Costimulation Blockade Disrupts CD4+ T Cell Memory Pathways and Uncouples Their Link to Decline in ß-Cell Function in Type 1 Diabetes.

We previously reported that costimulation blockade by abatacept limits the decline of ß-cell function and the frequency of circulating CD4+ central memory T cells (TCM) (CD45RO+CD62L+) in new-onset type 1 diabetes. In human subjects receiving placebo, we found a significant association between an increase in CD4+ TCM cells and the decline of ß-cell function. To extend and refine these findings, we examined changes in human CD4+ and CD8+ naive and memory T cell subsets at greater resolution using polychromatic flow and mass cytometry. In the placebo group, we successfully reproduced the original finding of a significant association between TCM and ß-cell function and extended this to other T cell subsets. Furthermore, we show that abatacept treatment significantly alters the frequencies of a majority of CD4+ conventional and regulatory T cell subsets; in general, Ag-naive subsets increase and Ag-experienced subsets decrease, whereas CD8+ T cell subsets are relatively resistant to drug effects, indicating a lesser reliance on CD28-mediated costimulation. Importantly, abatacept uncouples the relationship between changes in T cell subsets and ß-cell function that is a component of the natural history of the disease. Although these data suggest immunological markers for predicting change in ß-cell function in type 1 diabetes, the finding that abatacept blunts this relationship renders the biomarkers nonpredictive for this type of therapy. In sum, our findings point to a novel mechanism of action for this successful immunotherapy that may guide other disease-modifying approaches for type 1 diabetes.

Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated In Silico High-Throughput Sequencing Data Sets.

Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants' analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.

Weakly haemolytic variants of Brachyspira hyodysenteriae newly emerged in Europe belong to a distinct subclade with unique genetic properties.

Brachyspira (B.) hyodysenteriae is widespread globally, and can cause mucohaemorrhagic colitis (swine dysentery, SD) with severe economic impact in infected herds. Typical strains of B. hyodysenteriae are strongly haemolytic on blood agar, and the haemolytic activity is believed to contribute to virulence in vivo. However, recently there have been reports of atypical weakly haemolytic isolates of B. hyodysenteriae (whBh). In this study, 34 European whBh and 82 strongly haemolytic isolates were subjected to comparative genomic analysis. A phylogenetic tree constructed using core single nucleotide polymorphisms showed that the whBh formed a distinct sub-clade. All eight genes previously associated with haemolysis in B. hyodysenteriae were present in the whBh. No consistent patterns of amino acid substitutions for all whBh were found in these genes. In contrast, a genome region containing six coding sequences (CDSs) had consistent nucleotide sequence differences between strongly and whBh isolates. Two CDSs were predicted to encode ABC transporter proteins, and a TolC family protein, which may have a role in the export of haemolysins from B. hyodysenteriae. Another difference in this region was the presence of three CDSs in whBh that are pseudogenes in strongly haemolytic isolates. One of the intact CDSs from whBh encoded a predicted PadR-like transcriptional repressor that may play a role in repression of haemolysis functions. In summary, a sub-clade of whBh isolates has emerged in Europe, and several genomic differences, that potentially explain the weakly haemolytic phenotype, were identified. These markers may provide targets for discriminatory molecular tests needed in SD surveillance.

Occurrence and characterization of mcr-1-harbouring Escherichia coli isolated from pigs in Great Britain from 2013 to 2015.

