Flowering plant composition shapes pathogen infection intensity and reproduction in bumble bee colonies.

Pathogens pose significant threats to pollinator health and food security. Pollinators can transmit diseases during foraging, but the consequences of plant species composition for infection is unknown. In agroecosystems, flowering strips or hedgerows are often used to augment pollinator habitat. We used canola as a focal crop in tents and manipulated flowering strip composition using plant species we had previously shown to result in higher or lower bee infection in short-term trials. We also manipulated initial colony infection to assess impacts on foraging behavior. Flowering strips using high-infection plant species nearly doubled bumble bee colony infection intensity compared to low-infection plant species, with intermediate infection in canola-only tents. Both infection treatment and flowering strips reduced visits to canola, but we saw no evidence that infection treatment shifted foraging preferences. Although high-infection flowering strips increased colony infection intensity, colony reproduction was improved with any flowering strips compared to canola alone. Effects of flowering strips on colony reproduction were explained by nectar availability, but effects of flowering strips on infection intensity were not. Thus, flowering strips benefited colony reproduction by adding floral resources, but certain plant species also come with a risk of increased pathogen infection intensity.

Culex quinquefasciatus (Diptera: Culicidae) survivorship following the ingestion of bird blood infected with Haemoproteus sp. parasites.

Arthropod vectors are frequently exposed to a diverse assemblage of parasites, but the consequence of these infections on their biology and behavior are poorly understood. We experimentally evaluated whether the ingestion of a common protozoan parasite of avian hosts (Haemoproteus spp.; Haemosporida: Haemoproteidae) impacted the survivorship of Culex quinquefasciatus (Say) (Diptera: Culicidae). Blood was collected from wild northern cardinals (Cardinalis cardinalis) in College Station, Texas, and screened for the presence of Haemoproteus spp. parasites using microscopic and molecular methods. Experimental groups of Cx. quinquefasciatus mosquitoes were offered Haemoproteus-positive cardinal blood through an artificial feeding apparatus, while control groups received Haemoproteus-negative cardinal blood or domestic canary (Serinus canaria domestica) blood. Culex quinquefasciatus mosquitoes exposed to Haemoproteus infected cardinal blood survived significantly fewer days than mosquitoes that ingested Haemoproteus-negative cardinal blood. The survival of mosquitoes fed on positive cardinal blood had a median survival time of 18 days post-exposure and the survival of mosquitoes fed on negative cardinal blood exceeded 50% across the 30 day observation period. Additionally, mosquitoes that fed on canary controls survived significantly fewer days than cardinal negative controls, with canary control mosquitoes having a median survival time of 17 days. This study further supports prior observations that Haemoproteus parasites can be pathogenic to bird-biting mosquitoes, and suggests that Haemoproteus parasites may indirectly suppress the transmission of co-circulating vector-borne pathogens by modulating vector survivorship. Our results also suggest that even in the absence of parasite infection, bloodmeals from different bird species can influence mosquito survivorship.

Blood parasites of bird communities in Sri Lanka and their mosquito vectors.

Avian blood parasites have been shown to have significant health effects on avifauna worldwide. Sri Lanka, a tropical island rich with resident and migratory birds, has not been properly evaluated for avian blood parasites or their vectors. We investigated the presence of avian haemoparasites in Sri Lankan birds and the potential mosquito vectors of those pathogens. Blood samples were collected from local/migratory birds captured by standard mist nets from Anawilundawa bird sanctuary, Hanthana mountain range, and the University of Peradeniya park. Mosquitoes were collected from Halgolla forest reserve and the forest patches in Kurunegala and Gampola areas in addition to the above mist-netting localities. Part of the mitochondrial cytochrome b (cytb) gene was amplified and sequenced to detect the presence of haemoparasites from avian blood samples (86) and mosquito samples (480). Blood parasites of the two genera, i.e., Haemoproteus (4 species; Haemoproteus sp. 1-4) and Plasmodium (5 species; Plasmodium sp. 1-5) were identified from seven bird species (four resident and three migratory). Among these, three bird species (Red-vented bulbul (3/16), Asian Brown flycatcher (1/1), and India pitta (1/1)) were positive for Plasmodium spp., while four (Yellow-browed bulbul (1/4), oriental white-eye (1/4), brown-headed Barbet (1/4), and Indian blue robin (1/1)) were positive for Haemoproteus spp. Two mosquito species were also positive for Plasmodium (3) and Haemoproteus (1) species. Phylogenetic analysis and haplotype networks created using positive sequences of haemoparasites showed that a Plasmodium clade was shared by Cx nigropunctatus mosquitoes and the migratory bird, Indian pitta. The majority (85%) of the Plasmodium and Haemoproteus sequences of this study were not linked to the well-characterized species suggesting the distinct nature of the lineages. Associations between mosquito species and blood parasites of birds suggest the possible vector status of these mosquitoes.

