Molecular survey and genetic diversity of Plasmodium sp. infesting domestic poultry in northeastern Thailand.

Introduction: Haemosporidian parasites are prevalent worldwide and can cause economic losses in poultry production. These parasites are arousing interest in Thailand and are found in many avian species. There is insufficient information on the genetic diversity of these alveolates from the largest families - Plasmodidae, Haemoprotidae and Leucocytozoidae - specifically parasitising ducks, turkeys, and geese. Material and Methods: Blood samples from 116 backyard poultry (60 ducks, 36 turkeys and 20 geese) in northeastern Thailand were investigated for Plasmodium spp., Haemoproteus spp. and Leucocytozoon spp. infections using microscopic examination and molecular approaches. Results: A total of 37/116 birds (31.9%) had confirmed Plasmodium infections. The prevalence was 69.4% (25/36) in turkeys, 18.3% (11/60) in ducks, and 5.0% (1/20) in geese. Of these 37 positives, 86.5% were Plasmodium sp., 10.8% were P. gallinaceum and 2.7% were P. juxtanucleare. Sequence analysis based on the cytochrome b gene identified seven lineages, of which two were new lineages in backyard poultry. Conclusion: This is the first report on the prevalence of haemosporidian parasites in backyard poultry in northeastern Thailand. The results provide important data for better understanding the molecular epidemiology of haemosporidian parasites infection in poultry in this region, which will be helpful in controlling these blood parasites.

Differences in haemosporidian parasite prevalence and diversity in migratory and resident birds of prey species revealed by a non-invasive sampling method.

Avian haemosporidian parasites are widespread globally and infect numerous wild bird species. However, they have primarily been studied in passerine birds. Accordingly, the prevalence and diversity of these parasites in birds of prey remain understudied. This lack of research is primarily due to the international protection status of many of these species, their sparse distribution across ecosystems and difficulty to capture in the wild. The aim of this study was to examine the prevalence and diversity of haemosporidian parasites in two species of birds of prey, namely white-tailed eagle and lesser spotted eagle. To achieve this, a non-invasive approach was employed, involving the extraction of DNA from blood spots present in moulted feathers. Freshly moulted feathers were collected from the ground under nests or within the nests of these birds during the breeding season. A visible blood spot located at the junction of the calamus and rachis was removed and fixed in SET buffer for molecular analysis. The identification of haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) was conducted using PCR-based methods. Overall, parasite DNA was successfully detected in shed feathers that were kept in their original form at least decade prior to analysis. Among the studied individuals, resident white-tailed eagles showed significantly lower infection rates of haemosporidian parasites compared to migratory lesser spotted eagles. A total of nine genetic lineages of haemosporidian parasites were detected, with seven of them being new to science based on partial sequences of the cytb gene. Additionally, the phylogenetic relationships among these new lineages and previously described ones were established. These findings highlight the suitability of non-invasive sampling for investigating the prevalence and diversity of haemosporidian parasites in wild birds of prey populations. Moreover, this approach holds promise for studying other challenging-to-reach and protected bird species. According to our research, there is a greater chance of finding haemosporidian parasites in freshly gathered feathers.

High Abundance of Haemoproteus Parasites in Culicoides (Diptera, Ceratopogonidae), with a Confirmation of Culicoides reconditus as a New Vector of These Avian Blood Parasites.

Haemoproteus parasites are the most diverse among Haemosporida. However, their natural vectors (Culicoides) are still poorly investigated and were identified for only a few parasite species and lineages. The application of an integrative approach (insect dissection, microscopic analysis, and molecular-based methods) is necessary in these studies, which have been carried out by a few research groups, mainly in Europe. The aim of this study was (i) to determine the Culicoides species that are naturally infected by Haemoproteus parasites, and which can support its complete sporogonic development, and (ii) to investigate the prevalence of Culicoides species and Haemoproteus parasite lineages in different study sites. In total, 1953 parous Culicoides females, from 11 species, were collected in four different localities in Lithuania and were dissected and analyzed using an integrative approach. The most abundant was C. pictipennis (30.3%). Parasite DNA was found in 7.9% of all investigated Culicoides, of which ~30% had sporozoites in their salivary glands, confirming their vector competence for these parasites. The Botanical Garden presented the highest number of Culicoides parous females, Culicoides species, and parasite lineages, as well as the highest positivity for sporozoites. Culicoides reconditus was confirmed as a natural vector of Haemoproteus parasites, sporozoites of six Haemoproteus lineages were reported for the first time, and 12 new interactions between Haemoproteus parasite lineages and Culicoides species were identified. Haemoproteus parasites seem to be transmitted by a high number of Culicoides species, with C. kibunensis, C. pictipennis, and C. segnis being the most important vectors.

