Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites.

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.

Unresolved haemosporidia of the Australian skink, Egernia stokesii.

The Australian skink Egernia stokesii had been recognised as a host of two species of Plasmodium, Plasmodium mackerrasae and P. circularis; nevertheless, molecular data are available for only a single haemosporidian species of this host. Its sequences are labelled as "Plasmodium sp." or "Plasmodium mackerrasae", but morphological characteristics of this isolate are unavailable. Phylogenetic analyses of these sequences placed them into the clade of the genus Haemocystidium. In this study, blood samples of six E. stokesii were analysed by both, molecular and microscopic methods to clarify the haemosporidia of this lizard. Application of these approaches offered discordant results. Whereas sequence analysis clustered our isolates with lizard species of Haemocystidium, morphology of blood stages is more akin to Plasmodium than Haemocystidium. However, limited sampling, indistinguishable nuclei/merozoites and risk of possible hidden presence of mixed infection prevent reliable species identification of detected parasites or their description as new species of Haemocystidium.

A new long-read mitochondrial-genome protocol (PacBio HiFi) for haemosporidian parasites: a tool for population and biodiversity studies.

BACKGROUND: Studies on haemosporidian diversity, including origin of human malaria parasites, malaria's zoonotic dynamic, and regional biodiversity patterns, have used target gene approaches. However, current methods have a trade-off between scalability and data quality. Here, a long-read Next-Generation Sequencing protocol using PacBio HiFi is presented. The data processing is supported by a pipeline that uses machine-learning for analysing the reads. METHODS: A set of primers was designed to target approximately 6 kb, almost the entire length of the haemosporidian mitochondrial genome. Amplicons from different samples were multiplexed in an SMRTbell® library preparation. A pipeline (HmtG-PacBio Pipeline) to process the reads is also provided; it integrates multiple sequence alignments, a machine-learning algorithm that uses modified variational autoencoders, and a clustering method to identify the mitochondrial haplotypes/species in a sample. Although 192 specimens could be studied simultaneously, a pilot experiment with 15 specimens is presented, including in silico experiments where multiple data combinations were tested. RESULTS: The primers amplified various haemosporidian parasite genomes and yielded high-quality mt genome sequences. This new protocol allowed the detection and characterization of mixed infections and co-infections in the samples. The machine-learning approach converged into reproducible haplotypes with a low error rate, averaging 0.2% per read (minimum of 0.03% and maximum of 0.46%). The minimum recommended coverage per haplotype is 30X based on the detected error rates. The pipeline facilitates inspecting the data, including a local blast against a file of provided mitochondrial sequences that the researcher can customize. CONCLUSIONS: This is not a diagnostic approach but a high-throughput method to study haemosporidian sequence assemblages and perform genotyping by targeting the mitochondrial genome. Accordingly, the methodology allowed for examining specimens with multiple infections and co-infections of different haemosporidian parasites. The pipeline enables data quality assessment and comparison of the haplotypes obtained to those from previous studies. Although a single locus approach, whole mitochondrial data provide high-quality information to characterize species pools of haemosporidian parasites.

Molecular detection of blood protozoa and identification of black flies of the Simulium varicorne species group (Diptera: Simuliidae) in Thailand.

Species of the Simulium varicorne group in Thailand have veterinary significance as vectors of haemosporidian parasites. Accurate identification is, therefore, critical to the study of vectors and parasites. We used morphology and molecular markers to investigate cryptic genetic lineages in samples identified as Simulium chumpornense Takaoka & Kuvangkadilok, 2000. We also tested the efficiency of the nuclear internal transcribed spacer 2 (ITS2) marker for the identification of species in this group. Morphological examinations revealed that S. chumpornense lineage A is most similar to S. khelangense Takaoka, Srisuka & Saeung, 2022, with minor morphological differences. They are also genetically similar based on mitochondrial cytochrome c oxidase I (COI) sequences. Geographically, the sampling site where paratypes of S. khelangense were originally collected is <50 km from where S. chumpornense lineage A was collected. We concluded that cryptic lineage A of S. chumpornense is actually S. khelangense. COI sequences could not differentiate S. kuvangkadilokae Pramual and Tangkawanit, 2008 from S. chumpornense and S. khelangense. In contrast, ITS2 sequences provided perfect accuracy in the identification of these species. Molecular analyses of the blood protozoa Leucocytozoon and Trypanosoma demonstrated that S. khelangense carries L. shoutedeni, Leucocytozoon sp., and Trypanosoma avium. The Leucocytozoon sp. in S. khelangense differs genetically from that in S. asakoae Takaoka & Davies, 1995, signaling the possibility of vector-parasite specificity.

