Plasmodium spp.: an experimental study on vertebrate host susceptibility to avian malaria.

The interest in experimental studies on avian malaria caused by Plasmodium species has increased recently due to the need of direct information about host-parasite interactions. Numerous important issues (host susceptibility, development of infection, the resistance and tolerance to avian malaria) can be answered using experimental infections. However, specificity of genetically different lineages of malaria parasites and their isolates is largely unknown. This study reviews recent experimental studies and offers additional data about susceptibility of birds to several widespread cytochrome b (cyt b) lineages of Plasmodium species belonging to four subgenera. We exposed two domesticated avian hosts (canaries Serinus canaria and ducklings Anas platyrhynchos) and also 16 species of common wild European birds to malaria infections by intramuscular injection of infected blood and then tested them by microscopic examination and PCR-based methods. Our study confirms former field and experimental observations about low specificity and wide host-range of Plasmodium relictum (lineages SGS1 and GRW11) and P. circumflexum (lineage TURDUS1) belonging to the subgenera Haemamoeba and Giovannolaia, respectively. However, the specificity of different lineages and isolates of the same parasite lineage differed between species of exposed hosts. Several tested Novyella lineages were species specific, with a few cases of successful development in experimentally exposed birds. The majority of reported cases of mortality and high parasitaemia were observed during parasite co-infections. Canaries were susceptible mainly for the species of Haemamoeba and Giovannolaia, but were refractory to the majority of Novyella isolates. Ducklings were susceptible to three malaria infections (SGS1, TURDUS1 and COLL4), but parasitaemia was light (<0.01%) and transient in all exposed birds. This study provides novel information about susceptibility of avian hosts to a wide array of malaria parasite lineages, outlining directions for future experimental research on various aspects of biology and epidemiology of avian malaria.

A new method for isolation of purified genomic DNA from haemosporidian parasites inhabiting nucleated red blood cells.

During the last 10 years, whole genomes have been sequenced from an increasing number of organisms. However, there is still no data on complete genomes of avian and lizard Plasmodium spp. or other haemosporidian parasites. In contrast to mammals, bird and reptile red blood cells have nuclei and thus blood of these vertebrates contains high amount of host DNA; that complicates preparation of purified template DNA from haemosporidian parasites, which has been the main obstacle for genomic studies of these parasites. In the present study we describe a method that generates large amount of purified avian haemosporidian DNA. The method is based on a unique biological feature of haemosporidian parasites, namely that mature gametocytes in blood can be induced to exflagellate in vitro. This results in the development of numerous microgametes, which can be separated from host blood cells by simple centrifugation. Our results reveal that this straight forward method provides opportunities to collect pure parasite DNA material, which can be used as a template for various genetic analyses including whole genome sequencing of haemosporidians infecting birds and lizards.

Not all songbirds calibrate their magnetic compass from twilight cues: a telemetry study.

Migratory birds are able to use the sun and associated polarised light patterns, stellar cues and the geomagnetic field for orientation. No general agreement has been reached regarding the hierarchy of orientation cues. Recent data from naturally migrating North American Catharus thrushes suggests that they calibrate geomagnetic information daily from twilight cues. Similar results have been shown in caged birds in a few studies but not confirmed in others. We report that free-flying European migrants, song thrushes Turdus philomelos, released after pre-exposure to a horizontally rotated magnetic field, do not recalibrate their magnetic compass from solar cues, but rather show a simple domination of either the magnetic or the stellar compass. We suggest that different songbird species possess different hierarchies of orientation cues, depending on the geographic and ecological challenges met by the migrants.

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 (δ(13)C) and nitrogen (δ(15)N) 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 δ(13)C and δ(15)N values of feathers re-grown. The study shows that even heavy primary infections of malaria parasites do not affect feather δ(13)C and δ(15)N 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.

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.

Plasmodium relictum (lineage P-SGS1): Further observation of effects on experimentally infected passeriform birds, with remarks on treatment with Malarone.

Plasmodium relictum (lineage P-SGS1) is a widespread malaria parasite that causes disease of different severity in different species of birds. However, experimental studies on the effects of this parasite on avian hosts are uncommon. We investigated development of this lineage in experimentally infected greenfinches Carduelis chloris and compared the obtained data with the literature information about the virulence of the same parasite lineage for phylogenetically closely related bird species. We also used an opportunity to test the efficacy of the antimalarial drug Malarone in treatment of the experimental infection. The cryopreserved strain of the lineage P-SGS1 was multiplied in 4 experimentally infected chaffinches. Light parasitemia developed in these birds; the parasites were then inoculated to 6 uninfected recipient greenfinches. Six uninfected greenfinches were used as negative controls. Light parasitemia developed in all experimental greenfinches. There were no significant effects of malaria on the body mass of greenfinches, but haematocrit value was slightly lower in experimental birds than in control ones; the infection did not cause mortality or morbidity in these birds. According to available data, all investigated fringillid birds are susceptible to P. relictum (P-SGS1), but the same malaria parasite develops markedly differently in different bird species, even closely related hosts. Thus, the observed effects of the same malaria lineage on one species of bird cannot be generalized to others, even closely related ones. The cure with Malarone was highly efficient for blood stages of P. relictum, but exoerythrocytic stages were unaffected.

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.

Nested cytochrome b polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential.

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.