POECIVIRUS IS PRESENT IN INDIVIDUALS WITH BEAK DEFORMITIES IN SEVEN SPECIES OF NORTH AMERICAN BIRDS.

Avian keratin disorder (AKD), a disease of unknown etiology characterized by debilitating beak overgrowth, has increasingly affected wild bird populations since the 1990s. A novel picornavirus, poecivirus, is closely correlated with disease status in Black-capped Chickadees (Poecile atricapillus) in Alaska, US. However, our knowledge of the relationship between poecivirus and beak deformities in other species and other geographic areas remains limited. The growing geographic scope and number of species affected by AKD-like beak deformities require a better understanding of the causative agent to evaluate the population-level impacts of this epizootic. Here, we tested eight individuals from six avian species with AKD-consistent deformities for the presence of poecivirus: Mew Gull (Larus canus), Hairy Woodpecker (Picoides villosus), Black-billed Magpie (Pica hudsonia), American Crow (Corvus brachyrhynchos), Red-breasted Nuthatch (Sitta canadensis), and Blackpoll Warbler (Setophaga striata). The birds were sampled in Alaska and Maine (1999-2016). We used targeted PCR followed by Sanger sequencing to test for the presence of poecivirus in each specimen and to obtain viral genome sequence from virus-positive host individuals. We detected poecivirus in all individuals tested, but not in negative controls (water and tissue samples). Furthermore, we used unbiased metagenomic sequencing to test for the presence of other pathogens in six of these specimens (Hairy Woodpecker, two American Crows, two Red-breasted Nuthatches, Blackpoll Warbler). This analysis yielded additional viral sequences from several specimens, including the complete coding region of poecivirus from one Red-breasted Nuthatch, which we confirmed via targeted PCR followed by Sanger sequencing. This study demonstrates that poecivirus is present in individuals with AKD-consistent deformities from six avian species other than Black-capped Chickadee. While further investigation will be required to explore whether there exists a causal link between this virus and AKD, this study demonstrates that poecivirus is not geographically restricted to Alaska, but rather occurs elsewhere in North America.

Next-Generation Ecological Immunology.

The application of next-generation sequencing (NGS) to the study of whole genomes and transcriptomes is becoming commonplace in molecular biology and molecular medicine. Over the past decade, these technologies have become more accessible to the broader biological community as the cost of NGS has decreased significantly and the availability of ready-to-use library preparation kits and user-friendly bioinformatic tools has increased. Indeed, these technologies are starting to be deployed in the study of ecological systems and immune function and have already yielded new discoveries and insights. However, despite the power of these techniques, they remain underutilized by the ecological immunology (ecoimmunology) community. This is unfortunate because there are several key challenges faced by ecological immunologists that these technologies are particularly well suited to address: (1) to measure immune function in biologically meaningful ways, (2) to describe the complex relationship between immunology and other aspects of physiology in an ecological context, and (3) to relate ecoimmunology to disease ecology. Each of these three challenges confronting the ecoimmunology community is critical to understanding ecoimmunology, and each has been stymied by a lack of sufficient data to effectively tackle the questions at hand. Deploying NGS methods to tackle these challenges promises to revolutionize the field of ecoimmunology by facilitating the unbiased, broad-scale, and efficient discovery of novel immune genes and mechanisms; the quantification of variation in immune systems; the characterization of relevant physiological pathways; and the description of microbial communities.

Avian keratin disorder of Alaska black-capped chickadees is associated with Poecivirus infection.

BACKGROUND: Avian keratin disorder (AKD) is an epizootic of debilitating beak deformities, first documented in black-capped chickadees (Poecile atricapillus) in Alaska during the late 1990s. Similar deformities have now been recorded in dozens of species of birds across multiple continents. Despite this, the etiology of AKD has remained elusive, making it difficult to assess the impacts of this disease on wild populations. We previously identified an association between infection with a novel picornavirus, Poecivirus, and AKD in a small cohort of black-capped chickadees. METHODS: To test if the association between Poecivirus and AKD holds in a larger study population, we used targeted PCR followed by Sanger sequencing to screen 124 symptomatic and asymptomatic black-capped chickadees for Poecivirus infection. We further compared the efficacy of multiple non-terminal field sampling methods (buccal swabs, cloacal swabs, fecal samples, and blood samples) for Poecivirus screening. Finally, we used both in situ hybridization and a strand-specific expression assay to localize Poecivirus to beak tissue of AKD-positive individuals and to determine if virus is actively replicating in beak tissue. RESULTS: Poecivirus was detected in 28/28 (100%) individuals with AKD, but only 9/96 (9.4%) asymptomatic individuals with apparently normal beaks (p < 0.0001). We found that cloacal swabs are the most sensitive of these sample types for detecting Poecivirus in birds with AKD, but that buccal swabs should be combined with cloacal swabs in evaluating the infection status of asymptomatic birds. Finally, we used both in situ hybridization and a strand-specific expression assay to localize Poecivirus to beak tissue of AKD-positive individuals and to provide evidence of active viral replication. CONCLUSION: The data presented here show a strong, statistically significant relationship between Poecivirus infection and AKD, and provide evidence that Poecivirus is indeed an avian virus, infecting and actively replicating in beak tissue of AKD-affected BCCH. Taken together, these data corroborate and extend the evidence for a potential causal association between Poecivirus and AKD in the black-capped chickadee. Poecivirus continues to warrant further investigation as a candidate agent of AKD.

