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The Diplostomidae Poirier, 1886 is a large, globally distributed family of digeneans parasitic in intestines of their definitive hosts. Diplostomum and Tylodelphys spp. are broadly distributed, commonly reported, and the most often sequenced diplostomid genera. The majority of published DNA sequences from these genera originated from larval stages only, which typically cannot be identified to the species level based on morphology alone. We generated partial large ribosomal subunit (28S) rRNA and cytochrome c oxidase subunit 1 (cox1) mtDNA gene sequences from 14 species/species-level lineages of Diplostomum, six species/species-level lineages of Tylodelphys, two species/species-level lineages of Austrodiplostomum, one species previously assigned to Paralaria, two species/species-level lineages of Dolichorchis and one unknown diplostomid. Our DNA sequences of 11 species/species-level lineages of Diplostomum (all identified to species), four species/species-level lineages of Tylodelphys (all identified to species), Austrodiplostomum compactum, Paralaria alarioides and Dolichorchis lacombeensis originated from adult specimens. 28S sequences were used for phylogenetic inference to demonstrate the position of Paralaria alarioides and Dolichorchis spp. within the Diplostomoidea and study the interrelationships of Diplostomum, Tylodelphys and Austrodiplostomum. Our results demonstrate that two diplostomids from the North American river otter (P. alarioides and a likely undescribed taxon) belong within Diplostomum. Further, our results demonstrate the non-monophyly of Tylodelphys due to the position of Austrodiplostomum spp., based on our phylogenetic analyses and morphology. Furthermore, the results of phylogenetic analysis of 28S confirmed the status of Dolichorchis as a separate genus. The phylogenies suggest multiple definitive host-switching events (birds to otters and among major avian groups) and a New World origin of Diplostomum and Tylodelphys spp. Our DNA sequences from adult digeneans revealed identities of 10 previously published lineages of Diplostomum and Tylodelphys, which were previously identified to genus only. The novel DNA data from this work provide opportunities for future comparisons of larval diplostomines collected in ecological studies.

Host phylogenetic relatedness and ecological similarity are thought to contribute to parasite community assembly and infection rates. However, recent landscape level anthropogenic changes may disrupt host-parasite systems by impacting functional and phylogenetic diversity of host communities. We examined whether changes in host functional and phylogenetic diversity, forest cover, and minimum temperature influence the prevalence, diversity, and distributions of avian haemosporidian parasites (genera Haemoproteus and Plasmodium) across 18 avian communities in the Atlantic Forest. To explore spatial patterns in avian haemosporidian prevalence and taxonomic and phylogenetic diversity, we surveyed 2241 individuals belonging to 233 avian species across a deforestation gradient. Mean prevalence and parasite diversity varied considerably across avian communities and parasites responded differently to host attributes and anthropogenic changes. Avian malaria prevalence (termed herein as an infection caused by Plasmodium parasites) was higher in deforested sites, and both Plasmodium prevalence and taxonomic diversity were negatively related to host functional diversity. Increased diversity of avian hosts increased local taxonomic diversity of Plasmodium lineages but decreased phylogenetic diversity of this parasite genus. Temperature and host phylogenetic diversity did not influence prevalence and diversity of haemosporidian parasites. Variation in the diversity of avian host traits that promote parasite encounter and vector exposure (host functional diversity) partially explained the variation in avian malaria prevalence and diversity. Recent anthropogenic landscape transformation (reduced proportion of native forest cover) had a major influence on avian malaria occurrence across the Atlantic Forest. This suggests that, for Plasmodium, host phylogenetic diversity was not a biotic filter to parasite transmission as prevalence was largely explained by host ecological attributes and recent anthropogenic factors. Our results demonstrate that, similar to human malaria and other vector-transmitted pathogens, prevalence of avian malaria parasites will likely increase with deforestation.

Migration has an important impact on the transmission of pathogens. Migratory birds disperse parasites through their routes and may consequently introduce them to new areas and hosts. Hence, haemosporidian parasites, which are among the most prevalent, diverse and important bird pathogens, are potentially dispersed when infecting migrant hosts. Further, migrant hosts could enhance local parasite prevalence and richness by transporting new parasite strains to new areas. Here, we hypothesize and aim to evaluate if 1) migratory birds spread parasite lineages along their routes, and 2) localities crossed by more migratory birds have greater prevalence and richness of haemosporidians. For the first hypothesis, we tested whether parasite lineages found 1) in both migrants and residents, and 2) only in residents, differ in their frequencies of occurrence among localities. For the second hypothesis, we tested for a relationship among localities between the overall local haemosporidian parasite richness and prevalence, and the proportion of migratory bird individuals present in a locality. We combined a dataset on 13 200 bird samples with additional data from the MalAvi database (total: ~2800 sequenced parasites comprising 675 distinct lineages, from 506 host species and 156 localities) from South America, and used Bayesian multi‐level models to test our hypotheses. We demonstrate that parasites shared between resident and migratory species are the most spatially widespread, highlighting the potential of migrants to carry and transmit haemosporidians. Further, the presence of migrants in a locality was negatively related to local parasite richness, but not associated with local prevalence. Here, we confirm that migrants can contribute to parasite dispersal and visiting migrants are present in regions with lower Plasmodium prevalence. Also, we observed their presence might raise Haemoproteus community prevalence. Therefore, we demonstrate migrants enhance pathogens spread and their presence may influence parasite community transmission.

Studies contrasting parasite prevalence and host-parasite community structure between pristine and disturbed environments will improve our understanding of how deforestation affects disease transmission and parasite extinction. To determine how infection rates of a common and diverse group of avian blood parasites (Plasmodium and Haemoproteus) respond to changes in avian host composition after mining, we surveyed 25 bird communities from pristine forests (two forest types: plateau and hillside) and reforested sites in Northeast Amazonia. Infection rates and both parasite and avian host community structure exhibited considerable variation across the deforestation gradient. In opposition to the emerging pattern of lower avian haemosporidian prevalence in disturbed tropical forests in Africa, we show that secondary forests had higher haemosporidian prevalence in one of the largest mining areas of Amazonia. The dissimilarity displayed by bird communities may explain, in part, the higher prevalence of Haemoproteus in reforested areas owing to the tolerance of some bird species to open-canopy forest habitat. On the other hand, deforestation may cause local extinction of Plasmodium parasites due to the loss of their avian hosts that depend on closed-canopy primary forest habitats. Our results demonstrate that forest loss induced by anthropogenic changes can affect a host-parasite system and disturb both parasite transmission and diversity.

