Prevalence and composition of haemosporidians in an avian community from a World Heritage area: Associations with host foraging strata and forest regeneration.

Forest regeneration is becoming a powerful tool to combat land conversion which covers 30 % of the Neotropical territory. However, little is known about the effect of forest regeneration on vector-borne diseases. Here, we describe the haemosporidian lineage composition across a successional gradient within an Atlantic Forest bird community. We test whether forest successional stages, in addition to host life history traits affect haemosporidian infection probability. We sampled birds at 16 sampling units with different successional stages between 2017 and 2018 within a forest remnant located in Antonina, Paraná, Brazil. We captured bird individuals using mist-nets, identified them to the species level, and collected blood samples to detect and identify Plasmodium and Haemoproteus lineages based on molecular analysis. We used a Bayesian phylogenetic linear model with a Bernoulli distribution to test whether the haemosporidian infection probability is affected by nest type, foraging stratum, and forest successional stage. We captured 322 bird individuals belonging to 52 species and 21 families. We found 31 parasite lineages and an overall haemosporidian prevalence of 23.9 %, with most infections being caused by Plasmodium (21.7 % of prevalence). The Plasmodium probability of infection was associated with forest successional stage and bird foraging stratum. Birds from the secondary forest in an intermediate stage of succession are more likely to be infected by the parasites than birds from the primary forests (ß = 1.21, 95 % CI = 0.11 - 2.43), birds from upper strata exhibit a lower probability of infection than birds from lower foraging strata (ß = -1.81, 95 % CI = -3.80 - -0.08). Nest type did not affect the Plasmodium probability of infection. Our results highlight the relevance of forest succession on haemosporidian infection dynamics, which is particularly relevant in a world where natural regeneration is the main tool used in forest restoration.

Avian haemosporidians of the genera Plasmodium and Haemoproteus from resident and Neotropical migrant birds in Colombia.

Avian haemosporidians of the genera Plasmodium and Haemoproteus are a group of widely distributed blood parasites that can negatively affect the fitness of their hosts. Colombia contains the greatest diversity of birds on the planet, but knowledge about the associations between haemosporidian and its avifauna is scarce and fragmented. We collected blood samples from 255 birds (203 residents and 52 neotropical migrants) belonging to 27 families and 108 species. The study was conducted in six localities in the inter-Andean valleys of the Cauca and Magdalena rivers. Parasites of the genera Plasmodium and Haemoproteus were identified in the samples by morphological and molecular analysis of a fragment of the mitochondrial gene cyt b. Among the samples, 9.3% (n = 24) were positive for Plasmodium or Haemoproteus. Co-infection with Plasmodium and Haemoproteus was found in Red-eyed Vireo. Seventeen haemosporidian lineages were identified, five of which were reported for the first time in resident birds (Common Ground Dove, Checker-throated Stipplethroat, Tropical Kingbird, Pale-breasted Thrush, and Ruddy-breasted Seedeater) and one in the Summer Tanager (neotropical migrant). The research results confirm the wide diversity of haemosporidian present in tropical lowlands and the possible role of neotropical migratory birds in dissemination on haemosporidian along their migratory routes.

New morphological and molecular data for Haemoproteus (H.) paramultipigmentatus in the Atlantic Forest of Brazil.

Parasites of the genus Haemoproteus have been reported in almost all avian clades and zoogeographic regions, except Antarctica. However, despite the large number of reports worldwide, they are poorly studied in the Neotropical region, which includes the Atlantic Forest, a biodiversity hotspot with more than 890 bird species, of which 24% are endemic. Haemoproteus (Haemoproteus) paramultipigmentatus was described by morphological and molecular analyses in 2013 infecting Columbiform birds in Mexico. However, since the original description this parasite has not been studied in detail. Here, we investigate the prevalence of Haemoproteus spp. in Brazilian Columbiformes and conducted a taxonomic integrative study of the species Haemoproteus (Haemoproteus) paramultipigmentatus, including new morphological and molecular data from a Brazilian population. Moreover, we provide discussions about the geographic distribution and phylogenetic relationships between different lineages of this parasite. Our findings demonstrated a high prevalence of Haemoproteus spp. infection in Brazilian Columbiformes, which is in accordance with previous studies. Morphological characterization of H. paramultipigmentatus revealed minor differences from the original description. Through molecular and phylogenetic analyses we identified a new lineage of H. paramultipigmentatus that was added to the genetic databases. Our findings also suggest a new geographical distribution for this hemoparasite, including South American countries, and raise discussions about its current distribution.

