A highly invasive malaria parasite has expanded its range to non-migratory birds in North America

Parasite range expansions are a direct consequence of globalization and are an increasing threat to biodiversity. Here, we report a recent range expansion of the SGS1 strain of a highly invasive parasite, Plasmodium relictum, to two non-migratory passerines in North America. Plasmodium relictum is considered one of the world''s most invasive parasites and causes the disease avian malaria: this is the first reported case of SGS1 in wild non-migratory birds on the continent. Using a long-term database where researchers report avian malaria parasite infections, we summarized our current understanding of the geographical range of SGS1 and its known hosts. We also identified the most likely geographical region of this introduction event using the MSP1 allele. We hypothesize that this introduction resulted from movements of captive birds and subsequent spillover to native bird populations, via the presence of competent vectors and ecological fitting. Further work should be conducted to determine the extent to which SGS1 has spread following its introduction in North America.     

Plasmodium relictum (lineage SGS1) and Plasmodium ashfordi (lineage GRW2): the effects of the co-infection on experimentally infected passerine birds

The effects of avian malaria parasites of the genus Plasmodium on their hosts are insufficiently understood. This is particularly true for malarial co-infections, which predominant in many bird populations. We investigated effects of primary co-infection of Plasmodium relictum (lineage SGS1) and Plasmodium ashfordi (GRW2) on experimentally infected naive juveniles of siskin Spinus spinus, crossbill Loxia curvirostra and starling Sturnus vulgaris. All siskins and crossbills were susceptible but starlings resistant to both these infections. A general pattern of the co-infections was that heavy parasitemia (over 35% during peaks) of both parasites developed in both susceptible host species. There were no significant effects of the co-infections on mean body mass of the majority of infected birds. Mean haematocrit value decreased approximately 1.5 and 3 times in siskins and crossbills at the peak of parasitemia, respectively. Mortality was recorded among infected crossbills. We conclude that co-infections of P. relictum and P. ashfordi are highly virulent and act synergetically during primary infections in some but not all passerine birds.     

Associations between malaria and MHC genes in a migratory songbird

Malaria parasites are a widespread and species-rich group infecting many wild populations of mammals, birds and reptiles. Studies on humans have demonstrated that genetic factors play a key role in the susceptibility and outcome of malaria infections. Until the present study, it has not been examined whether genetic variation in hosts is important for the outcome of malaria infections in natural avian populations. We investigated associations between major histocompatibility complex (MHC) genes and prevalence of three different avian malaria parasites (Haemoproteus payevskyi (GRW1), Plasmodium sp. (GRW2) and Plasmodium sp. (GRW4)) in a long-term study of great reed warblers Acrocephalus arundinaceus. We hypothesized that the MHC genes could either give full protection against a malaria infection, or confer protection against lethal malaria and direct the infection towards being milder. We found a positive association between numbers of MHC class I alleles (a measure of level of heterozygosity) and prevalence of the GRW2 parasite, suggesting the latter scenario. There was also a positive association between a specific MHC allele (B4b), previously shown to be under frequency-dependent selection in the study population, and prevalence of GRW2. These associations suggest that individuals carrying either a large number of MHC alleles or a specific MHC allele are protected against lethal malaria infections.     

Are chronic avian haemosporidian infections costly in wild birds?

One group of commonly found parasites in birds, for which fitness consequences and effects on life history traits have been much debated are Haemosporidian blood parasites. In a long term study population of great reed warblers Acrocephalus arundinaceus in Sweden, previous studies have shown that the Haemosporidian blood parasites are in their chronic phase during the breeding season and that the fitness of infected and non‐infected birds are similar. In the present study, we quantified parasite intensity (parasitemia) in 718 adults great reed warblers sampled between 1987 and 1998 for the three most common parasite species; Haemoproteus payevskyi (lineage GRW1), Plasmodium ashfordi (GRW2) and Plasmodium relictum (GRW4). We verified that the q‐PCR method is accurately quantifying Haemoproteus payevskyi (GRW1) as it was highly correlated with the number of parasites seen under microscope. Frequency of mixed infections with two lineages was significantly higher than expected based on the prevalence of each of the three parasite lineages. The mean level of parasitemia was significantly different for the three lineages and individual birds had repeatable parasitemia levels between years. Females tended to have a higher parasitemia than males for all three parasite lineages combined. Females with higher GRW1 parasitemia tended to arrive later in spring to their breeding sites. There was a negative correlation between parasitemia and number of fledged offspring for GRW1, and a tendency for a negative correlation between GRW2 parasitemia and the proportion of recruiting offspring. Overall our results demonstrate that chronic Haemosporidian infections can have slight but significant effects on host life history traits, and therefore may act as important selective agents in wild bird populations.     

