Diversity of Culicoides in the middle belt of Ghana with Implications on the transmission of Mansonella perstans; a molecular approach.

BACKGROUND: Culicoides, also known as biting midges, carry pathogens which include Mansonella perstans. Mansonella perstans is a nematode parasite implicated in a number of disease outcomes. Even though a high prevalence of about 75% M. perstans infection has been recorded in some communities in the middle belt of Ghana, and a wide diversity of Culicoides species has been identified, the exact Culicoides species transmitting M. perstans in Ghana has not yet been deciphered. This study therefore aimed at assessing the species diversity of Culicoides and their role in the transmission of M. perstans in the middle belt of Ghana. METHODS: Culicoides species were sampled from 11 communities in the Asante-Akim North and Sene West districts in the middle belt of Ghana. Centre for Disease Control (CDC) UV light traps, as well as human bait (i.e. human landing catch and engorged catch) methods were used to assess the species abundance and diversity of Culicoides in the study communities in the wet and dry season. A colorimetric Loop-Mediated Isothermal Amplification (LAMP) assay was performed to assess the vector competence of the various Culicoides species. RESULTS: A total of 4810 Culicoides from 6 species were sampled. These included Culicoides inornatipennis, C. milnei, C. schultzei, C. grahamii, C. neavei, and C. imicola. Culicoides imicola was the most abundant species (56%) followed by C. grahamii (16%). Light traps sampled the most diverse species (6 species). Human landing catch and engorged catch methods identified three anthropophilic species, C. grahamii, C. milnei, and C. inornatipennis, with C. grahamii being the most anthropophilic with a peak biting time between the hours of 5 p.m. to 6 p.m. Generally, there was relatively higher species abundance in the wet than dry season. LAMP assay identified C. grahamii as the potential vector for M. perstans transmission in the middle belt of Ghana. CONCLUSIONS: For the first time, we have demonstrated that C. grahamii is the potential competent vector for M. perstans transmission in the middle belt of Ghana. It is more abundant in the rainy season and has a peak biting time between the hours of 5 and 6 p.m.

Visceral Haemoproteus minutus Infection in a Major Mitchell's Cockatoo (Lophochroa leadbeateri).

A 1-year-old major Mitchell's cockatoo (Lophochroa leadbeateri) was presented for evaluation of weakness, diarrhea with undigested seeds in the droppings, and weight loss. Leukocytosis with severe heterophilia, monocytosis, and lymphocytosis was noted on the complete blood count. Altered plasma biochemical parameters included a slight increase in creatine kinase and mild hypoproteinemia. Two blood smears before and after 2 days of treatment revealed mild polychromasia and anisocytosis but no blood parasites. Radiographic and computed tomographic imaging of the cockatoo were helpful in identifying airsacculitis, pneumonia, and gastrointestinal motility disorders. The patient died 5 days after treatment for the presenting clinical problems. On the gross postmortem examination, dark red foci in the ventricular muscle layers and 1-3-mm white foci in the myocardium, opaque air sacs, and dark lungs were identified. Histopathologic examination of submitted tissue samples found severe granulomatous ventriculitis and myocarditis with intralesional Haemoproteus species megalomeronts. Qualitative polymerase chain reaction testing for the cytochrome b (cyt b) gene performed on pooled heart, liver, kidney, and intestinal tissues identified 99.5% homology to Haemoproteus minutus. This case report demonstrates the expansion of the geographic range of H minutus to France and potentially to Belgium, which may compromise breeding and conservation of Australian parrots living outdoors. Challenging diagnosis, rapid disease progression, and the absence of validated treatment protocols for psittacine patients suggest that the use of preventive measures to reduce the presence of insect vectors such as hippoboscid flies and biting midges (Culicoides) should be considered. Haemoproteus minutus should be considered and potentially screened by polymerase chain reaction testing on blood samples, especially in the case of highly susceptible avian species (eg, Australian parrots in Europe) that present with sudden weakness, heterophilic leukocytosis, and monocytosis associated with mild anemia.

Exo-erythrocytic development of two Haemoproteus species (Haemosporida, Haemoproteidae), with description of Haemoproteus dumbbellus, a new blood parasite of bunting birds (Emberizidae).

