RNAscope in situ hybridization reveals microvascular sequestration of Plasmodium relictum pSGS1 blood stages but absence of exo-erythrocytic dormant stages during latent infection of Serinus canaria.

BACKGROUND: Birds chronically infected with avian malaria parasites often show relapses of parasitaemia after latent stages marked by absence of parasites in the peripheral circulation. These relapses are assumed to result from the activation of dormant exo-erythrocytic stages produced during secondary (post-erythrocytic) merogony of avian Plasmodium spp. Yet, there is no morphological proof of persistent or dormant tissue stages in the avian host during latent infections. This study investigated persistence of Plasmodium relictum pSGS1 in birds with latent infections during winter, with the goal to detect presumed persisting tissue stages using a highly sensitive RNAscope® in situ hybridization technology. METHODS: Fourteen domestic canaries were infected with P. relictum pSGS1 by blood-inoculation in spring, and blood films examined during the first 4 months post infection, and during winter and spring of the following year. After parasitaemia was no longer detectable, half of the birds were dissected, and tissue samples investigated for persisting tissue stages using RNAscope ISH and histology. The remaining birds were blood-checked and dissected after re-appearance of parasitaemia, and their tissues equally examined. RESULTS: Systematic examination of tissues showed no exo-erythrocytic stages in birds exhibiting latent infections by blood-film microscopy, indicating absence of dormant tissue stages in P. relictum pSGS1-infected canaries. Instead, RNAscope ISH revealed rare P. relictum blood stages in capillaries of various tissues and organs, demonstrating persistence of the parasites in the microvasculature. Birds examined after re-appearance of parasitemia showed higher numbers of P. relictum blood stages in both capillaries and larger blood vessels, indicating replication during early spring and re-appearance in the peripheral circulation. CONCLUSIONS: The findings suggest that persistence of P. relictum pSGS1 during latent infection is mediated by continuous low-level erythrocytic merogony and possibly tissue sequestration of infected blood cells. Re-appearance of parasitaemia in spring seems to result from increased erythrocytic merogony, therefore representing recrudescence and not relapse in blood-inoculated canaries. Further, the study highlights strengths and limitations of the RNAscope ISH technology for the detection of rare parasite stages in tissues, providing directions for future research on persistence and tissue sequestration of avian malaria and related haemosporidian parasites.

The 18S rRNA genes of Haemoproteus (Haemosporida, Apicomplexa) parasites from European songbirds with remarks on improved parasite diagnostics.

BACKGROUND: The nuclear ribosomal RNA genes of Plasmodium parasites are assumed to evolve according to a birth-and-death model with new variants originating by duplication and others becoming deleted. For some Plasmodium species, it has been shown that distinct variants of the 18S rRNA genes are expressed differentially in vertebrate hosts and mosquito vectors. The central aim was to evaluate whether avian haemosporidian parasites of the genus Haemoproteus also have substantially distinct 18S variants, focusing on lineages belonging to the Haemoproteus majoris and Haemoproteus belopolskyi species groups. METHODS: The almost complete 18S rRNA genes of 19 Haemoproteus lineages of the subgenus Parahaemoproteus, which are common in passeriform birds from the Palaearctic, were sequenced. The PCR products of 20 blood and tissue samples containing 19 parasite lineages were subjected to molecular cloning, and ten clones in mean were sequenced each. The sequence features were analysed and phylogenetic trees were calculated, including sequence data published previously from eight additional Parahaemoproteus lineages. The geographic and host distribution of all 27 lineages was visualised as CytB haplotype networks and pie charts. Based on the 18S sequence data, species-specific oligonucleotide probes were designed to target the parasites in host tissue by in situ hybridization assays. RESULTS: Most Haemoproteus lineages had two or more variants of the 18S gene like many Plasmodium species, but the maximum distances between variants were generally lower. Moreover, unlike in most mammalian and avian Plasmodium species, the 18S sequences of all but one parasite lineage clustered into reciprocally monophyletic clades. Considerably distinct 18S clusters were only found in Haemoproteus tartakovskyi hSISKIN1 and Haemoproteus sp. hROFI1. The presence of chimeric 18S variants in some Haemoproteus lineages indicates that their ribosomal units rather evolve in a semi-concerted fashion than according to a strict model of birth-and-death evolution. CONCLUSIONS: Parasites of the subgenus Parahaemoproteus contain distinct 18S variants, but the intraspecific variability is lower than in most mammalian and avian Plasmodium species. The new 18S data provides a basis for more thorough investigations on the development of Haemoproteus parasites in host tissue using in situ hybridization techniques targeting specific parasite lineages.