Haemosporidian infections in skylarks (<Emphasis Type="Italic">Alauda arvensis</Emphasis>): a comparative PCR-based and microscopy study on the parasite diversity and prevalence in southern Italy and the Netherlands

Changes in agricultural management have been identified as the most probable cause for the decline of Skylark (Alauda arvensis) populations in Europe. However, parasitic infections have not been considered as a possible factor influencing this process. Four hundred and thirty-four Skylarks from the Southern Italy and the Netherlands were screened for haemosporidian parasites (Haemosporida) using the microscopy and polymerase chain reaction (PCR)-based methods. The overall prevalence of infection was 19.5%; it was 41.8% in Italian birds and 8.3% in Dutch birds. The prevalence of Plasmodium spp. was 34.1% and 6.5% in Skylarks from Italy and Netherlands, respectively. Approximately 15% of all recorded haemosporidian infections were simultaneous infections both in Italian and Dutch populations. Six different mitochondrial cytochrome b (cyt b) lineages of Plasmodium spp. and three lineages of Haemoproteus tartakovskyi were found. The lineage SGS1 of Plasmodium relictum was the most prevalent at both study sites; it was recorded in 24.7% of birds in Italy and 5.5% in the Netherlands. The lineages SYAT05 of Plasmodium vaughani and GRW11 of P. relictum were also identified with a prevalence of <2% at both study sites. Two Plasmodium spp. lineages (SW2 and DELURB4) and three H. tartakovskyi lineages have been found only in Skylarks from Italy. Mitochondrial cyt b lineages SYAT05 are suggested for molecular identification of P. vaughani, a cosmopolitan malaria parasite of birds. This study reports the greatest overall prevalence of malaria infection in Skylarks during the last 100 years and shows that both Plasmodium and Haemoproteus spp. haemosporidian infections are expanding in Skylarks so it might contribute to a decrease of these bird populations in Europe.     

Does avian malaria infection affect feather stable isotope signatures?

It is widely accepted that stable isotope ratios in inert tissues such as feather keratin reflect the dietary isotopic signature at the time of the tissue synthesis. However, some elements such as stable nitrogen isotopes can be affected by individual physiological state and nutritional stress. Using malaria infection experiment protocols, we estimated the possible effect of malaria parasite infections on feather carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope signatures in juvenile common crossbills Loxia curvirostra. The birds were experimentally infected with Plasmodium relictum (lineage SGS1) and P. ashfordi (GRW2), two widespread parasites of passerines. Experimental birds developed heavy parasitemia of both parasites and maintained high levels throughout the experiment (33 days). We found no significant difference between experimental and control birds in both δ¹³C and δ¹⁵N values of feathers re-grown. The study shows that even heavy primary infections of malaria parasites do not affect feather δ¹³C and δ¹⁵N isotopic signatures. The results of this experiment demonstrate that feather isotope values of wild-caught birds accurately reflect the dietary isotopic sources at the time of tissue synthesis even when the animal’s immune system might be challenged due to parasitic infection.     

A rare case of Plasmodium (Haemamoeba) relictum infection in a free-living Red Knot (Calidris canutus rufa, Scolopacidae)

The Red Knot (Calidris canutus rufa) is a Nearctic migrant shorebird that breeds in the Canadian Arctic and spends the winter season in coastal sites in South America. A rare case of a blood protozoan was found by molecular analyses from an adult bird captured during spring migration at the last refuelling stopover in Delaware Bay USA in 2006. The parasite was identified as Plasmodium relictum belonging to subgenus Haemamoeba based on the shape of meronts, roundish gametocytes, and its position in the erythrocytes from the blood smears examination. A partial cytochrome b sequence was a 100% match to a sequence of Plasmodium relictum, sequence Genbank accession number: id DQ659543.1 (lineage code haplotype P5). This is the first report of avian malaria in a wild individual of C. c. rufa.     