Molecular and serological detection of arthropod-borne pathogens in carnivorous birds from Brazil.

Rickettsiales, Haemosporida and Rhizobiales agents can cause diseases that affect various animal species, including humans. Due to predation behaviour, carnivorous birds may play an important role in spreading these etiological agentes across geographically distant areas, specially if they are migratory. The aim of this study was to investigate the occurrence and to access the phylogenetic relations among Anaplasmataceae (Ehrlichia, Anaplasma, Neorickettsia), Bartonellaceae (Bartonella spp.), and Haemosporida (Plasmodium, Haemoproteus and Leucocytozoon) agents in blood samples from 121 carnivorous birds sampled in the states of São Paulo and Rio de Janeiro, Brazil. Inclusions resembling hemoparasites were not observed in Giemsa-stained preparations. While three animals were seropositive for E. chaffeensis (3.41% [3/88]; 95% CI:1.17-9.55%), five showed antibodies to A. phagocytophilum (5.68% [5/88]; 95% CI: 2.45-12.62%). Despite the detection of rrs gene fragments closely related to E. chaffeensis (4.13% [5/121]; 95% CI: 1.78-9.31%), no positivity was observed in the qPCR based on the genes vlpt for the organism. Similarly, 12 (9.91% [12/121]; 95% CI: 5.76-16.74%) samples were positive in the qPCR for Anaplasma spp. based on groEL gene, but negative in the qPCR for A. phagocytophilum based on msp-2 gene. Three samples were positive in the nPCR for E. canis based on rrs gene. Three samples were positive for Haemoproteus spp. and one for Plasmodium spp. in the nPCR based on cytB gene. Four birds (3.3% [4/121]; 95% CI: 1.29-8.19%) presented co-positivity by Ehrlichia sp. and Anaplasma sp. in molecular assays. One (0.82% [1/121]; 95% CI:0.15-4.53%) bird showed to be seropositive for E. chaffeensis and and positive in PCR for Haemoproteus sp. All birds were negative in the qPCR assay for Bartonella spp. (nuoG). The present work showed the occurrence of Anaplasmataceae agents and hemosporidians in carnivorous birds from southeastern Brazil. The role of these animals in the dispersion of Anaplasmataceae agents should be further investigated.

Mining increases the prevalence of avian haemosporidian parasites in Northeast Amazonia.

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.

Haemoproteus syrnii (Haemosporida: Haemoproteidae) in owls from Brazil: morphological and molecular characterization, potential cryptic species, and exo-erythrocytic stages.

Haemoproteus syrnii is a haemosporidian parasite found in owls. Although morphological and molecular data on the species is available, its exo-erythrocytic development was never researched. In this study, we provide the morphological, morphometric, and molecular characterization of H. syrnii populations found in owl species from Minas Gerais, southeast Brazil. We also characterized the coalescent species delimitation based on the molecular and histopathology data. Samples from 54 owls from six different species were analyzed, generating 11 sequences of the cyt b gene, from which six were new sequences. The overall prevalence of infection was high (72.22%). The H. syrnii sequences were grouped into two well-supported independent clades, which included other Haemoproteus (Parahaemoproteus) species. This was supported by both the coalescent species delimitation analysis and by the genetic divergence between lineages of these distinct clades. There were small morphological and morphometric differences within the population presented in this study. However, when compared with other studies, the molecular analysis demonstrated considerable intraspecific variation and suggests potential cryptic species. The histopathological analysis revealed, for the first time, that lungs and skeletal muscle are exo-erythrocytic stage location of H. syrnii, and that the parasite is linked to the histopathological changes found in owls. This study brings new data from Haemoproteus species biology and host infection, and improves host-parasite relationship understanding under an owl conservation perspective.

Molecular diversity and coalescent species delimitation of avian haemosporidian parasites in an endemic bird species of South America.