Avian haemosporidian parasites of accipitriform raptors.

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

Experimental study of newly described avian malaria parasite Plasmodium (Novyella) collidatum n. sp., genetic lineage pFANTAIL01 obtained from South Asian migrant bird.

BACKGROUND: Avian malaria parasites are microorganisms parasitizing erythrocytes and various tissues of the birds; they are common and distributed worldwide. These parasites are known to infect birds of different taxa and be the cause of the deaths of birds in the wild and in captivity. The species of parasites with the ability to colonize new territories and infect local non-migratory birds are of particular interest. This scenario is likely in temperate zones of Europe, because of climate change and its contribution in spreading vectors of southern origin, which can be involved in the transmission of malaria parasites. In the present study, a tropical Plasmodium parasite from a naturally infected long-distance migrant bird was isolated and tested for its ability to develop in common species of mosquitoes and European short-distance migrant birds. METHODS: Plasmodium sp. (pFANTAIL01) was isolated on the Curonian spit of the Baltic sea coast from the naturally infected Common rosefinch, Carpodacus erythrinus in June 2019. The parasite was described based on the morphological features of its blood stages, the partial mitochondrial cytochrome b gene and development after experimental infection of birds and mosquitoes. The parasite was inoculated into Eurasian siskins, Carduelis spinus. Parasitaemia, haematocrit and weight of birds were monitored. At the end of the survey, internal organs were collected to study exoerythrocytic stages of this parasite. Experimental infection of mosquitoes Culex pipiens form molestus and Culex quinquefasciatus was applied to study sporogonic development of the parasite. RESULTS: Based on morphological features, the parasite was described as a new species, Plasmodium collidatum n. sp., and attributed to subgenus Novyella. It was revealed that the obtained pFANTAIL01 lineage is a generalist parasite infecting a wide range of avian hosts and most likely is transmitted in South and Southeast (SE) Asia and Oceania. In Europe, this strain was recorded only in adult migratory birds wintering in South Asia. This parasite developed high parasitaemia in experimentally infected siskins and caused 25 % mortality. Exoerythrocytic stages of pFANTAIL01 were found in the lungs, liver, spleen and kidney of the deceased birds. Sporogonic development did not occur in Cx. pipiens form molestus and Cx. quinquefasciatus mosquitoes. CONCLUSIONS: Plasmodium collidatum is a highly virulent for Eurasian siskin and completes its development in these birds, which can be considered as a potential vertebrate host if the transmission of the infection starts occurring in Europe and temperate zones.

Geographic and host distribution of haemosporidian parasite lineages from birds of the family Turdidae.

BACKGROUND: Haemosporidians (Apicomplexa, Protista) are obligate heteroxenous parasites of vertebrates and blood-sucking dipteran insects. Avian haemosporidians comprise more than 250 species traditionally classified into four genera, Plasmodium, Haemoproteus, Leucocytozoon, and Fallisia. However, analyses of the mitochondrial CytB gene revealed a vast variety of lineages not yet linked to morphospecies. This study aimed to analyse and discuss the data of haemosporidian lineages isolated from birds of the family Turdidae, to visualise host and geographic distribution using DNA haplotype networks and to suggest directions for taxonomy research on parasite species. METHODS: Haemosporidian CytB sequence data from 350 thrushes were analysed for the present study and complemented with CytB data of avian haemosporidians gathered from Genbank and MalAvi database. Maximum Likelihood trees were calculated to identify clades featuring lineages isolated from Turdidae species. For each clade, DNA haplotype networks were calculated and provided with information on host and geographic distribution. RESULTS: In species of the Turdidae, this study identified 82 Plasmodium, 37 Haemoproteus, and 119 Leucocytozoon lineages, 68, 28, and 112 of which are mainly found in this host group. Most of these lineages cluster in the clades, which are shown as DNA haplotype networks. The lineages of the Leucocytozoon clades were almost exclusively isolated from thrushes and usually were restricted to one host genus, whereas the Plasmodium and Haemoproteus networks featured multiple lineages also recovered from other passeriform and non-passeriform birds. CONCLUSION: This study represents the first attempt to summarise information on the haemosporidian parasite lineages of a whole bird family. The analyses allowed the identification of numerous groups of related lineages, which have not been linked to morphologically defined species yet, and they revealed several cases in which CytB lineages were probably assigned to the wrong morphospecies. These taxonomic issues are addressed by comparing distributional patterns of the CytB lineages with data from the original species descriptions and further literature. The authors also discuss the availability of sequence data and emphasise that MalAvi database should be considered an extremely valuable addition to GenBank, but not a replacement.

