Insights into the Biology of Leucocytozoon Species (Haemosporida, Leucocytozoidae): Why Is There Slow Research Progress on Agents of Leucocytozoonosis?

Blood parasites of the genus Leucocytozoon (Leucocytozoidae) only inhabit birds and represent a readily distinct evolutionary branch of the haemosporidians (Haemosporida, Apicomplexa). Some species cause pathology and even severe leucocytozoonosis in avian hosts, including poultry. The diversity of Leucocytozoon pathogens is remarkable, with over 1400 genetic lineages detected, most of which, however, have not been identified to the species level. At most, approximately 45 morphologically distinct species of Leucocytozoon have been described, but only a few have associated molecular data. This is unfortunate because basic information about named and morphologically recognized Leucocytozoon species is essential for a better understanding of phylogenetically closely related leucocytozoids that are known only by DNA sequence. Despite much research on haemosporidian parasites during the past 30 years, there has not been much progress in taxonomy, vectors, patterns of transmission, pathogenicity, and other aspects of the biology of these cosmopolitan bird pathogens. This study reviewed the available basic information on avian Leucocytozoon species, with particular attention to some obstacles that prevent progress to better understanding the biology of leucocytozoids. Major gaps in current Leucocytozoon species research are discussed, and possible approaches are suggested to resolve some issues that have limited practical parasitological studies of these pathogens.

Keys to the avian Haemoproteus parasites (Haemosporida, Haemoproteidae).

BACKGROUND: Haemoproteus is a sister genus to malaria parasites (Plasmodium), which both belong to the order Haemosporida (Apicomplexa). Parasites of both genera are flourishing in birds, however, Haemoproteus species are noticeably less investigated. This is unfortunate because knowledge about close relatives of malaria pathogens is important for better understanding the evolutionary origin and basic biological features of the entire group of haemosporidian infections. Moreover, recent findings show that Haemoproteus species can cause severe damage of various bird organs due to megalomeronts and other exo-erythrocytic stages. These haemosporidians are remarkably diverse, but remain neglected partly due to difficulties in species identification. Hundreds of Haemoproteus genetic lineages have been reported in birds, and numerous new lineages are found each year, but most remain unidentified to the species level. Numerous new Haemoproteus pathogens were described during the past 20 years. However, keys for their identification are absent. Identification of Haemoproteus species remains a difficult task and is an obstacle for better understanding of the distribution and epidemiology of these parasites. This study aimed to develop comprehensive keys for the identification of described avian Haemoproteus species using morphological features of their blood stages (gametocytes). METHODS: Type and voucher preparations of avian Haemoproteus species were accessed in museums in Europe, Australia and the USA. Gametocytes of most described species were examined, and these data formed a background for this study. The data also were considered from published articles containing parasite species descriptions. The method of dichotomous keys was applied. The most difficult steps in the keys were accompanied with references to the corresponding parasite pictures. RESULTS: In all, 201 published articles were included in this review. Morphological diagnostic features of gametocytes of all described Haemoproteus species were analysed and compared. Illustrated keys for identification of these parasite species were developed. Available information about the molecular characterization of Haemoproteus parasites was provided. CONCLUSION: This review shows that 177 described species of avian Haemoproteus can be distinguished and identified in blood films using morphological characters of their gametocytes and host cells. These species were incorporated in the keys. Information about possible morphologically cryptic parasites was provided. Molecular markers are available for only 42% of the described Haemoproteus parasites, calling for researchers to fill this gap.

Increase of avian Plasmodium circumflexum prevalence, but not of other malaria parasites and related haemosporidians in northern Europe during the past 40 years.

