Asta Križanauskienė

A Comparative Analysis of Microscopy and PCR-Based Detection Methods for Blood Parasites

Abstract We compared information obtained by both microscopy and nested mitochondrial cytochrome b PCR in determining prevalence of haemosporidian infections in naturally infected birds. Blood samples from 472 birds of 11 species belonging to 7 families and 4 orders were collected in Europe, Africa, and North America. Skilled investigators investigated them using the PCR-based screening and microscopic examination of stained blood films. The overall prevalence of haemosporidian infections, which was determined by combining results of both these methods, was 60%. Both methods slightly underestimated the overall prevalence of infection, which was 54.2% after the PCR diagnostics and 53.6% after microscopic examination. Importantly, both these tools showed similar prevalence for Haemoproteus spp. (21% by PCR and 22% by microscopy), Plasmodium spp. (17% and 22%), and Leucocytozoon spp. (30% and 25%), verifying that microscopy is a reliable tool in determining patterns of distribution of blood haemosporidian parasites in naturally infected birds. We encourage using optical microscopy in studies of blood parasites in parallel to the now widely employed molecular methods. Microscopy is unlikely to result in false positives, which is a major concern in large-scale PCR studies. Moreover, it is relatively inexpensive and provides valuable information regarding the ways in which molecular methods can be further improved and most effectively applied, especially in the field studies of parasites. Importantly, blood films, which are used for microscopic examination, should be of good quality; they should be examined properly by skilled investigators. In spite of the substantial time investments associated with microscopy, such examination provides opportunities for simultaneous determination and verification of taxonomically different parasites. Presently, different PCR protocols must be used for the detection of parasites belonging to different genera; this is expensive and time consuming.

VARIATION IN HOST SPECIFICITY BETWEEN SPECIES OF AVIAN HEMOSPORIDIAN PARASITES: EVIDENCE FROM PARASITE MORPHOLOGY AND CYTOCHROME B GENE SEQUENCES

A parasites shift to a new host may have serious evolutionary consequences, since host switching usually is associated with a change in virulence and may lead to the evolution of emerging diseases. This phenomenon remains insufficiently studied in wildlife. Here, we combine microscopic examination of blood films and PCR-based methods to investigate the natural host specificity of Haemoproteus and Plasmodium spp. in birds of 4 families of the Passeriformes within a small geographic area. The material was collected on the Curonian Spit in the Baltic Sea between May and July in 2003–2004. A nested-PCR protocol was used for amplifying and sequencing a fragment of 480 nucleotides of the cytochrome b gene of the mtDNA of these parasites. Blood samples from 282 birds, which were positive both by microscopic examination of blood films and mtDNA amplification, were used in this study. We found that Haemoproteus majoris (lineages hPARUS1, hCCF5, hWW2, and hPHSIB1), Haemoproteus sp. (hWW1), Plasmodium (Haemamoeba) sp. (pSGS1), and Plasmodium (Haemamoeba) sp. (pGRW11) are capable of infecting birds belonging to different families of passeriform birds. Some species of Haemoproteus are less specific than have been traditionally believed. Haemoproteus majoris appears to have a genetic predisposition to have a broad host range. The level of host specificity varies markedly among different species of hemosporidian parasites of birds. The natural host range is thus not a reliable taxonomic character in the systematics of these parasites in the form in which it is still accepted in some recent taxonomic studies.

Within-Host Speciation of Malaria Parasites

Background Sympatric speciation—the divergence of populations into new species in absence of geographic barriers to hybridization—is the most debated mode of diversification of life forms. Parasitic organisms are prominent models for sympatric speciation, because they may colonise new hosts within the same geographic area and diverge through host specialization. However, it has been argued that this mode of parasite divergence is not strict sympatric speciation, because host shifts likely cause the sudden effective isolation of parasites, particularly if these are transmitted by vectors and therefore cannot select their hosts. Strict sympatric speciation would involve parasite lineages diverging within a single host species, without any population subdivision. Methodology/Principal Findings Here we report a case of extraordinary divergence of sympatric, ecologically distinct, and reproductively isolated malaria parasites within a single avian host species, which apparently occurred without historical or extant subdivision of parasite or host populations. Conclusions/Significance This discovery of within-host speciation changes our current view on the diversification potential of malaria parasites, because neither geographic isolation of host populations nor colonization of new host species are any longer necessary conditions to the formation of new parasite species.

