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The role of plumage ornamentation as a signal of parasite infection is a key issue in the evolution of animal behaviour. Despite the undeniable role of host–parasite interactions in sexual selection, it is still unclear how parasites influence the variance in the expression of females displaying multiple ornaments. Here, by taking advantage of a long-term and individual-based, monitored population of pied flycatchers, Ficedula hypoleuca , in a Mediterranean area, we investigated the variation in the expression of wing patch (size) and forehead patch (occurrence and size) in relation to infections with haemosporidian parasites in breeding adult females. Haemosporidian infection was related to wing patch size, and this association varied in direction and magnitude depending on the origin of birds (locally born versus immigrant) and the type of breeding habitat (oakwood versus pine plantation). Specifically, differences in wing patch size were most evident among immigrant females breeding in the pine plantation, with noninfected females expressing larger wing patches than infected ones. Regarding the forehead patch, its occurrence was also modulated by haemosporidian infection through two-way interactions with bird origin (lower occurrence in locally born females) and breeding habitat type (lower occurrence among infected than uninfected females in the oakwood, whereas the opposite was true in the pine plantation). Only bird age predicted forehead patch size. Therefore, in addition to haemosporidian infection, the expression of these ornaments was related to extrinsic (breeding habitat) and intrinsic (age, origin) factors, which suggests the existence of trade-offs between physiological responses to infection and the expression of secondary sexual traits. Overall, our results suggest that plumage ornaments provide independent and reliable information on the haemosporidian infection status of female pied flycatchers. • Whether plumage ornaments are a sign of parasitic infections in females is unclear. • We assessed the links between two female ornaments and haemosporidian infection. • The expression of both ornaments was related to infection, origin, habitat and age. • However, the correlations between these factors differed depending on the ornament. • This suggests different signals for ornamentation – infection response trade-offs. [ABSTRACT FROM AUTHOR]

Haemosporidian parasites, which encompass Haemoproteus, Plasmodium, and Leucocytozoon, are intracellular parasites commonly found in the blood and other tissues of birds worldwide. The prevalence of these parasites can vary depending on the bird species and the habitats they inhabit. Notably, the highest diversity of Haemosporidian parasites has been observed within the Passeriformes order of birds. In our study, we collected 145 blood samples from wild songbirds in the western region of Iran during the period from April to November 2021-2022. Blood smears were prepared using the push-slide method and subsequently stained with Giemsa. The stained smears were then examined under a light microscope, and all samples were screened using a nested PCR protocol that targeted a 479-base-pair segment of the mitochondrial cytochrome b (cytb) gene of Haemoproteus, Plasmodium, and Leucocytozoon species. Among the seven songbird families we sampled, we detected blood parasites in 31 individuals, representing an infection rate of 21.4% (95% CI: 0.1501 to 0.2896). These infected individuals belonged to five different families, and a total of 21 lineages were identified, including thirteen for Leucocytozoon spp, seven for Haemoproteus spp, and one for Plasmodium spp. Notably, the highest infection rates were observed in the Paridae family (9.7%, 95% CI: 0.0537 to 0.1567) and the Motacillidae family (4.8%, 95% CI: 0.0196 to 0.0970), while the lowest infection rates were recorded in the Corvidae family (1.4%, 95% CI: 0.0016 to 0.0490) and the Acrocephalidae family (1.4%, 95% CI: 0.0016 to 0.0490). Additionally, our study documented two new host species for Leucocytozoon spp. (Motacilla flava and Acrocephalus melanopogon), marking the first such observations globally. We also established five new host records (Motacilla flava, Pica pica, Acrocephalus melanopogon, Poecile lugubris, and Acrocephalus agricola) for avian haemosporidian parasites in the western region of Iran. These findings underscore the remarkable lineage diversity within the Leucocytozoon genus compared to other haemosporidian parasites in songbirds in western Iran, setting our study apart from previous research in Iran. [ABSTRACT FROM AUTHOR]

Abstract 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.

