Novel Rickettsia spp. in two common overwintering North American songbirds.

American robins and dark-eyed juncos migrate across North America and have been found to be competent hosts for some bacterial and viral pathogens, but their contributions to arthropod-borne diseases more broadly remain poorly characterized. Here, we sampled robins and juncos in multiple sites across North America for arthropod-borne bacterial pathogens of public health significance. We identified two novel Rickettsia spp. in one wintering migrant per bird species related to bellii, transitional, and spotted rickettsiae fever groups. Stable isotope analyses of feathers suggested spring migration of these common songbirds could disperse these novel rickettsiae hundreds-to-thousands of kilometers to host breeding grounds. Further work is needed to characterize zoonotic potential of these rickettsiae and host reservoir competence.

Eggshell texture but not odor treatment affects model egg rejection in American robins (Turdus migratorius).

To curb fitness costs associated with obligate avian brood parasitism, some hosts have evolved to reject foreign eggs in the nest. American robins (Turdus migratorius) are among the few hosts of the brown-headed cowbird (Molothrus ater) that mostly remove parasitic eggs from their nests. With the parasite's eggs looking nothing like their own, American robins likely rely most on visual cues when making rejection decisions. However, we still know little about the roles that tactile and olfactory cues play in robin's or other rejecter hosts' rejection decisions. Here, we conducted a set of experiments to test for the use of tactile or olfactory cues in egg rejection by robins. For the tactile experiment, we found that robins were more likely to reject rough rather than smooth eggs. However, our tactile model egg design was not able to fully discriminate between tactile and visual sensory modalities. In the olfaction experiment, we did not find a significant effect of egg scent treatment on rejection rates. Accordingly, future studies on egg rejection should attempt to fully distinguish between tactile and visual cues, as well as examine olfactory cues in other egg rejecter host species.

Cellular and immunological mechanisms influence host-adapted phenotypes in a vector-borne microparasite.

Predicting pathogen emergence and spillover risk requires understanding the determinants of a pathogens' host range and the traits involved in host competence. While host competence is often considered a fixed species-specific trait, it may be variable if pathogens diversify across hosts. Balancing selection can lead to maintenance of pathogen polymorphisms (multiple-niche-polymorphism; MNP). The causative agent of Lyme disease, Borrelia burgdorferi (Bb), provides a model to study the evolution of host adaptation, as some Bb strains defined by their outer surface protein C (ospC) genotype, are widespread in white-footed mice and others are associated with non-rodent vertebrates (e.g. birds). To identify the mechanisms underlying potential strain × host adaptation, we infected American robins and white-footed mice, with three Bb strains of different ospC genotypes. Bb burdens varied by strain in a host-dependent fashion, and strain persistence in hosts largely corresponded to Bb survival at early infection stages and with transmission to larvae (i.e. fitness). Early survival phenotypes are associated with cell adhesion, complement evasion and/or inflammatory and antibody-mediated removal of Bb, suggesting directional selective pressure for host adaptation and the potential role of MNP in maintaining OspC diversity. Our findings will guide future investigations to inform eco-evolutionary models of host adaptation for microparasites.

Pre-existing Microfilarial Infections of American Robins (Passeriformes: Turdidae) and Common Grackles (Passeriformes: Icteridae) Have Limited Impact on Enhancing Dissemination of West Nile Virus in Culex pipiens Mosquitoes (Diptera: Culicidae).

Microfilariae (MF) are the immature stages of filarial nematode parasites and inhabit the blood and dermis of all classes of vertebrates, except fish. Concurrent ingestion of MF and arboviruses by mosquitoes can enhance mosquito transmission of virus compared to when virus is ingested alone. Shortly after being ingested, MF penetrate the mosquito's midgut and may introduce virus into the mosquito's hemocoel, creating a disseminated viral infection much sooner than normal. This phenomenon is known as microfilarial enhancement. Both American Robins and Common Grackles harbor MF-that is, Eufilaria sp. and Chandlerella quiscali von Linstow (Spirurida: Onchocercidae), respectively. We compared infection and dissemination rates in Culex pipiens L. mosquitoes that fed on birds with and without MF infections that had been infected with West Nile virus (WNV). At moderate viremias, about 107 plaque-forming units (pfu)/ml of blood, there were no differences in infection or dissemination rates among mosquitoes that ingested viremic blood from a bird with or without microfilaremia. At high viremias, >108.5 pfu/ml, mosquitoes feeding on a microfilaremic Grackle with concurrent viremia had significantly higher infection and dissemination rates than mosquitoes fed on viremic Grackles without microfilaremia. Microfilarial enhancement depends on the specific virus, MF, and mosquito species examined. How virus is introduced into the hemocoel by MF differs between the avian/WNV systems described here (i.e., leakage) and various arboviruses with MF of the human filarid, Brugia malayi (Brug) (Spirurida: Onchocercidae) (i.e., cotransport). Additional studies are needed to determine if other avian species and their MF are involved in the microfilarial enhancement of WNV in nature.

