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.

Background: Blood parasites belonging to the Apicomplexa, Trypanosomatidae and Filarioidea are widespread in birds and have been studied extensively. Microscopical examination (ME) of stained blood films remains the gold standard method for the detection of these infections in birds, particularly because co-infections predominate in wildlife. None of the available molecular tools can detect all co-infections at the same time, but ME provides opportunities for this to be achieved. However, fixation, drying and staining of blood films as well as their ME are relatively time-consuming. This limits the detection of infected hosts during fieldwork when captured animals should be released soon after sampling. It is an obstacle for quick selection of donor hosts for parasite experimental, histological and other investigations in the field. This study modified, tested and described the buffy coat method (BCM) for quick diagnostics (~ 20 min/sample) of avian blood parasites.Methods: Blood of 345 birds belonging to 42 species was collected, and each sample was examined using ME of stained blood films and the buffy coat, which was examined after centrifugation in capillary tubes and after being transferred to objective glass slides. Parasite detection using these methods was compared using sensitivity, specificity, positive and negative predictive values and Cohen’s kappa index.Results: Haemoproteus,Leucocytozoon,Plasmodium, microfilariae,TrypanosomaandLankesterellaparasites were detected. BCM had a high sensitivity (> 90%) and specificity (> 90%) for detection ofHaemoproteusand microfilariae infections. It was of moderate sensitivity (57%) and high specificity (> 90%) forLankesterellainfections, but of low sensitivity (20%) and high specificity (> 90%) forLeucocytozooninfections.TrypanosomaandPlasmodiumparasites were detected only by BCM and ME, respectively. According to Cohen’s kappa index, the agreement between two diagnostic tools was substantial forHaemoproteus(0.80), moderate forLankesterella(0.46) and fair for microfilariae andLeucocytozoon(0.28) infections.Conclusions: BCM is sensitive and recommended as a quick and reliable tool to detectHaemoproteus,Trypanosomaand microfilariae parasites during fieldwork. However, it is not suitable for detection of species ofLeucocytozoonandPlasmodium. BCM is a useful tool for diagnostics of blood parasite co-infections. Its application might be extended to studies of blood parasites in other vertebrates during field studies.

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

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

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

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