Molecular phylogenetic analysis of circumnuclear hemoproteids (haemosporida, haemoproteidae) of sylviid birds, with description of haemoproteus parabelopolskyi sp. Nov.

Haemoproteus spp., with circumnuclear gametocytes and tentatively belonging to Haemoproteus belopolskyi, are widespread and prevalent in warblers belonging to the Sylviidae, with numerous mitochondrial cytochrome b (cyt b) lineages detected among them.We sampled the hemoproteids from 6 species of warblers adjacent to the Baltic Sea. Parasites were identified to species based on morphology of their gametocytes, and a segment of the parasites cyt b gene was sequenced. Sixteen mitochondrial cyt b lineages of hemoproteids with circumnuclear gametocytes were recorded. Two clades of lineages (clade A in species of Acrocephalus and Hippolais and clade B in species of Sylvia) with sequence divergence between their lineages >5% are distinguished in the phylogenetic tree. Within the clades A and B, the genetic distance between the lineages is ≤3.9 and ≤2.8%, respectively. We compared the morphology of gametocytes of 3 lineages (hHIICT1, hMW1, and hSYAT2) in detail. The lineages hHIICT1 and hMW1 (clade A) belong to the morphospecies H. belopolskyi. Parasites of the lineage hSYAT2 (clade B) are described as a new species Haemoproteus parabelopolskyi, which can be readily distinguished from H. belopolskyi by the significantly smaller nuclei of its macrogametocytes. Lineages closely related to H. belopolskyi and H. parabelopolskyi are identified. The sequence divergence between lineages of these 2 morphospecies ranges between 5.3 and 8.1%. It seems probable that avian Haemoproteus spp. with a genetic differentiation of ≥5% in mitochondrial cyt b gene might be morphologically differentiated at the stage of gametocytes. This study establishes the value of both PCR and morphology in identification of avian hemoproteids.

A New Morphologically Distinct Avian Malaria Parasite That Fails Detection By Established Polymerase Chain Reaction–Based Protocols for Amplification of the Cytochrome B Gene

Abstract: Plasmodium polymorphum n. sp. (Haemosporida, Plasmodiidae) was found in the skylark, Alauda arvensis (Passeriformes: Alaudidae), during autumnal migration in southern Italy. This organism is illustrated and described based on the morphology of its blood stages. The most distinctive feature of this malaria parasite is the clear preference of its blood stages (trophozoites, meronts, and gametocytes) for immature red blood cells, including erythroblasts. Based on preference of erythrocytic meronts for immature red blood cells, P. polymorphum is most similar to species of the subgenus Huffia. This parasite can be readily distinguished from all other bird malaria parasites, including Plasmodium (Huffia) spp., due to preferential development and maturation of its gametocytes in immature red blood cells, a unique character for avian Plasmodium spp. In addition, the margins of nuclei in blood stages of P. polymorphum are markedly smooth and distinct; this is also a distinct diagnostic feature of this parasite. Plasmodium polymorphum has been recorded only in the skylark; it is probably a rare parasite, whose host range and geographical distribution remain unclear. Microscopic examination detected a light infection of Plasmodium relictum (lineage GRW11, parasitemia of <0.01%) in the same sample with P. polymorphum; the latter parasite clearly predominated (3.5% parasitemia). However, experienced researchers were unable to detect sequences of mitochondrial cytochrome b gene (cyt b) of P. polymorphum from the microscopically positive sample by using published and newly designed primers for DNA amplification of avian Plasmodium spp. The light parasitemia of P. relictum was easily detectable using several polymerase chain reaction (PCR)–based assays, but P. polymorphum was undetectable in all applied assays. Quantitative PCR also showed the presence of light parasitemia (0.06%) of the lineage GRW11 in this sample. This supports the conclusion that the morphologically distinct parasite observed along with P. relictum and predominant in the sample is genetically dissimilar from the lineage GRW11 based on cyt b sequence. In samples with co-infections, general PCR protocols tend to favor the amplification of the parasite with the higher parasitemia or the amplification with the best matching sequence to the primers. Because the parasitemia of P. polymorphum was >50-fold higher than that of P. relictum and several different primers were tested, we suggest that the failure to amplify P. polymorphum is a more complex problem than why co-infections are commonly overlooked in PCR-based studies. We suggest possible explanations of these results and call for additional research on evolution of mitochondrial genome of hemosporidian parasites.

