Tatjana A. Iezhova

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.

Nested Cytochrome B Polymerase Chain Reaction Diagnostics Underestimate Mixed Infections of Avian Blood Haemosporidian Parasites: Microscopy is Still Essential

Numerous polymerase chain reaction (PCR)-based methods have been developed and used increasingly to screen vertebrate blood samples for the diagnosis of haemosporidian blood parasites (Sporozoa, Haemosporida), but a rigorous evaluation of the sensitivity of these methods for detecting mixed infections of different haemosporidian species belonging to the same and different genera and subgenera is lacking. This study links the information obtained by nested cytochrome b PCR and traditional microscopy in determining mixed haemosporidian infections in naturally infected birds. Samples from 83 individual passerine birds with single infections of Haemoproteus or Plasmodium spp., as determined by mitochondrial DNA amplification, also were investigated by microscopic examination of stained blood films. Thirty-six samples (43%) were found to harbor mixed Haemoproteus, or Plasmodium spp. infections, or both. Thus, the PCR assays alone underestimate the occurrence of mixed infections of haemosporidian parasites in naturally infected birds. To determine the true species composition of the haemosporidians in each individual host, PCR diagnostics need to be improved. Specific primers for Haemoproteus spp. and Plasmodium spp. should be developed. Ideally, a combination of the approaches of both microscopy and PCR-based methods is recommended for this purpose.

Prevalence and diversity patterns of avian blood parasites in degraded African rainforest habitats

Land use changes including deforestation, road construction and agricultural encroachments have been linked to the increased prevalence of several infectious diseases. In order to better understand how deforestation affects the prevalence of vector‐borne infectious diseases in wildlife, nine paired sites were sampled (disturbed vs. undisturbed habitats) in Southern Cameroon. We studied the diversity, prevalence and distribution of avian malaria parasites (Plasmodium spp.) and other related haemosporidians (species of Haemoproteus and Leucocytozoon) from these sites in two widespread species of African rainforest birds, the yellow‐whiskered greenbul (Andropadus latirostris, Pycnonotidae) and the olive sunbird (Cyanomitra olivacea, Nectariniidae). Twenty‐six mitochondrial cytochrome b lineages were identified: 20 Plasmodium lineages and 6 Haemoproteus lineages. These lineages showed no geographic specificity, nor significant differences in lineage diversity between habitat types. However, we found that the prevalence of Leucocytozoon and Haemoproteus infections were significantly higher in undisturbed than in deforested habitats (Leucocytozoon spp. 50.3% vs. 35.8%, Haemoproteus spp. 16.3% vs. 10.8%). We also found higher prevalence for all haemosporidian parasites in C. olivacea than in A. latirostris species (70.2% vs. 58.2%). Interestingly, we found one morphospecies of Plasmodium in C. olivacea, as represented by a clade of related lineages, showed increased prevalence at disturbed sites, while another showed a decrease, testifying to different patterns of transmission, even among closely related lineages of avian malaria, in relation to deforestation. Our work demonstrates that anthropogenic habitat change can affect host–parasite systems and result in opposing trends in prevalence of haemosporidian parasites in wild bird populations.

Nested Cytochrome B Polymerase Chain Reaction Diagnostics Detect Sporozoites of Hemosporidian Parasites in Peripheral Blood of Naturally Infected Birds

Abstract Some discrepancies between microscopy and PCR-based methods have been recently recorded in the diagnosis of Leucocytozoon spp. infection in naturally infected birds. To clarify this issue, blood samples from 109 yellow-whiskered greenbuls Andropadus latirostris were investigated using both the microscopic examination of blood films and a nested mitochondrial cytochrome b PCR. The overall prevalence of Leucocytozoon spp. infection was 4% after the standard microscopic examination and 17% using the PCR diagnostics. Samples from 9 randomly chosen birds that were microscopy negative, but PCR positive, were then examined microscopically by screening 2 entire blood films from each individual bird. Sporozoites of Leucocytozoon spp. were observed in 4 birds, and 1 gametocyte of the parasite was seen in each of 2 birds. We conclude that sensitive PCR-based diagnostics are able to detect extremely light parasitemias of circulating sporozoites and gametocytes of hemosporidian parasites. Because of the PCR detection of sporozoites of unknown fate in the peripheral circulation, conclusions regarding the distribution of hemosporidian lineages in wildlife should be made with caution. To be accepted as the lineages of successfully developing species of hemosporidians, such PCR-based information should be supported with the detection of blood stages of the parasites. The present study emphasizes the crucial need for a synthesis of information provided by the tools of traditional parasitology and molecular biology, particularly in field studies of blood parasites.