Haemoproteus spp. and Plasmodium spp. are blood parasites that occur in birds worldwide. Identifying the species within this group is complex, especially in wild birds that present low parasitemia when captured, making morphological identification very difficult. Thus, the use of alternative tools to identify species may be useful in the elucidation of the distribution of parasites that circulate in bird populations. The objectives of this study were to determine the prevalence and parasitemia of the genera Plasmodium and Haemoproteus in Tachyphonus coronatus in the Atlantic Forest, Brazil, and to evaluate the molecular diversity, geographic distribution, and specificity of these parasites based on coalescent species delimitation methods. Microscopic analysis, PCR, cyt b gene sequencing, phylogenetic analysis and coalescent species delimitation using single-locus algorithms were performed (Poisson tree process (PTP) and multi-rate Poisson tree process (MPTP) methods). The analyses were performed in 117 avian host individuals. The prevalence was 55.5% for Plasmodium and 1.7% for Haemoproteus, with a mean parasitemia of 0.06%. Twenty-five Plasmodium and two Haemoproteus lineages were recovered. The MPTP method recovered seven different evolutionarily significant units (ESUs) of Plasmodium and one of Haemoproteus, whereas PTP presented fourteen ESUs of Plasmodium and one of Haemoproteus. The MPTP was more consistent with current taxonomy, while PTP overestimated the number of lineages. These ESUs are widely distributed and have already been found in 22 orders of birds that, all together, inhabit every continent, except Antarctica. The computational methods of species delimitation proved to be effective in cases where the classification of Haemosporida based just on morphology is insufficient.

Using a multistate occupancy approach to determine molecular diagnostic accuracy and factors affecting avian haemosporidian infections.

The use of a sensitive and accurate parasite detection methodology is crucial in studies exploring prevalence of parasites in host populations or communities, and uncertainty in identifying parasite genera and/or lineages may limit the understanding of host-parasite interactions. Here, we used a multistate occupancy approach that accounts for imperfect detection to assess whether sex and breeding season influenced the prevalence of a specific Haemoproteus lineage (TARUF02) in a white-lined tanager population. Likewise, we explored whether the probability of detecting the target parasite in an infected bird using PCR and sequencing analyses may be influenced by season and host sex. We found little evidence that sex influenced the probability of an individual host being infected by a haemosporidian parasite. Conversely, we found that the probability of infection by Haemoproteus TARUF02 was ~30% higher during the breeding season, reflecting a higher prevalence of this parasite in this season. The probability that PCR detects DNA of haemosporidian parasite was higher for female birds, suggesting that they are more prone to be parasitized with parasitemia levels that are more successfully detected by molecular analysis. Sequencing successfully determined the Haemoproteus TARUF02 lineage in 60% of samples collected during the breeding season and 84% of samples collected during the non-breeding season. Understanding the ecology of hosts and aspects of their physiology that may influence the parasite infection is essential to better understanding of hemoparasite infections and how parasites influence their native hosts, through decreasing reproductive success, lifespan, and/or survival.

Haemosporidian parasites of resident and wintering migratory birds in The Bahamas.

In temperate regions, some avian haemosporidian parasites have evolved seasonal transmission strategies, with chronic infections relapsing during spring and transmission peaking during the hosts' breeding season. Because lineages with seasonal transmission strategies are unlikely to produce gametocytes in winter, we predicted that (1) resident birds living within wintering areas of Neotropical migrants would unlikely be infected with North American parasite lineages; and (2) if infected, wintering migratory birds would be more likely to harbor Plasmodium spp. rather than Parahaemoproteus spp. or Haemoproteus spp. parasites in their bloodstreams, as only Plasmodium produces life stages, other than gametocytes, that infect red blood cells. To test these predictions, we used molecular detection and microscopy to compare the diversity and prevalence of haemosporidian parasites among year-round residents and wintering migratory birds during February 2016, on three islands of The Bahamas archipelago, i.e., Andros, Grand Bahama, and Great Abaco. Infection prevalence was low and comparable between migratory (15/111) and resident (15/129) individuals, and it did not differ significantly among islands. Out of the 12 lineages detected infecting migratory birds, five were transmitted in North America; four lineages could have been transmitted during breeding, wintering, or migration; and three lineages were likely transmitted in The Bahamas. Resident birds mostly carried lineages endemic to the Caribbean region. All North American-transmitted parasite lineages detected among migratory birds were Plasmodium spp. Our findings suggest that haemosporidian parasites of migrants shift resource allocation seasonally, minimizing the production of gametocytes during winter, with low risk of infection spillover to resident birds.

