Malaria and related haemosporidioses in wild birds

Avian haemosporidia are blood parasites of birds that are transmitted by arthropod vectors. Three genera are known to infect birds, Plasmodium, Haemoproteus and Leucocytozoon, each of them containing numerous species. They occur throughout the world - except in the circumpolar regions - and infect a large proportion of wild birds. It has long been assumed that coevolution of parasite and host has resulted in perfect adaptation that prevents the hosts from becoming sick. When birds from cold climates are transferred to warmer regions they become infected and frequently suffer from severe disease (avian malaria) or even die. There is increasing evidence that wild birds native to the distribution areas of the parasites more frequently develop severe disease and even die due to malaria than previously assumed. We believe that avian haemosporidioses (including avian malaria) are grossly underestimated causes of severe diseases and mortalities in wild birds. There is a lack of comprehensive studies on wild bird mortalities and a lack of suitable methods for visualizing parasites of the different genera, subgenera and species of haemosporidians in samples of dead birds. We also believe that the variety of haemosporidian species with high virulence for certain species of wild as well as cage and aviary birds is wider than currently assumed. This is because the tissue stages of these parasites are morphologically similar and there are no tools that allow the exact identification of these parasites in tissue samples of dead birds, particularly in the case of mixed infections. We plan to test these ideas by visualizing defined genera and species of haemosporidia in tissue samples. This is intended with the aid of in situ hybridization using oligonucleotide probes against ribosomal RNA sequences. The sequences of the rRNA genes are largely unknown and need to be established by suitable amplification and sequencing strategies in unequivocal reference material.

Avian haemosporidian parasites belonging to the three genera Plasmodium, Haemoproteus and Leucocytozoon, have a nearly worldwide distribution and affect many bird species. Our primary aim was to look at these parasites from the perspective of their ability of causing diseases and mortalities in wild birds. Disease in birds is predominantly induced by tissue stages of the parasites that cannot be distinguished by morphological methods. PCR assays and subsequent sequence analysis have lead to significant advances in molecular barcoding of the parasites and have generated more than 3500 individual lineages. However, this technique does not allow to reliably assess the identity of a specific parasite responsible for tissue lesions due to frequently occurring mixed infections. Thus a method allowing to specifically identifying tissue stages at genus or species level was highly desired. Building on our experience with in situ hybridization and the successful design of an oligonucleotide probe for labelling parasites of the Plasmodium genus, we successfully generated and tested probes for identification of the genera Haemoproteus and Leucocytozoon. This was not a straightforward task, because the gene sequences of the ribosomal RNA of these parasites were unknown and had to be cloned and sequenced. Comparable to Plasmodium species causing human malaria several distinct variants of rRNA genes were identified in representatives of all three haemosporidian genera. However, based on these sequences robust and specific probes could be designed and were successfully used for identification of different types of blood and tissue stages. In addition, several species-specific probes were made. With the aid of these probes it was possible to find out that two common Plasmodium species in blackbirds, Plasmodium vaughani and P. matutinum were of different virulence for these birds. Infections with P. matutinum led to much more widespread and destructive multiplication of parasites in the birds tissues. While a large number of different Haemoproteus lineages seen in various bird species were mostly associated with low-grade tissue merogony, suggesting low pathogenic potential, several Leucocytozoon lineages, seen either in passerine birds as well as in owls or raptors were associated with megalomeront formation and significant tissue lesions. Thus it seems rewarding for future studies to place increasing emphasis on Leucocytozoon infections in wild birds in order to elucidate a larger number of cases of so far unknown causes of death. The current project served as basis for a related citizen science project and a three-year follow-up project with the aims to investigate the previously unknown tissue localizations of the enigmatic dormant stages of avian hemosporidiosis, to test experimentally whether different variants of rRNA can be found in the vector and in the host, and to investigate with contemporary methods whether traditional concepts for cellular localization of meronts prove to be correct.