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The fecal bacteria

The Fecal Bacteria offers a balanced, integrated discussion of fecal bacteria and their presence and ecology in the intestinal tract of mammals, in the environment, and in the food supply. This new volume covers their use in examining and assessing water quality in order to offer protection from illnesses related to swimming in or ingesting contaminated water, in addition to discussing their use in engineering considerations of water quality, modeling, monitoring, and regulations. Fecal bacteria are additionally used as indicators of contamination of ready-to-eat foods and fresh produce. The intestinal environment, the microbial community structure of the gut microbiota, and the physiology and genomics of this broad group of microorganisms are explored in the book. With contributions from an internationally recognized group of experts, the book integrates medicine, public health, environmental, and microbiological topics in order to provide a unique, holistic understanding of fecal bacteria. Moreover, it shows how the latest basic science and applied research findings are helping to solve problems and develop effective management strategies. For example, readers will discover how the latest tools and molecular approaches have led to our current understanding of fecal bacteria and enabled us to improve human health and water quality. The Fecal Bacteria is recommended for microbiologists, clinicians, animal scientists, engineers, environmental scientists, food safety experts, water quality managers, and students. It will help them better understand fecal bacteria and use their knowledge to protect human and environmental health. They can also apply many of the techniques and molecular tools discussed in this book to the study of a broad range of microorganisms in a variety of habitats.

Book↗

Extremophile hotspots linked to containerized industrial waste dumping in a deep-sea basin

Decaying barrels on the seafloor linked to DDT contamination have raised concerns about the public health implications of decades old industrial waste dumped off the coast of Los Angeles. To explore their contents, we collected sediment cores perpendicular to five deep-sea barrels. The concentration of DDT and its breakdown products were highly elevated relative to control sites yet did not vary with distance from the barrels, suggesting that they were not associated with the contamination. Sediment cores collected through white halos surrounding three barrels were enriched in calcite and had elevated pH. The associated microbial communities were low diversity and dominated by alkalophilic bacteria with metagenome-assembled genomes adapted to high pH. A solid concretion sampled between a white halo and barrel was composed of brucite, a magnesium hydroxide mineral that forms at high pH. Based on these findings, we postulate that leakage of containerized alkaline waste triggered the formation of mineral concretions that are slowly dissolving and raising the pH of the surrounding sediment pore water. This selects for taxa adapted to extreme alkalinity and drives the precipitation of “anthropogenic” carbonates forming white halos, which serve as a visual identifier of barrels that contained alkaline waste. Remarkably, containerized alkaline waste discarded >50 years ago represents a persistent pollutant creating localized mineral formations and microbial communities that resemble those observed at some hydrothermal systems. These formations were observed at one-third of the visually identified barrels in the San Pedro Basin and have unforeseen, long-term consequences for benthic communities in the region.

California↗

Laboratory assessment for recovery of porcine circovirus 2 and porcine reproductive and respiratory syndrome virus using two types of commercially available hollow-fiber ultrafilters

Groundwater near swine farms is an uninvestigated reservoir for porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCVs). Enteric microorganisms are often collected from groundwater via dead-end ultrafiltration, but recovery of PRRSV and PCV with this method has not been assessed. We recovered PRRSV2 and PCV2 by dead-end ultrafiltration followed by polyethylene glycol (PEG) precipitation, nucleic acid extraction, and reverse-transcription quantitative real-time PCR. We also compared 2 commercial hemodialysis ultrafilters (Asahi Kasei Rexeed-25A, Nipro Elisio-25H) and compared PRRSV2 recovery in these filters to other waterborne microorganisms. On average, 8 ± 1% of PRRSV2 was recovered by dead-end ultrafiltration and PEG precipitation, compared to 25 ± 6% for adenovirus 41. Full-process recovery of bacteria in the same filters was 5–15%; Cryptosporidium parvum recovery was 42 ± 12%. PCV2 was detected in 4 of 12 replicate filters, but low stock concentrations precluded quantitative recovery estimates. Elisio-25H ultrafilters performed similarly to Rexeed-25A filters for all organisms tested and is an effective replacement for the Rexeed-25A, which is no longer available in the United States. Our recovery of PRRSV2 and PCV2 by dead-end ultrafiltration in the laboratory suggests that PRRSV2 detection limits are as low as 3–50 genomic copies/L in sample volumes of 100–1,500 L. Based on quantitative microbial risk assessment, these concentrations are relevant to PRRSV2 infection rates in the U.S. swine herd.

