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Outside the box: Working with wildlife in biocontainment

Research with captive wildlife in Animal Biosafety Level 2 (ABSL2) and 3 (ABSL3) facilities is becoming increasingly necessary as emerging and re-emerging diseases involving wildlife have increasing impacts on human, animal, and environmental health. Utilizing wildlife species in a research facility often requires outside the box thinking with specialized knowledge, practices, facilities, and equipment. The USGS National Wildlife Health Center (NWHC) houses an ABSL3 facility dedicated to understanding wildlife diseases and developing tools to mitigate their impacts on animal and human health. This review presents considerations for utilizing captive wildlife for infectious disease studies, including, husbandry, animal welfare, veterinary care, and biosafety. Examples are drawn from primary literature review and collective 40-year experience of the NWHC. Working with wildlife in ABSL2 and ABSL3 facilities differs from laboratory animals in that typical laboratory housing systems, husbandry practices, and biosafety practices are not designed for work with wildlife. This requires thoughtful adaptation of standard equipment and practices, invention of customized solutions and development of appropriate enrichment plans using the natural history of the species and the microbiological characteristics of introduced and native pathogens. Ultimately, this task requires critical risk assessment, understanding of the physical and psychological needs of diverse species, creativity, innovation, and flexibility. Finally, continual reassessment and improvement are imperative in this constantly changing specialty area of infectious disease and environmental hazard research.

ILAR Journal

Transglobal spread of an ecologically relevant sea urchin parasite

Mass mortality of the dominant coral reef herbivore Diadema antillarum in the Caribbean in the early 1980s contributed to a persistent phase shift from coral- to algal-dominated reefs. In 2022, a scuticociliate most closely related to Philaster apodigitiformis caused further mass mortality of D. antillarum across the Caribbean, leading to >95% mortality at affected sites. Mortality was also reported in the related species Diadema setosum in the Mediterranean in 2022, though the causative agent of the Mediterranean outbreak has not yet been determined. In April 2023, mass mortality of Diadema setosum occurred along the Sultanate of Oman's coastline. Urchins displayed signs compatible with scuticociliatosis including abnormal behavior, drooping and loss of spines, followed by tissue necrosis and death. Here we report the detection of an 18S rRNA gene sequence in abnormal urchins from Muscat, Oman, that is identical to the Philaster strain responsible for D. antillarum mass mortality in the Caribbean. We also show that scuticociliatosis signs can be elicited in Diadema setosum by experimental challenge with the cultivated Philaster strain associated with Caribbean scuticociliatosis. These results demonstrate the Philaster sp. associated with D. antillarum mass mortality has rapidly spread to geographically distant coral reefs, compelling global-scale awareness and monitoring for this devastating condition through field surveys, microscopy, and molecular microbiological approaches, and prompting investigation of long-range transmission mechanisms.

ISME Journal

Integrating airborne remote sensing and field campaigns for ecology and Earth system science

In recent years, the availability of airborne imaging spectroscopy (hyperspectral) data has expanded dramatically. The high spatial and spectral resolution of these data uniquely enable spatially explicit ecological studies including species mapping, assessment of drought mortality and foliar trait distributions. However, we have barely begun to unlock the potential of these data to use direct mapping of vegetation characteristics to infer subsurface properties of the critical zone. To assess their utility for Earth systems research, imaging spectroscopy data acquisitions require integration with large, coincident ground‐based datasets collected by experts in ecology and environmental and Earth science. Without coordinated, well‐planned field campaigns, potential knowledge leveraged from advanced airborne data collections could be lost. Despite the growing importance of this field, documented methods to couple such a wide variety of disciplines remain sparse. We coordinated the first National Ecological Observatory Network Airborne Observation Platform (AOP) survey performed outside of their core sites, which took place in the Upper East River watershed, Colorado. Extensive planning for sample tracking and organization allowed field and flight teams to update the ground‐based sampling strategy daily. This enabled collection of an extensive set of physical samples to support a wide range of ecological, microbiological, biogeochemical and hydrological studies. We present a framework for integrating airborne and field campaigns to obtain high‐quality data for foliar trait prediction and document an archive of coincident physical samples collected to support a systems approach to ecological research in the critical zone. This detailed methodological account provides an example of how a multi‐disciplinary and multi‐institutional team can coordinate to maximize knowledge gained from an airborne survey, an approach that could be extended to other studies. The coordination of imaging spectroscopy surveys with appropriately timed and extensive field surveys, along with high‐quality processing of these data, presents a unique opportunity to reveal new insights into the structure and dynamics of the critical zone. To our knowledge, this level of co‐aligned sampling has never been undertaken in tandem with AOP surveys and subsequent studies utilizing this archive will shed considerable light on the breadth of applications for which imaging spectroscopy data can be leveraged.

