Germ-free Chickens Research Articles

The gut microbiota and its metabolite butyrate shape metabolism and antiviral immunity along the gut-lung axis in the chicken

The gut microbiota exerts profound influence on poultry immunity and metabolism through mechanisms that yet need to be elucidated. Here we used conventional and germ-free chickens to explore the influence of the gut microbiota on transcriptomic and metabolic signatures along the gut-lung axis in poultry. Our results demonstrated a differential regulation of certain metabolites and genes associated with innate immunity and metabolism in peripheral tissues of germ-free birds. Furthermore, we evidenced the gut microbiota’s capacity to regulate mucosal immunity in the chicken lung during avian influenza virus infection. Finally, by fine-analysing the antiviral pathways triggered by the short-chain fatty acid (SCFA) butyrate in chicken respiratory epithelial cells, we found that it regulates interferon-stimulated genes (ISGs), notably OASL, via the transcription factor Sp1. These findings emphasize the pivotal role of the gut microbiota and its metabolites in shaping homeostasis and immunity in poultry, offering crucial insights into the mechanisms governing the communication between the gut and lungs in birds.

Multiomics analysis reveals that microbiota regulate fat and muscle synthesis in chickens

Intestinal microbiota regulates the host metabolism, including fat metabolism and muscle development in mammals; however, studies on the interactions between the gut microbiome and in chickens with respect to fat metabolism and muscle development are still rare. We established a germ-free (GF) chicken model to determine the transcriptomes and metabolomes of GF and specific-pathogen-free (SPF) chickens. Transcriptome analysis showed 1282 differentially expressed genes (DEGs) in GF and SPF chickens. The expression levels of some genes related to muscle formation were very high in SPF chickens but low in GF chickens, suggesting that GF chickens had poorer muscle development ability. In contrast, the expression levels of some fat synthesis-related genes were very low in SPF chickens but high in GF chickens, suggesting that GF chickens had a more potent fat-synthesizing ability. Metabolome analysis revealed 62 differentially expressed metabolites (DEMs) in GF and SPF chickens, of which 35 were upregulated and 27 were downregulated. Furthermore, the Pearson correlation coefficient (PCC) was calculated, and an interaction network was constructed to visualize the crosstalk between the genes, metabolites, and gut microbiota in GF and SPF chickens. The top 10 gut microbiota were positively correlated with lipid metabolism including13(S)-HpODE and 9(S)-HpOTrE, and genes related to muscle development, while were negatively correlated with genes related to fat synthesis. In conclusion, this study indicated that chicken intestinal microbiota regulate host metabolism, inhibit fat synthesis, and may promote muscle development.

The caecal microbiota promotes the acute inflammatory response and the loss of the intestinal barrier integrity during severe Eimeria tenella infection.

Coccidiosis, a disease caused by intestinal apicomplexan parasites Eimeria, is a threat to poultry production. Eimeria tenella is one of the most pathogenic species, frequently causing a high prevalence of opportunistic infections. The objective of this study is to investigate the role of the microbiota in the pathogenesis of severe Eimeria tenella infection. We have previously shown that microbiota can promote parasite development. To study the effect of the microbiota on the pathogenesis of this infection, we used an experimental condition (inoculum of 10 000 oocysts E. tenella INRAE) in which the parasite load is similar between germ-free and conventional broilers at 7 days post-infection (pi). Thirteen conventional and 24 germ-free chickens were infected. Among this latter group, 12 remained germ-free and 12 received a microbiota from conventional healthy chickens at 4 days pi. Caeca and spleens were collected at 7 days pi. Our results demonstrated caecal lesions and epithelium damage in conventional chickens at 7 days pi but not in germ-free infected chickens. Administration of conventional microbiota to germ-free chickens partially restored these deleterious effects. At day 7 pi, both infected conventional and germ-free chickens exhibited increased gene expression of inflammatory mediators, including IL15, IFNγ, TNFα and the anti-inflammatory mediator SOCS1, whereas the inflammatory mediators CXCLi2, CCL20, IL18, CSF1, NOS2, PTGS2, IL1β, IL6, the receptor CCR2, and the anti-inflammatory mediators TGFβ1 and IL10 were upregulated only in infected conventional chickens. Notably, the IL18, PTGS2 gene expression was significantly higher in the infected conventional group. Overall, the inflammatory response enhanced by the microbiota might be in part responsible for higher lesion scores. Epithelial tight junction protein gene expression analysis revealed a significant upregulation of CLDN1 with the infection and microbiota, indicating a potential loss of the intestinal barrier integrity. These observations imply that, during E. tenella infection, the caecal microbiota could trigger an acute inflammatory response, resulting in a loss of intestinal integrity. Increase in bacterial translocation can then lead to the likelihood of opportunistic infections. Hence, modulating the microbiota may offer a promising strategy for improving poultry gut health and limiting caecal coccidiosis.

