Metagenomic characterisation of the gastrointestinal virome of neonatal pigs

Microorganisms that colonise the gastrointestinal tract are responsible for a large portion of the genetic diversity of the body. These microorganisms are of bacterial, archaeal and viral origin. The living space of these microorganisms, the microbiome, holds numerous interactions both between each other and the host. The viral part of the microbiome, the virome, consists of a multitude of virus species. These viruses infect and modulate cells from all three domains of life. Even though viruses have been acknowledged for their abilities to induce disease in its host, knowledge about the total diversity of viruses within the virome, and the role it plays in health and disease, is so far scarce. It is thought that the virome co-evolved with the host and that its establishment in mammals occurs early in life. The virome can be studied by the use of viral metagenomics, the study of all viral genetic material within a sample. Viral metagenomics was used in this thesis to generate datasets for comparative metagenomics. These datasets were then used for disease investigation and to compare similarities in the viromes of two mammalian species, pigs and humans. This thesis establishes a methodological framework for studying the virome in mammals, by use of viral metagenomics. A methodology for amplifying the metagenome prior to sequencing was assessed and a software for bioinformatics analysis of viral metagenomes was developed. With the methodologies developed herein, the eukaryotic virome of neonatal piglets suffering from diarrhoea was investigated. Several known enteric viruses were detected using viral metagenomics on healthy and diarrhoeic neonatal piglets. However, no virus was present exclusively within sick or healthy piglets and no virologial cause could be established for the neonatal diarrhoea. Comparative viral metagenomics was also used to establish if similarities existed between neonates of porcine and human origin, as well as between adults and neonates. Similarities were detected between adults of both species, who seems to be sharing a considerable part of their virome. There was also a notable difference between neonatal viromes and adult viromes, further supporting established theories about diversification over time of the virome

Identification of Transmission Routes of Campylobacter and On-Farm Measures to Reduce Campylobacter in Chicken

An in-depth analysis was performed on Swedish broiler producers that had delivered chickens with Campylobacter to slaughter over several years, in order to identify possible transmission routes and formulate effective measures to prevent chickens being colonized with Campylobacter. Between 2017 and 2019, 626 samples were collected at farm level and Campylobacter was isolated from 133 (21.2%). All C. jejuni and C. coli isolated from these samples were whole-genome sequenced, together with isolates from the corresponding cecum samples at slaughter (n = 256). Core genome multi-locus sequence typing (cgMLST) analysis, using schemes consisting of 1140 and 529 genes for C. jejuni and C. coli, respectively, revealed that nearby cattle, contaminated drinking water, water ponds, transport crates, and parent flocks were potential reservoirs of Campylobacter. A novel feature compared with previous studies is that measures were implemented and tested during the work. These contributed to a nationwide decrease in Campylobacter-positive flocks from 15.4% in 2016 to 4.6% in 2019, which is the lowest ever rate in Sweden. To conclude, there are different sources and routes of Campylobacter transmission to chickens from different broiler producers, and individual measures must be taken by each producer to prevent Campylobacter colonization of chickens

Field-Adapted Full Genome Sequencing of Peste-Des-Petits-Ruminants Virus Using Nanopore Sequencing

Peste-des-petits-ruminants virus (PPRV) is currently the focus of a control and eradication program. Full genome sequencing has the opportunity to become a powerful tool in the eradication program by improving molecular epidemiology and the study of viral evolution. PPRV is prevalent in many resource-constrained areas, with long distances to laboratory facilities, which can lack the correct equipment for high-throughput sequencing. Here we present a protocol for near full or full genome sequencing of PPRV. The use of a portable miniPCR and MinION brings the laboratory to the field and in addition makes the production of a full genome possible within 24 h of sampling. The protocol has been successfully used on virus isolates from cell cultures and field isolates from tissue samples of naturally infected goats

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole

Publisher's version (útgefin grein)Background: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10-9 M, 10-11 M, 10-13 M), pyrantel citrate (10-6 M, 10-8 M, 10-10 M), thiabendazole (10-5 M, 10-7 M, 10-9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system. Results: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10-9 M of ivermectin. Conclusions: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.[Figure not available: see fulltext.]This work was supported by the Swedish research council FORMAS (grant no. 942-2015-508).Peer Reviewe

Cross-Sectional Variations in Structure and Function of Coral Reef Microbiome With Local Anthropogenic Impacts on the Kenyan Coast of the Indian Ocean