Predicting West Nile virus transmission in North American bird communities using phylogenetic mixed effects models and eBird citizen science data.

BACKGROUND: West Nile virus (WNV) is a mosquito-transmitted disease of birds that has caused bird population declines and can spill over into human populations. Previous research has identified bird species that infect a large fraction of the total pool of infected mosquitoes and correlate with human infection risk; however, these analyses cover small spatial regions and cannot be used to predict transmission in bird communities in which these species are rare or absent. Here we present a mechanistic model for WNV transmission that predicts WNV spread (R0) in any bird community in North America by scaling up from the physiological responses of individual birds to transmission at the level of the community. We predict unmeasured bird species' responses to infection using phylogenetic imputation, based on these species' phylogenetic relationships with bird species with measured responses. RESULTS: We focused our analysis on Texas, USA, because it is among the states with the highest total incidence of WNV in humans and is well sampled by birders in the eBird database. Spatio-temporal patterns: WNV transmission is primarily driven by temperature variation across time and space, and secondarily by bird community composition. In Texas, we predicted WNV R0 to be highest in the spring and fall when temperatures maximize the product of mosquito transmission and survival probabilities. In the most favorable months for WNV transmission (April, May, September and October), we predicted R0 to be highest in the "Piney Woods" and "Oak Woods & Prairies" ecoregions of Texas, and lowest in the "High Plains" and "South Texas Brush County" ecoregions. Dilution effect: More abundant bird species are more competent hosts for WNV, and predicted WNV R0 decreases with increasing species richness. Keystone species: We predicted that northern cardinals (Cardinalis cardinalis) are the most important hosts for amplifying WNV and that mourning doves (Zenaida macroura) are the most important sinks of infection across Texas. CONCLUSIONS: Despite some data limitations, we demonstrate the power of phylogenetic imputation in predicting disease transmission in heterogeneous host communities. Our mechanistic modeling framework shows promise both for assisting future analyses on transmission and spillover in heterogeneous multispecies pathogen systems and for improving model transparency by clarifying assumptions, choices and shortcomings in complex ecological analyses.

Feeding Success and Host Selection by Culex quinquefasciatus Say Mosquitoes in Experimental Trials.

Arthropod vector feeding preferences are defined as an overutilization of a particular host species given its abundance in relationship to other species in the community. Numerous methods exist to quantify vector feeding preferences; however, controlled host choice experiments are generally an underutilized approach. In this report, we present results from controlled vector host choice experiments using Culex quinquefasciatus Say (Diptera: Culicidae) mosquitoes and wild avian hosts identified as important contributors to West Nile virus (WNv) transmission in Atlanta, Georgia, United States. In each experiment, we allowed lab-reared F1Cx. quinquefasciatus to feed freely overnight on two avian individuals of a different species (i.e., northern cardinals, American robins, blue jays, brown thrashers, and gray catbirds). We then estimated WNv transmission potential using vectorial capacity and R0. We found that mosquito blood feeding success was extremely variable among experimental replicates and that patterns of host choice only occasionally aggregated to a particular bird species. Vectorial capacity was highest for American robins and blue jays due to these species' higher reservoir competence for WNv and greater probabilities of mosquito selection of these species. Despite species-specific differences in vectorial capacity, total community capacity was similar among species pairs. R0 estimates were qualitatively similar to capacity, and R0 was below and above unity across species pairs. Our results provide empirical evidence that C. quinquefasciatus is an opportunistic blood feeder and highlight how variability in vector-host contact rates as well as host community composition can influence the likelihood of WNv transmission in avian communities.

Linking Bird and Mosquito Data to Assess Spatiotemporal West Nile Virus Risk in Humans.

West Nile virus (WNV; family Flaviviridae) causes a disease in humans that may develop into a deadly neuroinvasive disease. In North America, several peridomestic bird species can develop sufficient viremia to infect blood-feeding mosquito vectors without succumbing to the virus. Mosquito species from the genus Culex, Aedes and Ochlerotatus display variable host preferences, ranging between birds and mammals, including humans, and may bridge transmission among avian hosts and contribute to spill-over transmission to humans. In this study, we aimed to test the effect of density of three mosquito species and two avian species on WNV mosquito infection rates and investigated the link between spatiotemporal clusters of high mosquito infection rates and clusters of human WNV cases. We based our study around the city of Ottawa, Canada, between the year 2007 and 2014. We found a large effect size of density of two mosquito species on mosquito infection rates. We also found spatiotemporal overlap between a cluster of high mosquito infection rates and a cluster of human WNV cases. Our study is innovative because it suggests a role of avian and mosquito densities on mosquito infection rates and, in turn, on hotspots of human WNV cases.

