Morphological and molecular analyses of bird haemosporidian parasites and investigation on their specificity and pathogenicity

Dimitar Dimitrov

PhD thesis, Sofia 2011

Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia

Supervisor: Assoc. Prof. Vasil Golemansky Adviser: Dr. Pavel Zehtindjiev

Summary The aim of the present study was to characterize the genetic diversity, specificity and pathogenicity of avian malaria and related blood parasites (order ) in some less studied bird and regions. In addition, we aimed to link some of the DNA-sequences with their morphospecies. We used microscopy and a nested PCR protocol to examine the morphology and genetic diversity of cytochrome b (cyt b) in lineages from blood parasites of the genera , and in birds. In total, 1024 birds of 48 species and 15 families (mostly passerines) were examined for the presence of in Bulgaria, Russia, Italy and the Netherlands. Of them, 387 were recognised as infected with haemosporidian parasites. Besides the 44 individuals with mixed infections, 197 were positive for Plasmodium spp., 151 for Haemoproteus spp. and 16 for Leucocytozoon spp. We identified 72 genetic lineages of haemosporidian parasites: 46 of Haemoproteus, 16 of Plasmodium and 10 of Leucocytozoon. Fifteen new cyt b lineages of Haemoproteus and seven of Leucocytozoon were recorded and included in MalAvi database (http://mbio- serv4.mbioekol.lu.se/avianmalaria/). We also detected 27 new host records for previously known lineages in 147 birds from 16 species. We described morphologically two cyt b lineages from genus Plasmodium (GRW06 and SYAT05) and three from genus Haemoproteus (ALARV1, ALARV2 and ALARV3). Plasmodium (Huffia) elongatum and Plasmodium (Novyella) vaughani were linked with GRW06 and SYAT05 respectively. All three lineages from genus Haemoproteus were described as Haemoproteus (Parahaemoproteus) tartakovskyi. Other three lineages from genus Haemoproteus (DURB1, DURB2 and SYNIS2) showed more than 5% divergence from all lineages that have been described morphologically. These lineages probably represent at least 2 different morphospecies which remain to be identified. During the morphological description of GRW06 two lineages deposited in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) were found to be incorrectly identified. The lineage Plasmodium sp. (AF069611) was attributed to P. (Huffia) elongatum in GenBank and the lineage Plasmodium sp. (AY733088) was attributed to P. (Haemamoeba) relictum. Instead, these two lineages cluster well with lineages belonging to the subgenera Haemamoeba and Huffia, respectively. The results of this study are in accordance with our current knowledge on host specificity and distribution of parasite lineages. Plasmodium spp. are predominantly host generalists with a wide distribution, while Haemoproteus spp. and Leucocytozoon spp. exhibit narrower host

1 and geographical ranges. The most common lineage was SGS1 (Plasmodium relictum), which had a total prevalence of 34% and occurred in 10 host species belonging to 7 families. This study reports the greatest overall prevalence of malaria in skylarks (Alauda arvensis) during the last 100 years and shows that both Plasmodium and Haemoproteus spp. haemosporidian infections are expanding in this bird species, possibly contributing to its decrease in Europe. To investigate the specificity and pathogenicity of different parasites, we inoculated them into captive birds and followed the course of the infection. Nonspecific hosts from one short-distance migratory bird, the song thrush, (Turdus philomelos), and two sedentary species, the house and tree sparrow (Passer domesticus and Passer montanus) were resistant to Plasmodium ashfordi (lineage GRW02). This was demonstrated by the experimental inoculation and confirmed by microscopy and nested PCR. House sparrows in Bulgaria proved to be resistant when experimentally inoculated with lineage GRW04 of Plasmodium relictum, while this same lineage is common in the house sparrows that invaded North and South America. The test birds did not develop the infection that we had found in the great reed warbler (Acrocephalus arundinaceus), a trans-saharian migrant, used as donor in our experiment. These results indicate differences in the parasite specificity in Europe and America.

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