Colorado

Microbiome assembly in thawing permafrost and its feedbacks to climate

The physical and chemical changes that accompany permafrost thaw directly influence the microbial communities that mediate the decomposition of formerly frozen organic matter, leading to uncertainty in permafrost–climate feedbacks. Although changes to microbial metabolism and community structure are documented following thaw, the generality of post-thaw assembly patterns across permafrost soils of the world remains uncertain, limiting our ability to predict biogeochemistry and microbial community responses to climate change. Based on our review of the Arctic microbiome, permafrost microbiology, and community ecology, we propose that Assembly Theory provides a framework to better understand thaw-mediated microbiome changes and the implications for community function and climate feedbacks. This framework posits that the prevalence of deterministic or stochastic processes indicates whether the community is well-suited to thrive in changing environmental conditions. We predict that on a short timescale and following high-disturbance thaw (e.g., thermokarst), stochasticity dominates post-thaw microbiome assembly, suggesting that functional predictions will be aided by detailed information about the microbiome. At a longer timescale and lower-intensity disturbance (e.g., active layer deepening), deterministic processes likely dominate, making environmental parameters sufficient for predicting function. We propose that the contribution of stochastic and deterministic processes to post-thaw microbiome assembly depends on the characteristics of the thaw disturbance, as well as characteristics of the microbial community, such as the ecological and phylogenetic breadth of functional guilds, their functional redundancy, and biotic interactions. These propagate across space and time, potentially providing a means for predicting the microbial forcing of greenhouse gas feedbacks to global climate change.

Global Change Biology

Biodegradation in contaminated aquifers: Incorporating microbial/molecular methods

In order to evaluate natural attenuation in contaminated aquifers, there has been a recent recognition that a multidisciplinary approach, incorporating microbial and molecular methods, is required. Observed decreases in contaminant mass and identified footprints of biogeochemical reactions are often used as evidence of intrinsic bioremediation, but characterizing the structure and function of the microbial populations at contaminated sites is needed. In this paper, we review the experimental approaches and microbial methods that are available as tools to evaluate the controls on microbially mediated degradation processes in contaminated aquifers. We discuss the emerging technologies used in biogeochemical studies and present a synthesis of recent studies that serve as models of integrating microbiological approaches with more traditional geochemical and hydrogeologic approaches in order to address important biogeochemical questions about contaminant fate.

Groundwater

A wireline piston core barrel for sampling cohesionless sand and gravel below the water table

A coring device has been developed to obtain long and minimally disturbed samples of saturated cohesionless sand and gravel. The coring device, which includes a wireline and piston, was developed specifically for use during hollow-stem auger drilling but it also offers possibilities for cable tool and rotary drilling. The core barrel consists of an inner liner made of inexpensive aluminum or plastic tubing, a piston for core recovery, and an exterior steel housing that protects the liner when the core barrel is driven into the aquifer. The core barrel, which is approximately 1.6m (5.6 feet) long, is advanced ahead of the lead auger by hammering at the surface on drill rods that are attached to the core barrel. After the sampler has been driven 1.5m (5 feet), the drill rods are detached and a wireline is used to hoist the core barrel, with the sample contained in the aluminum or plastic liner, to the surface. A vacuum developed by the piston during the coring operation provides good recovery of both the sediment and aquifer fluids contained in the sediment. In the field the sample tubes can be easily split along their length for on-site inspection or they can be capped with the pore water fluids inside and transported to the laboratory. The cores are 5cm (2 inches) in diameter by 1.5m (5 feet) long. Core acquisition to depths of 35m (115 feet), with a recovery greater than 90 percent, has become routine in University of Waterloo aquifer studies. A large diameter (12.7cm [5 inch]) version has also been used successfully. Nearly continuous sample sequences from sand and gravel aquifers have been obtained for studies of sedimentology, hydraulic conductivity, hydrogeochemistry and microbiology.

