Ancient Plasmodium genomes shed light on the history of human malaria.

Malaria-causing protozoa of the genus Plasmodium have exerted one of the strongest selective pressures on the human genome, and resistance alleles provide biomolecular footprints that outline the historical reach of these species1. Nevertheless, debate persists over when and how malaria parasites emerged as human pathogens and spread around the globe1,2. To address these questions, we generated high-coverage ancient mitochondrial and nuclear genome-wide data from P. falciparum, P. vivax and P. malariae from 16 countries spanning around 5,500 years of human history. We identified P. vivax and P. falciparum across geographically disparate regions of Eurasia from as early as the fourth and first millennia BCE, respectively; for P. vivax, this evidence pre-dates textual references by several millennia3. Genomic analysis supports distinct disease histories for P. falciparum and P. vivax in the Americas: similarities between now-eliminated European and peri-contact South American strains indicate that European colonizers were the source of American P. vivax, whereas the trans-Atlantic slave trade probably introduced P. falciparum into the Americas. Our data underscore the role of cross-cultural contacts in the dissemination of malaria, laying the biomolecular foundation for future palaeo-epidemiological research into the impact of Plasmodium parasites on human history. Finally, our unexpected discovery of P. falciparum in the high-altitude Himalayas provides a rare case study in which individual mobility can be inferred from infection status, adding to our knowledge of cross-cultural connectivity in the region nearly three millennia ago.

Gut microbiota elicits a protective immune response against malaria transmission.

Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:ß-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galß1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.

Ape Origins of Human Malaria.

African apes harbor at least twelve Plasmodium species, some of which have been a source of human infection. It is now well established that Plasmodium falciparum emerged following the transmission of a gorilla parasite, perhaps within the last 10,000 years, while Plasmodium vivax emerged earlier from a parasite lineage that infected humans and apes in Africa before the Duffy-negative mutation eliminated the parasite from humans there. Compared to their ape relatives, both human parasites have greatly reduced genetic diversity and an excess of nonsynonymous mutations, consistent with severe genetic bottlenecks followed by rapid population expansion. A putative new Plasmodium species widespread in chimpanzees, gorillas, and bonobos places the origin of Plasmodium malariae in Africa. Here, we review what is known about the origins and evolutionary history of all human-infective Plasmodium species, the time and circumstances of their emergence, and the diversity, host specificity, and zoonotic potential of their ape counterparts.

Avian haemosporidians of the genera Plasmodium and Haemoproteus from resident and Neotropical migrant birds in Colombia.

Avian haemosporidians of the genera Plasmodium and Haemoproteus are a group of widely distributed blood parasites that can negatively affect the fitness of their hosts. Colombia contains the greatest diversity of birds on the planet, but knowledge about the associations between haemosporidian and its avifauna is scarce and fragmented. We collected blood samples from 255 birds (203 residents and 52 neotropical migrants) belonging to 27 families and 108 species. The study was conducted in six localities in the inter-Andean valleys of the Cauca and Magdalena rivers. Parasites of the genera Plasmodium and Haemoproteus were identified in the samples by morphological and molecular analysis of a fragment of the mitochondrial gene cyt b. Among the samples, 9.3% (n = 24) were positive for Plasmodium or Haemoproteus. Co-infection with Plasmodium and Haemoproteus was found in Red-eyed Vireo. Seventeen haemosporidian lineages were identified, five of which were reported for the first time in resident birds (Common Ground Dove, Checker-throated Stipplethroat, Tropical Kingbird, Pale-breasted Thrush, and Ruddy-breasted Seedeater) and one in the Summer Tanager (neotropical migrant). The research results confirm the wide diversity of haemosporidian present in tropical lowlands and the possible role of neotropical migratory birds in dissemination on haemosporidian along their migratory routes.

Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria.

Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, field-applicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. Our P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (5 P. falciparum and 10 P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections.

