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

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

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

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

Unexpected absence of exo-erythrocytic merogony during high gametocytaemia in two species of Haemoproteus (Haemosporida: Haemoproteidae), including description of Haemoproteus angustus n. sp. (lineage hCWT7) and a report of previously unknown residual bodies during in vitro gametogenesis.

Neglected avian blood parasites of the genus Haemoproteus (Haemoproteidae) have recently attracted attention due to the application of molecular diagnostic tools, which unravelled remarkable diversity of their exo-erythrocytic (or tissue) stages both regarding morphology and organ tropism levels. The development of haemoproteids might result in pathologies of internal organs, however the exo-erythrocytic development (EED) of most Haemoproteus species remains unknown. Seven individual birds - Curruca communis (1) and Phylloscopus trochilus (6) - with high gametocytaemia (between 1% and 24%) of Haemoproteus angustus n. sp. (hCWT7) and Haemoproteus palloris (lineage hWW1) were sampled in Lithuania, and their internal organs were examined extensively by parallel application of histology and chromogenic in situ hybridization methods. Tissue stages were apparently absent, suggesting that the parasitaemia was not accompanied by detectable tissue merogony. Haemoproteus angustus n. sp. was described and characterized morphologically and molecularly. Sexual process and ookinete development of the new species readily occurred in vitro, and a unique character for Haemoproteus parasites was discovered - the obligatory development of several tiny residual bodies, which were associated with intracellular transformation of both macrogametocytes and microgametocytes before their escape from the host cells and formation of gametes. A DNA haplotype network was constructed with lineages that cluster in one clade with the lineage hCWT7. This clade consists of lineages mostly found in Curruca birds, indicating specificity for birds of this genus. The lineage hCWT7 is mainly a parasite of C. communis. Most reports of this lineage came from Turkey, with only a few records in Europe, mostly in birds wintering in Africa where transmission probably occurs. This study highlights unexpected difficulties in the research of EED even when using sensitive molecular diagnostic tools and extends information about transformation in early stages of gametogenesis in haemosporidian parasites.

Molecular detection of Babesia vesperuginis in bats from Lithuania.

Babesia vesperuginis is an intraerythrocytic protozoan parasite that circulates among bats and ticks in many countries worldwide. However, the distribution of B. vesperuginis in the Baltic region has not been studied. A total of 86 dead bats from eight different species were collected and screened for Babesia spp. using real-time PCR. Overall, 52.3% (45/86) of the bats were found positive for Babesia spp. The prevalence of Babesia spp. in different organs varied, with the highest prevalence observed in heart tissues (37.0%) and the lowest in liver tissues (22.2%). However, the observed differences in prevalence among organs were not statistically significant. Blood samples from 125 bats of nine different species were also analyzed for Babesia spp. prevalence using real-time PCR and nested PCR. The results showed a prevalence of 35.2% and 22.4%, respectively. Moreover, 28.3% (17/60) of the examined blood samples were confirmed positive for Babesia spp. through blood smear analysis. The total of 32 partial sequences of the 18S rRNA gene derived in this study were 100% identical to B. vesperuginis sequences from GenBank. In eight species of bats, Pipistrellus nathusii, Pipistrellus pipistrellus, Pipistrellus pygmaeus, Vespertilio murinus, Eptesicus nilssonii, Eptesicus serotinus, Myotis daubentonii and Nyctalus noctula, Babesia parasites were identified. In E. nilssonii, Babesia spp. was identified for the first time.

Lankesterella (Apicomplexa, Lankesterellidae) Blood Parasites of Passeriform Birds: Prevalence, Molecular and Morphological Characterization, with Notes on Sporozoite Persistence In Vivo and Development In Vitro.

Recent studies confirmed that some Hepatozoon-like blood parasites (Apicomplexa) of birds are closely related to the amphibian parasite Lankesterella minima. Little is known about the biology of these pathogens in birds, including their distribution, life cycles, specificity, vectors, and molecular characterization. Using blood samples of 641 birds from 16 species, we (i) determined the prevalence and molecular diversity of Lankesterella parasites in naturally infected birds; (ii) investigated the development of Lankesterella kabeeni in laboratory-reared mosquitoes, Culex pipiens forma molestus and Aedes aegypti; and (iii) tested experimentally the susceptibility of domestic canaries, Serinus canaria, to this parasite. This study combined molecular and morphological diagnostic methods and determined 11% prevalence of Lankesterella parasites in Acrocephalidae birds; 16 Lankesterella lineages with a certain degree of host specificity and two new species (Lankesterella vacuolata n. sp. and Lankesterella macrovacuolata n. sp.) were found and characterized. Lankesterella kabeeni (formerly Hepatozoon kabeeni) was re-described. Serinus canaria were resistant after various experimental exposures. Lankesterella sporozoites rapidly escaped from host cells in vitro. Sporozoites persisted for a long time in infected mosquitoes (up to 42 days post exposure). Our study demonstrated a high diversity of Lankesterella parasites in birds, and showed that several avian Hepatozoon-like parasites, in fact, belong to Lankesterella genus.

