An automated computational data pipeline to rapidly acquire, score, and rank toxicological data for ecological hazard assessment.

Biological Evaluations support Endangered Species Act (ESA) consultation with the US Fish and Wildlife Service and National Marine Fisheries Service by federal action agencies, such as the USEPA, regarding impacts of federal activities on threatened or endangered species. However, they are often time-consuming and challenging to conduct. The identification of pollutant benchmarks or guidance to protect taxa for states and tribes when USEPA has not yet developed criteria recommendations is also of importance to ensure a streamlined approach to Clean Water Act program implementation. Due to substantial workloads, tight regulatory timelines, and the often-protracted length of ESA consultations, there is a need to streamline the development of biological evaluation toxicity assessments for determining the impact of chemical pollutants on ESA-listed species. Moreover, there is limited availability of species-specific toxicity data for many contaminants, further complicating the consultation process. New approach methodologies are being increasingly used in toxicology and chemical safety assessment to rapidly and cost-effectively provide data that can fill gaps in hazard and/or exposure characterization. Here, we present the development of an automated computational pipeline-RASRTox (Rapidly Acquire, Score, and Rank Toxicological data)-to rapidly extract and categorize ecological toxicity benchmark values from curated data sources (ECOTOX, ToxCast) and well-established quantitative structure-activity relationships (TEST, ECOSAR). As a proof of concept, points-of-departure (PODs) generated in RASRTox for 13 chemicals were compared against benchmark values derived using traditional methods-toxicity reference values (TRVs) and water quality criteria (WQC). The RASRTox PODs were generally within an order of magnitude of corresponding TRVs, though less concordant compared with WQC. The greatest utility of RASRTox, however, lies in its ability to quickly and systematically identify critical studies that may serve as a basis for screening value derivation by toxicologists as part of an ecological hazard assessment. As such, the strategy described in this case study can potentially be adapted for other risk assessment contexts and stakeholder needs. Integr Environ Assess Manag 2024;00:1-15. © 2024 Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Health of wild fish exposed to contaminants of emerging concern in freshwater ecosystems utilized by a Minnesota Tribal community.

Fish serve as indicators of exposure to contaminants of emerging concern (CECs)-chemicals such as pharmaceuticals, hormones, and personal care products-which are often designed to impact vertebrates. To investigate fish health and CECs in situ, we evaluated the health of wild fish exposed to CECs in waterbodies across northeastern Minnesota with varying anthropogenic pressures and CEC exposures: waterbodies with no human development along their shorelines, those with development, and those directly receiving treated wastewater effluent. Then, we compared three approaches to evaluate the health of fish exposed to CECs in their natural environment: a refined fish health assessment index, a histopathological index, and high-throughput (ToxCast) in vitro assays. Lastly, we mapped adverse outcome pathways (AOPs) associated with identified ToxCast assays to determine potential impacts across levels of biological organization within the aquatic system. These approaches were applied to subsistence fish collected from the Grand Portage Indian Reservation and 1854 Ceded Territory in 2017 and 2019. Overall, 24 CECs were detected in fish tissues, with all but one of the sites having at least one detection. The combined implementation of these tools revealed that subsistence fish exposed to CECs had histological and macroscopic tissue and organ abnormalities, although a direct causal link could not be established. The health of fish in undeveloped sites was as poor, or sometimes poorer, than fish in developed and wastewater effluent-impacted sites based on gross and histologic tissue lesions. Adverse outcome pathways revealed potential hazardous pathways of individual CECs to fish. A better understanding of how the health of wild fish harvested for consumption is affected by CECs may help prioritize risk management research efforts and can ultimately be used to guide fishery management and public health decisions. Integr Environ Assess Manag 2024;20:846-863. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Host gene expression is associated with viral shedding magnitude in blue-winged teals (Spatula discors) infected with low-path avian influenza virus.