Complete sporogony of the blood parasite Haemoproteus nucleocondensus in common biting midges: why is its transmission interrupted in Europe?

Haemoproteus species (Haemoproteidae) are widespread blood parasites and are transmitted by Culicoides biting midges and Hippoboscidae louse flies. Although these pathogens may cause morbidity or mortality, the vectors and patterns of transmission remain unknown for the great majority of avian haemoproteids. Haemoproteus nucleocondensus has been frequently reported in Europe in great reed warblers Acrocephalus arundinaceus after their arrival from African wintering grounds, but this infection has not been found in juveniles at the breeding sites. The factors that prevent its transmission remain unclear. This study was designed to test whether the sporogony of H. nucleocondensus (lineage hGRW8) can be completed in Culicoides impunctatus, one of the most abundant European biting midge species. Wild-caught females were infected with H. nucleocondensus from great reed warblers. Microscopic examination and PCR-based methods were used to detect sporogonic stages and to confirm species identity. This study showed that H. nucleocondensus completes sporogony in C. impunctatus, suggesting that there are no obstacles to its transmission from the point of view of vector availability and average temperature in Northern Europe. We discuss other ecological factors which should be considered to explain why the transmission of H. nucleocondensus and some other Southern origin haemosporidians are interrupted in North Europe.

Molecular detection of apicomplexan protozoa in Hokkaido brown bears (Ursus arctos yesoensis) and Japanese black bears (Ursus thibetanus japonicus).

Many tick-borne pathogens (TBPs) are present in wildlife. The objective of this study is to reveal the role of wild bears in maintaining TBPs. A total of 49 brown bears (Ursus arctos yesoensis) from Hokkaido, and 18 Japanese black bears (Ursus thibetanus japonicus) from Tochigi, and 66 Japanese black bears from Nagano were examined by two molecular methods, reverse line blot (RLB) hybridization, and nested PCR. A total of 5 TBPs (Hepatozoon ursi, Babesia sp. UR2-like group, Cytauxzoon sp. UR1, Babesia sp. UR1, and Babesia microti) were detected from bear blood DNA samples. B. microti was detected from blood DNA samples of Japanese black bear for the first time, with the prevalence of 6.0% (5/84). Out of detected pathogens, H. ursi, Babesia sp. UR2-like pathogens, and Cytauxzoon sp. UR1 were considered as three of the most prevalent TBPs in bears. The prevalence of H. ursi were significantly higher in Japanese black bear (0% vs 96.4%) while that of Babesia sp. UR2-like group was higher in Hokkaido brown bears (89.8% vs 40.5%). The prevalence of Babesia sp. UR1 were significantly higher in Japanese black bears from Tochigi (44.4%), comparing with those from Nagano (18.2%). The prevalence of the detected TBPs were significantly higher in adult bears, comparing with those in younger bears. The present study suggests that Japanese bear species contribute in the transmission of several TBPs in Japan. The expanding distribution of bears might cause the accidental transmission of TBPs to humans and domestic animals.

Molecular characterization and identification of Blastocystis and its subtypes from raccoon (Procyon lotor) in north of Iran.

