SAD rats: Effects of short photoperiod and carbohydrate consumption on sleep, liver steatosis, and the gut microbiome in diurnal grass rats.

Seasonal affective disorder (SAD) is a recurrent depression triggered by exposure to short photoperiods, with a subset of patients reporting hypersomnia, increased appetite, and carbohydrate craving. Dysfunction of the microbiota - gut - brain axis is frequently associated with depressive disorders, but its role in SAD is unknown. Nile grass rats (Arvicanthis niloticus) are potentially useful for exploring the pathophysiology of SAD, as they are diurnal and have been found to exhibit anhedonia and affective-like behavior in response to short photoperiods. Further, given grass rats have been found to spontaneously develop metabolic syndrome, they may be particularly susceptible to environmental triggers of metabolic dysbiosis. We conducted a 2 × 2 factorial design experiment to test the effects of short photoperiod (4 h:20 h Light:Dark (LD) vs. neutral 12:12 LD), access to a high concentration (8%) sucrose solution, and the interaction between the two, on activity, sleep, liver steatosis, and the gut microbiome of grass rats. We found that animals on short photoperiods maintained robust diel rhythms and similar subjective day lengths as controls in neutral photoperiods but showed disrupted activity and sleep patterns (i.e. a return to sleep after an initial bout of activity that occurs ~ 13 h before lights off). We found no evidence that photoperiod influenced sucrose consumption. By the end of the experiment, some grass rats were overweight and exhibited signs of non-alcoholic fatty liver disease (NAFLD) with micro- and macro-steatosis. However, neither photoperiod nor access to sucrose solution significantly affected the degree of liver steatosis. The gut microbiome of grass rats varied substantially among individuals, but most variation was attributable to parental effects and the microbiome was unaffected by photoperiod or access to sucrose. Our study indicates short photoperiod leads to disrupted activity and sleep in grass rats but does not impact sucrose consumption or exacerbate metabolic dysbiosis and NAFLD.

Nanopore adaptive sampling enriches for antimicrobial resistance genes in microbial communities.

Antimicrobial resistance (AMR) is a global public health threat. Environmental microbial communities act as reservoirs for AMR, containing genes associated with resistance, their precursors, and the selective pressures promoting their persistence. Genomic surveillance could provide insights into how these reservoirs change and impact public health. Enriching for AMR genomic signatures in complex microbial communities would strengthen surveillance efforts and reduce time-to-answer. Here, we tested the ability of nanopore sequencing and adaptive sampling to enrich for AMR genes in a mock community of environmental origin. Our setup implemented the MinION mk1B, an NVIDIA Jetson Xavier GPU, and Flongle flow cells. Using adaptive sampling, we observed consistent enrichment by composition. On average, adaptive sampling resulted in a target composition 4× higher than without adaptive sampling. Despite a decrease in total sequencing output, adaptive sampling increased target yield in most replicates. We also demonstrate enrichment in a diverse community using an environmental sample. This method enables rapid and flexible genomic surveillance.

Nanopore Adaptive Sampling Enriches for Antimicrobial Resistance Genes in Microbial Communities.

Antimicrobial resistance (AMR) is a global public health threat. Environmental microbial communities act as reservoirs for AMR, containing genes associated with resistance, their precursors, and the selective pressures to encourage their persistence. Genomic surveillance could provide insight into how these reservoirs are changing and their impact on public health. The ability to enrich for AMR genomic signatures in complex microbial communities would strengthen surveillance efforts and reduce time-to-answer. Here, we test the ability of nanopore sequencing and adaptive sampling to enrich for AMR genes in a mock community of environmental origin. Our setup implemented the MinION mk1B, an NVIDIA Jetson Xavier GPU, and flongle flow cells. We observed consistent enrichment by composition when using adaptive sampling. On average, adaptive sampling resulted in a target composition that was 4x higher than a treatment without adaptive sampling. Despite a decrease in total sequencing output, the use of adaptive sampling increased target yield in most replicates.

Epidemiological Characterization of African Swine Fever Dynamics in Ukraine, 2012-2023.