BACKGROUND: Malaria is a health problem not only in human and veterinary medicine, but also in wildlife. Several theoretical studies have suggested that avian malaria transmission might be increasing in Europe. However, there are few direct empirical observations. Research on the distribution of avian haemosporidian parasites was initiated around the Curonian Lagoon, Europe in 1976 and continues since. This has provided an opportunity to compare the prevalence and diversity of avian malaria parasites (genus Plasmodium) and related haemosporidians (genera Haemoproteus and Leucocytozoon) in the same bird species using similar methodology but examined in two groups 40 years apart. This study aimed to describe and discuss the available data on this subject. METHODS: Prevalence and diversity of haemosporidians was compared in two passeriform bird groups, which consisted of the same species that were sampled on the coast of the Curonian Lagoon (Russia, Lithuania) during the same season (September) in 1978-1983 (bird Group 1) and 2020 (bird Group 2). Blood films of the European robin, Coal tit, Great tit, Eurasian wren, and Eurasian jay were screened by microscopic examination. Parasites were identified using morphological characters of blood stages. PCR-based methods were applied to determine genetic lineages of the parasites found in birds of Group 2. RESULTS: No difference was discernible in the prevalence or diversity of haemosporidian parasites belonging to Haemoproteus, Leucocytozoon, Plasmodium (Haemamoeba) and Plasmodium (Novyella) between birds of Groups 1 and 2. This indicates a similar rate of transmission and relatively stable epidemiological situation in regard of these infections during the past 40 years. The prevalence of only one malaria parasite species, Plasmodium (Giovannolaia) circumflexum, increased remarkably, but only in Coal tit, Great tit, and Eurasian wren, with no significant prevalence change in European robin and Eurasian jay. CONCLUSION: Plasmodium circumflexum is spreading and seems to be a new invasive avian malaria pathogen in countries with cold climates. The exceptionally high prevalence of P. circumflexum in birds breeding in relatively close-nests suggests an important role of the nesting biology related to bird-vector interaction in this pathogen transmission. The epidemiological situation seems to be relatively stable in regard of other studied avian hosts and haemosporidian parasites in northern Europe.

Description and molecular characterization of novel Leucocytozoon parasite (Apicomplexa: Haemosporida: Leucocytozoidae), Leucocytozoon polynuclearis n. sp. found in North American woodpeckers.

We describe Leucocytozoon polynuclearis n. sp. (Haemosporida: Leucocytozoidae) from two North American woodpeckers, the northern flicker (Colaptes auratus Linnaeus) and white-headed woodpecker (Dryobates albolarvatus Boie, 1826), based on the morphology of its blood stages and portions of the mitochondrial cytochrome b gene. The most distinctive features of Leucocytozoon polynuclearis n. sp. development are the triangular-shaped host cell nuclei and position of host cell nuclei above gametocytes. This parasite inhabits thrombocytes. Leucocytozoon squamatus Nandi, 1986, the only other Leucocytozoon species detected from Picidae birds, lacks features that are commonly found with L. polynuclearis n. sp. infections. Phylogenetic analysis identified DNA lineages associated with L. polynuclearis n. sp. and showed that this parasite is more closely related to other North American Leucocytozoon species than to L. squamatus, whose initial description was from infected Old World Picidae species. Although there are reports of L. squamatus in North American Picidae species, these detections were based only on microscopic examinations, remain genetically non-characterized, and might be misidentifications with regards to L. polynuclearis n. sp. Available parasite distribution data indicate that L. polynuclearis n. sp. infects Picidae species throughout North America and L. squamatus distribution probably is restricted to Old World Piciformes birds.

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.

Molecular characterization of swallow haemoproteids, with description of one new Haemoproteus species.

Haemoproteus species (Haemosporida, Haemoproteidae) are cosmopolitan bird blood parasites, which often cause relatively benign infections in adapted avian hosts, but severe and even lethal haemoproteosis might develop due to internal organ damage if these pathogens inhabit non-adapted (wrong) hosts. Haemoproteids of swallows (Hirundinidae) remain fragmentarily investigated, with only two haemoproteid species reported in this bird family, which members are cosmopolitan, diverse and inhabit various terrestrial ecosystems, particularly in tropical countries. This study describes and provides molecular characterization of Haemoproteus parahirundinis n. sp. (cytochrome b lineage hHIRUS05), parasite of the most broadly distributed swallow, the Barn swallow Hirundo rustica. Gametocytes, gametes and ookinetes of the new species were examined and compared with other haemoproteids described in swallows. The phylogenetic analysis indicated the existence of a largely undescribed Haemoproteus species diversity in birds of the Hirundinidae and also suggests that all lineages of haemoproteids reported in swallows are transmitted by Culicoides biting midges, but not louse flies of the Hippoboscidae, which often inhabit their nests. The biting midges should be the first targets in vectors research of swallow haemoproteids. This study indicates existence of Haemoproteus species, which are readily distinct based on morphological characters of their blood and sporogonic stages, but differ only negligently in partial cytochrome b sequences, the main markers broadly used in molecular characterization of haemoproteids. That calls for further taxonomic research on haemoproteid in swallows, many species of which are endangered or even threatened with extinction because of habitat degradation.