Evolutionary Insights into the Microneme-Secreted, Chitinase-Containing High-Molecular-Weight Protein Complexes Involved in Plasmodium Invasion of the Mosquito Midgut.

While general mechanisms by which Plasmodium ookinetes invade the mosquito midgut have been studied, details regarding the interface of the ookinete, specifically its barriers to invasion, such as the proteolytic milieu, the chitin-containing, protein cross-linked peritrophic matrix, and the midgut epithelium, remain to be understood. Here, we review our knowledge of Plasmodium chitinases and the mechanisms by which they mediate ookinetes crossing the peritrophic matrix. The integration of new genomic insights into previous findings advances our understanding of Plasmodium evolution. Recently obtained Plasmodium species genomic data enable identification of the conserved residues in the experimentally demonstrated hetero-multimeric, high-molecular-weight complex comprised of a short chitinase covalently linked to binding partners, von Willebrand factor A domain-related protein (WARP) and secreted ookinete adhesive protein (SOAP). Artificial intelligence-based high-resolution structural modeling using the DeepMind AlphaFold algorithm yielded highly informative three-dimensional structures and insights into how short chitinases, WARP, and SOAP may interact at the atomic level to form the ookinete-secreted peritrophic matrix invasion complex. Elucidating the significance of the divergence of ookinete-secreted micronemal proteins among Plasmodium species may lead to a better understanding of the ookinete invasion machinery and the coevolution of Plasmodium-mosquito interactions.

Genomic and transcriptomic evidence for descent from Plasmodium and loss of blood schizogony in Hepatocystis parasites from naturally infected red colobus monkeys.

Hepatocystis is a genus of single-celled parasites infecting, amongst other hosts, monkeys, bats and squirrels. Although thought to have descended from malaria parasites (Plasmodium spp.), Hepatocystis spp. are thought not to undergo replication in the blood-the part of the Plasmodium life cycle which causes the symptoms of malaria. Furthermore, Hepatocystis is transmitted by biting midges, not mosquitoes. Comparative genomics of Hepatocystis and Plasmodium species therefore presents an opportunity to better understand some of the most important aspects of malaria parasite biology. We were able to generate a draft genome for Hepatocystis sp. using DNA sequencing reads from the blood of a naturally infected red colobus monkey. We provide robust phylogenetic support for Hepatocystis sp. as a sister group to Plasmodium parasites infecting rodents. We show transcriptomic support for a lack of replication in the blood and genomic support for a complete loss of a family of genes involved in red blood cell invasion. Our analyses highlight the rapid evolution of genes involved in parasite vector stages, revealing genes that may be critical for interactions between malaria parasites and mosquitoes.

Plasmodium simium: Population Genomics Reveals the Origin of a Reverse Zoonosis.

BACKGROUND: The population history of Plasmodium simium, which causes malaria in sylvatic Neotropical monkeys and humans along the Atlantic Coast of Brazil, remains disputed. Genetically diverse P vivax populations from various sources, including the lineages that founded the species P simium, are thought to have arrived in the Americas in separate migratory waves. METHODS: We use population genomic approaches to investigate the origin and evolution of P simium. RESULTS: We find a minimal genome-level differentiation between P simium and present-day New World P vivax isolates, consistent with their common geographic origin and subsequent divergence on this continent. The meagre genetic diversity in P simium samples from humans and monkeys implies a recent transfer from humans to non-human primates - a unique example of malaria as a reverse zoonosis of public health significance. Likely genomic signatures of P simium adaptation to new hosts include the deletion of >40% of a key erythrocyte invasion ligand, PvRBP2a, which may have favored more efficient simian host cell infection. CONCLUSIONS: New World P vivax lineages that switched from humans to platyrrhine monkeys founded the P simium population that infects nonhuman primates and feeds sustained human malaria transmission in the outskirts of major cities.

A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation.

