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Haemoproteus bobricklefsi sp. nov. (Haemosporida, Haemoproteidae) was found in the dunnock Prunella modularis and represents the first blood parasite described in accentor birds of the Prunellidae. The description is based on the morphology of blood stages and includes information about a barcoding segment of the mitochondrial cytochrome b gene (lineage hDUNNO01) and the full mitochondrial genome, which can be used for identification and diagnosis of this infection. The new parasite can be readily distinguished from described species of haemoproteids parasitizing passeriform birds due to markedly variable position of nuclei in advanced and fully grown macrogametocytes. Illustrations of blood stages of the new species are given, and phylogenetic analyses based on partial mitochondrial cytochrome b gene sequences and the full mitochondrial genome identified the closely related lineages. DNA haplotype networks showed that transmission occurs in Europe and North America. This parasite was found in the dunnock in Europe and several species of the Passerellidae in North America. It is probably of Holarctic distribution, with the highest reported prevalence in the UK. The parasite distribution seems to be geographically patchy, with preference for areas of relatively cool climates. Phylogenetic analysis suggests that H. bobricklefsi sp. nov. belongs to the Parahaemoproteus subgenus and is probably transmitted by biting midges belonging to Culicoides (Ceratopogonidae). The available data on molecular occurrence indicate that this pathogen is prone to abortive development, so worth attention in regard of consequences for bird health.

Migratory animals likely play an important role in the geographic spread of parasites. In fact, a common assumption is that parasites are potentially transmitted by migratory animals at temporary stopover sites along migratory routes, yet very few studies have assessed whether transmission at stopover sites can or does occur. We investigated the potential for a group of vector-transmitted parasites, the avian haemosporidians, to be transmitted during migratory stopover periods at Rushton Woods Preserve in Pennsylvania, USA. Using an analysis of 1454 sampled avian hosts, we found that while a core group of abundant haemosporidians was shared between local breeding birds and passing migrants, the parasite community of migratory birds at Rushton was distinct from that of local breeding birds and showed similarity to a previously sampled boreal forest haemosporidian community. Haemosporidians that were unique to passing migratory birds were associated with sampling sites in North America with cooler summer temperatures than haemosporidians that are transmitted at Rushton, suggesting that the transmission of these parasites may be restricted to high-latitude regions outside of our temperate stopover site. We also found that the abundance of mosquitoes in our study region is offset from that of migratory bird abundance during avian migratory periods, with the peak period of bird migration occurring during periods of low mosquito activity. Collectively, these findings suggest that although abundant haemosporidians are possibly transmitted between local and passing migratory birds, a combination of biotic and abiotic factors may constrain haemosporidian transmission during avian stopover at our study site.