Mixed Thymoma in an American Robin ( Turdus migratorius).

A mixed thymoma was diagnosed in a 15-year-old female American robin ( Turdus migratorius) that exhibited poor body condition, dysphagia, hyporexia, and depression. A 1.5-cm subcutaneous nodule was present in the cranio-ventral cervical region, which had been noticed by the owner 15 days before presentation. On cytologic evaluation of a fine-needle aspirate, well-differentiated lymphocytes were observed. Surgical excision was elected; however, the mass was firmly attached to the esophagus and the jugular vein, and the attempt at excision resulted in fatal hemorrhage. On histologic examination of the mass, small, well-differentiated lymphocytes were observed mixed with neoplastic reticular cells and Hassall's corpuscles. On immunohistochemical analysis, the cytoplasm of 80% of the reticular cells showed abundant detectable brown antigen binding with pancytokeratin staining, and most lymphoid cells showed detectable antigen in the cytoplasm by using CD3 antibodies. The cytologic, histopathologic, and immunohistochemical features of the neoplasm in this robin were consistent with a mixed thymoma.

Viral shedding of clade 2.3.4.4 H5 highly pathogenic avian influenza A viruses by American robins.

American robins (Turdus migratorius) are commonly associated with farmsteads in the United States and have shown previous evidence of exposure to an H5 avian influenza A virus (IAV) near a poultry production facility affected by a highly pathogenic (HP) H5 virus in Iowa, USA during 2015. We experimentally infected American robins with three clade 2.3.4.4 HP H5 viruses (H5N2 and H5N8). A total of 22/24 American robins shed virus, and all three strains were represented. The highest virus titres shed were 104.3 , 104.3 and 104.8 PFU/ml, associated respectively with viruses isolated from poultry, a captive gyrfalcon (Falco rusticolus), and a Northern pintail (Anas acuta). Of those birds that shed, viral shedding was initiated 1 or 2 days post-infection (DPI) and shedding ceased in all birds by 7 DPI. This study adds an additional synanthropic wildlife species to a growing list of animals that can successfully replicate and shed IAVs.

Does contrast between eggshell ground and spot coloration affect egg rejection?

Obligate avian brood parasitic species impose the costs of incubating foreign eggs and raising young upon their unrelated hosts. The most common host defence is the rejection of parasitic eggs from the nest. Both egg colours and spot patterns influence egg rejection decisions in many host species, yet no studies have explicitly examined the role of variation in spot coloration. We studied the American robin Turdus migratorius, a blue-green unspotted egg-laying host of the brown-headed cowbird Molothrus ater, a brood parasite that lays non-mimetic spotted eggs. We examined host responses to model eggs with variable spot coloration against a constant robin-mimetic ground colour to identify patterns of rejection associated with perceived contrast between spot and ground colours. By using avian visual modelling, we found that robins were more likely to reject eggs whose spots had greater chromatic (hue) but not achromatic (brightness) contrast. Therefore, egg rejection decision rules in the American robin may depend on the colour contrast between parasite eggshell spot and host ground coloration. Our study also suggests that egg recognition in relation to spot coloration, like ground colour recognition, is tuned to the natural variation of avian eggshell spot colours but not to unnatural spot colours.

Identification of Avian and Hemoparasite DNA in Blood-Engorged Abdomens of Culex pipiens (Diptera; Culicidae) from a West Nile Virus Epidemic region in Suburban Chicago, Illinois.

Multiple mosquito-borne parasites cocirculate in nature and potentially interact. To understand the community of parasites cocirculating with West Nile virus (WNV), we screened the bloodmeal content of Culex pipiens L. mosquitoes for three common types of hemoparasites. Blood-fed Cx. pipiens were collected from a WNV-epidemic area in suburban Chicago, IL, from May to September 2005 through 2010. DNA was extracted from dissected abdomens and subject to PCR and direct sequencing to identify the vertebrate host. RNA was extracted from the head or thorax and screened for WNV using quantitative reverse transcriptase PCR. Seventy-nine engorged females with avian host origin were screened using PCR and amplicon sequencing for filarioid nematodes, Haemosporida, and trypanosomatids. Filarioid nematodes were identified in 3.8% of the blooded abdomens, Plasmodium sp. in 8.9%, Haemoproteus in 31.6%, and Trypanosoma sp. in 6.3%. The sequences from these hemoparasite lineages were highly similar to sequences from birds in prior studies in suburban Chicago. Overall, 50.6% of blood-fed Culex pipiens contained hemoparasite DNA in their abdomen, presumably from current or prior bloodmeals. Additionally, we detected hemoparasite DNA in the blooded abdomen of three of 10 Cx. pipiens infected with WNV.