Haemosporidian Parasites of Chilean Ducks: The Importance of Biogeography and Nonpasserine Hosts.

Biogeography is known to have shaped the diversity and evolutionary history of avian haemosporidian parasites across the Neotropics. However, a paucity of information exists for the temperate Neotropics and especially from nonpasserine hosts. To understand the effect of biogeography in the temperate Neotropics on haemosporidians of nonpasserine hosts we screened ducks (Anseriformes) from central Chile for the presence of these parasites. Forty-two individuals of 4 duck species (Anas flavirostris, Anas georgica, Mareca sibilatrix, Spatula cyanoptera cyanoptera) were collected and assessed for haemosporidian parasite infections by real-time polymerase chain reaction screening and subsequent sequencing of the mitochondrial cytochrome b gene. Haemoproteus (subgenus Haemoproteus) and Plasmodium were detected in 2 host species, A. georgica and S. c. cyanoptera, with no Leucocytozoon found. Overall haemosporidian prevalence was low (14.2%), with the prevalence of Plasmodium (11.9%) being substantially greater than that of Haemoproteus (4.8%). Six haemosporidian cytochrome b lineages were recovered, 2 Haemoproteus and 4 Plasmodium, with all 6 lineages identified for the first time. In phylogenetic reconstruction, the Chilean Plasmodium lineages were more closely related to South American lineages from passerine birds than to known lineages from anseriforms. The subgenus Haemoproteus known from nonpasseriformes has never been identified from any anseriform host; however, we recovered 2 lineages from this subgenus, one from each A. georgica and S. c. cyanoptera. Further work is needed to determine if this presents true parasitism in ducks or only a spillover infection. The results of phylogenetic reconstruction demonstrate a unique evolutionary history of these Chilean parasites, differing from what is known for this host group. The unique geography of Chile, with a large part of the country being relatively isolated by the Atacama Desert in the north and the Andes in the east and south, would present opportunities for parasite diversification. Further work is needed to investigate how strongly the biogeographical isolation has shaped the haemosporidian parasites of this area. Our results add to the growing body of evidence that nonpasserine hosts support unique lineages of haemosporidian parasites, while also demonstrating the role of biogeography in haemosporidian parasite diversity in the temperate Neotropics.

Bird Tissues from Museum Collections Are Reliable for Assessing Avian Haemosporidian Diversity.

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.

Within-Lineage Divergence of Avian Haemosporidians: A Case Study to Reveal the Origin of a Widespread Haemoproteus Parasite.

Avian haemosporidian parasites are particularly diverse and widespread. To date, more than 3,000 distinct cytochrome b lineages have been recorded, of which some present extremely wide geographical distributions, even including multiple continents. Whether these isolates represent one or several cryptic species remains unknown. Here we carried out a case study of SISKIN1, a common haemosporidian parasite lineage belonging to the morphologically described species Haemoproteus tartakovskyi. To shed light on its evolutionary origin, we investigated the divergence between SISKIN1 isolates obtained from siskins and redpolls in Europe (Russia and Sweden) and house finches in North America (Mexico). First, we used sequence capture of a small data set (2 Russian isolates and 1 Mexican isolate) to investigate the genetic structure based on the full-length mitochondrial genome and ∼1,000 genes. The mitochondrial genomes of Russian isolates were identical with each other but differed from the Mexican one at 6 positions. The nuclear divergence between Russian and Mexican isolates was on average 2.8%, close to what has been observed between 2 species of malaria parasites that respectively infect humans (Plasmodium falciparum) and gorillas (Plasmodium praefalciparum). Second, we used the expanded data set (15 samples in total) to investigate the genetic structure in 3 genes known to be involved in host invasion. The European isolates were identical across all sequenced genes, whereas the Mexican isolates were highly diverse. The lack of shared alleles between European and Mexican populations suggests that they might have diverged in isolation without gene flow. From the MalAvi database we examined the lineages most similar to the SISKIN1 barcode fragment (part of the cyt b gene) and found that most of them had been recorded in North and South America. This suggests that the lineage SISKIN1 originated in North America and subsequently spread to Europe. Our analyses support that the cyt b gene barcoding region is a useful marker for identification of avian haemosporidian lineages that can classify them into clusters of closely related parasites, but to further investigate species limits and evolutionary history, molecular data from multiple faster-evolving genes are required.