Coral reefs face an increased number of environmental threats from anthropomorphic climate change and pollution from agriculture, industries and sewage. Because environmental changes lead to their compositional and functional shifts, coral reef microbial communities can serve as indicators of ecosystem impacts through development of rapid and inexpensive molecular monitoring tools. Little is known about coral reef microbial communities of the Western Indian Ocean (WIO). We compared taxonomic and functional diversity of microbial communities inhabiting near-coral seawater and sediments from Kenyan reefs exposed to varying impacts of human activities. Over 19,000 species (bacterial, viral and archaeal combined) and 4,500 clusters of orthologous groups of proteins (COGs) were annotated. The coral reefs showed variations in the relative abundances of ecologically significant taxa, especially copiotrophic bacteria and coliphages, corresponding to the magnitude of the neighboring human impacts in the respective sites. Furthermore, the near-coral seawater and sediment metagenomes had an overrepresentation of COGs for functions related to adaptation to diverse environments. Malindi and Mombasa marine parks, the coral reef sites closest to densely populated settlements were significantly enriched with genes for functions suggestive of mitigation of environment perturbations including the capacity to reduce intracellular levels of environmental contaminants and repair of DNA damage. Our study is the first metagenomic assessment of WIO coral reef microbial diversity which provides a much-needed baseline for the region, and points to a potential area for future research toward establishing indicators of environmental perturbations

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole

Background: Parascaris univalensis a pathogenic parasite of foals and yearlings worldwide. In recent years,Parascarisspp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adultP. univalensworms afterin vitroexposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways.Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10(-9) M, 10(-11) M, 10(-13) M), pyrantel citrate (10(-6) M, 10(-8) M, 10(-10) M), thiabendazole (10(-5) M, 10(-7) M, 10(-9) M) or without anthelmintics (control) at 37 degrees C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system.Results: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj &lt; 0.05 and log2 fold change >= 1 or &lt;= - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunitlgc-37, which was upregulated only in worms exposed to 10(-9) M of ivermectin.Conclusions: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described inP. univalensand future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance

MetLab: An In Silico Experimental Design, Simulation and Analysis Tool for Viral Metagenomics Studies.

Metagenomics, the sequence characterization of all genomes within a sample, is widely used as a virus discovery tool as well as a tool to study viral diversity of animals. Metagenomics can be considered to have three main steps; sample collection and preparation, sequencing and finally bioinformatics. Bioinformatic analysis of metagenomic datasets is in itself a complex process, involving few standardized methodologies, thereby hampering comparison of metagenomics studies between research groups. In this publication the new bioinformatics framework MetLab is presented, aimed at providing scientists with an integrated tool for experimental design and analysis of viral metagenomes. MetLab provides support in designing the metagenomics experiment by estimating the sequencing depth needed for the complete coverage of a species. This is achieved by applying a methodology to calculate the probability of coverage using an adaptation of Stevens' theorem. It also provides scientists with several pipelines aimed at simplifying the analysis of viral metagenomes, including; quality control, assembly and taxonomic binning. We also implement a tool for simulating metagenomics datasets from several sequencing platforms. The overall aim is to provide virologists with an easy to use tool for designing, simulating and analyzing viral metagenomes. The results presented here include a benchmark towards other existing software, with emphasis on detection of viruses as well as speed of applications. This is packaged, as comprehensive software, readily available for Linux and OSX users at https://github.com/norling/metlab

Cross-Sectional Variations in Structure and Function of Coral Reef Microbiome With Local Anthropogenic Impacts on the Kenyan Coast of the Indian Ocean

Coral reefs face an increased number of environmental threats from anthropomorphic climate change and pollution from agriculture, industries and sewage. Because environmental changes lead to their compositional and functional shifts, coral reef microbial communities can serve as indicators of ecosystem impacts through development of rapid and inexpensive molecular monitoring tools. Little is known about coral reef microbial communities of the Western Indian Ocean (WIO). We compared taxonomic and functional diversity of microbial communities inhabiting near-coral seawater and sediments from Kenyan reefs exposed to varying impacts of human activities. Over 19,000 species (bacterial, viral and archaeal combined) and 4,500 clusters of orthologous groups of proteins (COGs) were annotated. The coral reefs showed variations in the relative abundances of ecologically significant taxa, especially copiotrophic bacteria and coliphages, corresponding to the magnitude of the neighboring human impacts in the respective sites. Furthermore, the near-coral seawater and sediment metagenomes had an overrepresentation of COGs for functions related to adaptation to diverse environments. Malindi and Mombasa marine parks, the coral reef sites closest to densely populated settlements were significantly enriched with genes for functions suggestive of mitigation of environment perturbations including the capacity to reduce intracellular levels of environmental contaminants and repair of DNA damage. Our study is the first metagenomic assessment of WIO coral reef microbial diversity which provides a much-needed baseline for the region, and points to a potential area for future research toward establishing indicators of environmental perturbations