Aim: Macroecological analyses provide valuable insights into factors that influence how parasites are distributed across space and among hosts. Amid large uncertainties that arise when generalizing from local and regional findings, hierarchical approaches applied to global datasets are required to determine whether drivers of parasite infection patterns vary across scales. We assessed global patterns of haemosporidian infections across a broad diversity of avian host clades and zoogeographical realms to depict hotspots of prevalence and to identify possible underlying drivers. Location: Global. Time period: 1994–2019. Major taxa studied: Avian haemosporidian parasites (genera Plasmodium, Haemoproteus, Leucocytozoon and Parahaemoproteus). Methods: We amalgamated infection data from 53,669 individual birds representing 2,445 species world-wide. Spatio-phylogenetic hierarchical Bayesian models were built to disentangle potential landscape, climatic and biotic drivers of infection probability while accounting for spatial context and avian host phylogenetic relationships. Results: Idiosyncratic responses of the three most common haemosporidian genera to climate, habitat, host relatedness and host ecological traits indicated marked variation in host infection rates from local to global scales. Notably, host ecological drivers, such as migration distance for Plasmodium and Parahaemoproteus, exhibited predominantly varying or even opposite effects on infection rates across regions, whereas climatic effects on infection rates were more consistent across realms. Moreover, infections in some low-prevalence realms were disproportionately concentrated in a few local hotspots, suggesting that regional-scale variation in habitat and microclimate might influence transmission, in addition to global drivers. Main conclusions: Our hierarchical global analysis supports regional-scale findings showing the synergistic effects of landscape, climate and host ecological traits on parasite transmission for a cosmopolitan and diverse group of avian parasites. Our results underscore the need to account for such interactions, in addition to possible variation in drivers across regions, to produce the robust inference required to predict changes in infection risk under future scenarios.

Crassiphialinae Sudarikov, 1960 is a large subfamily of the Diplostomidae Poirier, 1886 with a complex taxonomic history. It includes a diversity of species parasitic in the intestines of avian and mammalian definitive hosts worldwide. Posthodiplostomum Dubois, 1936 is a large and broadly distributed crassiphialine genus notorious for its association with diseases in their fish second intermediate hosts. In this study, we generated partial 28S rDNA and cytochrome c oxidase subunit 1 (cox1) mtDNA gene sequences of digeneans beloning to seven crassiphialine genera. The 28S sequences were used to study the interrelationships among crassiphialines and their placement among other major diplostomoidean lineages. Our molecular phylogenetic analysis and review of morphology does not support subfamilies currently recognized in the Diplostomidae; therefore, we abandon the current subfamily system of the Diplostomidae. Molecular phylogenetic analyses suggest the synonymy of Posthodiplostomum, Ornithodiplostomum Dubois, 1936 and Mesoophorodiplostomum Dubois, 1936 ; morphological study of our well-fixed adult specimens and review of literature revealed lack of consistent differences among the three genera. Thus, we synonymize Ornithodiplostomum and Mesoophorodiplostomum with Posthodiplostomum. Our phylogenetic analyses suggest an Old World origin of Posthodiplostomum followed by multiple dispersal events among biogeographic realms. Furthermore, our analyses indicate that the ancestors of these digeneans likely parasitized ardeid definitive hosts. Four new species of Posthodiplostomum collected from birds in the New World as well as one new species of Posthodiplostomoides Williams, 1969 from Uganda are described.

As presently recognized, Herpetodiplostomum is a small genus of proterodiplostomid digeneans parasitic in crocodilians. Most members of Cheloniodiplostomum, a genus of proterodiplostomids that parasitize chelonians, were originally placed within Herpetodiplostomum. The 2 genera were distinguished based on the presence/absence of papillae on the holdfast organ and anterior extent of vitellarium. Our study of Herpetodiplostomum and Cheloniodiplostomum revealed a lack of consistent morphological differences between the genera. Therefore, we consider Cheloniodiplostomum to be a junior synonym of Herpetodiplostomum. Recent molecular phylogenetic studies included a number of proterodiplostomid taxa; however, DNA sequence data are not available for any Herpetodiplostomum or Cheloniodiplostomum species. Herein, we describe a new Herpetodiplostomum species from Geoffroy's side-necked turtle Phrynops geoffroanus from Mato Grosso State, Brazil. The new species can be distinguished from other Herpetodiplostomum species based on the distribution of vitellarium, level of development of holdfast papillae, and ratio of prosoma:opisthosoma width, among other characters. We provide the first molecular phylogeny of the Proterodiplostomidae that includes a Herpetodiplostomum species. The limited geographic distribution of Herpetodiplostomum spp. within the geographic ranges of caimans suggests a secondary host switching event from crocodilians to chelonian definitive hosts in the evolution of Herpetodiplostomum.

Neotropical birds are mostly parasitized by immature ticks and act as reservoir hosts of tick-borne pathogens of medical and veterinary interest. Hence, determining the factors that enable ticks to encounter these highly mobile hosts and increase the potential for tick dispersal throughout migratory flyways are important for understanding tick-borne disease transmission. We used 9682 individual birds from 572 species surveyed across Brazil and Bayesian models to disentangle possible avian host traits and climatic drivers of infestation probabilities, accounting for avian host phylogenetic relationships and spatiotemporal factors that may influence tick prevalence. Our models revealed that the probability of an individual bird being infested with tick larvae and nymphs was lower in partial migrant hosts and during the wet season. Notably, infestation probability increased in areas with a higher proportion of partial migrant birds. Other avian ecological traits known to influence tick prevalence (foraging habitat and body mass) and environmental condition that might constrain tick abundance (annual precipitation and minimum temperature) did not explain infestation probability. Our findings suggest that migratory flyways harbouring a greater abundance of migrant bird hosts also harbour a higher prevalence of immature ticks with potential to enhance the local transmission of tick-borne pathogens and spread across regions.

