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Background Accurate nestling age is valuable for studies on nesting strategies, productivity, and impacts on reproductive success. Most aging guides consist of descriptions and photographs that are time consuming to read and subjective to interpret. The Western Bluebird (Sialia mexicana) is a secondary cavity-nesting passerine that nests in coniferous and open deciduous forests. Nest box programs for cavity-nesting species have provided suitable nesting locations and opportunities for data collection on nestling growth and development. Methods We developed models for predicting the age of Western Bluebird nestlings from morphometric measurements using model training and validation. These were developed for mass, tarsus, and two different culmen measurements. Results Our models were accurate to within less than a day, and each model worked best for a specific age range. The mass and tarsus models can be used to estimate the ages of Western Bluebird nestlings 0–10 days old and were accurate to within 0.5 days for mass and 0.7 days for tarsus. The culmen models can be used to estimate ages of nestlings 0–15 days old and were also accurate to within less than a day. The daily mean, minimum, and maximum values of each morphometric measurement are provided and can be used in the field for accurate nestling age estimations in real time. Conclusions The model training and validation procedures used here demonstrate that this method can create aging models that are highly accurate. The methods can be applied to any passerine species provided sufficient nestling morphometric data are available.

Objective Currently, next generation sequencing (NGS) is widely used to decode potential novel or variant pathogens both in emergent outbreaks and in routine clinical practice. However, the efficient identification of novel or diverged pathogenomic compositions remains a big challenge. It is especially true for short DNA sequence fragments from NGS, since sequence similarity searching is vulnerable to false negatives or false positives, as is mismatching or matching with unrelated proteins. Therefore, this study aimed to establish a bioinformatics approach that can generate unique motif sequences for profiling searching, resulting in high specificity and sensitivity. Results In this study, we introduced a Shortest Unique Representative Hidden Markov Model (HMM) approach to identify bacterial toxin, virulence factor (VF), and antimicrobial resistance (AR) in short sequence reads. We first construct unique representative domain sequences of toxin genes, VFs, and ARs to avoid potential false positives, and then to use HMM models to accurately identify potential toxin, VF, and AR fragments. The benchmark shows this approach can achieve relatively high specificity and sensitivity if the appropriate cutoff value is applied. Our approach can be used to recognize the protein sequences of known toxins and pathogens, identifies their common characteristics and then searches for similar sequences in other organisms.

Background Estimates of the geographical distribution of Culex mosquitoes in the Americas have been limited to state and provincial levels in the United States and Canada and based on data from the 1980s. Since these estimates were made, there have been many more documented observations of mosquitoes and new methods have been developed for species distribution modeling. Moreover, mosquito distributions are affected by environmental conditions, which have changed since the 1980s. This calls for updated estimates of these distributions to understand the risk of emerging and re-emerging mosquito-borne diseases. Methods We used contemporary mosquito data, environmental drivers, and a machine learning ecological niche model to create updated estimates of the geographical range of seven predominant Culex species across North America and South America: Culex erraticus, Culex nigripalpus, Culex pipiens, Culex quinquefasciatus, Culex restuans, Culex salinarius, and Culex tarsalis. Results We found that Culex mosquito species differ in their geographical range. Each Culex species is sensitive to both natural and human-influenced environmental factors, especially climate and land cover type. Some prefer urban environments instead of rural ones, and some are limited to tropical or humid areas. Many are found throughout the Central Plains of the USA. Conclusions Our updated contemporary Culex distribution maps may be used to assess mosquito-borne disease risk. It is critical to understand the current geographical distributions of these important disease vectors and the key environmental predictors structuring their distributions not only to assess current risk, but also to understand how they will respond to climate change. Since the environmental predictors structuring the geographical distribution of mosquito species varied, we hypothesize that each species may have a different response to climate change. Graphical abstract

Long‐term data on host and parasite fitness are important for predicting how host–parasite interactions will be altered in an era of global change. Here, we use data collected from 1997 to 2013 to explore effects of changing environmental conditions on bird–blowfly interactions in northern New Mexico. The objectives of this study were to examine what climate variables influence blowfly prevalence and intensity and to determine whether blowflies and climate variables affect bird fledging success. We examined how temperature, precipitation, and drought affect two parasitic blowflies and their hosts, Western Bluebirds (Sialia mexicana) and Ash‐throated Flycatchers (Myiarchus cinerascens). We found that blowfly prevalence did not change over time. Blowfly intensity increased over time in bluebird nests, but not in flycatcher nests. More blowflies result in slightly higher fledging success in bluebirds, but not flycatchers. There was a significant interaction between blowflies and precipitation on bluebird fledging success. For flycatchers, there was a significant interaction between blowflies and temperature and between blowflies and drought severity on fledging success. Given that the southwest is projected to be hotter and have more frequent and prolonged droughts, we predict that flycatchers may be negatively impacted by blowflies if these trends continue. Future work should focus on investigating the role of both blowflies and climate on fledging success. Climate patterns may negatively impact host fitness through altered parasite pressure.

Objective: Currently, next generation sequencing (NGS) is widely used to decode potential novel or variant pathogens both in emergent outbreaks and in routine clinical practice. However, the efficient identification of novel or diverged pathogenomic compositions remains a big challenge. It is especially true for short DNA sequence fragments from NGS, since sequence similarity searching is vulnerable to false negatives or false positives, as mismatching or matching with unrelated proteins. Therefore, this study aimed to establish a bioinformatics approach that can generate unique motif sequences for profiling searching, resulting in high specificity and sensitivity. Results: In this study, we introduced a shortest unique representative hidden Markov model (HMM) approach to identify bacterial toxin, virulence factor (VF), and antimicrobial resistance (AR) in short sequence reads. We first construct unique representative domain sequences of toxin genes, VFs, and ARs to avoid potential false positives, and then to use HMM models to accurately identify potential toxin, VF, and AR fragments. The benchmark shows this approach can achieve relatively high specificity and sensitivity if the appropriate cutoff value is applied.

BackgroundIndividual 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 (AnasandSpatulaspp.), 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.ResultsIn 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.ConclusionsThe 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.