Avian haemosporidians are widespread parasites categorized into four families of the order Haemosporida (Apicomplexa). Species of the subgenus Parahaemoproteus (genus Haemoproteus) belong to the Haemoproteidae and are transmitted by Culicoides biting midges. Reports of death due to tissue damage during haemoproteosis in non-adapted birds have raised concerns about these pathogens, especially as their exo-erythrocytic development is known for only a few Haemoproteus spp. More research is needed to better understand the patterns of the parasites' development in tissues and their impact on avian hosts. Yellowhammers Emberiza citrinella (Emberizidae) and common house martins Delichon urbicum (Hirundinidae) were screened for Haemoproteus parasites by microscopic examination of blood films and PCR-based testing. Individuals with single infection were selected for histological investigations. H & E-stained sections were screened for detection and characterization of the exo-erythrocytic stages, while chromogenic in situ hybridization (CISH) and phylogenetic analysis were performed to confirm the Haemoproteus origin and their phylogenetic relationships. Haemoproteus dumbbellus n. sp. was discovered in Emberiza citrinella single-infected with the lineage hEMCIR01. Meronts of H. dumbbellus n. sp. developed in various organs of five of six tested individuals, a pattern which was reported in other Haemoproteus species clustering in the same clade, suggesting this could be a phylogenetic trait. By contrast, in Delichon urbicum infected with the Haemoproteus lineage hDELURB2, which was linked to the more distantly related parasite Haemoproteus hirundinis, only megalomeronts were found in the pectoral muscles of two of six infected individuals. All exo-erythrocytic stages were confirmed to be Haemoproteus parasites by CISH using a Haemoproteus genus-specific probe. While the development of meronts seems to be typical for species of the clade containing H. dumbbellus, further investigations and data from more species are needed to explore whether a phylogenetic pattern occurs in meront or megalomeront formation.

Effects of Artemisia annua on experimentally induced leucocytozoonosis in chickens.

The biting midge Culicoides arakawae is the vector for the parasite Leucocytozoon caulleryi. Birds infected with L. caulleryi develop leucocytozoonosis. Given the food safety concern regarding drug residue in eggs, discovering a natural alternative to antibiotics is a worthy of exploration. Thus, we investigated the effects of the antimalarial herb Artemisia annua on experimentally induced leucocytozoonosis in chickens. We reared C. arakawae in the laboratory. Eggs were cultured, developing into larvae, pupae, and imagoes. Female midges sucked the blood of sick chickens and then were ground into a solution injected into healthy chickens. The control group was given empty capsules daily, whereas the 2 experimental groups were given 40 mg/kg sulfadimethoxine or 0.5 g of A. annua powder. Leucocytozoon gametocytes were detected in chicken blood through Giemsa staining. PCR detected the cytochrome b gene of L. caulleryi in the infected chickens. No significant among-group differences in body weight gain were observed before d 14 postinoculation (P > 0.05). Body weight gain in the control group was significantly lower from day 14 to 28 postinoculation (P < 0.05). After day 14, rectal temperature in the experimental groups decreased significantly compared with that in the control group. Lower rates of pale comb and green feces were observed in the animals receiving treatment from day 0. The experimental groups had a higher recovery rate and recovered earlier than did the control group. By day 31, all the animals had recovered. PCR detected L. caulleryi in the infected chickens with high sensitivity and accuracy. The animals receiving A. annua exhibited increased weight gain and reduced parasite concentrations in the blood. This in turn reduced mortality and the occurrence of pale comb and green feces. The findings are informative for research on leucocytozoonosis.

Culicoides Latreille (Diptera: Ceratopogonidae) as potential vectors for Leishmania martiniquensis and Trypanosoma sp. in northern Thailand.

