   MeSH
• Disease Resistance * drug effects   MeSH
• Dietary Supplements analysis   MeSH
• Salmonella enteritidis * physiology   MeSH
• Salmonella Infections, Animal * immunology   microbiology   prevention & control   MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Monoglycerides * MeSH

The concept of competitive exclusion is well established in poultry and different products are used to suppress the multiplication of enteric pathogens in the chicken intestinal tract. While the effect has been repeatedly confirmed, the specific principles of competitive exclusion are less clear. The aim of the study was to compare metabolites in the cecal digesta of differently colonized chickens. Metabolites in the cecal contents of chickens treated with a commercial competitive exclusion product or with an experimental product consisting of 23 gut anaerobes or in control untreated chickens were determined by mass spectrometry. Extensive differences in metabolite composition among the digesta of all 3 groups of chickens were recorded. Out of 1,706 detected compounds, 495 and 279 were differently abundant in the chicks treated with a commercial or experimental competitive exclusion product in comparison to the control group, respectively. Soyasaponins, betaine, carnitine, glutamate, tyramine, phenylacetaldehyde, or 3-methyladenine were more abundant in the digesta of control chicks while 4-oxododecanedioic acid, nucleotides, dipeptides, amino acids (except for glutamate), and vitamins were enriched in the digesta of chickens colonized by competitive exclusion products. Metabolites enriched in the digesta of control chicks can be classified as of plant feed origin released in the digesta by degradative activities of the chicken. Some of these molecules disappeared from the digesta of chicks colonized by complex microbiota due to them being metabolized. Instead, nucleotides, amino acids, and vitamins increased in the digesta of colonized chicks as a consequence of the additional digestive potential brought to the cecum by microbiota from competitive exclusion products. It is therefore possible to affect metabolite profiles in the chicken cecum by its colonization with selected bacterial species.

• Keywords
• cecum, chicken, competitive exclusion, metabolome, microbiota,
• MeSH
• Cecum microbiology   MeSH
• Chickens * microbiology   MeSH
• Glutamic Acid MeSH
• Poultry Diseases * microbiology   MeSH
• Nucleotides MeSH
• Vitamin K MeSH
• Vitamins MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glutamic Acid MeSH
• Nucleotides MeSH
• Vitamin K MeSH
• Vitamins MeSH

An experimental group of one-day-old chicken from a commercial hatchery was given a defined mixture of 7 gut anaerobes. The next day the chicks were inoculated by an APEC strain O78:H4-ST117 resistant to ciprofloxacin, alongside with the control group and monitored for 4 wk after the inoculation for the presence of the colonizing strains and ciprofloxacin-resistant E. coli. Significant reduction of colonization rates in the first 2 wk was recorded in the experimental group for the numbers of ciprofloxacin-resistant E. coli. The results show that colonization of chicken by defined anaerobic mixtures may provide a decisive protection during the critical period of the chicken intestinal microflora development.

• Keywords
• avian pathogenic Escherichia coli (APEC), chicken, colonization, competitive exclusion, probiotics,
• MeSH
• Bacteroides MeSH
• Ciprofloxacin pharmacology   MeSH
• Escherichia coli MeSH
• Escherichia coli Infections * prevention & control   veterinary   MeSH
• Chickens MeSH
• Poultry Diseases * prevention & control   MeSH
• Probiotics * pharmacology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ciprofloxacin MeSH

In attempt to identify genes that are induced in chickens by Salmonella Enteritidis we identified a new highly inducible gene, interleukin 4 induced 1 gene (IL4I1). IL4I1 reached its peak expression (458× induction) in the cecum of newly hatched chickens 4 days post-infection and remained upregulated for an additional 10 days. IL4I1 was expressed and induced in macrophages and granulocytes, both at the mRNA and protein level. IL4I1 was expressed and induced also in CD4 and γδ T-lymphocytes though at a 50-fold lower level than in phagocytes. Expression of IL4I1 was not detected in CD8 T lymphocytes or B lymphocytes. Mutation of IL4I1 in chicken HD11 macrophages did not affect their bactericidal capacity against S. Enteritidis but negatively affected their oxidative burst after PMA stimulation. We therefore propose that IL4I1 is not directly involved in bactericidal activity of phagocytes and, instead, it is likely involved in the control of inflammatory response and signaling to T and B lymphocytes.