Novel Picornavirus Associated with Avian Keratin Disorder in Alaskan Birds.

UNLABELLED: Avian keratin disorder (AKD), characterized by debilitating overgrowth of the avian beak, was first documented in black-capped chickadees (Poecile atricapillus) in Alaska. Subsequently, similar deformities have appeared in numerous species across continents. Despite the widespread distribution of this emerging pathology, the cause of AKD remains elusive. As a result, it is unknown whether suspected cases of AKD in the afflicted species are causally linked, and the impacts of this pathology at the population and community levels are difficult to evaluate. We applied unbiased, metagenomic next-generation sequencing to search for candidate pathogens in birds affected with AKD. We identified and sequenced the complete coding region of a novel picornavirus, which we are calling poecivirus. Subsequent screening of 19 AKD-affected black-capped chickadees and 9 control individuals for the presence of poecivirus revealed that 19/19 (100%) AKD-affected individuals were positive, while only 2/9 (22%) control individuals were infected with poecivirus. Two northwestern crows (Corvus caurinus) and two red-breasted nuthatches (Sitta canadensis) with AKD-consistent pathology also tested positive for poecivirus. We suggest that poecivirus is a candidate etiological agent of AKD. IMPORTANCE: Avian keratin disorder (AKD) is an increasingly common disease of wild birds. This disease, characterized by beak overgrowth, was first described in the late 1990s and has been spreading rapidly both geographically and in terms of host species affected. AKD decreases host fitness and can be fatal. However, the cause of the disease has remained elusive, and its impact on host populations is poorly understood. We found a novel and divergent picornavirus in 19/19 AKD-affected black-capped chickadees that we examined but in only 2/9 control cases. We also found this virus in 4 individuals of 2 other passerine species that exhibited symptoms consistent with AKD. Our data suggest that this novel picornavirus warrants further investigation as the causative agent of AKD.

Common measures of immune function vary with time of day and sampling protocol in five passerine species.

Ecological immunology is a rapidly growing field of study that focuses on understanding variation in immune systems across species and how this relates to species ecology and evolution. Newly developed field methods aimed at studying variation in immune function in a field setting have yielded many insights. Nonetheless, there continues to be much debate regarding the interpretation of field measures of immune function. There is substantial evidence to suggest that handling stress could introduce variation into measures of immune function, yet no study has examined the impacts of incremental changes in handling times under 30 min on immune measures. Nor has any study examined variation in immune function with time of day, though other physiological measures, including glucocorticoids known to impact immune function, vary with time of day. Here, I used observational field data to test the hypothesis that innate immune function varies with handling stress. Furthermore, I tested the hypothesis that innate immune function changes over the course of the day. I show that measures of innate immune function vary with (1) handling stress over short time periods typical of sample collection in the field, and (2) the time of day that an individual is sampled. I discuss these findings from an ecological perspective and suggest that the observed variation is not random, but is likely to have important adaptive functions. I end with a summary of the practical implications of these findings for field studies of ecological immunology.

Variation with land use of immune function and prevalence of avian pox in Galapagos finches.

Introduced disease has been implicated in recent wildlife extinctions and population declines worldwide. Both anthropogenic-induced change and natural environmental features can affect pathogen spread. Furthermore, environmental disturbance can result in changes in stress physiology, nutrition, and social structure, which in turn can suppress immune system function. However, it remains unknown whether landscape variation results in heterogeneity in host resistance to pathogens. Avian pox virus, a pathogen implicated in avian declines and extinctions in Hawaii, was introduced to the Galapagos in the 1890 s, and prevalence (total number of current infections) has increased recently in finches. We tested whether prevalence and recovery trends in 7 species of Galapagos finches varied by elevation or human land use. To do so, we used infection data obtained from 545 wild-caught birds. In addition, we determined whether annual changes in 4 aspects of innate immune function (complement protein activity, natural antibody activity, concentration of PIT54 protein, and heterophil:lymphocyte ratio) varied by elevation or land use. Prevalence and recovery rates did not vary by elevation from 2008 to 2009. Avian pox prevalence and proportion of recovered individuals in undeveloped and urban areas did not change from 2008 to 2009. In agricultural areas, avian pox prevalence increased 8-fold (from 2% to 17% of 234 individuals sampled) and proportion of recovered individuals increased (11% to 19%) from 2008 to 2009. These results suggest high disease-related mortality. Variation in immune function across human land-use types correlated with variation in both increased prevalence and susceptibility, which indicates changes in innate immune function may underlie changes in disease susceptibility. Our results suggest anthropogenic disturbance, in particular agricultural practices, may underlie immunological changes in host species that themselves contribute to pathogen emergence.

House finches (Carpodacus mexicanus) balance investment in behavioural and immunological defences against pathogens.