Simple Summary: Feathers are essential for a bird's flight, insulation, communication, and camouflage. They degrade over time, so birds must moult regularly. This study examined how avian haemosporidian infection and the size of the uropygial gland affect feather quality and growth rate in two migratory bird species in southwestern Spain—the house martin (Delichon urbicum) and the sand martin (Riparia riparia). We found that house martins had the highest haemosporidian infection rates, likely due to their large colony size. Infection only decreased feather quality in house martins and did not affect the feather growth rate in any of the two hirundinids. Additionally, feather growth rate was positively linked to feather quality, but only in house martins. Finally, we found no connection between the uropygial gland size and feather quality or feather growth rate. These results show, for the first time, that avian haemosporidian parasites can negatively impact the feather quality of migratory birds, thus potentially affecting their flight and survival. Further research is needed to fully understand these relationships. Bird feathers have several functions, including flight, insulation, communication, and camouflage. Since feathers degrade over time, birds need to moult regularly to maintain these functions. However, environmental factors like food scarcity, stress, and parasite infections can affect feather quality and moult speed. This study examined the impact of avian haemosporidian infection and uropygial gland volume, as well as feather quality and feather growth rate in two migratory hirundine species captured in southwestern Spain—the house martin (Delichon urbicum) and sand martin (Riparia riparia). Our findings showed that the prevalence of infection varied among species, with house martins having the highest rates, possibly due to their larger colony size. Moreover, haemosporidian infection had a different impact on each species; infected house martins exhibited lower feather quality than healthy individuals, although this outcome was not observed in sand martins. Furthermore, no effect of infection on feather growth rate was observed in both hirundinids. Additionally, feather growth rate only correlated positively with feather quality in house martins. Finally, no link was observed between uropygial gland volume and feather quality or feather growth rate in any of the species in this study. These findings highlight the effect of haemosporidian infections on the plumage of migratory birds, marking, for the first time, how avian haemosporidian infection is shown to adversely impact feather quality. Even so, further research is needed to explore these relationships more deeply. [ABSTRACT FROM AUTHOR]

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 genusspecific 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Over thirty percent of bird species are undergoing population declines and are threatened with extinction in Portugal. Several reasons have been proposed to explain this decrease, such as the impact of human activities on natural environments and pathogens affecting the health of wildlife, domestic animals, and humans. Wildlife rescue and rehabilitation centres play an essential role in the conservation of endangered species. Despite wildlife rehabilitation centres providing valuable information on disease prevalence and transmission, the information on haemosporidian infection is still very scarce for birds admitted in these centres. In this study, we discovered new malaria parasites in birds admitted to wildlife rehabilitation centres in Portugal. We also revealed infection in bird species that were previously unknown to be infected with malaria parasites. Birds admitted to rehabilitation due to debilitating disease were more frequently infected with malaria. Furthermore, we demonstrate that the malaria infection extends the required period for medical treatment in these birds, which imposes additional economic costs for the rehabilitation and reduces the survival probabilities of the bird. These findings stress the importance of the study of malaria parasites in wildlife rehabilitation centres, also helping to design protocols and interventions to preserve endangered species. In the last decade, over 40% of bird species in Europe have experienced poor and bad conservation status, with more than 30% of bird species in mainland Portugal threatened with extinction. Along with anthropogenic factors, parasites and pathogens such as avian haemosporidians have been suggested to be responsible for these avian population declines. Wildlife rehabilitation centres play an essential role in species conservation and preservation. Moreover, animals admitted for rehabilitation can provide valuable information regarding transmission and pathogenicity of many diseases that affect wild birds that are rarely sampled in nature. However, reports of haemosporidians in captive birds are still limited. Here, we explored the prevalence and genetic diversity of avian haemosporidians in 89 birds from 29 species admitted to rehabilitation centres in Portugal, showing an overall infection prevalence of 30.3%. The prevalence of infection was higher in Strigiformes and in birds admitted to rehabilitation centres due to debilitating diseases. Remarkably, 30% of the infected bird species have not been found to harbour malaria parasites in preceding studies. We detected 15 different haemosporidian lineages infecting a third of bird species sampled. Notably, 2 out of these 15 detected haemosporidian lineages have not been obtained previously in other studies. Furthermore, we also identified nine new host–parasite interactions representing new host records for these haemosporidian parasites. Finally, our results revealed that birds infected with haemosporidians require longer rehabilitation treatments, which increase the economic costs for rehabilitation and may impair their survival prospects. These findings emphasise the importance of integrating haemosporidian infection considerations into rehabilitation protocols, highlighting the challenges posed by these infections in avian conservation and rehabilitation, including economic and logistical demands. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Haemoproteus parasites are one of the most studied avian blood parasites; however, their natural vectors, Culicoides biting midges, have been identified for only a small portion of them. The main reason for that might be due to the existence of a few research groups working with an integrative approach that allows not only the identification of parasite DNA in the insects, but also confirms the presence of the parasite infective stage (sporozoites) using microscopy. In this study, we aimed to identify the natural vectors of Haemoproteus parasites and to determine their prevalence in Culicoides biting midges in four different localities in Lithuania. Almost 2000 parous Culicoides females belonging to 11 species previously registered in the country were processed. The most abundant one was C. pictipennis. Parasite DNA was found in 7.9% of the analyzed biting midges, and sporozoites were present in almost 30% of these insects. The Botanical Garden was the locality with the highest abundance of biting midges and parasite species, as well as the place with the highest number of biting midges containing sporozoites. One new Haemoproteus vector (C. reconditus) was confirmed, and 12 new interactions between Haemoproteus parasite lineages and Culicoides biting midges of different species were identified. This study helps to understand the relationship between Haemoproteus parasites and Culicoides biting midges in the wild. 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. [ABSTRACT FROM AUTHOR]