Objectives: To determine the occurrence of mcr-1 -harbouring Escherichia coli in archived pig material originating in Great Britain (GB) from 2013 to 2015 and characterize mcr-1 plasmids. Methods: Enrichment and selective culture of 387 archived porcine caecal contents and recovery from archive of 1109 E. coli isolates to identify colistin-resistant bacteria by testing for the presence of mcr-1 by PCR and RT-PCR. mcr-1 -harbouring E. coli were characterized by WGS and compared with other available mcr-1 WGS. Results: Using selective isolation following enrichment, the occurrence of mcr-1 E. coli in caeca from healthy pigs at slaughter from unique farms in GB was 0.6% (95% CI 0%-1.5%) in 2015. mcr-1 E. coli were also detected in isolates from two porcine veterinary diagnostic submissions in 2015. All isolates prior to 2015 were negative. WGS analysis of the four mcr-1 -positive E. coli indicated no other antimicrobial resistance (AMR) genes were linked to mcr-1 -plasmid-bearing contigs, despite all harbouring multiple AMR genes. The sequence similarity between mcr-1 -plasmid-bearing contigs identified and those found in GB, Chinese and South African human isolates and Danish, French and Estonian livestock-associated isolates was 90%-99%. Conclusions: mcr-1- harbouring plasmids were diverse, implying transposable elements are involved in mcr-1 transmission in GB. The low number of mcr-1 -positive E. coli isolates identified suggested mcr-1 is currently uncommon in E. coli from pigs within GB. The high sequence similarity between mcr-1 plasmid draft genomes identified in pig E. coli and plasmids found in human and livestock-associated isolates globally requires further investigation to understand the full implications.

Lyssavirus in Indian Flying Foxes, Sri Lanka.

A novel lyssavirus was isolated from brains of Indian flying foxes (Pteropus medius) in Sri Lanka. Phylogenetic analysis of complete virus genome sequences, and geographic location and host species, provides strong evidence that this virus is a putative new lyssavirus species, designated as Gannoruwa bat lyssavirus.

Discovery of hantavirus circulating among Rattus rattus in French Mayotte island, Indian Ocean.

Hantaviruses are emerging zoonotic viruses that cause human diseases. In this study, sera from 642 mammals from La Réunion and Mayotte islands (Indian Ocean) were screened for the presence of hantaviruses by molecular analysis. None of the mammals from La Réunion island was positive, but hantavirus genomic RNA was discovered in 29/160 (18 %) Rattus rattus from Mayotte island. The nucleoprotein coding region was sequenced from the liver and spleen of all positive individuals allowing epidemiological and intra-strain variability analyses. Phylogenetic analysis based on complete coding genomic sequences showed that this Murinae-associated hantavirus is a new variant of Thailand virus. Further studies are needed to investigate hantaviruses in rodent hosts and in Haemorrhagic Fever with Renal Syndrome (HFRS) human cases.

Ecological and genetic determinants of plasmid distribution in Escherichia coli.

Bacterial plasmids are important carriers of virulence and antibiotic resistance genes. Nevertheless, little is known of the determinants of plasmid distribution in bacterial populations. Here the factors affecting the diversity and distribution of the large plasmids of Escherichia coli were explored in cattle grazing on semi-natural grassland, a set of populations with low frequencies of antibiotic resistance genes. Critically, the population genetic structure of bacterial hosts was chararacterized. This revealed structured E. coli populations with high diversity between sites and individuals but low diversity within cattle hosts. Plasmid profiles, however, varied considerably within the same E. coli genotype. Both ecological and genetic factors affected plasmid distribution: plasmid profiles were affected by site, E. coli diversity, E. coli genotype and the presence of other large plasmids. Notably 3/26 E. coli serotypes accounted for half the observed plasmid-free isolates indicating that within species variation can substantially affect carriage of the major conjugative plasmids. The observed population structure suggest that most of the opportunities for within species plasmid transfer occur between different individuals of the same genotype and support recent experimental work indicating that plasmid-host coevolution, and epistatic interactions on fitness costs are likely to be important in determining occupancy.

Colistin resistance in Salmonella and Escherichia coli isolates from a pig farm in Great Britain.