Individuals of migratory species may be more likely to become infected by parasites because they cross different regions along their route, thereby being exposed to a wider range of parasites during their annual cycle. Conversely, migration may have a protective effect since migratory behaviour allows hosts to escape environments presenting a high risk of infection. Haemosporidians are one of the best studied, most prevalent and diverse groups of avian parasites, however the impact of avian host migration on infection by these parasites remains controversial. We tested whether migratory behaviour influenced the prevalence and richness of avian haemosporidian parasites among South American birds. We used a dataset comprising ~ 11,000 bird blood samples representing 260 bird species from 63 localities and Bayesian multi-level models to test the impact of migratory behaviour on prevalence and lineage richness of two avian haemosporidian genera (Plasmodium and Haemoproteus). We found that fully migratory species present higher parasite prevalence and higher richness of haemosporidian lineages. However, we found no difference between migratory and non-migratory species when evaluating prevalence separately for Plasmodium and Haemoproteus, or for the richness of Plasmodium lineages. Nevertheless, our results indicate that migratory behaviour is associated with an infection cost, namely a higher prevalence and greater variety of haemosporidian parasites.

Determining the prevalence and local transmission dynamics of parasitic organisms are necessary to understand the ability of parasites to persist in host populations and disperse across regions, yet local transmission dynamics, diversity, and distribution of haemosporidian parasites remain poorly understood. We studied the prevalence, diversity, and distributions of avian haemosporidian parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon among resident and migratory birds in Serra do Mar, Brazil. Using 399 blood samples from 66 Atlantic Forest bird species, we determined the prevalence and molecular diversity of these pathogens across avian host species and described a new species of Haemoproteus. Our molecular and morphological study also revealed that migratory species were infected more than residents. However, vector infective stages (gametocytes) of Leucocytozoon spp., the most prevalent parasites found in the most abundant migrating host species in Serra do Mar (Elaenia albiceps), were not seen in blood films of local birds suggesting that this long-distance Austral migrant can disperse Leucocytozoon parasite lineages from Patagonia to the Atlantic Forest, but lineage sharing among resident species and local transmission cannot occur in this part of Brazil. Our study demonstrates that migratory species may harbor a higher diversity and prevalence of parasites than resident species, but transportation of some parasites by migratory hosts may not always affect local transmission.

Sphincterodiplostomum is a monotypic genus of diplostomid digeneans that parasitize fish-eating birds in the neotropics. The type species Sphincterodiplostomum musculosum has a unique, dorsal, tubular invagination in the opisthosoma with a muscular sphincter. Whereas larvae of S. musculosum are relatively commonly reported in Neotropical fish helminth surveys, adult specimens from birds are rarely collected. Prior to our study, no DNA sequence data for S. musculosum were available. Our molecular and morphological study of mature and immature adult Sphincterodiplostomum specimens from three species of birds and one species of crocodilian revealed the presence of at least two species of Sphincterodiplostomum in the neotropics. We provide the first molecular phylogeny of the Diplostomoidea that includes Sphincterodiplostomum. In addition, this is the first record of S. musculosum from caimans, along with the first record of fully mature adult S. musculosum from green kingfisher Chloroceryle americana. The new species of Sphincterodiplostomum (Sphincterodiplostomum joaopinhoi n. sp.) can be morphologically distinguished from S. musculosum based on the anterior extent of vitelline follicles, narrower prosoma, substantially smaller holdfast organ and structure of tegumental spines. Our data revealed 0.7% interspecific divergence in 28S and 10.6–11.7% divergence in cox1 sequences between the two Sphincterodiplostomum species.

Haemosporidian parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon are among the best studied parasites of Neotropical birds. Here, we describe variation in haemosporidian prevalence (i.e. the proportion of infected individuals in a sampled population) in Neotropical birds. We review correlates of haemosporidian prevalence (including several avian life-history traits, climate, and season) and the population and evolutionary consequences of infection for Neotropical birds. We find that prevalence varies among avian taxonomic families, genera, and even among species within the same genus, suggesting that prevalence reflects multiple factors, some of which (e.g., avian incubation period) are associated with avian family-level variation in prevalence while others (e.g., avian abundance) are associated with variation in prevalence among closely related host species. We find few correlates of prevalence that have been consistently demonstrated across studies. Prevalence in populations of many Neotropical birds has been estimated by microscopic examination of blood smears, which affords limited opportunity for comparison with data generated by molecular methods. However, at one site in the Brazilian Cerrado for which we have data, prevalence determined by microscopy does not correlate with prevalence determined by molecular methods for Plasmodium, but the 2 data types are positively correlated for Haemoproteus. Haemosporidians have been hypothesized to play a role in avian sexual selection, and we find the prevalence of Haemoproteus to be lower in polygynous species than in socially monogamous species in the Neotropics, confirming a pattern shown previously among Nearctic birds.LAY SUMMARYMany Neotropical birds are infected by insect-vectored blood parasites belonging to the order Haemosporida. These haemosporidian parasites, commonly known as “avian malaria” parasites, infect bird species to varying degrees.The proportion of birds in a population that are infected (parasite prevalence) has been related to variation in several avian traits, including length of incubation period and nesting height, climate, and other factors, but the patterns are not consistent among studies.We review proposed causes of variation in haemosporidian prevalence and potential consequences of infection for bird populations. Haemosporidian parasites have plausibly been involved in the evolution of colorful plumage and mating systems in birds.Here we find evidence for lower prevalence of haemosporidian infection associated with polygyny (where individual males mate with multiple females) in the Neotropics.Avian haemosporidian parasites exert an important influence on the health and adaptive responses of Neotropical birds.