The Absence of Gut Microbiota Alters the Development of the Apicomplexan Parasite Eimeria tenella

Coccidiosis is a widespread intestinal disease of poultry caused by a parasite of the genus Eimeria. Eimeria tenella, is one of the most virulent species that specifically colonizes the caeca, an organ which harbors a rich and complex microbiota. Our objective was to study the impact of the intestinal microbiota on parasite infection and development using an original model of germ-free broilers. We observed that germ-free chickens presented significantly much lower load of oocysts in caecal contents than conventional chickens. This decrease in parasite load was measurable in caecal tissue by RT-qPCR at early time points. Histological analysis revealed the presence of much less first (day 2pi) and second generation schizonts (day 3.5pi) in germ-free chickens than conventional chickens. Indeed, at day 3.5pi, second generation schizonts were respectively immature only in germ-free chickens suggesting a lengthening of the asexual phase of the parasite in the absence of microbiota. Accordingly to the consequence of this lengthening, a delay in specific gamete gene expressions, and a reduction of gamete detection by histological analysis in caeca of germ-free chickens were observed. These differences in parasite load might result from an initial reduction of the excystation efficiency of the parasite in the gut of germ-free chickens. However, as bile salts involved in the excystation step led to an even higher excystation efficiency in germ-free compared to conventional chickens, this result could not explain the difference in parasite load. Interestingly, when we shunted the excystation step in vivo by infecting chickens with sporozoites using the cloacal route of inoculation, parasite invasion was similar in germ-free and in conventional chickens but still resulted in significantly lower parasite load in germ-free chickens at day 7pi. Overall, these data highlighted that the absence of intestinal microbiota alters E. tenella replication. Strategies to modulate the microbiota and/or its metabolites could therefore be an alternative approach to limit the negative impact of coccidiosis in poultry.

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies.

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, which are considered the gold standard. Nesting birds are ideal models for the production of germ-free animals since there is no need to raise their relatives under sterile conditions. Germ-free chickens are mainly generated from specific-pathogen-free (SPF) experimental lines, which are poorly representative of commercial chicken lines.The method proposed here allowed the production of germ-free chickens from the fast growing broiler line Ross PM3, commonly used by the poultry industry.Eggs were quickly collected after laying at a broiler breeder farm.They underwent a strict decontamination process from the collection to the introduction in a sterile egg hatching isolator.The chicks have been hatched and kept in these sterile isolators during the period necessary to control their sterility. Originally developed for an experimental SPF white leghorn line, the present protocol has been adapted not only to the Ross PM3 broiler line but also to quails. It therefore represents a robust and readily adaptable procedure to other poultry species and nesting birds of economic, biological or ecological relevance.

Gut Microbial Dynamics during Conventionalization of Germfree Chicken.