How can we determine the molecular clock of malaria parasites

The association of contemporary hosts and their parasites might reflect either cospeciation or more recent shifts among existing hosts. Cospeciation implies that lineages of hosts and parasites diverge in parallel at the same time, but testing this prediction requires time-calibrated phylogenies, which are particularly difficult to obtain in organisms that leave few fossils. It has successively become clear that host shifts have been frequent in the evolutionary history of malaria parasites, but dating these host shifts cannot be done without calibrated phylogenies. Hence, it remains unresolved how long contemporary hosts and vectors have been coevolving with their malaria parasites. This review addresses conflicting rate estimates of molecular evolution and suggests research directions to aid dating diversification events in malaria parasites.

LOW HAEMOSPORIDIAN DIVERSITY AND ONE KEY-HOST SPECIES IN A BIRD MALARIA COMMUNITY ON A MID-ATLANTIC ISLAND (SÃO MIGUEL, AZORES)

When host species colonize new areas, the parasite assemblage infecting the hosts might change, with some parasite species being lost and others newly acquired. These changes would likely lead to novel selective forces on both host and its parasites. We investigated the avian blood parasites in the passerine bird community on the mid-Atlantic island of São Miguel, Azores, a bird community originating from continental Europe. The presence of haemosporidian blood parasites belonging to the genera Haemoproteus, Plasmodium, and Leucocytozoon was assessed using polymerase chain reaction. We found two Plasmodium lineages and two Leucocytozoon lineages in 11 bird species (84% of all breeding passerine species) on the island. These lineages were unevenly distributed across bird species. The Eurasian Blackbird (Turdus merula) was the key-host species (total parasite prevalence of 57%), harboring the main proportion of parasite infections. Except for Eurasian Blackbirds, all bird species had significantly lower prevalence and parasite diversity compared to their continental populations. We propose that in evolutionary novel bird communities, single species may act as key hosts by harboring the main part of the parasite fauna from which parasites ‘‘leak’’ into the other species. This would create very different host–parasite associations in areas recently colonized by hosts as compared to in their source populations.

IN VITRO HYBRIDIZATION OF HAEMOPROTEUS SPP.: AN EXPERIMENTAL APPROACH FOR DIRECT INVESTIGATION OF REPRODUCTIVE ISOLATION OF PARASITES

Abstract In spite of their potential as model organisms and their medical importance, parasite speciation processes have been insufficiently discussed in the general literature. Scarcity of experimental data regarding reproductive isolation of parasites is a serious obstacle. Toward this end we developed a method for the investigation of hybridization of hemosporidian parasites in vitro. Five species of Haemoproteus (Haemosporida, Haemoproteidae) were isolated from naturally infected passerine birds. They were identified to species based on morphology of their gametocytes and PCR amplification of a segment of the parasites mitochondrial cytochrome b gene. Hybridization of Haemoproteus spp. was initiated in vitro by mixing blood containing mature gametocytes of different species with a 3.7% solution of sodium citrate and exposure of the mixture to air. The following hybridization experiments were performed: (1) Haemoproteus minutus × Haemoproteus pallidus, (2) Haemoproteus balmorali × Haemoproteus tartakovskyi, and (3) Haemoproteus fringillae × H. tartakovskyi. The development of ookinetes of both species was blocked in the first experiment. Ookinetes of all species developed in other experiments, but presumed hybrids were distinguished only in the third experiment. Illustrations of ookinetes of all species are given. The present communication indicates that controlled experiments for direct hybridization of hemosporidians can be carried out in vitro. Such experimental research can be used to reconcile molecular and morphological data and to define biological species for this group of parasites.

Further Observations on In Vitro Hybridization of Hemosporidian Parasites: Patterns of Ookinete Development in Haemoproteus Spp.

