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Background: Migratory birds play an important part in the spread of parasites, with more or less impact on resident birds. Previous studies focus on the prevalence of parasites, but changes in infection intensity over time have rarely been studied. As infection intensity can be quantified by qPCR, we measured infection intensity during different seasons, which is important for our understanding of parasite transmission mechanisms., Methods: Wild birds were captured at the Thousand Island Lake with mist nets and tested for avian hemosporidiosis infections using nested PCR. Parasites were identified using the MalAvi database. Then, we used qPCR to quantify the infection intensity. We analyzed the monthly trends of intensity for all species and for different migratory status, parasite genera and sexes., Results: Of 1101 individuals, 407 were infected (37.0%) of which 95 were newly identified and mainly from the genus Leucocytozoon. The total intensity trend shows peaks at the start of summer, during the breeding season of hosts and during the over-winter season. Different parasite genera show different monthly trends. Plasmodium causes high prevalence and infection intensity of winter visitors. Female hosts show significant seasonal trends of infection intensity., Conclusions: The seasonal changes of infection intensity is consistent with the prevalence. Peaks occur early and during the breeding season and then there is a downward trend. Spring relapses and avian immunity are possible reasons that could explain this phenomenon. In our study, winter visitors have a higher prevalence and infection intensity, but they rarely share parasites with resident birds. This shows that they were infected with Plasmodium during their departure or migration and rarely transmit the disease to resident birds. The different infection patterns of different parasite species may be due to vectors or other ecological properties., (© 2023. The Author(s).)

Plasmodium parasites are present in a wide range of host species, some of which tend to be more susceptible than others, potentially as an outcome of evolved tolerance or resistance. Common starlings seem to cope with malaria infection while common crossbills are more susceptible to the same infections. That raises the question if the parasites rely on the same molecular mechanisms regardless of host species or do Plasmodium parasites change gene-expressions in accordance to the environment different hosts might provide? We used RNA-sequencing from starlings and crossbills, experimentally infected with Plasmodium homocircumflexum (lineage pCOLL4). The assembled transcriptome contained a total of 26,733 contigs. Parasite expression patterns differed between bird species. Parasites had higher expression of cell-invasion genes when infecting crossbills compared to starlings whereas in starlings genes related to apoptosis or/and oxidative stress showed higher expression levels. This article reveals how a Plasmodium parasite might adjust its expression and gene function depending on the host species infected., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Avian haemosporidian parasites are particularly diverse and widespread. To date, more than 3,000 distinct cytochrome b lineages have been recorded, of which some present extremely wide geographical distributions, even including multiple continents. Whether these isolates represent one or several cryptic species remains unknown. Here we carried out a case study of SISKIN1, a common haemosporidian parasite lineage belonging to the morphologically described species Haemoproteus tartakovskyi . To shed light on its evolutionary origin, we investigated the divergence between SISKIN1 isolates obtained from siskins and redpolls in Europe (Russia and Sweden) and house finches in North America (Mexico). First, we used sequence capture of a small data set (2 Russian isolates and 1 Mexican isolate) to investigate the genetic structure based on the full-length mitochondrial genome and ∼1,000 genes. The mitochondrial genomes of Russian isolates were identical with each other but differed from the Mexican one at 6 positions. The nuclear divergence between Russian and Mexican isolates was on average 2.8%, close to what has been observed between 2 species of malaria parasites that respectively infect humans ( Plasmodium falciparum ) and gorillas ( Plasmodium praefalciparum ). Second, we used the expanded data set (15 samples in total) to investigate the genetic structure in 3 genes known to be involved in host invasion. The European isolates were identical across all sequenced genes, whereas the Mexican isolates were highly diverse. The lack of shared alleles between European and Mexican populations suggests that they might have diverged in isolation without gene flow. From the MalAvi database we examined the lineages most similar to the SISKIN1 barcode fragment (part of the cyt b gene) and found that most of them had been recorded in North and South America. This suggests that the lineage SISKIN1 originated in North America and subsequently spread to Europe. Our analyses support that the cyt b gene barcoding region is a useful marker for identification of avian haemosporidian lineages that can classify them into clusters of closely related parasites, but to further investigate species limits and evolutionary history, molecular data from multiple faster-evolving genes are required.