Haemosporidians are a group of vector-borne parasites belonging to the order Haemosporida. These parasites infect avian hosts and require blood-sucking insects (Diptera) for transmission. The occurrence and diversity of haemosporidian parasites are shaped primarily by the specificity of the parasite and the susceptibility of the host/vector. In this study, the presence and distribution of haemosporidians in blood samples from birds in urbanized and natural habitats were estimated using microscopic and molecular approaches. Birds in urbanized habitats were infected with four different species of Plasmodium, P. relictum, P. vaughani, P. matutinum, and P. circumflexum, and one species of Haemoproteus, H. parabelopolskyi, and Leucocytozoon sp. The species H. attenuatus, H. concavocentralis, H. minutus, H. pallidus, H. noctuae, and H. tartakovskyi were additionally identified in birds in natural habitats. Typically, juvenile birds are essential markers of parasite species transmitted in the study area. The juveniles in the urbanized habitats carried P. relictum, P. vaughani, P. circumflexum, H. parabelopolskyi, and Leucocytozoon species. The most abundant parasite was H. parabelopolskyi, which was found in both habitat types. The prevalence of Haemoproteus/Plasmodium species determined by nested PCR in birds in natural habitats (43.80%; 53/121) was significantly greater than that in birds in urbanized habitats (21.94%; 43/196) (p < 0.05). There was no significant difference in the infection rate of Leucocytozoon sp. between the habitat types (p > 0.05; 10/121 vs. 19/196). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Meronts and megalomeronts of five co-infecting Haemoproteus spp. were described. • In situ hybridization combined with laser microdissection are effective in merogony research. • Lineage-specific tools revealed different tissue tropism in co-infecting parasites. • New pathologies during haemoproteosis were reported. • Haemoproteus magnus was molecularly characterized. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

Haemosporidians are a diverse group of vector-borne parasitic protozoa that have a global distribution and some of them live in the avian body as a benign parasite, with no symptoms. Avian Haemosporidians, include three main genera, Plasmodium, Haemoproteus, and Leucocytozoon. These blood parasites are transmitted across different regions through migratory birds. We studied the prevalence of avian haemosporidian parasites in both resident and migratory birds in the central and southern regions of Iraq. Our study represents pioneering research on Haemosporidian parasites in a wide range of avian species, including 248 samples from 47 species of both resident and migratory birds, collected from five cities in central and southern Iraq. We identified and determined the prevalence of three distinct haemosporidian genera, Haemoproteus spp., Leucocytozoon spp., and Plasmodium spp. Overall parasite infection was higher in resident hosts than in migrants. The prevalence of Plasmodium spp. was found to be highest among resident birds, with a rate of 20.1%, while the highest rate of Leucocytozoon spp. was observed in migratory birds, with a rate of 8.2%. Our study holds promising opportunities for understanding the impact of migratory and resident hosts on the dynamics of infectious diseases in wildlife.

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.

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.