OBJECTIVES: The objective of this study was to characterize colistin-resistant bacteria isolated from pigs on a farm in Great Britain following identification of a plasmid-borne colistin resistance mechanism in Escherichia coli from China. METHODS: Phenotypic antimicrobial susceptibility testing was undertaken by broth dilution and WGS was performed to detect the presence of genes encoding resistance and virulence. Transferable colistin resistance was investigated by conjugation. RESULTS: Two E. coli and one Salmonella Typhimurium variant Copenhagen were shown to be MDR, including resistance to colistin, with one E. coli and the Salmonella carrying the mcr-1 gene; all three harboured chromosomal mutations in genes conferring colistin resistance and both E. coli harboured ß-lactamase resistance. The Salmonella mcr-1 plasmid was highly similar to pHNSHP45, from China, while the E. coli mcr-1 plasmid only had the ISApII and mcr-1 genes in common. The frequency of mcr-1 plasmid transfer by conjugation to recipient Enterobacteriaceae from Salmonella was low, lying between 10(-7) and 10(-9) cfu/recipient cfu. We were unable to demonstrate mcr-1 plasmid transfer from the E. coli. Plasmid profiling indicated transfer of multiple plasmids from the Salmonella resulting in some MDR transconjugants. CONCLUSIONS: Identification of the mcr-1 gene in Enterobacteriaceae from pigs confirms its presence in livestock in Great Britain. The results suggest dissemination of resistance through different horizontally transferable elements. The in vitro transfer of multiple plasmids carrying colistin and other resistances from the Salmonella isolate underlines the potential for wider dissemination and recombination.

Host-microbiome interactions in human type 2 diabetes following prebiotic fibre (galacto-oligosaccharide) intake.

Aberrant microbiota composition and function have been linked to several pathologies, including type 2 diabetes. In animal models, prebiotics induce favourable changes in the intestinal microbiota, intestinal permeability (IP) and endotoxaemia, which are linked to concurrent improvement in glucose tolerance. This is the first study to investigate the link between IP, glucose tolerance and intestinal bacteria in human type 2 diabetes. In all, twenty-nine men with well-controlled type 2 diabetes were randomised to a prebiotic (galacto-oligosaccharide mixture) or placebo (maltodextrin) supplement (5·5 g/d for 12 weeks). Intestinal microbial community structure, IP, endotoxaemia, inflammatory markers and glucose tolerance were assessed at baseline and post intervention. IP was estimated by the urinary recovery of oral 51Cr-EDTA and glucose tolerance by insulin-modified intravenous glucose tolerance test. Intestinal microbial community analysis was performed by high-throughput next-generation sequencing of 16S rRNA amplicons and quantitative PCR. Prebiotic fibre supplementation had no significant effects on clinical outcomes or bacterial abundances compared with placebo; however, changes in the bacterial family Veillonellaceae correlated inversely with changes in glucose response and IL-6 levels (r -0·90, P=0·042 for both) following prebiotic intake. The absence of significant changes to the microbial community structure at a prebiotic dosage/length of supplementation shown to be effective in healthy individuals is an important finding. We propose that concurrent metformin treatment and the high heterogeneity of human type 2 diabetes may have played a significant role. The current study does not provide evidence for the role of prebiotics in the treatment of type 2 diabetes.

Whole-genome sequencing for national surveillance of Shiga toxin-producing Escherichia coli O157.

BACKGROUND: National surveillance of gastrointestinal pathogens, such as Shiga toxin-producing Escherichia coli O157 (STEC O157), is key to rapidly identifying linked cases in the distributed food network to facilitate public health interventions. In this study, we used whole-genome sequencing (WGS) as a tool to inform national surveillance of STEC O157 in terms of identifying linked cases and clusters and guiding epidemiological investigation. METHODS: We retrospectively analyzed 334 isolates randomly sampled from 1002 strains of STEC O157 received by the Gastrointestinal Bacteria Reference Unit at Public Health England, Colindale, in 2012. The genetic distance between each isolate, as estimated by WGS, was calculated and phylogenetic methods were used to place strains in an evolutionary context. RESULTS: Estimates of linked clusters representing STEC O157 outbreaks in England and Wales increased by 2-fold when WGS was used instead of traditional typing techniques. The previously unidentified clusters were often widely geographically distributed and small in size. Phylogenetic analysis facilitated identification of temporally distinct cases sharing common exposures and delineating those that shared epidemiological and temporal links. Comparison with multi locus variable number tandem repeat analysis (MLVA) showed that although MLVA is as sensitive as WGS, WGS provides a more timely resolution to outbreak clustering. CONCLUSIONS: WGS has come of age as a molecular typing tool to inform national surveillance of STEC O157; it can be used in real time to provide the highest strain-level resolution for outbreak investigation. WGS allows linked cases to be identified with unprecedented specificity and sensitivity that will facilitate targeted and appropriate public health investigations.