Intraspecific variation in host infectiousness affects disease transmission dynamics in human, domestic animal, and many wildlife host-pathogen systems including avian influenza virus (AIV); therefore, identifying host factors related to host infectiousness is important for understanding, controlling, and preventing future outbreaks. Toward this goal, we used RNA-seq data collected from low pathogenicity avian influenza virus (LPAIV)-infected blue-winged teal (Spatula discors) to determine the association between host gene expression and intraspecific variation in cloacal viral shedding magnitude, the transmissible fraction of virus. We found that host genes were differentially expressed between LPAIV-infected and uninfected birds early in the infection, host genes were differentially expressed between shed level groups at one-, three-, and five-days post-infection, host gene expression was associated with LPAIV infection patterns over time, and genes of the innate immune system had a positive linear relationship with cloacal viral shedding. This study provides important insights into host gene expression patterns associated with intraspecific LPAIV shedding variation and can serve as a foundation for future studies focused on the identification of host factors that drive or permit the emergence of high viral shedding individuals.

Using the Daphnia magna Transcriptome to Distinguish Water Source: Wetland and Stormwater Case Studies.

A major challenge in ecotoxicology is accurately and sufficiently measuring chemical exposures and biological effects given the presence of complex and dynamic contaminant mixtures in surface waters. It is impractical to quantify all chemicals in such matrices over space and time, and even if it were practical, concomitant biological effects would not be elucidated. Our study examined the performance of the Daphnia magna transcriptome to detect distinct responses across three water sources in Minnesota: laboratory (well) waters, wetland waters, and storm waters. Pyriproxyfen was included as a gene expression and male neonate production positive control to examine whether gene expression resulting from exposure to this well-studied juvenoid hormone analog can be detected in complex matrices. Laboratory-reared (<24 h) D. magna were exposed to a water source and/or pyriproxyfen for 16 days to monitor phenotypic changes or 96 h to examine gene expression responses using Illumina HiSeq 2500 (10 million reads per library, 50-bp paired end [2 × 50]). The results indicated that a unique gene expression profile was produced for each water source. At 119 ng/L pyriproxyfen (~25% effect concentration) for male neonate production, as expected, the Doublesex1 gene was up-regulated. In descending order, gene expression patterns were most discernable with respect to pyriproxyfen exposure status, season of stormwater sample collection, and wetland quality, as indicated by the index of biological integrity. However, the biological implications of the affected genes were not broadly clear given limited genome resources for invertebrates. Our study provides support for the utility of short-term whole-organism transcriptomic testing in D. magna to discern sample type, but highlights the need for further work on invertebrate genomics. Environ Toxicol Chem 2022;41:2107-2123. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Differential gene expression reveals host factors for viral shedding variation in mallards (Anas platyrhynchos) infected with low-pathogenic avian influenza virus.

Intraspecific variation in pathogen shedding impacts disease transmission dynamics; therefore, understanding the host factors associated with individual variation in pathogen shedding is key to controlling and preventing outbreaks. In this study, ileum and bursa of Fabricius tissues of wild-bred mallards (Anas platyrhynchos) infected with low-pathogenic avian influenza (LPAIV) were evaluated at various post-infection time points to determine genetic host factors associated with intraspecific variation in viral shedding. By analysing transcriptome sequencing data (RNA-seq), we found that LPAIV-infected wild-bred mallards do not exhibit differential gene expression compared to uninfected birds, but that gene expression was associated with cloacal viral shedding quantity early in the infection. In both tissues, immune gene expression was higher in high/moderate shedding birds compared to low shedding birds, and significant positive relationships with viral shedding were observed. In the ileum, expression for host genes involved in viral cell entry was lower in low shedders compared to moderate shedders at 1 day post-infection (DPI), and expression for host genes promoting viral replication was higher in high shedders compared to low shedders at 2 DPI. Our findings indicate that viral shedding is a key factor for gene expression differences in LPAIV-infected wild-bred mallards, and the genes identified in this study could be important for understanding the molecular mechanisms driving intraspecific variation in pathogen shedding.

Anthropogenic factors associated with contaminants of emerging concern detected in inland Minnesota lakes (Phase II).