Groundwater Monitoring & Remediation

Methane-derived marine carbonates of pleistocene age

In some calcium carbonate-bearing sandstones from the edge of the continental shelf off the northeast United States, the δC 13 range is from -30 and -60 per mil for both aragonite and high-magnesium calcite. The δC 13 of co-existing shells of Modiolus sp. is normal (+ 1.7 to -2.7 per mil). The δO 18 values of around + 3.5 per mil in all samples suggest deposition at temperatures around 0°C. Quaternary methane oxidized either chemically or microbiologically to carbon dioxide is the probable source of carbon in these carbonates.

Science

Chronic wasting disease (CWD) susceptibility of several North American rodents that are sympatric with cervid CWD epidemics

Chronic wasting disease (CWD) is a highly contagious always fatal neurodegenerative disease that is currently known to naturally infect only species of the deer family, Cervidae. CWD epidemics are occurring in free-ranging cervids at several locations in North America, and other wildlife species are certainly being exposed to infectious material. To assess the potential for transmission, we intracerebrally inoculated four species of epidemic-sympatric rodents with CWD. Transmission was efficient in all species; the onset of disease was faster in the two vole species than the two Peromyscus spp. The results for inocula prepared from CWD-positive deer with or without CWD-resistant genotypes were similar. Survival times were substantially shortened upon second passage, demonstrating adaptation. Unlike all other known prion protein sequences for cricetid rodents that possess asparagine at position 170, our red-backed voles expressed serine and refute previous suggestions that a serine in this position substantially reduces susceptibility to CWD. Given the scavenging habits of these rodent species, the apparent persistence of CWD prions in the environment, and the inevitable exposure of these rodents to CWD prions, our intracerebral challenge results indicate that further investigation of the possibility of natural transmission is warranted. Copyright ?? 2010, American Society for Microbiology. All Rights Reserved.

Journal of Virology

Frequent transmission of immunodeficiency viruses among bobcats and pumas

With the exception of human immunodeficiency virus (HIV), which emerged in humans after cross-species transmissions of simian immunodeficiency viruses from nonhuman primates, immunodeficiency viruses of the family Lentiviridae represent species-specific viruses that rarely cross species barriers to infect new hosts. Among the Felidae, numerous immunodeficiency-like lentiviruses have been documented, but only a few cross-species transmissions have been recorded, and these have not been perpetuated in the recipient species. Lentivirus seroprevalence was determined for 79 bobcats (Lynx rufus) and 31 pumas (Puma concolor) from well-defined populations in Southern California. Partial genomic sequences were subsequently obtained from 18 and 12 seropositive bobcats and pumas, respectively. Genotypes were analyzed for phylogenic relatedness and genotypic composition among the study set and archived feline lentivirus sequences. This investigation of feline immunodeficiency virus infection in bobcats and pumas of Southern California provides evidence that cross-species infection has occurred frequently among these animals. The data suggest that transmission has occurred in multiple locations and are most consistent with the spread of the virus from bobcats to pumas. Although the ultimate causes remain unknown, these transmission events may occur as a result of puma predation on bobcats, a situation similar to that which fostered transmission of HIV to humans, and likely represent the emergence of a lentivirus with relaxed barriers to cross-species transmission. This unusual observation provides a valuable opportunity to evaluate the ecological, behavioral, and molecular conditions that favor repeated transmissions and persistence of lentivirus between species. Copyright ?? 2007, American Society for Microbiology. All Rights Reserved.