A new morphologically distinct avian malaria parasite that fails detection by established polymerase chain reaction-based protocols for amplification of the cytochrome B gene

Plasmodium polymorphum n. sp. (Haemosporida, Plasmodiidae) was found in the skylark, Alauda arvensis (Passeriformes: Alaudidae), during autumnal migration in southern Italy. This organism is illustrated and described based on the morphology of its blood stages. The most distinctive feature of this malaria parasite is the clear preference of its blood stages (trophozoites, meronts, and gametocytes) for immature red blood cells, including erythroblasts. Based on preference of erythrocytic meronts for immature red blood cells, P. polymorphum is most similar to species of the subgenus Huffia . This parasite can be readily distinguished from all other bird malaria parasites, including Plasmodium ( Huffia ) spp., due to preferential development and maturation of its gametocytes in immature red blood cells, a unique character for avian Plasmodium spp. In addition, the margins of nuclei in blood stages of P. polymorphum are markedly smooth and distinct; this is also a distinct diagnostic feature of this parasite. Plasmodium polymorphum has been recorded only in the skylark; it is probably a rare parasite, whose host range and geographical distribution remain unclear. Microscopic examination detected a light infection of Plasmodium relictum (lineage GRW11, parasitemia of <0.01%) in the same sample with P. polymorphum ; the latter parasite clearly predominated (3.5% parasitemia). However, experienced researchers were unable to detect sequences of mitochondrial cytochrome b gene (cyt b ) of P. polymorphum from the microscopically positive sample by using published and newly designed primers for DNA amplification of avian Plasmodium spp. The light parasitemia of P. relictum was easily detectable using several polymerase chain reaction (PCR)-based assays, but P. polymorphum was undetectable in all applied assays. Quantitative PCR also showed the presence of light parasitemia (0.06%) of the lineage GRW11 in this sample. This supports the conclusion that the morphologically distinct parasite observed along with P. relictum and predominant in the sample is genetically dissimilar from the lineage GRW11 based on cyt b sequence. In samples with co-infections, general PCR protocols tend to favor the amplification of the parasite with the higher parasitemia or the amplification with the best matching sequence to the primers. Because the parasitemia of P. polymorphum was >50-fold higher than that of P. relictum and several different primers were tested, we suggest that the failure to amplify P. polymorphum is a more complex problem than why co-infections are commonly overlooked in PCR-based studies. We suggest possible explanations of these results and call for additional research on evolution of mitochondrial genome of hemosporidian parasites.     

Avian malaria: a new lease of life for an old experimental model to study the evolutionary ecology of Plasmodium

Avian malaria has historically played an important role as a model in the study of human malaria, being a stimulus for the development of medical parasitology. Avian malaria has recently come back to the research scene as a unique animal model to understand the ecology and evolution of the disease, both in the field and in the laboratory. Avian malaria is highly prevalent in birds and mosquitoes around the world and is amenable to laboratory experimentation at each stage of the parasite''s life cycle. Here, we take stock of 5 years of experimental laboratory research carried out using Plasmodium relictum SGS1, the most prevalent avian malaria lineage in Europe, and its natural vector, the mosquito Culex pipiens. For this purpose, we compile and analyse data obtained in our laboratory in 14 different experiments. We provide statistical relationships between different infection-related parameters, including parasitaemia, gametocytaemia, host morbidity (anaemia) and transmission rates to mosquitoes. This analysis provides a wide-ranging picture of the within-host and between-host parameters that may bear on malaria transmission and epidemiology.     

Local prevalence and transmission of avian malaria in the Alakai Plateau of Kauai,Hawaii, U.S.A.