BACKGROUND: The use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, including the two Plasmodium ovale species and zoonotic variants of the disease, will be important for the understanding of changing epidemiological patterns of the disease. METHODS: A qPCR-high resolution melting (HRM) method was to detect and differentiate all human Plasmodium species with one forward and one reverse primer set. The HRM detection method was further refined using a hydrolysis probe to specifically discriminate Plasmodium falciparum. RESULTS: Out of the 113 samples tested with the developed HRM-qPCR- P. falciparum probe assay, 96 (85.0 %) single infections, 12 (10.6 %) mixed infections, and 5 (4.4 %) were Plasmodium negative. The results were concordant with those of the nested PCR at 98.2 %. The assay limit of detection was varied from 21.47 to 46.43 copies /µl, equivalent to 1-2.11 parasites/µl. All P. falciparum infections were confirmed with the associated Taqman probe. CONCLUSIONS: Although the dependence on qPCR currently limits its deployment in resource-limited environments, this assay is highly sensitive and specific, easy to perform and convenient for Plasmodium mono-infection and may provide a novel tool for rapid and accurate malaria diagnosis also in epidemiological studies.

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.

Malaria parasite species composition of Plasmodium infections among asymptomatic and symptomatic school-age children in rural and urban areas of Kinshasa, Democratic Republic of Congo.

BACKGROUND: Malaria remains a major public health concern in the Democratic Republic of Congo (DRC), and school-age children are relatively neglected in malaria prevalence surveys and may constitute a significant reservoir of transmission. This study aimed to understand the burden of malaria infections in school-age children in Kinshasa/DRC. METHODS: A total of 634 (427 asymptomatic and 207 symptomatic) blood samples collected from school-age children aged 6 to 14 years were analysed by microscopy, RDT and Nested-PCR. RESULTS: The overall prevalence of Plasmodium spp. by microscopy, RDT and PCR was 33%, 42% and 62% among asymptomatic children and 59%, 64% and 95% in symptomatic children, respectively. The prevalence of Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale spp. by PCR was 58%, 20% and 11% among asymptomatic and 93%, 13% and 16% in symptomatic children, respectively. Among P. ovale spp., P. ovale curtisi, P. ovale wallikeri and mixed P. ovale curtisi + P. ovale wallikeri accounted for 75%, 24% and 1% of infections, respectively. All Plasmodium species infections were significantly more prevalent in the rural area compared to the urban area in asymptomatic infections (p < 0.001). Living in a rural as opposed to an urban area was associated with a five-fold greater risk of asymptomatic malaria parasite carriage (p < 0.001). Amongst asymptomatic malaria parasite carriers, 43% and 16% of children harboured mixed Plasmodium with P. falciparum infections in the rural and the urban areas, respectively, whereas in symptomatic malaria infections, it was 22% and 26%, respectively. Few children carried single infections of P. malariae (2.2%) and P. ovale spp. (1.9%). CONCLUSION: School-age children are at significant risk from both asymptomatic and symptomatic malaria infections. Continuous systematic screening and treatment of school-age children in high-transmission settings is needed.

Analysis of a two-strain malaria transmission model with spatial heterogeneity and vector-bias.

In this paper, we introduce a reaction-diffusion malaria model which incorporates vector-bias, spatial heterogeneity, sensitive and resistant strains. The main question that we study is the threshold dynamics of the model, in particular, whether the existence of spatial structure would allow two strains to coexist. In order to achieve this goal, we define the basic reproduction number [Formula: see text] and introduce the invasion reproduction number [Formula: see text] for strain [Formula: see text]. A quantitative analysis shows that if [Formula: see text], then disease-free steady state is globally asymptotically stable, while competitive exclusion, where strain i persists and strain j dies out, is a possible outcome when [Formula: see text] [Formula: see text], and a unique solution with two strains coexist to the model is globally asymptotically stable if [Formula: see text], [Formula: see text]. Numerical simulations reinforce these analytical results and demonstrate epidemiological interaction between two strains, discuss the influence of resistant strains and study the effects of vector-bias on the transmission of malaria.