A gnotobiotic Gallus gallus (chicken) model was developed to study the dynamics of intestinal microflora from hatching to 18 days of age employing metagenomics. Intestinal samples were collected from a local population of feral chickens and administered orally to germfree 3-day-old chicks. Animals were euthanized on days 9 and 18 postinoculation, and intestinal samples were collected and subjected to metagenomic analysis. On day 18, the five most prevalent phyla were Bacteroidetes (43.03 ± 3.19%), Firmicutes (38.51 ± 2.67%), Actinobacteria (6.77 ± 0.7%), Proteobacteria (6.38 ± 0.7%), and Spirochaetes (2.71 ± 0.55%). Principal-coordinate analysis showed that the day 18 variables clustered more closely than the day 9 variables, suggesting that the microbial communities had changed temporally. The Morista-Horn index values ranged from 0.7 to 1, indicating that the communities in the inoculum and in the day 9 and day 18 samples were more similar than dissimilar. The predicted functional profiles of the microbiomes of the inoculum and the day 9 and day 18 samples were also similar (values of 0.98 to 1). These results indicate that the gnotobiotic chicks stably maintained the phylogenetic diversity and predicted metabolic functionality of the inoculum community.IMPORTANCE The domestic chicken is the cornerstone of animal agriculture worldwide, with a flock population exceeding 40 billion birds/year. It serves as an economically valuable source of protein globally. The microbiome of poultry has important effects on chicken growth, feed conversion, immune status, and pathogen resistance. The aim of our research was to develop a gnotobiotic chicken model appropriate for the study chicken gut microbiota function. Our experimental model shows that young germfree chicks are able to colonize diverse sets of gut bacteria. Therefore, besides the use of this model to study mechanisms of gut microbiota interactions in the chicken gut, it could be also used for applied aspects such as determining the safety and efficacy of new probiotic strains derived from chicken gut microbiota.

Gene expression in the chicken caecum is dependent on microbiota composition

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.

Transient and Prolonged Response of Chicken Cecum Mucosa to Colonization with Different Gut Microbiota

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.

Differences in intestinal mucin dynamics between germ-free and conventionally reared chickens after mannan-oligosaccharide supplementation

A germ-free (GF) chicken model was used to test 2 hypotheses: 1. microbial colonization of the gastrointestinal tract (GIT) influences mucin gene expression and mucin types; and 2. mannan oligosaccharide (MOS) supplementation affects GIT cells directly, without bacteria mediation, compared with bacterial-mediated effect (i.e., indirectly). Gnotobiotic isolators were used: 1) GF, 2) with a single bacteria population, and 3) conventionalized by exposure to cecal bacterial contents. Each was divided to 2 diet groups: with or without MOS (2 kg/t) for 1 wk. Results show that the absence of bacteria in the GIT caused a reduction in neutral and acidic goblet cell (GC) number and density, an increase in sulfated mucin, absence of sialylated GC, and reduced mucin 2 mRNA expression in the small intestine of GF compared with conventional birds. These results indicate a reduced development of mucin production and secretion in the absence of GIT bacteria implying a less mature small intestine mucosa, supporting our first hypothesis. Results from the single bacteria population group were not conclusive and did not support any of the hypotheses. Supplementation of MOS, regardless of microbial presence, caused a reduction in neutral GC number and density but increased neutral GC area. The MOS caused different effects on acidic mucins in conventional and GF birds, causing a reduction in sialylated GC number (conventional) and a reduction in sulfated GC density (GF), all supporting a direct effect of MOS in GF animals, in addition to an indirect effect via gut microflora.

Regional and global changes in TCRαβ T cell repertoires in the gut are dependent upon the complexity of the enteric microflora

The repertoire of gut associated T cells is shaped by exposure to microbes, including the natural enteric microflora. Previous studies compared the repertoire of gut associated T cell populations in germ free (GF) and conventional mammals often focussing on intra-epithelial lymphocyte compartments. Using GF, conventional and monocolonised (gnotobiotic) chickens and chicken TCRβ-repertoire analysis techniques, we determined the influence of microbial status on global and regional enteric TCRβ repertoires. The gut of conventionally reared chickens exhibited non-Gaussian distributions of CDR3-lengths with some shared over-represented peaks in neighbouring gut segments. Sequence analysis revealed local clonal over-representation. Germ-free chickens exhibited a polyclonal, non-selected population of T cells in the spleen and in the gut. In contrast, gnotobiotic chickens exhibited a biased repertoire with shared clones evident throughout the gut. These data indicate the dramatic influence of enteric microflora complexity on the profile of TCRβ repertoire in the gut at local and global levels.

NUTRITIONAL studies with germ-free chickens.

NUTRITIONAL studies with germ-free chickens.

