Prevalence of avian malaria parasite in mosquitoes collected at a zoological garden in Japan

Several species of captive birds at zoological gardens of Japan were found to be infected with avian Plasmodium. However, incriminated vector mosquito species have not been identified yet. To indicate the competent vectors of avian malaria parasite, we collected mosquitoes at a zoological garden in Japan and examined for the avian malaria parasite DNA. Totally, 1,361 mosquitoes of 11 species were collected in the zoological garden of Kanagawa, the south of Tokyo in Japan in 2005. Captured mosquitoes were pooled by each species, date collected, and location and used for DNA extraction. Eight out of 169 DNA samples were positive for the nested PCR of avian Plasmodium cyt b gene. Estimated minimum infection rates of mosquitoes were 5.9 per 1,000. The PCR positive mosquito species were Culex pipiens group and Lutzia vorax. Some DNA sequences amplified from collected mosquitoes were identical to avian Plasmodium lineages detected from captive birds in the same zoological garden studied. Our results suggest that C. pipiens group and L. vorax could be incriminated vectors of avian malaria parasite transmitting in captive birds kept in the zoological garden in Japan.     

Malaria in penguins – current perceptions

Avian malaria is a mosquito-borne disease caused by protozoans of the genus Plasmodium, and it is considered one of the most important causes of morbidity and mortality in captive penguins, both in zoological gardens and rehabilitation centres. Penguins are known to be highly susceptible to this disease, and outbreaks have been associated with mortality as high as 50–80% of affected captive populations within a few weeks. The disease has also been reported in wild penguin populations, however, its impacts on the health and fitness of penguins in the wild is not clear. This review provides an overview of the aetiology, life cycle and epidemiology of avian malaria, and provides details on the strategies that can be employed for the diagnostic, treatment and prevention of this disease in captive penguins, discussing possible directions for future research.     

Entomological study on transmission of avian malaria parasites in a zoological garden in Japan: bloodmeal identification and detection of avian malaria parasite DNA from blood-fed mosquitoes

Several species of captive and wild birds have been found to be infected with various avian blood protozoa in Japan. We investigated the prevalence and transmission of avian malaria parasite and determined the bloodmeal hosts of mosquitoes collected in a zoological garden in Tokyo, Japan, by using the polymerase chain reaction. In total, 310 unfed and 140 blood-fed mosquitoes of seven species were collected by using sweep nets and CDC traps. Bloodmeal identification indicated that mosquitoes had fed on 17 avian and five mammalian species, including captive animals. The results of avian malaria parasite detection from mosquitoes with avian bloodmeals indicated that Culex pipiens pallens Coquillet is a main vector of avian Plasmodium in the current study site and that some captive and wild birds could be infected with avian malaria parasites. Furthermore, the distances between the collection site of blood-fed mosquitoes and the locations of their blood-source captive animals were estimated. Most females with fresh bloodmeals were found within 40 m of caged animals, whereas half-gravid and gravid females were found between 10 and 350 m from caged host animals. We demonstrated that blood-fed mosquitoes can provide useful information regarding the mosquito vector species of avian malaria parasites and allows for noninvasive detection of the presence of avian malaria parasites in bird populations.     

New malaria parasites of the subgenus <Emphasis Type="Italic">Novyella</Emphasis> in African rainforest birds,with remarks on their high prevalence,classification and diagnostics

Blood samples from 655 passerine birds were collected in rainforests of Ghana and Cameroon and examined both by microscopy and polymerase chain reaction (PCR)-based techniques. The overall prevalence of Plasmodium spp. was 46.6%, as determined by combining the results of both these diagnostic methods. In comparison to PCR-based diagnostics, microscopic examination of blood films was more sensitive in determining simultaneous infection of Plasmodium spp., but both detection methods showed similar trends of prevalence of malaria parasites in the same study sites. Plasmodium (Novyella) lucens n. sp., Plasmodium (Novyella) multivacuolaris n. sp. and Plasmodium (Novyella) parahexamerium n. sp. were found in the olive sunbird Cyanomitra olivacea (Nectariniidae), yellow-whiskered greenbul Andropadus latirostris (Picnonotidae), and white-tailed alethe Alethe diademata (Turdidae), respectively. These parasites are described based on the morphology of their blood stages and a segment of the mitochondrial cytochrome b (cyt b) gene, which can be used for molecular identification and diagnosis of these species. Illustrations of blood stages of new species are given, and phylogenetic analysis identifies DNA lineages closely related to these parasites. Malaria parasites of the subgenus Novyella with small erythrocytic meronts clearly predominate in African passerines. It is probable that the development of such meronts is a characteristic feature of evolution of Plasmodium spp. in African rainforest birds. Subgeneric taxonomy of avian Plasmodium spp. is discussed based on the recent molecular phylogenies of these parasites. It is concluded that a multi-genome phylogeny is needed before revising the current subgeneric classification of Plasmodium. We supported a hypothesis by Hellgren, Križanauskienė, Valkiūnas, Bensch (J Parasitol 93:889–896, 2007), according to which, haemosporidian species with a genetic differentiation of over 5% in mitochondrial cyt b gene are expected to be morphologically differentiated. This study emphasises the importance of employing both PCR-based and microscopic methods in taxonomic, ecological and evolutionary investigations of avian haemosporidian parasites.     