• MeSH
• Cecum immunology   MeSH
• Phagocytes immunology   MeSH
• Chickens * MeSH
• Leukocytes immunology   MeSH
• Poultry Diseases immunology   MeSH
• L-Amino Acid Oxidase metabolism   MeSH
• Avian Proteins metabolism   MeSH
• Salmonella enteritidis physiology   MeSH
• Salmonella Infections, Animal immunology   MeSH
• Spleen immunology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• L-Amino Acid Oxidase MeSH
• Avian Proteins MeSH

Gut microbiota is of considerable importance for each host. Despite this, germ-free animals can be obtained and raised to sexual maturity and consequences of the presence or absence of gut microbiota on gene expression of the host remain uncharacterised. In this study, we performed an unbiased study of protein expression in the caecum of germ-free and colonised chickens. The major difference between these two groups was in the expression of immunoglobulins which were essentially absent in the germ-free chickens. Microbiota also caused a minor decrease in the expression of focal adhesion and extracellular matrix proteins and an increase in the expression of argininosuccinate synthase ASS1, redox potential sensing, fermentative metabolic processes and detoxification systems represented by sulfotransferases SULT1C3 or SULT1E1. Since we also analysed expression in the caecum of E. coli Nissle and E. faecium DSM7134 mono-associated chickens, we concluded that at least immunoglobulin expression and expression of cystathionine synthase (CBS) was dependent on microbiota composition with E. coli Nissle stimulating more immunoglobulin and PIGR expression and E. faecium DSM7134 stimulating more CBS expression. Gut microbiota and its composition therefore affected protein expression in the chicken caecum though except for immunoglobulin production, the remaining differences were unexpectedly low.

• MeSH
• Cecum metabolism   microbiology   MeSH
• Enterococcus faecium physiology   MeSH
• Escherichia coli physiology   MeSH
• Gene Expression * MeSH
• Germ-Free Life MeSH
• Chickens genetics   microbiology   MeSH
• Avian Proteins metabolism   MeSH
• Gastrointestinal Microbiome physiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Avian Proteins MeSH

In this study we determined protein and gene expression in the caeca of newly hatched chickens inoculated with cecal contents sourced from hens of different ages. Over 250 proteins exhibited modified expression levels in response to microbiota inoculation. The most significant inductions were observed for ISG12-2, OASL, ES1, LYG2, DMBT1-L, CDD, ANGPTL6, B2M, CUZD1, IgM and Ig lambda chain. Of these, ISG12-2, ES1 and both immunoglobulins were expressed at lower levels in germ-free chickens compared to conventional chickens. In contrast, CELA2A, BRT-2, ALDH1A1, ADH1C, AKR1B1L, HEXB, ALDH2, ALDOB, CALB1 and TTR were expressed at lower levels following inoculation of microbiota. When chicks were given microbiota preparations from different age donors, the recipients mounted differential responses to the inoculation which also differed from the response profile in naturally colonised birds. For example, B2M, CUZD1 and CELA2A responded differently to the inoculation with microbiota of 4- or 40-week-old hens. The increased or decreased gene expression could be recorded 6 weeks after the inoculation of newly hatched chickens. To characterise the proteins that may directly interact with the microbiota we characterised chicken proteins that co-purified with the microbiota and identified a range of host proteins including CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda. We propose that induction of ISG12-2 results in reduced apoptosis of host cells exposed to the colonizing commensal microbiota and that CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda reduce contact of luminal microbiota with the gut epithelium thereby reducing the inflammatory response.

• Publication type
• Journal Article MeSH

Since poultry is a very common source of non-typhoid Salmonella for humans, different interventions aimed at decreasing the prevalence of Salmonella in chickens are understood as an effective measure for decreasing the incidence of human salmonellosis. One such intervention is the use of probiotic or competitive exclusion products. In this study we tested whether microbiota from donor hens of different age will equally protect chickens against Salmonella Enteritidis infection. Newly hatched chickens were therefore orally inoculated with cecal extracts from 1-, 3-, 16-, 28-, and 42-week-old donors and 7 days later, the chickens were infected with S. Enteritidis. The experiment was terminated 4 days later. In the second experiment, groups of newly hatched chickens were inoculated with cecal extracts of 35-week-old hens either on day 1 of life followed by S. Enteritidis infection on day 2 or were infected with S. Enteritidis infection on day 1 followed by therapeutic administration of the cecal extract on day 2 or were inoculated on day 1 of life with a mixture of the cecal extract and S. Enteritidis. This experiment was terminated when the chickens were 5 days old. Both Salmonella culture and chicken gene expression confirmed that inoculation of newly hatched chickens with microbiota from 3-week-old or older chickens protected them against S. Enteritidis challenge. On the other hand, microbiota from 1-week-old donors failed to protect chickens against S. Enteritidis challenge. Microbiota from 35-week-old hens protected chickens even 24 h after administration. However, simultaneous or therapeutic microbiota administration failed to protect chickens against S. Enteritidis infection. Gut microbiota can be used as a preventive measure against S. Enteritidis infection but its composition and early administration is critical for its efficacy.

