A quantitative PCR protocol for detecting specific Haemoproteus lineages: molecular characterization of blood parasites in a Sedge Warbler population from southern Poland

In evolutionary studies, blood parasites in avian populations are commonly used as a model of host-parasite interactions. The effect of mixed infections on avian hosts has recently drawn more interest, but the effects of infection with multiple blood parasites and specific parasite lineages are poorly known. A protocol for reliable detection and quantification of lineages is essential to this type of research. Here, we present a newly developed quantitative PCR (qPCR) assay using SYBR Green I to assess the infection intensity of SW1 and SW3 Haemoproteus belopolskyi lineages in a Sedge Warbler (Acrocephalus schoenobaenus) population from southern Poland. These Haemoproteus lineages are not specific to the Sedge Warbler, so the proposed protocol should prove useful for many avian malaria studies. The assay is based on nucleotide primers designed to amplify a fragment of the cytochrome b gene, allowing the two avian malaria lineages to be differentiated. Using this assay, specific host-parasite interactions can be identified and the impact of mixed infections on a host population can be assessed. Most of the parasitized birds in our study were in a low-intensity, chronic phase of infection. In those with mixed SW1/SW3 infections, we detected significantly higher parasitemia caused by the SW3 lineage, whose prevalence was underestimated by the commonly used method, nested PCR. The prevalence of avian malaria parasites in the studied population as estimated by nested PCR was 61 % and did not differ between years, though the prevalence of the SW1 lineage showed significant annual variation. Altogether, two Haemoproteus and five Plasmodium lineages were detected. The two Haemoproteus lineages (SW1, SW3) were most prevalent in the population and comprised 93 % of all infections. We detected significantly higher haemoparasite prevalence and intensity in males, which were sampled immediately after arrival from wintering grounds, suggesting decreased immunoprotection as a result of adaptive resource allocation during migration.