Therapeutic management of pseudomalaria in a flock of pigeons with chloroquine.

Two Indian rock pigeons aged 2-3 months presented to the Referral Veterinary Polyclinic and Teaching Veterinary Clinical Complex, Indian Veterinary Research Institute, Utter Pradesh with a history of decreased feed intake, twisting of the neck, and inability to fly. The same symptoms also caused the deaths of two other birds from the same flock. The bird seemed dull and depressed during a clinical examination, had ruffled feathers, a tilted head and circling. Examination of a faecal sample showed no intestinal parasites. Upon observation of a blood smear, many intracytoplasmic characteristic halter-shaped Hemoproteus columbae gametocytes could be detected. The case was diagnosed as pigeon pseudomalaria. The treatment was initiated with chloroquine@10 mg/kg body weight in drinking water for 5 days along with the multivitamin supplementation for one week. Permethrin spray was applied externally to the whole flock in the house to get rid of the fly vector. The clinical state of the birds was evaluated one week after initiation of the therapy. The pigeon had an uneventful recovery and the blood smear examination revealed no haemoparasites.

Brain parasites and misorientation of migratory birds.

Haemoproteus blood parasites of birds are thought to be relatively benign. Recent findings show that infections may develop in the brain of birds, possibly distorting their orientation sense. Misdirected migration may lead migrants outside their range where they are recognized as vagrants and can transmit parasites to novel hosts.

Novel phylogenetic clade of avian Haemoproteus parasites (Haemosporida, Haemoproteidae) from Accipitridae raptors, with description of a new Haemoproteus species.

Avian haemosporidian parasites (order Haemosporida, phylum Apicomplexa) are blood and tissue parasites transmitted by blood-sucking dipteran insects. Three genera (Plasmodium, Haemoproteus and Leucocytozoon) have been most often found in birds, with over 270 species described and named in avian hosts based mainly on the morphological characters of blood stages. A broad diversity of Haemoproteus parasites remains to be identified and characterized morphologically and molecularly, especially those infecting birds of prey, an underrepresented bird group in haemosporidian parasite studies. The aim of this study was to investigate and identify Haemoproteus parasites from a large sample comprising accipitriform raptors of 16 species combining morphological and new molecular protocols targeting the cytb genes of this parasite group. This study provides morphological descriptions and molecular characterizations of two Haemoproteus species, H. multivacuolatus n. sp. and H. nisi Peirce and Marquiss, 1983. Haemoproteus parasites of this group were so far found in accipitriform raptors only and might be classified into a separate subgenus or even genus. Cytb sequences of these parasites diverge by more than 15% from those of all others known avian haemosporidian genera and form a unique phylogenetic clade. This study underlines the importance of developing new diagnostic tools to detect molecularly highly divergent parasites that might be undetectable by commonly used conventional tools.