Journal of Veterinary Diagnostic Investigation↗

Gulf of Mexico blue hole harbors high levels of novel microbial lineages

Exploration of oxygen-depleted marine environments has consistently revealed novel microbial taxa and metabolic capabilities that expand our understanding of microbial evolution and ecology. Marine blue holes are shallow karst formations characterized by low oxygen and high organic matter content. They are logistically challenging to sample, and thus our understanding of their biogeochemistry and microbial ecology is limited. We present a metagenomic and geochemical characterization of Amberjack Hole on the Florida continental shelf (Gulf of Mexico). Dissolved oxygen became depleted at the hole’s rim (32 m water depth), remained low but detectable in an intermediate hypoxic zone (40–75 m), and then increased to a secondary peak before falling below detection in the bottom layer (80–110 m), concomitant with increases in nutrients, dissolved iron, and a series of sequentially more reduced sulfur species. Microbial communities in the bottom layer contained heretofore undocumented levels of the recently discovered phylum Woesearchaeota (up to 58% of the community), along with lineages in the bacterial Candidate Phyla Radiation (CPR). Thirty-one high-quality metagenome-assembled genomes (MAGs) showed extensive biochemical capabilities for sulfur and nitrogen cycling, as well as for resisting and respiring arsenic. One uncharacterized gene associated with a CPR lineage differentiated hypoxic from anoxic zone communities. Overall, microbial communities and geochemical profiles were stable across two sampling dates in the spring and fall of 2019. The blue hole habitat is a natural marine laboratory that provides opportunities for sampling taxa with under-characterized but potentially important roles in redox-stratified microbial processes.

Interational Society of Microbial Ecology (ISME) J↗

Metabolically diverse microorganisms mediate methylmercury formation under nitrate-reducing conditions in a dynamic hydroelectric reservoir

Brownlee Reservoir is a mercury (Hg)-impaired hydroelectric reservoir that exhibits dynamic hydrological and geochemical conditions and is located within the Hells Canyon Complex in Idaho, USA. Methylmercury (MeHg) contamination in fish is a concern in the reservoir. While MeHg production has historically been attributed to sulfate-reducing bacteria and methanogenic archaea, microorganisms carrying the hgcA gene are taxonomically and metabolically diverse and the major biogeochemical cycles driving mercury (Hg) methylation are not well understood. In this study, Hg speciation and redox-active compounds were measured throughout Brownlee Reservoir across the stratified period in four consecutive years (2016–2019) to identify the location where and redox conditions under which MeHg is produced. Metagenomic sequencing was performed on a subset of samples to characterize the microbial community with hgcA and identify possible links between biogeochemical cycles and MeHg production. Biogeochemical profiles suggested in situ water column Hg methylation was the major source of MeHg. These profiles, combined with genome-resolved metagenomics focused on hgcA -carrying microbes, indicated that MeHg production occurs in this system under nitrate- or manganese-reducing conditions, which were previously thought to preclude Hg-methylation. Using this multidisciplinary approach, we identified the cascading effects of interannual variability in hydrology on the redox status, microbial metabolic strategies, abundance and metabolic diversity of Hg methylators, and ultimately MeHg concentrations throughout the reservoir. This work expands the known conditions conducive to producing MeHg and suggests that the Hg-methylation mitigation efforts by nitrate or manganese amendment may be unsuccessful in some locations.

Idaho↗

Mixotrophic iron-oxidizing Thiomonas isolates from an acid mine drainage-affected creek

Natural attenuation of heavy metals occurs via coupled microbial iron cycling and metal precipitation in creeks impacted by acid mine drainage (AMD). Here, we describe the isolation, characterization, and genomic sequencing of two iron-oxidizing bacteria (FeOB) species: Thiomonas ferrovorans FB-6 and Thiomonas metallidurans FB-Cd, isolated from slightly acidic (pH 6.3), Fe-rich, AMD-impacted creek sediments. These strains precipitated amorphous iron oxides, lepidocrocite, goethite, and magnetite or maghemite and grew at a pH optimum of 5.5. While Thiomonas spp. are known as mixotrophic sulfur oxidizers and As oxidizers, the FB strains oxidized Fe, which suggests they can efficiently remove Fe and other metals via coprecipitation. Previous evidence for Thiomonas sp. Fe oxidation is largely ambiguous, possibly because of difficulty demonstrating Fe oxidation in heterotrophic/mixotrophic organisms. Therefore, we also conducted a genomic analysis to identify genetic mechanisms of Fe oxidation, other metal transformations, and additional adaptations, comparing the two FB strain genomes with 12 other Thiomonas genomes. The FB strains fall within a relatively novel group of Thiomonas strains that includes another strain (b6) with solid evidence of Fe oxidation. Most Thiomonas isolates, including the FB strains, have the putative iron oxidation gene cyc2 , but only the two FB strains possess the putative Fe oxidase genes mtoAB . The two FB strain genomes contain the highest numbers of strain-specific gene clusters, greatly increasing the known Thiomonas genetic potential. Our results revealed that the FB strains are two distinct novel species of Thiomonas with the genetic potential for bioremediation of AMD via iron oxidation.