Biting midges of genus Culicoides (Diptera: Ceratopogonidae) are the vectors of several pathogenic arboviruses and parasites of humans and animals. Several reports have suggested that biting midges might be a potential vector of Leishmania parasites. In this study, we screened for Leishmania and Trypanosoma DNA in biting midges collected from near the home of a leishmaniasis patient in Lamphun province, northern Thailand by using UV-CDC light traps. The identification of biting midge species was based on morphological characters and confirmed using the Cytochrome C oxidase subunit I (COI) gene. The detection of Leishmania and Trypanosoma DNA was performed by amplifying the internal transcribed spacer 1 (ITS1) and small subunit ribosomal RNA (SSU rRNA) genes, respectively. All the amplified PCR amplicons were cloned and sequenced. The collected 223 biting midges belonged to seven species (Culicoides mahasarakhamense, C. guttifer, C. innoxius, C. sumatrae, C. huffi, C. oxystoma, and C. palpifer). The dominant species found in this study was C. mahasarakhamense (47.53%). Leishmania martiniquensis DNA was detected in three samples of 106 specimens of C. mahasarakhamense tested indicating a field infection rate of 2.83%, which is comparable to reported rates in local phlebotomines. Moreover, we also detected Trypanosoma sp. DNA in one sample of C. huffi. To our knowledge, this is the first molecular detection of L. martiniquensis in C. mahasarakhamense as well as the first detection of avian Trypanosoma in C. huffi. Blood meal analysis of engorged specimens of C. mahasarakhamense, C. guttifer, and C. huffi revealed that all specimens had fed on avian, however, further studies of the host ranges of Culicoides are needed to gain a better insight of potential vectors of emerging leishmaniasis. Clarification of the vectors of these parasites is also important to provide tools to establish effective disease prevention and control programs in Thailand.

Experimental transmission of Leishmania (Mundinia) parasites by biting midges (Diptera: Ceratopogonidae).

Leishmania parasites, causative agents of leishmaniasis, are currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia. The recently established subgenus Mundinia has a wide geographical distribution and contains five species, three of which have the potential to infect and cause disease in humans. While the other Leishmania subgenera are transmitted exclusively by phlebotomine sand flies (Diptera: Psychodidae), natural vectors of Mundinia remain uncertain. This study investigates the potential of sand flies and biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to transmit Leishmania parasites of the subgenus Mundinia. Sand flies (Phlebotomus argentipes, P. duboscqi and Lutzomyia migonei) and Culicoides biting midges (Culicoides sonorensis) were exposed to five Mundinia species through a chicken skin membrane and dissected at specific time intervals post bloodmeal. Potentially infected insects were also allowed to feed on ear pinnae of anaesthetized BALB/c mice and the presence of Leishmania DNA was subsequently confirmed in the mice using polymerase chain reaction analyses. In C. sonorensis, all Mundinia species tested were able to establish infection at a high rate, successfully colonize the stomodeal valve and produce a higher proportion of metacyclic forms than in sand flies. Subsequently, three parasite species, L. martiniquensis, L. orientalis and L. sp. from Ghana, were transmitted to the host mouse ear by C. sonorensis bite. In contrast, transmission experiments entirely failed with P. argentipes, although colonisation of the stomodeal valve was observed for L. orientalis and L. martiniquensis and metacyclic forms of L. orientalis were recorded. This laboratory-based transmission of Mundinia species highlights that Culicoides are potential vectors of members of this ancestral subgenus of Leishmania and we suggest further studies in endemic areas to confirm their role in the lifecycles of neglected pathogens.

Vector incrimination and transmission of avian malaria at an aquarium in Japan: mismatch in parasite composition between mosquitoes and penguins.