The flying activity of biting midges (Ceratopogonidae: Culicoides) in Verkiai Regional Park, southeastern Lithuania.

Culicoides biting midges are small dipterous insects (Diptera: Ceratopogonidae) which are known to be vectors of arboviruses, bacteria, protozoan and helminth parasites that can cause disease and mortality in livestock and poultry globally. Detailed knowledge of the Culicoides species composition and biology is essential to assess the risk of the introduction and transmission of pathogens. The aim of this study was to obtain data on Culicoides species composition and flying activity in southeastern Lithuania and to determine the meteorological variables related to the abundance of Culicoides biting midges. Biting midges were collected in Verkiai Regional Park, southeastern Lithuania, using an Onderstepoort trap once a week from April to October 2016 and 2018, and from April to July 2019; 7332 Culicoides females belonging to 22 species were identified. Both morphology and DNA barcoding were used for identification. The number of specimens trapped was highest for the Obsoletus Group, followed by Culicoides kibunensis and Culicoides impunctatus. The highest relative abundance and diversity of biting midges were found in May and June. The number of trapped biting midges correlated positively with the mean air temperature. The first biting midges in spring were caught when the mean daily temperature rose higher than 10 °C. No Culicoides were detected when the air temperature dropped below 5 °C in autumn. Wind speed and air humidity had no statistically significant effect on Culicoides abundance.

Description of Haemoproteus asymmetricus n. sp. (Haemoproteidae), with remarks on predictability of the DNA haplotype networks in haemosporidian parasite taxonomy research.

Haemoproteus species (Haemosporida, Haemoproteidae) are cosmopolitan blood parasites, which have been neglected for over 100-years, but attracted attention recently due to reports of severe and even lethal haemoproteosis in birds and vectors. Approximately 150 species of avian Haemoproteus have been described and named, but molecular data suggest that hundreds of independently evolving molecular lineages might occur, indicating the existence of a remarkable undescribed species diversity. It is timely to develop a methodology, which allow the application of available genetic data in taxonomy of haemosporidians on species levels. This study aimed to test a hypothesis suggesting that DNA haplotype networks might aid in targeting genetically distinct, but still undescribed parasites, and might be used to direct taxonomic studies on haemosporidian species levels. Mainly, we tested a prediction that the lineage hTUPHI01, a common Haemoproteus parasite of Turdus philomelos, might be a new species, which is morphologically similar and genetically closely related to the parasites of Haemoproteus minutus group. Blood samples of T. philomelos naturally infected with this parasite lineage were collected and studied using microscopic examination of blood films and PCR-based methods. Haemoproteus asymmetricus n. sp. was found in this bird, described and characterised molecularly using partial cytochrome b (cytb) sequences. The new species shared some features with parasites of the H. minutus group, as was predicted by the DNA haplotype network. Due to the visualisation of closely related lineages as well as the evaluation of their host and geographic distributions, DNA haplotype networks can be recommended as the helpful methodology, able to direct and speed practical work on parasite species taxonomy and pathogen biodiversity. The combined molecular phylogenetic and morphological approaches showed that the well-supported clades in Bayesian phylogenetic trees based on the partial cytb gene sequences contain morphologically remarkably different Haemoproteus parasite species, which however, share some basic biological features. Phylogenetic analysis can be used for prediction of these basic features in still undescribed parasites. This study calls for further fusion of advanced molecular and microscopy approaches for better understanding haemosporidian parasite biology.

Development of Trypanosoma everetti in Culicoides biting midges.