Title: Nouveau clade phylogénétique de parasites de rapaces Accipitridae du genre Haemoproteus (Haemosporida, Haemoproteidae), avec description d'une nouvelle espèce d'Haemoproteus. Abstract: Les parasites hémosporidies aviaires (ordre Haemosporida, phylum Apicomplexa) sont des parasites sanguins et tissulaires transmis par des insectes diptères hématophages. Trois genres (Plasmodium, Haemoproteus et Leucocytozoon) ont été le plus souvent trouvés chez les oiseaux, avec plus de 270 espèces décrites et nommées chez les hôtes aviaires en fonction principalement des caractères morphologiques des stades sanguins. Une grande diversité des Haemoproteus reste à identifier et à caractériser morphologiquement et génétiquement, en particulier ceux qui infectent les oiseaux de proie, un groupe d'oiseaux sous-représenté dans les études sur les hémosporidies. Le but de cette étude était d'étudier et d'identifier les Haemoproteus à partir d'un large échantillon comprenant des rapaces accipitriformes de 16 espèces, en combinant des protocoles morphologiques et de nouveaux protocoles moléculaires ciblant les gènes cytb de ce groupe de parasites. Cette étude fournit des descriptions morphologiques et des caractérisations moléculaires de deux espèces d'Haemoproteus, H. multivacuolatus n. sp. et H. nisi Peirce and Marquiss, 1983. Les Haemoproteus de ce groupe n'ont jusqu'à présent été trouvés que chez les rapaces accipitriformes et pourraient être classés dans un sous-genre ou même un genre distinct. Les séquences cytb de ces parasites divergent de plus de 15 % de celles de tous les autres genres d'hémosporidies aviaires connus et forment un clade phylogénétique unique. Cette étude souligne l'importance de développer de nouveaux outils de diagnostic pour détecter des parasites moléculairement très divergents qui pourraient être indétectables par les outils conventionnels couramment utilisés.

Molecular characterization of Haemoproteus enucleator with emphasis on the host and geographic distribution.

Haemoproteus species (Haemosporida, Haemoproteidae) are cosmopolitan and highly diverse blood parasites of birds that have been neglected in avian medicine. However, recent discoveries based on molecular diagnostic markers show that these pathogens often cause marked damage to various internal organs due to exo-erythrocytic development, sometimes resulting in severe and even lethal avian haemoproteosis, including cerebral pathologies. Molecular markers are essential for haemoproteosis diagnostics, but the data is limited, particularly for parasites transmitted in tropical ecosystems. This study combined microscopic and molecular approaches to characterize Haemoproteus enucleator morphologically and molecularly. Blood samples were collected from the African pygmy kingfisher Ispidina picta in Cameroon, and the parasite was identified using morphological characters of gametocytes. The analysis of partial cytochrome b sequences (cytb) identified a new Haemoproteus lineage (hISPIC03), which was linked to the morphospecies H. enucleator. Illustrations of blood stages were provided and the phylogenetic analysis showed that the new lineage clustered with five other closely related lineages belonging to the same morphospecies (hALCLEU01, hALCLEU02, hALCLEU03, hISPIC01, and hALCQUA01), with a maximum genetic distance between these lineages of 1.5 % (7 bp difference) in the 478 bp cytb sequences. DNA haplotype network was developed and identified geographic and host distribution of all lineages belonging to H. enucleator group. These lineages were almost exclusively detected in African kingfishers from Gabon, Cameroon, South Africa, and Botswana. This study developed the molecular characterization of H. enucleator and provides opportunities for diagnostics of this pathogen at all stages of its life cycle, which remains undescribed in all its closely related lineages.

Molecular Survey of Haemosporidian Parasites in Procellariiformes Sampled in Southern Brazil, 2013-22.

The order Procellariiformes includes several species of seabirds that perform long-distance migrations crossing all the oceans. These movements may contribute to the dispersal and exchange of hemoparasites, such as haemosporidians. There is a lack of studies regarding the order Haemosporida in Procellariiformes, and, to date, only the genus Plasmodium has been reported. This survey investigated the occurrence of the three genera of haemosporidians, Plasmodium, Haemoproteus, and Leucocytozoon, in samples collected between 2013 and 2022 from 95 individuals of 14 species of Procellariiformes from southern Brazil, including live animals in rehabilitation centers, individuals caught as incidental bycatch, and carcasses found along the coast. A total of 171 samples of blood and fragments of liver and spleen were analyzed, with extracted DNA being subjected to a nested PCR followed by phylogeny analysis. All animals were negative for Plasmodium spp. and Leucocytozoon spp., but one Black-browed Albatross (Thalassarche melanophris) and one Manx Shearwater (Puffinus puffinus) specimen were positive for Haemoproteus spp. The sequences obtained from positive seabirds did not show 100% similarity with other known lineages available in the MalAvi database and thus were probably novel lineages. However, one sequence clustered together with Haemoproteus noctuae, a parasite from Strigiformes, while the other was grouped with Haemoproteus columbae, which is classically related to Columbiformes. These results suggest that both positive animals may have become infected when beached or in rehabilitation centers by a spillover of vectors from local birds. This highlights the importance of surveillance of the health of Procellariiformes regarding the possibility of dissemination of new pathogens in different bird populations.