African swine fever (ASF) is a viral disease, endemic to Africa, that causes high mortality when introduced into domestic pig populations. Since the emergence of p72-genotype II African swine fever virus (ASFV) in Georgia in 2007, an ASF epidemic has been spreading across Europe and many countries in Asia. The epidemic first reached Ukraine in 2012. To better understand the dynamics of spread of ASF in Ukraine, we analyzed spatial and temporal outbreak data reported in Ukraine between 2012 and mid-2023. The highest numbers of outbreaks were reported in 2017 (N = 163) and 2018 (N = 145), with overall peak numbers of ASF outbreaks reported in August (domestic pigs) and January (wild boars). While cases were reported from most of Ukraine, we found a directional spread from the eastern and northern borders towards the western and southern regions of Ukraine. Many of the early outbreaks (before 2016) were adjacent to the border, which is again true for more recent outbreaks in wild boar, but not for recent outbreaks in domestic pigs. Outbreaks prior to 2016 also occurred predominantly in areas with a below average domestic pig density. This new analysis suggests that wild boars may have played an important role in the introduction and early spread of ASF in Ukraine. However, in later years, the dynamic suggests human activity as the predominant driver of spread and a separation of ASF epizootics between domestic pigs and in wild boars. The decline in outbreaks since 2019 suggests that the implemented mitigation strategies are effective, even though long-term control or eradication remain challenging and will require continued intensive surveillance of ASF outbreak patterns.

Discovery of Avian Paramyxoviruses APMV-1 and APMV-6 in Shorebirds and Waterfowl in Southern Ukraine.

Emerging RNA virus infections are a growing concern among domestic poultry industries due to the severe impact they can have on flock health and economic livelihoods. Avian paramyxoviruses (APMV; avulaviruses, AaV) are pathogenic, negative-sense RNA viruses that cause serious infections in the respiratory and central nervous systems. APMV was detected in multiple avian species during the 2017 wild bird migration season in Ukraine and studied using PCR, virus isolation, and sequencing. Of 4090 wild bird samples collected, mostly from southern Ukraine, eleven isolates were grown in ovo and identified for APMV serotype by hemagglutinin inhibition test as: APMV-1, APMV-4, APMV-6, and APMV-7. To build One Health's capacity to characterize APMV virulence and analyze the potential risks of spillover to immunologically naïve populations, we sequenced virus genomes in veterinary research labs in Ukraine using a nanopore (MinION) platform. RNA was extracted and amplified using a multiplex tiling primer approach to specifically capture full-length APMV-1 (n = 5) and APMV-6 (n = 2) genomes at high read depth. All APMV-1 and APMV-6 fusion (F) proteins possessed a monobasic cleavage site, suggesting these APMVs were likely low virulence, annually circulating strains. Utilization of this low-cost method will identify gaps in viral evolution and circulation in this understudied but important critical region for Eurasia.

A Repeat Pattern of Founder Events for SARS-CoV-2 Variants in Alaska.

Alaska is a unique US state because of its large size, geographically disparate population density, and physical distance from the contiguous United States. Here, we describe a pattern of SARS-CoV-2 variant emergence across Alaska reflective of these differences. Using genomic data, we found that in Alaska, the Omicron sublineage BA.2.3 overtook BA.1.1 by the week of 27 February 2022, reaching 48.5% of sequenced cases. On the contrary, in the contiguous United States, BA.1.1 dominated cases for longer, eventually being displaced by BA.2 sublineages other than BA.2.3. BA.2.3 only reached a prevalence of 10.9% in the contiguous United States. Using phylogenetics, we found evidence of potential origins of the two major clades of BA.2.3 in Alaska and with logistic regression estimated how it emerged and spread throughout the state. The combined evidence is suggestive of founder events in Alaska and is reflective of how Alaska's unique dynamics influence the emergence of SARS-CoV-2 variants.

Pattern of SARS-CoV-2 variant B.1.1.519 emergence in Alaska.

Alaska has the lowest population density in the United States (US) with a mix of urban centers and isolated rural communities. Alaska's distinct population dynamics compared to the contiguous US may have contributed to unique patterns of SARS-CoV-2 variants observed in early 2021. Here we examined 2323 SARS-CoV-2 genomes from Alaska and 278,635 from the contiguous US collected from December 2020 through June 2021 because of the notable emergence and spread of lineage B.1.1.519 in Alaska. We found that B.1.1.519 was consistently detected from late January through June of 2021 in Alaska with a peak prevalence in April of 77.9% unlike the rest of the US at 4.6%. The earlier emergence of B.1.1.519 coincided with a later peak of Alpha (B.1.1.7) compared to the contiguous US. We also observed differences in variant composition over time between the two most populated regions of Alaska and a modest increase in COVID-19 cases during the peak incidence of B.1.1.519. However, it is difficult to disentangle how social dynamics conflated changes in COVID-19 during this time. We suggest that the viral characteristics, such as amino acid substitutions in the spike protein, likely contributed to the unique spread of B.1.1.519 in Alaska.