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.

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.

Keys to the avian malaria parasites.

BACKGROUND: Malaria parasites (genus Plasmodium) are widespread in birds. These pathogens cause pathology of blood and various organs, often resulting in severe avian malaria. Numerous recent studies have reported DNA sequences of avian malaria parasites, indicating rich genetic diversity and the possible existence of many undescribed species. However, the majority of reported Plasmodium lineages remain unidentified to species level, and molecular characterization is unavailable for the majority of described Plasmodium parasites. During the past 15 years, numerous new Plasmodium species have been described. However, keys for their identification are unavailable or incomplete. Identification of avian malaria parasites remains a difficult task even for experts, and this precludes development of avian malariology, particularly in wildlife. Here, keys for avian malaria parasites have been developed as a baseline for assisting academic and veterinary medicine researchers in identification of these pathogens. The main obstacles and future research priorities have been defined in the taxonomy of avian Plasmodium species. METHODS: The data were considered from published articles and type and voucher material, which was accessed in museums in Europe, the USA and Australia. Blood films containing various blood stages of the majority of described species were examined and used for the development of dichotomous keys for avian Plasmodium species. RESULTS: In all, 164 published articles were included in this review. Blood stages of avian Plasmodium parasites belonging to subgenera Haemamoeba, Giovannolaia, Novyella, Bennettinia and Huffia were analysed and compared. Illustrated keys for identification of subgenera and species of these parasites were developed. Lists of invalid and synonymous Plasmodium parasite names as well as names of doubtful identity were composed. CONCLUSION: This study shows that 55 described species of avian Plasmodium can be readily identified using morphological features of their blood stages. These were incorporated in the keys. Numerous synonymous names of Plasmodium species and also the names belonging to the category species inquirenda exist, and they can be used as reserves for future taxonomy studies. Molecular markers are unavailable for 58% of described Plasmodium parasites, raising a task for the current avian malaria researchers to fill up this gap.

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.

The widespread biting midge Culicoides impunctatus (Ceratopogonidae) is susceptible to infection with numerous Haemoproteus (Haemoproteidae) species.

BACKGROUND: Haemoproteus parasites are widespread, and some species cause disease in wild and domestic birds. However, the insect vectors remain unknown for the majority of species and genetic lineages of avian Haemoproteus. This information is crucial for better understanding the biology of haemoproteids, the epidemiology of haemoproteosis, and the development of morphological characters of sporogonic stages in wildlife haemosporidian parasites. It remains unclear whether the specificity of Haemoproteus parasites for vectors is broad or the transmission of a given parasite can be restricted to a single or few species of vectors. The aim of this study was to examine the sporogonic development of four species of common European avian haemoproteids in the common biting midge Culicoides impunctatus. METHODS: Wild-caught females of C. impunctatus were infected experimentally by allowing them to take blood meals on naturally infected Muscicapa striata, Cyanistes caeruleus, Ficedula hypoleuca and Motacilla flava harbouring mature gametocytes of Haemoproteus balmorali (genetic lineage hSFC9), H. majoris (hPARUS1), H. motacillae (hYWT1) and H. pallidus (hPFC1), respectively. Infected insects were collected, maintained under laboratory conditions and dissected daily in order to detect the development of ookinetes, oocysts and sporozoites. Microscopic examination and polymerase chain reaction based methods were used to detect the parasites. Bayesian analysis was applied to identify phylogenetic relationships among Haemoproteus lineages. RESULTS: All investigated parasites completed sporogony in C. impunctatus, indicating broad susceptibility of this biting midge for numerous Haemoproteus parasites. Ookinetes, oocysts and sporozoites were reported, described and compared morphologically. The investigated parasite species can be distinguished at the sporogony stage, particularly with regards to the morphology and rate of development of mature ookinetes. Analysis of data from the literature, and this study, shows that 12 genetically distantly related Haemoproteus parasites complete sporogony in C. impunctatus. CONCLUSIONS: Susceptibility of C. impunctatus is broad for Haemoproteus parasites, indicating that this biting midge is an important natural vector of numerous species of avian haemoproteids in Europe. Some Haemoproteus species can be readily distinguished using morphological characters of ookinetes and sporozoites, as well as the rate of ookinete development. These characters can be used for the identification of Haemoproteus species during sporogony in vectors, and are worth more attention in these parasite taxonomy studies at the species levels.

