Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

Gut dysbiosis might underlie the pathogenesis of type 1 diabetes. In mice of the non-obese diabetic (NOD) strain, we found that key features of disease correlated inversely with blood and fecal concentrations of the microbial metabolites acetate and butyrate. We therefore fed NOD mice specialized diets designed to release large amounts of acetate or butyrate after bacterial fermentation in the colon. Each diet provided a high degree of protection from diabetes, even when administered after breakdown of immunotolerance. Feeding mice a combined acetate- and butyrate-yielding diet provided complete protection, which suggested that acetate and butyrate might operate through distinct mechanisms. Acetate markedly decreased the frequency of autoreactive T cells in lymphoid tissues, through effects on B cells and their ability to expand populations of autoreactive T cells. A diet containing butyrate boosted the number and function of regulatory T cells, whereas acetate- and butyrate-yielding diets enhanced gut integrity and decreased serum concentration of diabetogenic cytokines such as IL-21. Medicinal foods or metabolites might represent an effective and natural approach for countering the numerous immunological defects that contribute to T cell-dependent autoimmune diseases.

Erratum: Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

This corrects the article DOI: 10.1038/ni.3713.

Campylobacter bilis sp. nov., isolated from chickens with spotty liver disease.

A novel species of Campylobacter was isolated from bile samples of chickens with spotty liver disease in Australia, making it the second novel species isolated from chickens with the disease, after Campylobacter hepaticus was isolated and described in 2016. Six independently derived isolates were obtained. They were Gram-stain-negative, microaerobic, catalase-positive, oxidase-positive and urease-negative. Unlike most other species of the genus Campylobacter, more than half of the tested strains of this novel species hydrolysed hippurate and most of them could not reduce nitrate. Distinct from C. hepaticus, many of the isolates were sensitive to 2,3,5-triphenyltetrazolium chloride (0.04%) and metronidazole (4 mg ml-1), and all strains were sensitive to nalidixic acid. Phylogenetic analysis using 16S rRNA and hsp60 gene sequences demonstrated that the strains formed a robust clade that was clearly distinct from recognized Campylobacter species. Whole genome sequence analysis of the strains showed that the average nucleotide identity and the genome blast distance phylogeny values compared to other Campylobacter species were less than 86 and 66%, respectively, which are below the cut-off values generally recognized for isolates of the same species. The genome of the novel species has a DNA G+C content of 30.6 mol%, while that of C. hepaticus is 27.9 mol%. Electron microscopy showed that the cells were spiral-shaped, with bipolar unsheathed flagella. The protein spectra generated from matrix-assisted laser desorption/ionization time of flight analysis demonstrated that they are different from the most closely related Campylobacter species. These data indicate that the isolates belong to a novel Campylobacter species, for which the name Campylobacter bilis sp. nov. is proposed. The type strain is VicNov18T (=ATCC TSD-231T=NCTC 14611T).

Design and Evaluation Challenges of Conversational Agents in Health Care and Well-being: Selective Review Study.

BACKGROUND: Health care and well-being are 2 main interconnected application areas of conversational agents (CAs). There is a significant increase in research, development, and commercial implementations in this area. In parallel to the increasing interest, new challenges in designing and evaluating CAs have emerged. OBJECTIVE: This study aims to identify key design, development, and evaluation challenges of CAs in health care and well-being research. The focus is on the very recent projects with their emerging challenges. METHODS: A review study was conducted with 17 invited studies, most of which were presented at the ACM (Association for Computing Machinery) CHI 2020 conference workshop on CAs for health and well-being. Eligibility criteria required the studies to involve a CA applied to a health or well-being project (ongoing or recently finished). The participating studies were asked to report on their projects' design and evaluation challenges. We used thematic analysis to review the studies. RESULTS: The findings include a range of topics from primary care to caring for older adults to health coaching. We identified 4 major themes: (1) Domain Information and Integration, (2) User-System Interaction and Partnership, (3) Evaluation, and (4) Conversational Competence. CONCLUSIONS: CAs proved their worth during the pandemic as health screening tools, and are expected to stay to further support various health care domains, especially personal health care. Growth in investment in CAs also shows the value as a personal assistant. Our study shows that while some challenges are shared with other CA application areas, safety and privacy remain the major challenges in the health care and well-being domains. An increased level of collaboration across different institutions and entities may be a promising direction to address some of the major challenges that otherwise would be too complex to be addressed by the projects with their limited scope and budget.

