Dataset Information

Lack of Evidence That Selenium-Yeast Improves Chicken Health and Modulates the Caecal Microbiota in the Context of Colonization by Campylobacter jejuni.

ABSTRACT: Faced with ever-increasing demand, the industrial production of food animals is under pressure to increase its production. In order to keep productivity, quality, and safety standards up while reducing the use of antibiotics, farmers are seeking new feed additives. In chicken production, one of these additives is selenium. This element is expected to confer some advantages in terms of animal health and productivity, but its impact on chicken intestinal microbiota as well as on the carriage of foodborne pathogens is unknown. In this study, chickens raised in a level 2 animal facility were fed or not 0.3 ppm of in-feed selenium-yeast until 35 days of age and were inoculated or not with the foodborne pathogen Campylobacter jejuni at the age of 14 days. At the end of the study, body weight, seric IgY, intestinal IgA, seric gluthatione peroxydase activity, the caecal microbiota (analyzed by MiSeq 16S rRNA gene sequencing), and C. jejuni caecal levels were analyzed. The experiment was completely replicated twice, with two independent batches of chickens. This study revealed that, for healthy chickens raised in very good hygienic conditions, selenium-yeast does not influence the bird's body weight and lowers their seric gluthatione peroxidase activity as well as their intestinal IgA concentrations. Furthermore, selenium-yeast did not modify the caecal microbiota or the colonization of C. jejuni. The results also showed that C. jejuni colonization does not impact any of the measured chicken health parameters and only slightly impacts the caecal microbiota. This study also clearly illustrated the need for true biological replication (independent animal trials) when assessing the microbiota shifts associated with treatments as the chickens microbiotas clearly clustered according to study replicate.

SUBMITTER: Thibodeau A

PROVIDER: S-EPMC5355472 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Lack of Evidence That Selenium-Yeast Improves Chicken Health and Modulates the Caecal Microbiota in the Context of Colonization by <i>Campylobacter jejuni</i>.

Thibodeau Alexandre A Letellier Ann A Yergeau Étienne É Larrivière-Gauthier Guillaume G Fravalo Philippe P

Frontiers in microbiology 20170317

Faced with ever-increasing demand, the industrial production of food animals is under pressure to increase its production. In order to keep productivity, quality, and safety standards up while reducing the use of antibiotics, farmers are seeking new feed additives. In chicken production, one of these additives is selenium. This element is expected to confer some advantages in terms of animal health and productivity, but its impact on chicken intestinal microbiota as well as on the carriage of fo ...[more]

PMID: 28367146

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Project description:Campylobacter jejuni is a zoonotic pathogen and is one of the leading causes of human gastroenteritis worldwide. C. jejuni IA3902 (representative of the sheep abortion clone) is genetically similar to C. jejuni W7 (representative of strain type NCTC 11168); however, there are significant differences in the ability of luxS mutants of these strains to colonize chickens. LuxS is essential for the activated methyl cycle and generates homocysteine for conversion to l-methionine. Comparative genomics identified differential distribution of the genes metA and metB, which function to convert homoserine for downstream production of l-methionine, between IA3902 and W7, which could enable a secondary pathway for l-methionine biosynthesis in a W7 ΔluxS but not in an IA3902 ΔluxS strain. To test the hypothesis that the genes metA and metB contribute to l-methionine production and chicken colonization by Campylobacter, we constructed two mutants for phenotypic comparison, the W7 ΔmetAB ΔluxS and IA3902 ΔluxS::metAB mutants. Quantitative reverse transcription-PCR and tandem mass spectrometry protein analysis were used to validate MetAB transcription and translation as present in the IA3902 ΔluxS::metAB mutant and absent in the W7 ΔmetAB ΔluxS mutant. Time-resolved fluorescence resonance energy transfer fluorescence assays demonstrated that l-methionine and S-adenosyl methionine concentrations decreased in the W7 ΔmetAB ΔluxS mutant and increased in the IA3902 ΔluxS::metAB mutant. Assessment of chicken colonization revealed that the IA3902 ΔluxS::metAB strain partially rescued the colonization defect of the IA3902 ΔluxS strain, while the W7 ΔmetAB ΔluxS strain showed significantly decreased colonization compared to that of the wild-type and the W7 ΔluxS strain. These results indicate that the ability to maintain l-methionine production in vivo, conferred by metA and metB in the absence of luxS, is critical for normal chicken colonization by C. jejuni.