Seasonal Variation in Detection of Haemosporidia in a Bird Community: A Comparison of Nested PCR and Microscopy.

In a 2-yr study on prevalence of Haemosporidia in an avian community in Ithaca, New York, USA, we tested the hypothesis that apparent seasonal variation in prevalence is influenced by the detection protocol. We confirmed a higher detection of Haemosporidia using a molecular diagnosis technique (PCR) than by microscopy; this further increased when the PCR test was triplicated. Microscopic examination and PCR techniques have different specificity and sensitivity and therefore different probabilities of detecting hemoparasites. Birds with chronic infections or sampled during winter often have very low parasitemia, and such infections may be missed by microscopy but detected by PCR. Haemosporidian prevalence was higher during the breeding season than during the nonbreeding season regardless of the method used. Detection of Leucocytozoon spp. infection from blood smears using microscopy was challenging.

Co-infecting Haemoproteus species (Haemosporida, Apicomplexa) show different host tissue tropism during exo-erythrocytic development in Fringilla coelebs (Fringillidae).

Avian haemosporidians of the genera Plasmodium, Haemoproteus, and Leucocytozoon are common blood parasites in wild birds all over the world. Despite their importance as pathogens potentially compromising host fitness and health, little is known about the exo-erythrocytic development of these parasites, particularly during co-infections which predominate in wildlife. This study aimed to address this issue using Haemoproteus parasites of Fringilla coelebs, a common bird species of the Western Palearctic and host to a variety of haemosporidian parasite lineages. Blood and tissue samples of 20 F. coelebs, positive for haemosporidians by blood film microscopy, were analysed by PCR and sequencing to determine cytochrome b lineages of the parasites. Tissue sections were examined for exo-erythrocytic stages by histology and in situ hybridization applying genus-, species-, and lineage-specific probes which target the 18S rRNA of the parasites. In addition, laser microdissection of tissue stages was performed to identify parasite lineages. Combined molecular results of PCR, laser microdissection, and in situ hybridization showed a high rate of co-infections, with Haemoproteus lineages dominating. Exo-erythrocytic meronts of five Haemoproteus spp. were described for the first known time, including Haemoproteus magnus hCCF6, Haemoproteus fringillae hCCF3, Haemoproteus majoris hCCF5, Haemoproteus sp. hROFI1, and Haemoproteus sp. hCCF2. Merogonic stages were observed in the vascular system, presenting a formerly unknown mode of exo-erythrocytic development in Haemoproteus parasites. Meronts and megalomeronts of these species were distinct regarding their morphology and organ distribution, indicating species-specific patterns of merogony and different host tissue tropism. New pathological aspects of haemoproteosis were reported. Furthermore, phylogenetic analysis of Haemoproteus spp. with regard to their exo-erythrocytic stages points towards separation of non-megalomeront-forming species from megalomeront-forming species, calling for further studies on exo-erythrocytic development of haemosporidian parasites to explore the phylogenetic character of this trait.

The association between haemosporidian infection and non-breeding moult location in great reed warblers revisited by combining feather stable isotope profiles and geolocator data.

Stable isotope analysis provides valuable insights into the ecology of long-distance migratory birds during periods spent away from a specific study site. In a previous study, Swedish great reed warblers (Acrocephalus arundinaceus) infected with haemosporidian parasites differed in feather isotope ratios compared to non-infected birds, suggesting that infected and non-infected birds spent the non-breeding season in different locations or habitats. Here, we use a novel dataset comprising geolocator data, isotopes, and haemosporidian infection status of 92 individuals from four Eurasian populations to investigate whether parasite transmission varies with geography or habitats. We found that the probability of harbouring Plasmodium and Leucocytozoon parasites was higher in birds moulting in the eastern region of the non-breeding grounds. However, no geographic pattern occurred for Haemoproteus infections or overall infection status. In contrast to the previous study, we did not find any relationship between feather isotope ratios and overall haemosporidian infection for the entire current dataset. Plasmodium-infected birds had lower feather δ15N values indicating that they occupied more mesic habitats. Leucocytozoon-infected birds had higher feather δ34S values suggesting more coastal sites or wetlands with anoxic sulphate reduction. As the composition and prevalence of haemosporidian parasites differed between the old and the current dataset, we suggest that the differences might be a consequence of temporal dynamics of haemosporidian parasites. Our results emphasize the importance of replicating studies conducted on a single population over a restricted time period, as the patterns can become more complex for data from wider geographical areas and different time periods.