Contaminants of emerging concern (CECs) include a variety of pharmaceuticals, personal care products, and hormones commonly detected in surface waters. Human activities, such as wastewater treatment and discharge, contribute to the distribution of CECs in water, but other sources and pathways are less frequently examined. This study aimed to identify anthropogenic activities and environmental characteristics associated with the presence of CECs, previously determined to be of high priority for further research and mitigation, in rural inland lakes in northeastern Minnesota, United States. The setting for this study consisted of 21 lakes located within both the Grand Portage Indian Reservation and the 1854 Ceded Territory, where subsistence hunting and fishing are important to the cultural heritage of the indigenous community. We used data pertaining to numbers of buildings, healthcare facilities, wastewater treatment plants, impervious surfaces, and wetlands within defined areas surrounding the lakes as potential predictors of the detection of high priority CECs in water, sediment, and fish. Separate models were run for each contaminant detected in each sample media. We used least absolute shrinkage and selection operator (LASSO) models to account for both predictor selection and parameter estimation for CEC detection. Across contaminants and sample media, the percentage of impervious surface was consistently positively associated with CEC detection. Number of buildings in the surrounding area was often negatively associated with CEC detection, though nonsignificant. Surrounding population, presence of wastewater treatment facilities, and percentage of wetlands in surrounding areas were positively, but inconsistently, associated with CECs, while catchment area and healthcare centers were generally not associated. The results of this study highlight human activities and environmental characteristics associated with CEC presence in a rural area, informing future work regarding specific sources and transport pathways. We also demonstrate the utility of LASSO modeling in the identification of these important relationships.

A chemical prioritization process: Applications to contaminants of emerging concern in freshwater ecosystems (Phase I).

Contaminants of emerging concern (CECs), such as pharmaceuticals, personal care products, and hormones, are frequently found in aquatic ecosystems around the world. Information on sublethal effects from exposure to commonly detected concentrations of CECs is lacking and the limited availability of toxicity data makes it difficult to interpret the biological significance of occurrence data. However, the ability to evaluate the effects of CECs on aquatic ecosystems is growing in importance, as detection frequency increases. The goal of this study was to prioritize the chemical hazards of 117 CECs detected in subsistence species and freshwater ecosystems on the Grand Portage Indian Reservation and adjacent 1854 Ceded Territory in Minnesota, USA. To prioritize CECs for management actions, we adapted Minnesota Pollution Control Agency's Aquatic Toxicity Profiles framework, a tool for the rapid assessment of contaminants to cause adverse effects on aquatic life by incorporating chemical-specific information. This study aimed to 1) perform a rapid-screening assessment and prioritization of detected CECs based on their potential environmental hazard; 2) identify waterbodies in the study region that contain high priority CECs; and 3) inform future monitoring, assessment, and potential remediation in the study region. In water samples alone, 50 CECs were deemed high priority. Twenty-one CECs were high priority among sediment samples and seven CECs were high priority in fish samples. Azithromycin, DEET, diphenhydramine, fluoxetine, miconazole, and verapamil were high priority in all three media. Due to the presence of high priority CECs throughout the study region, we recommend future monitoring of particular CECs based on the prioritization method used here. We present an application of a chemical hazard prioritization process and identify areas where the framework may be adapted to meet the objectives of other management-related assessments.

Sex ratio of Western Bluebirds Sialia mexicana is mediated by phenology and clutch size.

Mothers may produce more of one sex to maximize their fitness if there are differences in the cost of producing each sex or there are differences in their relative reproductive value. Breeding date and clutch size are known to influence offspring sex ratios in birds through sex differences in dispersal, social behaviours, differential mortality, and available food resources. We tested if breeding date, clutch size and drought conditions influenced offspring sex ratios in a sexually size-monomorphic species, the Western Bluebird, by interrogating a 21-year dataset. After controlling for differential mortality, we found that hatch dates late in the breeding season were associated with the production of more females, suggesting that the value of producing males declines as the breeding season progresses. When clutch size was taken into account, small clutches yielded significantly more females late in the breeding season compared to the early and middle parts of the breeding season that produced significantly more males. Large clutches early in the season tended to produce more females, although this was not significant. Drought severity was not correlated with sex ratio adjustment. We propose and discuss several explanations for these patterns, including male offspring, but not female offspring, acting as helpers, increased female nestling provisioning late in the breeding season, differences in food abundance, and egg-laying order. Future work will help to uncover the mechanisms leading to these patterns. Identifying patterns and mechanisms of sex ratio skew from long-term datasets is important for informing predictions regarding life-history trade-offs in wildlife populations.

The association between SAα2,3Gal occurrence frequency and avian influenza viral load in mallards (Anas platyrhynchos) and blue-winged teals (Spatula discors).