Blastocystis is a zoonotic protozoan parasite frequently identified in the intestinal tract of humans and a vast variety of animals, worldwide. Here, we assessed the prevalence of Blastocystis and its subtypes in stool samples of raccoons. Stool samples from 30 raccoons were collected. Total DNA was extracted, and the barcoding region of the small subunit ribosomal rRNA (SSU rRNA) gene was amplified and sequenced. Specific fragment for Blastocystis was successfully amplified in five samples (16.66%). Sequencing analysis revealed ST1, ST2, and ST3 among 1, 2, and 2 Blastocystis-positive samples. Our results documented the presence of Blastocystis subtypes 1-3 in raccoons. Subtype 1 showed higher similarity to the human isolates of Blastocystis. However, it seems that raccoons may emerge as reservoirs for Blastocystis and may be linked to zoonotic transmission of the protist.

Prevalence, molecular epidemiology and zoonotic risk of Entamoeba spp. from experimental macaques in Yunnan Province, southwestern China.

Amebiasis is a worldwide parasitic zoonosis, with symptoms of abdominal discomfort, indigestion, diarrhea, and even death. However, limited information about the prevalence of Entamoeba spp. in experimental nonhuman primates (NHPs) in southwestern China is available. The objective of the current study was to investigate the frequency and species identity of Entamoeba to evaluate potential zoonotic risk factors for Entamoeba spp. infection in experimental NHPs. A total of 505 fecal samples were collected from NHPs (macaques) and analyzed by PCR analysis the small subunit rRNA (SSU rRNA) gene of Entamoeba spp. Forty-seven specimens were positive for Entamoeba spp., and the prevalence of Entamoeba spp. was 9.31% (47/505). Significant differences in the prevalence rates among the three breeds (P = 0.002 < 0.01, df = 2, χ2 = 12.33) and feed types (P = 0.001 < 0.01, df = 1, χ2 = 10.12) were observed. Altogether, four Entamoeba species, including E. dispar (57.44%), E. chattoni (29.78%), E. histolytica (6.38%), and E. coli (6.38%), were identified by DNA sequence analysis. The results suggested a low prevalence but high diversity of Entamoeba species in experimental NHPs in Yunnan Province, southwestern China. Results of this study contribute to the knowledge of the genetic characteristics of Entamoeba spp. in NHPs.

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.

The global biogeography of avian haemosporidian parasites is characterized by local diversification and intercontinental dispersal.

The biogeographic histories of parasites and pathogens are infrequently compared with those of free-living species, including their hosts. Documenting the frequency with which parasites and pathogens disperse across geographic regions contributes to understanding not only their evolution, but also the likelihood that they may become emerging infectious diseases. Haemosporidian parasites of birds (parasite genera Plasmodium, Haemoproteus and Leucocytozoon) are globally distributed, dipteran-vectored parasites. To date, over 2000 avian haemosporidian lineages have been designated by molecular barcoding methods. To achieve their current distributions, some lineages must have dispersed long distances, often over water. Here we quantify such events using the global avian haemosporidian database MalAvi and additional records primarily from the Americas. We scored lineages as belonging to one or more global biogeographic regions based on infection records. Most lineages were restricted to a single region but some were globally distributed. We also used part of the cytochrome b gene to create genus-level parasite phylogenies and scored well-supported nodes as having descendant lineages in regional sympatry or allopatry. Descendant sister lineages of Plasmodium, Haemoproteus and Leucocytozoon were distributed in allopatry in 11, 16 and 15% of investigated nodes, respectively. Although a small but significant fraction of the molecular variance in cytochrome b of all three genera could be explained by biogeographic region, global parasite dispersal likely contributed to the majority of the unexplained variance. Our results suggest that avian haemosporidian parasites have faced few geographic barriers to dispersal over their evolutionary history.

The parasitic dinoflagellate Hematodinium perezi infecting mudflat crabs, Helice tientsinensis, in polyculture system in China.