• Keywords
• Salmonella Enteritidis, cecum, chicken, competitive exclusion, inflammation, microbiota,
• Publication type
• Journal Article MeSH

The response of chicken to non-typhoidal Salmonella infection is becoming well characterised but the role of particular cell types in this response is still far from being understood. Therefore, in this study we characterised the response of chicken embryo fibroblasts (CEFs) to infection with two different S. Enteritidis strains by microarray analysis. The expression of chicken genes identified as significantly up- or down-regulated (≥3-fold) by microarray analysis was verified by real-time PCR followed by functional classification of the genes and prediction of interactions between the proteins using Gene Ontology and STRING Database. Finally the expression of the newly identified genes was tested in HD11 macrophages and in vivo in chickens. Altogether 19 genes were induced in CEFs after S. Enteritidis infection. Twelve of them were also induced in HD11 macrophages and thirteen in the caecum of orally infected chickens. The majority of these genes were assigned different functions in the immune response, however five of them (LOC101750351, K123, BU460569, MOBKL2C and G0S2) have not been associated with the response of chicken to Salmonella infection so far. K123 and G0S2 were the only 'non-immune' genes inducible by S. Enteritidis in fibroblasts, HD11 macrophages and in the caecum after oral infection. The function of K123 is unknown but G0S2 is involved in lipid metabolism and in β-oxidation of fatty acids in mitochondria.

• MeSH
• Down-Regulation MeSH
• Fibroblasts cytology   metabolism   microbiology   MeSH
• Cells, Cultured MeSH
• Chickens genetics   metabolism   MeSH
• Chick Embryo MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Macrophages metabolism   microbiology   MeSH
• Poultry Diseases metabolism   pathology   MeSH
• Salmonella enteritidis physiology   MeSH
• Salmonella Infections, Animal metabolism   physiopathology   MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Transcriptome * MeSH
• Up-Regulation MeSH
• Animals MeSH
• Check Tag
• Chick Embryo MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Chickens can be infected with Salmonella enterica at any time during their life. However, infections within the first hours and days of their life are epidemiologically the most important, as newly hatched chickens are highly sensitive to Salmonella infection. Salmonella is initially recognized in the chicken caecum by TLR receptors and this recognition is followed by induction of chemokines, cytokines and many effector genes. This results in infiltration of heterophils, macrophages, B- and T-lymphocytes and changes in total gene expression in the caecal lamina propria. The highest induction in expression is observed for matrix metalloproteinase 7 (MMP7). Expression of this gene is increased in the chicken caecum over 4000 fold during the first 10 days after the infection of newly hatched chickens. Additional highly inducible genes in the caecum following S. Enteritidis infection include immune responsive gene 1 (IRG1), serum amyloid A (SAA), extracellular fatty acid binding protein (ExFABP), serine protease inhibitor (SERPINB10), trappin 6-like (TRAP6), calprotectin (MRP126), mitochondrial ES1 protein homolog (ES1), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), avidin (AVD) and transglutaminase 4 (TGM4). The induction of expression of these proteins exceeds a factor of 50. Similar induction rates are also observed for chemokines and cytokines such as IL1β, IL6, IL8, IL17, IL18, IL22, IFNγ, AH221 or iNOS. Once the infection is under control, which happens approx. 2 weeks after infection, expression of IgY and IgA increases to facilitate Salmonella elimination from the gut lumen. This review outlines the function of individual proteins expressed in chickens after infection with non-typhoid Salmonella serovars.

• MeSH
• Cecum metabolism   MeSH
• Gene Expression * MeSH
• Chickens * MeSH
• Poultry Diseases genetics   microbiology   MeSH
• Avian Proteins genetics   metabolism   MeSH
• Salmonella physiology   MeSH
• Salmonella Infections, Animal genetics   immunology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Avian Proteins MeSH