Trypanosoma species (Trypanosomatida, Kinetoplastea) are almost exclusively heteroxenous flagellated parasites, which have been extensively studied as the causative agents of severe trypanosomiasis in humans and domestic animals. However, the biology of avian trypanosomes remains insufficiently known, particularly in wildlife, despite information that some species might be pathogenic and affect the fitness of intensively infected individuals. Avian trypanosomes are cosmopolitans. Due to regular bird seasonal migrations, this host-parasite system might provide new insight for better understanding mechanisms of transcontinental dispersal of pathogens, their ecological plasticity, specificity and speciation. Trypanosoma everetti parasitizes numerous bird species globally, but data on its biology are scarce and its vectors remain unknown. This study aimed to test experimentally whether widespread Culicoides (Diptera: Ceratopogonidae) biting midges are susceptible to infection with this parasite. Two common house martins Delichon urbicum and two sedge warblers Acrocephalus schoenobaenus naturally infected with T. everetti were caught in the wild after arrival from African wintering grounds. Laboratory reared Culicoides nubeculosus and wild-caught Culicoides impunctatus biting midges were exposed by allowing them to take infected blood meals. The experimentally infected and control insects were maintained in the laboratory and dissected at intervals to follow the development of the parasite. Infections were determined using microscopic examination and PCR-based testing. Four closely related haplotypes of T. everetti were found, and each was present in different individual parasite-donor birds. These parasites readily developed and produced metacyclic trypomastigotes in C. nubeculosus and C. impunctatus biting midges. Molecular characterisation of T. everetti was developed. According to Bayesian phylogenetic analysis using a DNA fragment encoding 18S rRNA, the five species of small avian trypanosomes were closely related. Wild caught Culicoides biting midges were also collected and screened for the presence of natural infections. In all, 6.8% of wild-caught biting midges belonging to five Culicoides species were PCR-positive for kinetoplastids, including Trypanosoma species. Culicoides biting midges are readily susceptible and likely naturally transmit avian trypanosomes and thus, should be targeted in epidemiology research of avian trypanosomiasis.

A new methodology for sporogony research of avian haemoproteids in laboratory-reared Culicoides spp., with a description of the complete sporogonic development of Haemoproteus pastoris.

BACKGROUND: Haemosporidian parasites of the genus Haemoproteus (Haemoproteidae) are widespread and cause haemoproteosis in birds and therefore, their diversity, ecology and evolutionary biology have become subjects of intensive research. However, the vectors and transmission patterns of haemoproteids as well as the epidemiology of haemoproteosis remain insufficiently investigated. Several species of Culicoides (Ceratopogonidae) support complete sporogony of haemoproteids belonging to the subgenus Parahaemoproteus. However, experimental research with these fragile insects is difficult to design in the field, particularly because their abundance markedly depends on seasonality. This is an obstacle for continuous sampling of sufficient numbers of naturally infected or experimentally exposed midges from wildlife. We developed simple methodology for accessing sporogonic development of haemoproteids in laboratory-reared Culicoides nubeculosus. This study aimed to describe the mosaic of methods constituting this methodology, which was applied for investigation of the sporogonic development of Haemoproteus (Parahaemoproteus) pastoris, a widespread parasite of the common starling Sturnus vulgaris. METHODS: The methodology consists of the following main stages: (i) laboratory rearing of C. nubeculosus from the egg stage to adult insects; (ii) selection of naturally infected birds, the donors of mature gametocytes to expose biting midges; (iii) experimental exposure of insects and their laboratory maintenance; and (iv) dissection of exposed insects. Biting midges were exposed to H. pastoris (cytochrome b lineage hLAMPUR01) detected in one naturally infected common starling. Engorged insects were dissected at intervals in order to follow sporogony. Microscopic examination and PCR-based methods were used to identify the sporogonic stages and to confirm the presence of the parasite lineage in infected insects, respectively. RESULTS: Culicoides nubeculosus females were successfully reared and exposed to H. pastoris, which completed sporogonic development 7-9 days post-infection when sporozoites were observed in the salivary glands. CONCLUSIONS: The new methodology is easy to use and non-harmful for birds, providing opportunities to access the sporogonic stages of Parahaemoproteus parasites, which might be used in a broad range of parasitology and genetic studies. Culicoides nubeculosus is an excellent experimental vector of subgenus Parahaemoproteus and is recommended for various experimental studies aiming investigation of sporogony of these pathogens.

The life-cycle of the avian haemosporidian parasite Haemoproteus majoris, with emphasis on the exoerythrocytic and sporogonic development.

