Haemosporidian blood parasites in European birds of prey and owls

Avian blood parasites have been intensively studied using morphological methods with limited information on their host specificity and species taxonomic status. Now the analysis of gene sequences, especially the mitochondrial cytochrome b gene of the avian haemosporidian species of Haemoproteus, Plasmodium, and Leucocytozoon, offers a new tool to review the parasite specificity and status. By comparing morphological and genetic techniques, we observed nearly the same overall prevalence of haemosporidian parasites by microscopy (19.8%) and polymerase chain reaction (PCR) (21.8%) analyses. However, in contrast to the single valid Leucocytozoon species (L. toddi) in the Falconiformes we detected 4 clearly distinctive strains by PCR screening. In the Strigiformes, where the only valid Leucocytozoon species is L. danilewskyi, we detected 3 genetically different strains of Leucocytozoon spp. Two strains of Haemoproteus spp. were detected in the birds of prey and owls examined, whereas the strain found in the tawny owl belonged to the morphospecies Haemoproteus noctuae. Three Plasmodium spp. strains that had already been found in Passeriformes were also detected in the birds of prey and owls examined here, supporting previous findings indicating a broad and nonspecific host spectrum bridging different bird orders.     

Characterization of Sarcocystis from four species of hawks from Georgia, USA

During 2001 to 2004, 4 species of hawks (Buteo and Accipiter spp.) from Georgia were surveyed for Sarcocystis spp. infections by examining intestinal sections. In total, 159 of 238 (66.8%) hawks examined were infected with Sarcocystis spp. Samples from 10 birds were characterized by sequence analysis of a portion of the 18S rRNA gene (783 base pairs). Only 3 of the 10 sequences from the hawks were identical; the remainder differed by at least 1 nucleotide. Phylogenetic analysis failed to resolve the position of the hawk Sarcocystis species, but they were closely related several Sarcocystis species from raptors, rodents, and Sarcocystis neurona. The high genetic diversity of Sarcocystis suggests that more than 1 species infects these 4 hawk species; however, additional molecular or experimental work will be required to determine the speciation and diversity of parasites infecting these avian hosts. In addition to assisting with determining species richness of Sarcocystis in raptors, molecular analysis should be useful in the identification of potential intermediate hosts.     

Hematozoa of raptors from southern New Jersey and adjacent areas

Blood smears from 259 birds of 12 species, representing four families of raptors, from New Jersey, Pennsylvania, Delaware, and Virginia were examined for blood parasites. Infected birds constituted 59.1% of the total. Birds were infected with one or more of the following genera of protozoa: Leucocytozoon (43.2%); Haemoproteus (21.6%); Plasmodium (1.2%); and Trypanosoma (1.2%). Blood culture of 142 raptors of 11 species for Trypanosoma revealed a prevalence of 41.5%. Plasmodium circumflexum is reported for the first time in Accipiter striatus, and Trypanosoma sp. in Buteo jamaicensis.     

Avian hemosporidian parasites from northern California oak woodland and chaparral habitats

During spring-summer 2003-2004, the avian community was surveyed for hemosporidian parasites in an oak (Quercus spp.) and madrone (Arbutus spp.) woodland bordering grassland and chaparral habitats at a site in northern California, a geographic location and in habitat types not previously sampled for these parasites. Of 324 birds from 46 species (21 families) sampled (including four species not previously examined for hemosporidians), 126 (39%) were infected with parasites identified as species of one or more of the genera Plasmodium (3% of birds sampled), Haemoproteus (30%), and Leucocytozoon (11%). Species of parasite were identified by morphology in stained blood smears and were consistent with one species of Plasmodium, 11 species of Haemoproteus, and four species of Leucocytozoon. We document the presence of one of the parasite genera in seven new host species and discovered 12 new parasite species-host species associations. Hatching-year birds were found infected with parasites of all three genera. Prevalence of parasites for each genus differed significantly for the entire sample, and prevalence of parasites for the most common genus, Haemoproteus, differed significantly among bird families. Among families with substantial sample sizes, the Vireonidae (63%) and Emberizidae (70%) were most often infected with Haemoproteus spp. No evidence for parasite between-genus interaction, either positive or negative, was found. Overall prevalence of hemosporidians at the northern California sites and predominance of Haemoproteus spp. was similar to that reported in most other surveys for the USA, Canada, and the Caribbean islands.     

Leucocytozoon toddi and Haemoproteus tinnunculi (Protozoa: Haemosporina) in the Chimango caracara (Milvago chimango) in southern Chile.

Two species of blood protozoans were identified from blood smears collected from 15 specimens of the Chimango caracara (Milvago chimango) on Isla Grande de Chiloé in southern Chile. These included Leucocytozoon toddi in 13 birds, including all 5 of the 4-6 week old nestlings examined, and 8 of the subadults or adults. One of the nestlings also had a dual infection of L. toddi and Haemoproteus tinnunculi. These are the first reports of blood parasites from M. chimango.     

A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood

Many bird species host several lineages of apicomplexan blood parasites (Protista spp., Haemosporida spp.), some of which are shared across different host species. To understand such complex systems, it is essential to consider the fact that different lineages, species, and families of parasites can occur in the same population, as well as in the same individual bird, and that these parasites may compete or interact with each other. In this study, we present a new polymerase chain reaction (PCR) protocol that, for the first time, enables simultaneous typing of species from the 3 most common avian blood parasite genera (Haemoproteus, Plasmodium, and Leucocytozoon). By combining the high detection rate of a nested PCR with another PCR step to separate species of Plasmodium and Haemoproteus from Leucocytozoon, this procedure provides an easy, rapid, and accurate method to separate and investigate these parasites within a blood sample. We have applied this method to bird species with known infections of Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp. To obtain a higher number of parasite lineages and to test the repeatability of the method, we also applied it to blood samples from bluethroats (Luscinia svecica), for which we had no prior knowledge regarding the blood parasite infections. Although only a small number of different bird species were investigated (6 passerine species), we found 22 different parasite species lineages (4 Haemoproteus, 8 Plasmodium, and 10 Leucocytozoon).     

Respiratory nematodiases in raptors in Quebec

This is a retrospective study on wild raptors submitted to the Université de Montréal (Quebec, Canada) from 1989 to 1996. Cyathostoma spp. (Nematoda: Syngamidae) adults and/or eggs were found in air sacs, lungs, bronchi, and trachea of 12 raptors (Falconiformes and Strigiformes) from Quebec, Canada, belonging to eight different species, five of which are first host records for this parasite: barred owl (Strix varia), snowy owl (Nyctea scandiaca), northern harrier (Circus cyaneus), northern goshawk (Accipiter gentilis), and broad-winged hawk (Buteo platypterus). The infection was considered fatal in four birds, while no significant clinical signs were observed in the other cases. Major pathologic changes included diffuse pyogranulomatous air sacculitis, pneumonia, and bronchitis. A few unidentified larval nematodes embedded in a granuloma were found in the lungs of an additional Coopers' hawk (Accipiter cooperii); they were not considered clinically significant. A dead nematode, surrounded by necrotic inflammatory cells, was found in the air sac of a northern goshawk. The presence of nematodes in air sacs or lungs should be considered in wild raptors demonstrating respiratory problems.     

Prevalence of avian haemosporidian parasites and their host fidelity in the central Philippine islands

We examined the prevalence and host fidelity of avian haemosporidian parasites belonging to the genera Haemoproteus, Leucocytozoon and Plasmodium in the central Philippine islands by sampling 23 bird families (42 species). Using species-specific PCR assays of the mitochondrial cytochrome b gene (471base pairs, bp), we detected infections in 91 of the 215 screened individuals (42%). We also discriminated between single and multiple infections. Thirty-one infected individuals harbored a single Haemoproteus lineage (14%), 18 a single Leucocytozoon lineage (8%) and 12 a single Plasmodium lineage (6%). Of the 215 screened birds, 30 (14%) presented different types of multiple infections. Intrageneric mixed infections were generally more common (18 Haemoproteus/Haemoproteus, 3 Leucocytozoon/Leucocytozoon, and 1 Plasmodium/Plasmodium) than intergeneric mixed infections (7 Haemoproteus/Leucocytozoon and 1 Haemoproteus/Leucocytozoon/Plasmodium). We recovered 81 unique haemosporidian mitochondrial haplotypes. These clustered in three strongly supported monophyletic clades that correspond to the three haemosporidian genera. Related lineages of Haemoproteus and Leucocytozoon were more likely to derive from the same host family than predicted by chance; however, this was not the case for Plasmodium. These results indicate that switches between host families are more likely to occur in Plasmodium. We conclude that Haemoproteus has undergone a recent diversification across well-supported host-family specific clades, while Leucocytozoon shows a longer association with its host(s). This study supports previous evidence of a higher prevalence and stronger host-family specificity of Haemoproteus and Leucocytozoon compared to Plasmodium.     

Helminth community structure in birds of prey (Accipitriformes and Falconiformes) in southern Italy

We compared helminth communities in 6 species of birds of prey from the Calabria region of southern Italy. In total, 31 helminth taxa, including 17 nematodes, 9 digeneans, 3 acanthocephalans, and 2 cestodes, were found. All helminth species were observed in the gastrointestinal tract, except for 3 spirurid nematodes. Most of the parasite species were detected in at least 2 hosts, but 13 helminth species were found in only 1 host. At the infracommunity level, the overall species richness and Brillouin's index of diversity varied by host, with the highest values in a generalist feeder, the Eurasian buzzard (Buteo buteo), and the lowest in a specialist, the western honey buzzard (Pernis apivorus). Species richness was gender dependent only in the sparrow hawk (Accipiter nisus). The helminth communities were characterized by different dominant species, namely, Centrorhynchus spp. (Acanthocephala) in the Eurasian buzzard and common kestrel (Falco tinnunculus), Parastrigea intermedia (Digenea) in the marsh harrier (Circus aeruginosus), Physaloptera alata (Nematoda) in the sparrow hawk, Serratospiculum tendo (Nematoda) in the peregrine falcon (Falco peregrinus), and Strigea falconis (Digenea) in the western honey buzzard. Statistical analyses confirmed a highly significant difference of helminth infracommunity structure among host species. We conclude that in the Calabria region of southern Italy, each of the raptor species studied is distinct in terms of its helminth communities, and more diverse feeding habits of the host correspond with richer helminth communities.     

Prevalence and diversity patterns of avian blood parasites in degraded African rainforest habitats

Land use changes including deforestation, road construction and agricultural encroachments have been linked to the increased prevalence of several infectious diseases. In order to better understand how deforestation affects the prevalence of vector-borne infectious diseases in wildlife, nine paired sites were sampled (disturbed vs. undisturbed habitats) in Southern Cameroon. We studied the diversity, prevalence and distribution of avian malaria parasites ( Plasmodium spp.) and other related haemosporidians (species of Haemoproteus and Leucocytozoon ) from these sites in two widespread species of African rainforest birds, the yellow-whiskered greenbul ( Andropadus latirostris , Pycnonotidae) and the olive sunbird ( Cyanomitra olivacea , Nectariniidae). Twenty-six mitochondrial cytochrome b lineages were identified: 20 Plasmodium lineages and 6 Haemoproteus lineages. These lineages showed no geographic specificity, nor significant differences in lineage diversity between habitat types. However, we found that the prevalence of Leucocytozoon and Haemoproteus infections were significantly higher in undisturbed than in deforested habitats ( Leucocytozoon spp. 50.3% vs. 35.8%, Haemoproteus spp. 16.3% vs. 10.8%). We also found higher prevalence for all haemosporidian parasites in C. olivacea than in A. latirostris species (70.2% vs. 58.2%). Interestingly, we found one morphospecies of Plasmodium in C. olivacea , as represented by a clade of related lineages, showed increased prevalence at disturbed sites, while another showed a decrease, testifying to different patterns of transmission, even among closely related lineages of avian malaria, in relation to deforestation. Our work demonstrates that anthropogenic habitat change can affect host–parasite systems and result in opposing trends in prevalence of haemosporidian parasites in wild bird populations.     

Leucocytozoonosis in nestling bald eagles in Michigan and Minnesota.

Thirteen of 21 nestling bald eagles (Haliaeetus leucocephalus) examined for blood parasites in Michigan and Minnesota (USA) during June and July 1997 had patent infections of Leucocytozoon toddi. No other parasites were seen. The degree of parasitemia was light and varied from 1 to 2 on the Ashford Scale. Several of the infected nestlings appeared to have elevated levels of heterophils in their peripheral circulating blood. One of the infected nestlings also showed signs of severe anemia. We believe this is the first report of L. toddi in the bald eagle.     

Health of an ex situ population of raptors (Falconiformes and Strigiformes) in Mexico: diagnosis of internal parasites

Successful programs for ex situ and in situ conservation and management of raptors require detailed knowledge about their pathogens. The purpose of this study was to identify the internal parasites of some captive raptors in Mexico, as well as to verify their impact in the health status of infected birds. Birds of prey were confiscated and kept in captivity at the Centro de Investigación y Conservación de Vida Silvestre (CIVS) in Los Reyes La Paz, Mexico State. For this, fecal and blood samples from 74 birds of prey (66 Falconiformes and eight Strigiformes) of 15 species, juveniles and adults from both sexes (39 males and 35 female), were examined for the presence of gastrointestinal and blood parasites. Besides, the oropharyngeal cavity was macroscopically examined for the presence of lesions compatible with trichomoniasis. Among our results we found that lesions compatible with Trichomonas gallinae infection were detected only in two Red-tailed hawks (Buteo jamaicensis) (2.7%); nevertheless, infected birds were in good physical condition. Overall, gastrointestinal parasites were found in 10 (13.5%) raptors: nine falconiforms (13.6%) and one strigiform (12.5%), which mainly presented a single type of gastrointestinal parasite (90%). Eimeria spp. was detected in Harris's hawk (Parabuteo unicinctus), Swainson's hawk (Buteo swainsoni), Red-tailed hawk (B. jamaicensis) and Great horned owl (Bubo virginianus); whereas trematodes eggs were found in Peregrine falcon (Falco peregrinus) and Swainson's hawk (B. swainsoni). Furthermore, eggs of Capillaria spp. were found in one Swainson's hawk (B. swainsoni), which was also infected by trematodes. Hemoprotozoarian were detected in five (6.7%) falconiforms: Haemoproteus spp. in American kestrel (F. sparverius) and Leucocytozoon spp. in Red-tailed hawk (B. jamaicencis). Despite this, no clinical signs referable to gastrointestinal or blood parasite infection were observed in any birds. All parasites identified were recorded for the first time in raptors from Mexico. Furthermore, this represents the first report for T. gallinae, trematodes, Haemoproteus spp. and Leucocytozoon spp. in raptors from Latin America. Diagnosis and control of parasitic infections should be a part of the routine in health care evaluations for ex situ raptor populations. Finally, this information is also valuable for in situ conservation actions on these birds.     

Low haemosporidian diversity and one key-host species in a bird malaria community on a mid-Atlantic island (São Miguel, Azores)

When host species colonize new areas, the parasite assemblage infecting the hosts might change, with some parasite species being lost and others newly acquired. These changes would likely lead to novel selective forces on both host and its parasites. We investigated the avian blood parasites in the passerine bird community on the mid-Atlantic island of S?o Miguel, Azores, a bird community originating from continental Europe. The presence of haemosporidian blood parasites belonging to the genera Haemoproteus, Plasmodium, and Leucocytozoon was assessed using polymerase chain reaction. We found two Plasmodium lineages and two Leucocytozoon lineages in 11 bird species (84% of all breeding passerine species) on the island. These lineages were unevenly distributed across bird species. The Eurasian Blackbird (Turdus merula) was the key-host species (total parasite prevalence of 57%), harboring the main proportion of parasite infections. Except for Eurasian Blackbirds, all bird species had significantly lower prevalence and parasite diversity compared to their continental populations. We propose that in evolutionary novel bird communities, single species may act as key hosts by harboring the main part of the parasite fauna from which parasites "leak" into the other species. This would create very different host-parasite associations in areas recently colonized by hosts as compared to in their source populations.     

Hematozoa from Southern African vultures, with a description of Haemoproteus janovyi sp. n.

This study represents the first major survey of avian hematozoa from southern Africa and the only one dealing with blood parasites of vultures. Blood smears from 506 Rhodesian, Botswanan and South African vultures (Hooded, White-headed, Lappetfaced, Cape Griffon and Whitebacked Vultures) were examined for hematozoa. Haemoproteus janovyi sp. n. was observed in 35.2% of the vultures, Leucocytozoon toddi in 0.8%, Plasmodium fallax in 0.6%, Atoxoplasma sp. in 1.4% and microfilariae in 0.2%. Hematozoan prevalence increased with age of the vultures. Only 2 of 133 nestlings sampled during the dry season had patent parasitemias (L. toddi). Haemoproteid prevalence in immature vultures was depressed during the dry season, whereas it was stable throughout the year in adults. The only species which nests on cliffs (the Cape Griffon Vulture) did not harbor hematozoa whereas the other species which nest and roost in trees were infected with at least one hematozoan species.     

Blood parasites in owls with conservation implications for the Spotted Owl (Strix occidentalis)

The three subspecies of Spotted Owl (Northern, Strix occidentalis caurina; California, S. o. occidentalis; and Mexican, S. o. lucida) are all threatened by habitat loss and range expansion of the Barred Owl (S. varia). An unaddressed threat is whether Barred Owls could be a source of novel strains of disease such as avian malaria (Plasmodium spp.) or other blood parasites potentially harmful for Spotted Owls. Although Barred Owls commonly harbor Plasmodium infections, these parasites have not been documented in the Spotted Owl. We screened 111 Spotted Owls, 44 Barred Owls, and 387 owls of nine other species for haemosporidian parasites (Leucocytozoon, Plasmodium, and Haemoproteus spp.). California Spotted Owls had the greatest number of simultaneous multi-species infections (44%). Additionally, sequencing results revealed that the Northern and California Spotted Owl subspecies together had the highest number of Leucocytozoon parasite lineages (n = 17) and unique lineages (n = 12). This high level of sequence diversity is significant because only one Leucocytozoon species (L. danilewskyi) has been accepted as valid among all owls, suggesting that L. danilewskyi is a cryptic species. Furthermore, a Plasmodium parasite was documented in a Northern Spotted Owl for the first time. West Coast Barred Owls had a lower prevalence of infection (15%) when compared to sympatric Spotted Owls (S. o. caurina 52%, S. o. occidentalis 79%) and Barred Owls from the historic range (61%). Consequently, Barred Owls on the West Coast may have a competitive advantage over the potentially immune compromised Spotted Owls.     

Phylogeographic structuring of Plasmodium lineages across the North American range of the house finch (Carpodacus Mexicanus)

The determinants of the geographic distribution of avian hematozoa are poorly understood. Sampling parasites from one avian host species across a wide geographic range is an accepted approach to separate the potential influence of host species distribution from geographic effects not directly related to host species biology. We used polymerase chain reaction to screen samples for hematozoan infection from 490 house finches (Carpodacus mexicanus) collected at 8 sites spanning continental North America. To explore geographic patterns of parasite lineage distributions, we sequenced a portion of the mitochondrial cytochrome b gene of Plasmodium species infecting 77 house finches. We identified 5 distinct Plasmodium haplotypes representing 3 lineages that likely represent 3 species. One lineage was common at all sites where we detected Plasmodium species. The second lineage contained 3 haplotypes that showed phylogeographic structuring on a continent-wide scale, with 1 haplotype common in eastern North America and 2 common in western North America. The third divergent lineage was recovered from 1 individual host. Considered together, the partial phylogeographic structuring of Plasmodium cytochrome b lineages over the range of the house finch suggests that parasite lineage distribution is not solely dependent on host species distribution, and other factors such as arthropod vector competence and distribution may be important.     

The sensitivity of microscopy and PCR-based detection methods affecting estimates of prevalence of blood parasites in birds

Inferences about the evolution of host-parasitic relationships are often made based on the prevalence of avian malaria, which is usually estimated in a large sample of birds using either microscopic or molecular screening of blood samples. However, different techniques often have variable accuracy; thus, screening methodology can raise issues about statistical bias if method sensitivity varies systematically across parasites or hosts. To examine this possibility, published information was collected on the prevalence of species in 4 genera of avian blood parasites ( Plasmodium, Haemoproteus, Leucocytozoon, and Trypanosoma) from various sources that used different tools. The data were tested to determine if the application of different methods provided different estimates for the same hosts. In these comparisons between the main methodologies, the PCR-based molecular methods were generally found to provide higher estimates for Plasmodium spp. prevalence than microscopic tools, while there was no significant tendency for such a trend in species of Haemoproteus and Leucocytozoon. When analyzing intraspecific variance of prevalence within molecular studies, some studies provided consistently higher estimates for Haemoproteus spp. prevalence than others, indicating that differences between studies can affect detected estimates. Within microscopic studies, surveys that examined more microscopic fields were more likely to report higher prevalence for Plasmodium spp. than those relying on fewer microscopic fields. Consequently, studies making comparisons across parasite genera and/or host species from different sources need to consider several types of bias originating from variation in method sensitivity.     

A cautionary note on the use of nested PCR for parasite screening--an example from avian blood parasites

The use of new powerful nested polymerase chain reaction (PCR) techniques to identify and screen for prevalence of parasites has a huge potential. It allows for the detection and identification of low-intensity infections, but its high sensitivity and technical setup may also induce problems. Here, we report a cautionary note regarding misleading amplification of avian malaria species (Haemoproteus and Plasmodium) during Leucocytozoon spp. detection. We used a previously described nested PCR method for the molecular detection of avian malaria and Leucocytozoon spp. In the first step of the PCR protocol, these parasites are detected simultaneously; in the second PCR, Haemoproteus and Plasmodium spp. are separated from Leucocytozoon spp. However, in certain cases when a bird was infected with avian malaria, we obtained a slightly longer PCR product during the detection of Leucocytozoon spp. Our data imply that these "false" Leucocytozoon fragments are the consequences of strong amplification of certain malaria lineages in the first PCR, which can also be detected after the second PCR amplification that is specific to Leucocytozoon spp. parasites. Because these "false" Leucocytozoon fragments are slightly longer than the normal Leucocytozoon fragments, we suggest the use of well-separating agarose gels, several positive controls, and molecular standards to facilitate their separation. If one obtains a fragment that differs in length from the one expected for Leucocytozoon spp., sequencing is essential. More generally, in order to limit this type of problem with nested PCR protocols, we suggest that the first and the second primer pair be chosen so that they have different annealing temperatures.     

Host phylogeography and beta diversity in avian haemosporidian (Plasmodiidae) assemblages of the Lesser Antilles

1. We estimated the correlation between host phylogeographical structure and beta diversity of avian haemosporidian assemblages of passerine birds to determine the degree to which parasite communities change with host evolution, expressed as genetic divergence between island populations, and we investigated whether differences among islands in the haemosporidia of a particular host species reflect beta diversity in the entire parasite assemblage, beta diversity in vectors, turnover of bird species and/or geographical distance. 2. We used Mantel tests to assess the significance of partial correlations between host nucleotide difference (based on cytochrome b) and haemosporidian (Haemoproteus spp. and Plasmodium spp.) mitochondrial lineage beta diversity within a given host species and between Plasmodium mitochondrial lineage beta diversity and mosquito and bird species beta diversity (or turnover). Three abundant and widespread host species (Tiaris bicolor, Coereba flaveola and Loxigilla noctis/barbadensis) were included in the study. Haemosporidian lineage beta diversity among nine islands was assessed using the Chao-Jaccard, Chao-S?rensen and Morisita-Horn indices of community similarity. Beta diversity indices of mosquito species and turnover of bird species were calculated from data in published records and field guides. 3. In Loxigilla spp., we found a positive correlation with geographical distance and an unexpected negative correlation between haemosporidian beta diversity and host genetic distance. Tiaris bicolor exhibited a significant positive correlation between haemosporidian beta diversity and beta diversity within the entire parasite assemblage. We did not find significant correlations between parasite beta diversity and mosquito beta diversity or bird species turnover. 4. Host phylogeographical structure does not appear to drive within-host beta diversity of haemosporidian lineages. Instead, the array of parasites on one host can reflect the haemosporidian assemblage on other hosts.