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.

The experimental study on susceptibility of common European songbirds to Plasmodium elongatum (lineage pGRW6), a widespread avian malaria parasite.

BACKGROUND: Plasmodium elongatum (cytochrome b lineage pGRW6) is a widespread avian malaria parasite, often causing severe disease in non-adapted hosts. This parasite lineage is of global distribution however, its virulence remains insufficiently understood, particularly in wild birds. Surprisingly, this infection has never been reported in Common starlings Sturnus vulgaris and Common crossbills Loxia curvirostra, common European songbirds which were extensively sampled across Europe. A hypothesis was proposed that these birds might be resistant to the pGRW6 infection. The aim of this study was to test this hypothesis. METHODS: Lineage pGRW6 was isolated from a naturally infected Eurasian reed warbler, multiplied in vivo and inoculated in Common starlings and Common crossbills. Experimental and control groups (8 birds in each) were maintained in controlled conditions and examined microscopically every 4 days. Haematocrit value and body mass were monitored in parallel. At the end of the experiment (44 days post exposure), samples of internal organs were collected and examined using histological methods for possible presence of phanerozoites. RESULTS: All control birds remained uninfected. Experimental starlings were resistant. All exposed crossbills were susceptible and survived until the end of this study. Prepatent period was 12-16 days post exposure. Light parasitaemia (< 0.7%) developed in all birds, and only few phanerozoites were seen in bone marrow cells of 5 of 8 experimentally infected crossbills. Significant changes were reported only in haematocrit value but not body mass in the exposed crossbills compared to controls. CONCLUSION: Plasmodium elongatum (pGRW6) is of low virulence in Common crossbills and is unable to develop in Common starlings, indicating innate resistance of the later bird species. Low virulence in Common crossbills is likely due to the inability or low ability of this parasite lineage to develop phanerozoites resulting in light (if at all) damage of stem bone marrow cells. This study suggests that susceptibility of different bird species to the lineage pGRW6 is markedly variable. The global distribution of this parasite might be due to low virulence in wild adapted avian hosts, which survive this infection and serve as reservoirs host for non-adapted birds in whom this infection is often lethal.

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.

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.

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.

Description of Haemoproteus ciconiae sp. nov. (Haemoproteidae, Haemosporida) from the white stork Ciconia ciconia, with remarks on insensitivity of established polymerase chain reaction assays to detect this infection.

Haemoproteus ciconiae sp. nov. (Haemosporida, Haemoproteidae) was found in the white stork Ciconia ciconia (Ciconiiformes, Ciconiidae) after spring migration in Lithuania. This organism is illustrated and described based on the morphology of its blood stages. The new species can be readily distinguished from all other haemoproteids parasitizing ciconiiform birds due to the presence of large number (approximately 20 on average) small (<1 µm) pigment granules in its mature gametocytes. Growing and mature gametocytes of H. ciconiae were readily visible in all blood films (parasitemia of 0.001 %). However, experienced researchers were unable to detect sequences of its mitochondrial cytochrome b (cyt b) or apicoplast genes from the microscopically positive sample by using five established assays for polymerase chain reaction (PCR)-based detection of avian haemosporidian parasites. The white stork cyt b sequence was readily detectable, indicating the well-optimised PCR protocols and the good quality of total DNA in the sample containing the new species. The failure to amplify this parasite DNA indicates insufficient sensitivity of the currently used PCR-based assays in diagnostics of avian haemosporidian infections. We suggest possible explanations of this observation. To minimize number of the false negative PCR reports, we call for the continued use of optical microscopy in parallel with molecular diagnostics in studies of haemosporidian parasites, particularly in wildlife.

Plasmodium delichoni n. sp.: description, molecular characterisation and remarks on the exoerythrocytic merogony, persistence, vectors and transmission.