Spatially explicit predictions of blood parasites in a widely distributed African rainforest bird

Critical to the mitigation of parasitic vector-borne diseases is the development of accurate spatial predictions that integrate environmental conditions conducive to pathogen proliferation. Species of Plasmodium and Trypanosoma readily infect humans, and are also common in birds. Here, we develop predictive spatial models for the prevalence of these blood parasites in the olive sunbird (Cyanomitra olivacea). Since this species exhibits high natural parasite prevalence and occupies diverse habitats in tropical Africa, it represents a distinctive ecological model system for studying vector-borne pathogens. We used PCR and microscopy to screen for haematozoa from 28 sites in Central and West Africa. Species distribution models were constructed to associate ground-based and remotely sensed environmental variables with parasite presence. We then used machine-learning algorithm models to identify relationships between parasite prevalence and environmental predictors. Finally, predictive maps were generated by projecting model outputs to geographically unsampled areas. Results indicate that for Plasmodium spp., the maximum temperature of the warmest month was most important in predicting prevalence. For Trypanosoma spp., seasonal canopy moisture variability was the most important predictor. The models presented here visualize gradients of disease prevalence, identify pathogen hotspots and will be instrumental in studying the effects of ecological change on these and other pathogens.

Polymerase chain reaction-based identification of Plasmodium (Huffia) elongatum, with remarks on species identity of haemosporidian lineages deposited in GenBank

Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only 11 morphospecies of Plasmodium have been linked to these lineages. Such linking is important because it provides opportunities to combine the existing knowledge of traditional parasitology with novel genetic information of these parasites obtained by molecular techniques. This study linked one mitochondrial cytochrome b (cyt b) gene lineage with morphospecies Plasmodium (Huffia) elongatum, a cosmopolitan avian malaria parasite which causes lethal disease in some birds. One species of Plasmodium (mitochondrial cyt b gene lineage P-GRW6) was isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to one naive juvenile individual of the same host species. Heavy parasitaemia developed in the subinoculated bird, which enabled identification of the morphospecies and deposition of its voucher specimens. The parasite of this lineage belongs to P. elongatum. Illustrations of blood stages of this parasite are given. Other lineages closely related to P. elongatum were identified. The validity of the subgenus Huffia is supported by phylogenetic analysis. Mitochondrial cyt b gene lineages, with GenBank accession nos. AF069611 and AY733088, belong to Plasmodium cathemerium and P. elongatum, respectively; these lineages have been formerly attributed to P. elongatum and P. relictum, respectively. Some other incorrect species identifications of avian haematozoa in GenBank have been identified. We propose a strategy to minimise the number of such mistakes in GenBank in the future.

Spatial Variation of Haemosporidian Parasite Infection in African Rainforest Bird Species

Abstract Spatial heterogeneity influences the distribution, prevalence, and diversity of haemosporidian parasites. Previous studies have found complex patterns of prevalence with respect to habitat characteristics and parasite genotype, and their interactions, but there is little information regarding how parasitemia intensity and the prevalence of co-infections may vary in space. Here, using both molecular methods and microscopy, we report an analysis of the variation of parasitemia intensity and co-infections of avian haemosporidian parasites (Plasmodium and Haemoproteus species) in 2 common African birds species, the yellow-whiskered greenbul (Andropadus latirostris) and the olive sunbird (Cyanomitra olivacea), at 3 sites with distinct habitat characteristics in Ghana. First, we found an interaction between the site and host species for the prevalence of Plasmodium spp. and Haemoproteus spp. For the olive sunbird, the prevalence of Plasmodium spp., as well as the number of individuals with co-infections, varied significantly among the sites, but these measures remained constant for the yellow-whiskered greenbul. In addition, yellow-whiskered greenbuls infected with Haemoproteus spp. were found only at 1 site. Furthermore, for both bird species, the parasitemia intensity of Plasmodium spp. varied significantly among the 3 sites, but with opposing trends. These results suggest that spatial heterogeneity differently affects haemosporidian infection parameters in these vertebrate-hosts. Environmental conditions here can either favor or reduce parasite infection. We discuss the implications of these discrepancies for conservation and ecological studies of infectious diseases in natural populations.