Journal of Virology

Characterization of low-pathogenicity H5N1 avian influenza viruses from North America

Wild-bird surveillance in North America for avian influenza (AI) viruses with a goal of early identification of the Asian H5N1 highly pathogenic AI virus has identified at least six low-pathogenicity H5N1 AI viruses between 2004 and 2006. The hemagglutinin (HA) and neuraminidase (NA) genes from all 6 H5N1 viruses and an additional 38 North American wild-bird-origin H5 subtype and 28 N1 subtype viruses were sequenced and compared with sequences available in GenBank by phylogenetic analysis. Both HA and NA were phylogenetically distinct from those for viruses from outside of North America and from those for viruses recovered from mammals. Four of the H5N1 AI viruses were characterized as low pathogenicity by standard in vivo pathotyping tests. One of the H5N1 viruses, A/MuteSwan/MI/451072-2/06, was shown to replicate to low titers in chickens, turkeys, and ducks. However, transmission of A/MuteSwan/MI/451072-2/06 was more efficient among ducks than among chickens or turkeys based on virus shed. The 50% chicken infectious dose for A/MuteSwan/MI/451072-2/06 and three other wild-waterfowl-origin H5 viruses were also determined and were between 10 5.3 and 107.5 50% egg infective doses. Finally, seven H5 viruses representing different phylogenetic clades were evaluated for their antigenic relatedness by hemagglutination inhibition assay, showing that the antigenic relatedness was largely associated with geographic origin. Overall, the data support the conclusion that North American H5 wild-bird-origin AI viruses are low-pathogenicity wild-bird-adapted viruses and are antigenically and genetically distinct from the highly pathogenic Asian H5N1 virus lineage. Copyright ?? 2007, American Society for Microbiology. All Rights Reserved.

Journal of Virology

Deep drilling in the Chesapeake Bay impact structure - An overview

The late Eocene Chesapeake Bay impact structure lies buried at moderate depths below Chesapeake Bay and surrounding landmasses in southeastern Virginia, USA. Numerous characteristics made this impact structure an inviting target for scientific drilling, including the location of the impact on the Eocene continental shelf, its threelayer target structure, its large size (??85 km diameter), its status as the source of the North American tektite strewn field, its temporal association with other late Eocene terrestrial impacts, its documented effects on the regional groundwater system, and its previously unstudied effects on the deep microbial biosphere. The Chesapeake Bay Impact Structure Deep Drilling Project was designed to drill a deep, continuously cored test hole into the central part of the structure. A project workshop, funding proposals, and the acceptance of those proposals occurred during 2003-2005. Initial drilling funds were provided by the International Continental Scientific Drilling Program (ICDP) and the U.S. Geological Survey (USGS). Supplementary funds were provided by the National Aeronautics and Space Administration (NASA) Science Mission Directorate, ICDP, and USGS. Field operations were conducted at Eyreville Farm, Northampton County, Virginia, by Drilling, Observation, and Sampling of the Earth's Continental Crust (DOSECC) and the project staff during September-December 2005, resulting in two continuously cored, deep holes. The USGS and Rutgers University cored a shallow hole to 140 m in April-May 2006 to complete the recovered section from land surface to 1766 m depth. The recovered section consists of 1322 m of crater materials and 444 m of overlying postimpact Eocene to Pleistocene sediments. The crater section consists of, from base to top: basement-derived blocks of crystalline rocks (215 m); a section of suevite, impact melt rock, lithic impact breccia, and cataclasites (154 m); a thin interval of quartz sand and lithic blocks (26 m); a granite megablock (275 m); and sediment blocks and boulders, polymict, sediment-clast-dominated sedimentary breccias, and a thin upper section of stratified sediments (652 m). The cored postimpact sediments provide insight into the effects of a large continental-margin impact on subsequent coastal-plain sedimentation. This volume contains the first results of multidisciplinary studies of the Eyreville cores and related topics. The volume is divided into these sections: geologic column; borehole geophysical studies; regional geophysical studies; crystalline rocks, impactites, and impact models; sedimentary breccias; postimpact sediments; hydrologic and geothermal studies; and microbiologic studies. ?? 2009 The Geological Society of America.