Haemoproteus spp. are dipteran-borne protozoa that infect erythrocytes and reticulo-endothelial cells of birds. These parasites are not usually transmitted between birds belonging to different orders. The suborder Lari (order Charadriiformes) comprises ~170 avian species, the majority of which are aquatic, including gulls, terns, auklets, murres and skuas, among others. In spite of the diversity of this avian group, there is limited known diversity of haemosporidian parasites, with only 4 recorded Haemoproteus morphospecies thus far. We examined the blood smears of 21 kelp gulls (Larus dominicanus) captured at a breeding colony in South Africa, as well as Haemoproteus-positive archival blood smears of 15 kelp gulls and 1 Hartlaub's gull (Larus hartlaubii) sampled while under care at seabird rehabilitation facilities in South Africa. Haemoproteus sp. infection was detected in 19% of wild-caught kelp gulls. All parasites from the gulls were morphologically identified as Haemoproteus jenniae, a species previously recorded in Lari birds at the Galapagos Islands (Ecuador), Rocas Atoll (Brazil) and Poland. Gene sequencing uncovered a new cytochrome b lineage, LARDOM01, which was closely related to the previously reported H. jenniae lineage CREFUR01. Additionally, we evaluated a hapantotype blood smear of Haemoproteus skuae, which had been described infecting a brown skua (Catharacta antarctica) in South Africa. We provide a redescription of H. skuae and discuss the morphological characters distinguishing it from H. jenniae. Further research is necessary to improve our knowledge about the host and geographic distribution, health effects and phylogeny of H. jenniae and H. skuae.

Host–parasite 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 host–parasite relationships and the specialist–generalist 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 host–parasite 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 host–parasite systems, to provide reliable insights into the precise mechanisms underlying host–parasite interactions.

The relationships between host phylogenetics, functional traits and parasites in wildlife remain poorly understood in the Neotropics, especially in habitats with marked seasonal variation. Here, we examined the effect of seasonality and host functional traits on the prevalence of avian haemosporidians (Plasmodium and Haemoproteus) in the Brazilian Caatinga, a seasonally dry tropical forest. 933 birds were evaluated for haemosporidian infections. We found a high parasitism prevalence (51.2%), which was correlated with phylogenetic relatedness among avian species. Prevalence varied drastically among the 20 well-sampled species, ranging from 0 to 70%. Seasonality was the main factor associated with infections, but how this abiotic condition influenced parasite prevalence varied according to the host-parasite system. Plasmodium prevalence increased during the rainy season and, after excluding the large sample size of Columbiformes (n = 462/933), Plasmodium infection rate was maintained high in the wet season and showed a negative association with host body mass. No association was found between non-Columbiform bird prevalence and seasonality or body mass when evaluating both Plasmodium and Haemoproteus or only Haemoproteus infections. Parasite community was composed of 32 lineages including 7 new lineages. We evidenced that even dry domains can harbour a high prevalence and diversity of vector-borne parasites and pointed out seasonality as a ruling factor.

Abstract 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. Graphical abstract

[Display omitted] • Geographic range is more important than bird migratory behaviour for parasite diversity. • Patterns differed between Plasmodium , Haemoproteus and Leucocytozoon parasites. • Species resident in both Africa and Eurasia (RB) had higher diversity than all other groups. • RB and migrants had higher Haemoproteus diversity than single-continent residents. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Avian haemoproteids are prevalent parasites known to induce pathology or mortality in birds. Culicoides biting midges act as vectors for these parasites. Despite the annual arrival of various haemoproteids to Europe with migrant birds, not all these parasites undergo local transmission. The factors constraining the local spread of these pathogens remain incompletely understood. Our study investigates if the ecological isolation of birds from vectors, either temporally or spatially during the breeding period when transmission occurs, could lead to the cessation of transmission. Biting midges were systematically collected from two distinct habitats between May and September. A total of 1135 parous Culicoides females were identified and examined for the presence of Haemoproteus parasites using both microscopy and molecular tools. Notably, we report the first detection of Haemoproteus asymmetricus sporozoites in the salivary glands of Culicoides festivipennis females. The sporozoites of four Haemoproteus genetic lineages were also identified in Culicoides segnis, C. festivipennis, and C. kibunensis, further validating their status as potential Haemoproteus vectors. While the highest abundance of collected Culicoides females occurred in June, the peak prevalence of Haemoproteus parasites in biting midges was observed in July. Interestingly, the abundance of Culicoides was significantly greater in woodlands compared to reeds throughout the season. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 l ineages, including Plasmodium circumflexum (pTURDUS1), and Haemoproteus asymmetricus (hTUPHI01). We call for further research to better understand Leucocytozoon parasite exo-erythrocytic development. [Display omitted] • First report of Leucocytozoon exo-erythrocytic stages in song thrushes. • Meronts of Leucocytozoon infecting song thrushes are common in the kidneys. • A megalomeront-like structure of unusual morphology was found in one bird. • Chromogenic in situ hybridization is essential to confirm parasite identity. • Confirmed the transmission of three Leucocytozoon lineages (lTUPHI14, lSTUR1 and lTUPHI13) in Europe. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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.