Linkage between mitochondrial cytochrome b lineages and morphospecies of two avian malaria parasites, with a description of Plasmodium (Novyella) ashfordi sp. nov

Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only seven morphospecies have been linked to these lineages. This study linked two molecular sequences with morphospecies of malaria parasites. Two species of Plasmodium (mitochondrial cytochrome b gene lineages P-GRW2 and P-GRW4) were isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to naive juvenile individuals of the same host species. Heavy parasitemia developed in the subinoculated birds, which enable identification of the species and deposition of their voucher specimens. Parasites of the lineage P-GRW2 were described as a new species, Plasmodium (Novyella) ashfordi, which is characterized primarily by the fan-like mature erythrocytic meronts containing seven to eight merozoites and the terminal position of clumped pigment granules in the gametocytes. Illustrations of the blood stages of the new species and Plasmodium (Haemamoeba) relictum (lineage P-GRW4) are given. The parasites of both lineages are transmitted in Africa and probably not in northern Europe. Other lineages closely related to P. ashfordi and P. relictum are identified. This study establishes the value of PCR-based identification of avian malaria parasites.

Molecular characterization of five widespread avian haemosporidian parasites (Haemosporida), with perspectives on the PCR-based detection of haemosporidians in wildlife

Haemosporidians (Haemosporida) are cosmopolitan in birds. Over 250 species of these blood parasites have been described and named; however, molecular markers remain unidentified for the great majority of them. This is unfortunate because linkage between DNA sequences and identifications based on morphological species can provide important information about patterns of transmission, virulence, and evolutionary biology of these organisms. There is an urgent need to remedy this because few experts possess the knowledge to identify haemosporidian species and few laboratories are involved in training these taxonomic skills. Here, we describe new mitochondrial cytochrome b markers for the polymerase chain reaction (PCR)-based detection of four widespread species of avian Haemoproteus (Haemoproteus hirundinis, Haemoproteus parabelopolskyi, Haemoproteus pastoris, Haemoproteus syrnii) and 1 species of Plasmodium (Plasmodium circumflexum). Illustrations of blood stages of the reported species are given, and morphological and phylogenetic analyses identify the DNA lineages that are associated with these parasites. This study indicates that morphological characters, which have been traditionally used in taxonomy of avian haemosporidian parasites, have a phylogenetic value. Perspectives on haemosporidian diagnostics using microscopic and PCR-based methods are discussed, particularly the difficulties in detection of light parasitemia, coinfections, and abortive parasite development. We emphasize that sensitive PCR amplifies more infections than can be transmitted; it should be used carefully in epidemiology studies, particularly in wildlife parasitology research. Because molecular studies are describing remarkably more parasite diversity than previously expected, the need for traditional taxonomy and traditional biological knowledge is becoming all the more crucial. The linkage of molecular and morphological approaches is worth more of the attention of researchers because this approach provides new knowledge for better understanding insufficiently investigated lethal diseases caused by haemosporidian infections, particularly on the exoerythrocytic (tissue) and vector stages. That requires close collaboration between researchers from different fields with a common interest.

Haemosporidian Blood Parasites in European Birds of Prey and Owls

Avian blood parasites have been intensively studied using morphological methods with limited information on their host specificity and species taxonomic status. Now the analysis of gene sequences, especially the mitochondrial cytochrome b gene of the avian haemosporidian species of Haemoproteus, Plasmodium, and Leucocytozoon, offers a new tool to review the parasite specificity and status. By comparing morphological and genetic techniques, we observed nearly the same overall prevalence of haemosporidian parasites by microscopy (19.8%) and polymerase chain reaction (PCR) (21.8%) analyses. However, in contrast to the single valid Leucocytozoon species (L. toddi) in the Falconiformes we detected 4 clearly distinctive strains by PCR screening. In the Strigiformes, where the only valid Leucocytozoon species is L. danilewskyi, we detected 3 genetically different strains of Leucocytozoon spp. Two strains of Haemoproteus spp. were detected in the birds of prey and owls examined, whereas the strain found in the tawny owl belonged to the morphospecies Haemoproteus noctuae. Three Plasmodium spp. strains that had already been found in Passeriformes were also detected in the birds of prey and owls examined here, supporting previous findings indicating a broad and nonspecific host spectrum bridging different bird orders.