BACKGROUND: Haemoproteus parasites (Haemosporida, Haemoproteidae) are cosmopolitan in birds and recent molecular studies indicate enormous genetic diversity of these pathogens, which cause diseases in non-adapted avian hosts. However, life-cycles remain unknown for the majority of Haemoproteus species. Information on their exoerythrocytic development is particularly fragmental and controversial. This study aimed to gain new knowledge on life-cycle of the widespread blood parasite Haemoproteus majoris. METHODS: Turdus pilaris and Parus major naturally infected with lineages hPHYBOR04 and hPARUS1 of H. majoris, respectively, were wild-caught and the parasites were identified using microscopic examination of gametocytes and PCR-based testing. Bayesian phylogeny was used to determine relationships between H. majoris lineages. Exoerythrocytic stages (megalomeronts) were reported using histological examination and laser microdissection was applied to isolate single megalomeronts for genetic analysis. Culicoides impunctatus biting midges were experimentally exposed in order to follow sporogonic development of the lineage hPHYBOR04. RESULTS: Gametocytes of the lineage hPHYBOR04 are indistinguishable from those of the widespread lineage hPARUS1 of H. majoris, indicating that both of these lineages belong to the H. majoris group. Phylogenetic analysis supported this conclusion. Sporogony of the lineage hPHYBOR04 was completed in C. impunctatus biting midges. Morphologically similar megalomeronts were reported in internal organs of both avian hosts. These were big roundish bodies (up to 360 µm in diameter) surrounded by a thick capsule-like wall and containing irregularly shaped cytomeres, in which numerous merozoites developed. DNA sequences obtained from single isolated megalomeronts confirmed the identification of H. majoris. CONCLUSIONS: Phylogenetic analysis identified a group of closely related H. majoris lineages, two of which are characterized not only by morphologically identical blood stages, but also complete sporogonic development in C. impunctatus and development of morphologically similar megalomeronts. It is probable that other lineages belonging to the same group would bear the same characters and phylogenies based on partial cytb gene could be used to predict life-cycle features in avian haemoproteids including vector identity and patterns of exoerythrocytic merogony. This study reports morphologically unique megalomeronts in naturally infected birds and calls for research on exoerythrocytic development of haemoproteids to better understand pathologies caused in avian hosts.

Sporogony of four Haemoproteus species (Haemosporida: Haemoproteidae), with report of in vitro ookinetes of Haemoproteus hirundinis: phylogenetic inference indicates patterns of haemosporidian parasite ookinete development.

BACKGROUND: Haemoproteus (Parahaemoproteus) species (Haemoproteidae) are widespread blood parasites that can cause disease in birds, but information about their vector species, sporogonic development and transmission remain fragmentary. This study aimed to investigate the complete sporogonic development of four Haemoproteus species in Culicoides nubeculosus and to test if phylogenies based on the cytochrome b gene (cytb) reflect patterns of ookinete development in haemosporidian parasites. Additionally, one cytb lineage of Haemoproteus was identified to the species level and the in vitro gametogenesis and ookinete development of Haemoproteus hirundinis was characterised. METHODS: Laboratory-reared C. nubeculosus were exposed by allowing them to take blood meals on naturally infected birds harbouring single infections of Haemoproteus belopolskyi (cytb lineage hHIICT1), Haemoproteus hirundinis (hDELURB2), Haemoproteus nucleocondensus (hGRW01) and Haemoproteus lanii (hRB1). Infected insects were dissected at intervals in order to detect sporogonic stages. In vitro exflagellation, gametogenesis and ookinete development of H. hirundinis were also investigated. Microscopic examination and PCR-based methods were used to confirm species identity. Bayesian phylogenetic inference was applied to study the relationships among Haemoproteus lineages. RESULTS: All studied parasites completed sporogony in C. nubeculosus. Ookinetes and sporozoites were found and described. Development of H. hirundinis ookinetes was similar both in vivo and in vitro. Developing ookinetes of this parasite possess long outgrowths, which extend longitudinally and produce the apical end of the ookinetes. A large group of closely related Haemoproteus species with a similar mode of ookinete development was determined. Bayesian analysis indicates that this character has phylogenetic value. The species identity of cytb lineage hDELURB2 was determined: it belongs to H. hirundinis. CONCLUSIONS: Culicoides nubeculosus is susceptible to and is a likely natural vector of numerous species of Haemoproteus parasites, thus worth attention in haemoproteosis epidemiology research. Data about in vitro development of haemoproteids provide valuable information about the rate of ookinete maturation and are recommended to use as helpful step during vector studies of haemosporidian parasites, particularly because they guide proper dissection interval of infected insects for ookinete detection during in vivo experiments. Additionally, in vitro studies readily identified patterns of morphological ookinete transformations, the characters of which are of phylogenetic value in haemosporidian parasites.

