From spelling to reading: An intervention program with children at risk of reading failure.

This study aimed to assess the impact of an interactive spelling program on reading acquisition of children at risk of developing reading difficulties as well as to assess its effect on spelling and phonemic awareness. From an initial pool of 144 first-grade children attending four Portuguese primary schools, 53 children with low performances in letter knowledge and phonemic awareness tasks, and considered by their teachers to be at risk of developing reading difficulties, were selected. These children were randomly assigned to three groups: an experimental group that underwent an interactive spelling program, a comparison group that underwent a phonological awareness program, and a control group that underwent a copying program. The programs, conducted in pairs, comprised 12 sessions lasting 20 to 30 min twice a week. The pretest and posttest included word reading, word spelling, and phonemic awareness assessments. Data analysis showed that the spelling group significantly outperformed the other groups across all measures except in the phonemic awareness task, where there were no differences with the phonological group. The word copying group consistently yielded the lowest results. Unlike the other two groups, the posttest results of the experimental group also reached the class average in word reading. For ethical reasons, after the final assessments the control group underwent a version of the interactive spelling program. This study suggests that spelling activities can contribute significantly to reading acquisition and can serve as a valuable pedagogical tool to proactively address challenges in learning to read.

Molecular epidemiology and transmission dynamics of multi-drug resistant tuberculosis strains using whole genome sequencing in the Amhara region, Ethiopia.

BACKGROUND: Drug resistant Mycobacterium tuberculosis prevention and care is a major challenge in Ethiopia. The World health organization has designated Ethiopia as one of the 30 high burden multi-drug resistant tuberculosis (MDR-TB) countries. There is limited information regarding genetic diversity and transmission dynamics of MDR-TB in Ethiopia. OBJECTIVE: To investigate the molecular epidemiology and transmission dynamics of MDR-TB strains using whole genome sequence (WGS) in the Amhara region. METHODS: Forty-five MDR-TB clinical isolates from Amhara region were collected between 2016 and 2018, and characterized using WGS and 24-loci Mycobacterium Interspersed Repetitive Units Variable Number of Tandem Repeats (MIRU-VNTR) typing. Clusters were defined based on the maximum distance of 12 single nucleotide polymorphisms (SNPs) or alleles as the upper threshold of genomic relatedness. Five or less SNPs or alleles distance or identical 24-loci VNTR typing is denoted as surrogate marker for recent transmission. RESULTS: Forty-one of the 45 isolates were analyzed by WGS and 44% (18/41) of the isolates were distributed into 4 clusters. Of the 41 MDR-TB isolates, 58.5% were classified as lineage 4, 36.5% lineage 3 and 5% lineage 1. Overall, TUR genotype (54%) was the predominant in MDR-TB strains. 41% (17/41) of the isolates were clustered into four WGS groups and the remaining isolates were unique strains. The predominant cluster (Cluster 1) was composed of nine isolates belonging to lineage 4 and of these, four isolates were in the recent transmission links. CONCLUSIONS: Majority of MDR-TB strain cluster and predominance of TUR lineage in the Amhara region give rise to concerns for possible ongoing transmission. Efforts to strengthen TB laboratory to advance diagnosis, intensified active case finding, and expanded contact tracing activities are needed in order to improve rapid diagnosis and initiate early treatment. This would lead to the interruption of the transmission chain and stop the spread of MDR-TB in the Amhara region.

Disease management at the wildlife-livestock interface: Using whole-genome sequencing to study the role of elk in Mycobacterium bovis transmission in Michigan, USA.