Identification of a protein unique to the genus Plasmodium that contains a WD40 repeat domain and extensive low-complexity sequence.

Proteins containing WD40 domains play important roles in the formation of multiprotein complexes. Little is known about WD40 proteins in the malaria parasite. This report contains the initial description of a WD40 protein that is unique to the genus Plasmodium and possibly closely related genera. The N-terminal portion of this protein consists of seven WD40 repeats that are highly conserved in all Plasmodium species. Following the N-terminal region is a central region that is conserved within the major Plasmodium clades, such as parasites of great apes, monkeys, rodents, and birds, but partially conserved across all Plasmodium species. This central region contains extensive low-complexity sequence and is predicted to have a disordered structure. Proteins with disordered structure generally function in molecular interactions. The C-terminal region is semi-conserved across all Plasmodium species and has no notable features. This WD40 repeat protein likely functions in some aspect of parasite biology that is unique to Plasmodium and this uniqueness makes the protein a possible target for therapeutic intervention.

Blood parasites of bird communities in Sri Lanka and their mosquito vectors.

Avian blood parasites have been shown to have significant health effects on avifauna worldwide. Sri Lanka, a tropical island rich with resident and migratory birds, has not been properly evaluated for avian blood parasites or their vectors. We investigated the presence of avian haemoparasites in Sri Lankan birds and the potential mosquito vectors of those pathogens. Blood samples were collected from local/migratory birds captured by standard mist nets from Anawilundawa bird sanctuary, Hanthana mountain range, and the University of Peradeniya park. Mosquitoes were collected from Halgolla forest reserve and the forest patches in Kurunegala and Gampola areas in addition to the above mist-netting localities. Part of the mitochondrial cytochrome b (cytb) gene was amplified and sequenced to detect the presence of haemoparasites from avian blood samples (86) and mosquito samples (480). Blood parasites of the two genera, i.e., Haemoproteus (4 species; Haemoproteus sp. 1-4) and Plasmodium (5 species; Plasmodium sp. 1-5) were identified from seven bird species (four resident and three migratory). Among these, three bird species (Red-vented bulbul (3/16), Asian Brown flycatcher (1/1), and India pitta (1/1)) were positive for Plasmodium spp., while four (Yellow-browed bulbul (1/4), oriental white-eye (1/4), brown-headed Barbet (1/4), and Indian blue robin (1/1)) were positive for Haemoproteus spp. Two mosquito species were also positive for Plasmodium (3) and Haemoproteus (1) species. Phylogenetic analysis and haplotype networks created using positive sequences of haemoparasites showed that a Plasmodium clade was shared by Cx nigropunctatus mosquitoes and the migratory bird, Indian pitta. The majority (85%) of the Plasmodium and Haemoproteus sequences of this study were not linked to the well-characterized species suggesting the distinct nature of the lineages. Associations between mosquito species and blood parasites of birds suggest the possible vector status of these mosquitoes.

Plasmodium Genomics and Genetics: New Insights into Malaria Pathogenesis, Drug Resistance, Epidemiology, and Evolution.

Protozoan Plasmodium parasites are the causative agents of malaria, a deadly disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild or severe symptoms, or fatal, depending on many factors such as parasite virulence and host immune status. Malaria can be treated with various drugs, with artemisinin-based combination therapies (ACTs) being the first-line choice. Recent advances in genetics and genomics of malaria parasites have contributed greatly to our understanding of parasite population dynamics, transmission, drug responses, and pathogenesis. However, knowledge gaps in parasite biology and host-parasite interactions still remain. Parasites resistant to multiple antimalarial drugs have emerged, while advanced clinical trials have shown partial efficacy for one available vaccine. Here we discuss genetic and genomic studies of Plasmodium biology, host-parasite interactions, population structures, mosquito infectivity, antigenic variation, and targets for treatment and immunization. Knowledge from these studies will advance our understanding of malaria pathogenesis, epidemiology, and evolution and will support work to discover and develop new medicines and vaccines.