Since 2012, frequent outbreaks of Hematodinium diseases have significantly impacted sustainable culture of marine crabs Portunus trituberculatus in the coastal areas of Shandong Peninsula. The mechanisms of the Hematodinium parasite epizootics in polyculture pond systems remain to be explored and alternate crustacean hosts are speculated to play important roles in transmission and epizootiology of the disease. To investigate their possible role in transmission, the common wild mudflat crabs Helice tientsinensis were sampled from the waterway connecting to polyculture ponds in Huangdao, Qingdao, China and diagnosed for Hematodinium infection. Hematodinium infection was found in H. tientsinensis collected in the waterway from April-November 2018, with a prevalence of 5.8-31.7%. In addition, 23.1% of H. tientsinensis sampled from the adjacent polyculture pond were infected during the peak of the Hematodinium epizootic in July. Amoeboid trophonts or prespores were observed in the hemolymph of the Hematodinium-infected crabs and histopathological changes were observed in major organs (e.g. hepatopancreas, heart, gill and muscle). The ITS1 rRNA of Hematodinium sp. infecting H. tientsinensis shared 99-100% similarity to isolates infecting P. trituberculatus and Penaeus monodon in local polyculture ponds, and are included in a monophylogenetic clade, Hematodinium perezi genotype II, in the phylogenetic tree. The results further showed that this generalist parasite was infecting various types of marine crustaceans in the coastal waters of China, and that mudflat crabs H. tientsinensis may serve as an important alternate host during epizootics of Hematodinium parasites in the polyculture system.

Two Tales of Cytauxzoon felis Infections in Domestic Cats.

Cytauxzoonosis is an emerging infectious disease that affects wild felids as well as the domestic cat; it is caused by the apicomplexan protozoan parasites belonging to the genus Cytauxzoon. Cytauxzoonfelis is the species of major concern, whose transmission occurs via the bite of an infected tick. Cytauxzoonosis of the domestic cat has historically been considered uniformly fatal, with a short course of illness, and most domestic cats die within 9 to 15 days postinfection. However, increasing evidence of domestic cats surviving C. felis infection suggests the existence of different strains with various levels of pathogenicity. Although wild felids are considered natural reservoirs for this parasite, a number of studies suggest that domestic cats that have survived nonlethal infections may serve as an additional reservoir. The current article comprehensively reviews the parasite and its life cycle, geographic distribution, genetic variability, and pathogenesis, as well as host immunology and the diagnosis, treatment, and prevention of infection in the domestic cat. This information should provide a basis for better understanding the parasite as well as the pathogenesis of the disease.

Polychromophilus spp. (Haemosporida) in Malagasy bats: host specificity and insights on invertebrate vectors.

BACKGROUND: Bats are home to diverse haemosporidian parasites namely Plasmodium and Plasmodium-related. While information is available at a worldwide level, haemosporidian infection in bats from Madagascar is still scarce and recent changes in the taxonomy of the island's bat fauna, particularly the description of several new species, require a reassessment of previously described patterns, including blood parasite ecology and vectorial transmission. METHODS: A sample representing seven of the nine known bat families and 31 of the 46 currently recognized taxa from Madagascar and collected in the western and central portions of the island were screened by PCR for the presence of Polychromophilus. In addition, Nycteribiidae flies parasitizing Miniopteridae and Vespertilionidae were screened for parasites with the aim to better understand aspects of vector transmission. Phylogenetic reconstruction using the mitochondrial cytochrome b encoding gene was used in a Bayesian analysis to examine the relationship between Polychromophilus recovered from Malagasy bats and those identified elsewhere. RESULTS: Polychromophilus infection was restricted to Miniopterus spp. (Miniopteridae), Myotis goudoti (Vespertilionidae), and Paratriaenops furculus (Rhinonycteridae), with an overall infection rate of 13.5%. Polychromophilus melanipherus was found infecting Miniopterus spp. and P. furculus, whereas Polychromophilus murinus was only recovered from M. goudoti. These two protozoan parasites species were also detected in bat flies species known to parasitize Miniopterus spp. and M. goudoti, respectively. Generalized linear model analyses were conducted to elucidate the effect of species and sex on haemoparasites infection in Miniopterus spp., which revealed that males have higher risk of infection than females and prevalence differed according to the considered Miniopterus host. Molecular screening of nycteribiid flies revealed three positive species for Polychromophilus spp., including Penicillidia sp. (cf. fulvida), Penicillidia leptothrinax, and Nycteribia stylidiopsis. These three fly species are known to parasitize Miniopterus spp. and M. goudoti and should be considered as potential vectors of Polychromophilus spp. CONCLUSION: Phylogenetic analyses demonstrated the existence of at least four distinct clades within the genus Polychromophilus, two of which were documented in the present study. The screening of nycteribiid flies overlaid on the highly diversified genus Miniopterus, provides considerable insight into parasite transmission, with bat infection being associated with their roosting behaviour and the occurrence of specific arthropod vectors.