Avian malaria parasites or haemosporidia are found in bird species worldwide. This special issue focuses on 3 most commonly studied genera: Haemoproteus, Plasmodium and Leucocytozoon. Seven research articles and reviews are provided to illustrate the breadth of knowledge of the diversity of avian malaria parasites in different regional habitats and across bird species, and the use of avian haemosporidian systems to examine host–parasite eco-evolutionary questions.

Bird feathers have several functions, including flight, insulation, communication, and camouflage. Since feathers degrade over time, birds need to moult regularly to maintain these functions. However, environmental factors like food scarcity, stress, and parasite infections can affect feather quality and moult speed. This study examined the impact of avian haemosporidian infection and uropygial gland volume, as well as feather quality and feather growth rate in two migratory hirundine species captured in southwestern Spain—the house martin (Delichon urbicum) and sand martin (Riparia riparia). Our findings showed that the prevalence of infection varied among species, with house martins having the highest rates, possibly due to their larger colony size. Moreover, haemosporidian infection had a different impact on each species; infected house martins exhibited lower feather quality than healthy individuals, although this outcome was not observed in sand martins. Furthermore, no effect of infection on feather growth rate was observed in both hirundinids. Additionally, feather growth rate only correlated positively with feather quality in house martins. Finally, no link was observed between uropygial gland volume and feather quality or feather growth rate in any of the species in this study. These findings highlight the effect of haemosporidian infections on the plumage of migratory birds, marking, for the first time, how avian haemosporidian infection is shown to adversely impact feather quality. Even so, further research is needed to explore these relationships more deeply.

In the last decade, over 40% of bird species in Europe have experienced poor and bad conservation status, with more than 30% of bird species in mainland Portugal threatened with extinction. Along with anthropogenic factors, parasites and pathogens such as avian haemosporidians have been suggested to be responsible for these avian population declines. Wildlife rehabilitation centres play an essential role in species conservation and preservation. Moreover, animals admitted for rehabilitation can provide valuable information regarding transmission and pathogenicity of many diseases that affect wild birds that are rarely sampled in nature. However, reports of haemosporidians in captive birds are still limited. Here, we explored the prevalence and genetic diversity of avian haemosporidians in 89 birds from 29 species admitted to rehabilitation centres in Portugal, showing an overall infection prevalence of 30.3%. The prevalence of infection was higher in Strigiformes and in birds admitted to rehabilitation centres due to debilitating diseases. Remarkably, 30% of the infected bird species have not been found to harbour malaria parasites in preceding studies. We detected 15 different haemosporidian lineages infecting a third of bird species sampled. Notably, 2 out of these 15 detected haemosporidian lineages have not been obtained previously in other studies. Furthermore, we also identified nine new host–parasite interactions representing new host records for these haemosporidian parasites. Finally, our results revealed that birds infected with haemosporidians require longer rehabilitation treatments, which increase the economic costs for rehabilitation and may impair their survival prospects. These findings emphasise the importance of integrating haemosporidian infection considerations into rehabilitation protocols, highlighting the challenges posed by these infections in avian conservation and rehabilitation, including economic and logistical demands.