BACKGROUND: Individual heterogeneity in pathogen load can affect disease transmission dynamics; therefore, identifying intrinsic factors responsible for variation in pathogen load is necessary for determining which individuals are prone to be most infectious. Because low pathogenic avian influenza viruses (LPAIV) preferentially bind to alpha-2,3 sialic acid receptors (SAα2,3Gal) in the intestines and bursa of Fabricius in wild ducks (Anas and Spatula spp.), we investigated juvenile mallards (Anas platyrhyncos) and blue-winged teals (Anas discors) orally inoculated with A/northern pintail/California/44221-761/2006 (H5N9) and the virus titer relationship to occurrence frequency of SAα2,3Gal in the intestines and bursa. To test the natural variation of free-ranging duck populations, birds were hatched and raised in captivity from eggs collected from nests of free-ranging birds in North Dakota, USA. Data generated from qPCR were used to quantify virus titers in cloacal swabs, ileum tissue, and bursa of Fabricius tissue, and lectin histochemistry was used to quantify the occurrence frequency of SAα2,3Gal. Linear mixed models were used to analyze infection status, species, and sex-based differences. Multiple linear regression was used to analyze the relationship between virus titer and SAα2,3Gal occurrence frequency. RESULTS: In mallards, we found high individual variation in virus titers significantly related to high variation of SAα2,3Gal in the ileum. In contrast to mallards, individual variation in teals was minimal and significant relationships between virus titers and SAα2,3Gal were not determined. Collectively, teals had both higher virus titers and a higher occurrence frequency of SAα2,3Gal compared to mallards, which may indicate a positive association between viral load and SAα2,3Gal. Statistically significant differences were observed between infected and control birds indicating that LPAIV infection may influence the occurrence frequency of SAα2,3Gal, or vice versa, but only in specific tissues. CONCLUSIONS: The results of this study provide quantitative evidence that SAα2,3Gal abundance is related to LPAIV titers; thus, SAα2,3Gal should be considered a potential intrinsic factor influencing variation in LPAIV load.

Occurrence of contaminants of emerging concern in aquatic ecosystems utilized by Minnesota tribal communities.

Pharmaceuticals, personal care products, hormones, and other chemicals lacking water quality standards are frequently found in surface water. While evidence is growing that these contaminants of emerging concern (CECs) - those previously unknown, unrecognized, or unregulated - can affect the behavior and reproduction of fish and wildlife, little is known about the distribution of these chemicals in rural, tribal areas. Therefore, we surveyed the presence of CECs in water, sediment, and subsistence fish species across various waterbodies, categorized as undeveloped (i.e., no human development along shorelines), developed (i.e., human development along shorelines), and wastewater effluent-impacted (i.e., contain effluence from wastewater treatment plants), within the Grand Portage Indian Reservation and 1854 Ceded Territory in northeastern Minnesota, U.S.A. Overall, in 28 sites across three years (2016-2018), 117 of the 158 compounds tested were detected in at least one form of medium (i.e., water, sediment, or fish). CECs were detected most frequently at wastewater effluent-impacted sites, with up to 83 chemicals detected in one such lake, while as many as 17 were detected in an undeveloped lake. Although there was no statistically significant difference between the number of CECs present in developed versus undeveloped lakes, a range of 3-17 CECs were detected across these locations. Twenty-two CECs were detected in developed and undeveloped sites that were not detected in wastewater effluent-impacted sites. The detection of CECs in remote, undeveloped locations, where subsistence fish are harvested, raises scientific questions about the safety and security of subsistence foods for indigenous communities. Further investigation is warranted so that science-based solutions to reduce chemical risks to aquatic life and people can be developed locally and be informative for indigenous communities elsewhere.

Sublethal and Lethal Methods to Detect Recent Imidacloprid Exposure in Birds with Application to Field Studies.