Pathological and molecular characterization of avian malaria in captive Magellanic penguins (Spheniscus magellanicus) in South America

Avian malaria is a mosquito-borne disease that affects multiple avian species and is caused by protozoans of the genus Plasmodium. An avian malaria infection caused by Plasmodium sp. in Magellanic penguins (Spheniscus magellanicus) with high mortality is described in a zoo in Southern Brazil. Clinically, three birds presented signs of inappetence, anorexia, pale mucosa, dyspnea, and opisthotonus, with death in a clinical course of 5–8 h. At the necropsy, all birds exhibited pale mucosa, marked splenomegaly and hepatomegaly, in addition to moderate leptomeningeal blood vessels ingurgitation in the brain. Microscopically, multiple exoerythrocytic meronts were observed in the cytoplasm of endothelial cells in the spleen, liver, heart, lungs, brain, kidneys, and pancreas. The spleen had a multifocal perivascular inflammatory infiltrate of lymphocytes, plasma cells, and macrophages, which also exhibited hemosiderosis and erythrophagocytosis. The liver had a multifocal periportal inflammatory infiltrate of lymphocytes, macrophages, and plasma cells, in addition to marked hemosiderosis in the hepatic sinusoids. Fragments of spleen, liver, brain, skeletal muscle, and lung were tested by the polymerase chain reaction technique for the detection of a fragment of the cytochrome B gene from haemosporidians, which resulted positive for Plasmodium spp. After sequencing, the samples were phylogenetically associated to Plasmodium sp. detected in Turdus albicollis (KU562808) in Brazil and matched to the lineage TURALB01 previously detected in T. albicollis. Avian malaria infections caused by Plasmodium sp. of lineage TURALB01 may occur in S. magellanicus with high mortality, and, thus, it is essential to detect and characterize the agent involved to obtain the differential diagnosis of the condition.

     

Complete nucleotide sequences of the mitochondrial genomes of two avian malaria protozoa, <Emphasis Type="Italic">Plasmodium gallinaceum</Emphasis> and <Emphasis Type="Italic">Plasmodium juxtanucleare</Emphasis>

We analyzed mitochondrial genomes of two avian malaria protozoa, Plasmodium gallinaceum and Plasmodium juxtanucleare. Both mitochondrial genomes were estimated to be 6,002 and 6,014 bp in length, respectively, and to have the identical gene organization and contents to that of other Plasmodium species previously analyzed; three functional genes for cytochrome c oxidase subunit I, III, and cytochrome (cyt b), with following sets of discontinuous and scrambled 15 ribosomal subunit RNA (rRNA) genes. Similarities of the three protein-coding genes showed closer relationship within avian malaria protozoa rather than mammalian Plasmodium species. In addition, we showed the tandem repeated structure of each mitochondrial genome of both P. gallinaceum and P. juxtanucleare as well as previously found in mammalian Plasmodium species. This study revealed the complete sequences and structure of the mitochondrial genomes of avian malaria protozoa for the first time.     

Plasmodium (Haemamoeba) cathemerium gene sequences for phylogenetic analysis of malaria parasites

The DNA sequence information on avian malaria parasites of the genus Plasmodium is quite limited. At present, sequences of only 6 out of 34 valid species are available. However, sequence data of avian malaria parasites are particularly important with regard to the resolution of the phylogenetic relationships of the most virulent human malaria agent, Plasmodium falciparum. The question as to whether P. falciparum originates from avian or from mammalian parasites would contribute to our understanding of its biology and would probably facilitate the interpretation of experimental results. To add to the body of molecular data, we sequenced three genes (cytochrome b, 18 SSU rRNA, caseinolytic protease C) of different organellar origin of one of the most widespread avian malaria parasites, Plasmodium (Haemamoeba) cathemerium, which once used to be an important laboratory in vivo model in human malaria research. The analysis of the new P. cathemerium sequences in direct comparison with the rodent parasite P. berghei and the four human malaria parasites by pairwise distance calculation do not suggest a closer relationship of P. cathemerium to P. falciparum than to the other species involved.     