Leucocytozoon species are cosmopolitan and prevalent avian parasites, with some infections being lethal, mainly due to the exo-erythrocytic development of the parasite in bird tissues. The patterns of exo-erythrocytic development in Leucocytozoon spp. infections in wild birds remain poorly studied. This study investigated the development of Leucocytozoon spp. tissue stages in tits (Paridae). Great tits ( Parus major ), Blue tits ( Cyanistes caeruleus ), and Coal tits ( Periparus ater ) were screened for infections using an integrative approach that consisted of microscopic analysis of thin blood smears, histological techniques, chromogenic in situ hybridization (CISH), PCR-based methods, and phylogenetic analysis. In total, 41 individuals were analyzed (eight naturally infected that were selected and euthanized, and 33 found dead in the wild and opportunistically sampled). Among the naturally infected birds, all individuals that were microscopically positive for Leucocytozoon species were also PCR-positive for these parasites. Co-infections with Plasmodium spp. and Haemoproteus spp. were commonly found, mainly among the opportunistically sampled birds. Two morphotypes were identified, Leucocytozoon majoris (Laveran, 1902) and Leucocytozoon fringillinarum Woodcock, 1910. Tissue stages were present in three birds sampled exclusively during the non-breeding season, two of them with meronts developing in the kidneys and liver, and one individual with a megalomeront in the heart. All the exo-erythrocytic stages were confirmed to be Leucocytozoon spp. by CISH using a Leucocytozoon genus-specific probe. Phylogenetic analysis placed parasite lineages with different morphotypes in separate clades. The developmental patterns of exo-erythrocytic stages of Leucocytozoon spp. in naturally infected passerines are poorly understood, requiring further research. Les espèces de Leucocytozoon sont des parasites aviaires cosmopolites et répandus. Certaines infections sont mortelles, principalement en raison du développement exo-érythrocytaire du parasite dans les tissus des oiseaux. Les schémas de développement exo-érythrocytaire dans les infections à Leucocytozoon spp. chez les oiseaux sauvages restent peu étudiés. Cette étude a examiné le développement des stades tissulaires de Leucocytozoon spp. chez les mésanges (Paridae). Des mésanges charbonnières ( Parus major ), mésanges bleues ( Cyanistes caeruleus ) et mésanges noires ( Periparus ater ) ont été examinées pour détecter les infections à l’aide d’une approche intégrative consistant en une analyse microscopique de frottis sanguins minces, des techniques histologiques, une hybridation chromogène in situ (CISH), des méthodes basées sur la PCR et une analyse phylogénétique. Au total, 41 individus ont été analysés (huit naturellement infectés qui ont été sélectionnés et euthanasiés et 33 trouvés morts dans la nature et échantillonnés de manière opportuniste). Parmi les oiseaux naturellement infectés, tous les individus qui étaient microscopiquement positifs pour les espèces de Leucocytozoon étaient également positifs pour ces parasites par PCR. Des co-infections avec Plasmodium spp. et Haemoproteus spp. ont été couramment trouvées, principalement parmi les oiseaux échantillonnés de manière opportuniste. Deux morphotypes ont été identifiés, Leucocytozoon majoris (Laveran, 1902) et Leucocytozoon fringillinarum Woodcock, 1910. Des stades tissulaires étaient présents chez trois oiseaux échantillonnés exclusivement en dehors de la saison de reproduction, deux d’entre eux avec des mérontes se développant dans les reins et le foie, et un individu avec un mégaloméronte dans le cœur. Tous les stades exo-érythrocytaires ont été confirmés comme étant des Leucocytozoon spp. par CISH en utilisant une sonde spécifique au genre Leucocytozoon . L’analyse phylogénétique a placé les lignées de parasites avec différents morphotypes dans des clades distincts. Les modèles de développement des stades exo-érythrocytaires de Leucocytozoon spp. chez les passereaux naturellement infectés sont mal compris et nécessitent des recherches supplémentaires.

Blood parasites from the order Haemosporida infect many vertebrates and cause malaria-like diseases. In this study, a haemosporidian infection was detected in a sick grey crowned crane imported into China using a combination of morphological and molecular approaches. Blood samples were collected from the jugular vein and processed for morphological identification of infective parasites using stained blood smears and microscopy. No merogony occurs in the blood cells, and sporadic pigment granules were observed. Nested-PCR assays were employed for a molecular examination, which indicated that the cytb gene of this parasite had 94.1–94.9% identity to Haemoproteus antigonis . Subsequently, its mitochondrial genome structure was determined by high-throughput sequencing using the DNBSEQ-T7 platform. The determined structure was confirmed by the Sanger sequencing using amplicons. The mitochondrial genome obtained for this parasite exhibited a low CG content (32.0%) and possessed three protein-coding genes, encoding 1068 amino acids, which constituted 53.7% of the genome. Phylogenetic analysis indicated that this parasite clustered with Haemoproteus sp . is detected in grey crowned cranes from Africa. This parasite was likely acquired during importation of this animal; thus, strict quarantine of imported ornamental animals is required to prevent the entry of new pathogens.