Avian haemosporidians in the cattle egret (Bubulcus ibis) from central-western and southern Africa: High diversity and prevalence.

We described the geographic distribution of 82 haemosporidian lineages (Plasmodium, Haemoproteus, and Leucocytozoon) in the cattle egret sampled in five countries in central-western and southern Africa. Seventy-three lineages have not previously been reported. We determined the prevalence of three haemosporidians in the samples. We investigated the influence of the internal environment of the host and environmental variables on the Plasmodium diversity and whether environmental variables may explain spatial variations in the prevalence of Plasmodium. We screened DNA from 509 blood samples from nestlings in 15 African colonies for infection by sequencing the cytochrome b gene of parasites. The molecular phylogenetic analysis was performed using Bayesian methods and including sequences from the MalAvi and GeneBank databases. We found 62 new Plasmodium lineages in a clade with MYCAME02, which is a lineage described in waterbirds and recently identified in birds of prey as Plasmodium paranucleophilum. Two Haemoproteus lineages identified in cattle egret formed a distinct group with Haemoproteus catharti and MYCAMH1 (Haemoproteus spp.). Seven Leucocytozoon lineages found in the cattle egret clustered with Leucocytozoon californicus. We found different Plasmodium diversities among the colonies sampled, demonstrating that the internal environment of the host is not the primary determinant of diversity. A linear mixed-effects multivariate model showed that precipitation was positively associated with Plasmodium diversity when controlling for the effects of temperature, colony composition (mixed and non-mixed species) and country. Moreover, a generalized mixed model showed that temperature was positively associated with the prevalence of Plasmodium when controlling for precipitation, elevation and country. We conclude that the cattle egret is a good model for future haemosporidian studies, as we found a significant number of new lineages in this host, which occupies regions with different climate characteristics where environmental variables exert an influence on the diversity and prevalence of Plasmodium.

Polymorphism evidence in Plasmodium (Haemamoeba) lutzi Lucena, 1939 (Apicomplexa, Haemosporida) isolated from Brazilian wild birds.

Plasmodium parasites can infect great variety of bird species around the world inflicting the so called avian malaria, an illness that could be fatal in some cases and consequently, should be monitored and widely included into conservation programs. The aim of this study was to characterize two lineages of Plasmodium (Haemamoeba) lutzi found in some birds in the Atlantic Forest of Minas Gerais - Brazil, that were morphologically identified after blood smears analyses under light microscopy and molecularly by sequencing the mitochondrial cytochrome b gene (cyt b). Besides these two lineages could be clearly morphologically identified as P.(H.) lutzi, some variations in comparison with its original description were noticed: absence of meronts and gametocytes (early and fully grown) in polychromatic erythrocytes, the larger size of pigment granules in meronts and gametocytes, and the presence of small vacuoles between pigment accumulation in fully grow macrogametocytes. Moreover, a certain degree of genetic intraspecific diversity was also observed across the lineages of P. (H.) lutzi, indicating the existence of polymorphisms within this taxon, which is uncommon in Haemosporida. These results allow discussion about species boundaries within avian hemosporidians and highlight the importance of multidisciplinary approaches for a more efficient species identification and characterization.

Haemoproteus minutus is highly virulent for Australasian and South American parrots.

