Dataset Information

Oral administration of Lactobacillus reuteri expressing a 3D8 single-chain variable fragment (scFv) enhances chicken growth and conserves immune homeostasis.

ABSTRACT: The present study was aimed to investigate the effects of 3D8 scFv-secreting Probiotic Lactobacillus reuteri (L. reuteri) on growth performance, inflammatory responses, and intestinal microbial flora in chickens. To this end, a total of 14 healthy wild-type chickens were divided into two experimental groups. Each group was orally administrated with a daily dose of 109 colony-forming units (CFU) of 3D8 scFv-producing L. reuteri or wild-type (WT) for 35 days. Administration of L. reuteri/3D8 scFv significantly improved the body weight of chickens when compared to L. reuteri/WT group. The bacterial taxonomic composition of the fecal microbiota was determined by pyrosequencing of 16S rRNA gene amplicons. Firmicutes, Actinobacteria, and Proteobacteria were dominant phyla in two experimental groups. However, in 3D8 L. reuteri treatment groups at genus level, the Lactobacillus was highly abundant, being represented by 18.12%. In addition, serum levels of primary cytokines such as IL-6, IL-8, TNF-?, IFN-?, IL-4, and IGF1 were markedly reduced in the probiotic L. reuteri 3D8 group. In summary, our results indicate that the administration of L. reuteri expressing 3D8 scFv has a modulatory effect on inflammatory responses, improves weight gain while not affecting the common microbial composition of the chicken intestine.

SUBMITTER: Sureshkumar S

PROVIDER: S-EPMC6591341 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Oral administration of Lactobacillus reuteri expressing a 3D8 single-chain variable fragment (scFv) enhances chicken growth and conserves immune homeostasis.

Sureshkumar Shanmugam S Jung Sun Keun SK Kim Dongjun D Oh Keon Bong KB Yang Hyeon H Lee Hwi Cheul HC Jin Jo Yong JY Sun Lee Hae LH Lee Sukchan S Byun Sung June SJ

3 Biotech 20190624 7

The present study was aimed to investigate the effects of 3D8 scFv-secreting Probiotic Lactobacillus reuteri (L. reuteri) on growth performance, inflammatory responses, and intestinal microbial flora in chickens. To this end, a total of 14 healthy wild-type chickens were divided into two experimental groups. Each group was orally administrated with a daily dose of 109 colony-forming units (CFU) of 3D8 scFv-producing L. reuteri or wild-type (WT) for 35 days. Admini ...[more]

PMID: 31245246

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Project description:BACKGROUND: Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv). FINDINGS: Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3)pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with EPEC E2348/69. CONCLUSION: This study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int388-667) in purified form and the EPEC isolate.

Project description:Lactobacillus reuteri is a natural inhabitant of selected animal and human gastrointestinal tract (GIT). Certain strains have the capacity to transform glycerol to 3-hydroxypropionaldehyde (3-HPA), further excreted to form reuterin, a potent antimicrobial system. Reuterin-producing strains may be applied as a natural antimicrobial in feed to prevent pathogen colonization of animals, such as in chicken, and replace added antimicrobials. To date, only seven L. reuteri strains isolated from chicken have been characterized which limits phylogenetic studies and host-microbes interactions characterization. This study aimed to isolate L. reuteri strains from chicken GIT and to characterize their reuterin production and antimicrobial resistance (AMR) profiles using phenotypic and genetic methods. Seventy strains were isolated from faces, crops and ceca of six chicken from poultry farms and samples from slaughterhouse. Twenty-five strains were selected for further characterization. Draft genomes were generated for the new 25 isolates and integrated into a phylogenetic tree of 40 strains from different hosts. Phylogenetic analysis based on gene content as well as on core genomes showed grouping of the selected 25 L. reuteri chicken isolates within the poultry/human lineage VI. Strains harboring pdu-cob-cbi-hem genes (23/25) produced between 156 mM ± 11 and 330 mM ± 14 3-HPA, from 600 mM of glycerol, in the conditions of the test. All 25 chicken strains were sensitive to cefotaxime (MIC between 0.016 and 1 ?g/mL) and penicillin (MIC between 0.02 and 4 ?g/mL). Akin to the reference strains DSM20016 and SD2112, the novel isolates were resistant to penicillin, possibly associated with identified point mutations in ponA, pbpX, pbpF and pbpB. All strains resistant to erythromycin (4/27) carried the ermB gene, and it was only present in chicken strains. All strains resistant to tetracycline (5/27) harbored tetW gene. This study confirms the evolutionary history of poultry/human lineage VI and identifies pdu-cob-cbi-hem as a frequent trait but not always present in this lineage. L. reuteri chicken strains producing high 3-HPA yield may have potential to prevent enteropathogen colonization of chicken.

Dataset Information

Oral administration of Lactobacillus reuteri expressing a 3D8 single-chain variable fragment (scFv) enhances chicken growth and conserves immune homeostasis.

Publications

Oral administration of <i>Lactobacillus reuteri</i> expressing a 3D8 single-chain variable fragment (scFv) enhances chicken growth and conserves immune homeostasis.

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