Haemoproteus tartakovskyi (Haemoproteidae): Complete sporogony in Culicoides nubeculosus (Ceratopogonidae), with implications for avian haemoproteid experimental research.

Numerous recent studies have addressed the molecular characterization, distribution and genetic diversity of Haemoproteus spp. (Haemoproteidae). Some species of these blood parasites cause severe disease in birds, and heavy infections are often lethal in biting midges (Ceratopogonidae) and other blood-sucking insects. However, information about the vectors of haemoproteids is scarce. This presents an obstacle for better understanding the mechanisms of host-parasite interactions and the epidemiology of haemoproteosis. Here we investigated the sporogonic development of Haemoproteus tartakovskyi, a widespread bird parasite, in experimentally infected biting midges, Culicoides nubeculosus. These biting midges are widespread in the Europe. The insects were cultivated under laboratory conditions. Unfed females were allowed to take blood meals on wild caught siskins Carduelis spinus naturally infected with H. tartakovskyi (lineage hSISKIN1). Engorged females were maintained at 22-23 °C, dissected at intervals, and examined for sporogonic stages. Mature ookinetes of H. tartakovskyi were seen in the midgut content between 6 and 48 h post infection, oocysts were observed in the midgut wall 3-4 days post infection (dpi). Sporozoites were first reported in the salivary gland preparations 7 dpi. In accordance with microscopy data, polymerase chain reaction amplification and sequencing confirmed presence of the corresponding parasite lineage in experimentally infected biting midges. This study indicates that C. nubeculosus willingly takes blood meals on birds and is a vector of H. tartakovskyi. These biting midges are readily amenable to cultivation under laboratory conditions. Culicoides nubeculosus transmits Haemoproteus parasites infecting parrots, owls and siskins, birds belonging to different families and orders. Thus, this vector provides a convenient model for experimental research with avian haemoproteids.

Haemosporidian infections in the Tengmalm's Owl (Aegolius funereus) and potential insect vectors of their transmission.

Sedentary bird species are suitable model hosts for identifying potential vectors of avian blood parasites. We studied haemosporidian infections in the Tengmalm's Owl (Aegolius funereus) in the Ore Mountains of the Czech Republic using molecular detection methods. Sex of owl nestlings was scored using molecular sexing based on fragment analysis of PCR-amplified CHD1 introns. Observed infection prevalences in nestlings and adult owls were 51 and 86 %, respectively. Five parasite lineages were detected. Most of the infections comprised the Leucocytozoon AEFUN02 and STOCC06 lineages that probably refer to distinct Leucocytozoon species. Other lineages were detected only sporadically. Mixed infections were found in 49 % of samples. The main factor affecting the probability of infection was host age. No effect of individual sex on infection probability was evidenced. The youngest infected nestling was 12 days old. High parasite prevalence in the Tengmalm's Owl nestlings suggests that insect vectors must enter nest boxes to transmit parasites before fledging. Hence, we placed sticky insect traps into modified nest boxes, collected potential insect vectors, and examined them for the presence of haemosporidian parasites using molecular detection. We trapped 201 insects which were determined as biting midges from the Culicoides genus and two black fly species, Simulium (Nevermannia) vernum and Simulium (Eusimulium) angustipes. Six haemosporidian lineages were detected in the potential insect vectors, among which the Leucocytozoon lineage BT2 was common to the Tengmalm's Owl and the trapped insects. However, we have not detected the most frequently encountered Tengmalm's Owl Leucocytozoon lineages AEFUN02 and STOCC06 in insects.