The role of wildlife in the persistence and spread of livestock diseases is difficult to quantify and control. These difficulties are exacerbated when several wildlife species are potentially involved. Bovine tuberculosis (bTB), caused by Mycobacterium bovis, has experienced an ecological shift in Michigan, with spillover from cattle leading to an endemically infected white-tailed deer (deer) population. It has potentially substantial implications for the health and well-being of both wildlife and livestock and incurs a significant economic cost to industry and government. Deer are known to act as a reservoir of infection, with evidence of M. bovis transmission to sympatric elk and cattle populations. However, the role of elk in the circulation of M. bovis is uncertain; they are few in number, but range further than deer, so may enable long distance spread. Combining Whole Genome Sequences (WGS) for M. bovis isolates from exceptionally well-observed populations of elk, deer and cattle with spatiotemporal locations, we use spatial and Bayesian phylogenetic analyses to show strong spatiotemporal admixture of M. bovis isolates. Clustering of bTB in elk and cattle suggests either intraspecies transmission within the two populations, or exposure to a common source. However, there is no support for significant pathogen transfer amongst elk and cattle, and our data are in accordance with existing evidence that interspecies transmission in Michigan is likely only maintained by deer. This study demonstrates the value of whole genome population studies of M. bovis transmission at the wildlife-livestock interface, providing insights into bTB management in an endemic system.

Broadwick: a framework for computational epidemiology.

BACKGROUND: Modelling disease outbreaks often involves integrating the wealth of data that are gathered during modern outbreaks into complex mathematical or computational models of transmission. Incorporating these data into simple compartmental epidemiological models is often challenging, requiring the use of more complex but also more efficient computational models. In this paper we introduce a new framework that allows for a more systematic and user-friendly way of building and running epidemiological models that efficiently handles disease data and reduces much of the boilerplate code that usually associated to these models. We introduce the framework by developing an SIR model on a simple network as an example. RESULTS: We develop Broadwick, a modular, object-oriented epidemiological framework that efficiently handles large epidemiological datasets and provides packages for stochastic simulations, parameter inference using Approximate Bayesian Computation (ABC) and Markov Chain Monte Carlo (MCMC) methods. Each algorithm used is fully customisable with sensible defaults that are easily overridden by custom algorithms as required. CONCLUSION: Broadwick is an epidemiological modelling framework developed to increase the productivity of researchers by providing a common framework with which to develop and share complex models. It will appeal to research team leaders as it allows for models to be created prior to a disease outbreak and has the ability to handle large datasets commonly found in epidemiological modelling.

Patterns of Hemorrhagic Disease in White-Tailed Deer (Odocoileus virginianus) in the Great Plains of the USA, 1982-2020.

Hemorrhagic disease (HD) of deer is caused by epizootic hemorrhagic disease virus (EHDV) or bluetongue virus (BTV) and is considered one of the most important viral diseases of white-tailed deer (Odocoileus virginianus). Despite evidence of changing patterns of HD in the northeastern and upper midwestern US, the historical and current patterns of HD in the Great Plains remain poorly described. We used results from an annual survey documenting HD mortality to characterize historic and current patterns of HD in the northern and central Great Plains (North Dakota, South Dakota, Nebraska, Kansas, and Oklahoma), US, between 1982 and 2020. Further, we assessed temporal change using linear regression to determine change in annual reporting intensity (percentage of counties in a state with reported HD) and change in reporting frequency (the number of years a county or state reported HD) during each decade between 1982 and 2020. Across the 38-yr study period, HD reports expanded northeast across latitude and longitude. Intensity of HD reports significantly increased during this period for three (North Dakota, South Dakota, Kansas) of five states examined. Frequency of reports also increased for all five states. Such changes in northern latitudes might lead to increased deer mortality in regions where HD epizootics have been historically less frequent. Understanding how patterns of HD are changing on the landscape is important when considering future deer management in the face of other mortality factors.

The selection of software and database for metagenomics sequence analysis impacts the outcome of microbial profiling and pathogen detection.

Shotgun metagenomic sequencing analysis is widely used for microbial profiling of biological specimens and pathogen detection. However, very little is known about the technical biases caused by the choice of analysis software and databases on the biological specimen. In this study, we evaluated different direct read shotgun metagenomics taxonomic profiling software to characterize the microbial compositions of simulated mice gut microbiome samples and of biological samples collected from wild rodents across multiple taxonomic levels. Using ten of the most widely used metagenomics software and four different databases, we demonstrated that obtaining an accurate species-level microbial profile using the current direct read metagenomics profiling software is still a challenging task. We also showed that the discrepancies in results when different databases and software were used could lead to significant variations in the distinct microbial taxa classified, in the characterizations of the microbial communities, and in the differentially abundant taxa identified. Differences in database contents and read profiling algorithms are the main contributors for these discrepancies. The inclusion of host genomes and of genomes of the interested taxa in the databases is important for increasing the accuracy of profiling. Our analysis also showed that software included in this study differed in their ability to detect the presence of Leptospira, a major zoonotic pathogen of one health importance, especially at the species level resolution. We concluded that using different databases and software combinations can result in confounding biological conclusions in microbial profiling. Our study warrants that software and database selection must be based on the purpose of the study.