BACKGROUND: Captive populations of penguins outside of their natural distributions are often maintained in outdoor facilities, such as zoos and aquariums. Consequently, such penguins in captivity are constantly exposed to mosquito vectors and risk of avian malarial infection during their active period from spring to autumn, which can be lethal to these naïve birds. Previous studies have investigated parasite prevalence in mosquitoes or penguins, but simultaneous investigations, which would be crucial to monitor the transmission dynamics and cycle within a facility, have not been done. To identify dominant lineages and trends, multiple-year surveys are recommended. METHODS: Avian malaria parasites (Plasmodium spp.) and related haemosporidia were tested in penguins and mosquitoes at an aquarium in Japan through multiple years from 2011 to 2018. Prevalence and dynamics were confirmed, and molecular analyses targeting the protozoal cytb gene were used to reveal the transmission cycle. Blood meals of mosquitoes were also identified using molecular methods. RESULTS: Parasite detection in penguins tended to fluctuate within an individual. Two Plasmodium lineages were consistently detected in mosquitoes that had fed on penguins and wild birds observed around the aquarium. Plasmodium lineage CXPIP09 was detected from both mosquitoes and penguins, suggesting active transmission at this facility. However, Plasmodium cathemerium PADOM02 was only detected in mosquitoes, which may be due to host, vector or parasite-related factors, or detection methods and their limits. Additionally, Haemoproteus larae SPMAG12 was detected from penguins, suggesting active transmission via biting midges. CONCLUSIONS: The mismatch in parasite composition between penguins and mosquitoes shows that multiple aspects such as captive birds, wild birds and vector insects should be monitored in order to better understand and control avian malarial infection within ex-situ conservation facilities. Furthermore, morphological analyses would be needed to confirm competency and infection dynamics of avian malaria parasites.

Next-generation tools to control biting midge populations and reduce pathogen transmission.

Biting midges of the genus Culicoides transmit disease-causing agents resulting in a significant economic impact on livestock industries in many parts of the world. Localized control efforts, such as removal of larval habitat or pesticide application, can be logistically difficult, expensive and ineffective if not instituted and maintained properly. With these limitations, a population-level approach to the management of Culicoides midges should be investigated as a means to replace or supplement existing control strategies. Next-generation control methods such as Wolbachia- and genetic-based population suppression and replacement are being investigated in several vector species. Here we assess the feasibility and applicability of these approaches for use against biting midges. We also discuss the technical and logistical hurdles needing to be addressed for each method to be successful, as well as emphasize the importance of addressing community engagement and involving stakeholders in the investigation and development of these approaches.

Culicoides biting midges involved in transmission of haemoproteids.

BACKGROUND: Culicoides biting midges (Diptera, Ceratopogonidae) are known vectors of avian Haemoproteus parasites. These parasites cause diseases, pathology and even mortality in birds. The diversity of biting midges in Europe is great, but only four Culicoides species are known to be vectors of avian Haemoproteus parasites. In general, our knowledge about the role of the particular Culicoides species in the transmission of Haemoproteus parasites remains insufficient. Information gaps hinder a better understanding of parasite biology and the epizootiology of parasite-caused diseases. The aim of this study was to determine new Culicoides species involved in the transmission of Haemoproteus parasites. METHODS: Biting midges were collected using a UV trap as well as sticky traps installed in bird nest boxes. Individual parous females were diagnosed for the presence of haemoproteids using both PCR-based and microscopic methods. RESULTS: We collected and dissected 232 parous Culicoides females from 9 species using a UV trap and 293 females from 11 species from bird nest boxes. Culicoides obsoletus was the dominant species collected using a UV trap, and Culicoides kibunensis dominated among midges collected in nest boxes. PCR-based screening showed that 5.2% of parous biting midges collected using a UV trap and 4.4% of midges collected from nest boxes were infected with avian haemosporidian parasites. Haemoproteid DNA was detected in C. kibunensis, Culicoides pictipennis, Culicoides punctatus, Culicoides segnis and Culicoides impunctatus females. The sporozoites of Haemoproteus minutus (genetic lineages hTURDUS2 and hTUPHI01) were detected in the salivary glands of two C. kibunensis females using microscopy, and this finding was confirmed by PCR. CONCLUSIONS: Culicoides kibunensis was detected as a new natural vector of Haemoproteus minutus (hTURDUS2 and hTUPHI01). Haemoproteid DNA was detected in females from five Culicoides species. This study contributes to the epizootiology of avian Haemoproteus infections by specifying Culicoides species as vectors and species that are likely to be responsible for the transmission of haemoproteids in Europe.

A fatal case of a captive snowy owl (Bubo scandiacus) with Haemoproteus infection in Japan.

