Further insights into the pathogenesis of avian malaria

There is increasing evidence that avian haemosporidian parasites (genera of Plasmodium, Haemoproteus and Leucocytozoon) are not only benign commensals but are frequently responsible for lethal infections in birds. Pathogenicity is predominantly inferred by tissue stages of these parasites, which develop in different cell types and lead to blockage of blood vessels and tissue damage. Currently, there is only fragmented knowledge on long-term persistence of these infections and the existence of dormant parasite stages, on the cell types which are targets of replicative tissue stages, on the effect of mixed infections on tissue damage, and finally the question whether 18S ribosomal RNA is differentially expressed in certain developmental stages of the avian malaria parasites. This study sets out to test the following hypotheses: (1) In analogy to primate malaria parasites, avian haemosporidia also exhibit genetically different 18S ribosomal RNA variants in hosts (birds) and vectors (mosquitoes or biting midges). (2) Relapses in avian malaria are caused by dormant tissue stages which can be visualized by sufficiently sensitive molecular detection methods. (3) Exo- erythrocytic merogony of avian haemosporidian parasites is restricted to few specialized host cell types in different tissues and is more destructive in co-infections. First, differentially expressed 18 rRNA variants will be labelled by variant-specific probes in avian hosts and arthropod vectors. These results form the basis for the consecutive project parts. The search for dormant stages will be pursued by a highly sensitive in situ hybridization procedure in experimentally infected canaries. The host cell types supporting propagation of parasites in different tissues will be identified by double labelling approaches with antibodies for certain cell markers and molecular probes for haemosporidian meronts. In addition, the effect of mixed infections with two or more haemosporidian species will be tested by double staining procedures. The majority of the proposed research questions have not been addressed previously (dormant stages and differential expression of rRNAs in avian malaria) or were not explored in a comparably convincing manner, like the identification of cell types allowing propagation of avian malaria parasites, or the effects of mixed infections on the severity of tissue damage. Principal investigator is Herbert Weissenböck, professor and head of pathology at the University of Veterinary Medicine Vienna. He is an expert in molecular identification of pathogens in tissue samples and heads a research group on pathogenesis of avian haemosporidians. The project team will be complemented by a Postdoc and a PhD student. Certain project parts will be investigated in close cooperation with Gediminas Valkiunas from Nature Research Centre Vilnius, Lithuania, one of the world leading experts in avian haemosporidian research.

In the research field of avian haemosporidioses, there is currently only fragmented knowledge on long-term persistence of these infections and the existence of dormant parasite stages, on the effect of mixed infections on the formation of merogonic stages and tissue damage, and finally the question whether 18S ribosomal RNA is differentially expressed in certain developmental stages of the avian malaria parasites. This study intended to test the following hypotheses: (1) Relapses in avian malaria are caused by dormant tissue stages which can be visualized by sufficiently sensitive molecular detection methods. (2) In analogy to primate malaria parasites, avian haemosporidia also exhibit genetically different 18S ribosomal RNA variants in hosts (birds) and vectors (mosquitoes or biting midges). (3) Exo-erythrocytic merogony of avian haemosporidian parasites is restricted to few specialized host cell types in different tissues and is more destructive in co-infections. When searching for dormant stages, systematic examination of tissues of latently P. relictum pSGS1-infected canaries showed no exo-erythrocytic stages in birds exhibiting latent infections, indicating absence of dormant tissue stages in P. relictum pSGS1-infected canaries. Instead, there were rare P. relictum blood stages in capillaries of various tissues and organs, demonstrating persistence of the parasites in the microvasculature. The findings suggest that persistence of P. relictum pSGS1 during latent infection is mediated by continuous low-level erythrocytic merogony and possibly tissue sequestration of infected blood cells. By experimental infection of mosquitoes, biting midges and birds (canaries, siskins) we were able to show that Plasmodium relictum SGS1 expressed two 18S variants in both bird blood and mosquitoes, but their expression levels were different and reversed in the two hosts, with one variant being preferentially expressed over the other. Haemoproteus tartakovskyi SISKIN1, which features three distinct 18S variants, expressed 18S variant 1 in bird blood and variants 1 and 2 in the biting midges, while no signals were detected for variant 3. These results support the hypothesis that distinct 18S variants of the two parasite species are differentially expressed in the bird hosts and vectors. Combined molecular results of PCR, laser microdissection and in situ hybridization showed a high rate of co-infections in Fringilla coelebs, with Haemoproteus lineages dominating. Merogonic stages were observed in the vascular system, presenting a formerly unknown mode of exo-erythrocytic development in Haemoproteus parasites. Meronts and megalomeronts of these species were distinct regarding their morphology and organ distribution, indicating species-specific patterns of merogony and different host tissue tropism. Furthermore, phylogenetic analysis of Haemoproteus species with regard to their exo-erythrocytic stages points towards separation of non-megalomeront-forming species from megalomeront-forming species, calling for further studies on exo-erythrocytic development of haemosporidian parasites to explore the phylogenetic character of this trait.