We used domestic chickens (Gallus gallus domesticus) as a model for granivorous birds to identify methods to detect recent imidacloprid exposure in wild birds. We conducted dosing experiments of 1, 5, 10, and 20% of a reported median lethal dose for domestic chickens using repeated daily exposures over 7 d, at dosages equating to 1.04, 5.2, 10.4, and 20.8 mg/kg/d. We examined the parent compound and metabolites in serial collections of feces and blood during exposures and for 15 d after exposures. We also collected liver, kidney, brain, muscle, and spleen at the experiment end. Mean concentrations of parent compound at 15 d postexposure were highest in the feces and brain, followed by the liver, muscle, spleen, and kidney; but mean concentrations of metabolites 5-OH-imidacloprid and imidacloprid-olefin were highest in feces; then liver, spleen, muscle, and kidney; and then brain. Imidacloprid was rapidly cleared from blood, with only one individual in any dose group having detectable concentrations after 48 h. In contrast, fecal pellets had the highest frequency of imidacloprid detection after 15 d. Concentrations of metabolites were higher than those of the parent compound at all sampling times examined but provided no information about time since exposure. Feces may provide a reliable nonlethal method for detection of recent imidacloprid exposure in wild birds. Additional work is needed to disentangle exposure dose concentration and time since exposure in field-collected samples. Environ Toxicol Chem 2020;39:1355-1366. © 2020 SETAC.

De novo transcriptome assembly and data for the blue-winged teal (Spatula discors).

The blue-winged teal (Spatula discors) is a recreationally and ecologically important dabbling duck species in North America. Transcriptomic data of this species can be used in public and animal health studies given its role as a natural reservoir host for avian influenza, which can be a zoonotic disease of high concern. Ileum and bursa of Fabricius tissues were sampled from six captive raised blue-winged teals, four of the six who were experimentally infected with low-pathogenic avian influenza virus H5N9. RNAseq data were generated from extracted total mRNA from each tissue and pooled to create a de novo assembly of the transcriptome using Trinity. A total of 571,105 transcripts were identified at 449,956 unique unigenes that have been functionally annotated. This transcriptome will be useful for future blue-winged teal gene expression research, especially in hypothesis driven differential expression studies to determine the driving forces of avian influenza host-pathogen interactions, spatial distribution, and transmission.

Sialic acid on avian erythrocytes.

Understanding variation in physiological traits across taxa is a central question in evolutionary biology that has wide-ranging implications in biomedicine, disease ecology, and environmental protection. Sialic acid (Sia), and in particular, 5-N-acetylneuraminic acid (Neu5Ac), is chemically bound to galactose and the underlying glycan via α2-3 or α2-6 glycosidic linkage (i.e., Siaα2-3Galactose or Siaα2-6Galactose), conferring two different cell surface structures that affects cell to cell communication and interactions with foreign agents including microparasites and toxins. As an initial step towards understanding variation of Sia across the class Aves, we collected red blood cells (RBCs or erythrocytes) and measured Sia quantity in 76 species and 340 individuals using HPLC-MS/MS and glycosidic linkage type in 24 species and 105 individuals using hemagglutination assay. Although Sia quantity did not, α2-6 glycosidic linkage did exhibit a discernable phylogenetic pattern as evaluated by a phylogenetic signal (λ) value of 0.7. Sia quantity appeared to be higher in after hatch year birds than hatch year birds (P < 0.05); moreover, ~80% of the measured Sia across all individuals or species was expressed by ~20% of the individuals or species. Lastly, as expected, we detected a minimal presence of 5-N-glycolylneuraminic acid in the avian RBCs tested. These data provide novel insights and a large baseline dataset for further study on the variability of Sia in the class Aves which might be useful for understanding Sia dependent processes in birds.

An examination of the effect of aerosolized Permanone insecticide on zebra finch susceptibility to West Nile virus.

West Nile virus (WNV) is maintained cryptically primarily in avian (passerine) populations, where it is transmitted by Culex spp. mosquitoes. Mosquito-control measures currently include physical activities to reduce mosquito-breeding sites and the application of mosquito larvicides or aerosolized insecticides to kill adults (adulticides) when arboviral diseases such as WNV or Zika virus are detected in mosquito populations. Organochlorine, organophosphorus, carbamate, and pyrethroid insecticides are often used. Previous work suggests an effect of pyrethroids on the immune system in a variety of vertebrates. We examined the effects of exposure to aerosolized Permanone® 30:30 insecticide (permethrin and piperonyl butoxide in soy oil vehicle) at approximately 103 to 106 times potential environmental concentrations on the response of captive zebra finches (Taeniopygia guttata) to experimental challenge with WNV. Compared to vehicle control birds, WNV outcome was unchanged (65% of birds produced a viremia) in the "low" exposure (9.52 ± 3.13 mg/m3 standard deviation [SD] permethrin) group but reduced in the "high" exposure (mean 376.5 ± 27.9 mg/m3 SD permethrin) group (30% were viremic; p < 0.05). After clearing WNV infection, birds treated with Permanone regained less body mass than vehicle-treated birds (p < 0.001). The present study suggests that exposure to aerosolized Permanone insecticide at levels exceeding typical application rates has the potential to not change or to mildly enhance a bird's resistance to WNV. Environ Toxicol Chem 2017;36:3376-3386. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Nature and Consequences of Biological Reductionism for the Immunological Study of Infectious Diseases.