Polymerase chain reaction-based identification of <Emphasis Type="Italic">Plasmodium</Emphasis> (<Emphasis Type="Italic">Huffia</Emphasis>) <Emphasis Type="Italic">elongatum</Emphasis>, with remarks on species identity of haemosporidian lineages deposited in GenBank

Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only 11 morphospecies of Plasmodium have been linked to these lineages. Such linking is important because it provides opportunities to combine the existing knowledge of traditional parasitology with novel genetic information of these parasites obtained by molecular techniques. This study linked one mitochondrial cytochrome b (cyt b) gene lineage with morphospecies Plasmodium (Huffia) elongatum, a cosmopolitan avian malaria parasite which causes lethal disease in some birds. One species of Plasmodium (mitochondrial cyt b gene lineage P-GRW6) was isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to one naive juvenile individual of the same host species. Heavy parasitaemia developed in the subinoculated bird, which enabled identification of the morphospecies and deposition of its voucher specimens. The parasite of this lineage belongs to P. elongatum. Illustrations of blood stages of this parasite are given. Other lineages closely related to P. elongatum were identified. The validity of the subgenus Huffia is supported by phylogenetic analysis. Mitochondrial cyt b gene lineages, with GenBank accession nos. AF069611 and AY733088, belong to Plasmodium cathemerium and P. elongatum, respectively; these lineages have been formerly attributed to P. elongatum and P. relictum, respectively. Some other incorrect species identifications of avian haematozoa in GenBank have been identified. We propose a strategy to minimise the number of such mistakes in GenBank in the future.     

A retrospective survey into the presence of Plasmodium spp. and Toxoplasma gondii in archived tissue samples from New Zealand raptors: New Zealand falcons (Falco novaeseelandiae), Australasian harriers (Circus approximans) and moreporks (Ninox novaeseelandiae)

Human colonisation of New Zealand has resulted in the introduction of emerging diseases, such as avian malaria and toxoplasmosis, which arrived with their exotic avian and mammalian hosts. Plasmodium spp. and Toxoplasma gondii have a wide host range, and several species of endemic New Zealand birds have developed a fatal disease following infection with either pathogen. However, no reports of either toxoplasmosis or avian malaria in New Zealand raptors, namely, the New Zealand falcons (Falco novaeseelandiae), Australasian harriers (Circus approximans) and moreporks (Ninox novaeseelandiae) exist in the literature. Therefore, this study was designed to determine if these two pathogens are present in these raptors through a retrospective analysis of archived tissue samples. Detection and isolate identification of these pathogens was determined using established histological and molecular techniques. All three species of New Zealand raptors tested positive for the presence of Plasmodium spp. (10/117; 8.5%) and an atypical genotype of T. gondii (9/117; 7.7%). Plasmodium lineages identified include P. elongatum GRW6, P. relictum SGS1, P. relictum PADOM02 and Plasmodium sp. LINN1. Two Australasian harriers and one morepork tested positive for the presence of both Plasmodium spp. and T. gondii. However, the pathogenicity of these organisms to the raptors is unclear as none of the tissues showed histological evidence of clinical disease associated with Plasmodium spp. and T. gondii infections. Thus, these results demonstrate for the first time that these two potential pathogens are present in New Zealand’s raptors; however, further research is required to determine the prevalence and pathogenicity of these organisms among the living populations of these birds in the country.

     

Genetic characterization of avian malaria (Protozoa) in the endangered lesser kestrel, <Emphasis Type="Italic">Falco naumanni</Emphasis>

We genetically analyzed avian malaria (Protozoa) isolated from lesser kestrels (Falco naumanni) breeding in La Mancha, Central Spain. A total of 586 adult individuals were screened for blood parasites using a very efficient polymerase chain reaction approach that amplifies a partial segment (498 bp) of the cytochrome b gene of avian malaria of the genera Haemoproteus and Plasmodium. The prevalence of Plasmodium was 8.2%, and the prevalence of Haemoproteus was 4.1%. Sequence analyses revealed six unique lineages of avian malaria, three Plasmodium (LK5, LK6, RTSR1) and three Haemoproteus (LK2, LK3, LK4). According to sequence divergence, these lineages seem to correspond to at least three different species, although all recovered lineages could be independent evolutionary units. The third most common lineage (RTSR1) has been previously retrieved from two other avian host species, including a resident African bird species and a trans-Saharan migrant passerine, suggesting that lesser kestrels could acquire this Plasmodium lineage at their winter quarters in Africa.     