Leptospira transcriptome sequencing using long-read technology reveals unannotated transcripts and potential polyadenylation of RNA molecules.

IMPORTANCE: Leptospirosis, caused by the spirochete bacteria Leptospira, is a zoonotic disease of humans and animals, accounting for over 1 million annual human cases and over 60,000 deaths. We have characterized operon transcriptional units, identified novel RNA coding regions, and reported evidence of potential posttranscriptional polyadenylation in the Leptospira transcriptomes for the first time using Oxford Nanopore Technology RNA sequencing protocols. The newly identified RNA coding regions and operon transcriptional units were detected only in the pathogenic Leptospira transcriptomes, suggesting their significance in virulence-related functions. This article integrates bioinformatics, infectious diseases, microbiology, molecular biology, veterinary sciences, and public health. Given the current knowledge gap in the regulation of leptospiral pathogenicity, our findings offer valuable insights to researchers studying leptospiral pathogenicity and provide both a basis and a tool for researchers focusing on prokaryotic molecular studies for the understanding of RNA compositions and prokaryotic polyadenylation for their organisms of interest.

The devil you know and the devil you don't: current status and challenges of bovine tuberculosis eradication in the United States.

Having entered into its second century, the eradication program for bovine tuberculosis (bTB, caused by Mycobacterium bovis) in the United States of America occupies a position both enviable and daunting. Excepting four counties in Michigan comprising only 6109 km2 (0.06% of US land area) classified as Modified Accredited, as of April 2022 the entire country was considered Accredited Free of bTB by the US Department of Agriculture for cattle and bison. On the surface, the now well-described circumstances of endemic bTB in Michigan, where white-tailed deer (Odocoileus virginianus) serve as a free-ranging wildlife maintenance host, may appear to be the principal remaining barrier to national eradication. However, the situation there is unique in the U.S., and far-removed from the broader issues of bTB control in the remainder of the country. In Michigan, extensive surveillance for bTB in deer over the last quarter century, and regulatory measures to maximize the harvest of publicly-owned wildlife, have been implemented and sustained. Prevalence of bTB in deer has remained at a low level, although not sufficiently low to eliminate cattle herd infections. Public attitudes towards bTB, cattle and deer, and their relative importance, have been more influential in the management of the disease than any limitations of biological science. However, profound changes in the demographics and social attitudes of Michigan's human population are underway, changes which are likely to force a critical reevaluation of the bTB control strategies thus far considered integral. In the rest of the U.S. where bTB is not self-sustaining in wildlife, changes in the scale of cattle production, coupled with both technical and non-technical issues have created their own substantial challenges. It is against this diverse backdrop that the evolution of whole genome sequencing of M. bovis has revolutionized understanding of the history and ecology of bTB in Michigan, resolved previously undiscernible epidemiological puzzles, provided insights into zoonotic transmission, and unified eradication efforts across species and agencies. We describe the current status of bTB eradication in the U.S., how circumstances and management have changed, what has been learned, and what remains more elusive than ever.

Genomic epidemiology of Mycobacterium bovis infection in sympatric badger and cattle populations in Northern Ireland.