Human induced changes on landscape can alter the biotic and abiotic factors that influence the transmission of vector-borne parasites. To examine how infection rates of vector-transmitted parasites respond to changes on natural landscapes, we captured 330 Blue-black Grassquits (Volatinia jacarina) in Brazilian biomes and assessed the prevalence and diversity of avian haemosporidian parasites (Plasmodium and Haemoproteus) across avian host populations inhabiting environment under different disturbance and climatic conditions. Overall prevalence in Blue-black Grassquits was low (11%) and infection rates exhibited considerable spatial variation, ranging from zero to 39%. Based on genetic divergence of cytochrome b gene, we found two lineages of Haemoproteus (Parahaemoproteus) and 10 of Plasmodium. We showed that Blue-black Grassquit populations inhabiting sites with higher proportion of native vegetation cover were more infected across Brazil. Other landscape metrics (number of water bodies and distance to urban areas) and climatic condition (temperature and precipitation) known to influence vector activity and promote avian malaria transmission did not explain infection probability in Blue-black Grassquit populations. Moreover, breeding season did not explain prevalence across avian host populations. Our findings suggest that avian haemosporidian prevalence and diversity in Blue-black Grassquit populations are determined by recent anthropogenic changes in vegetation cover that may alter microclimate, thus influencing vector activity and parasite transmission.

Ticks are ectoparasites that feed on blood of a broad taxonomic range of terrestrial and flying vertebrates and are distributed across a wide range of environmental conditions. Here, we explore the biotic and abiotic factors on infestation probability of ticks of the genus Amblyomma and assess the degree of host specificity based on analysis of 1028 birds surveyed across Brazil. We show that tick infestation rates exhibited considerable variation across the 235 avian species analyzed and that the probability of an individual bird being parasitized by immature ticks (larvae and nymphs) increased with annual precipitation. Host phylogeny and two host ecological traits known to promote tick exposure (body mass and foraging behavior) did not predict infestation probability. Moreover, immature ticks displayed a low degree of host specificity at the family level. Lastly, tick occurrence in birds carrying infection with avian malaria and related parasites did not differ from those free of these haemosporidian parasites, indicating a lack of parasite avoidance by immature ticks. Our findings demonstrate that tick occurrence in birds across Brazilian biomes responds to environmental factors rather than ecological and evolutionary host attributes.

Ticks are among the best studied parasitic groups as they spread important pathogens of medical and veterinary importance worldwide. Migratory birds can play an important role in transporting ticks infected with pathogens across wide geographic regions. It is therefore important to understand which factors promote tick parasitism rates across their avian hosts and the associated potential for disease spread. Here, we identified the host attributes of infestation probability of ticks from the genus Amblyomma in 955 birds from Pantanal, Brazil. Infestation rates exhibited considerable variation across the 129 avian species surveyed and were explained by both host ecological traits and evolutionary history. The probability of an individual bird being infested with immature ticks (larvae and/or nymphs) was higher across resident bird species that forage at ground level and during the wet season. Bird species that feed on vertebrates were less likely to be infested by ticks. Other ecological traits known to promote tick exposure (age, body mass, social behavior, and sex) did not predict infestation probability. Our findings demonstrate that tick occurrence in Pantanal birds is determined by avian host attributes, but tick occurrence throughout the year constrains exposure to host-seeking ticks. Moreover, the ecology of the avian host might prevent the potential spread of tick-borne diseases outside Pantanal as migratory hosts are likely less infested by ticks.

Geographic variation in environmental conditions as well as host traits that promote parasite transmission may impact infection rates and community assembly of vector-transmitted parasites. Identifying the ecological, environmental and historical determinants of parasite distributions and diversity is therefore necessary to understand disease outbreaks under changing environments. Here, we identified the predictors and contributions of infection probability and phylogenetic diversity of Leucocytozoon (an avian blood parasite) at site and species levels across the New World. To explore spatial patterns in infection probability and lineage diversity for Leucocytozoon parasites, we surveyed 69 bird communities from Alaska to Patagonia. Using phylogenetic Bayesian hierarchical models and high-resolution satellite remote-sensing data, we determined the relative influence of climate, landscape, geography and host phylogeny on regional parasite community assembly. Infection rates and parasite diversity exhibited considerable variation across regions in the Americas. In opposition to the latitudinal gradient hypothesis, both the diversity and prevalence of Leucocytozoon parasites decreased towards the equator. Host relatedness and traits known to promote vector exposure neither predicted infection probability nor parasite diversity. Instead, the probability of a bird being infected with Leucocytozoon increased with increasing vegetation cover (NDVI) and moisture levels (NDWI), whereas the diversity of parasite lineages decreased with increasing NDVI. Infection rates and parasite diversity also tended to be higher in cooler regions and higher latitudes. Whereas temperature partially constrains Leucocytozoon diversity and infection rates, landscape features, such as vegetation cover and water body availability, play a significant role in modulating the probability of a bird being infected. This suggests that, for Leucocytozoon, the barriers to host shifting and parasite host range expansion are jointly determined by environmental filtering and landscape, but not by host phylogeny. Our results show that integrating host traits, host ancestry, bioclimatic data and microhabitat characteristics that are important for vector reproduction are imperative to understand and predict infection prevalence and diversity of vector-transmitted parasites. Unlike other vector-transmitted diseases, our results show that Leucocytozoon diversity and prevalence will likely decrease with warming temperatures. Fil: Fecchio, Alan. Universidade Federal de Mato Grosso; Brasil Fil: Bell, Jeffrey. University of North Dakota; Estados Unidos Fil: Bosholn, Mariane. Ministério da Ciência, Tecnologia, Inovações. Instituto Nacional de Pesquisas da Amazônia; Brasil Fil: Vaughan, Jefferson. University Of North Dakota; Estados Unidos Fil: Tkach, Vasyl. University Of North Dakota; Estados Unidos Fil: Lutz, Holly. Field Museum Of Natural History; Estados Unidos. University of Chicago; Estados Unidos Fil: Cueto, Víctor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Centro de Investigación Esquel de Montaña y Estepa Patagóica. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Esquel. Centro de Investigación Esquel de Montaña y Estepa Patagónica; Argentina Fil: Gorosito, Cristian Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Centro de Investigación Esquel de Montaña y Estepa Patagóica. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Esquel. Centro de Investigación Esquel de Montaña y Estepa Patagónica; Argentina Fil: González-Acuña, Daniel. Universidad de Concepción; Chile Fil: Stromlund, Chad. University of North Dakota; Estados Unidos Fil: Kvasager, Danielle. University of North Dakota; Estados Unidos Fil: Comiche, Kiba. Universidade de Sao Paulo; Brasil Fil: Kirchgatter, Karin. Universidade de Sao Paulo; Brasil Fil: Pinho, João. Universidade Federal de Mato Grosso; Brasil Fil: Berv, Jacob. Cornell University; Estados Unidos Fil: Anciães, Marina. Ministério da Ciência, Tecnologia, Inovações. Instituto Nacional de Pesquisas da Amazônia; Brasil Fil: Fontana, Carla S.. Pontificia Universidade Católica do Rio Grande do Sul; Brasil Fil: Zyskowski, Kristof. University of Yale; Estados Unidos Fil: Sampaio, Sidnei. Universidade Federal da Bahia; Brasil Fil: Dispoto, Janice. Academy Of Natural Sciences Philadelphia; Estados Unidos Fil: Galen, Spencer. Academy Of Natural Sciences Philadelphia; Estados Unidos Fil: Weckstein, Jason. Academy Of Natural Sciences Philadelphia; Estados Unidos. Drexel University; Estados Unidos Fil: Clark, Nicholas. The University Of Queensland; Australia