Exo-erythrocytic development of avian malaria and related haemosporidian parasites.

BACKGROUND: Avian malaria parasites (Plasmodium spp.) and related haemosporidians (Haemosporida) are responsible for diseases which can be severe and even lethal in avian hosts. These parasites cause not only blood pathology, but also damage various organs due to extensive exo-erythrocytic development all over the body, which is not the case during Plasmodium infections in mammals. However, exo-erythrocytic development (tissue merogony or schizogony) remains the most poorly investigated part of life cycle in all groups of wildlife haemosporidian parasites. In spite of remarkable progress in studies of genetic diversity, ecology and evolutionary biology of avian haemosporidians during the past 20 years, there is not much progress in understanding patterns of exo-erythrocytic development in these parasites. The purpose of this review is to overview the main information on exo-erythrocytic development of avian Plasmodium species and related haemosporidian parasites as a baseline for assisting academic and veterinary medicine researchers in morphological identification of these parasites using tissue stages, and to define future research priorities in this field of avian malariology. METHODS: The data were considered from peer-reviewed articles and histological material that was accessed in zoological collections in museums of Australia, Europe and the USA. Articles describing tissue stages of avian haemosporidians were included from 1908 to the present. Histological preparations of various organs infected with the exo-erythrocytic stages of different haemosporidian parasites were examined. RESULTS: In all, 229 published articles were included in this review. Exo-erythrocytic stages of avian Plasmodium, Fallisia, Haemoproteus, Leucocytozoon, and Akiba species were analysed, compared and illustrated. Morphological characters of tissue stages that can be used for diagnostic purposes were specified. CONCLUSION: Recent molecular studies combined with histological research show that avian haemosporidians are more virulent than formerly believed. The exo-erythrocytic stages can cause severe disease, especially in non-adapted avian hosts, suggesting the existence of a group of underestimated malignant infections. The development of a given haemosporidian strain can be markedly different in different avian hosts, resulting in significantly different virulence. A methodology combining the traditional histology techniques with molecular diagnostic tools is essential to speed research in this field of avian malariology.

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.

Deforestation does not affect the prevalence of a common trypanosome in African birds.

In spite of numerous reports of avian Trypanosoma spp. in birds throughout the world, patterns of the distribution and prevalence of these blood parasites remains insufficiently understood. It is clear that spatial heterogeneity influences parameters of parasite distributions in natural populations, but data regarding avian trypanosomes are scarce. Using microscopy and molecular diagnostic methods, we analysed the variation of prevalence of avian Trypanosoma parasites in two widespread African bird species, the yellow-whiskered greenbul Andropadus latirostris and the olive sunbird Cyanomitra olivacea. In all, 353 birds were captured in pristine forests and agroforest sites in Cameroon and Ghana. Overall, the prevalence of avian trypanosomes was 51.3%. Five morphospecies were reported (Trypanosoma everetti, T. anguiformis, T. avium, T. naviformis, T. ontarioensis). Trypanosoma everetti predominated, representing 98% of all Trypanosoma spp. reports, and it was present in both avian hosts. The prevalence of T. everetti was significantly less in the yellow-whiskered greenbul (19%) than olive sunbird (83%), and the same pattern of prevalence was reported in these avian hosts at different study sites. We found no interaction between sites and the prevalence of T. everetti. For both avian hosts, the prevalence did not differ significantly between pristine forests and agroforests. This indicates the same pattern of transmission at sites with different levels of deforestation and suggests that spatial heterogeneity related to deforestation does not affect the prevalence of avian Trypanosoma infections. It is likely that host-related factors, but not environmental conditions favour or reduce these parasite infections in forests of sub-Saharan Africa. Microscopic and PCR-based diagnostics showed the same sensitivity in diagnostics of T. everetti. We discuss the implications of these findings for the epidemiology of avian trypanosomiasis in natural populations.