Bovine tuberculosis (bTB) is a costly, epidemiologically complex, multi-host, endemic disease. Lack of understanding of transmission dynamics may undermine eradication efforts. Pathogen whole-genome sequencing improves epidemiological inferences, providing a means to determine the relative importance of inter- and intra-species host transmission for disease persistence. We sequenced an exceptional data set of 619 Mycobacterium bovis isolates from badgers and cattle in a 100 km2 bTB 'hotspot' in Northern Ireland. Historical molecular subtyping data permitted the targeting of an endemic pathogen lineage, whose long-term persistence provided a unique opportunity to study disease transmission dynamics in unparalleled detail. Additionally, to assess whether badger population genetic structure was associated with the spatial distribution of pathogen genetic diversity, we microsatellite genotyped hair samples from 769 badgers trapped in this area. Birth death models and TransPhylo analyses indicated that cattle were likely driving the local epidemic, with transmission from cattle to badgers being more common than badger to cattle. Furthermore, the presence of significant badger population genetic structure in the landscape was not associated with the spatial distribution of M. bovis genetic diversity, suggesting that badger-to-badger transmission is not playing a major role in transmission dynamics. Our data were consistent with badgers playing a smaller role in transmission of M. bovis infection in this study site, compared to cattle. We hypothesize, however, that this minor role may still be important for persistence. Comparison to other areas suggests that M. bovis transmission dynamics are likely to be context dependent, with the role of wildlife being difficult to generalize.

Selective sweep sites and SNP dense regions differentiate Mycobacterium bovis isolates across scales.

Mycobacterium bovis, a bacterial zoonotic pathogen responsible for the economically and agriculturally important livestock disease bovine tuberculosis (bTB), infects a broad mammalian host range worldwide. This characteristic has led to bidirectional transmission events between livestock and wildlife species as well as the formation of wildlife reservoirs, impacting the success of bTB control measures. Next Generation Sequencing (NGS) has transformed our ability to understand disease transmission events by tracking variant sites, however the genomic signatures related to host adaptation following spillover, alongside the role of other genomic factors in the M. bovis transmission process are understudied problems. We analyzed publicly available M. bovis datasets collected from 700 hosts across three countries with bTB endemic regions (United Kingdom, United States, and New Zealand) to investigate if genomic regions with high SNP density and/or selective sweep sites play a role in Mycobacterium bovis adaptation to new environments (e.g., at the host-species, geographical, and/or sub-population levels). A simulated M. bovis alignment was created to generate null distributions for defining genomic regions with high SNP counts and regions with selective sweeps evidence. Random Forest (RF) models were used to investigate evolutionary metrics within the genomic regions of interest to determine which genomic processes were the best for classifying M. bovis across ecological scales. We identified in the M. bovis genomes 14 and 132 high SNP density and selective sweep regions, respectively. Selective sweep regions were ranked as the most important in classifying M. bovis across the different scales in all RF models. SNP dense regions were found to have high importance in the badger and cattle specific RF models in classifying badger derived isolates from livestock derived ones. Additionally, the genes detected within these genomic regions harbor various pathogenic functions such as virulence and immunogenicity, membrane structure, host survival, and mycobactin production. The results of this study demonstrate how comparative genomics alongside machine learning approaches are useful to investigate further the nature of M. bovis host-pathogen interactions.

Monocytic-Myeloid Derived Suppressor Cells Suppress T-Cell Responses in Recovered SARS CoV2-Infected Individuals.

Coronavirus disease 2019 (COVID-19) caused by SARS Coronavirus 2 (CoV2) is associated with massive immune activation and hyperinflammatory response. Acute and severe CoV2 infection is characterized by the expansion of myeloid derived suppressor cells (MDSC) because of cytokine storm, these MDSC suppress T cell functions. However, the presence of MDSC and its effect on CoV2 antigen specific T cell responses in individuals long after first detection of CoV2 and recovery from infection has not been studied. We and others have previously shown that CD11b+CD33+CD14+HLA-DR-/lo monocytic MDSC (M-MDSC) are present in individuals with clinical recovery from viral infection. In this study, we compared the frequency, functional and transcriptional signatures of M-MDSC isolated from CoV2 infected individuals after 5-months of the first detection of the virus (CoV2+) and who were not infected with CoV2 (CoV2-). Compared to CoV2- individuals, M-MDSC were present in CoV2+ individuals at a higher frequency, the level of M-MDSC correlated with the quantity of IL-6 in the plasma. Compared to CoV2-, increased frequency of PD1+, CD57+ and CX3CR1+ T effector memory (TEM) cell subsets was also present in CoV2+ individuals, but these did not correlate with M-MDSC levels. Furthermore, depleting M-MDSC from peripheral blood mononuclear cells (PBMC) increased T cell cytokine production when cultured with the peptide pools of immune dominant spike glycoprotein (S), membrane (M), and nucleocapsid (N) antigens of CoV2. M-MDSC suppressed CoV2 S- antigen-specific T cell in ROS, Arginase, and TGFß dependent manner. Our gene expression, RNA-seq and pathway analysis studies further confirm that M-MDSC isolated from CoV2+ individuals are enriched in pathways that regulate both innate and adaptive immune responses, but the genes regulating these functions (HLA-DQA1, HLA-DQB1, HLA-B, NLRP3, IL1ß, CXCL2, CXCL1) remained downregulated in M-MDSC isolated from CoV2+ individuals. These results demonstrate that M-MDSC suppresses recall responses to CoV2 antigens long after recovery from infection. Our findings suggest M-MDSC as novel regulators of CoV2 specific T cell responses, and should be considered as target to augment responses to vaccine.

