Prevalence and genetic diversity of avian haemosporidian parasites in wild bird species of the order Columbiformes.

Diseases can play a role in species decline. Among them, haemosporidian parasites, vector-transmitted protozoan parasites, are known to constitute a risk for different avian species. However, the magnitude of haemosporidian infection in wild columbiform birds, including strongly decreasing European turtle doves, is largely unknown. We examined the prevalence and diversity of haemosporidian parasites Plasmodium, Leucocytozoon and subgenera Haemoproteus and Parahaemoproteus in six species of the order Columbiformes during breeding season and migration by applying nested PCR, one-step multiplex PCR assay and microscopy. We detected infections in 109 of the 259 screened individuals (42%), including 15 distinct haemosporidian mitochondrial cytochrome b lineages, representing five H. (Haemoproteus), two H. (Parahaemoproteus), five Leucocytozoon and three Plasmodium lineages. Five of these lineages have never been described before. We discriminated between single and mixed infections and determined host species-specific prevalence for each parasite genus. Observed differences among sampled host species are discussed with reference to behavioural characteristics, including nesting and migration strategy. Our results support previous suggestions that migratory birds have a higher prevalence and diversity of blood parasites than resident or short-distance migratory species. A phylogenetic reconstruction provided evidence for H. (Haemoproteus) as well as H. (Parahaemoproteus) infections in columbiform birds. Based on microscopic examination, we quantified parasitemia, indicating the probability of negative effects on the host. This study provides a large-scale baseline description of haemosporidian infections of wild birds belonging to the order Columbiformes sampled in the northern hemisphere. The results enable the monitoring of future changes in parasite transmission areas, distribution and diversity associated with global change, posing a potential risk for declining avian species as the European turtle dove.

Patterns of Haemoproteus majoris (Haemosporida, Haemoproteidae) megalomeront development.

Blood parasites of the genus Haemoproteus (Haemosporida, Haemoproteidae) are cosmopolitan and prevalent in birds. Numerous species and lineages of these pathogens have been identified. Some of the infections are lethal in avian hosts mainly due to damage of organs by tissue stages, which remain insufficiently investigated. Several closely related lineages of Haemoproteus majoris, a common parasite of passeriform birds, have been identified. One recent study described megalomeronts of unique morphology in the lineages hPHYBOR04 and hPARUS1 of H. majoris and suggested that the similar tissues stages might also be features in other phylogenetically closely related lineages of the same parasite species. This study aimed to test if (i) megalomeronts are present during the development of the lineage hPHSIB1 of H. majoris and if (ii) they are similar to the other investigated lineages of this species in regard of their morphology and location in organs. One adult wood warbler Phylloscopus sibilatrix, an Afrotropical migrant, naturally infected with H. majoris lineage hPHSIB1 was wild-caught after seasonal spring migration and screened using microscopic examination of blood films and histological sections of organs as well as using PCR-based testing. Bayesian phylogenetic analysis placed the lineages hPHSIB1, hPHYBOR04 and hPARUS1 in one, well-supported clade. Parasitaemia was high (6.5%) in the examined wood warbler, numerous megalomeronts were found in kidneys, and a few in the intestine. Megalomeronts of the lineage hPHSIB1 were morphologically hardly distinguishable from those of lineages hPHYBOR04 and hPARUS1; only negligible differences in the maturation stage of the cytomeres were seen. The kidneys were the main location site of the megalomeronts in all three lineages of this parasite species. This study shows that closely related lineages of H. majoris produce megalomeronts of similar morphology and predominant location in kidneys, while the normal function of this organ may be affected by the presence of numerous large megalomeronts. Megalomeronts of different avian Haemoproteus species are markedly variable in morphology and location, but phylogenetically closely related lineages possess cryptic megalomeronts. This finding suggests that phylogenies based on partial cytb gene could provide information for prediction of patterns of exo-erythrocytic development of closely related Haemoproteus parasites and are worthy of attention in planning haemosporidian parasite tissue stage research.

Seasonal population dynamics and a role as natural vector of Leucocytozoon of black fly, Simulium chumpornense Takaoka & Kuvangkadilok.