Haemosporidian parasite infections of Malagasy Philepittidae and Nectariniidae are driven by phylogeny rather than ecology.

The nectarivorous common sunbird asity (Neodrepanis coruscans) is phylogenetically closely related to the frugivorous velvet asity (Philepitta castanea), yet it shares similar habitat and foraging behaviour as the Malagasy sunbirds (Cinnyris spp.). As ecological factors have been shown to influence blood parasite prevalence, it should be tested whether parasite abundance, prevalence and diversity of N. coruscans are more similar to the sunbirds than to its relative. Therefore, blood samples (n = 156) and smears (n = 60) were tested for different blood parasites (Haemosporida, trypanosomes, filarioid nematodes) using molecular and microscopic methods. High prevalence of haemosporidian parasites was observed in all bird taxa, with rates ranging from 23% in N. coruscans to 84.6% in C. notatus. The Malagasy Cinnyris spp. exhibited a high occurrence of mixed haemosporidian infections (>76%) with various specialized lineages. Within the Philepittidae family, no Haemoproteus infection was detected and just a few cases of mixed infections. Nectariniidae species predominantly had specialized haemosporidian lineages, while Philepittidae had infections mainly caused by generalist lineages. These findings emphasize the diverse range of blood parasites in Nectariniidae, while additionally highlighting the high diversity of trypanosomes and filarioid nematodes in Philepittidae. Additionally, several newly discovered haemosporidian lineages, Trypanosoma isolates and filarioid nematode isolates were identified. Notably, Philepittidae exhibited a lower prevalence of avian haemosporidian parasites compared to Nectariniidae, possibly due to potential resistance mechanisms. Despite N. coruscans sharing similar habitat and behavioural ecology with both Cinnyris spp., it closely resembles its relative, P. castanea, in all aspects of haemosporidian parasitism.

Contribution to ecological environmental factors and the occurrence of haemosporidians in birds in Zhongar Alatau National Park, Kazakhstan.

In addition to the presence of a suitable host and vector, the prevalence of haemosporidians is influenced by several important factors, including the environmental conditions of the habitat, which depend on broader geographic characteristics. The aim of this study is to perform a preliminarily assessment of the distribution of blood parasites in birds from the mountainous area of Zhongar Alatau NP and to find potential new sites for research on their ecology in Kazakhstan. The results of this research constitute the first report on the occurrence of blood parasites from this area. A total of 58 birds, from the order Passeriformes and one individual from the order Caprimulgiformes, were examined during the study. The overall prevalence of infections caused by haemosporidian parasites (Haemoproteus, Leucocytozoon) was 18.6%. Neither the genus Plasmodium nor the presence of trypanosomes and microfilariae was detected in the birds examined. Three birds (5.1% prevalence) were infected with parasites of the genus Haemoproteus, in all eleven positive birds the analyses showed the presence of parasites of the genus Leucocytozoon (18.6% prevalence). The presence of parasites genus Haemoproteus was detected only in birds that were also infected with Leucocytozoon parasites. More infections with parasites of the genus Leucocytozoon are predicted due to the higher altitude and ecological factors at the capture sites, which are more favourable for the development of vectors of this genus. The species Haemoproteus majoris was detected in the host Emberiza cioides and species Haemoproteus minutus in host Turdus merula. Other species of this genus in the hosts Cyanistes cyanus and Turdus atrogularis were not determined. The species Leucocytozoon fringilinarum was detected in the hosts Cyanistes cyanus and Parus major, Leucocytozoon dubreuili was detected in Turdus atrogularis and Turdus merula. In the other host species Aegithalos caudatus, Emberiza cioides and Periparus aterus, it was not possible to dermine the species of the genus Leucocytozoon.