Quantification of parasite shedding and horizontal transmission parameters in Histomonas meleagridis-infected turkeys determined by real-time quantitative PCR.

To gain more insight into the within flock transmission of Histomonas meleagridis, the shedding of parasites was quantified by a newly developed real-time quantitative (q)PCR and the basic reproduction number (R0) and the mean number of secondary infections per infectious bird per day in a susceptible population (ß) of H. meleagridis in the absence of heterakis were assessed. Forty turkeys were divided into two groups of 10 and 30 birds at 14 days of age. Birds of the first group were inoculated with 200,000 histomonads each, the second group served as a susceptible contact group. Cloacal swabs were taken at -1, 1, 4, 7, 9, 11, 14, 18 and 21 days post inoculation (p.i.) to assess the shedding of the parasite by the qPCR (detection limit 330 histomonads/ml droppings). The experiment ended at 28 days p.i. Mortality was 100% in the inoculated birds and started at day 12 p.i., while in the contacts, it was 83% and started at 16 days p.i. Shedding started 1 day after the inoculation in both groups. The mean shedding levels (and 95% CI) expressed as parasite equivalents per gram cloacal content on a log10 scale in the inoculated, contact birds that died and contact birds alive were 2.0 (1.6-2.4), 1.6 (1.4-1.9) and 1.2 (0.5-2.0), respectively. Birds that died shed histomonas more often and were infectious for 13.4 days; in contrast, those that recovered were infectious for 5.7 days. R0 was estimated to be 8.4 and ß 0.70. Simulations made with the parameters obtained were in agreement with the experimental results, confirming their validity.

Host preferences of ornithophilic biting midges of the genus Culicoides in the Eastern Balkans.

Many biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) are competent vectors of a diverse number of pathogens. The identification of their feeding behaviour and of vector-host associations is essential for understanding their transmission capacity. By applying two different nested polymerase chain reaction (PCR) assays, of which one targeted the avian cyt b gene and the other targeted the COI gene of a wide range of vertebrates, we identified the blood hosts of six biting midge species including Culicoides circumscriptus, Culicoides festivipennis, Culicoides punctatus, Culicoides pictipennis, Culicoides alazanicus and Culicoides cf. griseidorsum, the latter two of which are reported in Bulgaria for the first time. Bird DNA was found in 50.6% of 95 investigated bloodmeals, whereas mammalian DNA was identified in 13.7%. Two Culicoides species were found to feed on both birds and mammals. There was remarkable diversity in the range of avian hosts: 23 species from four orders were identified in the abdomens of four Culicoides species. The most common bird species identified was the magpie, Pica pica (n = 7), which was registered in all four ornithophilic biting midge species. Six bloodmeals from the great tit, Parus major, were recorded only in C. alazanicus. None of the studied species of Culicoides appeared to be restricted to a single avian host.

Trypanosomes and haemosporidia in the buzzard (Buteo buteo) and sparrowhawk (Accipiter nisus): factors affecting the prevalence of parasites.