Avian malaria is among the most important threats to native Hawaiian forest birds. It is caused by the parasite Plasmodium relictum and is transmitted by the introduced mosquito vector Culex quinquefasciatus. Temperature increases and precipitation declines due to climate change over the last decade may be responsible for the observed recent expansion in the range and prevalence of avian malaria on the Alakai Plateau, Kauai Island. To examine the hypothesis that conditions are now favorable for transmission of malaria on the Plateau, mosquitoes were sampled with CO₂ and Reiter oviposition traps at three sites (Kawaikoi, Halepa'akai, and Koke'e) on several occasions between October, 2013 and April, 2014. P. relictum infection was assessed by PCR or dissection under a microscope. We also surveyed mosquito larvae along Halepa'akai and Kawaikoi streams. We observed that Cx. quinquefasciatus is well established on the Alakai Plateau, as mosquitoes were caught on all field trips, except in April at Halepa'akai, and larvae were found throughout the year. We observed differences in adult abundance among sites and microhabitats (stream vs ridge lines).     

Plasmodium relictum (lineage P-SGS1): effects on experimentally infected passerine birds

We evaluated the effects of Plasmodium relictum (lineage P-SGS1), which is a host generalist, to five species of passerine birds. Light infection of P. relictum was isolated from a naturally infected adult reed warbler Acrocephalus scirpaceus. The parasites were inoculated to naive juveniles of the chaffinch Fringilla coelebs, common crossbill Loxia curvirostra, house sparrow Passer domesticus, siskin Spinus spinus and starling Sturnus vulgaris. Susceptibility of these birds to the infection of P. relictum was markedly different. This parasite developed in birds belonging to the Fringillidae and Passeridae but the starlings (Sturnidae) were resistant. Only 50% of experimental house sparrows were susceptible to the infection. The intensity of parasitemia varied markedly inside and between different susceptible bird species. There were no effects of the infection on body mass or temperature of experimentally infected birds. Infection of P. relictum leads to the significant decrease of haematocrit value and hypertrophy of spleen and liver in heavily infected common crossbills and siskins. This study shows that infection of the same lineage of P. relictum causes diseases of different severity in different avian hosts; that might have different evolutionary consequences and should be taken in consideration in conservation projects.     

Parallel evolution of gene classes,but not genes: Evidence from Hawai'ian honeycreeper populations exposed to avian malaria

Adaptation in nature is ubiquitous, yet characterizing its genomic basis is difficult because population demographics cause correlations with nonadaptive loci. Introduction events provide opportunities to observe adaptation over known spatial and temporal scales, facilitating the identification of genes involved in adaptation. The pathogen causing avian malaria, Plasmodium relictum, was introduced to Hawai'i in the 1930s and elicited extinctions and precipitous population declines in native honeycreepers. After a sharp initial population decline, the Hawai'i ‘amakihi (Chlorodrepanis virens) has evolved tolerance to the parasite at low elevations where P. relictum exists, and can sustain infection without major fitness consequences. High‐elevation, unexposed populations of ‘amakihi display little to no tolerance. To explore the genomic basis of adaptation to P. relictum in low‐elevation ‘amakihi, we genotyped 125 ‘amakihi from the island of Hawai'i via hybridization capture to 40,000 oligonucleotide baits containing SNPs and used the reference ‘amakihi genome to identify genes potentially under selection from malaria. We tested for outlier loci between low‐ and high‐elevation population pairs and identified loci with signatures of selection within low‐elevation populations. In some cases, genes commonly involved in the immune response (e.g., major histocompatibility complex) were associated with malaria presence in the population. We also detected several novel candidate loci that may be implicated in surviving malaria infection (e.g., beta‐defensin, glycoproteins and interleukin‐related genes). Our results suggest that rapid adaptation to pathogens may occur through changes in different immune genes, but in the same classes of genes, across populations.     