Molecular characterization of six widespread avian haemoproteids, with description of three new Haemoproteus species.

Species of Haemoproteus (Haemosporida, Haemoproteidae) are widespread and often prevalent blood parasites of birds all over the word. They are particularly diverse in tropical countries. Due to limited knowledge of life cycles, these pathogens usually have been considered relatively benign and were neglected in veterinary medicine and bird management. However, recent molecular studies provided evidence that Haemoproteus parasites might cause severe diseases if they infect non-adapted (wrong) avian hosts due to marked damage of organs by exo-erythrocytic stages (megalomeronts). Additionally, high Haemoproteus infections are lethal to blood-sucking insects. Molecular markers are essential for reliable detection and species identification both at tissue stages in vertebrates and sporogonic stages in arthropods however, remain insufficiently developed for wildlife haemosporidian parasites. This study combined PCR-based and microscopic approaches and reported cytochrome b gene (cytb) and apicoplast gene (clpc) markers for characterization of six widespread species of haemoproteids parasitizing common birds wintering in tropics and subtropics of the Old World. Three new Haemoproteus species were described using morphological and molecular markers. Molecular characterization of haemoproteids parasitizing falcons was developed. Morphological and phylogenetic characterization of Haemoproteus tinnunculi (cytb lineage hFALSUB01), H. brachiatus (hLK03), H. parabelopolskyi (hSYAT1), H. homogeneae n. sp. (hSYAT16), H. homopicae n. sp. (hGAGLA07) and H. homominutus n. sp. (hCUKI1) was performed and provides clues for infections diagnostics. This study adds three species to the group of morphologically readily distinct Haemoproteus parasites, which differ in few base pairs (< 1%) in their partial cytb sequences, indicating that low genetic difference in such sequences often show between-species divergence and should be carefully applied in taxonomic biodiversity studies of haemosporidian parasites. Bayesian phylogenetic analysis identified the position of detected lineages in regard of other Haemoproteus species, suggesting that all reported parasites belong to subgenus Parahaemoproteus and likely are transmitted by Culicoides biting midges. Importance of clpc gene sequences was specified in haemosporidian parasite taxonomy on species levels.

Patterns of Plasmodium homocircumflexum virulence in experimentally infected passerine birds.

BACKGROUND: Avian malaria parasites (genus Plasmodium) are cosmopolitan and some species cause severe pathologies or even mortality in birds, yet their virulence remains fragmentally investigated. Understanding mechanisms and patterns of virulence during avian Plasmodium infections is crucial as these pathogens can severely affect bird populations in the wild and cause mortality in captive individuals. The goal of this study was to investigate the pathologies caused by the recently discovered malaria parasite Plasmodium homocircumflexum (lineage pCOLL4) in four species of European passeriform birds. METHODS: One cryopreserved P. homocircumflexum strain was multiplied and used for experimental infections. House sparrows (Passer domesticus), common chaffinches (Fringilla coelebs), common crossbills (Loxia curvirostra) and common starlings (Sturnus vulgaris) were exposed by subinoculation of infected blood. Experimental and control groups (8 individuals in each) were observed for over 1 month. Parasitaemia, haematocrit value and body mass were monitored. At the end of the experiment, samples of internal organs were collected and examined using histological and chromogenic in situ hybridization methods. RESULTS: All exposed birds were susceptible, with similar average prepatent period and maximum parasitaemia, yet virulence was different in different bird species. Mortality due to malaria was reported in chaffinches, house sparrows and crossbills (7, 5 and 3 individuals died respectively), but not in starlings. Exoerythrocytic meronts (phanerozoites) were observed in the brain of all dead experimental birds. Blockage of blood vessels in the brain led to cerebral ischaemia, invariably causing brain damage, which is likely the main reason of mortality. Phanerozoites were observed in parenchymal organs, heart and muscles of all infected individuals, except starlings. CONCLUSION: This study shows that P. homocircumflexum is generalist and the same lineage caused similar parasitaemia-related pathologies in different host species. Additionally, the mode of exo-erythrocytic development is different in different birds, resulting in different mortality rates. This should be taken into consideration in studies addressing pathology during avian malaria infections.