Conserved associations between G-quadruplex-forming DNA motifs and virulence gene families in malaria parasites.

BACKGROUND: The Plasmodium genus of malaria parasites encodes several families of antigen-encoding genes. These genes tend to be hyper-variable, highly recombinogenic and variantly expressed. The best-characterized family is the var genes, exclusively found in the Laveranian subgenus of malaria parasites infecting humans and great apes. Var genes encode major virulence factors involved in immune evasion and the maintenance of chronic infections. In the human parasite P. falciparum, var gene recombination and diversification appear to be promoted by G-quadruplex (G4) DNA motifs, which are strongly associated with var genes in P. falciparum. Here, we investigated how this association might have evolved across Plasmodium species - both Laverania and also more distantly related species which lack vars but encode other, more ancient variant gene families. RESULTS: The association between var genes and G4-forming motifs was conserved across Laverania, spanning ~ 1 million years of evolutionary time, with suggestive evidence for evolution of the association occurring within this subgenus. In rodent malaria species, G4-forming motifs were somewhat associated with pir genes, but this was not conserved in the Laverania, nor did we find a strong association of these motifs with any gene family in a second outgroup of avian malaria parasites. Secondly, we compared two different G4 prediction algorithms in their performance on extremely A/T-rich Plasmodium genomes, and also compared these predictions with experimental data from G4-seq, a DNA sequencing method for identifying G4-forming motifs. We found a surprising lack of concordance between the two algorithms and also between the algorithms and G4-seq data. CONCLUSIONS: G4-forming motifs are uniquely strongly associated with Plasmodium var genes, suggesting a particular role for G4s in recombination and diversification of these genes. Secondly, in the A/T-rich genomes of Plasmodium species, the choice of prediction algorithm may be particularly influential when studying G4s in these important protozoan pathogens.

Rapid phenotyping towards personalized malaria medicine.

Malaria is major public health concerns which continues to claim the lives of more than 435,000 people each year. The challenges with anti-malarial drug resistance and detection of low parasitaemia forms an immediate barrier to achieve the fast-approaching United Nations Sustainable Development Goals of ending malaria epidemics by 2030. In this Opinion article, focusing on the recent published technologies, in particularly the nuclear magnetic resonance (NMR)-based diagnostic technologies, the authors offer their perspectives and highlight ways to bring these point-of-care technologies towards personalized medicine. To this end, they advocate an open sourcing initiative to rapidly close the gap between technological innovations and field implementation.

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden.

BACKGROUND: Passerine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects. METHODS: For molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite burden was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin-eosin-stained sections. RESULTS: By PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses. CONCLUSION: This study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.

Plasmodium transmission differs between mosquito species and parasite lineages.

Factors such as the particular combination of parasite-mosquito species, their co-evolutionary history and the host's parasite load greatly affect parasite transmission. However, the importance of these factors in the epidemiology of mosquito-borne parasites, such as avian malaria parasites, is largely unknown. Here, we assessed the competence of two mosquito species [Culex pipiens and Aedes (Ochlerotatus) caspius], for the transmission of four avian Plasmodium lineages (Plasmodium relictum SGS1 and GRW11 and Plasmodium cathemerium-related lineages COLL1 and PADOM01) naturally infecting wild house sparrows. We assessed the effects of parasite identity and parasite load on Plasmodium transmission risk through its effects on the transmission rate and mosquito survival. We found that Cx. pipiens was able to transmit the four Plasmodium lineages, while Ae. caspius was unable to transmit any of them. However, Cx. pipiens mosquitoes fed on birds infected by P. relictum showed a lower survival and transmission rate than those fed on birds infected by parasites related to P. cathemerium. Non-significant associations were found with the host-parasite load. Our results confirm the existence of inter- and intra-specific differences in the ability of Plasmodium lineages to develop in mosquito species and their effects on the survival of mosquitoes that result in important differences in the transmission risk of the different avian malaria parasite lineages studied.