Genome Sequence of Erysipelothrix sp. Strain Poltava, Isolated from Acute Septic Erysipelas of Swine in Ukraine.

The complete genome of Erysipelothrix sp. strain Poltava, isolated from fatal acute septic erysipelas of swine in Ukraine, was assembled using Nanopore sequences. One circular chromosome of 1,794,858 bp (N50, 1,794,858 bp) encodes 16 putative antibiotic resistance genes and secreted virulence factors, highlighting the risk of cross-species livestock and human infection.

A repeat pattern of founder events for SARS-CoV-2 variants in Alaska.

Alaska is a unique US state because of its large size, geographically disparate population density, and physical distance from the contiguous United States. Here, we describe a pattern of SARS-CoV-2 variant emergence across Alaska reflective of these differences. Using genomic data, we found that in Alaska the Omicron sublineage BA.2.3 overtook BA.1.1 by the week of 2022-02-27, reaching 48.5% of sequenced cases. On the contrary in the contiguous United States, BA.1.1 dominated cases for longer, eventually being displaced by BA.2 sublineages other than BA.2.3. BA.2.3 only reached a prevalence of 10.9% in the contiguous United States. Using phylogenetics, we found evidence of potential origins of the two major clades of BA.2.3 in Alaska and with logistic regression estimated how it emerged and spread throughout the state. The combined evidence is suggestive of founder events in Alaska and is reflective of how Alaska’s unique dynamics influence the emergence of SARS-CoV-2 variants.

Unearthing Shifts in Microbial Communities Across a Soil Disturbance Gradient.

Permafrost, an important source of soil disturbance, is particularly vulnerable to climate change in Alaska where 85% of the land is underlained with discontinuous permafrost. Boreal forests, home to plants integral to subsistence diets of many Alaska Native communities, are not immune to the effects of climate change. Soil disturbance events, such as permafrost thaw, wildfires, and land use change can influence abiotic conditions, which can then affect active layer soil microbial communities. In a previous study, we found negative effects on boreal plants inoculated with microbes impacted by soil disturbance compared to plants inoculated with microbes from undisturbed soils. Here, we identify key shifts in microbial communities altered by soil disturbance using 16S rRNA gene sequencing and make connections between microbial community changes and previously observed plant growth. Additionally, we identify further community shifts in potential functional mechanisms using long read metagenomics. Across a soil disturbance gradient, microbial communities differ significantly based on the level of soil disturbance. Consistent with the earlier study, the family Acidobacteriaceae, which consists of known plant growth promoters, was abundant in undisturbed soil, but practically absent in most disturbed soil. In contrast, Comamonadaceae, a family with known agricultural pathogens, was overrepresented in most disturbed soil communities compared to undisturbed. Within our metagenomic data, we found that soil disturbance level is associated with differences in microbial community function, including mechanisms potentially involved in plant pathogenicity. These results indicate that a decrease in plant growth can be linked to changes in the microbial community and functional composition driven by soil disturbance and climate change. Together, these results build a genomic understanding of how shifting soil microbiomes may affect plant productivity and ecosystem health as the Arctic warms.

Genetic Diversity of Porcine Circovirus 2 in Wild Boar and Domestic Pigs in Ukraine.