Thuringia↗

Identification, characterization and genetic mapping of TLR1 loci in rainbow trout (Oncorhynchus mykiss)

Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5' UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam2CSK4) and triacylated lipoprotein (Pam3CSK4). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are critical for future elucidation of omTLR1 functions.

Fish and Shellfish Immunology↗

Mercury methylation genes identified across diverse anaerobic microbial guilds in a eutrophic sulfate-enriched lake

Mercury (Hg) methylation is a microbially mediated process that converts inorganic Hg into bioaccumulative, neurotoxic methylmercury (MeHg). The metabolic activity of methylating organisms is highly dependent on biogeochemical conditions, which subsequently influences MeHg production. However, our understanding of the ecophysiology of methylators in natural ecosystems is still limited. Here, we identified potential locations of MeHg production in the anoxic, sulfidic hypolimnion of a freshwater lake. At these sites, we used shotgun metagenomics to characterize microorganisms with the Hg-methylation gene hgcA . Putative methylators were dominated by hgcA sequences divergent from those in well-studied, confirmed methylators. Using genome-resolved metagenomics, we identified organisms with hgcA (hgcA+) within the Bacteroidetes and the recently described Kiritimatiellaeota phyla. We identified hgcA+ genomes derived from sulfate-reducing bacteria, but these accounted for only 22% of hgcA+ genome coverage. The most abundant hgcA+ genomes were from fermenters, accounting for over half of the hgcA gene coverage. Many of these organisms also mediate hydrolysis of polysaccharides, likely from cyanobacterial blooms. This work highlights the distribution of the Hg-methylation genes across microbial metabolic guilds and indicate that primary degradation of polysaccharides and fermentation may play an important but unrecognized role in MeHg production in the anoxic hypolimnion of freshwater lakes.

Environmental Science and Technology↗

ArxA, a new clade of arsenite oxidase within the DMSO reductase family of molybdenum oxidoreductases

Arsenotrophy, growth coupled to autotrophic arsenite oxidation or arsenate respiratory reduction, occurs only in the prokaryotic domain of life. The enzymes responsible for arsenotrophy belong to distinct clades within the DMSO reductase family of molybdenum-containing oxidoreductases: specifically arsenate respiratory reductase, ArrA, and arsenite oxidase, AioA (formerly referred to as AroA and AoxB). A new arsenite oxidase clade, ArxA, represented by the haloalkaliphilic bacterium Alkalilimnicola ehrlichii strain MLHE-1 was also identified in the photosynthetic purple sulfur bacterium Ectothiorhodospira sp. strain PHS-1. A draft genome sequence of PHS-1 was completed and an arx operon similar to MLHE-1 was identified. Gene expression studies showed that arx A was strongly induced with arsenite. Microbial ecology investigation led to the identification of additional arx A-like sequences in Mono Lake and Hot Creek sediments, both arsenic-rich environments in California. Phylogenetic analyses placed these sequences as distinct members of the ArxA clade of arsenite oxidases. ArxA-like sequences were also identified in metagenome sequences of several alkaline microbial mat environments of Yellowstone National Park hot springs. These results suggest that ArxA-type arsenite oxidases appear to be widely distributed in the environment presenting an opportunity for further investigations of the contribution of Arx-dependent arsenotrophy to the arsenic biogeochemical cycle.