The black fly, Simulium chumpornense Takaoka & Kuvangkadilok, is suspected to be a natural vector of Leucocytozoon. In this study, seasonal variation in abundance of the adult fly and molecular detection of Leucocytozoon in S. chumpornense and in domestic chicken was investigated in northeastern Thailand. A total of 10,416 female adults were obtained by monthly collection for one year at six sampling sites. There was a peak of abundance in the dry season (March - May) when more than 74% of the specimens were collected. An increased number of suitable habitats for immature stages during the dry season is potentially a factor driving dry season abundance. Molecular genetic investigations revealed that S. chumpornense harbored the infectious stage of Leucocytozoon sp. indicating that it is a natural vector of this parasite. Blood smear screening for the parasite in domestic chickens found a considerable prevalence (52%) of Leucocytozoon spp. infections. The cytochrome b sequences of this parasite in domestic chickens comprised two lineages. One lineage was closely related to Leucocytozoon sp. found in S. chumpornense and another matched with L. schoutedeni. Therefore, S. chumpornense is a vector of Leucocytozoon sp. while L. schoutedeni is possibly transmitted by other ornithophilic black fly species.

A new methodology for sporogony research of avian haemoproteids in laboratory-reared Culicoides spp., with a description of the complete sporogonic development of Haemoproteus pastoris.

BACKGROUND: Haemosporidian parasites of the genus Haemoproteus (Haemoproteidae) are widespread and cause haemoproteosis in birds and therefore, their diversity, ecology and evolutionary biology have become subjects of intensive research. However, the vectors and transmission patterns of haemoproteids as well as the epidemiology of haemoproteosis remain insufficiently investigated. Several species of Culicoides (Ceratopogonidae) support complete sporogony of haemoproteids belonging to the subgenus Parahaemoproteus. However, experimental research with these fragile insects is difficult to design in the field, particularly because their abundance markedly depends on seasonality. This is an obstacle for continuous sampling of sufficient numbers of naturally infected or experimentally exposed midges from wildlife. We developed simple methodology for accessing sporogonic development of haemoproteids in laboratory-reared Culicoides nubeculosus. This study aimed to describe the mosaic of methods constituting this methodology, which was applied for investigation of the sporogonic development of Haemoproteus (Parahaemoproteus) pastoris, a widespread parasite of the common starling Sturnus vulgaris. METHODS: The methodology consists of the following main stages: (i) laboratory rearing of C. nubeculosus from the egg stage to adult insects; (ii) selection of naturally infected birds, the donors of mature gametocytes to expose biting midges; (iii) experimental exposure of insects and their laboratory maintenance; and (iv) dissection of exposed insects. Biting midges were exposed to H. pastoris (cytochrome b lineage hLAMPUR01) detected in one naturally infected common starling. Engorged insects were dissected at intervals in order to follow sporogony. Microscopic examination and PCR-based methods were used to identify the sporogonic stages and to confirm the presence of the parasite lineage in infected insects, respectively. RESULTS: Culicoides nubeculosus females were successfully reared and exposed to H. pastoris, which completed sporogonic development 7-9 days post-infection when sporozoites were observed in the salivary glands. CONCLUSIONS: The new methodology is easy to use and non-harmful for birds, providing opportunities to access the sporogonic stages of Parahaemoproteus parasites, which might be used in a broad range of parasitology and genetic studies. Culicoides nubeculosus is an excellent experimental vector of subgenus Parahaemoproteus and is recommended for various experimental studies aiming investigation of sporogony of these pathogens.

The life-cycle of the avian haemosporidian parasite Haemoproteus majoris, with emphasis on the exoerythrocytic and sporogonic development.