High susceptibility of the laboratory-reared biting midges Culicoides nubeculosus to Haemoproteus infections, with review on Culicoides species that transmit avian haemoproteids.

Haemosporidian parasites belonging to Haemoproteus cause avian diseases, however, vectors remain unidentified for the majority of described species. We used the laboratory-reared biting midges Culicoides nubeculosus to determine if the sporogonic development of three widespread Haemoproteus parasites completes in this insect. The midges were reared and fed on one common blackbird, white wagtail and thrush nightingale naturally infected with Haemoproteus minutus, Haemoproteus motacillae and Haemoproteus attenuatus, respectively. The engorged females were dissected in order to follow their sporogonic development. Microscopic examination was used to identify sporogonic stages. Bayesian phylogeny based on partial cytochrome b gene was constructed in order to determine phylogenetic relationships among Culicoides species-transmitted haemoproteids. All three parasites completed sporogony. Phylogenetic analysis placed Culicoides species transmitted haemoproteids in one well-supported clade, proving that such analysis readily indicates groups of dipteran insects transmitting avian haemoproteids. Available data show that 11 species of Culicoides have been proved to support complete sporogony of 18 species of avian haemoproteids. The majority of Culicoides species can act as vectors for many Haemoproteus parasites, indicating the low specificity of these parasites to biting midges, whose are globally distributed. This calls for control of haemoproteid infections during geographical translocation of infected birds.

De novo transcriptome assembly and preliminary analyses of two avian malaria parasites, Plasmodium delichoni and Plasmodium homocircumflexum.

Parasites of the genus Plasmodium infect a wide array of hosts, causing malaria in all major groups of terrestrial vertebrates including primates, reptiles, and birds. Molecular mechanisms explaining why some Plasmodium species are virulent, while other closely related malaria pathogens are relatively benign in the same hosts, remain unclear. Here, we present the RNA sequencing and subsequent transcriptome assembly of two avian Plasmodium parasites which can eventually be used to better understand the genetic mechanisms underlying Plasmodium species pathogenicity in an avian host. Plasmodium homocircumflexum, a cryptic, pathogenic species that often causes mortality and Plasmodium delichoni, a newly described, relatively benign malaria parasite that does not kill its hosts, were used to experimentally infect two Eurasian siskins (Carduelis spinus). RNA extractions were performed and RNA sequencing was completed using high throughput Illumina sequencing. Using established bioinformatics pipelines, the sequencing data from both species were used to generate transcriptomes using published Plasmodium species genomes as a scaffold. The finalized transcriptome of P. homocircumflexum contained 21,612 total contigs while that of P. delichoni contained 12,048 contigs. We were able to identify many genes implicated in erythrocyte invasion actively expressed in both P. homocircumflexum and P. delichoni, including the well described vaccine candidates Apical Membrane Antigen-1 (AMA-1) and Merozoite Surface Protein 1 (MSP1). This work acts as a stepping stone to increase available data on avian Plasmodium parasites, thus enabling future research into the evolution of pathogenicity in malaria.

A new blood parasite of leaf warblers: molecular characterization, phylogenetic relationships, description and identification of vectors.