Natural Vector of Avian Haemoproteus asymmetricus Parasite and Factors Altering the Spread of Infection.

Avian haemosporidians (Apicomplexa, Haemosporida) are widespread blood protists, often causing severe haemosporidiosis, pathology, or even mortality in their hosts. Migrant birds regularly bring various haemosporidian parasites from wintering grounds to European breeding areas. Some haemosporidian parasites are prevalent in breeding sites and complete their life cycles in temperate climate zones and can be transmitted, but others do not. The factors altering the spread of these haemosporidians are not fully understood. Culicoides biting midges (Diptera: Ceratopogonidae) play an important role in the transmission of worldwide distributed avian haemosporidian parasites belonging to the genus Haemoproteus, but this information is particularly scarce and insufficient. The key factors limiting the spread of these pathogens in temperate climate zones, which we suspect and aim to study, are the absence of susceptible vectors and the ecological isolation of birds from vectors during the breeding period when transmission occurs. The primary objective of this study was to evaluate how the habitats of biting midges and bird breeding sites influence parasite transmission while also seeking to expand our understanding of the natural vectors for these parasites. Biting midges were collected using UV traps on the Curonian Spit, Lithuania, in different habitats, such as woodland and reeds, from May to September. Parous Culicoides females were identified, dissected, and investigated for the presence of Haemoproteus parasites using both microscopy and PCR-based tools. Among the dissected 1135 parous Culicoides females, the sporozoites of Haemoproteus asymmetricus (genetic lineage hTUPHI01) have been detected for the first time in the salivary glands of Culicoides festivipennis. The sporozoites of four Haemoproteus lineages were detected in Culicoides segnis, C. festivipennis, and Culicoides kibunensis biting midges. PCR-based screening showed that the females of seven Culicoides species were naturally infected with Haemoproteus parasites. The DNA of the parasite of owls, Haemoproteus syrnii (hSTAL2), was detected for the first time in Culicoides punctatus. The highest abundance of collected Culicoides females was in June, but the highest prevalence of Haemoproteus parasites in biting midges was in July. The abundance of Culicoides was higher in the woodland compared with reeds during the season. The acquired findings indicate the varied abundance and diversity of biting midges throughout the season and across distinct habitats. This variability could potentially impact the transmission of Haemoproteus parasites among birds with diverse breeding site ecologies. These outcomes hold the potential to enhance our understanding of the epizootiology of Haemoproteus infections within temperate climatic zones.

Unravelling the mosquito-haemosporidian parasite-bird host network in the southwestern Iberian Peninsula: insights into malaria infections, mosquito community and feeding preferences.

BAKGROUND: Vector-borne diseases affecting humans, wildlife and livestock have significantly increased their incidence and distribution in the last decades. Because the interaction among vectors-parasite-vertebrate hosts plays a key role driving vector-borne disease transmission, the analyses of the diversity and structure of vector-parasite networks and host-feeding preference may help to assess disease risk. Also, the study of seasonal variations in the structure and composition of vector and parasite communities may elucidate the current patterns of parasite persistence and spread as well as facilitate prediction of how climate variations may impact vector-borne disease transmission. Avian malaria and related haemosporidian parasites constitute an exceptional model to understand the ecology and evolution of vector-borne diseases. However, the characterization of vector-haemosporidian parasite-bird host assemblages is largely unknown in many regions. METHODS: Here, we analyzed 5859 female mosquitoes captured from May to November in five localities from southwestern Spain to explore the composition and seasonal variation of the vector-parasite-vertebrate host network. RESULTS: We showed a gradual increase in mosquito abundance, peaking in July. A total of 16 different haemosporidian lineages were found infecting 13 mosquito species. Of these assemblages, more than 70% of these vector-parasite associations have not been described in previous studies. Moreover, three Haemoproteus lineages were reported for the first time in this study. The prevalence of avian malaria infections in mosquitoes varied significantly across the months, reaching a maximum in November. Mosquito blood-feeding preference was higher for mammals (62.5%), whereas 37.5% of vectors fed on birds, suggesting opportunistic feeding behavior. CONCLUSION: These outcomes improve our understanding of disease transmission risk and help tovector control strategies.