Identifying the mechanisms driving the distribution and diversity of parasitic organisms and characterizing the structure of parasite assemblages are critical to understanding host-parasite evolution, community dynamics, and disease transmission risk. Haemosporidian parasites of the genera Plasmodium and Haemoproteus are a diverse and cosmopolitan group of bird pathogens. Despite their global distribution, the ecological and historical factors shaping the diversity and distribution of these protozoan parasites across avian communities and geographic regions remains unclear. Here we used a region of the mitochondrial cytochrome b gene to characterize the diversity, biogeographical patterns and phylogenetic relationships of Plasmodium and Haemoproteus infecting Amazonian birds. Specifically, we asked whether, and how, host community similarity and geography (latitude and area of endemism) structure parasite assemblages across 15 avian communities in the Amazon Basin. We identified 265 lineages of haemosporidians recovered from 2661 sampled birds from 330 species. Infection prevalence varied widely among host species, avian communities, areas of endemism, and latitude. Composition analysis demonstrated that both malarial parasites and host communities differed across areas of endemism and as a function of latitude. Thus, areas with similar avian community composition were similar in their parasite communities. Our analyses, within a regional biogeographic context, imply that host switching is the main event promoting diversification in malarial parasites. Although dispersal of haemosporidian parasites was constrained across six areas of endemism, these pathogens are not dispersal-limited among communities within the same area of endemism. Our findings indicate that the distribution of malarial parasites in Amazonian birds is largely dependent on local ecological conditions and host evolutionary relationships. This article is protected by copyright. All rights reserved.

Aim: Migrating birds transport their parasites, often over long distances, but little is known about the transfer of these parasites to resident hosts in either the wintering or breeding ranges of the migratory host populations. We investigated the haemosporidian parasite faunas of migratory and resident birds to determine connections among distant parasite faunas, plausibly brought about by migratory host populations. Location: Samples were obtained, primarily during or shortly after the local breeding season, throughout the Americas, from the United States through the Caribbean Basin and into northern South America. Methods: Infections were identified by PCR and sequencing of parasite DNA in avian blood samples. The analyses were based on c. 4700 infections representing 79 parasite lineages of Plasmodium and Haemoproteus spp. Geographical connections of lineages between regions in the Americas were compared to those in the Euro-African migration system, where migration distances are longer for many host species and the migrant and resident avifaunas in the wintering areas are phylogenetically more divergent. Results: Haemosporidian lineages exhibited considerable variation in distribution in the Americas, and patterns of distribution differ markedly between the Americas and the Euro-African migration system. In particular, few lineages were recovered from resident species in both temperate and tropical latitudes, particularly in the Euro-African system, in which a large proportion of lineages were restricted to migrants. Parasite lineages in the Euro-African system exhibited considerable phylogenetic conservatism in their distributions, that is, a tendency of related lineages to exhibit similar geographical distributions. In contrast, clades of parasites in the Americas displayed more geographical diversity, with four of 12 clades exhibiting all four of the distribution types representing the combinations of resident and migrant host species in both temperate and tropical latitudes. Main conclusions: Long-distance migrants connect communities of avian haemosporidian parasites in breeding and wintering areas with disparate avifaunas and different vector communities. The degree of parasite lineage sharing between migrants and residents in breeding and wintering areas appears to reflect, to a large degree, the taxonomic similarity of migrants to the resident species in both areas. (Less)

Crassiphiala is a monotypic genus of diplostomid digeneans and is the type genus of the subfamily Crassiphialinae. The type species Crassiphiala bulboglossa parasitizes kingfishers in the Nearctic and has a Neascus-type metacercaria that encysts on fish intermediate hosts, often causing black spot disease. While recent molecular phylogenetic studies included some members of the Crassiphialinae, no DNA sequence data of Crassiphiala is currently available. Our molecular and morphological study of adult and larval crassiphialines from the Americas revealed the presence of at least three lineages of Crassiphiala from the Nearctic and two lineages from the Neotropics. This is the first record of Crassiphiala from the Neotropics. Herein, we provide the first molecular phylogeny of the Diplostomoidea that includes Crassiphiala. Our data revealed 0.2–2.4% divergence among 28S sequences and 11–19.8% among CO1 sequences of lineages of Crassiphiala. The results of our analyses did not support the monophyly of Crassiphialinae. Our results clearly demonstrated that the diversity of Crassiphiala has been underestimated.