BACKGROUND: Haemoproteus and Plasmodium species are widespread avian blood parasites. Several Plasmodium species are known for their high virulence and have caused significant declines in naïve bird populations. The impact of closely related Haemoproteus parasites is largely unknown. Recently we reported a lethal disease in two parrot aviaries caused by Haemoproteus parasites. RESULTS: Here we show that the causative pathogen Haemoproteus minutus is responsible for further 17 lethal outbreaks in parrot aviaries in Denmark, Germany and Great Britain. All affected parrots are endemic to Australasia and South America. We sequenced the cytochrome b gene from megalomeront-infected muscle tissue of 21 parrots and identified the two lineages TUPHI01 and TURDUS2 as causative agents, commonly naturally infecting the common blackbird (Turdus merula) and the song thrush (Turdus philomelos), respectively, in the Palaearctic. No intraerythrocytic parasite stages were found in any of the parrots. We failed to detect H. minutus in invasive Indian ring-necked parakeets (Psittacula krameri) in Germany. Together this suggests that abortive infections with two virulent lineages of H. minutus are lethal for naïve parrot species from Australasia and South America. We asked whether we could detect H. minutus in New Zealand, where its Turdus hosts were introduced in the 1800s. We therefore tested invasive blackbirds and song thrushes, and the co-existing endemic red-fronted parakeet (Cyanoramphus novaezelandiae) population on three New Zealand islands. No Haemoproteus spp. DNA was detected in all blood samples, indicating absence of transmission. CONCLUSIONS: The results of this study show that captive parrots in Europe are threatened by two lineages of an otherwise benign parasite of Turdus spp. Aviary collections of parrots should be protected from Culicoides spp. vectors in Europe. Animal trade and climate changes extending the current vector and parasite distribution have to be considered as potential risk factors for the introduction of the disease in naïve parrot populations.

Spatial distribution, prevalence and diversity of haemosporidians in the rufous-collared sparrow, Zonotrichia capensis.

BACKGROUND: Parasite prevalence and diversity are determined by the distribution of hosts and vectors and by the interplay among a suite of environmental factors. Distributions of parasite lineages vary based on host susceptibility and geographical barriers. Hemoparasites of the genera Haemoproteus and Plasmodium have wide distributions, and high prevalence and genetic diversity within perching birds (Order Passeriformes). The rufous-collared sparrow (Zonotrichia capensis) is widely distributed in Central and South America across an immense diversity of environments from sea level to more than 4000 meters above sea level. It therefore provides an excellent model to investigate whether altitudinal and latitudinal gradients influence the distribution, prevalence and diversity of haemosporidian parasites, their population structure and the biogeographical boundaries of distinct parasite lineages. RESULTS: We assembled samples from 1317 rufous-collared sparrows spanning 75 locales from across Central and South America (between 9.5°N and 54°S; 10-4655 meters above sea level). We used DNA sequence data from a fragment of the mitochondrial cytochrome b gene (cytb) of Haemoproteus and Plasmodium from 325 positive samples and found prevalences of 22 and 3%, respectively. Haemoproteus exhibited a higher prevalence than Plasmodium but with comparatively lower genetic diversity. We detected a relationship of Plasmodium and Haemoproteus prevalence with altitude and latitude; however, altitude and latitude did not influence parasite diversity. CONCLUSIONS: Parasite lineages showed a phylogeographical boundary coincident with the Andes Mountains, although we also observed a north-south disjunction in Peru for Haemoproteus. Haemosporidian distribution was not homogeneous but differed based on latitude and altitude. This is most probably due to environmental factors that have influenced both vector distribution and abundance, as well as parasite development. Our study provides key insights on the distribution of haemoparasite lineages and parasite dynamics within hosts.

Genomic sequence capture of haemosporidian parasites: Methods and prospects for enhanced study of host-parasite evolution.

Avian malaria and related haemosporidians (Plasmodium, [Para]Haemoproteus and Leucocytoozoon) represent an exciting multihost, multiparasite system in ecology and evolution. Global research in this field accelerated after the publication in 2000 of PCR protocols to sequence a haemosporidian mitochondrial (mtDNA) barcode and the development in 2009 of an open-access database to document the geographic and host ranges of parasite mtDNA haplotypes. Isolating haemosporidian nuclear DNA from bird hosts, however, has been technically challenging, slowing the transition to genomic-scale sequencing techniques. We extend a recently developed sequence capture method to obtain hundreds of haemosporidian nuclear loci from wild bird samples, which typically have low levels of infection, or parasitemia. We tested 51 infected birds from Peru and New Mexico and evaluated locus recovery in light of variation in parasitemia, divergence from reference sequences and pooling strategies. Our method was successful for samples with parasitemia as low as ~0.02% (2 of 10,000 blood cells infected) and mtDNA divergence as high as 15.9% (one Leucocytozoonsample), and using the most cost-effective pooling strategy tested. Phylogenetic relationships estimated with >300 nuclear loci were well resolved, providing substantial improvement over the mtDNA barcode. We provide protocols for sample preparation and sequence capture including custom probe sequences and describe our bioinformatics pipeline using atram 2.0, phyluce and custom Perl/Python scripts. This approach can be applied to thousands of avian samples that have already been found to have haemosporidian infections of at least moderate intensity, greatly improving our understanding of parasite speciation, biogeography and evolutionary dynamics.