Changes in the Earth’s climate and weather continue to impact the planet’s ecosystems, including the interface of infectious disease agents with their hosts and vectors. Environmental disasters, natural and human-made activities raise risk factors that indirectly facilitate infectious disease outbreaks. Subsequently, changes in habitat, displaced populations, and environmental stresses that affect the survival of species are amplified over time. The recurrence and spread of vector-borne (e.g., mosquito, tick, aphid) human, animal, and plant pathogens to new geographic locations are also influenced by climate change. The distribution and range of humans, agricultural animals and plants, wildlife and native plants, as well as vectors, parasites, and microbes that cause neglected diseases of the tropics as well as other global regions are also impacted. In addition, genomic sequencing can now be applied to detect signatures of infectious pathogens as they move into new regions. Molecular detection assays complement metagenomic sequencing to help us understand the microbial community found within the microbiomes of hosts and vectors, and help us uncover mechanistic relationships between climate variability and pathogen transmission. Our understanding of, and responses to, such complex dynamics and their impacts can be enhanced through effective, multi-sectoral One Health engagement coupled with applications of both traditional and novel technologies. Concerted efforts are needed to further harness and leverage technology that can identify and track these impacts of climate changes in order to mitigate and adapt to their effects.

Background Individual heterogeneity in pathogen load can impact 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. Since 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 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 percentage of SAα2,3Gal in the intestines and bursa. To evaluate 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 real time RT-PCR used to quantify virus titers in cloacal swabs, ileum tissue, and bursa of Fabricius tissue and lectin histochemistry used to quantify percentage of SAα2,3Gal was analyzed through a series of multiple linear regression and mixed models. Results In mallards, we found high individual variation in virus titers significantly related to high variation of SAα2,3Gal in the ileum, with sex being a significant factor. 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 percentage 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 percentage of SAα2,3Gal, or vice versa, but only in specific tissues.Conclusions The results of this study provide quantitative evidence that SAα2,3Gal is related to LPAIV titers; thus, SAα2,3Gal should be considered a potential intrinsic factor of variation in LPAIV load.

An increase in tree mortality is expected to occur worldwide due to climate-induced drought and increasing temperatures. The 2000–2002 drought in the southwestern United States was one of the most severe in the last 50 years. It led to a severe outbreak of bark beetles that resulted in high mortality of pinon pine (Pinus edulis) trees on the Pajarito Plateau in Northern New Mexico beginning in 2002. Many areas in pinon-juniper habitat had entire stands of pinon die leaving only juniper (Juniperus spp.). Point count surveys were used to determine avian responses to tree mortality from 2003 to 2013. We also tested whether birds responded differently in sites that were mechanically thinned in 2002 and 2003 on Los Alamos National Laboratory property compared to sites not thinned. Junipers and dead pinon pines due to bark beetles and drought were removed on thinned sites. Richness, diversity, and abundance steadily declined after 2003. There was a 73% decrease in abundance and a 45% decrease in richness from 2003 to 2013. There was no difference in community composition between thinned and unthinned sites. Bird abundance and species richness declined faster in thinned sites than unthinned sites, but diversity decreased similarly in both treatments. Several species disappeared over time and some declined substantially. Our results suggest a delay in bird responses to tree mortality on the Pajarito Plateau. Pinon mortality may be a significant threat to bird communities in the southwestern U.S., and tree thinning to control fire may be an added risk.

Around the world, scavenging birds such as vultures and condors have been experiencing drastic population declines. Scavenging birds have a distinct digestive process to deal with higher amounts of bacteria in their primary diet of carcasses in varying levels of decay. These observations motivate us to present an analysis of captive and healthy California condor (Gymnogyps californianus) microbiomes to characterize a population raised together under similar conditions. Shotgun metagenomic DNA sequences were analyzed from fecal and cloacal samples of captive birds. Classification of shotgun DNA sequence data with peptide signatures using the Sequedex package provided both phylogenetic and functional profiles, as well as individually annotated reads for targeted confirmatory analysis. We observed bacterial species previously associated with birds and gut microbiomes, including both virulent and opportunistic pathogens such as Clostridium perfringens, Propionibacterium acnes, Shigella flexneri, and Fusobacterium mortiferum, common flora such as Lactobacillus johnsonii, Lactobacillus ruminus, and Bacteroides vulgatus, and mucosal microbes such as Delftia acidovorans, Stenotrophomonas maltophilia, and Corynebacterium falsnii. Classification using shotgun metagenomic reads from phylogenetic marker genes was consistent with, and more specific than, analysis based on 16S rDNA data. Classification of samples based on either phylogenetic or functional profiles of genomic fragments differentiated three types of samples: fecal, mature cloacal and immature cloacal, with immature birds having approximately 40% higher diversity of microbes.

Los Alamos National Laboratory (LANL) was established in 1943 as part of the Manhattan Project to build nuclear weapons, and currently operates as a national research laboratory. As part of an ongoing assessment of site-related ecological risk at LANL, western bluebird (Sialia mexicana) and ash-throated flycatcher (Myiarchus cinerascens) eggs were collected from 1997 to 2012 and analyzed for 18 inorganic elements. Concentrations of many inorganic elements in eggs were below reporting limits. Between species comparisons revealed that western bluebird eggs had higher levels of barium while ash-throated flycatcher eggs had higher levels of mercury. No statistically significant differences were observed in concentrations of inorganic elements in western bluebird eggs collected from the study area (which consists of areas within the current and historic LANL boundary) and from a non-industrial reference site; nor were any statistically significant differences observed between two canyons of interest, known to have received effluents and storm water runoff from LANL facilities, and the non-industrial reference site. Inorganic element levels detected in western bluebirds were typically within the range measured in eggs of other passerine in the published literature. These data suggest that concentrations of inorganic elements in passerine eggs collected from the study area appear to be at levels causing negligible risks to local bird populations.

Simple Summary Environmental changes such as climate change have affected wildlife species behavior and geographic ranges globally. We analyzed nesting data of western bluebirds to determine whether the link between geographic range shifts of a western bluebird population in New Mexico, USA is due to individual adaptations or changes occurring at a larger scale. We looked at location data of marked and recaptured nestlings and adults that nested within our study area. We found that individual choices have no impact on the geographic range shift being observed in this population, suggesting that population-level processes, such as emigration and immigration, may be the main cause of these shifts. Abstract Geographic ranges of plants and animals are shifting due to environmental change. While some species are shifting towards the poles and upslope in elevation, the processes leading to these patterns are not well known. We analyzed 22 years of western bluebird (Sialia mexicana) data from a large nest box network in northern New Mexico at elevations between 1860 m and 2750 m. This population has shifted to higher elevations over time, but whether this is due to changes in nesting behavior and preference for higher elevation within the population or driven by immigration is unclear. We banded adults and nestlings from nest boxes and examined nesting location and elevation for individual birds captured two or more times. Most recaptured birds nested at the same nest boxes in subsequent years, and the number of birds that moved upslope did not significantly differ from the number that moved downslope. Fledglings moved greater distances and elevations than adults, but these movements were not upslope specific. Female fledglings showed greater changes in elevation and distance compared to male fledglings, but again, movements were not consistently upslope. The upslope shift in this population may be due to birds immigrating into the population and not from changes in individual nesting behavior.