Absorption of methionine and 2-hydroxy-4-methylthiobutoanic acid in conventional and germ-free chickens

The apparent absorption of 3H-labeled L-Met and L-2-hydroxy-4-methylthiobutoanic acid (MHA-FA) was compared in germ-free and conventional broiler chickens to determine the effect of intestinal bacteria on the absorption of Met and MHA-FA. The two diets contained 0.236% of added Met or MHA-FA. Nineteen germ-free birds were maintained in two isolators and fed diets that had been sterilized by gamma irradiation (50 kilogreys). Nineteen conventional birds were reared in batteries and received nonirradiated feed. Diets were fed ad libitum for 3 wk. On d 21 of the experiment, the birds fasted overnight and were refed the experimental diets to which 1.11 × 10(7) Bq of 1-[methyl3H]MHA-FA or 1-[methyl3H]Met/kg of feed had been added. 51CrCl3 (1.11 × 10(7) Bq/kg of feed) was added as an indigestible marker. After 3 h the birds were euthanized, and their intestinal tracts were removed and partitioned into six sections. Residual Met and MHA-FA in digesta were calculated as the ratio of 3H:51Cr in each sample divided by the ratio of 3H:51Cr in the feed. The residual MHA-FA in the distal ileum of germ-free broilers was lower than in conventional birds (4.7 and 10.2% respectively; P < 0.05). In contrast the residual Met in the distal ileum of germ-free broilers was not different than in conventional birds (3.0 and 3.7% respectively; P > 0.05). This study demonstrates that intestinal bacteria significantly reduce the apparent absorption of MHA-FA from the intestinal tract of broiler chickens.

Colonization ability and pathogenic properties of a fim− mutant of an avian strain of Escherichia coli

Several studies suggest that the expression of type 1 fimbriae is involved in the virulence of Escherichia coli in chickens, by promoting adhesion of bacteria to the respiratory tract, which is most probably the first step to occur in the infection, and by interacting with the immune response. In order to determine to what extent type 1 fimbriae were involved in the pathogenic process, the fim cluster of an avian pathogenic strain of E. coli, MT78 (O2:K1:H+), was modified in vitro and reintroduced in the parent strain via allele exchange using suicide vector pCVD442. The mutant strain thus generated (DM34) had its entire fim cluster removed. Its pathogenic properties were compared to those of the parent strain in an experimental reproduction of avian colibacillosis in 15-day-old chickens, after primary infection with infectious bronchitis virus followed by intratracheal inoculation of the challenge strain. In specific-pathogen-free (SPF) animals, mutant DM34 was less pathogenic than the parent strain and colonized the lungs of infected animals to a lower level. In germ-free chickens, although DM34 was less pathogenic than MT78 according to the differences in weight gains, it colonized the trachea, lungs and internal organs to the same extent as MT78. Our results suggest that, whereas type 1 fimbriae are not strictly required in colonization of the respiratory tract of germ-free chickens, they might be important in establishing a bacterial population in the lungs of SPF animals. The difference regularly observed in weight gains between mutant- and wild-type-inoculated chickens reflects a decreased pathogenicity of the fim − mutant. However, the isolation of E. coli in target organs and the observation of colibacillosis symptoms and lesions in mutant-inoculated chickens suggest that type 1 fimbriae by themselves play a limited role in pathogenicity.

α-Galactosides are poorly digested by germ-free chickens

α-Galactosides are poorly digested by germ-free chickens

Increase of mannose residues, as Salmonella typhimurium-adhering factor, on the cecal mucosa of germ-free chickens infected with Eimeria tenella

