Gediminas Valkiūnas

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.

Dynamics of parasitemia of malaria parasites in a naturally and experimentally infected migratory songbird, the great reed warbler Acrocephalus arundinaceus

Little is known about the development of infection of malaria parasites of the genus Plasmodium in wild birds. We used qPCR, targeting specific mitochondrial lineages of Plasmodium ashfordi (GRW2) and Plasmodium relictum (GRW4), to monitor changes in intensities of parasitemia in captive great reed warblers Acrocephalus arundinaceus from summer to spring. The study involved both naturally infected adults and experimentally infected juveniles. The experiment demonstrated that P. ashfordi and P. relictum lineages differ substantially in several life-history traits (e.g. prepatent period and dynamics of parasitemia) and that individual hosts show substantial differences in responses to these infections. The intensity of parasitemia of lineages in mixed infections co-varied positively, suggesting a control mechanism by the host that is general across the parasite lineages. The intensity of parasitemia for individual hosts was highly repeatable suggesting variation between the host individuals in their genetic or acquired control of the infections. In future studies, care must be taken to avoid mixed infections in wild caught donors, and when possible use mosquitoes for the experiments as inoculation of infectious blood ignores important initial stages of the contact between the bird and the parasite.

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.

Nonspecific patterns of vector, host and avian malaria parasite associations in a central African rainforest.

Malaria parasites use vertebrate hosts for asexual multiplication and Culicidae mosquitoes for sexual and asexual development, yet the literature on avian malaria remains biased towards examining the asexual stages of the life cycle in birds. To fully understand parasite evolution and mechanism of malaria transmission, knowledge of all three components of the vector‐host‐parasite system is essential. Little is known about avian parasite–vector associations in African rainforests where numerous species of birds are infected with avian haemosporidians of the genera Plasmodium and Haemoproteus. Here we applied high resolution melt qPCR‐based techniques and nested PCR to examine the occurrence and diversity of mitochondrial cytochrome b gene sequences of haemosporidian parasites in wild‐caught mosquitoes sampled across 12 sites in Cameroon. In all, 3134 mosquitoes representing 27 species were screened. Mosquitoes belonging to four genera (Aedes, Coquillettidia, Culex and Mansonia) were infected with twenty‐two parasite lineages (18 Plasmodium spp. and 4 Haemoproteus spp.). Presence of Plasmodium sporozoites in salivary glands of Coquillettidia aurites further established these mosquitoes as likely vectors. Occurrence of parasite lineages differed significantly among genera, as well as their probability of being infected with malaria across species and sites. Approximately one‐third of these lineages were previously detected in other avian host species from the region, indicating that vertebrate host sharing is a common feature and that avian Plasmodium spp. vector breadth does not always accompany vertebrate–host breadth. This study suggests extensive invertebrate host shifts in mosquito–parasite interactions and that avian Plasmodium species are most likely not tightly coevolved with vector species.

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.

EVIDENCE FOR CRYPTIC SPECIATION OF LEUCOCYTOZOON SPP. (HAEMOSPORIDA, LEUCOCYTOZOIDAE) IN DIURNAL RAPTORS

Species of Leucocytozoon (Haemosporida, Leucocytozoidae) traditionally have been described based on morphological characters of their blood stages and host cells, with limited information on their avian host specificity. Based on the current taxonomy, Leucocytozoon toddi is the sole valid species of leucocytozoids parasitizing falconiform birds. Using a nested polymerase chain reaction protocol, we determined the prevalence of Leucocytozoon infection in 5 species of diurnal raptors from California. Of 591 birds tested, 177 (29.9%) were infected with Leucocytozoon toddi. Subsequent phylogenetic analysis of the cytochrome b gene revealed that distinct haplotypes are present in hawks of these genera. Haplotypes present in Buteo spp. are not found in Accipiter spp., and there is a 10.9% sequence divergence between the 2 lineage clades. In addition, Leucocytozoon sp. from Accipiter spp. from Europe group more closely with parasites found in Accipiter spp. from California than the same California Accipiter species do with their sympatric Buteo spp. Similarly, a Leucocytozoon haplotype from a Common Buzzard (Buteo buteo) from Kazakhstan forms a monophyletic lineage with a parasite from B. jamaicensis from California. These results suggest that Leucocytozoon toddi is most likely a group of cryptic species, with 1 species infecting Buteo spp. and 1 or more species, or subspecies, infecting Accipiter spp.

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.

Blood Parasites in Owls with Conservation Implications for the Spotted Owl (Strix occidentalis)

The three subspecies of Spotted Owl (Northern, Strix occidentalis caurina; California, S. o. occidentalis; and Mexican, S. o. lucida) are all threatened by habitat loss and range expansion of the Barred Owl (S. varia). An unaddressed threat is whether Barred Owls could be a source of novel strains of disease such as avian malaria (Plasmodium spp.) or other blood parasites potentially harmful for Spotted Owls. Although Barred Owls commonly harbor Plasmodium infections, these parasites have not been documented in the Spotted Owl. We screened 111 Spotted Owls, 44 Barred Owls, and 387 owls of nine other species for haemosporidian parasites (Leucocytozoon, Plasmodium, and Haemoproteus spp.). California Spotted Owls had the greatest number of simultaneous multi-species infections (44%). Additionally, sequencing results revealed that the Northern and California Spotted Owl subspecies together had the highest number of Leucocytozoon parasite lineages (n = 17) and unique lineages (n = 12). This high level of sequence diversity is significant because only one Leucocytozoon species (L. danilewskyi) has been accepted as valid among all owls, suggesting that L. danilewskyi is a cryptic species. Furthermore, a Plasmodium parasite was documented in a Northern Spotted Owl for the first time. West Coast Barred Owls had a lower prevalence of infection (15%) when compared to sympatric Spotted Owls (S. o. caurina 52%, S. o. occidentalis 79%) and Barred Owls from the historic range (61%). Consequently, Barred Owls on the West Coast may have a competitive advantage over the potentially immune compromised Spotted Owls.

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.