Wildlife populations can respond to changes in climate conditions by either adapting or moving to areas with preferred climate regimes. We studied nesting responses of two bird species, western bluebird (Sialia mexicana) and ash-throated flycatcher (Myiarchus cinerascens), to changing climate conditions (i.e., rising temperatures and increased drought stress) over 21 years in northern New Mexico. We used data from 1649 nests to assess whether the two species responded to changing climate conditions through phenological shifts in breeding time or shifts in nesting elevation. We also examined changes in reproductive output (i.e., clutch size). Our data show that western bluebirds significantly increased nesting elevation over a 19-year period by approximately 5 m per year. Mean spring temperature was the best predictor of western bluebird nesting elevation. Higher nesting elevations were not correlated with hatch dates or clutch sizes in western bluebirds, suggesting that nesting at higher elevations does not affect breeding time or reproductive output. We did not observe significant changes in nesting elevation or breeding dates in ash-throated flycatchers. Nesting higher in elevation may allow western bluebirds to cope with the increased temperatures and droughts. However, this climate niche conservatism may pose a risk for the conservation of the species if climate change and habitat loss continue to occur. The lack of significant changes detected in nesting elevation, breeding dates, and reproductive output in ash-throated flycatchers suggests a higher tolerance for changing environmental conditions in this species. This is consistent with the population increases reported for flycatchers in areas experiencing dramatic climate changes.

Investigating disease pathogenesis and personalized prognostics are major biomedical needs. Because patients sharing the same diagnosis can experience different outcomes, such as survival or death, physicians need new personalized tools, including those that rapidly differentiate several inflammatory phases. To address these topics, a pattern recognition-based method (PRM) that follows an inverse problem approach was designed to assess, in

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.

Viral proteins evade host immune function by molecular mimicry, often achieved by short linear motifs (SLiMs) of three to ten consecutive amino acids (AAs). Motif mimicry tolerates mutations, evolves quickly to modify interactions with the host, and enables modular interactions with protein complexes. Host cells cannot easily coordinate changes to conserved motif recognition and binding interfaces under selective pressure to maintain critical signaling pathways. SLiMs offer potential for use in synthetic biology, such as better immunogens and therapies, but may also present biosecurity challenges. We survey viral uses of SLiMs to mimic host proteins, and information resources available for motif discovery. As the number of examples continues to grow, knowledge management tools are essential to help organize and compare new findings., Highlights • Short linear motifs (SLiMs) are patterns of three to ten consecutive AAs used by eukaryotic cells for tasks that include: signaling, localization, degradation, and proteolytic cleavage. • Viruses use SLiMs to their advantage, including interference with antiviral innate immune pathways. • Viral SLiMs can tolerate mutations, evolve quickly to modify host interactions, and co-occur in a modular manner or involve multiprotein complexes. • SLiMs are useful in synthetic biology, where minor edits can alter target specificity, modulate persistence, reprogram interactions with cell-signaling domains, and alter protein function in myriad other ways. • Aside from possible beneficial uses, for example, to produce better immunogens and develop therapeutic interventions against infectious disease, SLiMs may help characterize new and emerging threats to global health.

Infectious disease emergence into humans from animals or the environment occurs primarily due to genetic changes in the microbe through mutation or re-assortment making it either more transmissible or virulent or through a change in the disease “ecosystem”. Research into infectious disease emergence can be grouped into different strategic approaches. One strategic approach is to study a specific or model disease system to understand the ecology of an infectious disease and how is transmitted and propagated through the environment and different hosts and then extrapolate that disease system knowledge to related pathogens. The other strategic approach follows the genomics and phylogenetics—tracking how pathogens are evolving and changing at the amino acid level. Here we argue that for understanding complex zoonotic diseases and for the purposes of preventing emergence and re-emergence into humans, that the Return on Investment be considered for the best research strategy.

In recent years, the bacteria-killing assay (BKA) has become a popular technique among ecoimmunologists. New variations of that assay allow researchers to use smaller volumes of blood, an important consideration for those working on small-bodied animals. However, this version of the assay requires access to a lab with a nanodrop spectrophotometer, something that may not be available in the field. One possible solution is to freeze plasma for transport; however, this assumes that frozen plasma samples will give comparable results to fresh ones. We tested this assumption using plasma samples from three species of birds: chickens (Gallus gallus), ash-throated flycatchers (Myiarchus cinerascens), and western bluebirds (Sialia mexicana). Chicken plasma samples lost most or all of their bacterial killing ability after freezing. This did not happen in flycatchers and bluebirds; however, frozen plasma did not produce results comparable to those obtained using fresh plasma. We caution researchers using the BKA to use fresh samples whenever possible, and to validate the use of frozen samples on a species-by-species basis.

In 1943, Los Alamos National Laboratory (LANL) was established as part of the Manhattan project to design atomic weapons. LANL now operates as a multidisciplinary research institution. As part of an ongoing assessment of siterelated ecological risk, organochlorine pesticides, their metabolites, polycyclic aromatic hydrocarbons, polychlorinated biphenyls (PCBs), and 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) were evaluated in western bluebird (Sialia mexicana) and ash-throated flycatcher (Myiarchus cinerascens) eggs relative to a developed but non-industrial reference area; PCBs and TEQs were also evaluated in nestlings. Chemicals were below detection limits in the majority of samples. Western bluebird eggs collected from the study area had significantly lower concentrations of dieldrin, oxychlordane, and trans-nonachlor when compared with eggs from the reference area. No differences were observed in concentrations of dichlorodiphenyldichloroethylene (DDE), dichlorodiphenyltrichloroethane (DDT), and heptachlor epoxide. Ash-throated flycatcher eggs contained higher total TEQ concentrations when compared with western bluebird eggs; however, no differences in concentrations of DDE, DDT, dieldrin, or total PCBs were observed. No differences were observed in total PCBs or TEQs in nestlings between the two species. Western bluebird eggs contained higher levels of total PCBs and TEQs when compared with nestlings; no differences were observed in total PCBs or TEQs between ash-throated flycatcher eggs and nestlings. Chemical concentrations detected in eggs of both species were below levels that are associated with adverse effects reported in the scientific literature, suggesting that concentrations of organic chemicals observed here appear to be at levels causing negligible risks to local bird populations.