BACKGROUND: Haemoproteus parasites (Haemosporida, Haemoproteidae) are cosmopolitan in birds and recent molecular studies indicate enormous genetic diversity of these pathogens, which cause diseases in non-adapted avian hosts. However, life-cycles remain unknown for the majority of Haemoproteus species. Information on their exoerythrocytic development is particularly fragmental and controversial. This study aimed to gain new knowledge on life-cycle of the widespread blood parasite Haemoproteus majoris. METHODS: Turdus pilaris and Parus major naturally infected with lineages hPHYBOR04 and hPARUS1 of H. majoris, respectively, were wild-caught and the parasites were identified using microscopic examination of gametocytes and PCR-based testing. Bayesian phylogeny was used to determine relationships between H. majoris lineages. Exoerythrocytic stages (megalomeronts) were reported using histological examination and laser microdissection was applied to isolate single megalomeronts for genetic analysis. Culicoides impunctatus biting midges were experimentally exposed in order to follow sporogonic development of the lineage hPHYBOR04. RESULTS: Gametocytes of the lineage hPHYBOR04 are indistinguishable from those of the widespread lineage hPARUS1 of H. majoris, indicating that both of these lineages belong to the H. majoris group. Phylogenetic analysis supported this conclusion. Sporogony of the lineage hPHYBOR04 was completed in C. impunctatus biting midges. Morphologically similar megalomeronts were reported in internal organs of both avian hosts. These were big roundish bodies (up to 360 µm in diameter) surrounded by a thick capsule-like wall and containing irregularly shaped cytomeres, in which numerous merozoites developed. DNA sequences obtained from single isolated megalomeronts confirmed the identification of H. majoris. CONCLUSIONS: Phylogenetic analysis identified a group of closely related H. majoris lineages, two of which are characterized not only by morphologically identical blood stages, but also complete sporogonic development in C. impunctatus and development of morphologically similar megalomeronts. It is probable that other lineages belonging to the same group would bear the same characters and phylogenies based on partial cytb gene could be used to predict life-cycle features in avian haemoproteids including vector identity and patterns of exoerythrocytic merogony. This study reports morphologically unique megalomeronts in naturally infected birds and calls for research on exoerythrocytic development of haemoproteids to better understand pathologies caused in avian hosts.

Experimental characterization of the complete life cycle of Haemoproteus columbae, with a description of a natural host-parasite system used to study this infection.

Characterization of complete life cycles of haemoparasites requires the maintenance of suitable susceptible vertebrate hosts and vectors for long periods in captivity, in order to follow the complete parasitic cycle in definitive and intermediate hosts. Currently, there are few host-parasite models established in avian haemosporidian research, and those have been developed mainly for species of Passeriformes and their parasites. This study aimed to develop an experimental methodology to access the complete life cycle of Haemoproteus columbae (cytb lineage HAECOL1), which parasitizes the Rock Pigeon (Columba livia) and louse fly (Pseudolynchia canariensis). A colony of louse flies, which are the natural vectors of this parasite, was established. Thirty newly emerged insects were exposed to H. columbae infection and used to infect naïve Rock Pigeons. The peak of parasitaemia (acute stage) was seen between 27 and 32 days p.i. when up to 70.8% of red blood cells were infected. The crisis occurred approximately 1 week after the peak, and the long-lasting chronic parasitaemia stage followed. Exo-erythrocytic meronts were seen mainly in the lungs where extensive tissue damage was reported, but also in the kidneys and spleen. In the vector, the sporogonic cycle of H. columbae was completed between 13 and 16 days p.i., at an average temperature ranging between 12 and 15 °C. This host-parasite model is tractable for maintenance in captivity. It is recommended for use in studies aiming for detailed characterization of host-parasite relationships in areas such as physiology, pathology, immunobiology, genetics, as well as for evaluative treatments and to follow the infection in any stage of parasite development both in the vertebrate or invertebrate host.

Effects of Eugenol on Haemoproteus columbae in domestic pigeons (Columba livia domestica) from Riyadh, Saudi Arabia.