High prevalence of haemosporidian parasites in Eurasian jays.

Avian haemosporidians are vector-borne parasites, infecting a great variety of birds. The order Passeriformes has the highest average infection probability; nevertheless, some common species of Passeriformes have been rather poorly studied. We investigated haemosporidians in one such species, the Eurasian jay Garrulus glandarius (Corvidae), from a forest population in Hesse, Central Germany. All individuals were infected with at least one haemosporidian genus (overall prevalence: 100%). The most common infection pattern was a mixed Haemoproteus and Leucocytozoon infection, whereas no Plasmodium infection was detected. Results on lineage diversity indicate a rather pronounced host-specificity of Haemoproteus and Leucocytozoon lineages infecting birds of the family Corvidae.

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.

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 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.

Avian haemosporidians of breeding birds in the Davis Mountains sky-islands of west Texas, USA.

Avian haemosporidians are protozoan parasites transmitted by insect vectors that infect birds worldwide, negatively impacting avian fitness and survival. However, the majority of haemosporidian diversity remains undescribed. Quantifying this diversity is critical to determining parasite­host relationships and host-switching potentials of parasite lineages as climate change induces both host and vector range shifts. In this study, we conducted a community survey of avian haemosporidians found in breeding birds on the Davis Mountains sky islands in west Texas, USA. We determined parasite abundance and host associations and compared our results to data from nearby regions. A total of 265 birds were screened and infections were detected in 108 birds (40.8%). Most positive infections were identified as Haemoproteus (36.2%), followed by Plasmodium (6.8%) and Leucocytozoon (0.8%). A total of 71 haemosporidian lineages were detected of which 39 were previously undescribed. We found that regional similarity influenced shared lineages, as a higher number of lineages were shared with avian communities in the sky islands of New Mexico compared to south Texas, the Texas Gulf Coast and central Mexico. We found that migratory status of avian host did not influence parasite prevalence, but that host phylogeny is likely an important driver.

Very low prevalence of haemosporidian parasites in two species of marsh terns.

Vector-transmitted haemosporidians are among the most common parasites in birds, but our knowledge of the inter-specific patterns of infection rates and the parasite community composition is far from complete because of the unequal distribution of the screening effort across bird families and genera. To assess infection rates and the diversity of haemosporidians from the genera Plasmodium, Haemoproteus, and Leucocytozoon in marsh terns, which represent poorly explored in this regard genus of the family gulls, terns, and skimmers (Laridae), we screened two species: the Whiskered Tern (Chlidonias hybrida) and the Black Tern (Chlidonias niger). We sampled these long-distance migratory birds on breeding grounds: the Whiskered Tern in south-central Poland and north-central Ukraine, and the Black Tern-in north-central Ukraine. We found that birds from both species were infected only sporadically, with prevalence at the population level not exceeding 3.4%. Only parasites from the genera Plasmodium and Leucocytozoon were detected. There was neither an inter-specific difference nor a difference between populations of the Whiskered Tern in infection rates. In total, we registered three lineages-one Plasmodium and two Leucocytozoon-that were previously recorded in other bird species, and two unidentified Plasmodium infections. One of the lineages (Leucocytozoon LARCAC02) represents a specialist parasite with the host range restricted to larids and geographic range restricted to Poland, and two others (Plasmodium SGS1 and Leucocytozoon CIAE02) represent generalist parasites with very broad host and geographic ranges. This study reinforces the existing evidence that terns host parasites from genera Haemoproteus, Plasmodium, and Leucocytozoon only sporadically.