A central assumption in ecological immunology is that immune responses are costly, with costs manifesting directly (e.g., increases in metabolic rate and increased amino acid usage) or as tradeoffs with other life processes (e.g., reduced growth and reproductive success). Across taxa, host longevity, timing of maturity, and reproductive effort affect the organization of immune systems. It is reasonable, therefore, to expect that these and related factors should also affect immune activation costs. Specifically, species that spread their breeding efforts over a long lifetime should experience lower immune costs than those that mature and breed quickly and die comparatively early. Likewise, body mass should affect immune costs, as body size affects the extent to which hosts are exposed to parasites as well as how hosts can combat infections (via its effects on metabolic rates and other factors). Here, we used phylogenetic meta-regression to reveal that, in general, animals incur costs of immune activation, but small species that are relatively long-lived incur the largest costs. These patterns probably arise because of the relative need for defense when infection risk is comparatively high and fitness can only be realized over a comparatively long period. However, given the diversity of species considered here and the overall modest effects of body mass and life history on immune costs, much more research is necessary before generalizations are appropriate.

Parasites can impose heavy costs on their hosts, and females may benefit from selecting mates that are parasite resistant and/or have a stronger immune response. Trade-offs between immune response and sexual signaling have been proposed as a mechanism to ensure signal honesty. Much of the work on sexual signaling and immune response does not consider parasites directly and thus cannot confirm whether a stronger immune response necessarily results in lower parasite burdens. Also, immunity is costly, and these costs can lower the overall fitness of individuals with too strong of an immune response. Males with immune responses of intermediate strength are therefore expected to be preferred by females and have the highest reproductive success. We tested whether immune response and blood parasite loads relate to sexual signaling and mating preferences in western bluebirds (Sialia mexicana). Immunity did not predict male reproductive success when considering either within- or extra-pair offspring, although a stronger immune response was correlated with lower parasite loads. However, uninfected males were more likely to sire extra-pair offspring than males infected with avian malaria. Thus, females were more likely to mate with uninfected males but not necessarily males with a stronger immune response. Our results may indicate that females select parasite-resistant males as mates to gain resistance genes for their offspring or that infected males are less likely to pursue extra-pair copulations.

Coloration in birds can act as an important sexual signal in males, yet in many species, both sexes display bright colors. Social selection may account for this pattern, with more brightly colored individuals pairing together on the best territories. Mutual mate choice may also explain this, as males investing a great deal of parental care in the offspring should be choosy about their social mates. It is less clear whether this pattern of mate choice can apply to extra-pair partners as well. We examined western bluebirds (Sialia mexicana) to determine whether more colorful individuals tended to pair with one another, both in social pairs and between females and their extra-pair partners. Both male and female western bluebirds display both UV-blue structural plumage and a melanin-based chestnut breast patch, although females are duller than males. Social pairs mated assortatively with regard to UV-blue brightness, but not chestnut coloration. There was no evidence that extra-pair partners mated assortatively, but males with brighter UV-blue coloration had fewer extra-pair offspring in their nests. Older males were more successful at siring extra-pair offspring, despite displaying no differences in coloration compared to younger males. Coloration did not play a role in determining extra-pair male success. These results suggest that coloration plays a role in the formation of social pairs, but not mate choice for extra-pair partners.

Lead (Pb) is a metabolic poison that can negatively influence biological processes, leading to illness and mortality across a large spectrum of North American avifauna (.120 species) and other organisms. Pb poisoning can result from numerous sources, including ingestion of bullet fragments and shot pellets left in animal carcasses, spent ammunition left in the field, lost fishing tackle, Pb-based paints, large-scale mining, and Pb smelting activities. Although Pb shot has been banned for waterfowl hunting in the United States (since 1991) and Canada (since 1999), Pb exposure remains a problem for many avian species. Despite a large body of scientific literature on exposure to Pb and its toxicological effects on birds, controversy still exists regarding its impacts at a population level. We explore these issues and highlight areas in need of investigation: (1) variation in sensitivity to Pb exposure among bird species; (2) spatial extent and sources of Pb contamination in habitats in relation to bird exposure in those same locations; and (3) interactions between avian Pb exposure and other landscape-level stressors that synergistically affect bird demography. We explore multiple paths taken to reduce Pb exposure in birds that (1) recognize common ground among a range of affected interests; (2) have been applied at local to national scales; and (3) engage governmental agencies, interest groups, and professional societies to communicate the impacts of Pb ammunition and fishing tackle, and to describe approaches for reducing their availability to birds. As they have in previous times, users of fish and wildlife will play a key role in resolving the Pb poisoning issue.

Marine cone snails are carnivorous gastropods that use peptide toxins called conopeptides both as a defense mechanism and as a means to immobilize and kill their prey. These peptide toxins exhibit a large chemical diversity that enables exquisite specificity and potency for target receptor proteins. This diversity arises in terms of variations both in amino acid sequence and length, and in posttranslational modifications, particularly the formation of multiple disulfide linkages. Most of the functionally characterized conopeptides target ion channels of animal nervous systems, which has led to research on their therapeutic applications. Many facets of the underlying molecular mechanisms responsible for the specificity and virulence of conopeptides, however, remain poorly understood. In this review, we will explore the chemical diversity of conopeptides from a computational perspective. First, we discuss current approaches used for classifying conopeptides. Next, we review different computational strategies that have been applied to understanding and predicting their structure and function, from machine learning techniques for predictive classification to docking studies and molecular dynamics simulations for molecular-level understanding. We then review recent novel computational approaches for rapid high-throughput screening and chemical design of conopeptides for particular applications. We close with an assessment of the state of the field, emphasizing important questions for future lines of inquiry.