Porcine circovirus type 2 (PCV2) is responsible for a number of porcine circovirus-associated diseases (PCVAD) that can severely impact domestic pig herds. For a non-enveloped virus with a small genome (1.7 kb ssDNA), PCV2 is remarkably diverse, with eight genotypes (a-h). New genotypes of PCV2 can spread through the migration of wild boar, which are thought to infect domestic pigs and spread further through the domestic pig trade. Despite a large swine population, the diversity of PCV2 genotypes in Ukraine has been under-sampled, with few PCV2 genome sequences reported in the past decade. To gain a deeper understanding of PCV2 genotype diversity in Ukraine, samples of blood serum were collected from wild boars (n = 107) that were hunted in Ukraine during the November-December 2012 hunting season. We found 34/107 (31.8%) prevalence of PCV2 by diagnostic PCR. For domestic pigs, liver samples (n = 16) were collected from a commercial market near Kharkiv in 2019, of which 6 out of 16 (37%) samples were positive for PCV2. We sequenced the genotyping locus ORF2, a gene encoding the PCV2 viral capsid (Cap), for 11 wild boar and six domestic pig samples in Ukraine using an Oxford Nanopore MinION device. Of 17 samples with resolved genotypes, the PCV2 genotype b was the most common in wild boar samples (10 out of 11, 91%), while the domestic pigs were infected with genotypes b and d. We also detected genotype b/d and b/a co-infections in wild boars and domestic pigs, respectively, and for the first time in Ukraine we detected genotype f in a wild boar from Poltava. Building a maximum-likelihood phylogeny, we identified a sublineage of PCV2 genotype b infections in both wild and domestic swine, suggesting a possible epizootic cluster and an ecological interaction between wild boar and domestic pig populations in northeastern Ukraine.

Accuracy and Completeness of Long Read Metagenomic Assemblies.

Microbes influence the surrounding environment and contribute to human health. Metagenomics can be used as a tool to explore the interactions between microbes. Metagenomic assemblies built using long read nanopore data depend on the read level accuracy. The read level accuracy of nanopore sequencing has made dramatic improvements over the past several years. However, we do not know if the increased read level accuracy allows for faster assemblers to make as accurate metagenomic assemblies as slower assemblers. Here, we present the results of a benchmarking study comparing three commonly used long read assemblers, Flye, Raven, and Redbean. We used a prepared DNA standard of seven bacteria as our input community. We prepared a sequencing library using a VolTRAX V2 and sequenced using a MinION mk1b. We basecalled with Guppy v5.0.7 using the super-accuracy model. We found that increasing read depth benefited each of the assemblers, and nearly complete community member chromosomes were assembled with as little as 10× read depth. Polishing assemblies using Medaka had a predictable improvement in quality. We found Flye to be the most robust across taxa and was the most effective assembler for recovering plasmids. Based on Flye's consistency for chromosomes and increased effectiveness at assembling plasmids, we would recommend using Flye in future metagenomic studies.

Complete Genome Sequence of Brucella abortus 68, Isolated from Aborted Fetal Sheep in Ukraine.

The complete genome sequence of Brucella abortus 68, isolated from an aborted sheep fetus in Luhansk, Ukraine, was assembled using Nanopore sequences. Two circular chromosomes totaling 3,281,317 bp (N 50, 2,124,943 bp) comprised the complete genome sequence. The strain encodes the fosfomycin antibiotic resistance gene fosX, highlighting the risk of cross-species livestock and human infection.

Soil Disturbance Affects Plant Productivity via Soil Microbial Community Shifts.

Recent advances in climate research have discovered that permafrost is particularly vulnerable to the changes occurring in the atmosphere and climate, especially in Alaska where 85% of the land is underlain by mostly discontinuous permafrost. As permafrost thaws, research has shown that natural and anthropogenic soil disturbance causes microbial communities to undergo shifts in membership composition and biomass, as well as in functional diversity. Boreal forests are home to many plants that are integral to the subsistence diets of many Alaska Native communities. Yet, it is unclear how the observed shifts in soil microbes can affect above ground plant communities that are relied on as a major source of food. In this study, we tested the hypothesis that microbial communities associated with permafrost thaw affect plant productivity by growing five plant species found in Boreal forests and Tundra ecosystems, including low-bush cranberry and bog blueberry, with microbial communities from the active layer soils of a permafrost thaw gradient. We found that plant productivity was significantly affected by the microbial soil inoculants. Plants inoculated with communities from above thawing permafrost showed decreased productivity compared to plants inoculated with microbes from undisturbed soils. We used metagenomic sequencing to determine that microbial communities from disturbed soils above thawing permafrost differ in taxonomy from microbial communities in undisturbed soils above intact permafrost. The combination of these results indicates that a decrease in plant productivity can be linked to soil disturbance driven changes in microbial community membership and abundance. These data contribute to an understanding of how microbial communities can be affected by soil disturbance and climate change, and how those community shifts can further influence plant productivity in Boreal forests and more broadly, ecosystem health.