The prevalences of heteroxenous parasites are influenced by the interplay of three main actors: hosts, vectors, and the parasites themselves. We studied blood protists in the nesting populations of raptors in two different areas of the Czech Republic. Altogether, 788 nestlings and 258 adult Eurasian sparrowhawks (Accipiter nisus) and 321 nestlings and 86 adult common buzzards (Buteo buteo) were screened for parasites by the microscopic examination of blood smears and by cultivation. We examined the role of shared vectors and parasite phylogenetic relationships on the occurrence of parasites. In different years and hosts, trypanosome prevalence ranged between 1.9 and 87.2 %, that of Leucocytozoon between 1.9 and 100 %, and Haemoproteus between 0 and 72.7 %. Coinfections with Leucocytozoon and Trypanosoma, phylogenetically distant parasites but both transmitted by blackflies (Simuliidae), were more frequent than coinfections with Leucocytozoon and Haemoproteus, phylogenetically closely related parasites transmitted by different vectors (blackflies and biting midges (Ceratopogonidae), respectively). For example, 16.6 % buzzard nestlings were coinfected with Trypanosoma and Leucocytozoon, while only 4.8 % with Leucocytozoon and Haemoproteus and 0.3 % with Trypanosoma and Haemoproteus. Nestlings in the same nest tended to have the same infection status. Furthermore, prevalence increased with the age of nestlings and with Julian date, while brood size had only a weak negative/positive effect on prevalence at the individual/brood level. Prevalences in a particular avian host species also varied between study sites and years. All these factors should thus be considered while comparing prevalences from different studies, the impact of vectors being the most important. We conclude that phylogenetically unrelated parasites that share the same vectors tend to have similar distributions within the host populations of two different raptor species.

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.

Description of Leucocytozoon quynzae sp. nov. (Haemosporida, Leucocytozoidae) from hummingbirds, with remarks on distribution and possible vectors of leucocytozoids in South America.

We describe Leucocytozoon quynzae sp. nov. (Haemosporida, Leucocytozoidae), which is the first Leucocytozoon parasite identified to species level in hummingbirds. It was found in the Amethyst-throated Sunangel (Heliangelus amethysticollis, Trochilidae, Apodiformes) captured in the Palacio Forest, which belongs to the damping zone of Chingaza National Natural Park, Cundinamarca, Colombia, at 2,900 m above sea level where the transmission occurs; the new species were found both in the high Andean forest and Paramo ecosystem. This parasite is described based on the morphology of its blood stages, a fragment of the mitochondrial cytochrome b gene, and the complete mitochondrial genome. Illustrations of blood stages of the new species are given, and the phylogenetic analysis places this lineage in a well-supported clade with other lineages of unidentified to species level leucocytozoids reported in the Trochilidae birds elsewhere. The new species possess gametocytes in roundish host cells; it can be readily distinguished from other similar leucocytozoids, primarily due to (1) a comma-like shape of the host cell nucleus, which extended one half or less of the circumference of the gametocyte and (2) a large number of prominent volutin granules in the cytoplasm. Identical mitochondrial cytochrome b sequence of Leucocytozoon quynzae was found in different hummingbird species at the type locality and also was reported in one passerine bird at the highlands of Peru. Leucocytozoon quynzae is the first leucocytozoid parasite described from South American birds; its transmission occurs both at low temperatures and high elevations. We discuss some patterns of distribution of avian leucocytozoids in South America and the role of Gigantodax spp. (Diptera, Simuliidae) as potential vectors of Leucocytozoon parasites in the Andean Region.

Degree of associations among vectors of the genus Culicoides (Diptera: Ceratopogonidae) and host bird species with respect to haemosporidian parasites in NE Bulgaria.

The occurrence of haemosporidians in biting midges of the genus Culicoides is examined in North-East Bulgaria in order to reveal their potential role for parasite transmission. A PCR-based technique amplifying part of the mitochondrial cytochrome b gene of the parasite is applied on naturally infected biting midges. Totally, 640 parous individuals of four species and 95 blood-fed individuals of six species of Culicoides are examined for the presence of DNA of haemosporidians. Haemosporidian genetic lineages are identified in individuals of three insect species: Culicoides alazanicus (12 lineages, nine lineages of Haemoproteus and three lineages of Plasmodium), Culicoides festivipennis and Culicoides circumscriptus (with two and one lineages of Haemoproteus, respectively). Two genetic lineages of Haemoproteus are recorded in more than one vector species. These results demonstrate variations in the specificity of Haemoproteus genetic lineages to their potential vectors, since some lineages are recorded in a single vector species and others occur in two or more vector species.