Corticosterone (CORT) is the main glucocorticoid hormone of amphibians, reptiles, birds and some mammals. This hormone may have evolved as an adaptive metabolic mechanism, in part because increased concentrations of CORT are essential for individuals to manage energy resources and thus cope with negative perturbations such as predation and storms. The benefits of CORT are offset by costs, because elevated levels can suppress inflammatory responses of individuals, making them more susceptible to parasites and pathogens. In this study, we investigated the relationships between feather CORT levels, infection status and diversity of haemosporidian parasites in the Blue-crowned Manakin Lepidothrix coronata, considering possible effects related to the sex and age of individuals. We predicted higher levels of feather CORT in infected individuals. We observed that feather CORT levels were similar among individuals of different sexes and ages. Although haemosporidian infection status did not vary among sexes, occurrence probability was higher among younger individuals, which may indicate that the less developed immune system of these individuals makes them more susceptible to avian malaria. Contrary to expectations, we found that feather CORT levels were not associated with the infection status and diversity of haemosporidian parasites. That haemosporidian occurrence probability does not increase with elevated feather CORT levels suggests that individuals are not immunosuppressed by elevated levels of this hormone, at least to the extent that feather CORT truly reflects individual differences in the level of this hormone., The field data collection was based upon research supported by the Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (UNIVERSAL number 471092/2012-6 Faixa C to M.A.) and by Fundacao de Amparo a Pesquisa do Estado do Amazonas (PRONEX proj. 003/2009, number 653/2009 to M.A. and Albertina Lima). Hormone analysis was funded by Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (Ciencia sem Fronteiras Chamada No. 71/2013 to M.A., D.G. and Regina Macedo). Haemosporidian laboratory work was funded by NSF DEB-1503804 to J.D.W. During the project, M.B. received a fellowship from Fundacao de Amparo a Pesquisa do Estado do Amazonas (FAPEAM) and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq). A.F. was supported by a postdoctoral fellowship (PNPD scholarship) from Coordenacao de Aperfeicoamento de Pessoal de Nıvel Superior (CAPES).

Parasites with low host specificity (e.g. infecting a large diversity of host species) are of special interest in disease ecology, as they are likely more capable of circumventing ecological or evolutionary barriers to infect new hosts than are specialist parasites. Yet for many parasites, host specificity is not fixed and can vary in response to environmental conditions. Using data on host associations for avian malaria parasites (Apicomplexa: Haemosporida), we develop a hierarchical model that quantifies this environmental dependency by partitioning host specificity variation into region- and parasite-level effects. Parasites were generally phylogenetic host specialists, infecting phylogenetically clustered subsets of available avian hosts. However, the magnitude of this specialization varied biogeographically, with parasites exhibiting higher host specificity in regions with more pronounced rainfall seasonality and wetter dry seasons. Recognizing the environmental dependency of parasite specialization can provide useful leverage for improving predictions of infection risk in response to global climate change.

The removal of ectoparasites is a common behavior found across animal taxa and is a determinant to avoid the negative effects of parasites’ presence. Eventually, the elimination of ectoparasites is associated with mutualistic interactions. Cleaner birds remove ectoparasites, providing benefits to its mutualistic host by reducing parasite burden while they obtain a protein food source. Here we report some evidence that giant cowbirds (Molothrus oryzivorus) may have an important role as a cleaner bird. We found 74 adult ticks inside the ventriculus of one male giant cowbird. The ticks belonged to three different species: Amblyomma dubitatum, A. sculptum and A. triste. We found that the sex-ratio of the consumed adult ticks was not different from 1:1. Although additional data are necessary, the large number of ticks found suggests that the giant cowbird may have developed a mutualistic association with large, social mammals such as capybaras (Hydrochoerus hydrochaeris), since this animal is an important host species for the three tick species found in the present study.

Haemosporidian parasites of the genera Plasmodium, Leucocytozoon, and Haemoproteus are one of the most prevalent and widely studied groups of parasites infecting birds. Plasmodium is the most well-known haemosporidian as the avian parasite Plasmodium relictum was the original transmission model for human malaria and was also responsible for catastrophic effects on native avifauna when introduced to Hawaii. The past two decades have seen a dramatic increase in research on avian haemosporidian parasites as a model system to understand evolutionary and ecological parasite-host relationships. Despite haemosporidians being one the best studied groups of avian parasites their specialization among avian hosts and variation in prevalence amongst regions and host taxa are not fully understood. In this review we focus on describing the current phylogenetic and morphological diversity of haemosporidian parasites, their specificity among avian and vector hosts, and identifying the determinants of haemosporidian prevalence among avian species. We also discuss how these parasites might spread across regions due to global climate change and the importance of avian migratory behavior in parasite dispersion and subsequent diversification.

Plumage coloration deriving from carotenoid and melanin pigments can be a quality signal in birds, and can be under conspecific inspection in social interactions. For example, parasite load and immune system status can be inferred through plumage color intensity, and can influence the choice of sexual partners. Here, we evaluated two plumage ornaments in the Yellow-billed Cardinal [Paroaria capitata (d’Orbigny & Lafresnaye 1837)]: the carotenoid-based coloration of the cap and the melanin-based coloration of the bib. We evaluated whether these ornaments were related to blood parasite burden, immunological status and body condition, and whether they could reveal individual sex and age. Cardinals were mist-netted in a Brazilian wetland, and 12 individuals were infected with malaria parasites. Both carotenoid and melanin colorations were related to age, but only carotenoids reflected immunological status. Adult cardinals presented redder caps and darker bibs in comparison to juveniles, and redder caps were a...

Despite many studies on avian blood parasites, we still have a limited understanding of the mechanisms that drive patterns of haemosporidian infection among tropical birds, including effects associated with sex, age, and seasonality. Using molecular and morphological methods for blood-parasite detection, we found that juvenile White-banded Tanagers (Neothraupis fasciata (Lichtenstein, 1823)) had lower haemosporidian prevalence than adults in a population within central Brazil. However, no sex or seasonal differences were detected. Of the 92 White-banded Tanagers analyzed, 67 individuals (72.8% prevalence) were infected with either Haemoproteus or Plasmodium (phylum Sporozoa, class Coccidea, order Haemosporida). Sequencing of a portion of the mitochondrial cytochrome b gene revealed six haemosporidian lineages: two lineages within the genus Haemoproteus and four within the genus Plasmodium. The prevalences of Plasmodium and Haemoproteus parasites were 43.5% and 17.4%, respectively. Our results suggest that this species maintains chronic infections all year round and individuals are able to sustain high parasite pressure.