Background The order Accipitriformes comprises the largest group of birds of prey with 260 species in four families. So far, 21 haemosporidian parasite species have been described from or reported to occur in accipitriform birds. Only five of these parasite species have been characterized molecular genetically. The first part of this study involved molecular genetic screening of accipitriform raptors from Austria and Bosnia-Herzegovina and the first chromogenic in situ hybridization approach targeting parasites in this host group. The aim of the second part of this study was to summarize the CytB sequence data of haemosporidian parasites from accipitriform raptors and to visualize the geographic and host distribution of the lineages. Methods Blood and tissue samples of 183 accipitriform raptors from Austria and Bosnia-Herzegovina were screened for Plasmodium , Haemoproteus and Leucocytozoon parasites by nested PCR, and tissue samples of 23 PCR-positive birds were subjected to chromogenic in situ hybridization using genus-specific probes targeting the parasites’ 18S rRNAs. All published CytB sequence data from accipitriform raptors were analysed, phylogenetic trees were calculated, and DNA haplotype network analyses were performed with sequences from clades featuring multiple lineages detected in this host group. Results Of the 183 raptors from Austria and Bosnia-Herzegovina screened by PCR and sequencing, 80 individuals (44%) were infected with haemosporidian parasites. Among the 39 CytB lineages detected, 18 were found for the first time in the present study. The chromogenic in situ hybridization revealed exo-erythrocytic tissue stages of Leucocytozoon parasites belonging to the Leucocytozoon toddi species group in the kidneys of 14 infected birds. The total number of CytB lineages recorded in accipitriform birds worldwide was 57 for Leucocytozoon , 25 for Plasmodium , and 21 for Haemoproteus . Conclusion The analysis of the DNA haplotype networks allowed identifying numerous distinct groups of lineages, which have not yet been linked to morphospecies, and many of them likely belong to yet undescribed parasite species. Tissue stages of Leucocytozoon parasites developing in accipitriform raptors were discovered and described. The majority of Leucocytozoon and Haemoproteus lineages are specific to this host group, but most Plasmodium lineages were found in birds of other orders. This might indicate local transmission from birds kept at the same facilities (raptor rescue centres and zoos), likely resulting in abortive infections. To clarify the taxonomic and systematic problems, combined morphological and molecular genetic analyses on a wider range of accipitriform host species are needed.