Special Paper of the Geological Society of America

Microbial precipitation of dolomite in methanogenic groundwater

We report low-temperature microbial precipitation of dolomite in dilute natural waters from both field and laboratory experiments. In a freshwater aquifer, microorganisms colonize basalt and nucleate nonstoichiometric dolomite on cell walls. In the laboratory, ordered dolomite formed at near-equilibrium conditions from groundwater with molar Mg:Ca ratios of <1; dolomite was absent in sterile experiments. Geochemical and microbiological data suggest that methanogens are the dominant metabolic guild in this system and are integral to dolomite precipitation. We hypothesize that the attached microbial consortium reacts with the basalt surface, releasing Mg and Ca into solution, which drives dolomite precipitation via nucleation on the cell wall. These findings provide insight into the long-standing dolomite problem and suggest a fundamental role for microbial processes in the formation of dolomite across a wide range of environmental conditions.

Geology

Predicting paleoclimate from compositional data using multivariate Gaussian process inverse prediction

Multivariate compositional count data arise in many applications including ecology, microbiology, genetics and paleoclimate. A frequent question in the analysis of multivariate compositional count data is what underlying values of a covariate(s) give rise to the observed composition. Learning the relationship between covariates and the compositional count allows for inverse prediction of unobserved covariates given compositional count observations. Gaussian processes provide a flexible framework for modeling functional responses with respect to a covariate without assuming a functional form. Many scientific disciplines use Gaussian process approximations to improve prediction and make inference on latent processes and parameters. When prediction is desired on unobserved covariates given realizations of the response variable, this is called inverse prediction. Because inverse prediction is often mathematically and computationally challenging, predicting unobserved covariates often requires fitting models that are different from the hypothesized generative model. We present a novel computational framework that allows for efficient inverse prediction using a Gaussian process approximation to generative models. Our framework enables scientific learning about how the latent processes co-vary with respect to covariates while simultaneously providing predictions of missing covariates. The proposed framework is capable of efficiently exploring the high dimensional, multi-modal latent spaces that arise in the inverse problem. To demonstrate flexibility, we apply our method in a generalized linear model framework to predict latent climate states given multivariate count data. Based on cross-validation, our model has predictive skill competitive with current methods while simultaneously providing formal, statistical inference on the underlying community dynamics of the biological system previously not available.

Annals of Applied Statistics

Occupancy modeling for improved accuracy and understanding of pathogen prevalence and dynamics

Most pathogen detection tests are imperfect, with a sensitivity < 100%, thereby resulting in the potential for a false negative, where a pathogen is present but not detected. False negatives in a sample inflate the number of non-detections, negatively biasing estimates of pathogen prevalence. Histological examination of tissues as a diagnostic test can be advantageous as multiple pathogens can be examined and providing important information on associated pathological changes to the host. However, it is usually less sensitive than molecular or microbiological tests for specific pathogens. Our study objectives were to 1) develop a hierarchical occupancy model to examine pathogen prevalence in spring Chinook salmon Oncorhynchus tshawytscha and their distribution among host tissues 2) use the model to estimate pathogen-specific test sensitivities and infection rates, and 3) illustrate the effect of using replicate within host sampling on sample sizes required to detect a pathogen. We examined histological sections of replicate tissue samples from spring Chinook salmon O. tshawytscha collected after spawning for common pathogens seen in this population: Apophallus/ echinostome metacercariae, Parvicapsula minibicornis, Nanophyetus salmincola/ metacercariae, and Renibacterium salmoninarum . A hierarchical occupancy model was developed to estimate pathogen and tissue-specific test sensitivities and unbiased estimation of host- and organ-level infection rates. Model estimated sensitivities and host- and organ-level infections rates varied among pathogens and model estimated infection rate was higher than prevalence unadjusted for test sensitivity, confirming that prevalence unadjusted for test sensitivity was negatively biased. The modeling approach provided an analytical approach for using hierarchically structured pathogen detection data from lower sensitivity diagnostic tests, such as histology, to obtain unbiased pathogen prevalence estimates with associated uncertainties. Accounting for test sensitivity using within host replicate samples also required fewer individual fish to be sampled. This approach is useful for evaluating pathogen or microbe community dynamics when test sensitivity is <100%.