Using 3D printed eggs to examine the egg-rejection behaviour of wild birds.

The coevolutionary relationships between brood parasites and their hosts are often studied by examining the egg rejection behaviour of host species using artificial eggs. However, the traditional methods for producing artificial eggs out of plasticine, plastic, wood, or plaster-of-Paris are laborious, imprecise, and prone to human error. As an alternative, 3D printing may reduce human error, enable more precise manipulation of egg size and shape, and provide a more accurate and replicable protocol for generating artificial stimuli than traditional methods. However, the usefulness of 3D printing technology for egg rejection research remains to be tested. Here, we applied 3D printing technology to the extensively studied egg rejection behaviour of American robins, Turdus migratorius. Eggs of the robin's brood parasites, brown-headed cowbirds, Molothrus ater, vary greatly in size and shape, but it is unknown whether host egg rejection decisions differ across this gradient of natural variation. We printed artificial eggs that encompass the natural range of shapes and sizes of cowbird eggs, painted them to resemble either robin or cowbird egg colour, and used them to artificially parasitize nests of breeding wild robins. In line with previous studies, we show that robins accept mimetically coloured and reject non-mimetically coloured artificial eggs. Although we found no evidence that subtle differences in parasitic egg size or shape affect robins' rejection decisions, 3D printing will provide an opportunity for more extensive experimentation on the potential biological or evolutionary significance of size and shape variation of foreign eggs in rejection decisions. We provide a detailed protocol for generating 3D printed eggs using either personal 3D printers or commercial printing services, and highlight additional potential future applications for this technology in the study of egg rejection.

Host group formation decreases exposure to vector-borne disease: a field experiment in a 'hotspot' of West Nile virus transmission.

Animals can decrease their individual risk of predation by forming groups. The encounter-dilution hypothesis extends the potential benefits of gregariousness to biting insects and vector-borne disease by predicting that the per capita number of insect bites should decrease within larger host groups. Although vector-borne diseases are common and can exert strong selective pressures on hosts, there have been few tests of the encounter-dilution effect in natural systems. We conducted an experimental test of the encounter-dilution hypothesis using the American robin (Turdus migratorius), a common host species for the West Nile virus (WNV), a mosquito-borne pathogen. By using sentinel hosts (house sparrows, Passer domesticus) caged in naturally occurring communal roosts in the suburbs of Chicago, we assessed sentinel host risk of WNV exposure inside and outside of roosts. We also estimated per capita host exposure to infected vectors inside roosts and outside of roosts. Sentinel birds caged inside roosts seroconverted to WNV more slowly than those outside of roosts, suggesting that social groups decrease per capita exposure to infected mosquitoes. These results therefore support the encounter-dilution hypothesis in a vector-borne disease system. Our results suggest that disease-related selective pressures on sociality may depend on the mode of disease transmission.

Prevalence of filarioid nematodes and trypanosomes in American robins and house sparrows, Chicago USA.

Hosts are commonly infected with a suite of parasites, and interactions among these parasites can affect the size, structure, and behavior of host-parasite communities. As an important step to understanding the significance of co-circulating parasites, we describe prevalence of co-circulating hemoparasites in two important avian amplification hosts for West Nile virus (WNV), the American robin (Turdus migratorius) and house sparrow (Passer domesticus), during the 2010-2011 in Chicago, Illinois, USA. Rates of nematode microfilariemia were 1.5% of the robins (n = 70) and 4.2% of the house sparrows (n = 72) collected during the day and 11.1% of the roosting robins (n = 63) and 0% of the house sparrows (n = 11) collected at night. Phylogenetic analysis of nucleotide sequences of the 18S rRNA and cytochrome oxidase subunit I (COI) genes from these parasites resolved two clades of filarioid nematodes. Microscopy revealed that 18.0% of American robins (n = 133) and 16.9% of house sparrows (n = 83) hosted trypanosomes in the blood. Phylogenetic analysis of nucleotide sequences from the 18s rRNA gene revealed that the trypanosomes fall within previously described avian trypanosome clades. These results document hemoparasites in the blood of WNV hosts in a center of endemic WNV transmission, suggesting a potential for direct or indirect interactions with the virus.