Unearthing Antibiotic Resistance Associated with Disturbance-Induced Permafrost Thaw in Interior Alaska.

Monitoring antibiotic resistance genes (ARGs) across ecological niches is critical for assessing the impacts distinct microbial communities have on the global spread of resistance. In permafrost-associated soils, climate and human driven disturbances augment near-surface thaw shifting the predominant bacteria that shape the resistome in overlying active layer soils. This thaw is of concern in Alaska, because 85% of land is underlain by permafrost, making soils especially vulnerable to disturbances. The goal of this study is to assess how soil disturbance, and the subsequent shift in community composition, will affect the types, abundance, and mobility of ARGs that compose the active layer resistome. We address this goal through the following aims: (1) assess resistance phenotypes through antibiotic susceptibility testing, and (2) analyze types, abundance, and mobility of ARGs through whole genome analyses of bacteria isolated from a disturbance-induced thaw gradient in Interior Alaska. We found a high proportion of isolates resistant to at least one of the antibiotics tested with the highest prevalence of resistance to ampicillin. The abundance of ARGs and proportion of resistant isolates increased with disturbance; however, the number of ARGs per isolate was explained more by phylogeny than isolation site. When compared to a global database of soil bacteria, RefSoil+, our isolates from the same genera had distinct ARGs with a higher proportion on plasmids. These results emphasize the hypothesis that both phylogeny and ecology shape the resistome and suggest that a shift in community composition as a result of disturbance-induced thaw will be reflected in the predominant ARGs comprising the active layer resistome.

Complete Genome Sequence of Salmonella enterica subsp. enterica Serovar Kottbus Strain Kharkiv, Isolated from a Commercial Pork Production Facility in Ukraine.

The complete genome of Salmonella enterica subsp. enterica serovar Kottbus strain Kharkiv (serogroup C2-C3), which was isolated from a commercial pork production facility in Kharkiv, Ukraine, was assembled using long-read Nanopore sequences. A single circular contig (4,799,045 bp) comprised a complete chromosome encoding antibiotic resistance, highlighting the risk of cross-species livestock and human infection.

Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function.

BACKGROUND: Riverine ecosystems are biogeochemical powerhouses driven largely by microbial communities that inhabit water columns and sediments. Because rivers are used extensively for anthropogenic purposes (drinking water, recreation, agriculture, and industry), it is essential to understand how these activities affect the composition of river microbial consortia. Recent studies have shown that river metagenomes vary considerably, suggesting that microbial community data should be included in broad-scale river ecosystem models. But such ecogenomic studies have not been applied on a broad "aquascape" scale, and few if any have applied the newest nanopore technology. RESULTS: We investigated the metagenomes of 11 rivers across 3 continents using MinION nanopore sequencing, a portable platform that could be useful for future global river monitoring. Up to 10 Gb of data per run were generated with average read lengths of 3.4 kb. Diversity and diagnosis of river function potential was accomplished with 0.5-1.0 ⋅ 106 long reads. Our observations for 7 of the 11 rivers conformed to other river-omic findings, and we exposed previously unrecognized microbial biodiversity in the other 4 rivers. CONCLUSIONS: Deeper understanding that emerged is that river microbial consortia and the ecological functions they fulfil did not align with geographic location but instead implicated ecological responses of microbes to urban and other anthropogenic effects, and that changes in taxa manifested over a very short geographic space.

Complete Genome Sequences of Seven Strains of Pseudomonas spp. Isolated from Boreal Forest Soil in Interior Alaska.

Here, we describe the complete genome assemblies of seven Pseudomonas sp. isolates collected from a boreal forest soil on the University of Alaska Fairbanks campus. Using the VolTRAX v2 multiplex library preparation for Nanopore sequencing and Illumina reads for polishing, we assembled complete genome sequences for each of the isolates.

Complete Genome Sequence of a Virulent African Swine Fever Virus from a Domestic Pig in Ukraine.

Here, we report the complete genome sequence of an African swine fever (ASF) virus (ASFV/Kyiv/2016/131) isolated from the spleen of a domestic pig in Ukraine with a lethal case of African swine fever. Using only long-read Nanopore sequences, we assembled a full-length genome of 191,911 base pairs in a single contig.