Environmental Microbiology↗

Evidence for free-living Bacteroides in Cladophora along the shores of the Great Lakes

Bacteroides is assumed to be restricted to the alimentary canal of animals and humans and is considered to be non-viable in ambient environments. We hypothesized that Bacteroides could persist and replicate within beach-stranded Cladophora glomerata mats in southern Lake Michigan, USA. Mean Bacteroides concentration (per GenBac3 Taqman quantitative PCR assay) during summer 2012 at Jeorse Park Beach was 5.2 log calibrator cell equivalents (CCE) g -1 dry weight (dw), ranging from 3.7 to 6.7. We monitored a single beach-stranded mat for 3 wk; bacterial concentrations increased by 1.6 log CCE g -1 dw and correlated significantly with ambient temperature (p = 0.003). Clonal growth was evident, as observed by >99% nucleotide sequence similarity among clones. In in vitro studies, Bacteroides concentrations increased by 5.5 log CCE g -1 after 7 d (27°C) in fresh Cladophora collected from rocks. Partial sequencing of the 16S rRNA gene of 36 clones from the incubation experiment showed highly similar genotypes (≥97% sequence overlap). The closest enteric Bacteroides spp. from the National Center for Biotechnology Information database were only 87 to 91% similar. Genomic similarity, clonality, growth, and persistence collectively suggest that putative, free-living Bacteroides inhabit Cladophora mats of southern Lake Michigan. These findings may have important biological, medical, regulatory, microbial source tracking, and public health implications.

southern Lake Michigan↗

Dryland biological soil crust cyanobacteria show unexpected decreases in abundance under long-term elevated CO 2

Biological soil crusts (biocrusts) cover soil surfaces in many drylands globally. The impacts of 10 years of elevated atmospheric CO 2 on the cyanobacteria in biocrusts of an arid shrubland were examined at a large manipulated experiment in Nevada, USA. Cyanobacteria-specific quantitative PCR surveys of cyanobacteria small-subunit (SSU) rRNA genes suggested a reduction in biocrust cyanobacterial biomass in the elevated CO 2 treatment relative to the ambient controls. Additionally, SSU rRNA gene libraries and shotgun metagenomes showed reduced representation of cyanobacteria in the total microbial community. Taxonomic composition of the cyanobacteria was similar under ambient and elevated CO 2 conditions, indicating the decline was manifest across multiple cyanobacterial lineages. Recruitment of cyanobacteria sequences from replicate shotgun metagenomes to cyanobacterial genomes representing major biocrust orders also suggested decreased abundance of cyanobacteria sequences across the majority of genomes tested. Functional assignment of cyanobacteria-related shotgun metagenome sequences indicated that four subsystem categories, three related to oxidative stress, were differentially abundant in relation to the elevated CO 2 treatment. Taken together, these results suggest that elevated CO 2 affected a generalized decrease in cyanobacteria in the biocrusts and may have favoured cyanobacteria with altered gene inventories for coping with oxidative stress.

Nevada↗

Tick control: Trapping, bio-control, host management and other alternative strategies

Biology of Ticks is the most comprehensive work on tick biology and tick-borne diseases. This second edition is a multi-authored work, featuring the research and analyses of renowned experts across the globe. Spanning two volumes, the book examines the systematics, biology, structure, ecological adaptations, evolution, genomics and the molecular processes that underpin the growth, development and survival of these important disease-transmitting parasites. Also discussed is the remarkable array of diseases transmitted (or caused) by ticks, as well as modern methods for their control. This book should serve as a modern reference for students, scientists, physicians, veterinarians and other specialists. Volume II includes chapters on the ecology of non-nidicolous and nidicolous ticks, genetics and genomics (including the genome of the Lyme disease vector Ixodes scapularis) and immunity, including host immune responses to tick feeding and tick-host interactions, as well as the tick's innate immune system that prevents and/or controls microbial infections. Six chapters cover in depth the many diseases caused by the major tick-borne pathogens, including tick-borne protozoa, viruses, rickettsiae of all types, other types of bacteria (e.g., the Lyme disease agent) and diseases related to tick paralytic agents and toxins. The remaining chapters are devoted to tick control using vaccines, acaricides, repellents, biocontrol, and, finally, techniques for breeding ticks in order to develop tick colonies for scientific study.