Summary To study increase of the Salmonella population in the cecum of chickens infected with Eimeria te-nella, changes in mannose residues on the cecal mucosa were investigated. Inhibition of S typhimurium adherence to the cecum by a 2% carbohydrate (d-mannose, d-galactose, l-fucose, α-methyl- d-glucoside) in phosphate-buffered saline solution was examined. Only d-mannose had inhibitory effects. Whereas d-galactose had somewhat enhancing effects on adherence of S typhimurium to the cecal mucosa of uninfected germ-free chickens. In infected and uninfected chickens, d-mannose inhibited adherence of Styphimurium. d-Mannose significantly (P&lt; 0.05) increased adherence of Bacteroides sp. In infected and uninfected chickens, d-mannose did not have any effect on adherence of Clostridium perfringens and Bifidobacterium thermophilum. Under microscopic observation, only concanavalin A and Lens culinaris agglutinin, of 8 lectins examined, were recognized as lectin-positive staining lines or spots in the cecal mucosa, indicating presence of mannose residues on the cecal mucosa. In E tenella-infected chickens, lectin-positive staining was seen strongly on the coarse surface of damaged cells and at the bottom of the crypts. These results indicate that coccidial infection may induce increase of mannose residues on the intestinal surface and allow adhesion of more salmonellae to the intestine.

The role of the O-antigen lipopolysaccharide on the colonization in vivo of the germfree chicken gut by Klebsiella pneumoniae

We isolated lipopolysaccharide and capsular polysaccharide (K antigen)-defective mutants from two Klebsiella pneumoniae parental strains, and compared their ability to colonize in vivo the germfree chicken gut. The high-molecular weight lipopolysaccharide (LPS) (O antigen) was found necessary for the colonization while the capsular polysaccharide (K2 or K29) was not of importance.

Effect of bacterial inoculation on neutral detergent fibre digestion and energy availability in germ-free chickens

Effect of bacterial inoculation on neutral detergent fibre digestion and energy availability in germ-free chickens

Adhesion of bacteria to the cecal mucosal surface of conventional and germ-free chickens infected with Eimeria tenella

Summary When Salmonella typhimurium and Clostridium perfringens were tested in conventional chickens, larger numbers of Styphimurium and C perfringens adhered to Eimeriatenella-infected ceca than to uninfected ceca. In germ-free chickens, Styphimurium and C perfringens adhered to the E tenella-infected cecal mucosa more than to the uninfected cecal mucosa, but fewer Bacteroides vulgatus and Bifidobacterium thermophilum adhered to the E tenella-infected ceca than to the uninfected ceca. Many bacteria adhered to the lesions caused by E tenella as observed by scanning electron microscopy. On the basis of our findings, we suggest that infection with E tenella upsets the balance of competitive adherence of bacteria, allowing more colonization of Styphimurium and C perfringens.

Influence of Bacteria on Clostridium perfringens Infections in Young Chickens

When monoflora chickens with Lactobacillus acidophilus or Streptococcus faecalis were inoculated with Clostridium perfringens either in broth culture or resuspended in Gifu anaerobic medium broth or supernatant fluid, few or no chickens died. Approximately 50% of germ-free chickens died after inoculation of C. perfringens culture, whereas no conventional birds died after inoculation of broth culture. C. perfringens in the contents of duodenum from germ-free chickens numbered about 10(4) colony-forming units (CFU)/g after inoculation 10(8) CFU broth culture per bird, but in gnotobiotic and conventional chickens this organism decreased or was not detected. When C. perfringens was cultured in intestinal contents collected from germ-free chickens, C. perfringens proliferated but alpha toxin was not detected. These findings indicate that the pathogenicity of C. perfringens was suppressed by L. acidophilus or S. faecalis administered previously or inhibited by normal intestinal flora.

Possibilities of Bacteremia and Toxemia in Death of Chickens Infected with Eimeria tenella

The possibility that bacteremia and toxemia were the causes of death in cases of cecal coccidiosis was investigated. Germ-free and ordinary chickens with microflora were inoculated with sporulated oocysts of Eimeria tenella. At 5 days postinoculation, cecal lesions in ordinary chickens were more severe than those in germ-free ones. Cardiac blood, spleen, and liver were examined in ordinary chickens for bacteremia and endotoxemia, and small numbers of bacteria were recovered from both infected and uninfected birds. Endotoxin levels in plasma of E. tenella-infected birds were low and not different from the levels of uninfected controls. To examine unknown toxic factors, a large volume of serum from infected chickens was injected intravenously into uninfected birds. No significant clinical signs were observed. It is concluded that the intestinal bacteria increase the severity of coccidial lesions without bacteremia and toxemia.