Description, molecular characterisation, diagnostics and life cycle of Plasmodium elongatum (lineage pERIRUB01), the virulent avian malaria parasite.

Plasmodium elongatum causes severe avian malaria and is distributed worldwide. This parasite is of particular importance due to its ability to develop and cause lethal malaria not only in natural hosts, but also in non-adapted endemic birds such as the brown kiwi and different species of penguins. Information on vectors of this infection is available but is contradictory. PCR-based analysis indicated the possible existence of a cluster of closely related P. elongatum lineages which might differ in their ability to develop in certain mosquitoes and birds. This experimental study provides information about molecular and morphological characterisation of a virulent P. elongatum strain (lineage pERIRUB01) isolated from a naturally infected European robin, Erithacus rubecula. Phylogenetic analysis based on partial cytochrome b gene sequences showed that this parasite lineage is closely related to P. elongatum (lineage pGRW6). Blood stages of both parasite lineages are indistinguishable, indicating that they belong to the same species. Both pathogens develop in experimentally infected canaries, Serinus canaria, causing death of the hosts. In both these lineages, trophozoites and erythrocytic meronts develop in polychromatic erythrocytes and erythroblasts, gametocytes parasitize mature erythrocytes, exoerythrocytic stages develop in cells of the erythrocytic series in bone marrow and are occasionally reported in spleen and liver. Massive infestation of bone marrow cells is the main reason for bird mortality. We report here on syncytium-like remnants of tissue meronts, which slip out of the bone marrow into the peripheral circulation, providing evidence that the syncytia can be a template for PCR amplification. This finding contributes to better understanding positive PCR amplifications in birds when parasitemia is invisible and improved diagnostics of abortive haemosporidian infections. Sporogony of P. elongatum (pERIRUB01) completes the cycle and sporozoites develop in widespread Culex quinquefasciatus and Culex pipiens pipiens form molestus mosquitoes. This experimental study provides information on virulence and within species lineage diversity in a single pathogenic species of haemosporidian parasite.

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.

Molecular characterisation of three avian haemoproteids (Haemosporida, Haemoproteidae), with the description of Haemoproteus (Parahaemoproteus) palloris n. sp.

DNA barcoding (molecular characterisation) is a useful tool for describing the taxonomy and systematics of organisms. Over 250 species of avian haemosporidian parasites have been described using morphological characters, yet molecular techniques based on polymerase chain reaction (PCR) suggest this diversity is underestimated. Moreover, molecular techniques are particularly useful for the detection of chronic infections and tissue stages of these parasites. Species delimitation is problematic among haemosporidians, and many questions about the mechanisms and patterns of speciation, host specificity and pathogenicity are still unresolved. Accumulation of additional genetic and morphological information is needed to approach these questions. Here, we combine microscopic examination with PCR-based methods to develop molecular characterisation of Haemoproteus (Parahaemoproteus) manwelli Bennett, 1978 and Haemoproteus (Parahaemoproteus) gavrilovi Valkiunas & Iezhova, 1990, both of which parasitise the bee-eater Merops apiaster L. We also describe a new species, Haemoproteus (Parahaemoproteus) palloris n. sp., from the blood of the willow warbler Phylloscopus trochilus (L.). We performed phylogenetic analyses with a set of mitochondrial cytochrome b (cyt b) gene lineages, which have been linked to parasite morphospecies and are available in the MalAvi database. Our findings show that morphological characters, which have been traditionally used in the description of haemosporidians, exhibit phylogenetic congruence. This study contributes to a better understanding of avian haemosporidian diversity and provides new molecular markers (cyt b and apicoplast gene sequences) for the diagnostics of inadequately investigated haemosporidian infections.

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.