Eugenol was investigated for the treatment of Haemoproteus columbae (H. columbae) infected squabs (young domestic pigeons, Columba domestica). Thirty naturally-infected squabs were divided into three groups of 10 each. One group was treated with Eugenol, while the positive and negative control groups were administered buparvaquone (Butalex®) and distilled water, respectively. The number of infected red blood cells (RBCs) was calculated in all groups before and after treatment at 4-day intervals for 16 days. The results showed a significant therapeutic effect of Eugenol, with a progressive decrease in the number of infected RBCs from 89.20 ± 2.11 before treatment to 0.90 ± 0.31 at the end of treatment (P≤0.05). Butalex® was able to suppress the number of infected RBCs from 93.70 ± 1.72 before treatment to 0.90 ± 0.35 at the end of the experiment (P≤0.05). Eugenol showed therapeutic effects against H. columbae and may be regarded as a candidate for further studies to develop new drugs against blood parasites, in both animals and humans.

High susceptibility of the laboratory-reared biting midges Culicoides nubeculosus to Haemoproteus infections, with review on Culicoides species that transmit avian haemoproteids.

Haemosporidian parasites belonging to Haemoproteus cause avian diseases, however, vectors remain unidentified for the majority of described species. We used the laboratory-reared biting midges Culicoides nubeculosus to determine if the sporogonic development of three widespread Haemoproteus parasites completes in this insect. The midges were reared and fed on one common blackbird, white wagtail and thrush nightingale naturally infected with Haemoproteus minutus, Haemoproteus motacillae and Haemoproteus attenuatus, respectively. The engorged females were dissected in order to follow their sporogonic development. Microscopic examination was used to identify sporogonic stages. Bayesian phylogeny based on partial cytochrome b gene was constructed in order to determine phylogenetic relationships among Culicoides species-transmitted haemoproteids. All three parasites completed sporogony. Phylogenetic analysis placed Culicoides species transmitted haemoproteids in one well-supported clade, proving that such analysis readily indicates groups of dipteran insects transmitting avian haemoproteids. Available data show that 11 species of Culicoides have been proved to support complete sporogony of 18 species of avian haemoproteids. The majority of Culicoides species can act as vectors for many Haemoproteus parasites, indicating the low specificity of these parasites to biting midges, whose are globally distributed. This calls for control of haemoproteid infections during geographical translocation of infected birds.

Exo-erythrocytic development of avian malaria and related haemosporidian parasites.

BACKGROUND: Avian malaria parasites (Plasmodium spp.) and related haemosporidians (Haemosporida) are responsible for diseases which can be severe and even lethal in avian hosts. These parasites cause not only blood pathology, but also damage various organs due to extensive exo-erythrocytic development all over the body, which is not the case during Plasmodium infections in mammals. However, exo-erythrocytic development (tissue merogony or schizogony) remains the most poorly investigated part of life cycle in all groups of wildlife haemosporidian parasites. In spite of remarkable progress in studies of genetic diversity, ecology and evolutionary biology of avian haemosporidians during the past 20 years, there is not much progress in understanding patterns of exo-erythrocytic development in these parasites. The purpose of this review is to overview the main information on exo-erythrocytic development of avian Plasmodium species and related haemosporidian parasites as a baseline for assisting academic and veterinary medicine researchers in morphological identification of these parasites using tissue stages, and to define future research priorities in this field of avian malariology. METHODS: The data were considered from peer-reviewed articles and histological material that was accessed in zoological collections in museums of Australia, Europe and the USA. Articles describing tissue stages of avian haemosporidians were included from 1908 to the present. Histological preparations of various organs infected with the exo-erythrocytic stages of different haemosporidian parasites were examined. RESULTS: In all, 229 published articles were included in this review. Exo-erythrocytic stages of avian Plasmodium, Fallisia, Haemoproteus, Leucocytozoon, and Akiba species were analysed, compared and illustrated. Morphological characters of tissue stages that can be used for diagnostic purposes were specified. CONCLUSION: Recent molecular studies combined with histological research show that avian haemosporidians are more virulent than formerly believed. The exo-erythrocytic stages can cause severe disease, especially in non-adapted avian hosts, suggesting the existence of a group of underestimated malignant infections. The development of a given haemosporidian strain can be markedly different in different avian hosts, resulting in significantly different virulence. A methodology combining the traditional histology techniques with molecular diagnostic tools is essential to speed research in this field of avian malariology.