Leptospira enrichment culture followed by ONT metagenomic sequencing allows better detection of Leptospira presence and diversity in water and soil samples.

BACKGROUND: Leptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are the major transmission sources in humans and animals. Clusters of disease outbreaks are common during rainy seasons. METHODOLOGY/PRINCIPAL FINDINGS: In this study, to detect the presence of Leptospira, we applied PCR, direct metagenomic sequencing, and enrichment culture followed by PCR and metagenomic sequencing on water and soil samples. Direct sequencing and enrichment cultures followed by PCR or sequencing effectively detected pathogenic and nonpathogenic Leptospira compared to direct PCR and 16S amplification-based metagenomic sequencing in soil or water samples. Among multiple culture media evaluated, Ellinghausen-McCullough-Johnson-Harris (EMJH) media containing antimicrobial agents was superior in recovering and detecting Leptospira from the environmental samples. Our results show that enrichment culture followed by PCR can be used to confirm the presence of pathogenic Leptospira in environmental samples. Additionally, metagenomic sequencing on enrichment cultures effectively detects the abundance and diversity of Leptospira spp. from environmental samples. CONCLUSIONS/SIGNIFICANCE: The selection of methodology is critical when testing environmental samples for the presence of Leptospira. Selective enrichment culture improves Leptospira detection efficacy by PCR or metagenomic sequencing and can be used successfully to understand the presence and diversity of pathogenic Leptospira during environmental surveillance.

Whole Genome Sequencing Refines Knowledge on the Population Structure of Mycobacterium bovis from a Multi-Host Tuberculosis System.

Classical molecular analyses of Mycobacterium bovis based on spoligotyping and Variable Number Tandem Repeat (MIRU-VNTR) brought the first insights into the epidemiology of animal tuberculosis (TB) in Portugal, showing high genotypic diversity of circulating strains that mostly cluster within the European 2 clonal complex. Previous surveillance provided valuable information on the prevalence and spatial occurrence of TB and highlighted prevalent genotypes in areas where livestock and wild ungulates are sympatric. However, links at the wildlife-livestock interfaces were established mainly via classical genotype associations. Here, we apply whole genome sequencing (WGS) to cattle, red deer and wild boar isolates to reconstruct the M. bovis population structure in a multi-host, multi-region disease system and to explore links at a fine genomic scale between M. bovis from wildlife hosts and cattle. Whole genome sequences of 44 representative M. bovis isolates, obtained between 2003 and 2015 from three TB hotspots, were compared through single nucleotide polymorphism (SNP) variant calling analyses. Consistent with previous results combining classical genotyping with Bayesian population admixture modelling, SNP-based phylogenies support the branching of this M. bovis population into five genetic clades, three with apparent geographic specificities, as well as the establishment of an SNP catalogue specific to each clade, which may be explored in the future as phylogenetic markers. The core genome alignment of SNPs was integrated within a spatiotemporal metadata framework to further structure this M. bovis population by host species and TB hotspots, providing a baseline for network analyses in different epidemiological and disease control contexts. WGS of M. bovis isolates from Portugal is reported for the first time in this pilot study, refining the spatiotemporal context of TB at the wildlife-livestock interface and providing further support to the key role of red deer and wild boar on disease maintenance. The SNP diversity observed within this dataset supports the natural circulation of M. bovis for a long time period, as well as multiple introduction events of the pathogen in this Iberian multi-host system.

