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

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

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.

Exo-erythrocytic development of Plasmodium matutinum (lineage pLINN1) in a naturally infected roadkill fieldfare Turdus pilaris.

BACKGROUND: Species of Plasmodium (Haemosporida, Plasmodiidae) are remarkably diverse haemoparasites. Information on genetic diversity of avian malaria pathogens has been accumulating rapidly, however exo-erythrocytic development of these organisms remains insufficiently addressed. This is unfortunate because, contrary to Plasmodium species parasitizing mammals, the avian malaria parasites undergo several cycles of exo-erythrocytic development, often resulting in damage of various organs. Insufficient knowledge on the exo-erythrocytic development in most described Plasmodium species precludes the understanding of mechanisms of virulence during avian malaria. This study extends information on the exo-erythrocytic development of bird malaria parasites. METHODS: A roadkill fieldfare (Turdus pilaris) was sampled in Switzerland and examined using pathologic, cytologic, histologic, molecular and microbiologic methods. Avian malaria was diagnosed, and erythrocytic and exo-erythrocytic stages of the parasite were identified using morphologic characteristics and barcode DNA sequences of the cytochrome b gene. The species-specific characteristics were described, illustrated, and pathologic changes were reported. RESULTS: An infection with Plasmodium matutinum lineage pLINN1 was detected. Parasitaemia was relatively low (0.3%), with all erythrocytic stages (trophozoites, meronts and gametocytes) present in blood films. Most growing erythrocytic meronts were markedly vacuolated, which is a species-specific feature of this parasite's development. Phanerozoites at different stages of maturation were seen in leukocytes, macrophages, and capillary endothelial cells in most organs examined; they were particularly numerous in the brain. Like the erythrocytic meronts, growing phanerozoites were markedly vacuolated. Conspicuous exo-erythrocytic development and maturation in leucocytes suggests that this fieldfare was not adapted to the infection and the parasite was capable to escape from cellular immunity. CONCLUSIONS: This is the first report of exo-erythrocytic development of the malaria parasite lineage pLINN1 during single infection and the first report of this lineage in the fieldfare. The findings of multiple phanerozoites in brain, skeletal muscle, and eye tissue in combination with signs of vascular blockage and thrombus formation strongly suggest an impaired vision and neuromuscular responsiveness as cause of the unexpected collision with a slowly moving car. Further studies on exo-erythrocytic stages of haemosporidian parasites are pivotal to understand the true level of populational damage of avian malaria in wild birds.

Massive Infection of Lungs with Exo-Erythrocytic Meronts in European Robin Erithacus rubecula during Natural Haemoproteus attenuatus Haemoproteosis.

Haemoproteus species are widespread avian blood parasites belonging to Haemoproteidae (Haemosporida). Blood stages of these pathogens have been relatively well-investigated, though exo-erythrocytic (tissue) stages remain unidentified for the majority of species. However, recent histopathological studies show that haemoproteins markedly affect bird organs during tissue merogony. This study investigated the exo-erythrocytic development of Haemoproteus (Parahaemoproteus) attenuatus (lineage hROBIN1), the common parasite of flycatchers (Muscicapidae). Naturally infected European robins Erithacus rubecula were examined. Parasite species and lineage were identified using microscopic examination of blood stages and DNA sequence analysis. Parasitaemia intensity varied between 0.8 and 26.5% in seven host individuals. Organs of infected birds were collected and processed for histological examination. Tissues stages (meronts) were seen in six birds and were present only in the lungs. The parasites were usually located in groups and were at different stages of maturation, indicating asynchronous exo-erythrocytic development. In most parasitized individuals, 100 meronts were observed in 1 cm2 section of lungs. The largest meronts reached 108 µm in length. Mature meronts contained numerous roundish merozoites of approximately 0.8 µm in diameter. Megalomeronts were not observed. Massive merogony and resulting damage of lungs is a characteristic feature during H. attenuatus infections and might occur in related parasite lineages, causing haemoproteosis.

Effects of land use change (rural-urban) on the diversity and epizootiological parameters of avian Haemosporida in a widespread neotropical bird.

Transformation of natural environments for livestock, agriculture and human settlements modifies the diversity of organisms, usually decreasing in highly disturbed land uses. Like their hosts, parasites have to adapt to novel human impacted landscapes, in which the abiotic and biotic conditions are radically different from those of conserved natural environments. We evaluated the diversity (alpha and beta taxonomic and phylogenetic diversity) of haemosporidians (mtDNA cyt b lineages) in the common chlorospingus (Chlorospingus flavopectus) at five land use types. We further analyzed the response of prevalence, parasitaemia and parasite aggregation to land use types and seasonality. Parasite lineage richness (i.e., haplotypes) and abundance (no. infected hosts) decreased with disturbance. Parasite assemblages were commonly dominated by either one of two lineages, one dominant in the urban greenspace (pBAEBIC02) and the other dominant in well-preserved mountain cloud forest (hCHLFLA01). Beta diversity was mainly explained by lineage turnover. Phylo beta diversity was low (i.e., lineages are closely related). Overall prevalence increased in wet season that coincides with host's breeding season. Haemoproteus and Plasmodium prevalence presented the opposite response to urbanization (negative and positive, respectively). Parasitaemia presented similar values across land uses for both genera and seasons, while Plasmodium aggregation decreased with urbanization. Thus, some parasite lineages (pBAEBIC02) will benefit from the urbanization process, while others will entirely disappear from cities (hCHLFLA01).

Combining morphological and molecular data to reconstruct the phylogeny of avian Haemosporida.

The traditional classification of avian Haemosporida is based mainly on morphology and life history traits. Recently, molecular hypotheses have challenged the traditional classification, leading to contradictory opinions on whether morphology is phylogenetically informative. However, the morphology has never been used to reconstruct the relationships within the group. We inferred the phylogeny of avian Haemosporida from 133 morphological characters present in blood stages. We included all species with at least one mitochondrial gene characterized (n = 93). The morphological hypothesis was compared with the one retrieved from mitochondrial DNA (mtDNA) nucleotide sequences and a hypothesis that used a combination of morphological and molecular data (i.e., total evidence). In order to recover the evolutionary history and identify phylogenetically and taxonomically informative characters, they were mapped on the total evidence phylogeny. The morphological hypothesis presented more polytomies than the other two, especially within Haemoproteus. In the molecular hypothesis, the two Haemoproteus subgenera are paraphyletic, and some relationships within Parahaemoproteus were resolved. By combining the morphological and molecular data, we were able to resolve the majority of polytomies and posterior probabilities increased. We identified a unique combination of morphological traits, clearly differentiating avian Haemosporida genera, sub-genera of Leucocytozoon and Haemoproteus, and some Plasmodium sub-genera. Plasmodium had the highest number of synapomorphies. Furthermore, 86% of the species presented a unique combination of taxonomically informative characters. A limiting factor was the mismatch of traits characterized in species descriptions, leading to a morphological matrix with a considerable amount of missing data, particularly for the stages of early young and young gametocytes (67% of all missing data). Characters lacking information for the majority of species included the colour of pigment granules, the cytoplasm appearance, and the presence and dimensions of vacuoles. According to our results, the combination of morphology and mtDNA proved to be a robust alternative to reconstruct the relationships among avian Haemosporida, obtaining a resolution and support similar to that obtained using full mitochondrial genome sequences for over 100 lineages.