Mechanisms of mortality in Culicoides biting midges due to Haemoproteus infection.

We examined the effects of Haemoproteus infection on the survival and pathology caused in the biting midges. Forty-six females of Culicoides impunctatus were exposed experimentally by allowing them to feed on a naturally infected red-backed shrike infected with Haemoproteus lanii (lineage hRB1, gametocytaemia 5·2%). Seventeen females were fed on an uninfected bird (controls). Dead insects were collected, counted and used for dissection, histological examination and polymerase chain reaction-based testing. Parasites were present in all experimentally infected biting midges, but absent from control insects. Survivorship differed significantly between the control and infected groups. Twelve hours post-exposure (PE), 45 (98%) experimentally infected midges were dead, but all control midges remained alive, and many of them survived until 7 day PE. The migrating ookinetes of H. lanii overfilled midgut, markedly damaged the midgut wall, entered the haemocoel and overfilled the abdomen and thorax of exposed biting midges. Massive infection by migrating ookinetes led to damage of abdomen and thorax of biting midges. The parasites often present in large clumps in the haemocoel in abdomen and thorax, leading to the interruption of the haemolymph circulation. These are the main reasons for rapid death of biting midges after feeding on high-intensity infections of Haemoproteus parasites.

Molecular and morphological description of Haemoproteus (Parahaemoproteus) bukaka (species nova), a haemosporidian associated with the strictly Australo-Papuan host subfamily Cracticinae.

Linking morphological studies with molecular phylogeny is important to understanding cryptic speciation and the evolution of host-parasite relationships. Haemosporidian parasites of the Australo-Papuan bird family Artamidae are relatively unstudied. Only one parasite species from the subfamily Cracticinae has been described, and this was based solely on morphological description. This is despite many Cracticinae species being easily observed and abundant over large ranges and in close proximity to human populations. We used morphological and molecular methods to describe a new Haemoproteus species (H. bukaka sp. nov.) from an endemic Butcherbird host (Cracticus louisiadensis) in a relatively unstudied insular area of high avian endemism (Papua New Guinea's Louisiade Archipelago). Phylogenetic reconstructions using parasite cyt-b gene sequences placed the proposed Haemoproteus bukaka sp. nov. close to other host-specialist Haemoproteus species that infect meliphagid honeyeater hosts in the region, e.g. H. ptilotis. Distinct morphological characters of this haemosporidian include macrogametocytes with characteristic large vacuoles opposing a subterminal nucleus on the host cell envelope. Among 27 sampled individuals, prevalence of H. bukaka sp.nov. was high (74 % infection rate) but strongly variable across four islands in the archipelago (ranging from 0 to 100 % prevalence). Parasitaemia levels were low across all infected individuals (0.1-0.6 %). We suspect host density may play a role in maintaining high prevalence given the close proximity and similar physical environments across islands. The findings are discussed in the context of the host genus Cracticus and theory relating to parasite-host evolution and its conservation implications in Papua New Guinea.

Hemosporidian parasites of free-living birds in the São Paulo Zoo, Brazil

Numerous studies addressed the diversity of bird Plasmodium and Haemoproteus parasites. However, a few have been carried out in continental avian hotspot regions such as Brazil, a country with markedly different biomes, including Amazon, Brazilian Savanna, Atlantic Forest,Caatinga, Pantanal, and Pampas. We present the first study on hemosporidian (Haemosporida) parasites in free-living birds from an Atlantic Forest fragment where more than 80 avian species have been reported. Within this area, the São Paulo Zoo locates, and it is the fourth largest zoo in the world and the largest in Latin America. A total of 133 free-living bird samples representing 12 species were collected in the zoo, with the overall hemosporidian prevalence of 18 % by PCR based diagnostics. Twenty-four positive PCR signals were reported from four different bird species, including migratory ones. Columba livia, an urban species, considered nowadays a pest in big cities, showed 100% prevalence of Haemoproteusspp., mainly Haemoproteus columbae...

Diverse sampling of East African haemosporidians reveals chiropteran origin of malaria parasites in primates and rodents.