Avian haemosporidian parasites are widespread and infect birds from a broad variety of avian families with diverse consequences ranging from subclinical infections to severe and fatal disease. This study aimed to determine the occurrence and diversity of avian haemosporidia including associated clinical signs and pathomorphological lesions in captive and free-ranging, wild birds from two zoos and the near environment in Switzerland. Blood samples from 475 birds, including 230 captive and 245 free-ranging, wild individuals belonging to 42 different avian species from 15 orders were examined for the presence of avian haemosporidian DNA by a one-step multiplex PCR designed to simultaneously detect and discriminate the genera Plasmodium, Haemoproteus and Leucocytozoon by targeting mitochondrial genome sequences. Positive samples were additionally tested using a nested PCR targeting the cytochrome b gene of Plasmodium and Haemoproteus. The obtained amplicons were bidirectionally sequenced. This study revealed haemosporidian DNA in 42 samples, belonging to ten host species. The most commonly detected lineage was Plasmodium relictum SGS1, which was identified in 29 birds (Phoenicopterus roseus: n = 24, Alectoris graeca: n = 1, Lamprotornis superbus: n = 1, Somateria mollissima: n = 1, Spheniscus demersus: n = 1, Tetrao urogallus crassirostris: n = 1), followed by Haemoproteus sp. STRURA03 in six avian hosts (Bubo bubo: n = 5, Bubo scandiacus = 1), Plasmodium relictum GRW11 in four individuals (Phoenicopterus roseus: n = 3, Spheniscus demersus: n = 1) and Plasmodium elongatum GRW06 in one Alectura lathami lathami. A Phalacrocorax carbo was infected with Plasmodium relictum, but the exact lineage could not be determined. One mixed infection with P. relictum and Haemoproteus sp. was detected in a Bubo scandiacus. Only five individuals (Spheniscus demersus: n = 2, Somateria mollissima: n = 1, Bubo scandiacus: n = 1, Alectoris graeca: n = 1) showed clinical and pathomorphological evidence of a haemosporidian infection.

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

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

Abstract The Brown-capped Rosy-Finch is an endangered high alpine specialist that breeds on cliffs in the Rocky Mountains of North America. We know little about the parasites that infect these birds but filling this knowledge gap is important for understanding their population decline. The aim of this study was to survey haemosporidian parasite infections in Brown-capped Rosy-Finches. We sampled 104 Brown-capped Rosy-Finches during their breeding season at six sampling sites spanning the Colorado Rocky Mountains where they are nearly endemic. We used nested PCR methods to screen birds for parasite infections, and Sanger sequencing data were used to identify parasite lineages. Four of the sampling sites had birds with infections. Females were more often infected than males (prevalence = 15.6% and 9.7%, respectively). We observed twice as many infected birds sampled in July compared to birds sampled in June (prevalence = 12.5% and 6.25%, respectively). The percent of infected birds by sampling site ranged from 0 to over 20%. In total we identified eight different genetic lineages of haemosporidian parasites infecting Brown-capped Rosy-Finches—seven were in the genus Leucocytozoon, and one was in the genus Haemoproteus. Network analysis clearly separates Haemoproteus from Leucocytozoon, with Leucocytozoon lineages comprising two major clusters. Based on reports made to the MalAvi database, all lineages in Cluster 1 have the same reported insect vector, Simulium silvestre. We report that Brown-capped Rosy-Finches experience infections with haemosporidian parasites and that the percentage of infected birds differed markedly between sampling sites. We hypothesize that vector ecology and associated variation in climate at sampling sites explain our observations.

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.

Haemosporidians are a group of vector-borne parasites belonging to the order Haemosporida. These parasites infect avian hosts and require blood-sucking insects (Diptera) for transmission. The occurrence and diversity of haemosporidian parasites are shaped primarily by the specificity of the parasite and the susceptibility of the host/vector. In this study, the presence and distribution of haemosporidians in blood samples from birds in urbanized and natural habitats were estimated using microscopic and molecular approaches. Birds in urbanized habitats were infected with four different species of Plasmodium, P. relictum, P. vaughani, P. matutinum, and P. circumflexum, and one species of Haemoproteus, H. parabelopolskyi, and Leucocytozoon sp. The species H. attenuatus, H. concavocentralis, H. minutus, H. pallidus, H. noctuae, and H. tartakovskyi were additionally identified in birds in natural habitats. Typically, juvenile birds are essential markers of parasite species transmitted in the study area. The juveniles in the urbanized habitats carried P. relictum, P. vaughani, P. circumflexum, H. parabelopolskyi, and Leucocytozoon species. The most abundant parasite was H. parabelopolskyi, which was found in both habitat types. The prevalence of Haemoproteus/Plasmodium species determined by nested PCR in birds in natural habitats (43.80%; 53/121) was significantly greater than that in birds in urbanized habitats (21.94%; 43/196) (p < 0.05). There was no significant difference in the infection rate of Leucocytozoon sp. between the habitat types (p > 0.05; 10/121 vs. 19/196).

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.

