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Patterns and drivers in malaria-like infections: meta and infra-population dynamics of specialised parasites

Final Report Summary - AVIANMALARIAGENETICS (Patterns and drivers in malaria-like infections: meta and infra-population dynamics of specialised parasites)

Final publishable summary report:
The project AvianMalariaGenetics aims to understand the between- and within-host dynamics of parasite populations under the influence of the immune systems of genetically different hosts. To approach this question the project has five main objectives: 1.Organization of a Raptor sample exchange network (RSEN), including scientific and conservationist working groups and rescue centres across Europe. 2. Extending of the longitudinal sampling of common buzzard infections in Eastern Westphalia during two field seasons. 3. Establishment of multiplex PCRs for genotyping Leucocytozoon antigenic and buzzard immunity-related candidate genes. 4. Laser capture microdissection and genotyping of single parasite strains. 5. Genotyping of infections for host immune-related genes and Leucocytozoon antigenic loci for longitudinal and spatial population genetic and host-parasite co-selection analyses.
The objectives of the project were pursued as follows: Raptor researchers across Europe were contacted and offered a collaboration within the Raptor Sample Exchange Network with perspective for additional information on their study population and joint publications. A large proportion of the project time was spent in practical field work during two field seasons in Eastern Westphalia, including intensive repeated sampling of buzzard nestlings infected with Leucocytozoon. During this work special slides were used for preparing blood smears for laser-capture microdissection. Massive multiplex PCRs assays covering more than 90 loci for the buzzard host and the Leucocytozoon parasite, each were designed, tested multiple times and finally a number of samples were analysed with these assays and high-throughput sequencing.

A Raptor Sample Exchange Network (RSEN) was organized including researchers and wildlife rescue centres from Germany, Spain, Portugal, Finland, Bulgaria, Greece and additional samples from Mexico, South Africa and Mongolia. These samples were genotyped for the presence of Leucocytozoon and the mitochondrial genetic lineage of the infection. Common buzzards appeared to be the most highly infected core hosts, with lineages typical of buzzards also occurring in kites and vultures. On the opposite, goshawk lineages were not found in other raptor genera.
The sampling of the buzzard study population in eastern Westphalia was further intensified in the two years of the project, with some infections being sampled up to seven times over their course and with complete spatial coverage of L.buteonis hosts of an area of 300 km2. Thanks to this effort the study now covers 16 years with nearly complete spatial coverage of the host-parasite populations with over 3000 samples and a Leucocytozoon infection prevalence of 44%.
The multiplex PCR-based assays for the genotyping of both host and parasite, based on a draft genome assembly of the common buzzard and its Leucocytozoon parasites. This assays delivers the first and most detailed dataset of its type for candidate genes with particular bearing to the host-parasite arms race and coexistence. Tests during the development of the multiplex assay showed instability of performance and increasing sensitivity to template contamination with increasing primer numbers.
The presence of non-interbreeding parasite strains was addressed by several different approaches: single haploid parasitic cells were microdissected with a laser-capture microdissection microscope and infections of single strains were used as reference for delineation of strains in mixed infections.
Finally, the samples gathered through RSEN and through intensified sampling of the Westphalian buzzard population were genotyped with the established multiplex assays to find population dynamics and selection patterns, and relate them to the genotypes of hosts and parasites. This revealed several loci which are suitable for population genetic analyses of both host and parasite. A set of other loci delivered signatures of selection on the parasite population during the course of single infections. Overall, we found support for the hypothesis that under given conditions in solitary species vector-transmitted diseases can spread from parent to offspring while this pattern becomes weaker with increasing coloniality of the hosts.