Malaria parasite Plasmodium (Novyella) delichoni n. sp. (Haemosporida, Plasmodiidae) was found in a widespread Eurasian songbird, the common house martin Delichon urbicum (Hirundinidae). It is described based on the morphology of its blood stages and segments of the mitochondrial cytochrome b and apicoplast genes, which can be used for molecular identification of this species. Erythrocytic meronts and gametocytes are strictly nucleophilic, and mature gametocytes possess pigment granules of markedly variable size, including large ones (1 µm in length). Due to these features, P. delichoni can be readily distinguished from all described species of avian malaria parasites belonging to subgenus Novyella. Additionally, mature erythrocytic merozoites contain a dense clump of chromatin, a rare character in avian malaria parasites. Erythrocytic merogony is asynchronous. Illustrations of blood stages of the new species are given, and phylogenetic analysis identifies DNA lineages closely related to this parasite. Domestic canary Serinus canaria and Eurasian siskin Carduelis spinus were infected after subinoculation of infected blood obtained from the house martin. Parasitemia was long lasting in both these hosts, but it was high (up to 70 %) in Eurasian siskins and low (up to 1 %) in canaries. Mortality was not observed, and histological examination and chromogenic in situ hybridisation did not reveal secondary exoerythrocytic meronts (phanerozoites) in the exposed birds. It is likely that persistence of this infection occurs due to long-lasting parasitemia in avian hosts. Sporogony was abortive in mosquitoes Culex pipiens pipiens form molestus, Culex quinquefasciatus and Aedes aegypti at gametogenesis or ookinete stages. The new species is absent from juvenile birds at breeding sites in Europe, indicating that transmission occurs at African wintering grounds.

Plasmodium spp.: an experimental study on vertebrate host susceptibility to avian malaria.

The interest in experimental studies on avian malaria caused by Plasmodium species has increased recently due to the need of direct information about host-parasite interactions. Numerous important issues (host susceptibility, development of infection, the resistance and tolerance to avian malaria) can be answered using experimental infections. However, specificity of genetically different lineages of malaria parasites and their isolates is largely unknown. This study reviews recent experimental studies and offers additional data about susceptibility of birds to several widespread cytochrome b (cyt b) lineages of Plasmodium species belonging to four subgenera. We exposed two domesticated avian hosts (canaries Serinus canaria and ducklings Anas platyrhynchos) and also 16 species of common wild European birds to malaria infections by intramuscular injection of infected blood and then tested them by microscopic examination and PCR-based methods. Our study confirms former field and experimental observations about low specificity and wide host-range of Plasmodium relictum (lineages SGS1 and GRW11) and P. circumflexum (lineage TURDUS1) belonging to the subgenera Haemamoeba and Giovannolaia, respectively. However, the specificity of different lineages and isolates of the same parasite lineage differed between species of exposed hosts. Several tested Novyella lineages were species specific, with a few cases of successful development in experimentally exposed birds. The majority of reported cases of mortality and high parasitaemia were observed during parasite co-infections. Canaries were susceptible mainly for the species of Haemamoeba and Giovannolaia, but were refractory to the majority of Novyella isolates. Ducklings were susceptible to three malaria infections (SGS1, TURDUS1 and COLL4), but parasitaemia was light (<0.01%) and transient in all exposed birds. This study provides novel information about susceptibility of avian hosts to a wide array of malaria parasite lineages, outlining directions for future experimental research on various aspects of biology and epidemiology of avian malaria.

The evidence for rapid gametocyte viability changes in the course of parasitemia in Haemoproteus parasites.

Avian haemosporidian parasites of the genus Haemoproteus (Haemoproteidae, Haemosporida) are widespread, and some species cause diseases both in vertebrate hosts and blood-sucking insects. Parasitemia of Haemoproteus species usually is long-lasting, with gametocytes present in the circulation for several months. However, the viability of gametocytes and their ability to produce sexual cells have been insufficiently understood in the course of parasitemia. We initiated the sexual development in vitro conditions and calculated proportions of normal and anomalous ookinetes, which developed in two species of Haemoproteus. Mature gametocytes of the parasites were obtained from naturally infected avian hosts at different days of parasitemia. Haemoproteus (Parahaemoproteus) lanii (cytochrome b lineage hRB1) was isolated from one red-backed shrike Lanius collurio. Two isolates of Haemoproteus (Parahaemoproteus) tartakovskyi (cytochrome b lineage hSISKIN1) were used: one was obtained from a siskin Carduelis spinus and one from a common crossbill Loxia curvirostra. The wild-caught birds were kept indoors under controlled conditions, and blood was taken from them every 1 or 2 days during 10-14 days. After each blood sampling, the sexual process and ookinete development were initiated in vitro by exposure of infected blood containing mature gametocytes to air. Smears were prepared at intervals of 15 min, 3 h, and 12 h after the exposure; they were examined microscopically. In all, 25 experiments were performed; each experiment was repeated two times. The ratios of macro- and microgametocytes did not change in all experimental infections during this study. Sexual process occurred, and both normal and anomalous ookinetes developed in all parasites. The proportion of normal ookinetes did not change significantly in both isolates of H. tartakovskyi. Between 8 and 10 days of observation, the proportion of normal ookinetes of H. lanii decreased 6 times compared to the beginning of the experiment. That was accompanied with the rapid decrease of parasitemia and the inability of the majority of mature gametocytes to escape from erythrocytes and produce gametes, indicating disorder of the gametogenesis. There was clear difference in the gametogenesis between H. tartakovskyi and H. lanii from this point of view. This study shows that the viability of Haemoproteus gametocytes might change dramatically in the course of parasitemia within 1-2 days, and the presence of mature gametocytes in the circulation does not necessarily indicate their ability to exflagellate and produce ookinetes. We predict that this finding is important epidemiologically due to relationship with sporogony success.