Porcine Epidemic Diarrhea Virus among Farmed Pigs, Ukraine.

An outbreak of porcine epidemic diarrhea occurred in the summer of 2014 in Ukraine, severely affecting piglets <10 days of age; the mortality rate approached 100%. Full genome sequencing showed the virus to be closely related to strains reported from North America, showing a sequence identity of up to 99.8%.

Genetic Characterization of Highly Pathogenic Avian Influenza (H5N8) Virus from Domestic Ducks, England, November 2014.

Genetic sequences of a highly pathogenic avian influenza (H5N8) virus in England have high homology to those detected in mainland Europe and Asia during 2014. Genetic characterization suggests this virus is an avian-adapted virus without specific affinity for zoonoses. Spatio-temporal detections of H5N8 imply a role for wild birds in virus spread.

A fetus with de novo 2q33.2q35 deletion including MAP2 with brain anomalies, esophageal atresia, and laryngeal stenosis.

Deletions of the long arm of chromosome 2 are rare. Few cases of interstitial deletions of the 2q33q35 region have been reported. Individuals with deletions in this region have growth retardation, psychomotor retardation, micrognathia, microcephaly, and apparently low-set ears. We describe a female fetus with a de novo deletion of 2q33.2 to q35 with delayed gyral formation with widespread neuronal heterotopia of the white matter, small cerebellum, esophageal atresia, laryngeal stenosis, micrognathia, and intrauterine growth retardation. With the use of karyotyping and high-resolution array comparative genomic hybridization the boundaries and gene content of the deletion were identified. Our aim was to determine whether a candidate gene for the brain phenotype was present in the deletion. By this means and based on literature we pinpointed the microtubule associated gene MAP2 as a candidate for the brain anomalies.

Double-negative B cells and DNASE1L3 colocalise with microbiota in gut-associated lymphoid tissue.

Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the subepithelial dome. The initial response to antigens and how bacteria are handled is incompletely understood. By iterative application of spatial transcriptomics and multiplexed single-cell technologies, we identify that the double negative 2 subset of B cells, previously associated with autoimmune diseases, is present in the subepithelial dome in health. We show that in this location double negative 2 B cells interact with dendritic cells co-expressing the lupus autoantigens DNASE1L3 and C1q and microbicides. We observe that in humans, but not in mice, dendritic cells expressing DNASE1L3 are associated with sampled bacteria but not DNA derived from apoptotic cells. We propose that fundamental features of autoimmune diseases are microbiota-associated, interacting components of normal intestinal immunity.

Next generation sequencing of viral RNA genomes.

BACKGROUND: With the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform. RESULTS: As representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers' minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed. CONCLUSIONS: The approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources.

Complete genome sequence of Ikoma lyssavirus.

Lyssaviruses (family Rhabdoviridae) constitute one of the most important groups of viral zoonoses globally. All lyssaviruses cause the disease rabies, an acute progressive encephalitis for which, once symptoms occur, there is no effective cure. Currently available vaccines are highly protective against the predominantly circulating lyssavirus species. Using next-generation sequencing technologies, we have obtained the whole-genome sequence for a novel lyssavirus, Ikoma lyssavirus (IKOV), isolated from an African civet in Tanzania displaying clinical signs of rabies. Genetically, this virus is the most divergent within the genus Lyssavirus. Characterization of the genome will help to improve our understanding of lyssavirus diversity and enable investigation into vaccine-induced immunity and protection.

Pasteurella sp. associated with fatal septicaemia in six African elephants.