Mitochondrial genome amplification of avian haemosporidian parasites from single-infected wildlife samples using a novel nested PCR approach.

Haemosporidian parasites that infect birds (Apicomplexa: Haemosporida) are blood parasites that require an invertebrate host (vector) and a vertebrate host for their lifecycle and cause malaria-like diseases. This group of parasites has provided valuable insights into host specificity, virulence, and parasite dispersal. Additionally, they have played a significant role in reshaping our understanding of the evolutionary history of apicomplexans. In order to accurately identify species and to address phylogenetic questions such as the timing of the haemosporidian radiation, the use of a sufficiently large genetic data set is crucial. However, acquiring this genetic data poses significant challenges. In this research, a sensitive nested PCR assay was developed. This assay allows for the easy amplification of complete mitochondrial genomes of haemosporidian parasites in birds, even during the chronic stage of infection. The effectiveness of this new nested PCR assay was evaluated using blood and tissue samples of birds with verified single parasite infections from previous studies. The approach involves amplifying four overlapping fragments of the mitochondrial genome and requires DNA extracts from single-infected samples. This method successfully amplified the complete mitochondrial genomes of 24 distinct haemosporidian parasite lineages found in various bird species. This data is invaluable for conducting phylogenetic analyses and accurately defining species. Furthermore, this study proposes the existence of at least 15 new haemosporidian parasite species based on the genetic information obtained. Data regarding pGRW04, previously categorized as Plasmodium relictum like pSGS1 and pGRW11, indicates that the pGRW04 lineage is actually a separate, hidden Plasmodium species.

Exo-erythrocytic development of Leucocytozoon parasites (Haemosporida, Leucocytozoidae) in song thrushes Turdus philomelos.

Leucocytozoon parasites (Haemosporida, Leucocytozoidae) are haemosporidians whose diversity, exo-erythrocytic development and potential vectors are the least studied. The knowledge about their exo-erythrocytic development and pathogenicity is fragmentary, resulting in an incomplete comprehension of the impact of these parasites on avian hosts. For a long time, Leucocytozoon infections were considered benign to wild birds, even though they were virulent in poultry and responsible for some wild bird population declines. This study aimed to investigate the presence of Leucocytozoon species exo-erythrocytic stages in song thrushes Turdus philomelos using conventional histological techniques (sections stained by H&E) and chromogenic in situ hybridization (CISH). Tissues from ten birds (seven naturally infected and three opportunistic samplings) were examined using both methods. Parasite lineages were identified from blood samples using PCR-based techniques. Leucocytozoon species meronts were found in five individuals (in four birds using H&E staining protocol, and in three in CISH-treated histological sections). Meronts were found mainly in the kidneys, but some meronts were also present in the lungs. It was possible to observe different maturation stages of meronts in the same bird individual, indicating an asynchronous development. Cytomeres were readily visible in developing meronts. One megalomeront-like structure was present close to a blood vessel in the heart. It was covered with a prominent capsular-like wall. No inflammatory reaction or necrosis was seen in the tissues surrounding the meronts or the megalomeront-like structure. We could confirm the transmission of three Leucocytozoon lineages (lTUPHI14, lSTUR1 and lTUPHI13) in Europe, and add evidence of the transmission of two Plasmodium lineages, including Plasmodium circumflexum (pTURDUS1), and Haemoproteus asymmetricus (hTUPHI01). We call for further research to better understand Leucocytozoon parasite exo-erythrocytic development.

Avian Haemosporidians Infecting Short- and Long-Distance Migratory Old World Flycatcher Species and the Variation in Parasitaemia After Endurance Flights.