The Many Hosts of Mycobacteria 8 (MHM8): A conference report.

Mycobacteria are important causes of disease in human and animal hosts. Diseases caused by mycobacteria include leprosy, tuberculosis (TB), nontuberculous mycobacteria (NTM) infections and Buruli Ulcer. To better understand and treat mycobacterial disease, clinicians, veterinarians and scientists use a range of discipline-specific approaches to conduct basic and applied research, including conducting epidemiological surveys, patient studies, wildlife sampling, animal models, genetic studies and computational simulations. To foster the exchange of knowledge and collaboration across disciplines, the Many Hosts of Mycobacteria (MHM) conference series brings together clinical, veterinary and basic scientists who are dedicated to advancing mycobacterial disease research. Started in 2007, the MHM series recently held its 8th conference at the Albert Einstein College of Medicine (Bronx, NY). Here, we review the diseases discussed at MHM8 and summarize the presentations on research advances in leprosy, NTM and Buruli Ulcer, human and animal TB, mycobacterial disease comorbidities, mycobacterial genetics and 'omics, and animal models. A mouse models workshop, which was held immediately after MHM8, is also summarized. In addition to being a resource for those who were unable to attend MHM8, we anticipate this review will provide a benchmark to gauge the progress of future research concerning mycobacteria and their many hosts.

Mechanistic analysis of the search behaviour of Caenorhabditis elegans.

A central question in movement research is how animals use information and movement to promote encounter success. Current random search theory identifies reorientation patterns as key to the compromise between optimizing encounters for both nearby and faraway targets, but how the balance between intrinsic motor programmes and previous environmental experience determines the occurrence of these reorientation behaviours remains unknown. We used high-resolution tracking and imaging data to describe the complete motor behaviour of Caenorhabditis elegans when placed in a novel environment (one in which food is absent). Movement in C. elegans is structured around different reorientation behaviours, and we measured how these contributed to changing search strategies as worms became familiar with their new environment. This behavioural transition shows that different reorientation behaviours are governed by two processes: (i) an environmentally informed 'extrinsic' strategy that is influenced by recent experience and that controls for area-restricted search behaviour, and (ii) a time-independent, 'intrinsic' strategy that reduces spatial oversampling and improves random encounter success. Our results show how movement strategies arise from a balance between intrinsic and extrinsic mechanisms, that search behaviour in C. elegans is initially determined by expectations developed from previous environmental experiences, and which reorientation behaviours are modified as information is acquired from new environments.

Escrita Inventada e Aquisição da Leitura em Crianças de Idade Pré-escolar / Invented Spelling and Reading Acquisition in Pre-school-age Children

RESUMOVários investigadores mostraram que actividades de escrita inventada com crianças em idade pré-escolar contribuem para a aquisição da literacia, tendo um impacto positivo na evolução da sua escrita e consciência fonológica. O nosso objectivo foi avaliar o impacto de um programa de escrita inventada na aquisição da leitura. Participaram 60 crianças Portuguesas de 5 anos que não sabiam ler nem escrever. Foram aleatoriamente divididas em dois grupos, experimental e controlo, equivalentes quanto às letras conhecidas, consciência fonológica e inteligência. O grupo experimental participou num programa de escrita inventada e o grupo de controlo num programa de leitura de histórias. A leitura de palavras foi avaliada num pré-teste, pós-teste e pós-teste postergado. O grupo experimental teve resultados superiores nos dois pós-testes.

ABSTRACTSeveral researchers have shown that invented spelling activities with pre-school-age children contribute to literacy acquisition, having a positive impact on the evolution of children's writing and phonological awareness. Our aim was to assess the impact of an invented spelling programme on reading acquisition. The participants were 60 Portuguese 5-year-old children who were not able to read or write. Children were randomly divided in two groups, experimental and control, equivalent in terms of knowledge of letters, phonological abilities and intelligence. The experimental group participated in an invented spelling programme and the control group in a story reading programme. Children's word reading was evaluated in a pre-test, a post-test and a differed post-test. Experimental group had better results in both post-tests.