Prevalence of Leucocytozoon infection in domestic birds in Ghana.

Leucocytozoon is a haemosporidian parasite known to cause leucocytozoonosis in domestic and wild birds in most parts of the world. It is an important pathogen, as some species can be pathogenic, especially in domestic birds. One of the factors affecting poultry health management worldwide is parasitism. However, the study of haemosporidian parasites in Ghana is still lacking. This study sought to assess the prevalence and diversity of Leucocytozoon parasites in domestic birds in Ghana. Blood samples were collected from domestic birds in Ghana's Bono and Eastern regions to screen for Leucocytozoon parasites. Thin blood smears were prepared for microscopy and DNA was extracted from whole blood kept in ethylenediaminetetraacetic acid (EDTA) tubes for PCR. Due to the large number of samples, real-time PCR was performed to amplify the conserved rDNA gene. Two different nested PCR protocols were performed on the positive samples obtained from real-time PCR results, to amplify a partial region of the mitochondrial cytochrome b gene and the amplicons were sequenced. Sequencing revealed six new lineages of Leucocytozoon sp. recovered in 976 individual domestic birds and these sequences were deposited in the National Center for Biotechnology Information (NCBI) GenBank. An overall Leucocytozoon prevalence of 11.6% was reported in all birds sampled. The most prevalent lineage LGHA146 (GenBank accession no. OM643346) (93.8%) was found infecting 3 bird species, Gallus gallus, Meleagris gallopavo, and Anas platyrhynchos. Phylogenetic analysis revealed that the new lineages (GenBank accession nos. OM643342, OM643343, OM643344, OM643345, OM643346, and OM643347), reported in this study were closely related to Leucocytozoon schoutedeni. We suggest that further studies be conducted to evaluate the effect of these parasite species on the general well-being of poultry in Ghana.

Complete mitochondrial genome analyses confirm that bat Polychromophilus and ungulate Plasmodium constitute a distinct clade independent of other Plasmodium species.

In recent phylogenetic studies, bat Polychromophilus and ungulate Plasmodium, two relatively understudied haemosporidian parasites within the Apicomplexa phylum, have often been overlooked. Instead, the focus has been primarily on haemosporidian parasites in primates, rodents, and birds. Several phylogenetic analyses of bat Polychromophilus have relied on limited datasets and short informative DNA sequences. As a result of these inherent limitations, the substantiation of their evolutionary stance has encountered a diminished degree of robust validation. This study successfully obtained complete mitochondrial genome sequences from 11 Polychromophilus parasites originating from Hipposideros gentilis and Myotis siligoensis bats for the first time. Additionally, the authors have sequenced the apicoplast caseinolytic protease C genes from Polychromophilus murinus and a potentially new Polychromophilus species. These mitochondrial genomes range in length from 5994 to 6001 bp and consist of three protein-coding genes (PCGs), seven small subunit ribosomal RNA genes (SSU rRNA), 12 large subunit ribosomal RNA genes (LSU rRNA), and seven miscellaneous RNA genes. Phylogenetic analyses using Bayesian Inference and Maximum Likelihood methods indicated robust support for the grouping of ungulate Plasmodium and bat Polychromophilus in a single clade separate from other Plasmodium spp., confirming previous reports, albeit with stronger evidence in this study. The divergence between Polychromophilus in bats and Plasmodium in ungulates occurred approximately 29.61 to 55.77 million years ago (Mya), with a node age estimated at 40.63 Mya. These findings highlight that the genus Plasmodium, which includes species found in ungulates, birds, reptiles, and other mammals, does not form a monophyletic group. By incorporating Polychromophilus in bats and Plasmodium in ungulates, this study contributes significantly to understanding the phylogenetic relationships within the Haemosporida order. It provides valuable insights into the evolutionary history and interconnections among these diverse parasites, thereby expanding knowledge in this field.

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.

Avian Plasmodium spp. and Haemoproteus spp. parasites in mosquitoes in Germany.