Abstract:  Increasingly frequent outbreaks of zoonotic infections call for studies of wildlife parasites to reach a better understanding of the mechanisms of host switch, leading to the evolution of new diseases. However, speciation processes have been insufficiently addressed in experimental parasitology studies, primarily due to difficulties in determining and measuring mate-recognition signals in parasites. We investigated patterns of sexual process and ookinete development in avian Haemoproteus (Parahaemoproteus) spp. (Haemosporida, Haemoproteidae) using in vitro experiments on between-lineage hybridization. Eleven mitochondrial cytochrome b (cyt b) lineages belonging to 9 species of hemoproteid were isolated from naturally infected passerine birds. The parasites were identified to species on the basis of morphology of their gametocytes and polymerase chain reaction amplification of segments of the cyt b gene. Sexual process and ookinete development were initiated in vitro by mixing blood containing mature gametocytes with a 3.7% solution of sodium citrate and exposing the mixture to air. Ookinetes of all lineages except Haemoproteus payevskyi (lineage hRW1) and Haemoproteus nucleocondensus (hGRW1) developed; the 2 latter species did not exflagellate. Between-lineage hybridization was initiated by mixing blood containing mature gametocytes of 2 different parasites; the following experiments were performed: (1) Haemoproteus pallidus (lineage hPFC1) × Haemoproteus minutus (lineage hTURDUS2); (2) H. pallidus (hPFC1) × Haemoproteus tartakovskyi (hSISKIN1); (3) Haemoproteus belopolskyi (hHIICT3) × Haemoproteus lanii (hRB1); (4) Haemoproteus balmorali (hSFC1) × H. pallidus (hPFC1); (5) H. belopolskyi (hHIICT1) × Haemoproteus parabelopolskyi (hSYBOR1); (6) H. tartakovskyi (hHAWF1) × H. tartakovskyi (hSISKIN1); (7) H. pallidus (hPFC1) × H. lanii (hRB1); (8) H. tartakovskyi (hHAWF1) × H. parabelopolskyi (hSYBOR1). We report 4 patterns of between-lineage interactions that seem to be common and might prevent mixing lineages during simultaneous sexual process in wildlife: (1) the blockage of ookinete development of both parasites; (2) the development of ookinetes of 1 parasite and blockage of ookinete development of the other; (3) selective within-lineage mating resulting in ookinete development of both parent species and absence of hybrid organisms; (4) absence of selective within-lineage mating resulting in presence of ookinetes of both parents and also development of hybrid organisms with unclear potential for further sporogony. The present study indicates directions for collection of source material in the investigation of mechanisms of reproductive isolation leading to speciation in these parasites. The next steps in these studies should be the development of nuclear markers for distinguishing hemosporidian hybrid organisms and the experimental observation of further development of hybrid ookinetes in vectors.

Haemoproteus nucleocondensus n. sp. (Haemosporida, Haemoproteidae) from a Eurasian sogbird, the Great Reed Warbler Acrocephalus arundinaceus

Haemoproteus (Parahaemoproteus) nucleocondensus n. sp. (Haemosporida, Haemoproteidae) is described from a Eurasian songbird, the Great Reed Warbler Acrocephalus arundinaceus based on the morphology of its blood stages and partial sequences of the mitochondrial cytochrome b gene. Transmission of this haemoproteid occurs in Africa, where the Great Reed Warblers are infected. Due to unclear reasons, its transmission does not occur at European breeding grounds of this bird, resulting in absence of the parasites in juvenile birds before and during seasonal migration. Haemoproteus nucleocondensus can be readily distinguished from other avian haemoproteids by tiny, compact microgametocyte nuclei that are significantly smaller than macrogametocyte nuclei, a rare character of haemosporidian parasites. By this morphological feature, the new species is similar to Haemoproteus payevskyi, Haemoproteus micronuclearis, and Haemoproteus nucleofascialis. Haemoproteus nucleocondensus can be readily distinguished from both H. payevskyi and H. micronuclearis, primarily due to its numerous growing gametocytes (size greater than erythrocyte nuclei), which do not touch the envelope of erythrocytes along their entire margin; such gametocytes do not develop in Haemoproteus payevskyi and H. micronuclearis, but present in H. nucleofascialis. However, microgametocyte nuclei are band-like in shape in the latter parasite; that is not a case in H. nucleocondensus. Illustrations of blood stages of the new species are given, and morphological and phylogenetic analyses identify the DNA lineages that are associated with these parasites. Genetic distance between cyt b gene sequences of the new species and H. payevskyi, H. micronuclearis, and H. nucleofascialis is 4.4%, 5.9%, and 8.1%, respectively. Cytochrome b gene sequences (GenBank no. JX026901) can be used for bar-coding in studies of H. nucleocondensus.