BACKGROUND: Blood parasites of the genus Haemoproteus Kruse, 1890 are cosmopolitan, might be responsible for mortality in non-adapted birds, and often kill blood-sucking insects. However, this group remains insufficiently investigated in the wild. This is particularly true for the parasites of leaf warblers of the Phylloscopidae Alström, Ericson, Olsson & Sundberg the common small Old World passerine birds whose haemoproteid parasite diversity and vectors remain poorly studied. This study reports a new species of Haemoproteus parasitizing leaf warblers, its susceptible vector and peculiar phylogenetic relationships with other haemoproteids. METHODS: Wood warblers (Phylloscopus sibilatrix Bechstein) were caught in Lithuania during spring migration, and blood films were examined microscopically. Laboratory reared Culicoides nubeculosus Meigen were exposed experimentally by allowing them to take blood meals on one individual harbouring mature gametocytes of the new Haemoproteus species (lineage hPHSIB2). To follow sporogonic development, the engorged insects were dissected at intervals. The parasite lineage was distinguished using sequence data, and morphological analysis of blood and sporogonic stages was carried out. Bayesian phylogeny was constructed in order to determine the phylogenetic relationships of the new parasite with other haemoproteids. RESULTS: Haemoproteus (Parahaemoproteus) homopalloris n. sp. was common in wood warblers sampled after arrival to Europe from their wintering grounds in Africa. The new parasite belongs to a group of avian haemoproteid species with macrogametocytes possessing pale staining cytoplasm. All species of this group clustered together in the phylogenetic analysis, indicating that intensity of the cytoplasm staining is a valuable phylogenetic character. Laboratory-reared biting midges C. nubeculosus readily supported sporogony of new infections. Phylogenetic analysis corroborated vector experiments, placing the new parasite in the clade of Haemoproteus (Parahaemoproteus) parasites transmitted by biting midges. CONCLUSIONS: Haemoproteus homopalloris n. sp. is the third haemoproteid, which is described from and is prevalent in wood warblers. Phylogenetic analysis identified a clade containing seven haemoproteids, which are characterised by pale staining of the macrogametocyte cytoplasm and with ookinetes maturing exceptionally rapidly (between 1 to 1.5 h after exposure to air). Both these features may represent valuable phylogenetic characters. Studies targeting mechanisms of sporogonic development of haemoproteids remain uncommon and should be encouraged. Culicoides nubeculosus is an excellent experimental vector of the new parasite species.

Characterization of Plasmodium relictum, a cosmopolitan agent of avian malaria.

BACKGROUND: Microscopic research has shown that Plasmodium relictum is the most common agent of avian malaria. Recent molecular studies confirmed this conclusion and identified several mtDNA lineages, suggesting the existence of significant intra-species genetic variation or cryptic speciation. Most identified lineages have a broad range of hosts and geographical distribution. Here, a rare new lineage of P. relictum was reported and information about biological characters of different lineages of this pathogen was reviewed, suggesting issues for future research. METHODS: The new lineage pPHCOL01 was detected in Common chiffchaff Phylloscopus collybita, and the parasite was passaged in domestic canaries Serinus canaria. Organs of infected birds were examined using histology and chromogenic in situ hybridization methods. Culex quinquefasciatus mosquitoes, Zebra finch Taeniopygia guttata, Budgerigar Melopsittacus undulatus and European goldfinch Carduelis carduelis were exposed experimentally. Both Bayesian and Maximum Likelihood analyses identified the same phylogenetic relationships among different, closely-related lineages pSGS1, pGRW4, pGRW11, pLZFUS01, pPHCOL01 of P. relictum. Morphology of their blood stages was compared using fixed and stained blood smears, and biological properties of these parasites were reviewed. RESULTS: Common canary and European goldfinch were susceptible to the parasite pPHCOL01, and had markedly variable individual prepatent periods and light transient parasitaemia. Exo-erythrocytic and sporogonic stages were not seen. The Zebra finch and Budgerigar were resistant. Neither blood stages nor vector stages of all examined P. relictum lineages can be distinguished morphologically. CONCLUSION: Within the huge spectrum of vertebrate hosts, mosquito vectors, and ecological conditions, different lineages of P. relictum exhibit indistinguishable, markedly variable morphological forms. Parasites of same lineages often develop differently in different bird species. Even more, the variation of biological properties (parasitaemia dynamics, blood pathology, prepatent period) in different isolates of the same lineage might be greater than the variation in different lineages during development in the same species of birds, indicating negligible taxonomic value of such features. Available lineage information is excellent for parasite diagnostics, but is limited in predictions about relationships in certain host-parasite associations. A combination of experiments, field observations, microscopic and molecular diagnostics is essential for understanding the role of different P. relictum lineages in bird health.

Molecular characterization and distribution of Plasmodium matutinum, a common avian malaria parasite.