Parasites of the genus Haemoproteus are vector-borne avian haemosporidia commonly found in bird species of the world. Haemoproteus infections are typically considered relatively benign in birds. However, some Haemoproteus species cause severe disease and mortality, especially for captive birds removed from their original habitat. In September 2018, a captive 15-year-old snowy owl (Bubo scandiacus), kept in a zoological garden of Japan, died subacutely after presenting leg dysfunction. This case showed significantly low PCV and elevated AST, ALT, CK, and LDH values. Many megalomeronts with prominent morphological characteristics of Haemoproteus were observed in the left leg muscles. Those megalomeronts exhibited multilocular structures and were internally filled with merozoites. A new lineage of Haemoproteus was detected by subsequent PCR for the cytochrome b (cytb) gene of avian haemosporidia from DNA extracted from several organ tissues. The detected lineage was classified in the subgenus Parahaemoproteus and was similar to those from the wild birds inhabiting the region including the study area, suggesting that this snowy owl likely acquired its infection from wild birds. This is the first report of a fatal case of a captive bird with a locally transmitted Haemoproteus infection in Japan. We considered the pathogenicity of this infection in conjunction with the clinical course and hematology results. We surmise that snowy owls may be particularly susceptible to infection with Haemoproteus parasites, and warming northern temperatures may exacerbate the overall health of these and other high latitude birds. Further research into the prevalence of Haemoproteus in wild birds near zoological gardens and potential biting midge vectors is necessary for the ex situ conservation of introduced birds.

Development of Trypanosoma everetti in Culicoides biting midges.

Trypanosoma species (Trypanosomatida, Kinetoplastea) are almost exclusively heteroxenous flagellated parasites, which have been extensively studied as the causative agents of severe trypanosomiasis in humans and domestic animals. However, the biology of avian trypanosomes remains insufficiently known, particularly in wildlife, despite information that some species might be pathogenic and affect the fitness of intensively infected individuals. Avian trypanosomes are cosmopolitans. Due to regular bird seasonal migrations, this host-parasite system might provide new insight for better understanding mechanisms of transcontinental dispersal of pathogens, their ecological plasticity, specificity and speciation. Trypanosoma everetti parasitizes numerous bird species globally, but data on its biology are scarce and its vectors remain unknown. This study aimed to test experimentally whether widespread Culicoides (Diptera: Ceratopogonidae) biting midges are susceptible to infection with this parasite. Two common house martins Delichon urbicum and two sedge warblers Acrocephalus schoenobaenus naturally infected with T. everetti were caught in the wild after arrival from African wintering grounds. Laboratory reared Culicoides nubeculosus and wild-caught Culicoides impunctatus biting midges were exposed by allowing them to take infected blood meals. The experimentally infected and control insects were maintained in the laboratory and dissected at intervals to follow the development of the parasite. Infections were determined using microscopic examination and PCR-based testing. Four closely related haplotypes of T. everetti were found, and each was present in different individual parasite-donor birds. These parasites readily developed and produced metacyclic trypomastigotes in C. nubeculosus and C. impunctatus biting midges. Molecular characterisation of T. everetti was developed. According to Bayesian phylogenetic analysis using a DNA fragment encoding 18S rRNA, the five species of small avian trypanosomes were closely related. Wild caught Culicoides biting midges were also collected and screened for the presence of natural infections. In all, 6.8% of wild-caught biting midges belonging to five Culicoides species were PCR-positive for kinetoplastids, including Trypanosoma species. Culicoides biting midges are readily susceptible and likely naturally transmit avian trypanosomes and thus, should be targeted in epidemiology research of avian trypanosomiasis.

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.

Survey of infection and determination of the transmission vector of Onchocerca fasciata in camels (Camelus bactrianus) in Inner Mongolia, China.

Onchocerciasis in camels is caused by adult Onchocerca spp. and results in great economic losses to the camel industry. However, only a few studies on Onchocerca have been conducted, especially regarding the intermediate host and vector(s). In the present study, 192 camels were examined from December and January during 2013 and 2016, and the filarial larvae suspected to be Onchocerca spp. were further identified. Furthermore, aquatic dipteran insects in the living environment of camels were collected from May to September between 2013 and 2017 and dissected. Eventually, onchocercal lesions were observed in 95 of 192 (49%) camels and the captured insects were classified into 49 species from 42 genera in 21 families, among which 18 species were newly recorded in Inner Mongolia and 14 were haematophagous species. The filarial larvae were found in Culicoides puncticollis and identified as Onchocerca fasciata, indicating that C. puncticollis is the vector of O. fasciata in Inner Mongolia. These findings provide an estimate of onchocerciasis infection in camels and an alternative method of identifying insects and screening vectors using molecular methods. Important data are also provided for the diagnosis and control of onchocerciasis, thereby further filling the gap in knowledge regarding transmission vectors in China.