SummaryMost infectious disease surveillance methods are not well fit for early detection.To address such limitation, here we evaluated a ratio- and Systems Biology-basedmethod that does not require prior knowledge on the identity of an infectiveagent. Using a reference group of birds experimentally infected with West Nilevirus (WNV) and a problem group of unknown health status (except that theywere WNV-negative and displayed inflammation), both groups were followedover 22 days and tested with a system that analyses blood leucocyte ratios. To testthe ability of the method to discriminate small data sets, both the reference group(n = 5) and the problem group (n = 4) were small. The questions of interest wereas follows: (i) whether individuals presenting inflammation (disease-positive orD+) can be distinguished from non-inflamed (disease-negative or D ) birds, (ii)whether two or more D+ stages can be detected and (iii) whether sample sizeinfluences detection. Within the problem group, the ratio-based method distin-guished the following: (i) three (one D and two D+) data classes; (ii) two (earlyand late) inflammatory stages; (iii) fast versus regular or slow responders; and(iv) individuals that recovered from those that remained inflamed. Because ratiosdiffered in larger magnitudes (up to 48 times larger) than percentages, it is sug-gested that data patterns are likely to be recognized when disease surveillancemethods are designed to measure inflammation and utilize ratios.IntroductionNew infectious disease surveillance methods are needed(McClintock et al., 2010). The fact that most surveillancemethods are not designed for early detection (Bravataet al., 2004) justifies the development of new methods. Tothat end, new systems could be designed to facilitateprompt responsiveness – a concept associated with both aminimal data set (the smallest data set that can supportdiagnosis and decisions) and a ‘critical response time’ (theminimal time required to deploy interventions, Rivas et al.,2003; Kloeze et al., 2012).However, there are additional reasons why newinfectious disease surveillance systems are needed, includinga particular challenge: to choose an appropriate method,the identity of the infectious agent should be known –which cannot be known unless a specific test is used. Thecircularity of this problem leads to the following question:Is it possible to develop generic disease surveillance meth-ods, even when the infecting cause of disease is unknown?One way to answer this question is considering the factthat the immune system displays a ‘mirror image’ of theinfection – an ‘image’ that, although imperfect and not spe-cific, can tell, rapidly, whether leucocyte profiles departfrom the healthy profile. In the absence of an alternativeexplanation, when an inflammatory or immune response isdetected, such information may indicate an infectious pro-cess. Assessments of the immune response are used whenmicrobiological test results display a poor sensitivity, forexample, leucocyte counts and differential percentages arecurrently used to monitor pulmonary infections in children(Brennan et al., 2008). Similarly, early inflammatoryresponses are also used to evaluate vaccines (Mastelic et al.,2013).

In many bird species, double brooding (producing a second brood after successfully rearing a first) is a common reproductive strategy that can greatly increase reproductive output. However, the proportion of breeding pairs that produce a second brood varies considerably from year to year. Here, we examine how environmental factors such as weather patterns and individual factors such as clutch size, the female's body mass, and date of breeding affect the rate of double brooding in a population of the Western Bluebird (Sialia mexicana) in New Mexico. More pairs tended to produce second broods in years in which freezing ended earlier and there was warm, dry weather during the breeding season, but this pattern was apparent only when we excluded years with wildfires from the analysis. In addition, pairs that bred earlier were more likely to produce a second brood, and in these pairs females tended to be heavier. Thus both the length of the breeding season and the weather during that time influence ra...

Immunological reagents for wild, non-model species are limited or often non-existent for many species.In this study, we compare the reactivity of a new anti-passerine IgY secondary antibody with existing secondary antibodies developed for use with birds. Samples from 41 species from the following six avian orders were analysed: Anseriformes (1 family, 1 species), Columbiformes (1 family, 2 species), Galliformes (1 family, 1 species), Passeriformes (16 families, 34 species), Piciformes (1 family, 2 species) and Suliformes (1 family, 1 species). Direct ELISAs were performed to detect total IgY using goat anti-passerine IgY, goat anti-chicken IgY or goat anti-bird IgY secondary antibodies.The anti-passerine antibody exhibited significantly higher IgY reactivity compared to the anti-chicken and/or anti-bird antibodies in 80% of the passerine families tested. Birds in the order Piciformes (woodpeckers) and order Suliformes (cormorants) were poorly detected by all three secondary antibodies. A comparison of serum and plasma IgY levels was made within the same individuals for two passerine species (house finch and white-crowned sparrow), and serum exhibited significantly more IgY than the plasma for all three secondary antibodies. This result indicates that serum may be preferred to plasma when measuring total antibody levels in blood.This study indicates that the anti-passerine IgY secondary antibody can effectively be used in immunological assays to detect passerine IgY for species in most passerine families and is preferred over anti-chicken and anti-bird secondary antibodies for the majority of passerine species. This anti-passerine antibody will allow for more accurate detection and quantification of IgY in more wild bird species than was possible with previously available secondary antibodies.

We present a metagenomic analysis of California condor (Gymnogyps californianus) microbiomes with a goal of developing indicators of condor and ecosystem health. 16S ribosomal DNA (rDNA) and shotgun metagenomic DNA sequences were acquired from fecal and cloacal samples of captive birds. Detailed classification of shotgun DNA sequence data with the Sequedex analysis package provided both phylogenetic and functional profiles of all samples. We observed numerous bacterial species previously associated with birds and gut microbiomes, including both virulent and opportunistic pathogens such as Clostridium perfringens, Propionibacterium acnes, Shigella flexneri, and Fusobacterium mortiferum, probiotics such as Lactobacillus johnsonii, Lactobacillus ruminus, and Bacteroides vulgatus, and mucosal microbes such as Delftia acidovorans, Stenotrophomonas maltophilia, and Corynebacterium falsnii. Classification using shotgun metagenomic reads from phylogenetic marker genes was consistent with, but more specific than, analysis based on 16S data. Classification of samples based on functional profiles of genomic fragments differentiated three distinct types of samples: fecal, mature cloacal, and immature cloacal. Statistical analysis of the determinants of sample classification indicated that fecal samples were enriched in metabolism-related genes involved in glycolysis, methionine degradation, maltose utilization, and butyrate production, while cloacal samples were enriched in aromatic metabolism, virulence genes, and membrane transporters. Our analysis was further supported by the recovery of protein sequences for phylogenetic analysis, such as the RNA polymerase of Delftia, a Shigella toxin, a nitrate reductase of Delftia, and beta lactamases from several species. Our work identifies several strategies by which metagenomic sequencing of condor microbiomes may inform us about condor diet, infections, intoxication, and other types of stress.

Numerous bird species are highly susceptible to North American strains of West Nile virus (WNV), and although domestic chickens are relatively resistant to WNV-associated disease, this species currently represents the most practical avian model for immune responses to WNV infection. Knowledge of the immunomodulation of susceptibility to WNV in birds is important for understanding taxonomic differences in infection outcomes. While focusing on immunophenotyping of CD3+, CD4+, CD8+, and CD45+ lymphocyte subpopulations, we compared lymphocyte subpopulations, blood chemistries, cloacal temperatures, IgM and IgG antibody titers, and differential whole-blood cell counts of WNV-infected and uninfected hens. Total blood calcium and lymphocyte numbers were lower in WNV-infected chickens compared with uninfected chickens. The heterophil-to-lymphocyte ratio increased over time from 2 to 22 d postinoculation (DPI) in uninfected chickens and from 2 to 8 DPI in WNV-infected chickens, although levels declined from 8 to 22 DPI in the latter group. No significant differences were found in the remaining immunological and hematological variables of the WNV-infected and uninfected groups. Our results reaffirm that chickens are resistant to WNV infection, and demonstrated that the heterophil-to-lymphocyte ratio differed between groups, allowing for sorting of infection status. Similar patterns in immune responses over time in both infected and uninfected hens may be related to age (i.e., 10 wk) and associated immune development.