PURPOSE: Avian haemosporidians are widespread parasites, occurring in many bird families and causing pathologies ranging from rather benign infections to highly virulent diseases. The state of knowledge about lineage-specific intensities of haemosporidian infection (i.e., parasitaemia) is mainly based on infection experiments conducted under laboratory conditions. The levels and range of parasitaemia in natural host-parasite associations as well as their influencing factor remain largely unexplored. METHODS: Thus, we explored the parasitaemia of four songbird species (i.e., European Robins, Black and Common Redstarts and Whinchats) during migration by screening individuals upon landing on an insular passage site after extensive endurance flights to (1) describe their natural host-parasite associations, (2) quantify parasitaemia and (3) explore potential host- and parasite-related factors influencing parasitaemia. RESULTS: We found 68% of Whinchats to be infected with haemosporidians, which is more frequent than any other of the studied host species (30-34%). Furthermore, we confirmed that parasitaemia of Haemoproteus infections was higher than average Plasmodium infections. Median parasitaemia levels were rather low (parasite cells in 0.01% of hosts' red blood cells) and varied largely among the different parasite lineages. However, we found four individuals hosting infections with parasitaemia higher than typical chronic infections. CONCLUSIONS: Based on the known transmission areas of the respective lineages, we argue that these higher intensity infections might be relapses of consisting infections rather than acute phases of recent primary infections.

Haemoproteus parasites and passerines: the effect of local generalists on inferences of host-parasite co-phylogeny in the British Isles.

Host­parasite associations provide a benchmark for investigating evolutionary arms races and antagonistic coevolution. However, potential ecological mechanisms underlying such associations are difficult to unravel. In particular, local adaptations of hosts and/or parasites may hamper reliable inferences of host­parasite relationships and the specialist­generalist definitions of parasite lineages, making it problematic to understand such relationships on a global scale. Phylogenetic methods were used to investigate co-phylogenetic patterns between vector-borne parasites of the genus Haemoproteus and their passeriform hosts, to infer the ecological interactions of parasites and hosts that may have driven the evolution of both groups in a local geographic domain. As several Haemoproteus lineages were only detected once, and given the occurrence of a single extreme generalist, the effect of removing individual lineages on the co-phylogeny pattern was tested. When all lineages were included, and when all singly detected lineages were removed, there was no convincing evidence for host­parasite co-phylogeny. However, when only the generalist lineage was removed, strong support for co-phylogeny was indicated, and ecological interactions could be successfully inferred. This study exemplifies the importance of identifying locally abundant lineages when sampling host­parasite systems, to provide reliable insights into the precise mechanisms underlying host­parasite interactions.

Bird species with wider geographical ranges have higher blood parasite diversity but not prevalence across the African-Eurasian flyway.

Avian blood parasites, from the genera Plasmodium, Haemoproteus and Leucocytozoon, are predicted to alter their range and prevalence as global temperatures change, and host and vector ranges shift. Understanding large-scale patterns in the prevalence and diversity of avian malaria and malaria-like parasites is important due to an incomplete understanding of their effects in the wild, where studies suggest even light parasitaemia can potentially cause rapid mortality, especially in naïve populations. We conducted phylogenetically controlled analyses to test for differences in prevalence and lineage diversity of haemoparasite infection (for Plasmodium, Haemoproteus and Leucocytozoon) in and between resident and migratory species along the African-Eurasian flyway. To test whether migratory strategy or range size drives differences in parasite prevalence and diversity between resident and migrant species, we included three categories of resident species: Eurasian only (n = 36 species), African only (n = 41), and species resident on both continents (n = 17), alongside intercontinental migrants (n = 64), using a subset of data from the MalAvi database comprising 27,861 individual birds. We found that species resident on both continents had a higher overall parasite diversity than all other categories. Eurasian residents had lower Plasmodium diversity than all other groups, and both migrants and species resident on both continents had higher Haemoproteus diversity than both African and Eurasian residents. Leucocytozoon diversity did not differ between groups. Prevalence patterns were less clear, with marked differences between genera. Both Plasmodium and Leucocytozoon prevalence was higher in species resident on both continents and African residents than in migrants and Eurasian residents. Haemoproteus prevalence was lower in Eurasian residents than species resident on both continents. Our findings contrast with previous findings in the North-South American flyway, where long-distance migrants had higher parasite diversity than residents and short-distance migrants, although we found contrasting patterns for parasite diversity to those seen for parasite prevalence. Crucially, our results suggest that geographic range may be more important than migratory strategy in driving parasite diversity within species along the African-Palaearctic flyway. Our findings differ between the three parasite genera included in our analysis, suggesting that vector ecology may be important in determining these large-scale patterns. Our results add to our understanding of global patterns in parasite diversity and abundance, and highlight the need to better understand the influence of vector ecology to understand the drivers of infection risk and predict responses to environmental change.