Evaluation of serum chemistry values associated with avian malaria infections in African black-footed penguins (Spheniscus demersus)

The value profiles of 5 intracellular enzymes, 15 metabolites (with 2 associated ratios), and 3 electrolytes were monitored over time in 9 captive-reared African black-footed penguins (Spheniscus demersus) with different avian malaria clinical status: uninfected, subclinically infected, and clinically infected with fatal outcome. Fatal infections were caused byPlasmodium relictum. Numerous schizonts were visible in the lungs, liver, spleen, and interstitial tissue of the kidneys. The feference ranges of 23 serum clinical chemistry parameters and 2 ratios were established forS. demersus. The mean values obtained for 8 of 23 parameters of the infected penguins were signficantly different from those recorded for the uninfected birds, indicating impaired renal function, hepatic dysfunction, and nonspecific tissue damage related to the infestation with exoerythrocytic schizonts. Analysis of sensitivity, specificity, and negative and positive predictive values (PPVs) showed that gamma-glutamyltranspeptidase (GGTP), alanine aminotransferase (ALT), and creatinine reached PPVs and a specificity over 57% for avian malaria infections in penguins. Creatinine, ALT, and GGTP values should be consulted in evaluation of the clinical malaria status ofS. demersus.     

Characteristics of naturally acquired avian malaria infections in naive juvenile African black-footed penguins (Spheniscus demersus)

Antibody responses to naturally acquiredPlasmodium relictum andP. elongatum infections, blood parasitemia, and disease signs were investigated in 23 naive juvenile African black-footed penguins (Spheniscus demersus). Anti-Plasmodium spp. immunoglobulins were detected by enzyme-linked immunosorbent assay (ELISA) usingP. falciparum antigens. All birds rapidly developed antibody toP. relictum andP. elongatum. Five penguins showed detectable parasitemia and signs of the disease. Parasitemia was not related to the timing of the maximal antibody response or to the antibody titer. Two of the five parasitemic birds died and gross examination revealed splenomegaly, hepatomegaly, and congested, edematous lungs. Although the other 17 birds were clearly exposed to the disease, none showed signs of infection. No subsequent episode of parasitemia was observed in individual penguins. A comparison of the fate of 1993 penguins with those from other years showed a great variability in the proportion of birds exhibiting signs of malaria.     

Genetic characterization,distribution and prevalence of avian pox and avian malaria in the Berthelot’s pipit (<Emphasis Type="Italic">Anthus berthelotii</Emphasis>) in Macaronesia

Exotic pathogens have been implicated in the decline and extinction of various native-island-bird species. Despite the fact that there is increasing concern about the introduction of diseases in island ecosystems, little is known about parasites in the islands of Macaronesia. We focus on Berthelot’s pipit (Anthus berthelotii), an endemic and widespread Macaronesian bird species, using a combination of field studies and molecular techniques to determine: (1) the range and prevalence of avian pox and malaria in Berthelot’s pipits throughout the species’ distribution, (2) the genetic characterization of both parasites in order to ascertain the level of host specificity. We sampled 447 pipits across the 12 islands inhabited by this species. Overall, 8% of all individuals showed evidence of pox lesions and 16% were infected with avian malaria, respectively. We observed marked differences in the prevalence of parasites among islands both within and between archipelagos. Avian pox prevalence varied between 0–54% within and between archipelagos and avian malaria prevalence varied between 0–64% within and between archipelagos. The diversity of pathogens detected was low: only two genetic lineages of avian malaria and one lineage of avian pox were found to infect the pipit throughout its range. Interestingly, both avian malaria parasites found were Plasmodium spp. that had not been previously reported in the Macaronesian avifauna (but that had been observed in the lesser kestrel Falco naumannii), while the avian pox was a host specific lineage that had previously been reported on two of the Canary Islands.     

Investigation of a mortality cluster in wild adult yellow-eyed penguins (Megadyptes antipodes) at Otago Peninsula,New Zealand

We investigated an epidemic mortality cluster of yellow-eyed penguins (Megadyptes antipodes) that involved 67 moribund or dead birds found on various beaches of the Otago Peninsula, New Zealand, between 21 January and 20 March 2013. Twenty-four carcases were examined post-mortem. Histological lesions of pulmonary, hepatic and splenic erythrophagocytosis and haemosiderosis were found in 23 of 24 birds. Fifteen birds also had haemoglobin-like protein droplets within renal tubular epithelial cells. Despite consistent histological lesions, a cause of death could not be established. