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BackgroundThe role of zoos in conservation programmes has increased significantly in last decades, and the health of captive animals is essential to guarantee success of such programmes. However, zoo birds suffer from parasitic infections, which often are caused by malaria parasites and related haemosporidians. Studies determining the occurrence and diversity of these parasites, aiming better understanding infection influence on fitness of captive birds, are limited.MethodsIn 2011–2015, the prevalence and diversity of Plasmodium spp. and Haemoproteus spp. was examined in blood samples of 677 captive birds from the São Paulo Zoo, the largest zoo in Latin America. Molecular and microscopic diagnostic methods were used in parallel to detect and identify these infections.ResultsThe overall prevalence of haemosporidians was 12.6%. Parasites were mostly detected by the molecular diagnosis, indicating that many birds harbour subclinical or abortive infections. In this project, birds of 17 orders (almost half of all the orders currently accepted in taxonomy of birds), 29 families, and 122 species, were tested, detecting positive individuals in 27% of bird species. Birds from the Anatidae were the most prevalently infected (64.7% of all infected animals). In all, infections with parasites of the genus Plasmodium (overall prevalence 97.6%) predominated when compared to those of the genus Haemoproteus (2.4%). In total, 14 cytochrome b (cytb) lineages of Plasmodium spp. and 2 cytb lineages of Haemoproteus spp. were recorded. Eight lineages were new. One of the reported lineages was broad generalist while others were reported in single or a few species of birds. Molecular characterization of Haemoproteus ortalidum was developed.ConclusionThis study shows that many species of birds are at risk in captivity. It is difficult to stop haemosporidian parasite transmission in zoos, but is possible to reduce the infection rate by treating the infected animals or/and while keeping them in facilities free from mosquitoes. Protocols of quarantine should be implemented whenever an animal is transferred between bird maintaining institutions. This is the first survey of haemosporidians in captive birds from different orders maintained in zoos. It is worth emphasizing the necessity of applying practices to control these parasites in management and husbandry of animals in captivity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1729-8) contains supplementary material, which is available to authorized users.

Numerous studies addressed the diversity of bird Plasmodium and Haemoproteus parasites. However, a few have been carried out in continental avian hotspot regions such as Brazil, a country with markedly different biomes, including Amazon, Brazilian Savanna, Atlantic Forest, Caatinga, Pantanal, and Pampas. We present the first study on hemosporidian (Haemosporida) parasites in free-living birds from an Atlantic Forest fragment where more than 80 avian species have been reported. Within this area, the São Paulo Zoo locates, and it is the fourth largest zoo in the world and the largest in Latin America. A total of 133 free-living bird samples representing 12 species were collected in the zoo, with the overall hemosporidian prevalence of 18 % by PCR-based diagnostics. Twenty-four positive PCR signals were reported from four different bird species, including migratory ones. Columba livia, an urban species, considered nowadays a pest in big cities, showed 100 % prevalence of Haemoproteus spp., mainly Haemoproteus columbae. We discuss the epidemiological importance of new parasites introduced by migratory birds in the São Paulo Zoo area and the risk it poses to the captive species, which are natives or exotics. We also warn about the influence these parasites can have on the biodiversity and the structure of host populations by altering the competitive interaction between the free-living and the captive birds.

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BackgroundHaemoproteus parasites (Haemosporida, Haemoproteidae) are cosmopolitan in birds and recent molecular studies indicate enormous genetic diversity of these pathogens, which cause diseases in non-adapted avian hosts. However, life-cycles remain unknown for the majority of Haemoproteus species. Information on their exoerythrocytic development is particularly fragmental and controversial. This study aimed to gain new knowledge on life-cycle of the widespread blood parasite Haemoproteus majoris.MethodsTurdus pilaris and Parus major naturally infected with lineages hPHYBOR04 and hPARUS1 of H. majoris, respectively, were wild-caught and the parasites were identified using microscopic examination of gametocytes and PCR-based testing. Bayesian phylogeny was used to determine relationships between H. majoris lineages. Exoerythrocytic stages (megalomeronts) were reported using histological examination and laser microdissection was applied to isolate single megalomeronts for genetic analysis. Culicoides impunctatus biting midges were experimentally exposed in order to follow sporogonic development of the lineage hPHYBOR04.ResultsGametocytes of the lineage hPHYBOR04 are indistinguishable from those of the widespread lineage hPARUS1 of H. majoris, indicating that both of these lineages belong to the H. majoris group. Phylogenetic analysis supported this conclusion. Sporogony of the lineage hPHYBOR04 was completed in C. impunctatus biting midges. Morphologically similar megalomeronts were reported in internal organs of both avian hosts. These were big roundish bodies (up to 360 μm in diameter) surrounded by a thick capsule-like wall and containing irregularly shaped cytomeres, in which numerous merozoites developed. DNA sequences obtained from single isolated megalomeronts confirmed the identification of H. majoris.ConclusionsPhylogenetic analysis identified a group of closely related H. majoris lineages, two of which are characterized not only by morphologically identical blood stages, but also complete sporogonic development in C. impunctatus and development of morphologically similar megalomeronts. It is probable that other lineages belonging to the same group would bear the same characters and phylogenies based on partial cytb gene could be used to predict life-cycle features in avian haemoproteids including vector identity and patterns of exoerythrocytic merogony. This study reports morphologically unique megalomeronts in naturally infected birds and calls for research on exoerythrocytic development of haemoproteids to better understand pathologies caused in avian hosts.