Book chapter↗

Metaproteomics and metagenomics reveal microbial pathways of organic matter degradation and methanogenesis in a marginally producing natural gas well

The expansion of natural gas production and utilization worldwide has led to the decline of many once-productive wells, eventually resulting in costly well-plugging and unused infrastructure. However, in areas like the Michigan basin, MI, where the majority of natural gas is biogenically produced, microbial communities could potentially be stimulated to generate additional methane, increasing gas supply and reducing the need to drill new wells. In this study, we performed metaproteomic, metagenomic, and geochemical analyses of Antrim Shale formation water from a marginally producing natural gas well to evaluate resident microbial community functions in the context of potential bioenergy production. Functional proteins involved in methanogenesis, degradation/catabolism (including organic matter degradation), biosynthesis, energy utilization, transmembrane transport, and stress response were among the most commonly identified groups. Three metagenome-assembled genomes (MAGs) were characterized, including Methanomicrobiaceae, Methanothrix , and Smithella . For each, the identified proteins involved in methanogenesis and the degradation of diverse organic compounds, strongly suggest their role in utilizing shale-derived organic matter. These findings provide an increased understanding of the microorganisms and their metabolisms generating natural gas in the Antrim Shale and establish a foundation for future stimulation efforts aimed at enhancing biogenic methane production in marginal gas wells.

Michigan↗

Discovery and genomic characterization of a novel hepadnavirus from asymptomatic anadromous alewife (Alosa pseudoharengus)

The alewife ( Alosa pseudoharengus) is an anadromous herring that inhabits waters of northeastern North America. This prey species is a critical forage for piscivorous birds, mammals, and fishes in estuarine and oceanic ecosystems. During a discovery project tailored to identify potentially emerging pathogens of this species, we obtained the full genome of a novel hepadnavirus (ApHBV) from clinically normal alewives collected from the Maurice River, Great Egg Harbor River, and Delaware River in New Jersey, USA during 2015–2018. This previously undescribed hepadnavirus contained a circular DNA genome of 3146 nucleotides. Phylogenetic analysis of the polymerase protein placed this virus in the clade of metahepadnaviruses (family: Hepadnaviridae ; genus: Metahepadnavirus ). There was no evidence of pathology in the internal organs of infected fish and virions were not observed in liver tissues by electron microscopy. We developed a Taqman-based quantitative (qPCR) assay and screened 182 individuals collected between 2015 and 2018 and detected additional qPCR positives (n = 6). An additional complete genome was obtained in 2018 and it has 99.4% genome nucleotide identity to the first virus. Single-nucleotide polymorphisms were observed between the two genomes, including 7/9 and 12/8 synonymous vs nonsynonymous mutations across the polymerase and surface proteins, respectively. While there was no evidence that this virus was associated with disease in this species, alewives are migratory interjurisdictional fishes of management concern. Identification of microbial agents using de novo sequencing and other advanced technologies is a critical aspect of understanding disease ecology for informed population management.

New Jersey↗

A tetrapod-like repertoire of innate immune receptors and effectors for coelacanths

The recent availability of both robust transcriptome and genome resources for coelacanth ( Latimeria chalumnae ) has led to unique discoveries for coelacanth immunity such as the lack of IgM, a central component of adaptive immunity. This study was designed to more precisely address the origins and evolution of gene families involved in the initial recognition and response to microbial pathogens, which effect innate immunity. Several multigene families involved in innate immunity are addressed, including: Toll-like receptors (TLRs), retinoic acid inducible gene 1 (RIG1)-like receptors (RLRs), the nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs), diverse immunoglobulin domain-containing proteins (DICP) and modular domain immune-type receptors (MDIRs). Our analyses also include the tripartite motif-containing proteins (TRIM), which are involved in pathogen recognition as well as the positive regulation of antiviral immunity. Finally, this study addressed some of the downstream effectors of the antimicrobial response including IL-1 family members, type I and II interferons (IFN) and IFN-stimulated effectors (ISGs). Collectively, the genes and gene families in coelacanth that effect innate immune functions share characteristics both in content, structure and arrangement with those found in tetrapods but not in teleosts. The findings support the sister group relationship of coelacanth fish with tetrapods.

Journal of Experimental Zoology Part B: Molecular ↗

Metagenomic analysis of a permafrost microbial community reveals a rapid response to thaw

Permafrost contains an estimated 1672 Pg carbon (C), an amount roughly equivalent to the total currently contained within land plants and the atmosphere1,2,3. This reservoir of C is vulnerable to decomposition as rising global temperatures cause the permafrost to thaw2. During thaw, trapped organic matter may become more accessible for microbial degradation and result in greenhouse gas emissions4,5. Despite recent advances in the use of molecular tools to study permafrost microbial communities6,7,8,9, their response to thaw remains unclear. Here we use deep metagenomic sequencing to determine the impact of thaw on microbial phylogenetic and functional genes, and relate these data to measurements of methane emissions. Metagenomics, the direct sequencing of DNA from the environment, allows the examination of whole biochemical pathways and associated processes, as opposed to individual pieces of the metabolic puzzle. Our metagenome analyses reveal that during transition from a frozen to a thawed state there are rapid shifts in many microbial, phylogenetic and functional gene abundances and pathways. After one week of incubation at 5 °C, permafrost metagenomes converge to be more similar to each other than while they are frozen. We find that multiple genes involved in cycling of C and nitrogen shift rapidly during thaw. We also construct the first draft genome from a complex soil metagenome, which corresponds to a novel methanogen. Methane previously accumulated in permafrost is released during thaw and subsequently consumed by methanotrophic bacteria. Together these data point towards the importance of rapid cycling of methane and nitrogen in thawing permafrost.