Evolution has conserved "economic" systems that perform many functions, faster or better, with less. For example, three to five leukocyte types protect from thousands of pathogens. To achieve so much with so little, biological systems combine their limited elements, creating complex structures. Yet, the prevalent research paradigm is reductionist. Focusing on infectious diseases, reductionist and non-reductionist views are here described. The literature indicates that reductionism is associated with information loss and errors, while non-reductionist operations can extract more information from the same data. When designed to capture one-to-many/many-to-one interactions-including the use of arrows that connect pairs of consecutive observations-non-reductionist (spatial-temporal) constructs eliminate data variability from all dimensions, except along one line, while arrows describe the directionality of temporal changes that occur along the line. To validate the patterns detected by non-reductionist operations, reductionist procedures are needed. Integrated (non-reductionist and reductionist) methods can (i) distinguish data subsets that differ immunologically and statistically; (ii) differentiate false-negative from -positive errors; (iii) discriminate disease stages; (iv) capture in vivo, multilevel interactions that consider the patient, the microbe, and antibiotic-mediated responses; and (v) assess dynamics. Integrated methods provide repeatable and biologically interpretable information.

SURVEY FOR WEST NILE VIRUS ANTIBODIES IN WILD DUCKS, 2004-06, USA.

Detection of West Nile virus (WNV) in ducks has been reported in North America in isolated cases of mortality in wild waterbirds and following outbreaks in farmed ducks. Although the virus has been noted as an apparent incidental finding in several species of ducks, little is known about the prevalence of exposure or the outcome of infection with WNV in wild ducks in North America. From 2004-06, we collected sera from 1,406 wild-caught American Wigeon ( Anas americana ), Mallard ( Anas platyrhynchos ), and Northern Pintail ( Anas acuta ) ducks at national wildlife refuges (NWRs) in North Dakota and Wood Ducks ( Aix sponsa ) at NWRs in South Carolina and Tennessee. We measured the prevalence of previous exposure to WNV in these ducks by measuring WNV antibodies and evaluated variation in exposure among species, age, and year. Additionally, we evaluated the performance of a commercial antibody to wild bird immunoglobulin in duck species that varied in their phylogenetic relatedness to the bird species the antibody was directed against. As determined by a screening immunoassay and a confirmatory plaque reduction neutralization assay, the prevalence of WNV antibody was 10%. In light of experimental studies that show ducks to be relatively resistant to mortality caused by WNV, the antibody prevalence we detected suggests that wild ducks may be less-frequently exposed to WNV than expected for birds inhabiting wetlands where they may acquire infection from mosquitoes.

Birds shed RNA-viruses according to the pareto principle.

A major challenge in disease ecology is to understand the role of individual variation of infection load on disease transmission dynamics and how this influences the evolution of resistance or tolerance mechanisms. Such information will improve our capacity to understand, predict, and mitigate pathogen-associated disease in all organisms. In many host-pathogen systems, particularly macroparasites and sexually transmitted diseases, it has been found that approximately 20% of the population is responsible for approximately 80% of the transmission events. Although host contact rates can account for some of this pattern, pathogen transmission dynamics also depend upon host infectiousness, an area that has received relatively little attention. Therefore, we conducted a meta-analysis of pathogen shedding rates of 24 host (avian) - pathogen (RNA-virus) studies, including 17 bird species and five important zoonotic viruses. We determined that viral count data followed the Weibull distribution, the mean Gini coefficient (an index of inequality) was 0.687 (0.036 SEM), and that 22.0% (0.90 SEM) of the birds shed 80% of the virus across all studies, suggesting an adherence of viral shedding counts to the Pareto Principle. The relative position of a bird in a distribution of viral counts was affected by factors extrinsic to the host, such as exposure to corticosterone and to a lesser extent reduced food availability, but not to intrinsic host factors including age, sex, and migratory status. These data provide a quantitative view of heterogeneous virus shedding in birds that may be used to better parameterize epidemiological models and understand transmission dynamics.