Infection with parasites and pathogens is costly for hosts, causing loss of nutritional resources, reproductive potential, tissue integrity and even life. In response, animals have evolved behavioural and immunological strategies to avoid infection by pathogens and infestation by parasites. Scientists generally study these strategies in isolation from each other; however, since these defences entail costs, host individuals should benefit from balancing investment in these strategies, and understanding of infectious disease dynamics would benefit from studying the relationship between them. Here, we show that Carpodacus mexicanus (house finches) avoid sick individuals. Moreover, we show that individuals investing less in behavioural defences invest more in immune defences. Such variation has important implications for the dynamics of pathogen spread through populations, and ultimately the course of epidemics. A deeper understanding of individual- and population-level disease defence strategies will improve our ability to understand, model and predict the outcomes of pathogen spread in wildlife.

The value, limitations, and challenges of employing local experts in conservation research.

Evidence suggests that the involvement of local people in conservation work increases a project's chances of success. Involving citizen scientists in research, however, raises questions about data quality. As a tool to better assess potential participants for conservation projects, we developed a knowledge gradient, K, along which community members occupy different positions on the basis of their experience with and knowledge of a research subject. This gradient can be used to refine the citizen-science concept and allow researchers to differentiate between community members with expert knowledge and those with little knowledge. We propose that work would benefit from the inclusion of select local experts because it would allow researchers to harness the benefits of local involvement while maintaining or improving data quality. We used a case study from the DeHoop Nature Preserve, South Africa, in which we conducted multiple interviews, identified and employed a local expert animal tracker, evaluated the expert's knowledge, and analyzed the data collected by the expert. The expert animal tracker J.J. created his own sampling design and gathered data on mammals. He patrolled 4653 km in 214 days and recorded 4684 mammals. He worked from a central location, and his patrols formed overlapping loops; however, his data proved neither spatially nor temporally autocorrelated. The distinctive data collected by J.J. are consistent with the notion that involving local experts can produce reliable data. We developed a conceptual model to help identify the appropriate participants for a given project on the basis of research budget, knowledge or skills needed, technical literacy requirements, and scope of the project.

Resumen: Las evidencias sugieren que la participación de habitantes locales en el trabajo de conservación incrementa la probabilidad de éxito de un proyecto. Sin embargo, involucrar a científicos ciudadanos genera interrogantes sobre la calidad de los datos. Como una herramienta para evaluar a potenciales participantes en proyectos de conservación, desarrollamos un gradiente de conocimiento, K, en el que los miembros de la comunidad ocupan diferentes posiciones con base en su experiencia y conocimiento de un tema de investigación. Este gradiente puede ser utilizado para refinar el concepto de ciencia-ciudadana y permite que los investigadores diferencien a los miembros de la comunidad con conocimiento experto de los que tienen poco conocimiento. Proponemos que el trabajo se beneficiaría con la inclusión de expertos locales selectos porque permitiría que los investigadores aprovechen los beneficios de la participación local al mismo tiempo que mantienen o incrementan la calidad de los datos. Utilizamos un caso de estudio de la Reserva Natural DeHoop, Sudáfrica, donde realizamos múltiples entrevistas, identificamos y empleamos a un experto local en el rastreo de animales, evaluamos el conocimiento del experto y analizamos los datos recolectados por el experto. El rastreador experto de animales, J.J., creó su propio diseño de muestreo y recolectó datos de mamíferos. J.J. recorrió 4653 km en 214 días y registró 4684 mamíferos. Trabajaba en una localidad central, y sus recorridos formaron círculos sobrepuestos; sin embargo, sus datos no estuvieron autocorrelacionados espacial ni temporalmente. Los datos recolectados por J.J., el experto, son consistentes con la idea de que los expertos locales pueden producir datos confiables. Desarrollamos un modelo conceptual para identificar a participantes apropiados para un proyecto determinado basado en el presupuesto, el conocimiento o habilidades requeridas, los requerimientos de conocimientos técnicos y el alcance del proyecto.

Microarray-based detection and genotyping of viral pathogens.

The detection of viral pathogens is of critical importance in biology, medicine, and agriculture. Unfortunately, existing techniques to screen for a broad spectrum of viruses suffer from severe limitations. To facilitate the comprehensive and unbiased analysis of viral prevalence in a given biological setting, we have developed a genomic strategy for highly parallel viral screening. The cornerstone of this approach is a long oligonucleotide (70-mer) DNA microarray capable of simultaneously detecting hundreds of viruses. Using virally infected cell cultures, we were able to efficiently detect and identify many diverse viruses. Related viral serotypes could be distinguished by the unique pattern of hybridization generated by each virus. Furthermore, by selecting microarray elements derived from highly conserved regions within viral families, individual viruses that were not explicitly represented on the microarray were still detected, raising the possibility that this approach could be used for virus discovery. Finally, by using a random PCR amplification strategy in conjunction with the microarray, we were able to detect multiple viruses in human respiratory specimens without the use of sequence-specific or degenerate primers. This method is versatile and greatly expands the spectrum of detectable viruses in a single assay while simultaneously providing the capability to discriminate among viral subtypes.