Infections of avian haemosporidian parasites are regularly identified by molecular methods including multiplex PCR, which allows researchers to distinguish mixed infections of parasites from multiple genera. Here we extend the utility of a previously designed multiplex PCR by designing a primer set specific to parasites of the subgenus Haemoproteus (genus: Haemoproteus). The updated one-step multiplex PCR protocol we describe here allows for the detection of the genera Plasmodium and Leucocytozoon and the two subgenera (Haemoproteus and Parahaemoproteus) of the genus Haemoproteus. A sensitivity analysis showed that the multiplex PCR could amplify DNA of parasites in the subgenus Haemoproteus at very low levels of infection. We used this multiplex PCR to identify haemosporidian infections in 250 adult domestic pigeons (Columba livia) in Turkey. All samples were also screened by microscopy and a widely used nested PCR to compare with the results of multiplex PCR, to detect low levels of parasitemia, and to identify possible abortive infections. In total, 71 pigeons (28.4%) were found to be infected by all three methods. The multiplex PCR protocol successfully detected and discriminated both subgenera Haemoproteus and Parahaemoproteus infections. We compared our results with previous host species records to assess the host specificity of the parasite lineages we found. Our findings provide novel data on the prevalence of avian haemosporidians in domestic pigeons and demonstrate the utility of the new one-step multiplex PCR protocol for the determination of mixed avian haemosporidian infections. We expect that this protocol will contribute to a better understanding of the distribution, epizootiology, and ecology of avian haemosporidians. [ABSTRACT FROM AUTHOR]

The commonly occurring haemoparasites in pigeons include Plasmodium, Leucocytozoon and Haemoproteus which cause decrease in production and performance of the birds. Due to the dearth of knowledge on prevalence of these parasites in pigeons in Pakistan, current survey-based study was planned to assess the prevalence of above-mentioned parasites in pigeons in selected rural areas of District Faisalabad, Punjab, Pakistan. The morphological identification of parasites was done under microscope by comparing it with standard keys. Out of 384 samples, 56.51% were found positive for haemoparasites. According to town-wise distribution, Jinnah town, Iqbal town, Madina town and Lyallpur town prevalence was 61.24, 48.86, 55.56, and 59.26%, respectively. According to genus-wise distribution, 48.14, 48.21, and 64.92% prevalence of mixed infection, genera Plasmodium/Haemoproteus and Leucocytozoon were observed, respectively. The prevalence was found high in female birds (65.9%) as compared to male birds (51.18%). The prevalence was higher in Cheena (69.02%) and Golden breeds (76.57%) as compared to Sialkoti (32.89%) and Dub wali (37.17%). The prevalence was found higher in squabs (74.15%) as compared to adults (49.6%) and growers (52.56%), in scavengers feeding pattern (77.31%) as compared to feeders (35.78%), and in open coop type (76.68%) as compared to closed coop type (36.12%). The values of Hb, PCV, and RBC's count were lower, while WBC's counts were higher in infected pigeons. The values of uric acid, creatinine, albumin and bilirubin were lower while gamma-glutamyl transferase (GGT), alanine transaminase (ALT), aspartate transaminase (AST) and glucose were higher in infested birds as compared to non- infected birds. The study indicated the impact of haemoparasites on the hematological parameters of the birds and the data generated could help in formulating effective control strategies against haemoparasites particularly Plasmodium, Leucocytozoon and Haemoproteus. [ABSTRACT FROM AUTHOR]