The historical and environmental forces involved in determining the geographic location of plant hybrid zones have long been of interest. Often hybrid zones appear to be intimately tied to the environment, yet because many abiotic factors covary it is often difficult to understand which are truly the most important in maintaining the position and structure of a hybrid zone. This study uses empirical data and modeling analyses to examine whether abiotic factors are responsible for the location and structure of an oak (Quercus) hybrid zone and, if so, which factors are the most important. Specifically, leaf trait measurements and ecological niche models were used to test the hypothesis that water availability plays a primary role in promoting and maintaining the location of hybridization between 2 species. Leaf trait analyses and ecological niche models both supported the hypothesis that water availability determines the location and structure of the hybrid zone. These findings lend support for the...

Regional droughts have far-reaching, substantial, and easily recognizable impacts on popula- tions and the environment. One component of these impacts that is not widely recognized is impairment of immune function by drought-related physiological stress. We studied cell-mediated immune function of cavity- nestling Western Bluebirds (Sialia mexicana), Ash-throated Flycatchers (Myiarchus cinerascens), and Violet- green Swallows (Tachycineta thalassina) at Los Alamos, New Mexico. There was a dramatic decrease in the cell-mediated immune responsiveness of developing nestlings associated with unusually dry conditions. Adult Western Bluebirds captured in 2002 weighed 7% less than in all previous years and average clutch size for all three species was reduced by 21% in 2002. Nestling body mass was also reduced for flycatcher and bluebird nestlings in 2002 compared to all other years. Survival to fledging age was lower overall during the drought years of 2000-2002 compared to the first 3 years of the study. Received 17 April 2006. Accepted 14 February 2008.

The validity of the haematocrit or packed cell volume as an indicator of condition in wild birds has recently been questioned. We reviewed over 300 published papers on haematocrit values for wild birds. These studies show that changes in haematocrit could be caused by a number of different natural factors that include age, sex, geographical elevation, energy expenditure, parasitism, nutrition and genetics. Haematocrit also increased with age from hatching, due to increased erythropoiesis, so that adult birds generally have greater haematocrit values than nestlings or juveniles. Haematocrit values were either independent of elevation or increased with elevation. A meta-analysis of 36 studies showed no difference in haematocrit between the sexes. Relationships between haematocrit value and both energy expenditure and parasitic infection vary between studies. In temperate climates, haematocrit tended to be higher in winter than in summer, which may be due to dehydration or increased oxygen demand caused by thermogenesis, moult or acquisition of reproductive status. Our review indicates that the use of haematocrit as a sole indicator of condition or health could lead to incorrect conclusions if natural factors that can affect haematocrit are not taken into consideration.

Determining optimal surveillance networks for an emerging pathogen is difficult since it is not known beforehand what the characteristics of a pathogen will be or where it will emerge. The resources for surveillance of infectious diseases in animals and wildlife are often limited and mathematical modeling can play a supporting role in examining a wide range of scenarios of pathogen spread. We demonstrate how a hierarchy of mathematical and statistical tools can be used in surveillance planning help guide successful surveillance and mitigation policies for a wide range of zoonotic pathogens. The model forecasts can help clarify the complexities of potential scenarios, and optimize biosurveillance programs for rapidly detecting infectious diseases. Using the highly pathogenic zoonotic H5N1 avian influenza 2006-2007 epidemic in Nigeria as an example, we determined the risk for infection for localized areas in an outbreak and designed biosurveillance stations that are effective for different pathogen strains and a range of possible outbreak locations. We created a general multi-scale, multi-host stochastic SEIR epidemiological network model, with both short and long-range movement, to simulate the spread of an infectious disease through Nigerian human, poultry, backyard duck, and wild bird populations. We chose parameter ranges specific to avian influenza (but not to a particular strain) and used a Latin hypercube sample experimental design to investigate epidemic predictions in a thousand simulations. We ranked the risk of local regions by the number of times they became infected in the ensemble of simulations. These spatial statistics were then complied into a potential risk map of infection. Finally, we validated the results with a known outbreak, using spatial analysis of all the simulation runs to show the progression matched closely with the observed location of the farms infected in the 2006-2007 epidemic.

During the past two decades, a critical need has developed to determine how exposure to contaminants in the environment affects individual and population processes. In this study, the immunocompetence of Western Bluebirds (Sialia mexicana) and Ash- throated Flycatchers (Myiarchus cinerascens) was studied on a landscape-soil contaminant gradient at Los Alamos National Laboratory (LANL) in New Mexico during 1997-1999. A variety of contaminants (heavy metals, chemicals, insecticides, polychlorinated biphenyls, and radioactive isotopes) range across different spatial scales and concentrations on LANL land. The two species have similar life-history traits, except that the Ash-throated Flycatcher has a faster rate of development and fledges 4-5 days earlier than the bluebird. The number of active nest boxes increased from 1997 to 1998 and 1999 for the Western Bluebird, but not for the Ash-throated Flycatcher. Survival of nestling flycatchers was lower in areas within 60 m of a potential contaminant release site, with a higher survivorship function associated with boxes farther away. The two species did not differ in their response to antigens, and there was no difference between locations for immunocompetence for either species. Flycatcher nestlings had a higher average cell-mediated response than bluebird nestlings, as predicted by the faster rate of development of the flycatchers. Phytohemag- glutinin response varied between locations for both species. The cell-mediated effects were dynamic in that, in general, the same locations showed similar patterns for each year. Hematocrits steadily increased with age for both species and varied between locations for the bluebird, but not the flycatcher.

The tradeoff between immunocompetence and development in avian species could have long-term consequences relating to fitness. This study investigated the developmental cost of mounting a humoral immune response in growing mountain chickadees, Poecile gambeli. We immunologically challenged nestling chickadees using nonpathogenic antigens of either Newcastle disease virus vaccine or sheep red blood cells. Body mass, tarsal length, haematocrit, total plasma protein, and survival did not vary significantly between antigen treatment groups and control groups. Developmental stability was assessed using fluctuating asymmetry (FA) between the right and left ninth primary feather length. FA was increased significantly in the antigen-challenged birds. This result indicates that the up-regulation of the humoral immune response was developmentally costly to the nestlings by causing an increase in FA.