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BackgroundBlood parasites of the genus Haemoproteus Kruse, 1890 are cosmopolitan, might be responsible for mortality in non-adapted birds, and often kill blood-sucking insects. However, this group remains insufficiently investigated in the wild. This is particularly true for the parasites of leaf warblers of the Phylloscopidae Alström, Ericson, Olsson & Sundberg the common small Old World passerine birds whose haemoproteid parasite diversity and vectors remain poorly studied. This study reports a new species of Haemoproteus parasitizing leaf warblers, its susceptible vector and peculiar phylogenetic relationships with other haemoproteids.MethodsWood warblers (Phylloscopus sibilatrix Bechstein) were caught in Lithuania during spring migration, and blood films were examined microscopically. Laboratory reared Culicoides nubeculosus Meigen were exposed experimentally by allowing them to take blood meals on one individual harbouring mature gametocytes of the new Haemoproteus species (lineage hPHSIB2). To follow sporogonic development, the engorged insects were dissected at intervals. The parasite lineage was distinguished using sequence data, and morphological analysis of blood and sporogonic stages was carried out. Bayesian phylogeny was constructed in order to determine the phylogenetic relationships of the new parasite with other haemoproteids.ResultsHaemoproteus (Parahaemoproteus) homopalloris n. sp. was common in wood warblers sampled after arrival to Europe from their wintering grounds in Africa. The new parasite belongs to a group of avian haemoproteid species with macrogametocytes possessing pale staining cytoplasm. All species of this group clustered together in the phylogenetic analysis, indicating that intensity of the cytoplasm staining is a valuable phylogenetic character. Laboratory-reared biting midges C. nubeculosus readily supported sporogony of new infections. Phylogenetic analysis corroborated vector experiments, placing the new parasite in the clade of Haemoproteus (Parahaemoproteus) parasites transmitted by biting midges.ConclusionsHaemoproteus homopalloris n. sp. is the third haemoproteid, which is described from and is prevalent in wood warblers. Phylogenetic analysis identified a clade containing seven haemoproteids, which are characterised by pale staining of the macrogametocyte cytoplasm and with ookinetes maturing exceptionally rapidly (between 1 to 1.5 h after exposure to air). Both these features may represent valuable phylogenetic characters. Studies targeting mechanisms of sporogonic development of haemoproteids remain uncommon and should be encouraged. Culicoides nubeculosus is an excellent experimental vector of the new parasite species.

Background: Blood parasites belonging to the Apicomplexa, Trypanosomatidae and Filarioidea are widespread in birds and have been studied extensively. Microscopical examination (ME) of stained blood films remains the gold standard method for the detection of these infections in birds, particularly because co-infections predominate in wildlife. None of the available molecular tools can detect all co-infections at the same time, but ME provides opportunities for this to be achieved. However, fixation, drying and staining of blood films as well as their ME are relatively time-consuming. This limits the detection of infected hosts during fieldwork when captured animals should be released soon after sampling. It is an obstacle for quick selection of donor hosts for parasite experimental, histological and other investigations in the field. This study modified, tested and described the buffy coat method (BCM) for quick diagnostics (~ 20 min/sample) of avian blood parasites. Methods: Blood of 345 birds belonging to 42 species was collected, and each sample was examined using ME of stained blood films and the buffy coat, which was examined after centrifugation in capillary tubes and after being transferred to objective glass slides. Parasite detection using these methods was compared using sensitivity, specificity, positive and negative predictive values and Cohen's kappa index. Results: Haemoproteus, Leucocytozoon, Plasmodium, microfilariae, Trypanosoma and Lankesterella parasites were detected. BCM had a high sensitivity (> 90%) and specificity (> 90%) for detection of Haemoproteus and microfilariae infections. It was of moderate sensitivity (57%) and high specificity (> 90%) for Lankesterella infections, but of low sensitivity (20%) and high specificity (> 90%) for Leucocytozoon infections. Trypanosoma and Plasmodium parasites were detected only by BCM and ME, respectively. According to Cohen's kappa index, the agreement between two diagnostic tools was substantial for Haemoproteus (0.80), moderate for Lankesterella (0.46) and fair for microfilariae and Leucocytozoon (0.28) infections. Conclusions: BCM is sensitive and recommended as a quick and reliable tool to detect Haemoproteus, Trypanosoma and microfilariae parasites during fieldwork. However, it is not suitable for detection of species of Leucocytozoon and Plasmodium. BCM is a useful tool for diagnostics of blood parasite co-infections. Its application might be extended to studies of blood parasites in other vertebrates during field studies.