Rasa Bernotienė

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.

Description of the first cryptic avian malaria parasite, Plasmodium homocircumflexum n. sp., with experimental data on its virulence and development in avian hosts and mosquitoes.

For over 100 years studies on avian haemosporidian parasite species have relied on similarities in their morphology to establish a species concept. Some exceptional cases have also included information about the life cycle and sporogonic development. More than 50 avian Plasmodium spp. have now been described. However, PCR-based studies show a much broader diversity of haemosporidian parasites, indicating the possible existence of a diverse group of cryptic species. In the present study, using both similarity and phylogenetic species definition concepts, we believe that we report the first characterised cryptic speciation case of an avian Plasmodium parasite. We used sequence information on the mitochondrial cytochrome b gene and constructed phylogenies of identified Plasmodium spp. to define their position in the phylogenetic tree. After analysis of blood stages, the morphology of the parasite was shown to be identical to Plasmodium circumflexum. However, the geographic distribution of the new parasite, the phylogenetic information, as well as patterns of development of infection, indicate that this parasite differs from P. circumflexum. Plasmodium homocircumflexum n. sp. was described based on information about genetic differences from described lineages, phylogenetic position and biological characters. This parasite develops parasitemia in experimentally infected birds - the domestic canary Serinus canaria domestica, siskin Carduelis spinus and crossbill Loxia curvirostra. Anaemia caused by high parasitemia, as well as cerebral paralysis caused by exoerythrocytic stages in the brain, are the main reasons for mortality. Exoerythrocytic stages also form in other organs (heart, kidneys, liver, lungs, spleen, intestines and pectoral muscles). DNA amplification was unsuccessful from faecal samples of heavily infected birds. The sporogonic development initiates, but is abortive, at the oocyst stage in two common European mosquito species, Culex pipiens pipiens (forms pipiens and molestus) and Aedes vexans. Vectors of this Plasmodium sp. remain unknown.

Plasmodium spp.: An experimental study on vertebrate host susceptibility to avian malaria.

The interest in experimental studies on avian malaria caused by Plasmodium species has increased recently due to the need of direct information about host-parasite interactions. Numerous important issues (host susceptibility, development of infection, the resistance and tolerance to avian malaria) can be answered using experimental infections. However, specificity of genetically different lineages of malaria parasites and their isolates is largely unknown. This study reviews recent experimental studies and offers additional data about susceptibility of birds to several widespread cytochrome b (cyt b) lineages of Plasmodium species belonging to four subgenera. We exposed two domesticated avian hosts (canaries Serinus canaria and ducklings Anas platyrhynchos) and also 16 species of common wild European birds to malaria infections by intramuscular injection of infected blood and then tested them by microscopic examination and PCR-based methods. Our study confirms former field and experimental observations about low specificity and wide host-range of Plasmodium relictum (lineages SGS1 and GRW11) and P. circumflexum (lineage TURDUS1) belonging to the subgenera Haemamoeba and Giovannolaia, respectively. However, the specificity of different lineages and isolates of the same parasite lineage differed between species of exposed hosts. Several tested Novyella lineages were species specific, with a few cases of successful development in experimentally exposed birds. The majority of reported cases of mortality and high parasitaemia were observed during parasite co-infections. Canaries were susceptible mainly for the species of Haemamoeba and Giovannolaia, but were refractory to the majority of Novyella isolates. Ducklings were susceptible to three malaria infections (SGS1, TURDUS1 and COLL4), but parasitaemia was light (<0.01%) and transient in all exposed birds. This study provides novel information about susceptibility of avian hosts to a wide array of malaria parasite lineages, outlining directions for future experimental research on various aspects of biology and epidemiology of avian malaria.

Plasmodium relictum (lineages pSGS1 and pGRW11): Complete synchronous sporogony in mosquitoes Culex pipiens pipiens

Plasmodium relictum is a widespread invasive agent of avian malaria, responsible for acute, chronic and debilitating diseases in many species of birds. Recent PCR-based studies revealed astonishing genetic diversity of avian malaria parasites (genus Plasmodium), with numerous genetic lineages deposited in GenBank. Many studies addressed distribution and evolutionary relationships of avian Plasmodium lineages, but information about patterns of development of different lineages in mosquito vectors remains insufficient. Here we present data on sporogonic development of 2 widespread mitochondrial cytochrome b lineages (cyt b) of P. relictum (pSGS1 and pGRW11) in mosquito Culex pipiens pipiens. Genetic distance between these lineages is 0.2%; they fall in a well-supported clade in the phylogenetic tree. Three P. relictum strains were isolated from common crossbill (Loxia curvirostra, lineage pSGS1), domestic canary (Serinus canaria domestica, pSGS1) and house sparrow (Passer domesticus, pGRW11). These strains were multiplied in domestic canaries and used as donors of malarial gametocytes to infect C. p. pipiens. Mosquitoes were allowed to take blood meal on infected canaries and then dissected on intervals to study development of sporogonic stages. All 3 strains developed synchronously and completed sporogony in this vector, with infective sporozoites reported in the salivary glands on the day 14 after infection. Ookinetes, oocysts and sporozoites of all strains were indistinguishable morphologically. This study shows that patterns of sporogonic development of the closely related lineages pSGS1 and pGRW11 and different strains of the lineage pSGS1 of P. relictum are similar indicating that phylogenetic trees based on the cyt b gene likely can be used for predicting sporogonic development of genetically similar avian malaria lineages in mosquito vectors.

Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections.

Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections.

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.

