Loss of αß but not γδ T cells in chickens causes a severe phenotype.

The availability of genetically modified mice has facilitated the study of mammalian T cells. No model has yet been developed to study these cells in chickens, an important livestock species with a high availability of γδ T cells. To investigate the role of γδ and αß T cell populations in birds, we generated chickens lacking these T cell populations. This was achieved by genomic deletion of the constant region of the T cell receptor γ or ß chain, leading to a complete loss of either γδ or αß T cells. Our results show that a deletion of αß T cells but not γδ T cells resulted in a severe phenotype in KO chickens. The αß T cell KO chickens exhibited granulomas associated with inflammation of the spleen and the proventriculus. Immunophenotyping of αß T cell KO chickens revealed a significant increase in monocytes and expectedly the absence of CD4+ T cells including FoxP3+ regulatory T cells. Surprisingly there was no increase of γδ T cells. In addition, we observed a significant decrease in immunoglobulins, B lymphocytes, and changes in the bursa morphology. Our data reveal the consequences of T cell knockouts in chickens and provide new insights into their function in vertebrates.

Allocoprobacillus halotolerans gen. nov., sp. nov and Coprobacter tertius sp. nov., isolated from human gut microbiota.

Two novel bacterial isolates were cultured from faecal samples of patients attending the Breast Care clinic at the Norwich and Norfolk University Hospital. Strain LH1062T was isolated from a 58-year-old female diagnosed with invasive adenocarcinoma with ductal carcinoma in situ. Strain LH1063T was isolated from a healthy 51-year-old female. Isolate LH1062T was predicted to be a potential novel genus most closely related to Coprobacillus, whilst LH1063T was predicted to be a novel species belonging to Coprobacter. Both strains were characterized by polyphasic approaches including 16S rRNA gene analysis, core-genome analysis, average nucleotide identity (ANI) comparisons and phenotypic analysis. Initial screening of the 16S rRNA gene of LH1062T returned a nucleotide identity of 93.4 % to Longibaculum muris. For LH1063T, nucleotide identity was a 92.6 % to Coprobacter secundus. Further investigations showed that LH1062T had a genome size of 2.9 Mb and G+C content of 31.3 mol %. LH1063T had a genome size of 3.3Mb and G+C content of 39.2 mol %. Digital DNA-DNA hybridization (dDDH) and ANI values of LH1062T with its closest relative, Coprobacillus cateniformis JCM 10604T, were 20.9 and 79.54 %, respectively. For LH1063T, the dDDH and ANI values with its closest relative, Coprobacter secundus 177T, were 19.3 and 77.81 %, respectively. Phenotypic testing confirmed that LH1062T could not be matched to a known validly published isolate in any database; thereby indicating a novel genus for which the name Allocoprobacillus gen. nov. is now proposed with LH1062T (=DSM 114537T=NCTC 14686T) being the type strain of the proposed novel species Allocoprobacillus halotolerans sp. nov. Strain LH1063T (=DSM 114538T=NCTC 14698T) fits within the genus Coprobacter and, it being the third species within this genus, the name Coprobacter tertius sp. nov. is proposed.

The proteome of bacterial membrane vesicles in Escherichia coli-a time course comparison study in two different media.

Introduction: Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown. Methods: To gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1ß and IL-8 in the human monocyte cell line THP-1 upon bMV treatment. Results: Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1ß and IL-8 expressions. Conclusion: Our study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

Noninvasive Fecal Cytokine and Microbiota Profiles Predict Commencement of Necrotizing Enterocolitis in a Proof-of-Concept Study.

Background and Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell's staging is routinely used to guide diagnosis, but early diagnostic signs are nonspecific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed. We investigated fecal cytokine levels, coupled with gut microbiota profiles, as a noninvasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers. Methods: Premature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary's or Queen Charlotte's & Chelsea) between January 2011 and December 2012. During the NICU stay, expert neonatologists grouped individuals by modified Bell's staging (healthy, NEC1, NEC2/3) and fecal samples from diapers were collected consecutively. Microbiota profiles were assessed by 16S rRNA gene amplicon sequencing and cytokine concentrations were measured by V-Plex multiplex assays. Results: Early evaluation of microbiota profiles revealed only minor differences. However, at later time points, significant changes in microbiota composition were observed for Bacillota (adj. P = .0396), with Enterococcus being the least abundant in Bell stage 2/3 NEC. Evaluation of fecal cytokine levels revealed significantly higher concentrations of IL-1α (P = .045), IL-5 (P = .0074), and IL-10 (P = .032) in Bell stage 1 NEC compared to healthy individuals. Conclusion: Differences in certain fecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles contribute to improving knowledge about NEC pathogenesis.

Processing Matters in Nutrient-Matched Laboratory Diets for Mice-Microbiome.

The composition of the microbiome is subject to the host's diet. In commercial laboratory mouse diets, different physical forms of the same diets are available, containing-according to their labels-identical ingredients and nutrient compositions. However, variations in nutrient composition and starch gelatinization due to production processes and their impact on digestibility have been described. In this study, a total of 48 C57BL/J6 mice were assigned to two equal groups and were fed diets (produced with different processes-extruded vs. pelleted) for eight weeks in two biological replicates. At the end of the experiment, samples were collected from five different gastrointestinal regions, including the stomach, small intestine, cecum, large intestine, and an extracorporeal region (feces), and the microbiome was analyzed with 16S rRNA gene amplicon sequencing. The replicates in both experiments differed significantly in their relative abundances of Muribaculaceae species. Furthermore, the gastrointestinal content of pellet-fed mice contained larger numbers of Lactobacillus species. These results indicate that starch gelatinization and ingredient composition significantly influence microbial makeup. In conclusion, different feed processing methods may affect fundamental digestive and metabolic processes, impacting animal experiments and biasing microbiome data.

Early-Life Immune System Maturation in Chickens Using a Synthetic Community of Cultured Gut Bacteria.

The gut microbiome is crucial for both maturation of the immune system and colonization resistance against enteric pathogens. Although chicken are important domesticated animals, the impact of their gut microbiome on the immune system is understudied. Therefore, we investigated the effect of microbiome-based interventions on host mucosal immune responses. Increased levels of IgA and IgY were observed in chickens exposed to maternal feces after hatching compared with strict hygienic conditions. This was accompanied by increased gut bacterial diversity as assessed by 16S rRNA gene amplicon sequencing. Cultivation work allowed the establishment of a collection of 43 bacterial species spanning 4 phyla and 19 families, including the first cultured members of 3 novel genera and 4 novel species that were taxonomically described. This resource is available at www.dsmz.de/chibac A synthetic community consisting of nine phylogenetically diverse and dominant species from this collection was designed and found to be moderately efficient in boosting immunoglobulin levels when provided to chickens early in life.IMPORTANCE The immune system plays a crucial role in sustaining animal health. Its development is markedly influenced by early microbial colonization of the gastrointestinal tract. As chicken are fully dependent on environmental microbes after hatching, extensive hygienic measures in production facilities are detrimental to the microbiota, resulting in low colonization resistance against pathogens. To combat enteric infections, antibiotics are frequently used, which aggravates the issue by altering gut microbiota colonization. Intervention strategies based on cultured gut bacteria are proposed to influence immune responses in chicken.