BACKGROUND: Although haemosporidian parasites may cause considerable health and economic problems in aviaries, there is limited understanding of the vectors transmitting them. Mosquito-borne Plasmodium species are responsible for the deaths of numerous exotic (= immunologically naïve) birds in zoos every year, while native birds are adapted to the parasites and largely protected by an effective immune response. METHODS: Mosquitoes were collected in bird/animal parks, wetlands and private gardens in various regions of Germany from 2020 to 2022. Females were pooled with up to 10 specimens according to taxon, location and date. Extracted DNA was screened for avian Haemosporida-specific mitochondrial rDNA using real-time polymerase chain reaction (PCR). Positive samples were amplified by a Plasmodium/Haemoproteus-specific nested PCR targeting the partial cytochrome b gene, followed by sequencing of the PCR product for species identification. Sequences were checked against GenBank and MalAvi databases. RESULTS: PCR of 2633 pools with 8834 female mosquitoes signalled infection with Plasmodium in 46 pools and with Haemoproteus in one pool. Further amplification and sequencing demonstrated the occurrence of Haemoproteus majoris lineage PARUS1 (n = 1) as well as several Plasmodium species and lineages, including Plasmodium relictum SGS1 (n = 16) and GRW11 (n = 1), P. matutinum LINN1 (n = 13), P. vaughani SYAT05 (n = 10), P. circumflexum TURDUS01 (n = 3), P. cathemerium PADOM02 (n = 1) and Plasmodium sp. SYBOR02 (n = 1) and PLOPRI01 (n = 1). The infections were detected in Culex pipiens sensu lato (n = 40), Culiseta morsitans/fumipennis (n = 6) and Aedes cinereus/geminus (n = 1). CONCLUSIONS: Although the overall Plasmodium minimum infection rate (5.2) appears to be low, the results demonstrated not only the ongoing circulation of Plasmodium parasites in the German mosquito population, but also the occurrence of eight distinct Plasmodium lineages, with three of them (PADOM02, SYBOR02, PLOPRI01) being detected in Germany for the first time. This study highlights the importance of conducting mosquito-borne pathogen surveillance studies simultaneously targeting vectors and vertebrate hosts, as certain species may be detected more readily in their vectors than in their vertebrate hosts, and vice versa.

A non-invasive feather-based methodology for the detection of blood parasites (Haemosporida).

Blood parasite (haemosporidian) infections are conventionally detected using blood samples; this implies capturing and handling birds to obtain them, which induces stress and causes pain. Feathers have blood vessels, and some blood could be preserved in the feather's shaft after moulting. We used feather DNA for detecting haemosporidians by PCR testing in diverse scenarios. First, haemosporidian DNA was detected in feathers from carcasses of infected birds, proving the feasibility of the approach. Storage temperature affected DNA recovery, with maximum retrieval and haemosporidian detection at the lowest temperature (- 20 °C). All feather types from infected birds kept at optimal conditions yielded haemosporidian DNA. Parasite detection by PCR was correlated with DNA yield, which was significantly higher in heavier birds, flight feathers, and more feathers per pool. Lastly, haemosporidians were detected employing feathers moulted from wild and captive birds to estimate infection prevalence. We show for the first time that using blood from feather shafts for haemosporidian detection can be an advantageous and less invasive alternative to blood sampling if feathers are optimally preserved. This method could contribute to uncovering haemosporidian infections in endangered and elusive birds, and it might facilitate routine screening in captive birds, thereby improving infection detection, prevention, and control.

Leucocytozoon cariamae n. sp. and Haemoproteus pulcher coinfection in Cariama cristata (Aves: Cariamiformes): first mitochondrial genome analysis and morphological description of a leucocytozoid in Brazil.