Phylogenies of parasites provide hypotheses on the history of their movements between hosts, leading to important insights regarding the processes of host switching that underlie modern-day epidemics. Haemosporidian (malaria) parasites lack a well resolved phylogeny, which has impeded the study of evolutionary processes associated with host-switching in this group. Here we present a novel phylogenetic hypothesis that suggests bats served as the ancestral hosts of malaria parasites in primates and rodents. Expanding upon current taxon sampling of Afrotropical bat and bird parasites, we find strong support for all major nodes in the haemosporidian tree using both Bayesian and maximum likelihood approaches. Our analyses support a single transition of haemosporidian parasites from saurian to chiropteran hosts, and do not support a monophyletic relationship between Plasmodium parasites of birds and mammals. We find, for the first time, that Hepatocystis and Plasmodium parasites of mammals represent reciprocally monophyletic evolutionary lineages. These results highlight the importance of broad taxonomic sampling when analyzing phylogenetic relationships, and have important implications for our understanding of key host switching events in the history of malaria parasite evolution.

In vitro development of Haemoproteus columbae (Haemosporida: Haemoproteidae), with perspectives for genomic studies of avian haemosporidian parasites.

The evolutionary origin of wildlife and human malaria parasites (Plasmodium spp.) has been discussed for several decades. The lack of genomic data about species of wildlife haemosporidian parasites related to Plasmodium limits the number of taxa available for phylogenetic analysis. Genomic data about avian parasites of the genus Haemoproteus parasites, the sister genus to Plasmodium are still not available, mainly due to difficulties in obtaining pure DNA of parasites inhabiting nucleated avian host cells. Recent studies show that microgametes of Haemoproteus (Parahaemoproteus) spp. develop in vitro and can be isolated by simple centrifugation, allowing the isolation of pure parasite DNA for genomic studies. However, in vitro development of Haemoproteus (Haemoproteus) spp. has not been investigated, and it is unclear if microgametes of these parasites also can be obtained under in vitro conditions. Here, we provide the first data about the in vitro development of Haemoproteus (Haemoproteus) columbae, a widespread avian haemosporidian parasite, which is specific to pigeons and doves (Columbiformes) and is transmitted by hippoboscid flies (Diptera, Hippoboscidae). In vitro gametogenesis and ookinete development of H. columbae were studied using a strain isolated from a feral Rock Pigeon (Columba livia) in Bogotá-Colombia. The morphological events leading to exflagellation, fertilization and ookinete formation, as well as the rate of development of these stages were followed in vitro at 40 °C, 19 °C and 15 °C for 48 h. Macrogametes, microgametes, zygotes and initial stages of ookinete development were observed in all temperatures, but mature ookinetes were seen only at 40 °C. The largest diversity of sporogonic stages of H. columbae were present at 40 °C however, exflagellation, fertilization of macrogametes and development of immature ookinetes were also observed at 15 °C and 19 °C. Morphological and morphometric features of these stages in vitro were described and illustrated. This study demonstrates a requirement of high temperature for the successful development of mature ookinetes of H. columbae, but not gametes. We show that 1) parasites of the H. (Haemoproteus) subgenus exflagellate in vitro at 15-19 °C, as is the case in H. (Parahaemoproteus) spp. and 2) in vitro exflagellation can be used to obtain pure DNA for genomic studies.

In vitro development of Haemoproteus parasites: the efficiency of reproductive cells increase during simultaneous sexual process of different lineages.