Effect of repeated exposure to Plasmodium relictum (lineage SGS1) on infection dynamics in domestic canaries

Parasites are known to exert strong selection pressures on their hosts and, as such, favour the evolution of defence mechanisms. The negative impact of parasites on their host can have substantial consequences in terms of population persistence and the epidemiology of the infection. In natural populations, however, it is difficult to assess the cost of infection while controlling for other potentially confounding factors. For instance, individuals are repeatedly exposed to a variety of parasite strains, some of which can elicit immunological memory, further protecting the host from subsequent infections. Cost of infection is, therefore, expected to be particularly strong for primary infections and to decrease for individuals surviving the first infectious episode that are re-exposed to the pathogen. We tested this hypothesis experimentally using avian malaria parasites (Plasmodium relictum-lineage SGS1) and domestic canaries (Serinus canaria) as a model. Hosts were infected with a controlled dose of P. relictum as a primary infection and control birds were injected with non-infected blood. The changes in haematocrit and body mass were monitored during a 20 day period. A protein of the acute phase response (haptoglobin) was assessed as a marker of the inflammatory response mounted in response to the infection. Parasite intensity was also monitored. Surviving birds were then re-infected 37 days post primary infection. In agreement with the predictions, we found that primary infected birds paid a substantially higher cost in terms of infection-induced reduction in haematocrit compared with re-exposed birds. After the secondary infection, re-exposed hosts were also able to clear the infection at a faster rate than after the primary infection. These results have potential consequences for the epidemiology of avian malaria, since birds re-exposed to the pathogen can maintain parasitemia with low fitness costs, allowing the persistence of the pathogen within the host population.     

Seasonal dynamics in mosquito abundance and temperature do not influence avian malaria prevalence in the Himalayan foothills

We examined seasonal prevalence in avian haemosporidians (Plasmodium and Haemoproteus) in migrant and resident birds in western Himalaya, India. We investigated how infection with haemosporidians in avian hosts is associated with temporal changes in temperature and mosquito abundance along with host abundance and life‐history traits (body mass). Using molecular methods for parasite detection and sequencing partial cytochrome b gene, 12 Plasmodium and 27 Haemoproteus lineages were isolated. Our 1‐year study from December 2008 to December 2009 in tropical Himalayan foothills revealed a lack of seasonal variation in Plasmodium spp. prevalence in birds despite a strong correlation between mosquito abundance and temperature. The probability of infection with Plasmodium decreased with increase in temperature. Total parasite prevalence and specifically Plasmodium prevalence showed an increase with average avian body mass. In addition, total prevalence exhibited a U‐shaped relationship with avian host abundance. There was no difference in prevalence of Plasmodium spp. or Haemoproteus spp. across altitudes; parasite prevalence in high‐altitude locations was mainly driven by the seasonal migrants. One Haemoproteus lineage showed cross‐species infections between migrant and resident birds. This is the first molecular study in the tropical Himalayan bird community that emphasizes the importance of studying seasonal variation in parasite prevalence. Our study provides a basis for further evolutionary study on the epidemiology of avian malaria and spread of disease across Himalayan bird communities, which may not have been exposed to vectors and parasites throughout the year, with consequential implications to the risk of infection to naïve resident birds in high altitude.     

Host specialization and geographic localization of avian malaria parasites: a regional analysis in the Lesser Antilles

We recovered 26 genetically distinct avian malaria parasite lineages, based on cytochrome b sequences, from a broad survey of terrestrial avifauna of the Lesser Antilles. Here we describe their distributions across host species within a regional biogeographic context. Most parasite lineages were recovered from a few closely related host species. Specialization on one host species and distribution across many hosts were both rare. Geographic patterns of parasite lineages indicated limited dispersal and frequent local extinction. The central islands of the archipelago share similar parasite lineages and patterns of infection. However, the peripheral islands harbor well-differentiated parasite communities, indicating long periods of isolation. Nonetheless, 20 of 26 parasite lineages were recovered from at least one of three other geographic regions, the Greater Antilles, North America, and South America, suggesting rapid dispersal relative to rate of differentiation. Six parasite lineages were restricted to the Lesser Antilles, primarily to endemic host species. Host differences between populations of the same parasite lineage suggest that host preference may evolve more rapidly than mitochondrial gene sequences. Taken together, distributions of avian malarial parasites reveal evidence of coevolution, host switching, extinction, and periodic recolonization events resulting in ecologically dynamic as well as evolutionarily stable patterns of infection.