Avian haemosporidian parasites are widespread and infect birds from a broad variety of avian families with diverse consequences ranging from subclinical infections to severe and fatal disease. This study aimed to determine the occurrence and diversity of avian haemosporidia including associated clinical signs and pathomorphological lesions in captive and free-ranging, wild birds from two zoos and the near environment in Switzerland. Blood samples from 475 birds, including 230 captive and 245 free-ranging, wild individuals belonging to 42 different avian species from 15 orders were examined for the presence of avian haemosporidian DNA by a one-step multiplex PCR designed to simultaneously detect and discriminate the genera Plasmodium , Haemoproteus and Leucocytozoon by targeting mitochondrial genome sequences. Positive samples were additionally tested using a nested PCR targeting the cytochrome b gene of Plasmodium and Haemoproteus. The obtained amplicons were bidirectionally sequenced. This study revealed haemosporidian DNA in 42 samples, belonging to ten host species. The most commonly detected lineage was Plasmodium relictum SGS1, which was identified in 29 birds (Phoenicopterus roseus : n = 24, Alectoris graeca : n = 1, Lamprotornis superbus : n = 1, Somateria mollissima : n = 1, Spheniscus demersus : n = 1, Tetrao urogallus crassirostris : n = 1), followed by Haemoproteus sp. STRURA03 in six avian hosts (Bubo bubo : n = 5, Bubo scandiacus = 1), Plasmodium relictum GRW11 in four individuals (Phoenicopterus roseus : n = 3, Spheniscus demersus : n = 1) and Plasmodium elongatum GRW06 in one Alectura lathami lathami. A Phalacrocorax carbo was infected with Plasmodium relictum , but the exact lineage could not be determined. One mixed infection with P. relictum and Haemoproteus sp. was detected in a Bubo scandiacus. Only five individuals (Spheniscus demersus : n = 2, Somateria mollissima : n = 1, Bubo scandiacus : n = 1, Alectoris graeca : n = 1) showed clinical and pathomorphological evidence of a haemosporidian infection. [Display omitted] • Avian heamosporidian parasites were detected in captive birds in Switzerland. • Plasmodium relictum , P. elongatum and Haemoproteus were found in 10 bird species. • Subclinical and clinical infections were recorded. • Genus Spheniscus, Somateria, Bubo and Alectoris showed clinical avian malaria. • Leucocytozoon was not detected in any of the blood samples. [ABSTRACT FROM AUTHOR]

Pigeons are reared as a symbol of peace, love, gentleness and spirit messengers and now-a-days also for meat, as ornamental pet bird or as an experimental animal. There are about 400 varieties of fancy pigeons and little is known about the diseases and management of these birds. Heavy mortality has been observed in the pigeons that shows neurological manifestations. The present study is aimed at diagnosis of infectious diseases causing neurological signs in pigeons. The study was conducted in the different pigeon lofts of four districts in Kerala and a total of 44 pigeons comprising 21 fancy breeds were included. These birds were screened for major infectious etiologies causing neurological signs like paramyxoviral infection, salmonellosis and Haemoproteus infection. The birds were also screened for presence of ectoparasites by visual examination. The study showed 63.63 per cent positive for paramyxoviral infection using antigen NDV kits. The occurrence of Haemoproteus infection was 54.7 per cent in Giemsa-stained blood smears and 63.63 per cent in PCR amplifying cyt b gene. Pseudolynchia canariensis could be detected in pigeons. Isolation and identification of Salmonella from faecal samples were done by culture and biochemical tests and found to be 22.2 per cent. Combined infections of paramyxovirus infection with Haemoproteus, paramyxovirus infection with Salmonella, Haemoproteus with Salmonella and infection of all the three etiologies were the interesting finding of the study. The present study is one of its kind in Kerala and also in India. The study warrants the prevention and control of these infectious diseases which has great zoonotic potential.

Host–parasite distribution and interactions depend on geography, evolutionary associations, species composition, and their response to environmental variables. In this sense, host specificity as a key parasite's life history trait may determine parasite community composition but can be difficult to identify in nature. The haemosporidian–avian host communities of tropical mountain systems offer an opportunity to better understand how network interactions influence parasite–host specialization. By using frequency (i.e., prevalence) and interaction intensity (i.e., number of parasites per infected host) data, we calculated two quantitative indices to evaluate haemosporidian host specialization at both community (H2′) and haemosporidian lineages and bird species levels (d′). Additionally, we evaluated alpha (SI) and beta diversity (βWN) of haemosporidian–bird interactions along four distinct elevations (representing different vegetation types) in Central Veracruz, Mexico. We recorded 607 birds from 88 species in two sampling years, registering 78 haemosporidian cytochrome b lineages by PCR in 125 positive samples from 38 bird species. Haemosporidian–bird interactions showed high specialization and modular network structure for each vegetation type. The degree of specialization and modularity was stronger when considering interaction intensity (i.e., parasitemia). Species‐level specialization was higher for birds than for haemosporidians at the two vegetation types with highest interaction richness (i.e., pine–oak forest and tropical deciduous forest). There were high interaction beta diversity values (~1) among vegetation types, reflecting turnover in both avian and parasite communities. Our findings suggest that vegetation type (proxy for the local environmental conditions) constrains the distribution of hosts and parasites, and that intraspecies infection intensity plays an important role in the transmission dynamics and interactions of the haemosporidian community. [ABSTRACT FROM AUTHOR]