Stable Recombinant-Gene Expression from a Ligilactobacillus Live Bacterial Vector via Chromosomal Integration.

Disease control in animal production systems requires constant vigilance. Historically, the application of in-feed antibiotics to control bacteria and improve performance has been a much-used approach to maintain animal health and welfare. However, the widespread use of in-feed antibiotics is thought to increase the risk of antibiotic resistance developing. Alternative methods to control disease and maintain productivity need to be developed. Live vaccination is useful in preventing colonization of mucosa-dwelling pathogens by inducing a mucosal immune response. Native poultry isolate Ligilactobacillus agilis La3 (previously Lactobacillus agilis) has been identified as a candidate for use as a live vector to deliver therapeutic proteins such as bacteriocins, phage endolysins, or vaccine antigens to the gastrointestinal tract of chickens. In this study, the complete genome sequence of L. agilis La3 was determined and transcriptome analysis was undertaken to identify highly expressed genes. Predicted promoter regions and ribosomal binding sites from constitutively expressed genes were used to construct recombinant protein expression cassettes. A series of double-crossover shuttle plasmids were constructed to facilitate rapid selectable integration of expression cassettes into the Lagilis La3 chromosome via homologous recombination. Inserts showed 100% stable integration over 100 generations without selection. A positive relationship was found between protein expression levels and the predicted strength of the promoters. Using this system, stable chromosomal expression of a Clostridium perfringens antigen, rNetB, was demonstrated without selection. Finally, two recombinant strains, Lagilis La3::P eft -rnetB and Lagilis La3::P cwah -rnetB, were constructed and characterized, and they showed potential for future application as live vaccines in chickens.IMPORTANCE Therapeutic proteins such as antigens can be used to prevent infectious diseases in poultry. However, traditional vaccine delivery by intramuscular or subcutaneous injection generally has not proven effective for mucosa-dwelling microorganisms that live within the gastrointestinal tract. Utilizing live bacteria to deliver vaccine antigens directly to the gut immune system can overcome some of the limitations of conventional vaccination. In this work, Ligilactobacillus agilis La3, an especially effective gut colonizer, has been analyzed and engineered with modular and stable expression systems to produce recombinant proteins. To demonstrate the effectiveness of the system, expression of a vaccine antigen from poultry pathogen Clostridium perfringens was monitored over 100 generations without selection and found to be completely stable. This study demonstrates the development of genetic tools and novel constitutive expression systems and further development of L. agilis La3 as a live delivery vehicle for recombinant proteins.

Temporal dynamics of gut microbiota in caged laying hens: a field observation from hatching to end of lay.

Gut health has major implications for the general health of food-producing animals such as the layer birds used in the egg industry. In order to modulate gut microbiota for the benefit of gut health, an understanding of the dynamics and details of the development of gut microbiota is critical. The present study investigated the phylogenetic composition of the gut microbiota of a commercial layer flock raised in cages from hatch to the end of the production cycle. This study also aimed to understand the establishment and development of gut microbiota in layer chickens. Results showed that the faecal microbiota was dominated by phyla Firmicutes and Proteobacteria in the rearing phase, but Bacteroidetes in mid lay and late lay phase. The gut microbiota composition changed significantly during the transfer of the flock from the rearing to the production shed. The richness and diversity of gut microbiota increased after week 6 of the flocks age and stabilized in the mid and late lay phase. The overall dynamics of gut microbiota development was similar to that reported in earlier studies, but the phylogenetic composition at the phylum and family level was different. The production stage of the birds is one of the important factors in the development of gut microbiota. This study has contributed to a better understanding of baseline gut microbiota development over the complete life cycles in layer chickens and will help to develop strategies to improve the gut health. KEY POINTS: • Faecal microbiota of caged hens was dominated by phyla Firmicutes and Proteobacteria in the rearing phase. • The gut microbiota composition changed significantly during the transfer of the flock from the rearing to the production shed. • The richness and diversity of gut microbiota increased after week 6 of the flocks age and stabilized in the mid and late lay phase.

The Gut Microbiota of Laying Hens and Its Manipulation with Prebiotics and Probiotics To Enhance Gut Health and Food Safety.