Complete sporogony of Plasmodium relictum (lineage pGRW4) in mosquitoes Culex pipiens pipiens, with implications on avian malaria epidemiology.

Plasmodium relictum (lineage pGRW4) causes malaria in birds and is actively transmitted in countries with warm climates and also temperate regions of the New World. In Europe, the lineage pGRW4 has been frequently reported in many species of Afrotropical migrants after their arrival from wintering grounds, but is rare in European resident birds. Obstacles for transmission of this parasite in Europe have not been identified. Culex quinquefasciatus is an effective vector of pGRW4 malaria, but this mosquito is absent from temperate regions of Eurasia. It remains unclear if the lineage pGRW4 completes sporogony in European species of mosquitoes. Here we compare the sporogonic development of P. relictum (pGRW4) in experimentally infected mosquitoes Culex pipiens pipiens form molestus, C. quinquefasciatus, and Ochlerotatus cantans. The pGRW4 parasite was isolated from a garden warbler Sylvia borin, multiplied, and used to infect laboratory-reared Culex spp. and wild-caught Ochlerotatus mosquitoes by allowing them to take blood meals on infected birds. The exposed females were maintained at a mean laboratory temperature of 19 °C, which ranged between 14 °C at night and 24 °C during daytime. They were dissected on intervals to study the development of sporogonic stages. Only ookinetes developed in O. cantans; sporogonic development was abortive. The parasite completed sporogony in both Culex species, with similar patterns of development, and sporozoites were reported in the salivary glands 16 days after infection. The presence of sporogonic stages of the lineage pGRW4 in mosquitoes was confirmed by PCR-based testing of (1) the sporozoites present in salivary glands and (2) the single oocysts, which were obtained by laser microdissection from infected mosquito midguts. This study shows that P. relictum (pGRW4) completes sporogony in C. p. pipiens at relatively low temperatures. We conclude that there are no restrictions for spreading this bird infection in Europe from the point of view of vector availability and temperature necessary for sporogony. Other factors should be considered and were discussed for the explanation of rare reports of this malaria parasite in Europe.

In vitro development of Haemoproteus parasites: the efficiency of reproductive cells increase during simultaneous sexual process of different lineages.

Recent in vitro experimental studies reported the complex patterns of haemosporidian (Haemosporida) between-lineage interactions, which prevent mixing of lineages during simultaneous sexual process. Numerous anomalous ookinetes have been observed; these are not involved in sporogony. Massive development of such ookinetes might influence parasite transmission but is insufficiently investigated. The simultaneous sexual process of several lineages is a common phenomenon in vectors due to high prevalence of haemosporidian co-infections in wildlife. It remains unclear if the number of anomalous ookinetes changes during dual-infection sporogony in comparison with the single-infection process. We calculated proportions of the anomalous and normal ookinetes, which developed during single-infection (control) and dual-infection experiments in vitro conditions. Three mitochondrial cytochrome b lineages belonging to three Haemoproteus spp. (Haemosporida, Haemoproteidae) were isolated from naturally infected passerine birds. Sexual process and ookinete development were initiated in vitro by mixing blood containing mature gametocytes of two different parasites; the following experiments were performed: (1) Haemoproteus tartakovskyi (lineage hSISKIN1) × Haemoproteus lanii (lineage hRBS4) and (2) Haemoproteus belopolskyi (hHIICT3) × H. lanii (hRBS4). Genetic difference between lineages was 5.0-5.9%. Normal and anomalous ookinetes developed in all control and dual-infection experiments. The number of anomalous ookinetes markedly decreased, and normal ookinetes increased in all dual-infection experiments in comparison with those in controls, except for H. belopolskyi, in which proportion of the anomalous and normal ookinetes did not change. This study shows that simultaneous sexual process of two genetically distant lineages of haemosporidian parasites might increase the efficiency of reproductive cells, resulting in the development of a greater number of normal ookinetes. The marked increase of the number of normal ookinetes, which is involved in sporogony, indicates the success of sporogony in dual infections. Some haemosporidian lineages might benefit from simultaneous sporogony. Widespread avian Haemoproteus spp. are convenient and laboratory-friendly organisms for in vitro experimental research addressing between-lineage interaction in parasites during the sexual process.