Nature↗

Identification of novel hepaciviruses and Sylvilagus-associated viruses via metatranscriptomics in North American lagomorphs

Cottontails ( Sylvilagus spp.) and jackrabbits ( Lepus spp.) within the Leporidae family are native to North America and are found in a wide range of habitats, including deserts, forests, and grasslands. Although there is a growing body of research describing the arrival of the highly virulent rabbit haemorrhagic disease virus 2 (RHDV2, GI.2) on this continent, and its impact on native lagomorphs, information about the natural virome and microbiome of healthy and deceased American lagomorphs is relatively limited. In this study, we used a meta-transcriptomics approach to conduct whole pathogen profiling on healthy and deceased animals in the USA. We analysed 48 matched liver and lung sample pools from apparently healthy cottontails and jackrabbits in Texas and an additional 48 liver samples from deceased animals from nine other US states. This approach enabled the discovery of three distinct new viruses and revealed additional new insights into the lung and liver microbiomes of North American lagomorphs. Of the three new viruses, a tetnovirus and a novel picorna-like virus were likely of insect origin and therefore considered environmental contaminants. Of particular interest was a new species of hepacivirus, with around 50% sequence identity to a known hepacivirus from a xeric four-striped grass rat ( Rhabdomys pumilio ). Phylogenetic analysis from 41 individual hepacivirus genomes recovered from our lagomorph samples revealed two distinct clades, corresponding with different cottontail species. No hepaciviruses were detected in any of the jackrabbit samples. This is the first description of a hepacivirus in lagomorphs. Our findings extend the Hepacivirus genus, provide new insights into its evolution, and describe the first baseline on microbial diversity in North American lagomorphs, an important step towards understanding the role of potential pathogens for population management and conservation.

Arizona, California, Iowa, Massachusetts, Montana,↗

Two risk assessments: Evaluating the use of indicator HF183 Bacteroides versus pathogen measurements for modelling recreational illness risks in an urban watershed

The purpose of this study was to evaluate the performance of HF183 Bacteroides for estimating pathogen exposures during recreational water activities. We compared the use of Bacteroides -based exposure assessment to exposure assessment that relied on pathogen measurements. We considered two types of recreational water sites: those impacted by combined sewer overflows (CSOs) and those not impacted by CSOs. Samples from CSO-impacted and non-CSO-impacted urban creeks were analysed by quantitative polymerase chain reaction (qPCR) for HF183 Bacteroides and eight human gastrointestinal pathogens. Exposure assessment was conducted two ways for each type of site (CSO-impacted vs. non-CSO impacted): 1) by estimating pathogen concentrations from HF183 Bacteroides concentrations using published ratios of HF183 and pathogens in sewage and 2) by estimating pathogen concentrations from qPCR measurements. QMRA (quantitative microbial risk assessment) was then conducted for swimming, wading, and fishing exposures. Overall, mean risk estimates varied from 0.27 to 53 illnesses per 1,000 recreators depending on exposure assessment, site, activity, and norovirus dose-response model. HF183-based exposure assessment identified CSO-impacted sites as higher risk, and the recommended HF183 risk-based threshold of 525 genomic copies per 100 mL was generally protective of public health at the CSO-impacted sites but was not as protective at the non-CSO-impacted sites. In the context of our urban watershed, HF183-based exposure assessment over- and under-estimated risk relative to exposure assessment based on pathogen measurements and the etiology of predicted pathogen-specific illnesses differed significantly. Across all sites, the HF183 model overestimated risk for norovirus, adenovirus, and Campylobacter jejuni , and it underestimated risk for E. coli and Cryptosporidium . To our knowledge, this study is the first to directly compare health risk estimates using HF183 and empirical pathogen measurements from the same waterways. Our work highlights the importance of site-specific hazard identification and exposure assessment to decide whether HF183 is applicable for monitoring risk.

Pennsylvania↗