Low occurrence of hemosporidian parasites in the Neotropic cormorant (Phalacrocorax brasilianus) in Chile.

Hemosporidian parasites rarely infect aquatic birds. Few studies have been conducted in South America identifying some lineages of the genera Plasmodium, Leucocytozoon, and Haemoproteus, but none has been done in the Neotropic cormorant (Phalacrocorax brasilianus). This species is widely distributed through the American continent, from Southern USA to Tierra del Fuego, using a wide range of aquatic habitats. We molecularly studied the occurrence and diversity of hemosporidian lineages infecting individuals of Neotropic cormorant across a broad latitudinal gradient in Chile (Arica to Tierra del Fuego). As expected, a very low occurrence of individuals infected by Plasmodium sp. (4/123, 3.3%) and Leucocytozoon sp. (2/123, 1.6%) was detected. We found no evidence of Haemoproteus sp. We identified one lineage of Plasmodium (ZEMAC01) and one new lineage of Leucocytozoon (PHABRA01) infecting cormorants. Individuals infected by Plasmodium sp. were birds from only one site (i.e., Chillán), whereas Leucocytozoon sp. was found in one bird from Valdivia and another one from Tierra del Fuego. Our results expand the known range of hemosporidian parasite lineages in aquatic birds providing an essential baseline data that contribute to a better understanding of the geographic range of hemosporidian parasites infecting Phalacrocoracidae in South America.

First Record of Leucocytozoon (Haemosporida: Leucocytozoidae) in Amazonia: Evidence for Rarity in Neotropical Lowlands or Lack of Sampling for This Parasite Genus?

Birds harbor an astonishing diversity of haemosporidian parasites belonging to the genera Haemoproteus, Leucocytozoon, and Plasmodium. Currently there are more than 250 morphologically described avian haemosporidian species and 2,828 unique lineages found in virtually all avian clades and zoogeographic regions, except for Antarctica. Our report is based on PCR and microscopic screening of 1,302 individual avian samples from Brazil to detect the underrepresented genus Leucocytozoon. This survey primarily focuses on passerine birds collected from Amazonia, the Atlantic Rain Forest, and Pantanal. We also summarize studies conducted in Brazil that report haemosporidian prevalence using both microscopy and molecular tools and present for the first time a record of Leucocytozoon infecting an avian host population in Amazonia. Based on our findings, we suggest that high average temperatures may be constraining both the distribution and diversity of Leucocytozoon in lowland tropical South America.

The malaria parasite Plasmodium relictum in the endemic avifauna of eastern Cuba.

Island populations are vulnerable to introduced pathogens, as evidenced by extinction or population decline of several endemic Hawaiian birds caused by the malaria parasite, Plasmodium relictum (order Haemosporida). We analyzed blood samples from 363 birds caught near Guantánamo Bay, Cuba, for the presence of haemosporidian infections. We characterized parasite lineages by determining nucleotide variation of the parasite's mitochondrial cyt b gene. Fifty-nine individuals were infected, and we identified 7 lineages of haemosporidian parasites. Fifty individuals were infected by 6 Haemoproteus sp. lineages, including a newly characterized lineage of Haem. (Parahaemoproteus) sp. CUH01. Nine individuals carried the P. relictum lineage GRW4, including 5 endemic Cuban Grassquits (Tiaris canorus) and 1 migratory Cape May Warbler (Setophaga tigrina). A sequence of the merozoite surface protein gene from one Cuban Grassquit infected with GRW4 matched that of the Hawaiian haplotype Pr9. Our results indicate that resident and migratory Cuban birds are infected with a malaria lineage that has severely affected populations of several endemic Hawaiian birds. We suggest GRW4 may be associated with the lack of several bird species on Cuba that are ubiquitous elsewhere in the West Indies. From the standpoint of avian conservation in the Caribbean Basin, it will be important to determine the distribution of haemosporidian parasites, especially P. relictum GRW4, in Cuba as well as the pathogenicity of this lineage in species that occur and are absent from Cuba.