Molecular characterization of five widespread avian haemosporidian parasites (Haemosporida), with perspectives on the PCR-based detection of haemosporidians in wildlife.

Haemosporidians (Haemosporida) are cosmopolitan in birds. Over 250 species of these blood parasites have been described and named; however, molecular markers remain unidentified for the great majority of them. This is unfortunate because linkage between DNA sequences and identifications based on morphological species can provide important information about patterns of transmission, virulence, and evolutionary biology of these organisms. There is an urgent need to remedy this because few experts possess the knowledge to identify haemosporidian species and few laboratories are involved in training these taxonomic skills. Here, we describe new mitochondrial cytochrome b markers for the polymerase chain reaction (PCR)-based detection of four widespread species of avian Haemoproteus (Haemoproteus hirundinis, Haemoproteus parabelopolskyi, Haemoproteus pastoris, Haemoproteus syrnii) and 1 species of Plasmodium (Plasmodium circumflexum). Illustrations of blood stages of the reported species are given, and morphological and phylogenetic analyses identify the DNA lineages that are associated with these parasites. This study indicates that morphological characters, which have been traditionally used in taxonomy of avian haemosporidian parasites, have a phylogenetic value. Perspectives on haemosporidian diagnostics using microscopic and PCR-based methods are discussed, particularly the difficulties in detection of light parasitemia, coinfections, and abortive parasite development. We emphasize that sensitive PCR amplifies more infections than can be transmitted; it should be used carefully in epidemiology studies, particularly in wildlife parasitology research. Because molecular studies are describing remarkably more parasite diversity than previously expected, the need for traditional taxonomy and traditional biological knowledge is becoming all the more crucial. The linkage of molecular and morphological approaches is worth more of the attention of researchers because this approach provides new knowledge for better understanding insufficiently investigated lethal diseases caused by haemosporidian infections, particularly on the exoerythrocytic (tissue) and vector stages. That requires close collaboration between researchers from different fields with a common interest.

First experimental observation on biology of the avian malaria parasite Plasmodium (Novyella) homonucleophilum (lineage pSW2), with remarks on virulence and distribution.

Species of subgenus Novyella remain most fragmentarily studied amongst avian malaria agents. Transmission of the recently described Plasmodium (Novyella) homonucleophilum (lineage pSW2) occurs broadly in the Old World, including Europe, however biology of this pathogen remains insufficiently investigated. This study provided the first data on the development of P. homonucleophilum in the experimentally infected Eurasian siskins Spinus spinus exposed by inoculation of infected blood. The parasite strain was isolated from a naturally infected song thrush Turdus philomelos, multiplied in vivo, and inoculated to six Eurasian siskins. The same number of birds were used as negative controls. All exposed birds were susceptible, and the controls remained uninfected during the entire study (172 days). Prepatent period was 8-12 days post exposure (dpe). Maximum parasitaemia reached 50-90 % of infected erythrocytes between 20 and 44 dpe. Then, parasitaemia decreased but remained relatively high during the entire observation. Three of six exposed birds died, indicating high virulence of this infection. The parasitaemia increase coincided with a decline of haematocrit value, indicating anaemia. Polychromasia was evident in all infected birds but not in controls. Body mass of exposed birds increased, coinciding with increased food intake. The latter probably is an adaptation to compensate energy loss of hosts due to the long-lasting parasitism. Exo-erythrocytic stages were not found, suggesting that long-lasting parasitaemia was entirely due to erythrocytic merogony. The lineage pSW2 has been reported broadly in the Old World and is likely a generalist infection. Neglected avian Novyella malaria parasites are worth more attention of researchers due to their cosmopolitan distribution and high virulence.