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.

Development of Leishmania orientalis in the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) and the biting midge Culicoides soronensis (Diptera: Ceratopogonidae).

Leishmania (Mundinia) orientalis is a newly described species causing human leishmaniasis in Thailand whose natural vector is unknown. L. orientalis infections in sand flies and/or biting midges under laboratory conditions have not been previously investigated. In this study, the development of L. orientalis in two experimental vectors, Lutzomyia longipalpis sand flies and Culicoides sonorensis biting midges was investigated for the first time using light microscopy, scanning electron microscopy, and histological examination. The results showed that L. orientalis was unable to establish infection in Lu. longipalpis. No parasites were found in the sand fly gut 4 days post-infected blood meal (PIBM). In contrast, the parasite successfully established infection in C. sonorensis. The parasites differentiated from amastigotes to procyclic promastigotes in the abdominal midgut (AMG) on day 1 PIBM. On day 2 PIBM, nectomonad promastigotes were observed in the AMG and migrated to the thoracic midgut (TMG). Leptomonad promastigotes appeared at the TMG on day 3 PIBM. Clusters of leptomonad promastigotes and metacyclic promastigotes colonized around the stomodeal valve with the accumulation of a promastigote secretory gel-like material from day 3 PIBM onwards. Haptomonad-like promastigotes were observed from day 5 PIBM, and the proportion of metacyclic promastigotes reached 23% on day 7 PIBM. The results suggest that biting midges or other sand fly genera or species might be vectors of L. orientalis.

Identification of a new vector species of avian haemoproteids, with a description of methodology for the determination of natural vectors of haemosporidian parasites.

BACKGROUND: Haemosporidian parasites are transmitted by dipteran blood-sucking insects but certain vectors remain unidentified for the great majority of described species. Sensitive PCR-based methods are often used for the detection of haemosporidian infection in wild-caught insects. However, this approach alone cannot distinguish between different sporogonic stages and thus is insufficient to demonstrate that the parasites produce the infective stage (sporozoite), which is essential for transmission. To prove that PCR-positive insects could act as vectors, the record of sporozoites is needed. We developed a methodology for the determination of natural vectors of avian Haemoproteus species and other haemosporidians. The essence of this approach is to apply PCR-based and microscopic diagnostic tools in parallel for sporozoite detection in insects. METHODS: Culicoides biting midges transmit avian Haemoproteus parasites, but certain insect species, which are involved in transmission, remain insufficiently investigated. Biting midges were collected in the wild and identified; parous females were dissected and preparations of thorax content containing salivary glands were prepared. Remnants of the dissected midges were screened using PCR-based methods. Only thorax preparations of PCR-positive biting midges were examined microscopically. RESULTS: In total, 460 parous females belonging to 15 species were collected and dissected. DNA of haemosporidians was detected in 32 (7%) of dissected insects belonging to 7 species. Of the thorax samples PCR-positive for Haemoproteus parasites, two preparations were microscopically positive for sporozoites. Both biting midges were Culicoides kibunensis. Haemoproteus pallidus (hPFC1) was identified, indicating that transmission of this infection occurs at the study site. It was proved that seven species of biting midges take bird blood meals naturally in the wild. CONCLUSIONS: Culicoides kibunensis is a new vector species of avian haemoproteids and is a natural vector of H. pallidus. Numerous studies have identified vectors of Haemoproteus parasites experimentally; however, this is the first direct identification of a natural vector of Haemoproteus infection in the Old World. We suggest using the described methodology for vector research of Haemoproteus and other haemosporidians in the wild.

Culicoides species community composition and infection status with parasites in an urban environment of east central Texas, USA.