The distribution of avian haemosporidians of the genus Leucocytozoon in the Neotropics remains poorly understood. Recent studies confirmed their presence in the region using molecular techniques alone, but evidence for gametocytes and data on putative competent hosts for Leucocytozoon are still lacking outside highland areas. We combined morphological and molecular data to characterize a new Leucocytozoon species infecting a non-migratory red-legged seriema (Cariama cristata), the first report of a competent host for Leucocytozoon in Brazil. Leucocytozoon cariamae n. sp. is distinguished from the Leucocytozoon fringillinarum group by its microgametocytes that are not strongly appressed to the host cell nucleus. The bird studied was coinfected with Haemoproteus pulcher, and we present a Bayesian phylogenetic analysis based on nearly complete mitochondrial genomes of these 2 parasites. Leucocytozoon cariamae n. sp. morphology is consistent with our phylogenetic analysis indicating that it does not share a recent common ancestor with the L. fringillinarum group. Haemoproteus pulcher and Haemoproteus catharti form a monophyletic group with Haemocystidium parasites of Reptilia, supporting the polyphyly of the genus Haemoproteus. We also discussed the hypothesis that H. pulcher and H. catharti may be avian Haemocystidium, highlighting the need to study non-passerine parasites to untangle the systematics of Haemosporida.

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.

Molecular investigation of Haemoproteus and Plasmodium species of some raptors in Hatay province: new CytB lineages for raptors of Accipitriformes in Turkey.

Accipitriform raptors are significant indicators of biodiversity and environmental health. Currently, most of the studies on avian haemosporidian parasites are on passerine birds, and data on raptors is constricted, with similarities both around the world and in Turkey. This study aimed to investigate the presence of Haemoproteus and Plasmodium spp. in raptors by microscopy and nested PCR technique. The study material consisted of 47 accipitriform raptors (Buteo buteo: 14, Buteo rufinus: 7, Clanga pomarina: 8, Circaetus gallicus: 12, Milvus migrans: 6). The prevalence of haemosporidian infection was 12.8% (6/47, 1 from Buteo buteo, 4 from Clanga pomarina, 1 from Milvus migrans) microscopically and 14.9% (7/47) molecularly. One Circaetus gallicus, microscopically found to be negative, probably due to low parasitemia, was molecularly found to be positive. All PCR-positive amplicons were bidirectionally sequenced, and the identification of lineages of the isolates and phylogenetic analysis were performed using the MalAvi and GenBank databases. The study revealed H-MILANS02 lineage in Buteo buteo, H-MILANS02 and P-CIAE1 lineages in Clanga pomarina, P-GRW06 lineage in Circaetus gallicus, and P-RTSR1 lineage in Milvus migrans, respectively. While this study removes the uncertainty regarding the reporting of the H-MILANS02 lineage in Turkey, it is also the first report to reveal 3 different Plasmodium spp. CytB lineages in raptors. Moreover, the fact that the P-GRW06 lineage (Plasmodium elongatum) detected in passerine birds was detected in a raptor, Circaetus gallicus, draws attention to the need for further investigations on host-parasite interaction and gives clues about the host-shifting ability of this parasite.

Comparative performance of microscopy, nested PCR, and real-time PCR for screening avian haemosporidian parasites in Afrotropical starlings (family Sturnidae).

Prevalence studies of avian haemosporidian parasites frequently use microscopy and the nested polymerase chain reaction (PCR) protocols for detecting infections. Newer PCR protocols to detect parasites are being developed, with the distinct advantage of reducing screening cost and time, as well as increasing efficiency and sensitivity. The detection ability of microscopy and nested PCR was compared against a real-time PCR (qPCR) protocol using genomic DNA extracted from 240 bird blood samples collected from three starling species (Cape Starling, the Greater Blue-eared Starling, and the Wattled Starling; family Sturnidae) in the Kruger national park, South Africa. All three protocols successfully detected avian haemosporidian parasites with the qPCR having a considerable edge against the other two methods. Fifteen unique cytochrome b lineages were identified of which seven were new lineages. Microscopy and nested PCR recorded similar prevalence (32.92% and 35.42% respectively). The qPCR protocol used here, although more sensitive (52.92% prevalence), is not able to differentiate between parasite genera but provides the opportunity to screen a large number of samples in large-scale studies within a specific region. This study recommends the development and adoption of new molecular protocols with increased sensitivity and accuracy in prevalence studies. Nevertheless, microscopy remains essential for the morphological description of parasites and for distinguishing between abortive and successful chronic infections. The PCR-based method displays the detection of the parasitic genome but does not reveal whether parasites have or will develop into a successful infection.