Complete Sporogony of Plasmodium relictum (lineages pSGS1 and pGRW11) in Mosquito Culex pipiens pipiens form molestus, with Implications to Avian Malaria Epidemiology

Abstract:  Plasmodium parasites (Plasmodiidae) cause malaria in many species of terrestrial vertebrates and are transmitted mainly by mosquitoes (Culicidae). Avian malaria is often caused by Plasmodium relictum, a cosmopolitan hemosporidian infection. Numerous genetic lineages of P. relictum have been described. However, it remains unclear if these lineages can be transmitted by Culex pipiens pipiens form molestus, which is widespread but has been insufficiently investigated as a possible vector of avian malaria. The aim of this study was to test experimentally if 2 common P. relictum lineages complete sporogony in the experimentally infected insects. The mosquitoes were cultivated under laboratory conditions. Unfed females were allowed to take blood meals on domestic canaries experimentally infected with the lineages pSGS1 and pGRW11 of P. relictum. These lineages are widespread and cause malaria in many species of birds. Infected female flies were examined for sporogonic development of each parasite lineage. Both exposed groups were maintained under the same laboratory conditions. Mosquitoes were dissected at intervals, and the midguts and salivary glands were prepared in order to detect sporogonic stages. Numerous ookinetes, oocysts, and sporozoites of both parasite lineages were observed. Polymerase chain reaction confirmed the presence of corresponding parasite lineages in the experimentally infected insects. Sporogonic stages of both lineages were morphologically similar and developed synchronously in this mosquito; however, the lineage pGRW11 developed a significantly greater number of oocysts than did the lineage pSGS1. This study shows that Culex p. pipiens f. molestus is susceptible to 2 widespread lineages of P. relictum and worth more attention in avian malaria epidemiology. We recommend C. p. pipiens f. molestus for experimental research of avian malaria parasites, principally because it willingly takes blood meals on birds and because it is easy to establish and maintain new colonies of this insect under laboratory conditions using wild-sampled eggs, larvae, or imago.

Description, molecular characterisation, diagnostics and life cycle of Plasmodium elongatum (lineage pERIRUB01), the virulent avian malaria parasite.

Plasmodium elongatum causes severe avian malaria and is distributed worldwide. This parasite is of particular importance due to its ability to develop and cause lethal malaria not only in natural hosts, but also in non-adapted endemic birds such as the brown kiwi and different species of penguins. Information on vectors of this infection is available but is contradictory. PCR-based analysis indicated the possible existence of a cluster of closely related P. elongatum lineages which might differ in their ability to develop in certain mosquitoes and birds. This experimental study provides information about molecular and morphological characterisation of a virulent P. elongatum strain (lineage pERIRUB01) isolated from a naturally infected European robin, Erithacus rubecula. Phylogenetic analysis based on partial cytochrome b gene sequences showed that this parasite lineage is closely related to P. elongatum (lineage pGRW6). Blood stages of both parasite lineages are indistinguishable, indicating that they belong to the same species. Both pathogens develop in experimentally infected canaries, Serinus canaria, causing death of the hosts. In both these lineages, trophozoites and erythrocytic meronts develop in polychromatic erythrocytes and erythroblasts, gametocytes parasitize mature erythrocytes, exoerythrocytic stages develop in cells of the erythrocytic series in bone marrow and are occasionally reported in spleen and liver. Massive infestation of bone marrow cells is the main reason for bird mortality. We report here on syncytium-like remnants of tissue meronts, which slip out of the bone marrow into the peripheral circulation, providing evidence that the syncytia can be a template for PCR amplification. This finding contributes to better understanding positive PCR amplifications in birds when parasitemia is invisible and improved diagnostics of abortive haemosporidian infections. Sporogony of P. elongatum (pERIRUB01) completes the cycle and sporozoites develop in widespread Culex quinquefasciatus and Culex pipiens pipiens form molestus mosquitoes. This experimental study provides information on virulence and within species lineage diversity in a single pathogenic species of haemosporidian parasite.

Haemoproteus minutus and Haemoproteus belopolskyi (Haemoproteidae): Complete sporogony in the biting midge Culicoides impunctatus (Ceratopogonidae), with implications on epidemiology of haemoproteosis

Species of Haemoproteus (Haemoproteidae) are cosmopolitan haemosporidian parasites, some of which cause severe diseases in birds. Numerous recent studies address molecular characterization, distribution and genetic diversity of haemoproteids. However, the information about their vectors is scarce. We investigated sporogonic development of two widespread species of Haemoproteus (Haemoproteus minutus and Haemoproteus belopolskyi) in the experimentally infected biting midge Culicoides impunctatus. Wild-caught flies were allowed to take blood meals on naturally infected common blackbirds Turdus merula and icterine warblers Hippolais icterina harboring mature gametocytes of H. minutus (lineage hTURDUS2) and H. belopolskyi (hHIICT1), respectively. The engorged flies were collected, transported to the laboratory, held at 15-18°C, and dissected daily in order to obtain ookinetes, oocysts and sporozoites. Mature ookinetes of H. minutus developed blisteringly rapidly; they were numerous in the midgut content between 1 and 4 h post exposure. Ookinetes of H. belopolskyi developed slower and were reported 1 day post exposure (dpe). Oocysts of both parasites were seen in the midgut wall 3-4 dpe. Sporozoites of H. minutus and H. belopolskyi were first observed in the salivary glands preparations 7 dpe. The percentage of experimentally infected flies with sporozoites of H. minutus was 82.1% and 91.7% with H. belopolskyi. In accordance with microscopy data, polymerase chain reaction amplification and sequencing confirmed presence of the corresponding parasite lineages in experimentally infected biting midges. Sporogonic stages of these parasites were described and illustrated. This study indicates that C. impunctatus is involved in the transmission of deadly H. minutus, which kills captive parrots in Europe. This biting midge is an important vector of avian haemoproteids and worth more attention in epidemiology research of avian haemoproteosis.

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.