The microbiota plays a vital role in maintaining gut health and influences the overall performance of chickens. Most gut microbiota-related studies have been performed in broilers, which have different microbial communities compared to those of layers. The normal gut microbiota of laying chickens is dominated by Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Actinobacteria at the phylum level. The composition of the gut microbiota changes with chicken age, genotype, and production system. The metabolites of gut microbiota, such as short-chain fatty acids, indole, tryptamine, vitamins, and bacteriocins, are involved in host-microbiota cross talk, maintenance of barrier function, and immune homeostasis. Resident gut microbiota members also limit and control the colonization of foodborne pathogens. In-feed supplementations of prebiotics and probiotics strengthen the gut microbiota for improved host performance and colonization resistance to gut pathogens, such as Salmonella and Campylobacter The mechanisms of action of prebiotics and probiotics come through the production of organic acids, activation of the host immune system, and production of antimicrobial agents. Probiotic candidates, including Lactobacillus, Bifidobacterium, Bacillus, Saccharomyces, and Faecalibacterium isolates, have shown promising results toward enhancing food safety and gut health. Additionally, a range of complex carbohydrates, including mannose oligosaccharides, fructo-oligosaccharides, and galacto-oligosaccharides, and inulin are promising candidates for improving gut health. Here, we review the potential roles of prebiotics and probiotics in the reshaping of the gut microbiota of layer chickens to enhance gut health and food safety.

Development of an enzyme-linked immunosorbent assay for detecting Campylobacter hepaticus specific antibodies in chicken sera - a key tool in Spotty Liver Disease screening and vaccine development.

Spotty Liver Disease (SLD) is an emerging disease of serious concern in the egg production industry, as it causes significant egg loss and mortality in layer hens. The causative agent is a newly identified Gram-negative bacterium, Campylobacter hepaticus, and knowledge about C. hepaticus pathogenesis and the potential for vaccine development is still in its infancy. Current detection methods for SLD, such as PCR and culturing, only detect an active infection and will not give any indication of a past infection from which the bacteria have been cleared. An immunological assay, on the other hand, can provide information on previous infections and therefore is crucial in vaccine development against SLD. In the present study, we have developed the first immunoassay capable of detecting C. hepaticus-specific antibodies present in the sera of infected birds. The assay uses C. hepaticus total protein extract (TPE) as the antigen coating on enzyme-linked immunosorbent assay (ELISA) plates. The cross reactivity of C. hepaticus antibodies with closely related C. jejuni and C. coli antigens was successfully overcome by pre-absorbing the sera using C. jejuni cell extracts. The assay was validated using sera samples from both naturally- and experimentally-infected birds, birds vaccinated with formalin-killed bacteria, and serum samples from SLD-negative birds (control group). The optimized ELISA assay had 95.5% specificity and 97.6% sensitivity. The immunoassay provides a useful tool for monitoring the exposure of poultry flocks to C. hepaticus infection and can be used to direct and support vaccine development. HIGHLIGHTS The first immunoassay developed for Spotty Liver Disease (SLD). A useful method for detecting C. hepaticus-specific antibodies in birds. Highly specific (95.5%) and sensitive (97.6%) assay. A key tool for use in epidemiological studies and vaccine development.

An intermittent hypercaloric diet alters gut microbiota, prefrontal cortical gene expression and social behaviours in rats.

Objectives: Excessive consumption of high fat and high sugar (HFHS) diets alters reward processing, behaviour, and changes gut microbiota profiles. Previous studies in gnotobiotic mice also provide evidence that these gut microorganisms may influence social behaviour. To further investigate these interactions, we examined the impact of the intermittent access to a HFHS diet on social behaviour, gene expression and microbiota composition in adolescent rats. Methods: Male rats were permitted intermittent daily access (2 h / day) to a palatable HFHS chow diet for 28 days across adolescence. Social interaction, social memory and novel object recognition were assessed during this period. Following testing, RT-PCR was conducted on hippocampal and prefrontal cortex (PFC) samples. 16S ribosomal RNA amplicon sequencing was used for identification and relative quantification of bacterial taxa in faecal samples. Results: We observed reduced social interaction behaviours, impaired social memory and novel object recognition in HFHS diet rats compared to chow controls. RT-PCR revealed reduced levels of monoamine oxidase A (Maoa), catechol-O-methyltransferase (Comt) and brain derived neurotrophic factor (Bdnf) mRNA in the PFC of HFHS diet rats. Faecal microbiota analysis demonstrated that the relative abundance of a number of specific bacterial taxa differed significantly between the two diet groups, in particular, Lachnospiraceae and Ruminoccoceae bacteria. Discussion: Intermittent HFHS diet consumption evoked physiological changes to the brain, particularly expression of mRNA associated with reward and neuroplasticity, and gut microbiome. These changes may underpin the observed alterations to social behaviours.