Genomic sequencing of avian haemosporidian parasites (Haemosporida) has been challenging due to excessive contamination from host DNA. In this study, we developed a cost-effective protocol to obtain parasite sequences from naturally infected birds, based on targeted sequence capture and next generation sequencing. With the genomic data of Haemoproteus tartakovskyi as a reference, we successfully sequenced up to 1000 genes from each of the 15 selected samples belonging to nine different cytochrome b lineages, eight of which belong to Haemoproteus and one to Plasmodium. The targeted sequences were enriched to ∼10 4 -fold, and mixed infections were identified as well as the proportions of each mixed lineage. We found that the total number of reads and the proportions of exons sequenced decreased when the parasite lineage became more divergent from the reference genome. For each of the samples, the recovery of sequences from different exons varied with the function and GC content of the exon. From the obtained sequences, we detected within-lineage variation in both mitochondrial and nuclear genes, which may be a result of local adaptation to different host species and environmental conditions. This targeted sequence capture protocol can be applied to a broader range of species and will open a new door for further studies on disease diagnostics and comparative analysis of haemosporidians evolution., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Knowing the natural dynamics of pathogens in migratory birds is important, for example, to understand the factors that influence the transport of pathogens to and their transmission in new geographical areas, whereas the transmission of other pathogens might be restricted to a specific area. We studied haemosporidian blood parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon in a migratory bird, the garden warbler Sylvia borin. Birds were sampled in spring, summer and early autumn at breeding grounds in Sweden, on migration at Capri, Italy and on arrival and departure from wintering staging areas in West Africa: mapping recoveries of garden warblers ringed in Fennoscandia and Capri showed that these sites are most probably on the migratory flyway of garden warblers breeding at Kvismaren. Overall, haemosporidian prevalence was 39%, involving 24 different parasite lineages. Prevalence varied significantly over the migratory cycle, with relatively high prevalence of blood parasites in the population on breeding grounds and at the onset of autumn migration, followed by marked declines in prevalence during migration both on spring and autumn passage. Importantly, we found that when examining circannual variation in the different lineages, significantly different prevalence profiles emerged both between and within genera. Our results suggest that differences in prevalence profiles are the result of either different parasite transmission strategies or coevolution between the host and the various parasite lineages. When separating parasites into common vs. rare lineages, we found that two peaks in the prevalence of rare parasites occur; on arrival at Swedish breeding grounds, and after the wintering period in Africa. Our results stress the importance of appropriate taxonomic resolution when examining host-parasite interactions, as variation in prevalence both between and within parasite genera can show markedly different patterns., (© 2013 The Authors. Journal of Evolutionary Biology © 2013 European Society For Evolutionary Biology.)

The blackcap (Sylvia atricapilla) is a common Palearctic migratory warbler, and haemosporidian parasites are common in this species. However, genetic and phenotypic diversity of haemosporidians in warblers has been insufficiently investigated and poorly linked. We addressed this issue by combining molecular and microscopy data for detection of pigment-forming haemosporidians of the genera Haemoproteus and Plasmodium. Blood samples from 498 blackcaps were collected at 7 different sites in Europe and investigated for these parasites by polymerase chain reaction (PCR)-based techniques and microscopic examination. In all, 56% of the birds were infected by at least 1 out of 25 distinct mitochondrial cytochrome b (cyt b) gene lineages of these haemosporidians. It is concluded that the blackcap is infected not only with blackcap specific haemosporidians, but also with Haemoproteus majoris, which is a host generalist and common in birds belonging to the Paridae. Haemoproteus pallidulus sp. nov. is described based on morphology of its blood stages and segments of the cyt b and dihydrofolate reductase/thymidylate synthase (DHFR-TS) genes. This study provides evidence that genetic diversity of haemosporidian parasites might be positively correlated with migratory strategies of their avian hosts; it also contributes to the value of both microscopy and molecular diagnostics of avian blood parasites.

During a parasite survey, we collected data on the presence and distribution of feather mites, intestinal parasites, and blood parasites of small ground finches (Geospiza fuliginosa) from 4 islands in the Galapagos. We recorded 4 species of feather mites, with the most common species, Trouessartia geospiza, present on the majority (77% [308/400]) of individuals. Birds with high loads of T. geospiza came from larger islands and had higher body masses. We identified 3 species of intestinal Isospora (Isospora fragmenta, Isospora temeraria, and Isospora exigua) in fecal samples that showed a diurnal pattern of oocyst release. Among samples collected in the afternoon, infection prevalence was 61% (11/18), while only 0.5% (1/192) contained oocysts in the morning. We screened 40 individuals from one island (Isabela) for blood parasites using molecular markers. Although no parasites of Haemoproteus, Leucocytozoon, or Plasmodium were detected, a high proportion of birds (80% [32/40]) had systemic Isospora spp. infections. A high infection prevalence (74% [20/27]), but low infection intensity, was confirmed using optical microscopy. This result could either be due to the detection of a previously unidentified systemic Isospora sp. parasite, or a result of the previously described Isospora spp. parasites causing systemic infections.