Feedback-based, system-level properties of vertebrate-microbial interactions.

BACKGROUND: Improved characterization of infectious disease dynamics is required. To that end, three-dimensional (3D) data analysis of feedback-like processes may be considered. METHODS: To detect infectious disease data patterns, a systems biology (SB) and evolutionary biology (EB) approach was evaluated, which utilizes leukocyte data structures designed to diminish data variability and enhance discrimination. Using data collected from one avian and two mammalian (human and bovine) species infected with viral, parasite, or bacterial agents (both sensitive and resistant to antimicrobials), four data structures were explored: (i) counts or percentages of a single leukocyte type, such as lymphocytes, neutrophils, or macrophages (the classic approach), and three levels of the SB/EB approach, which assessed (ii) 2D, (iii) 3D, and (iv) multi-dimensional (rotating 3D) host-microbial interactions. RESULTS: In all studies, no classic data structure discriminated disease-positive (D+, or observations in which a microbe was isolated) from disease-negative (D-, or microbial-negative) groups: D+ and D- data distributions overlapped. In contrast, multi-dimensional analysis of indicators designed to possess desirable features, such as a single line of observations, displayed a continuous, circular data structure, whose abrupt inflections facilitated partitioning into subsets statistically significantly different from one another. In all studies, the 3D, SB/EB approach distinguished three (steady, positive, and negative) feedback phases, in which D- data characterized the steady state phase, and D+ data were found in the positive and negative phases. In humans, spatial patterns revealed false-negative observations and three malaria-positive data classes. In both humans and bovines, methicillin-resistant Staphylococcus aureus (MRSA) infections were discriminated from non-MRSA infections. CONCLUSIONS: More information can be extracted, from the same data, provided that data are structured, their 3D relationships are considered, and well-conserved (feedback-like) functions are estimated. Patterns emerging from such structures may distinguish well-conserved from recently developed host-microbial interactions. Applications include diagnosis, error detection, and modeling.

Using unfixed, frozen tissues to study natural mucin distribution.

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight(1-3). Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract(3,4). In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins(5-9). The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms(8,10-12). As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media. Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods(13). Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo. In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods. We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.

Testing independent and interactive effects of corticosterone and synergized resmethrin on the immune response to West Nile virus in chickens.

Public health agencies utilize aerial insecticides to interrupt an active West Nile virus (WNV) transmission cycle, which may expose WNV-infected birds to these agents. Although resmethrin has been considered benign to birds, no studies have evaluated whether the environmentally employed form of resmethrin with PBO synergist (synergized resmethrin (SR)) can suppress avian immunity to WNV infection and enhance a bird's host competence. Recognizing that wild birds confront toxicological stressors in the context of various physiological states, we exposed four groups (n=9-11) of 9-week-old chickens (Gallus domesticus) to drinking water with either SR (three alternate days at 50 microg/l resmethrin+150 microg/l piperonyl butoxide), CORT (10 days at 20mg/l to induce subacute stress), the combination of SR and CORT, or 0.10% ethanol vehicle coincident with WNV infection. Compared to controls, SR treatment did not magnify but extended viremia by 1 day, and depressed IgG; CORT treatment elevated (mean, 4.26 log(10)PFU/ml) and extended viremia by 2 days, enhanced IgM and IgG, and increased oral virus. The combination of SR and CORT increased the number of chickens that shed oral virus compared to those treated with CORT alone. None of the chickens developed a readily infectious viremia to mosquitoes (none >or=5 log(10)PFU/ml), but viremia in a CORT-exposed chicken was up to 4.95 log(10)PFU/ml. Given that SR is utilized during WNV outbreaks, continued work toward a complete risk assessment of the potential immunotoxic effects of SR is warranted. This would include parameterization of SR exposures with immunological consequences in wild birds using both replicating (in the laboratory) and non-replicating (in the field) antigens. As a start, this study indicates that SR can alter some immunological parameters, but with limited consequences to primary WNV infection outcome, and that elevated CORT mildly enhances SRs immunotoxicity in chickens.