Project description:BackgroundCampylobacter jejuni is the leading cause of bacterial gastroenteritis in humans and the handling or consumption of contaminated poultry meat is a key source of infection. Selective breeding of poultry that exhibit elevated resistance to Campylobacter is an attractive control strategy. Here we studied the global transcriptional response of inbred chicken lines that differ in resistance to C. jejuni colonisation at a key site of bacterial persistence.ResultsThree-week-old chickens of line 61 and N were inoculated orally with C. jejuni strain M1 and caecal contents and tonsils were sampled at 1 and 5 days post-infection. Caecal colonisation was significantly lower in line 61 compared to line N at 1 day post-infection, but not 5 days post-infection. RNA-Seq analysis of caecal tonsils of both lines revealed a limited response to C. jejuni infection compared to age-matched uninfected controls. In line N at days 1 and 5 post-infection, just 8 and 3 differentially expressed genes (DEGs) were detected (fold-change > 2 and false-discovery rate of < 0.05) relative to uninfected controls, respectively. In the relatively resistant line 61, a broader response to C. jejuni was observed, with 69 DEGs relating to immune regulation, cell signalling and metabolism at 1 day post-infection. However, by day 5 post-infection, no DEGs were detected. By far, the greatest number of DEGs were between uninfected birds of the two lines implying that differential resistance to C. jejuni is intrinsic. Of these genes, several Major Histocompatibility Complex class I-related genes (MHCIA1, MHCBL2 and MHCIY) and antimicrobial peptides (MUC2, AvBD10 and GZMA) were expressed to a greater extent in line N. Two genes within quantitative trait loci associated with C. jejuni colonisation were also more highly expressed in line N (ASIC4 and BZFP2). Quantitative reverse-transcriptase PCR analysis of a subset of transcripts confirmed the RNA-Seq results.ConclusionsOur data indicate a limited transcriptional response in the caecal tonsils of inbred chickens to intestinal colonisation by Campylobacter but identify a large number of differentially transcribed genes between lines 61 and N that may underlie variation in heritable resistance to C. jejuni.

Project description:Two batch cultures of wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 500 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10% Oxygen, 10% Carbon dioxide, 80% Nitrogen) from Don Whitley Scientific, Ltd, which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached, a custom-made diffusion capsule (as described in Pirt, 1971) containing chicken caecal contents was placed for 10, 30, or 60 minutes. After the exposure, samples of both treated and untreated cells were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen's RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Batch cultures of wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 500 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10% Oxygen, 10% Carbon dioxide, 80% Nitrogen) from Don Whitley Scientific, Ltd, which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached, a custom-made diffusion capsule (as described in Pirt, 1971) containing chicken caecal contents was placed for 10, 30, or 60 minutes. After the exposure, 30 ml samples of both treated and untreated cells were mixed immediately on ice with 3.56 ml 100% ethanol and 185 M-BM-5l phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. Equivalent amounts of RNA (15 M-NM-<g) from control and test cultures were used as template for synthesis of labelled cDNA. Labelling was done by using dCTP nucleotide analogues containing either Cy3 or Cy5 fluorescent dyes. RNA was primed with 9 M-NM-<g pd(N)6 random hexamers (Amersham Biosciences). For annealing, the mixture was incubated for 10 min at 65oC and then 10 min at room temperature. Each reaction mixture (0.5 mM dATP, dTTP and dGTP, 0.2 mM dCTP, 0.1 mM DTT (Invitrogen) and 1 mM Cy3-dCTP or Cy5-dCTP, total volume 25ul) was incubated for 3 h at 42 oC with 200 U of Superscript III RNase-H Reverse Transcriptase (Invitrogen). The reaction was terminated by the addition of 5ul 1 mM NaOH and heating the tube to 65 oC for 10 min to hydrolyse the RNA. Then it was neutralised with 5ul 1 M HCl and 1 M TE (pH 8). Purification of cDNA was done with a PCR purification kit (Qiagen). The cDNA was eluted and resuspended in 30 M-NM-<l elution buffer (Qiagen, supplied in kit). Each slide set (control slide and dye-swap) was prepared as follows: For the control slide, Cy3-dCTP labelled control cDNA was mixed with Cy5-dCTP labelled test cDNA. For the dye-swap slide, Cy5-dCTP labelled control cDNA was mixed with Cy3-dCTP labelled test cDNA. This is made to compensate for possible differences in the labelled nucleotides incorporation. The slides used were C. jejuni OciChipM-BM-. arrays from Ocimum Biosolutions. The cDNA mixture for each slide was dried by evaporation for approximately 35 min in a SPD 121P SpeedVacM-BM-. (Thermo Savant, Waltham, MA, USA). The dry cDNA was resuspended in pre-warmed (42M-BM-:C) salt-based hybridisation buffer (Ocimum Biosolutions) and was heated to 95 M-BM-0C for 3 min and then placed on ice for 3 min. The spotted area of the slide (located with an array finder) was enclosed within a gene frame (MWG/Ocimum). The cDNA suspension was distributed through the inner space of the gene frame and enclosed with an air-tight coverslip. The slides were incubated for 16-24 hours at 42M-BM-: C in sealed MWG hybridisation chambers shaken in a water bath. After incubation, gene frames and coverslips were removed and slides washed sequentially in 2x, 1x, 0.2x y 0.1x SSC buffer, by shaking for 5 minutes at 80 rpm at 37M-BM-: C pre-warmed buffer. (2x buffer was supplemented with 1% SDS). Then the slides were dried by centrifugation at 250 x g for 5 min. Slides were scanned using an Affymetrix 428 scanner. The processing of images and quantification of the microarrays signal was done using software from Biodiscovery Inc. (ImaGene, version 4.0 and GeneSight, version 4.0). Spots with signal intensity lower than background or other significant blemishes were eliminated from subsequent processing. Mean values from each channel were then log2 transformed and normalised using the Subtract by Mean method to remove intensity-dependent effects in the log2 (ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalised values. Significance analysis of the data used the Student's t test to determine the probability that the average of the experimental replicates was significantly different from the average of the control replicates. p-values for the data were calculated by treating each slide as a repeat using Genesight 4. Genes differentially regulated M-bM-^IM-% 2-fold and displaying a p-value of M-bM-^IM-$ 0.05 were defined as being statistically significant and differentially transcribed.