Species of Haemoproteus (Haemosporida: Haemoproteidae), avian haemosporidians, have traditionally been described based on morphology of their gametocytes and on limited experimental information on their vertebrate host specificity. We investigated to what extent the morphological species are represented by monophyletic groups based on DNA sequence data using 2 different fragment lengths of the cytochrome b (cyt. b) gene. Phylogenetic reconstructions of obtained cyt. b lineages from 6 morphospecies of Haemoproteus showed that all lineages formed monophyletic clusters matching the morphospecies. Comparing our data with a recently published study showed that this is not always the case; the morphospecies H. belopolskyi consists of 2 distinct clusters of lineages that apparently have converged in morphology. However, the overall broad congruence between the molecular and morphological clustering of lineages will facilitate the integration of the knowledge obtained by traditional and molecular parasitology. Mean between morphospecies variation was 10-fold higher than the within species variation (5.5% vs. 0.54%), suggesting that Haemoproteus lineages with a genetic differentiation >5% are expected to be morphologically differentiated in most cases. When investigate the utility of 2 different fragment sizes of the cyt. b gene, the partial, 479-bp, cyt. b protocol picked up all mitochondrial (mt)DNA lineages that are found when using the full cyt. b gene, 1073 bp, suggesting that this protocol is sufficient for identification of most mtDNA lineages. All of the mtDNA lineages were associated with unique alleles when amplification was possible at a nuclear locus, strengthening the hypothesis that the designation of lineages based on mtDNA is largely genome-wide representative. We, therefore, propose the use of a cyt. b fragment of this length as a standard gene fragment for a DNA bar-coding system for avian Haemoproteus species.

Haemoproteus spp., with circumnuclear gametocytes and tentatively belonging to Haemoproteus belopolskyi, are widespread and prevalent in warblers belonging to the Sylviidae, with numerous mitochondrial cytochrome b (cyt b) lineages detected among them. We sampled the hemoproteids from 6 species of warblers adjacent to the Baltic Sea. Parasites were identified to species based on morphology of their gametocytes, and a segment of the parasite's cyt b gene was sequenced. Sixteen mitochondrial cyt b lineages of hemoproteids with circumnuclear gametocytes were recorded. Two clades of lineages (clade A in species of Acrocephalus and Hippolais and clade B in species of Sylvia) with sequence divergence between their lineages >5% are distinguished in the phylogenetic tree. Within the clades A and B, the genetic distance between the lineages is < or = 3.9 and < and = 2.8%, respectively. We compared the morphology of gametocytes of 3 lineages (hHIICT1, hMW1, and hSYAT2) in detail. The lineages hHIICTI and hMW1 (clade A) belong to the morphospecies H. belopolskyi. Parasites of the lineage hSYAT2 (clade B) are described as a new species Haemoproteus parabelopolskyi, which can be readily distinguished from H. belopolskyi by the significantly smaller nuclei of its macrogametocytes. Lineages closely related to H. belopolskyi and H. parabelopolskyi are identified. The sequence divergence between lineages of these 2 morphospecies ranges between 5.3 and 8.1%. It seems probable that avian Haemoproteus spp. with a genetic differentiation of > or =5% in mitochondrial cyt b gene might be morphologically differentiated at the stage of gametocytes. This study establishes the value of both PCR and morphology in identification of avian hemoproteids.

We investigated the degree of geographical shifts of transmission areas of vector-borne avian blood parasites (Plasmodium, Haemoproteus and Leucocytozoon) over ecological and evolutionary timescales. Of 259 different parasite lineages obtained from 5886 screened birds sampled in Europe and Africa, only two lineages were confirmed to have current transmission in resident bird species in both geographical areas. We used a phylogenetic approach to show that parasites belonging to the genera Haemoproteus and Leucocytozoon rarely change transmission area and that these parasites are restricted to one resident bird fauna over a long evolutionary time span and are not freely spread between the continents with the help of migratory birds. Lineages of the genus Plasmodium seem more freely spread between the continents. We suggest that such a reduced transmission barrier of Plasmodium parasites is caused by their higher tendency to infect migratory bird species, which might facilitate shifting of transmission area. Although vector-borne parasites of these genera apparently can shift between a tropical and a temperate transmission area and these areas are linked with an immense amount of annual bird migration, our data suggest that novel introductions of these parasites into resident bird faunas are rather rare evolutionary events.

Numerous polymerase chain reaction (PCR)-based methods have been developed and used increasingly to screen vertebrate blood samples for the diagnosis of haemosporidian blood parasites (Sporozoa, Haemosporida), but a rigorous evaluation of the sensitivity of these methods for detecting mixed infections of different haemosporidian species belonging to the same and different genera and subgenera is lacking. This study links the information obtained by nested cytochrome b PCR and traditional microscopy in determining mixed haemosporidian infections in naturally infected birds. Samples from 83 individual passerine birds with single infections of Haemoproteus or Plasmodium spp., as determined by mitochondrial DNA amplification, also were investigated by microscopic examination of stained blood films. Thirty-six samples (43%) were found to harbor mixed Haemoproteus, or Plasmodium spp. infections, or both. Thus, the PCR assays alone underestimate the occurrence of mixed infections of haemosporidian parasites in naturally infected birds. To determine the true species composition of the haemosporidians in each individual host, PCR diagnostics need to be improved. Specific primers for Haemoproteus spp. and Plasmodium spp. should be developed. Ideally, a combination of the approaches of both microscopy and PCR-based methods is recommended for this purpose.