PLoS ONE

The rising tide of ocean diseases: Unsolved problems and research priorities

New studies have detected a rising number of reports of diseases in marine organisms such as corals, molluscs, turtles, mammals, and echinoderms over the past three decades. Despite the increasing disease load, microbiological, molecular, and theoretical tools for managing disease in the world's oceans are under-developed. Review of the new developments in the study of these diseases identifies five major unsolved problems and priorities for future research: (1) detecting origins and reservoirs for marine diseases and tracing the flow of some new pathogens from land to sea; (2) documenting the longevity and host range of infectious stages; (3) evaluating the effect of greater taxonomic diversity of marine relative to terrestrial hosts and pathogens; (4) pinpointing the facilitating role of anthropogenic agents as incubators and conveyors of marine pathogens; (5) adapting epidemiological models to analysis of marine disease.

Frontiers in Ecology and the Environment

Interaction and influence of two creeks on Escherichia coli concentrations of nearby beaches: Exploration of predictability and mechanisms

The impact of river outfalls on beach water quality depends on numerous interacting factors. The delivery of contaminants by multiple creeks greatly complicates understanding of the source contributions, especially when pollution might originate up- or down-coast of beaches. We studied two beaches along Lake Michigan that are located between two creek outfalls to determine the hydrometeorologic factors influencing near-shore microbiologic water quality and the relative impact of the creeks. The creeks continuously delivered water with high concentrations of Escherichia coli to Lake Michigan, and the direction of transport of these bacteria was affected by current direction. Current direction reversals were associated with elevated E. coli concentrations at Central Avenue beach. Rainfall, barometric pressure, wave height, wave period, and creek specific conductance were significantly related to E. coli concentration at the beaches and were the parameters used in predictive models that best described E. coli variation at the two beaches. Multiple inputs to numerous beaches complicates the analysis and understanding of the relative relationship of sources but affords opportunities for showing how these complex creek inputs might interact to yield collective or individual effects on beach water quality.

Journal of Environmental Quality

Fecal indicator and pathogenic bacteria and their antibiotic resistance in alluvial groundwater of an irrigated agricultural region with dairies

Surveys of microbiological groundwater quality were conducted in a region with intensive animal agriculture in California, USA. The survey included monitoring and domestic wells in eight concentrated animal feeding operations (CAFOs) and 200 small (domestic and community supply district) supply wells across the region. Campylobacter was not detected in groundwater, whereas Escherichia coli O157:H7 and Salmonella were each detected in 2 of 190 CAFO monitoring well samples. Nonpathogenic generic E. coli and Enterococcus spp. were detected in 24.2% (46/190) and 97.4% (185/190) groundwater samples from CAFO monitoring wells and in 4.2% (1/24) and 87.5% (21/24) of CAFO domestic wells, respectively. Concentrations of both generic E. coli and Enterococcus spp. were significantly associated with well depth, season, and the type of adjacent land use in the CAFO. No pathogenic bacteria were detected in groundwater from 200 small supply wells in the extended survey. However, 4.5 to 10.3% groundwater samples were positive for generic E. coli and Enterococcus. Concentrations of generic E. coli were not significantly associated with any factors, but concentrations of Enterococcus were significantly associated with proximity to CAFOs, seasons, and concentrations of potassium in water. Among a subset of E. coli and Enterococcus isolates from both surveys, the majority of E. coli (63.6%) and Enterococcus (86.1%) isolates exhibited resistance to multiple (≥3) antibiotics. Findings confirm significant microbial and antibiotic resistance loading to CAFO groundwater. Results also demonstrate significant attenuative capacity of the unconfined alluvial aquifer system with respect to microbial transport.

California