Recent in vitro experimental studies reported the complex patterns of haemosporidian (Haemosporida) between-lineage interactions, which prevent mixing of lineages during simultaneous sexual process. Numerous anomalous ookinetes have been observed; these are not involved in sporogony. Massive development of such ookinetes might influence parasite transmission but is insufficiently investigated. The simultaneous sexual process of several lineages is a common phenomenon in vectors due to high prevalence of haemosporidian co-infections in wildlife. It remains unclear if the number of anomalous ookinetes changes during dual-infection sporogony in comparison with the single-infection process. We calculated proportions of the anomalous and normal ookinetes, which developed during single-infection (control) and dual-infection experiments in vitro conditions. Three mitochondrial cytochrome b lineages belonging to three Haemoproteus spp. (Haemosporida, Haemoproteidae) were isolated from naturally infected passerine birds. Sexual process and ookinete development were initiated in vitro by mixing blood containing mature gametocytes of two different parasites; the following experiments were performed: (1) Haemoproteus tartakovskyi (lineage hSISKIN1) × Haemoproteus lanii (lineage hRBS4) and (2) Haemoproteus belopolskyi (hHIICT3) × H. lanii (hRBS4). Genetic difference between lineages was 5.0-5.9%. Normal and anomalous ookinetes developed in all control and dual-infection experiments. The number of anomalous ookinetes markedly decreased, and normal ookinetes increased in all dual-infection experiments in comparison with those in controls, except for H. belopolskyi, in which proportion of the anomalous and normal ookinetes did not change. This study shows that simultaneous sexual process of two genetically distant lineages of haemosporidian parasites might increase the efficiency of reproductive cells, resulting in the development of a greater number of normal ookinetes. The marked increase of the number of normal ookinetes, which is involved in sporogony, indicates the success of sporogony in dual infections. Some haemosporidian lineages might benefit from simultaneous sporogony. Widespread avian Haemoproteus spp. are convenient and laboratory-friendly organisms for in vitro experimental research addressing between-lineage interaction in parasites during the sexual process.

Abortive long-lasting sporogony of two Haemoproteus species (Haemosporida, Haemoproteidae) in the mosquito Ochlerotatus cantans, with perspectives on haemosporidian vector research.

Haemoproteus spp. are cosmopolitan vector-born haemosporidian parasites, some species of which cause diseases in non-adapted birds. Recent polymerase chain reaction (PCR)-based studies have detected mitochondrial cytochrome b gene lineages of these Haemoproteus parasites in blood-sucking mosquitoes and speculated about possible involvement of these insects in transmission of avian haemoproteids. However, development of Haemoproteus lineages has not been documented in mosquitoes. We infected 304 individuals of Ochlerotatus cantans, a widespread Eurasian mosquito, with Haemoproteus tartakovskyi (lineage hSISKIN1) and Haemoproteus balmorali (lineage hROBIN1). Mosquitoes were allowed to take non-infected and infected blood meals and maintained in the laboratory until 17 days post-infection (dpi). They were tested for presence of sporogonic stages by microscopic and PCR-based methods. Microscopic examination revealed partial development of both parasites in the infected insects. Numerous ookinetes were seen in the gut area and adjacent tissues located in the head, thorax and abdomen of mosquitoes between 1 and 5 dpi. Numerous oocysts were seen in the midgut wall between 4 and 15 dpi; they were also present in the head and thorax of infected mosquitoes testifying to the active movement of ookinetes throughout the body. Oocysts degenerated between 11 and 17 dpi. Sporozoites were not seen in oocysts or mosquito salivary glands, indicating abortive sporogonic development at the oocyst stage. In accordance with microscopy data, PCR and sequencing revealed presence of the lineages hSISKIN1 and hROBIN1 in experimental mosquitoes as long as 15 and 17 dpi, respectively, demonstrating relatively long survival of Haemoproteus parasites in the resistant insects without DNA degeneration. The present study shows that PCR-based diagnostics should be carefully used in vector studies of haemosporidians because it detects parasites in insects for several weeks after initial infection, but does not distinguish abortive parasite development. Demonstration of infective sporozoites in insects is essential for definitively demonstrating the insects are vectors.

Molecular characterization of haemosporidian parasites (Haemosporida) in yellow wagtail (Motacillaflava), with description of in vitro ookinetes of Haemoproteus motacillae.