Leucocytozoon species are common in countries with warm climates but are an often neglected blood parasite in poultry. Although Leucocytozoon macleani is less virulent than Leucocytozoon caulleryi , it can still negatively impact production performance. In Thailand, the available reports indicate a high prevalence of Leucocytozoon spp. , but detailed morphological characteristics of the parasites remain insufficiently known. In this study, Giemsa-stained blood smears and extracted genomic (g) DNA were obtained from 60 domestic chickens ( Gallus gallus domesticus ). Blood smears were examined for the presence of Leucocytozoon species and their morphological characteristics were examined. A total of 60 gDNA samples were used for nested-PCR amplification of the cytochrome b gene of Leucocytozoon species, followed by sequencing and phylogenetic analysis. The microscopic and molecular examinations revealed prevalence of leucocytozoonosis in chickens of 85% and 90%, respectively. Sequence analysis indicated that several infected chickens harboured multiple Leucocytozoon lineages. Leucocytozoon macleani was morphologically identified in nine samples and could be linked to the lineages GALLUS17, GALLUS34, and the new lineages GALLUS63. The found gametocytes of L. macleani morphologically resembled those reported previously, but exhibited some distinct characteristics. Phylogenetically, the lineages of L. macleani isolated in this study grouped separately from some other L. macleani lineages deposited in GenBank. In conclusion, the prevalence of Leucocytozoon infection in chickens from Northeastern Thailand was high, with frequent co-infections by multiple lineages. Leucocytozoon macleani may exhibit cryptic specification. This study is the first report of L. macleani lineages described using MalAvi database nomenclature, alongside their morphological characteristics. Les espèces de Leucocytozoon sont courantes dans les pays à climat chaud, mais sont souvent négligées comme parasites sanguins chez les volailles. Bien que Leucocytozoon macleani soit moins virulent que Leucocytozoon caulleryi , il peut néanmoins avoir un impact négatif sur les performances de production. En Thaïlande, les rapports disponibles indiquent une prévalence élevée de Leucocytozoon spp., mais les caractéristiques morphologiques détaillées des parasites restent insuffisamment connues. Dans cette étude, des frottis sanguins colorés au Giemsa et de l’ADN génomique (g) extrait ont été obtenus chez 60 poulets domestiques ( Gallus gallus domesticus ). Les frottis sanguins ont été examinés pour détecter la présence d’espèces de Leucocytozoon et leurs caractéristiques morphologiques ont été étudiées. Soixante échantillons d’ADNg ont été utilisés pour l’amplification par PCR nichée du gène du cytochrome b des espèces de Leucocytozoon , suivie d’un séquençage et d’une analyse phylogénétique. Les examens microscopiques et moléculaires ont révélé respectivement une prévalence de leucocytozoonose chez les poulets de 85% et 90%. L’analyse des séquences a indiqué que plusieurs poulets infectés hébergeaient plusieurs lignées de Leucocytozoon . Leucocytozoon macleani a été identifié morphologiquement dans neuf échantillons et pourrait être lié aux lignées GALLUS17, GALLUS34 et aux nouvelles lignées GALLUS63. Les gamétocytes de L. macleani trouvés ressemblaient morphologiquement à ceux rapportés précédemment, mais présentaient certaines caractéristiques distinctes. Phylogénétiquement, les lignées de L. macleani isolées dans cette étude se sont regroupées séparément de certaines autres lignées de L. macleani déposées dans GenBank. En conclusion, la prévalence de l’infection à Leucocytozoon chez les poulets du nord-est de la Thaïlande était élevée, avec de fréquentes co-infections par plusieurs lignées. Leucocytozoon macleani pourrait présenter une spécification cryptique. Cette étude est le premier rapport sur les lignées de L. macleani décrites à l’aide de la nomenclature de la base de données MalAvi, ainsi que sur leurs caractéristiques morphologiques.

Leucocytozoonosis is a vector-borne blood protozoal disease that affects birds worldwide. Although Egypt has a large-scale poultry industry, data on Leucocytozoon infections in broilers remain limited. This study reports the first detection of leucocytozoonosis in 24 broiler farms (180,000 birds) in El-Sharkia governorate, Egypt. The affected broiler flocks were raised in open poultry farms located near agricultural rice fields and water sources during the late summer season, which are conditions favorable for vector proliferation. Clinically, infected birds exhibited depression, anorexia, weight loss, and anemia, with mortality rates ranging from 0.5% to 2%. Postmortem examination revealed widespread hemorrhages across multiple organs and a large pool of blood in the abdominal cavity. Microscopic examination of blood smears and cell block preparations showed the presence of various gametocytes within leukocytes and erythrocytes. Histopathological examination identified characteristic megaloschizonts in multiple organs, including the kidneys, liver, lungs, muscles, heart, intestine, pancreas, and spleen, accompanied by hemorrhages, necrosis, and mononuclear cell infiltration, primarily lymphocytes and macrophages. Molecular identification using real-time PCR confirmed L. caulleryi infection, corroborating the hematological and pathological findings. This study provides key insights into L. caulleryi diagnosis, promotes awareness among veterinarians and poultry producers, and highlights the need for improved biosecurity.