Eggshell quality, clutch size, sex ratio, and hatching success of western bluebirds (Sialia mexicana) and ash-throated flycatchers (Myiarchus cinerascens) were studied on a landscape-soil contaminant gradient at Los Alamos National Laboratory (LANL) in New Mexico from 1997 to 1999. A variety of contaminants (heavy metals, chemicals, insecticides, polychlorinated biphenyls (PCBs), organochlorines, and radioactive isotopes) range across different spatial scales and concentrations on LANL land. This study is an example of a monitoring program over a large area with varying degree of contamination that is used to highlight locations of concern for future research. There were two locations where the flycatcher had a lower hatching success. The bluebirds at Sandia wetland, a location of concern for PCBs. had a thinner eggshell thickness index (RATCLIFFE) and the eggs were smaller than at other locations. The flycatcher had thinner eggshells than bluebirds, which could add to sensitivity to exposure to contaminants. There was no variation in clutch size or sex ratio between locations or areas closer to contaminant release sites for both species. Percent females in the clutch ranged from 0 to 100% in the WEBL and from 33 to 67% for ATFL.

We investigated the interacting effects of a nonpathogenic immunological challenge and exposure to lead shot early in the development of nestling western bluebirds (Sialia mexicana). Nestlings were randomly assigned to each of six treatments of an incomplete block design with two antigen treatments, Newcastle disease virus (NDV) or sheep red blood cells (SRBC), and four lead shot treatments (no shot, one shot – 0.05 g, 2 shots – 0.1 g, or three shots – 0.15 g). Survival functions did not differ between the lead treatment groups, and there were no effects of lead treatments on weight, growth rates, fluctuating asymmetry (FA), or antibody response. NDV- and SRBC-treated birds survived better than control birds which may be due to an adjuvant-activation of the entire immune system. However, FA was greater in individuals in the NDV and SRBC treatment groups, suggesting a tradeoff between growth and immunocompetence. Cell-mediated response to phytohemagglutinin of the high-lead treatment groups was significantly less than other groups. Hematocrit increased with age and weight, and was not affected by lead or antigen. While in this study the shape of the growth curve, FA, cell-mediated immunity, and behavior were affected by the higher dose of lead shot, actual lead concentrations in blood are needed to verify this process. Antigenic stress was documented in nestling developmental stability and there were no measured effect of both the lead shot and immunological challenge in combination.

Exposure to the combination of a contaminant and an immunological challenge during development may greatly increase the impact of either or both of these stressors on an individual. This study investigated the interacting effects of a nonpathogenic immunological challenge and lead shot exposure early in the development of a precocial species. Seventy-one quail (Coturnix coturnix japonica) chicks orally received either one #9 lead shot (0.05 g), four lead shots (0.2 g), or no lead at the age of 8 days. A third of each of these groups of chicks were intraperitoneally injected with either 0.075 ml of 10% chukar (Alectoris graeca) red blood cells (CRBCs), Newcastle disease virus (NDV), or a placebo oil vaccine at 13 and 35 days of age. There was no difference in the survival between any of the lead or antigen treatment groups. Lead concentrations in blood were greater for the lead-dosed groups on day 49 but not on day 128. Growth curves were approximated by Weibull functions; growth parameters did not differ between lead-treated and antigen-treated birds. CRBC antigen increases fluctuating asymmetry (FA) for primary feather five, while NDV reduced FA. Lead did not affect antibody production or cell-mediated immune response. White blood cell numbers increased 7 days after antigen injection on days 27 and 49 of age. Granulocyte numbers were significantly higher for the lead-treated quail than the control quail, and both antigen-treated groups had lower granulocyte numbers than control quail. The treatment groups of combining NDV and lead shot had an average plasma protein 14% lower than other groups. Lead shot in the highest of these doses lowered asymptotic weights, increased hematocrits, lowered plasma protein, and increased granulocyte numbers of quail.

We investigated the effect of blow fly larvae (Protocalliphora spatulata) on nestling savannah sparrows (Passerculus sandwichensis) in Alaska subject to biennially fluctuating prey abundance. Grasshopper populations in some regions of interior Alaska can show a strong biennial periodicity. Growth of nestling savannah sparrows, numbers of blow fly larvae in nests and on nestlings, and grasshopper populations were monitored during four breeding seasons (1990–1993) on the Delta Agriculture Project site near Delta Junction, Alaska. Growth parameters for parasitized and nonparasitized nestlings were compared within and between years. There were no significant differences in growth parameters between parasitized and nonparasitized nestlings between any years. There were also no differences in growth parameters within years, except for tarsus length in 1991 and nestling mass in 1993 (low grasshopper years); in 1993, parasitized nestlings were heavier and the sample size was small. Additionally, there were no differences in fledging success within years. Although parasitism combined with other factors (e.g., food limitation) may have an impact on avian reproduction, our data do not support this.

We used stable isotope ratios to determine the metabolic routing fraction of carbon and nitrogen in feathers in addition to faecal analysis to estimate diet overlap of six sympatric species of manakins in the eastern lowland forest of Ecuador. Collectively, all species varied from-23.7 to-32.7 ‰ for δ(13)C, and from 6.0 to 9.9‰ for δ(15)N, with Machaeropterus regulus showing isotopic differences from the other species. We developed a mixing model that explicitly addresses the routing of carbon and nitrogen to feathers. Interestingly, these results suggest a higher proportion of nitrogen and carbon derived from insects than anticipated based on feeding observations and faecal analysis. A concentration-dependent mixing isotopic model was also used to look at dietary proportions. While larvae and arachnids had higher δ(15)N values, these two groups may also be preferred prey of manakins and may be more assimilated into tissues, leading to a potential overestimation of the contribution to diet. This study supports the finding that manakin species, previously thought be primarily frugivorous, contain a significant amount of arthropods in their diet.

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.