Project description:Campylobacter jejuni is a major foodborne pathogen that causes gastroenteritis in humans. Chickens act as the reservoir host for C. jejuni, wherein the pathogen asymptomatically colonizes the ceca leading to contamination of carcasses during slaughter. The major colonization factors in C. jejuni include motility, intestinal epithelial attachment, acid/bile tolerance, and quorum sensing. Reducing the expression of the aforementioned factors could potentially reduce C. jejuni colonization in chickens. This study investigated the efficacy of subinhibitory concentration (SIC; compound concentration not inhibiting bacterial growth) of carvacrol in reducing the expression of C. jejuni colonization factors in vitro. Moreover, the effect of carvacrol on the expression of C. jejuni proteome was investigated using liquid chromatography-tandem mass spectrometry. The motility assay was conducted at 42°C, and the motility zone was measured after 24 h of incubation. For the adhesion assay, monolayers of primary chicken enterocytes (?105 cells/well) were inoculated with C. jejuni (6 log cfu/well) either in the presence or absence of carvacrol, and the adhered C. jejuni were enumerated after 90 min of incubation at 42°C. The effect of carvacrol on C. jejuni quorum sensing and susceptibility to acid/bile stress was investigated using a bioluminescence assay and an acid-bile survival assay, respectively. The SIC (0.002%) of carvacrol reduced the motility of C. jejuni strains S-8 and NCTC 81-176 by ?50 and 35%, respectively (P < 0.05). Carvacrol inhibited C. jejuni S-8 and NCTC 81-176 adhesion to chicken enterocytes by ?0.8 and 1.5 log cfu/mL, respectively (P < 0.05). Moreover, carvacrol reduced autoinducer-2 activity and increased the susceptibility of C. jejuni to acid and bile in both the strains (P < 0.05). Liquid chromatography-tandem mass spectrometry revealed that the SIC of carvacrol reduced the expression of selected C. jejuni colonization proteins critical for motility (methyl-accepting chemotaxis protein), adhesion (GroL), growth and metabolism (AspA, AcnB, Icd, Fba, Ppa, AnsA, Ldh, Eno, PurB-1), and anaerobic respiration (NapB, HydB, SdhA, NrfA) (P < 0.05). Results suggest the mechanisms by which carvacrol could reduce C. jejuni colonization in chickens.

Dataset Information

Lack of Evidence That Selenium-Yeast Improves Chicken Health and Modulates the Caecal Microbiota in the Context of Colonization by Campylobacter jejuni.

Publications

Lack of Evidence That Selenium-Yeast Improves Chicken Health and Modulates the Caecal Microbiota in the Context of Colonization by <i>Campylobacter jejuni</i>.

Similar Datasets

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