SUMMARYParasites of the generaPlasmodiumandHaemoproteus(Apicomplexa: Haemosporida) are a diverse group of pathogens that infect birds nearly worldwide. Despite their ubiquity, the ecological and evolutionary factors that shape the diversity and distribution of these protozoan parasites among avian communities and geographic regions are poorly understood. Based on a survey throughout the Neotropics of the haemosporidian parasites infecting manakins (Pipridae), a family of Passerine birds endemic to this region, we asked whether host relatedness, ecological similarity and geographic proximity structure parasite turnover between manakin species and local manakin assemblages. We used molecular methods to screen 1343 individuals of 30 manakin species for the presence of parasites. We found no significant correlations between manakin parasite lineage turnover and both manakin species turnover and geographic distance. Climate differences, species turnover in the larger bird community and parasite lineage turnover in non-manakin hosts did not correlate with manakin parasite lineage turnover. We also found no evidence that manakin parasite lineage turnover among host species correlates with range overlap and genetic divergence among hosts. Our analyses indicate that host switching (turnover among host species) and dispersal (turnover among locations) of haemosporidian parasites in manakins are not constrained at this scale.

Uvulifer Yamaguti, 1934, is a genus of diplostomoidean digeneans that parasitizes kingfishers worldwide. Species have a Neascus-type metacercaria that encysts in or on fish intermediate ho...

Birds harbor a diverse group of haemosporidian parasites that reproduce and develop in the host blood cells, muscle tissue, and various organs, which can cause negative effects on the survival and reproduction of their avian hosts. Characterization of the diversity, distribution, host specificity, prevalence patterns, and phylogenetic relationships of these parasites is critical to the study of avian host-parasite ecology and evolution and for understanding and preventing epidemics in wild bird populations. Here, we tested whether muscle and liver samples collected as part of standard ornithological museum expeditions can be examined to study the diversity and distributions of haemosporidians in the same way as blood collected from individual birds that are typically banded and released. We used a standard molecular diagnostic screening method for mitochondrial DNA (cytochrome b) of the parasites and found that blood, muscle, and liver collected from the same host individual provide similar estimates of prevalence and diversity of haemosporidians from the genera Parahaemoproteus and Leucocytozoon. Although we found higher prevalence for the genus Plasmodium when we screened blood vs. liver and muscle samples, the estimates of the diversity of Plasmodium from different tissue types are not affected at the community level. Given these results, we conclude that for several reasons existing museum genetic resources collections are valuable data sources for the study of haemosporidians. First, ornithological museum collections around the world house tens of thousands of vouchered tissue samples collected from remote regions of the world. Second, the host specimens are vouchered and thus host identification and phenotype are permanently documented in databased archives with a diversity of associated ancillary data. Thus, not only can identifications be confirmed but also a diversity of morphological measurements and data can be measured and accessed for these host specimens in perpetuity.

SUMMARYStudies on avian haemosporidia are on the rise, but we still lack a basic understanding of how ecological and evolutionary factors mold the distributions of haemosporidia among species in the same bird community. We studied the structure and organization of a local avian haemosporidian assemblage (generaPlasmodiumandHaemoproteus) in the Cerrado biome of Central Brazil for 5 years. We obtained 790 blood samples from 54 bird species of which 166 (21%) were infected with haemosporidians based on molecular diagnostics. Partial sequences of the parasite cytochrome b gene revealed 18 differentiated avian haemosporidian lineages. We also analysed the relationship of life-history traits (i.e., nesting height, migration status, nest type, sociality, body mass, and embryo development period) of the 14 most abundant bird species with the prevalence of avian haemosporidia. It was found that host species that bred socially presented a higher prevalence ofHaemoproteus(Parahaemoproteus) than bird species that bred in pairs. Thus, aspects of host behaviour could be responsible for differential exposure to vectors. The assemblage of avian haemosporidia studied here also confirms a pattern that is emerging in recent studies using molecular markers to identify avian haemosporidians, namely that many lineages are host generalists.

Parasitos podem levar espécies de aves à extinção, afetar as dinâmicas temporais e espaciais dos hospedeiros, alterar a estrutura de comunidades e o status social de indivíduos. Avaliou-se a prevalência e a intensidade de parasitos em famílias e espécies de 925 aves capturadas, entre 2000 e 2001, na Mata Atlântica de Minas Gerais. Foram coradas com Giemsa extensões de sangue para detectar parasitos hematozoários. As aves (n= 15,8%) 11 famílias estavam infectadas por pelo menos um gênero de parasito, especialmente Muscicapidae (28,3%) e Conopophagidae (25%). Entre as 146 aves infectadas, Plasmodium foi detectado em todas as famílias e possuiu a maior prevalência (54,8%). Trypanosoma,Haemoproteus e microfilaria possuíram baixas prevalências (23,3, 23,3 e 2,1%, respectivamente). Aves capturadasdurante a estação chuvosa estavam mais infectadas do que aves capturadas durante a estação seca. A baixa prevalência geral de parasitos do sangue das aves é semelhante aos padrões encontrados em outras localidades da região Neotropical. Parasites may lead bird species to extinction, affect host temporal and spatial population dynamics, alter community structure and alter individuals social status. We evaluated blood parasite prevalence and intensity according to bird families and species, among 925 birds that were caught in 2000 and 2001, in the Atlantic Forest in the State of Minas Gerais, Brazil. We applied Giemsa staining to thin blood smears, to detect blood parasites. The birds (n = 15.8%) in 11 families, were infected by at least one parasite genus, especially Muscicapidae (28.3%) and Conopophagidae (25%). Among the 146 infected birds, Plasmodium was detected in all bird families and had the highest prevalence (54.8%). Trypanosoma, Haemoproteus and microfilaria had lower prevalence rates (23.3, 23.3 and 2.1%, respectively). Birds caught during the rainy season were more infected than birds caught during the dry season. The overall low prevalence of blood parasites in birds is similar to the patterns found elsewhere in the Neotropical region.