BACKGROUND: Despite their importance as vectors of zoonotic parasites that can impact human and animal health, Culicoides species distribution across different habitat types is largely unknown. Here we document the community composition of Culicoides found in an urban environment including developed and natural sites in east central Texas, a region of high vector diversity due to subtropical climates, and report their infection status with haemoparasites. RESULTS: A total of 251 individual Culicoides were collected from May to June 2016 representing ten Culicoides species, dominated by C. neopulicaris followed by C. crepuscularis. We deposited 63 sequences to GenBank among which 25 were the first deposition representative for six Culicoides species: C. arboricola (n = 1); C. nanus (n = 4); C. debilipalpis (n = 2); C. haematopotus (n = 14); C. edeni (n = 3); and C. hinmani (n = 1). We also record for the first time the presence of C. edeni in Texas, a species previously known to occur in the Bahamas, Florida and South Carolina. The urban environments with natural area (sites 2 and 4) had higher species richness than sites more densely populated or in a parking lot (sites 1 and 3) although a rarefaction analysis suggested at least two of these sites were not sampled sufficiently to characterize species richness. We detected a single C. crepuscularis positive for Onchocercidae gen. sp. DNA and another individual of the same species positive for Haemoproteus sacharovi DNA, yielding a 2.08% prevalence (n = 251) for both parasites in this species. CONCLUSIONS: We extend the knowledge of the Culicoides spp. community in an urban environment of Texas, USA, and contribute to novel sequence data for these species. Additionally, the presence of parasite DNA (Onchocercidae gen. sp. and H. sacharovi) from C. crepuscularis suggests the potential for this species to be a vector of these parasites.

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.

A new blood parasite of leaf warblers: molecular characterization, phylogenetic relationships, description and identification of vectors.

BACKGROUND: Blood parasites of the genus Haemoproteus Kruse, 1890 are cosmopolitan, might be responsible for mortality in non-adapted birds, and often kill blood-sucking insects. However, this group remains insufficiently investigated in the wild. This is particularly true for the parasites of leaf warblers of the Phylloscopidae Alström, Ericson, Olsson & Sundberg the common small Old World passerine birds whose haemoproteid parasite diversity and vectors remain poorly studied. This study reports a new species of Haemoproteus parasitizing leaf warblers, its susceptible vector and peculiar phylogenetic relationships with other haemoproteids. METHODS: Wood warblers (Phylloscopus sibilatrix Bechstein) were caught in Lithuania during spring migration, and blood films were examined microscopically. Laboratory reared Culicoides nubeculosus Meigen were exposed experimentally by allowing them to take blood meals on one individual harbouring mature gametocytes of the new Haemoproteus species (lineage hPHSIB2). To follow sporogonic development, the engorged insects were dissected at intervals. The parasite lineage was distinguished using sequence data, and morphological analysis of blood and sporogonic stages was carried out. Bayesian phylogeny was constructed in order to determine the phylogenetic relationships of the new parasite with other haemoproteids. RESULTS: Haemoproteus (Parahaemoproteus) homopalloris n. sp. was common in wood warblers sampled after arrival to Europe from their wintering grounds in Africa. The new parasite belongs to a group of avian haemoproteid species with macrogametocytes possessing pale staining cytoplasm. All species of this group clustered together in the phylogenetic analysis, indicating that intensity of the cytoplasm staining is a valuable phylogenetic character. Laboratory-reared biting midges C. nubeculosus readily supported sporogony of new infections. Phylogenetic analysis corroborated vector experiments, placing the new parasite in the clade of Haemoproteus (Parahaemoproteus) parasites transmitted by biting midges. CONCLUSIONS: Haemoproteus homopalloris n. sp. is the third haemoproteid, which is described from and is prevalent in wood warblers. Phylogenetic analysis identified a clade containing seven haemoproteids, which are characterised by pale staining of the macrogametocyte cytoplasm and with ookinetes maturing exceptionally rapidly (between 1 to 1.5 h after exposure to air). Both these features may represent valuable phylogenetic characters. Studies targeting mechanisms of sporogonic development of haemoproteids remain uncommon and should be encouraged. Culicoides nubeculosus is an excellent experimental vector of the new parasite species.