Common ravens ( Corvus corax ) are intelligent scavengers that adapt to diverse environments, playing a key ecological role, but their health and ecosystem contributions can be affected by parasites. This study investigates the prevalence and diversity of blood parasite infections in common ravens using molecular techniques. Blood samples ( n  = 42) were collected from dead common ravens in Germany and screened for filarioid nematodes, trypanosomatids, and haemosporidian parasites. The results showed that 26.2% of the common ravens were PCR-positive for at least one parasite, with some cases of mixed infections. Filarioid nematodes were found in 16.7%, trypanosomatids in 4.8%, and haemosporidian parasites in 16.7% of the common ravens. Sequencing revealed the presence of four Leucocytozoon CytB lineages and one Haemoproteus lineage. The findings suggest that common ravens in Germany are often infected with diverse avian blood parasites, with a higher prevalence of filarioid nematodes. Further research is needed to confirm the circulation of these parasites in the common raven population and to identify the specific filarioid nematode species present in Germany.

Biodiversity surveys remain a critical tool for characterizing the global species richness of parasites. In high-latitude regions of the world, characterizing parasite biodiversity is of particular importance due to the rapid rate at which the climate is changing and potentially shifting parasite distributions and abundances. We sampled a bird community on Prince of Wales Island in southern Alaska, United States, to test for the abundance and richness of haemosporidian and trypanosomatid parasites in this understudied region. We tested for parasites in 67 archived tissue samples of 18 bird species, of which five had not been previously tested for haemosporidians and 11 had not been sampled for trypanosomatids using molecular methods. We recovered two novel mitochondrial genetic lineages of haemosporidians (genera Haemoproteus and Leucocytozoon ), and three novel 18S rRNA genotypes belonging to Trypanosoma of uncertain species-level affinities. Surprisingly, we also identified a trypanosomatid from the genus Zelonia , a group of monoxenous parasites of insects, from an avian tissue. While this anomalous record may have been the result of environmental contamination, it nonetheless reflects the first record of Zelonia in the entire Nearctic region.

Background Haemosporidians (Haemosporida, Apicomplexa), which include malaria parasites, are found in nearly all terrestrial ecosystems. Avian haemosporidians have been extensively studied; however, there is limited information on parasites in owls (order Strigiformes). Here, haemosporidians infecting North American owls were characterized using an integrative methodology. Methods Taking advantage of injured/dead owls from rehabilitation centres in the central and northeastern USA, 53 individuals of Bubo virginianus , Strix varia , Megascops asio , Bubo scandiacus , Aegolius acadicus , and Tyto furcata were screened using polymerase chain reaction for all samples and microscopy for those with available blood smears. Parasite mitochondrial genomes were obtained using a long-read sequencing method (PacBio HiFi), which efficiently detects multiple infections in a single host. The relationships between parasite lineages were estimated using phylogenetic and haplotype network methods. Results In total, 21 individuals from three species were positive by PCR: B. virginianus (14/17, 82.4%), M. asio (3/5, 60%), and S. varia (4/8, 50%). Two Plasmodium , three Haemoproteus , and four Leucocytozoon lineages were identified infecting these hosts, with one Haemoproteus and one Leucocytozoon being new to science. All positive individuals were infected with Haemoproteus parasites, and two B. virginianus had a mixed infection with Leucocytozoon and Haemoproteus species. The hSTVAR01 cytochrome b ( cytb ) lineage common in North America is linked for the first time to Haemoproteus syrnii . Haemoproteus syrnii was found in all 14 positive B. virginianus and two S. varia . Notably, all the cytb lineages from previously identified H. syrnii , based on erythrocytic stages, were not monophyletic, indicating the existence of an undescribed species. The pPADOM11 cytb lineage was recognized as an allele of Plasmodium elongatum. Conclusion Long reads enabled the detection of mixed/co-infections. The link between genetic data and morphospecies was established in two cases. Several Leucocytozoon clades were observed; however, only one morphospecies, Leucocytozoo danilewskyi , has been described in owls. Thus, there is a need for a detailed analysis of blood stages to determine whether different owl Haemoproteus and Leucocytozoon parasites exhibit morphological differences or represent cryptic species. Overall, this study underscores the importance of high-quality molecular data in characterizing the biodiversity of haemosporidian parasites. Graphical Abstract