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[Display omitted] • Haemoproteus hirundinis (lineage hDELURB2) develops megalomeronts in pectoral muscle of Delichon urbicum. • Haemoproteus dumbbellus n. sp. (lineage hEMCIR01) develops meronts in various organs of Emberiza citrinella. • Chromogenic in situ hybridization confirmed the Haemoproteus origin of exo-erythrocytic stages. • Phylogenetic analysis suggests formation of meronts is typical for species close to H. dumbbellus. • Haemoproteus dumbbellus n. sp. (hEMCIR01) is described from the blood of E. citrinella. Avian haemosporidians are widespread parasites categorized into four families of the order Haemosporida (Apicomplexa). Species of the subgenus Parahaemoproteus (genus Haemoproteus) belong to the Haemoproteidae and are transmitted by Culicoides biting midges. Reports of death due to tissue damage during haemoproteosis in non-adapted birds have raised concerns about these pathogens, especially as their exo-erythrocytic development is known for only a few Haemoproteus spp. More research is needed to better understand the patterns of the parasites' development in tissues and their impact on avian hosts. Yellowhammers Emberiza citrinella (Emberizidae) and common house martins Delichon urbicum (Hirundinidae) were screened for Haemoproteus parasites by microscopic examination of blood films and PCR-based testing. Individuals with single infection were selected for histological investigations. H & E-stained sections were screened for detection and characterization of the exo-erythrocytic stages, while chromogenic in situ hybridization (CISH) and phylogenetic analysis were performed to confirm the Haemoproteus origin and their phylogenetic relationships. Haemoproteus dumbbellus n. sp. was discovered in Emberiza citrinella single - infected with the lineage hEMCIR01. Meronts of H. dumbbellus n. sp. developed in various organs of five of six tested individuals, a pattern which was reported in other Haemoproteus species clustering in the same clade, suggesting this could be a phylogenetic trait. By contrast, in Delichon urbicum infected with the Haemoproteus lineage hDELURB2, which was linked to the more distantly related parasite Haemoproteus hirundinis , only megalomeronts were found in the pectoral muscles of two of six infected individuals. All exo-erythrocytic stages were confirmed to be Haemoproteus parasites by CISH using a Haemoproteus genus-specific probe. While the development of meronts seems to be typical for species of the clade containing H. dumbbellus , further investigations and data from more species are needed to explore whether a phylogenetic pattern occurs in meront or megalomeront formation. [ABSTRACT FROM AUTHOR]

Haemoproteus parasites (Apicomplexa, Haemosporida) are widespread pathogens of birds, with a rich genetic (about 1900 lineages) and morphospecies (178 species) diversity. Nonetheless, their life cycles are poorly understood. The exo-erythrocytic stages of three Haemoproteus majoris (widespread generalist parasite) lineages have been previously reported, each in a different bird species. We aimed to further study and compare the development of five H. majoris lineages—hCCF5, hCWT4, hPARUS1, hPHSIB1, and hWW2—in a wider selection of natural avian hosts. A total of 42 individuals belonging to 14 bird species were sampled. Morphospecies and parasitemia were determined by microscopy of blood films, lineages by DNA-barcoding a 478 bp section of the cytochrome b gene, and exo-erythrocytic stages by histology and chromogenic in situ hybridization. The lineage hCWT4 was morphologically characterized as H. majoris for the first time. All lineage infections exclusively featured megalomeronts. The exo-erythrocytic stages found in all examined bird species were similar, particularly for the lineages hCCF5, hPARUS1, and hPHSIB1. Megalomeronts of the lineages hWW2 and hCWT4 were more similar to each other than to the former three lineages. The kidneys and gizzard were most often affected, followed by lungs and intestines; the site of development showed variation depending on the lineage. [ABSTRACT FROM AUTHOR]

Haemoproteus parasites (Apicomplexa, Haemosporida) are widespread pathogens of birds, with a rich genetic (about 1900 lineages) and morphospecies (178 species) diversity. Nonetheless, their life cycles are poorly understood. The exo-erythrocytic stages of three Haemoproteus majoris (widespread generalist parasite) lineages have been previously reported, each in a different bird species. We aimed to further study and compare the development of five H. majoris lineages—hCCF5, hCWT4, hPARUS1, hPHSIB1, and hWW2—in a wider selection of natural avian hosts. A total of 42 individuals belonging to 14 bird species were sampled. Morphospecies and parasitemia were determined by microscopy of blood films, lineages by DNA-barcoding a 478 bp section of the cytochrome b gene, and exo-erythrocytic stages by histology and chromogenic in situ hybridization. The lineage hCWT4 was morphologically characterized as H. majoris for the first time. All lineage infections exclusively featured megalomeronts. The exo-erythrocytic stages found in all examined bird species were similar, particularly for the lineages hCCF5, hPARUS1, and hPHSIB1. Megalomeronts of the lineages hWW2 and hCWT4 were more similar to each other than to the former three lineages. The kidneys and gizzard were most often affected, followed by lungs and intestines; the site of development showed variation depending on the lineage.

Abstract Background Haemoproteus 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. Methods Turdus 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. Results Gametocytes 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. Conclusions Phylogenetic 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.