In studies of haemosporidian systematics and taxonomy, a combination of microscopic examination and molecular identification has been recommended. The yellow wagtail (Motacillaflava) is a frequently used species in studies of host-parasite interactions; blood parasites of this bird have been well studied using microscopic examination of blood films, but data on molecular characterization of the parasites are scattered. Here we present the first study linking several haemosporidian cyt b sequences with their morphospecies parasitizing the yellow wagtail, combining light microscopy and nested PCR methods. The lineage hYWT1 of Haemoproteus motacillae was the most prevalent parasite; it was recorded in 38% of the birds. The latter lineage and the lineages hYWT2, hYWT3, hYWT5 and hYWT6 form a well-supported clade on the phylogenetic tree and likely represent intraspecific genetic variation of H. motacillae, with genetic divergence of 0.3 - 1.5 % among these lineages. Microscopic examination of smears prepared during an in vitro experiment, which was designed for observation of developing ookinetes, showed that H. motacillae ookinetes were present 5 hours after exposure of blood to air at 18-20° C. Ookinetes of this parasite belong to a group of large Haemoproteus spp. ookinetes (19.9 µm in length on average). Illustrations of the parasites are given. Two of reported Plasmodium lineages, pCOLLI and pYWT4 are phylogenetically closely related to the widespread host generalist parasites Plasmodium relictun (cyt b lineages pSGS 1, pGRW4 and pGRW11), but it was not possible to establish this morphologically. This study contributes to the molecular identification of avian haemosporidian parasites and provides information on morphology of H. motacillae ookinetes, which is additional information assisting the microscopic identification of this species.

Molecular characterization of Haemoproteus sacharovi (Haemosporida, Haemoproteidae), a common parasite of columbiform birds, with remarks on classification of haemoproteids of doves and pigeons.

Haemoproteus (Haemosporida, Haemoproteidae) is the largest genus of avian haemosporidian parasites, some species of which cause lethal diseases in birds. Subgenera Parahaemoproteus and Haemoproteus are usually accepted in this genus; these parasites are transmitted by biting midges (Ceratopogonidae) and hippoboscid flies (Hippoboscidae), respectively. As of yet, species of Parahaemoproteus have not been reported to infect doves and pigeons (Columbiformes), parasites of these birds have not been reported to be transmitted by biting midges (Ceratopogonidae). Applying microscopy and PCR based methods, we identified mitochondrial cytochrome b (cyt b) sequences of Haemoproteus sacharovi, a wide-spread parasite of doves and pigeons. Phylogenetic relationships of dove haemoproteids, which traditionally have been classified in the subgenus Haemoproteus, showed that H. sacharovi and H. turtur, common parasites of doves, branch in the clade with Parahaemoproteus species, indicating that these haemoproteids may belong to this subgenus and are likely transmitted by biting midges. This study provides barcodes for H. sacharovi, clarifies the taxonomic positions of H. sacharovi and H. turtur, and indicates directions for development of classification of avian haemoproteid species. Our analysis shows that the current subgeneric classification of avian haemoproteids is generally effective, but the position of some species may need to be revised.

Further observations on in vitro hybridization of hemosporidian parasites: patterns of ookinete development in Haemoproteus spp.

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.

Use of flow cytometry to separate Leucocytozoon caulleryi gametocytes from avian blood.

The highly pathogenic avian protozoan Leucocytozoon caulleryi infects host chicken cells, and interference by the host genome results in difficulty in obtaining protozoal DNA for genetic analysis. We used flow cytometry analysis to separate expelled L. caulleryi gametocytes from infected chicken blood and to analyse cell populations and sorting by FACS efficiency. Infected blood cells stained with SYTO-24 showed a specific area on 2-dimensional scattergrams compared to uninfected blood. The specific area was sorted, and approximately 85% of the sorted cells were identified as L. caulleryi gametocytes by microscopic observation. DNA was also extracted from the sorted fraction, and a clear increase in polymerase chain reaction (PCR) amplification of protozoal DNA was observed compared to infected blood without sorting. Host-derived DNA was also detected by PCR; however, its amplification was decreased compared to that in unsorted infected blood. This is the first report of the separation of L. caulleryi gametocytes from infected host blood using flow cytometry. This method may be applied to further genetic analyses such as studies of the dynamics of stage-specific L. caulleryi gene expression.