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. peerReviewed

Background Diagnostic errors can occur, in infectious diseases, when anti-microbial immune responses involve several temporal scales. When responses span from nanosecond to week and larger temporal scales, any pre-selected temporal scale is likely to miss some (faster or slower) responses. Hoping to prevent diagnostic errors, a pilot study was conducted to evaluate a four-dimensional (4D) method that captures the complexity and dynamics of infectious diseases. Methods Leukocyte-microbial-temporal data were explored in canine and human (bacterial and/or viral) infections, with: (i) a non-structured approach, which measures leukocytes or microbes in isolation; and (ii) a structured method that assesses numerous combinations of interacting variables. Four alternatives of the structured method were tested: (i) a noise-reduction oriented version, which generates a single (one data point-wide) line of observations; (ii) a version that measures complex, three-dimensional (3D) data interactions; (iii) a non-numerical version that displays temporal data directionality (arrows that connect pairs of consecutive observations); and (iv) a full 4D (single line-, complexity-, directionality-based) version. Results In all studies, the non-structured approach revealed non-interpretable (ambiguous) data: observations numerically similar expressed different biological conditions, such as recovery and lack of recovery from infections. Ambiguity was also found when the data were structured as single lines. In contrast, two or more data subsets were distinguished and ambiguity was avoided when the data were structured as complex, 3D, single lines and, in addition, temporal data directionality was determined. The 4D method detected, even within one day, changes in immune profiles that occurred after antibiotics were prescribed. Conclusions Infectious disease data may be ambiguous. Four-dimensional methods may prevent ambiguity, providing earlier, in vivo, dynamic, complex, and personalized information that facilitates both diagnostics and selection or evaluation of anti-microbial therapies.

There is currently special concern for the effects of range grasshopper control on insectivorous grassland birds, such as upland shorebirds. The direct and indirect effects on killdeer (Charadrius vociferus) of carbaryl applications for grasshopper control were investigated in North Dakota in 1992. Specimens were collected from two rangeland sites aerially sprayed with 1.44 and 1.12 kg/ha of Sevin-4-Oil (0.56 and 0.45 kg/ha carbaryl active ingredient, respectively) and two untreated areas. Potential exposure to carbaryl, estimated from pesticide residue concentrations of whole-body and gizzard contents, was highest 2 d after treatment and then decreased over time. Whole-body carbaryl residues averaged significantly higher (p < 0.05) in specimens from sprayed areas, particularly from sites where the higher carbaryl application was used. There was no difference in the mean brain AChE activity and whole-body lipid content of killdeer from treated and untreated areas. The relative abundance of arthropods (principal prey of killdeer) was also estimated on the treatment area. As expected, grasshopper densities declined significantly posttreatment but the spray caused a temporary increase in the availability of affected grasshoppers. The two application rates caused similar reductions in grasshopper numbers. Killdeer did not exhibit a functional response of a prey switch as a result of the spray, but they did demonstrate an increased arthropod capture rate for a short period (8 d) after treatment. Although exposure was documented on the higher application rate area, this exposure was apparently not high enough to cause an impact on the parameters we measured in this study. These results support the conclusions of other studies that carbaryl, applied at the rate of 1.12 kg/ha, has little impact on birds and is equally as effective in reducing grasshopper numbers as the higher application rate.

Background To effectively control the geographical dissemination of infectious diseases, their properties need to be determined. To test that rapid microbial dispersal requires not only susceptible hosts but also a pre-existing, connecting network, we explored constructs meant to reveal the network properties associated with disease spread, which included the road structure. Methods Using geo-temporal data collected from epizoonotics in which all hosts were susceptible (mammals infected by Foot-and-mouth disease virus, Uruguay, 2001; birds infected by Avian Influenza virus H5N1, Nigeria, 2006), two models were compared: 1) ‘connectivity’, a model that integrated bio-physical concepts (the agent’s transmission cycle, road topology) into indicators designed to measure networks (‘nodes’ or infected sites with short- and long-range links), and 2) ‘contacts’, which focused on infected individuals but did not assess connectivity. Results The connectivity model showed five network properties: 1) spatial aggregation of cases (disease clusters), 2) links among similar ‘nodes’ (assortativity), 3) simultaneous activation of similar nodes (synchronicity), 4) disease flows moving from highly to poorly connected nodes (directionality), and 5) a few nodes accounting for most cases (a “20∶80″ pattern). In both epizoonotics, 1) not all primary cases were connected but at least one primary case was connected, 2) highly connected, small areas (nodes) accounted for most cases, 3) several classes of nodes were distinguished, and 4) the contact model, which assumed all primary cases were identical, captured half the number of cases identified by the connectivity model. When assessed together, the synchronicity and directionality properties explained when and where an infectious disease spreads. Conclusions Geo-temporal constructs of Network Theory’s nodes and links were retrospectively validated in rapidly disseminating infectious diseases. They distinguished classes of cases, nodes, and networks, generating information usable to revise theory and optimize control measures. Prospective studies that consider pre-outbreak predictors, such as connecting networks, are recommended.

Hairworms (Nematomorpha: Gordiida) are internal parasites that alter the behavior of their terrestrial insect host, forcing it to enter the water to reach its reproductive habitat. After reproduction of the free-living adults, the larvae encyst in aquatic insects and are retained upon metamorphosis of the insect into an adult fly. This paratenic host links the aquatic and terrestrial environments after its consumption by omnivorous or predatory insects. Therefore, hairworms are usually only associated with invertebrates, and few reports discuss hairworm interactions with vertebrate species. Here, we report on the finding of horsehair worms in nests of a cavity-nesting bird species in Los Alamos County, New Mexico. From 2004 to 2008, 7 nests within nest boxes occupied by the western bluebird (Sialia mexicana) contained 8 hairworms that were identified as Gordius robustus. All of the nest boxes with worms were less than 100 m from stagnant or low-flowing streams. The most likely explanation for the presence of the worms in the nests is that worms engaged in anti-predator avoidance after their insect hosts were collected and before digestion by nestling birds.

T-cell lymphocyte populations can be delineated into subsets based on expression of cell surface proteins that can be measured in peripheral blood by monoclonal antibodies and flow cytometry percentages of the lymphocyte subpopulations. In order to accurately assess immunocompetence in birds, natural variability in both avian immune function and the methodology must be understood. Our objectives were to (1) further develop flow cytometry for estimating subpopulations of lymphocytes in peripheral blood from poultry, (2) estimate repeatability and variability in the methodology with respect to poultry in a free-range and environmentally diverse situation, and (3) estimate the best antibody and cell marker combination for estimating lymphocyte subpopulations. This work demonstrated the repeatability of using flow cytometry for measurements of peripheral blood in chickens using anti-chicken antibodies for lymphocyte subpopulations. Immunofluorescence staining of cells isolated from peripheral blood revealed that the CD3(+) antibodies reacted with an average of approximately 12-24% of the lymphoid cells in the blood, depending on the fluorescence type. The CD4(+) and CD8(+) molecules were expressed in a range of 4-31% and 1-10% of the lymphoid cells in the blood, respectively. Both fluorescence label and antibody company contribute to the variability of results and should be considered in future flow cytometry studies in poultry.