Species of Plasmodium (Plasmodiidae, Haemosporida) are widespread and cause malaria, which can be severe in avian hosts. Molecular markers are essential to detect and identify parasites, but still absent for many avian malaria and related haemosporidian species. Here, we provide first molecular characterization of Plasmodium matutinum, a common agent of avian malaria. This parasite was isolated from a naturally infected thrush nightingale Luscinia luscinia (Muscicapidae). Fragments of mitochondrial, apicoplast and nuclear genomes were obtained. Domestic canaries Serinus canaria were susceptible after inoculation of infected blood, and the long-lasting light parasitemia developed in two exposed birds. Clinical signs of illness were not reported. Illustrations of blood stages of P. matutinum (pLINN1) are given, and phylogenetic analysis identified the closely related avian Plasmodium species. The phylogeny based on partial cytochrome b (cyt b) sequences suggests that this parasite is most closely related to Plasmodium tejerai (cyt b lineage pSPMAG01), a common malaria parasite of American birds. Both these parasites belong to subgenus Haemamoeba, and their blood stages are similar morphologically, particularly due to marked vacuolization of the cytoplasm in growing erythrocytic meronts. Molecular data show that transmission of P. matutinum (pLINN1) occurs broadly in the Holarctic, and the parasite likely is of cosmopolitan distribution. Passeriform birds and Culex mosquitoes are common hosts. This study provides first molecular markers for detection of P. matutinum.

Mechanisms of mortality in Culicoides biting midges due to Haemoproteus infection.

We examined the effects of Haemoproteus infection on the survival and pathology caused in the biting midges. Forty-six females of Culicoides impunctatus were exposed experimentally by allowing them to feed on a naturally infected red-backed shrike infected with Haemoproteus lanii (lineage hRB1, gametocytaemia 5·2%). Seventeen females were fed on an uninfected bird (controls). Dead insects were collected, counted and used for dissection, histological examination and polymerase chain reaction-based testing. Parasites were present in all experimentally infected biting midges, but absent from control insects. Survivorship differed significantly between the control and infected groups. Twelve hours post-exposure (PE), 45 (98%) experimentally infected midges were dead, but all control midges remained alive, and many of them survived until 7 day PE. The migrating ookinetes of H. lanii overfilled midgut, markedly damaged the midgut wall, entered the haemocoel and overfilled the abdomen and thorax of exposed biting midges. Massive infection by migrating ookinetes led to damage of abdomen and thorax of biting midges. The parasites often present in large clumps in the haemocoel in abdomen and thorax, leading to the interruption of the haemolymph circulation. These are the main reasons for rapid death of biting midges after feeding on high-intensity infections of Haemoproteus parasites.

Mortality and pathology in birds due to Plasmodium (Giovannolaia) homocircumflexum infection, with emphasis on the exoerythrocytic development of avian malaria parasites.

BACKGROUND: Species of avian malaria parasites (Plasmodium) are widespread, but their virulence has been insufficiently investigated, particularly in wild birds. During avian malaria, several cycles of tissue merogony occur, and many Plasmodium spp. produce secondary exoerythrocytic meronts (phanerozoites), which are induced by merozoites developing in erythrocytic meronts. Phanerozoites markedly damage organs, but remain insufficiently investigated in the majority of described Plasmodium spp. Avian malaria parasite Plasmodium (Giovannolaia) homocircumflexum (lineage pCOLL4) is virulent and produces phanerozoites in domestic canaries Serinus canaria, but its pathogenicity in wild birds remains unknown. The aim of this study was to investigate the pathology caused by this infection in species of common European birds. METHODS: One individual of Eurasian siskin Carduelis spinus, common crossbill Loxia curvirostra and common starling Sturnus vulgaris were exposed to P. homocircumflexum infection by intramuscular sub-inoculation of infected blood. The birds were maintained in captivity and parasitaemia was monitored until their death due to malaria. Brain, heart, lungs, liver, spleen, kidney, and a piece of breast muscle were examined using histology and chromogenic in situ hybridization (ISH) methods. RESULTS: All exposed birds developed malaria infection, survived the peak of parasitaemia, but suddenly died between 30 and 38 days post exposure when parasitaemia markedly decreased. Numerous phanerozoites were visible in histological sections of all organs and were particularly easily visualized after ISH processing. Blockage of brain capillaries with phanerozoites may have led to cerebral ischaemia, causing cerebral paralysis and is most likely the main reason of sudden death of all infected individuals. Inflammatory response was not visible around the brain, heart and muscle phanerozoites, and it was mild in parenchymal organs. The endothelial damage likely causes dysfunction and failure of parenchymal organs. CONCLUSION: Plasmodium homocircumflexum caused death of experimental passerine birds due to marked damage of organs by phanerozoites. Patterns of phanerozoites development and pathology were similar in all exposed birds. Mortality was reported when parasitaemia decreased or even turned into chronic stage, indicating that the light parasitaemia is not always indication of improved health during avian malaria. Application of traditional histological and ISH methods in parallel simplifies investigation of exoerythrocytic development and is recommended in avian malaria research.