The sudden mortality of African elephants (Loxodonta africana) in Botswana and Zimbabwe in 2020 provoked considerable public interest and speculation. Poaching and malicious poisoning were excluded early on in the investigation. Other potential causes included environmental intoxication, infectious diseases, and increased habitat stress due to ongoing drought. Here we show evidence of the mortalities in Zimbabwe as fatal septicaemia associated with Bisgaard taxon 45, an unnamed close relative of Pasteurella multocida. We analyse elephant carcasses and environmental samples, and fail to find evidence of cyanobacterial or other intoxication. Post-mortem and histological findings suggest a bacterial septicaemia similar to haemorrhagic septicaemia caused by P. multocida. Biochemical tests and 16S rDNA analysis of six samples and genomic analysis of one sample confirm the presence of Bisgaard taxon 45. The genome sequence contains many of the canonical P. multocida virulence factors associated with a range of human and animal diseases, including the pmHAS gene for hyaluronidase associated with bovine haemorrhagic septicaemia. Our results demonstrate that Bisgaard taxon 45 is associated with a generalised, lethal infection and that African elephants are susceptible to opportunistically pathogenic Pasteurella species. This represents an important conservation concern for elephants in the largest remaining metapopulation of this endangered species.

Genome sequence of Mycoplasma hyorhinis strain GDL-1.

Mycoplasma hyorhinis impacts swine health and production in many countries, either as a primary pathogen or as a component of a polymicrobial infection. Isolates of this species are also common contaminants of tissue culture lines. The genome sequence of the cell culture isolate M. hyorhinis GDL-1 is presented herein.

Environmental factors determining the epidemiology and population genetic structure of the Bacillus cereus group in the field.

Bacillus thuringiensis (Bt) and its insecticidal toxins are widely exploited in microbial biopesticides and genetically modified crops. Its population biology is, however, poorly understood. Important issues for the safe, sustainable exploitation of Bt include understanding how selection maintains expression of insecticidal toxins in nature, whether entomopathogenic Bt is ecologically distinct from related human pathogens in the Bacillus cereus group, and how the use of microbial pesticides alters natural bacterial populations. We addressed these questions with a MLST scheme applied to a field experiment in which we excluded/added insect hosts and microbial pesticides in a factorial design. The presence of insects increased the density of Bt/B. cereus in the soil and the proportion of strains expressing insecticidal toxins. We found a near-epidemic population structure dominated by a single entomopathogenic genotype (ST8) in sprayed and unsprayed enclosures. Biopesticidal ST8 proliferated in hosts after spraying but was also found naturally associated with leaves more than any other genotype. In an independent experiment several ST8 isolates proved better than a range of non-pathogenic STs at endophytic and epiphytic colonization of seedlings from soil. This is the first experimental demonstration of Bt behaving as a specialized insect pathogen in the field. These data provide a basis for understanding both Bt ecology and the influence of anthropogenic factors on Bt populations. This natural population of Bt showed habitat associations and a population structure that differed markedly from previous MLST studies of less ecologically coherent B. cereus sample collections. The host-specific adaptations of ST8, its close association with its toxin plasmid and its high prevalence within its clade are analogous to the biology of Bacillus anthracis. This prevalence also suggests that selection for resistance to the insecticidal toxins of ST8 will have been stronger than for other toxin classes.

Nocturnal plant respiration is under strong non-temperature control.

Most biological rates depend on the rate of respiration. Temperature variation is typically considered the main driver of daily plant respiration rates, assuming a constant daily respiration rate at a set temperature. Here, we show empirical data from 31 species from temperate and tropical biomes to demonstrate that the rate of plant respiration at a constant temperature decreases monotonically with time through the night, on average by 25% after 8 h of darkness. Temperature controls less than half of the total nocturnal variation in respiration. A new universal formulation is developed to model and understand nocturnal plant respiration, combining the nocturnal decrease in the rate of plant respiration at constant temperature with the decrease in plant respiration according to the temperature sensitivity. Application of the new formulation shows a global reduction of 4.5 -6 % in plant respiration and an increase of 7-10% in net primary production for the present-day.