Blood parasites play a fundamental role in the ecology and evolution of passerine birds because they are able to affect the fitness and survival of their hosts. The prevalence of avian malarial parasites among host species can vary from 0 to 100% but the ecological and evolutionary reasons for this variation are not clear. In this study we tested if height or type of nest, body mass or social system, which we believe are variables associated with exposure of hosts to vectors, could explain the variation in the prevalence of blood parasites in a bird community from the Cerrado biome of central Brazil. We found a significant positive correlation between nest-height and prevalence of Haemoproteus, which is consistent with the hypothesis linking prevalence of blood parasites with nesting stratum in North American birds. We also found evidence for increased levels of parasitism by Haemoproteus in neotropical birds that live in groups and breed cooperatively and increased levels of parasitism by Plasmodium in species that nest in cavities or closed cups. We suggest that reproductive and behavioural parameters of hosts may be responsible for their differential exposure to vectors and that these parameters may therefore be able to indicate interspecific variation in the prevalence of blood parasites in other bird communities.

Species that are introduced to novel environments can lose their native pathogens and parasites during the process of introduction. The escape from the negative effects associated with these natural enemies is commonly employed as an explanation for the success and expansion of invasive species, which is termed the enemy release hypothesis (ERH). In this study, nested PCR techniques and microscopy were used to determine the prevalence and intensity (respectively) of Plasmodium spp. and Haemoproteus spp. in introduced house sparrows and native urban birds of central Brazil. Generalized linear mixed models were fitted by Laplace approximation considering a binomial error distribution and logit link function. Location and species were considered as random effects and species categorization (native or non-indigenous) as fixed effects. We found that native birds from Brazil presented significantly higher parasite prevalence in accordance with the ERH. We also compared our data with the literature, and found that house sparrows native to Europe exhibited significantly higher parasite prevalence than introduced house sparrows from Brazil, which also supports the ERH. Therefore, it is possible that house sparrows from Brazil might have experienced a parasitic release during the process of introduction, which might also be related to a demographic release (e.g. release from the negative effects of parasites on host population dynamics).

Psitacídeos neotropicais usualmente exploram o dossel das florestas em busca de alimentos como néctar, flores, folhas, polpa e sementes de frutos. Como essas aves não estabelecem territórios, movimentam-se através de mosaicos de vegetação no sentido de utilizar recursos alimentares, produzidos massivamente, à medida que se tornam disponíveis. Neste estudo, nós examinamos a utilização de uma mata ciliar, no Pantanal Sul (Brasil), por uma comunidade de psitacídeos, constituída por um gradiente de formas que incluiu desde o pequeno Brotogeris chiriri até a grande Ara chloroptera. Os recursos vegetais, importantes para os psitacídeos, foram produzidos massivamente durante a estação chuvosa (frutos carnosos), subseqüentemente durante as cheias anuais (também frutos carnosos) e, finalmente, na estação seca (flores). Tanto as pequenas quanto as grandes espécies consumiram tais frutos, no entanto os periquitos utilizaram predominantemente a polpa, enquanto as espécies maiores consumiram em proporções similares a polpa e as sementes. Durante a estação seca os periquitos utilizaram extraordinariamente néctar das flores, sobretudo produzido por Inga vera, que anualmente floresceu massivamente ao final da estação seca, período mais rigoroso do ano. Dentre as espécies maiores, apenas Propyrrhura auricollis utilizou substancialmente néctar. A intensa produção de frutos, tipicamente dispersos por peixes ou pela água durante a cheia anual, foi amplamente utilizada pelos psitacídeos. Portanto, ao contrário dos padrões de frutificação da maioria das matas secas neotropicais, a ocorrência de um pico de frutificação durante as cheias emerge como um evento marcante de disponibilidade de frutos, num período (transição da estação úmida para a seca) em que tipicamente tendem a declinar. Nesse sentido, a peculiar produção massiva de flores e frutos, nessa mata ciliar, potencialmente contribui para manutenção das grandes populações de psitacídeos, ainda presentes, no Pantanal Sul. ABSTRACT - Neotropical parrots usually forage in forest canopies for nectar, flowers, leaves, fruit pulp, and seeds. As they have no all-purpose territories, these birds usually exploit vegetation mosaics in order to use plentiful resources as they become available. In this study we examine the use of a gallery forest in the southern Pantanal (Brazil) by a diverse parrot community that ranged from Brotogeris chiriri (a small species) to Ara chloroptera (a large one). Plant food resources principally used by parrots were abundantly available during the rainy season (fleshy fruits), the annual floods (fleshy fruits), and the dry season (flowers). While both smaller and larger species foraged on fruits, parakeets largely consumed the pulp, while larger parrot species used pulp and seeds. In the dry season parakeets foraged extensively on nectar, especially Inga vera nectar that was abundantly available during the last two months of the dry season, the harshest period of the year. Among larger parrots, only Propyrrhura auricollis frequently harvested nectar. Fruits maturing during floods, despite being fish- or water- dispersed were extensively used by the parrots. Hence, unlike what happens in most other Neotropical dry forests, occurrence of a fruiting peak during the annual flooding, which occurs in the transition from the wet to the dry season, constitutes an extra and significant episode of food availability, since in this period, fruit production normally declines. Therefore, the unique and abundant availability of flowers and fruits in this gallery forest may account for the presence of large parrot populations in the southern Pantanal.

The Amazon biome harbors the richest avifauna in the world. However, in the Brazilian Amazon only a single previous study has systematically evaluated wild birds for ticks. During 2012, wild birds were captured in two areas of the Brazilian Amazon forest, one located in the State of Amazonas, and another in the State of Para. Overall, 581 wild birds representing 118 species were captured, but only 18 individuals (3.1% prevalence) were infested by ticks, comprising immature stages of Amblyomma calcaratum Neumann, 1899, Amblyomma geayi Neumann, 1899, Amblyomma humerale Koch, 1844, and Amblyomma longirostre (Koch, 1844). The only previous study of birds in the Brazilian Amazon reported 40.2% tick prevalence. Such contrasting prevalence values may stem from seasonal differences or differences in forest disturbance at the two sites.

Background Birds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction. Methods Using available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR). Results The real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ2 = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples. Conclusions Our real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research.