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We used a nested PCR protocol to examine the genetic diversity of cytochrome b (cyt b) lineages from blood parasites of the genera Plasmodium and Haemoproteus in birds in Bulgaria. In total, 460 birds of 43 species and 14 families (mostly passerines) were examined for the presence of infections. Of them, 267 were recognised as infected with haemosporidian parasites. Mixed infections were recorded in 24 individuals (9%). Besides the 24 individuals with mix infections, 114 (43%) were positive for Plasmodium spp. and 129 (48%) for Haemoproteus spp. We identified 52 genetic lineages of haemosporidian parasites: 38 of Haemoproteus and 14 of Plasmodium. Twelve new cyt b lineages of Haemoproteus were recorded; they occurred in the following hosts: grey-faced woodpecker (Picus canus), golden oriole (Oriolus oriolus), jay (Garrulus glandarius), barred warbler (Sylvia nisoria), song thrush (Turdus philomelos), spotted flycatcher (Muscicapa striata), spanish sparrow (Passer hispaniolensis), hawfinch (Coccothraustes coccothraustes), and cirl bunting (Emberiza cirlus). We also detected 22 new host records for previously known lineages. The most common lineage was SGS1 (Plasmodium relictum), which had a total prevalence of 14% and occurred in 8 host species belonging to 5 families. Three of the cyt b lineages of genus Haemoproteus (DURB1, DURB2 and SYNIS2) showed more than 5% divergence from all described morphologically lineages. These lineages probably represent at least 2 different morphospecies which remains to be identified.
This study presents data from a molecular survey of the species of the genus Culicoides from the region of Kalimok Field Station (NE Bulgaria) and haemosporidian parasites occurring in them in order to investigate the host-parasite specificity of haemosporidians to their dipteran vectors. The identification of Culicoides spp. was carried out by morphological and molecular-genetic methods. We collected and analysed 230 individuals of the genus Culicoides. Nine species were found. Eight species were identified morphologically; Culicoides obsoletus, C. riethi, C. newsteadi, C. circumscriptus, C. festivipennis, C. punctatus, C. pictipennis and C. puncticollis. The ninth species might be classified as either of C. nubeculosus or C. riethi and its identification needs additional investigations. The total prevalence of Haemoproteus in the examined biting midges was 2.17%. Three individuals of C. pictipennis were infected with the Haemoproteus lineage TURDUS2 (prevalence 16.67%), a common parasite of thrushes (Turdidae). Two individuals of C. circumscriptus contained Haemoproteus lineages (prevalence 2.78%); these were the lineage HAWF2 (previously reported from Coccothraustes coccothraustes) and a new lineage CULCIR1 not previously reported in the literature.
The blood parasite diversity was studied in paddyfield warblers (Acrocephalus agricola) breeding in NE Bulgaria, SW Russia and S. Kazakhstan. Nine cytochrome b gene lineages were recorded, 4 belonging to Haemoproteus spp. and 5 to Plasmodium spp. The overall prevalence of haemosporidians was 33.3%. The composition of parasites varied geographically, with six lineages recorded in Russia, five lineages in Bulgaria and two lineages in Kazakhstan. Two lineages are described for the first time, i.e. ACAGR1 (belonging to Plasmodium sp. and recorded from a single bird in Russia) and ACAGR2 (belonging to Haemoproteus sp., recorded from Bulgaria and Russia). The latter lineage is the most widespread parasite in the Bulgarian population, scarce in Russia and absent in Kazakhstan. It is supposed that ACAGR2 has originated from the widespread lineage ACDUM1 differing from it by a single nucleotide. One lineage only (ACDUM2) occurs in all the three populations studied and is a nonspecific parasite known from various passerines. Six of the registered lineages have been found in a single population of A. agricola and also represent non-specific parasites occurring in a wide range of passerine birds. Their records in A. agricola may indicate the high transmission rate of these parasites in the habitats where this host co-occurs with other passerines. The variation of the composition of the haemosporidian parasite communities through the breeding range of A. agricola makes up heterogeneous selection pressures that may drive intraspecific variation in important life-history traits.
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