Species of Haemoproteus (Haemoproteidae, Haemosporida) are common bird pathogens. Recent molecular studies combined with histopathology research have reported development of megalomeronts of these parasites in various organs, sometimes resulting in the death of the avian host. Five Common starlings (Sturnus vulgaris) were found naturally infected with Haemoproteus pastoris lineage hLAMPUR01. The parasite was identified using microscopic examination of blood films and DNA sequences. Infected bird organs were investigated histologically for (i) the presence of exo-erythrocytic stages and (ii) the patterns of development (morphology and localization) in different host individuals. For the first time, megalomeronts of Haemoproteus parasites were seen developing in the brain, while numerous others at different stages of maturation were found in the intestine, pancreas, kidneys, lungs, esophagus, spleen, gizzard, and trachea. Megalomeronts were predominantly roundish or oval, up to 800 μm, they were surrounded by a capsular-like wall and developed asynchronously in the same bird individual. After megalomeront maturation and rupture, a massive infiltration of blood cells occurred, indicating the hemorrhagic processes. Review of available data showed that different Haemoproteus species produce markedly different megalomeronts, morphology of which can probably be predicted using phylogenetic analysis based on partial sequences of cytochrome b gene.

Simple Summary: Birds are hosts to diverse blood parasites belonging to many taxonomic groups. Among them, numerous haemosporidian parasites of the genus Haemoproteus are transmitted globally. These pathogens develop in the blood and internal organs of birds. The blood stages (gametocytes) are known for about 150 described species, but the tissues stages or exo-erythrocytic stages (meronts and megalomeronts) are known only fragmentarily for about 10% of the described species. Knowledge on merogony is important in avian medicine for better understanding of pathologies during haemoproteosis. This study reported and characterized the megalomeronts of Haemoproteus pastoris, a parasite of the widespread Common starling (Sturnus vulgaris). Parasites were identified using molecular and microscopy examination tools. Five individual naturally infected birds were sampled, and their organs were examined histologically. Megalomeronts were found in eight different organs. The parasites were described and illustrated. The largest megalomeront, of all observed forms and shapes, reached 800 μm in length. Importantly, Haemoproteus megalomeronts were reported in the brain of avian hosts for the first time, indicating non-described pathology during avian haemoproteosis. This study contributes to a better understanding of the life cycle of avian haemoproteids and opens new perspectives in pathology research during avian haemoproteosis, which is important for birds' health. Species of Haemoproteus (Haemoproteidae, Haemosporida) are common bird pathogens. Recent molecular studies combined with histopathology research have reported development of megalomeronts of these parasites in various organs, sometimes resulting in the death of the avian host. Five Common starlings (Sturnus vulgaris) were found naturally infected with Haemoproteus pastoris lineage hLAMPUR01. The parasite was identified using microscopic examination of blood films and DNA sequences. Infected bird organs were investigated histologically for (i) the presence of exo-erythrocytic stages and (ii) the patterns of development (morphology and localization) in different host individuals. For the first time, megalomeronts of Haemoproteus parasites were seen developing in the brain, while numerous others at different stages of maturation were found in the intestine, pancreas, kidneys, lungs, esophagus, spleen, gizzard, and trachea. Megalomeronts were predominantly roundish or oval, up to 800 μm, they were surrounded by a capsular-like wall and developed asynchronously in the same bird individual. After megalomeront maturation and rupture, a massive infiltration of blood cells occurred, indicating the hemorrhagic processes. Review of available data showed that different Haemoproteus species produce markedly different megalomeronts, morphology of which can probably be predicted using phylogenetic analysis based on partial sequences of cytochrome b gene. [ABSTRACT FROM AUTHOR]

• Megalomeronts of Haemoproteus majoris (hPHSIB1) were discovered and described. • Megalomeronts of this parasite predominantly develop in kidneys. • Morphology of megalomeronts was similar in all investigated H. majoris lineages. • Development of megalomeronts was similar in all investigated H. majoris lineages. • Phylogenetic analysis predicts patterns of megalomeront development in Haemoproteus. Blood parasites of the genus Haemoproteus (Haemosporida, Haemoproteidae) are cosmopolitan and prevalent in birds. Numerous species and lineages of these pathogens have been identified. Some of the infections are lethal in avian hosts mainly due to damage of organs by tissue stages, which remain insufficiently investigated. Several closely related lineages of Haemoproteus majoris , a common parasite of passeriform birds, have been identified. One recent study described megalomeronts of unique morphology in the lineages hPHYBOR04 and hPARUS1 of H. majoris and suggested that the similar tissues stages might also be features in other phylogenetically closely related lineages of the same parasite species. This study aimed to test if (i) megalomeronts are present during the development of the lineage hPHSIB1 of H. majoris and if (ii) they are similar to the other investigated lineages of this species in regard of their morphology and location in organs. One adult wood warbler Phylloscopus sibilatrix , an Afrotropical migrant, naturally infected with H. majoris lineage hPHSIB1 was wild-caught after seasonal spring migration and screened using microscopic examination of blood films and histological sections of organs as well as using PCR-based testing. Bayesian phylogenetic analysis placed the lineages hPHSIB1, hPHYBOR04 and hPARUS1 in one, well-supported clade. Parasitaemia was high (6.5%) in the examined wood warbler, numerous megalomeronts were found in kidneys, and a few in the intestine. Megalomeronts of the lineage hPHSIB1 were morphologically hardly distinguishable from those of lineages hPHYBOR04 and hPARUS1; only negligible differences in the maturation stage of the cytomeres were seen. The kidneys were the main location site of the megalomeronts in all three lineages of this parasite species. This study shows that closely related lineages of H. majoris produce megalomeronts of similar morphology and predominant location in kidneys, while the normal function of this organ may be affected by the presence of numerous large megalomeronts. Megalomeronts of different avian Haemoproteus species are markedly variable in morphology and location, but phylogenetically closely related lineages possess cryptic megalomeronts. This finding suggests that phylogenies based on partial cytb gene could provide information for prediction of patterns of exo-erythrocytic development of closely related Haemoproteus parasites and are worthy of attention in planning haemosporidian parasite tissue stage research. [ABSTRACT FROM AUTHOR]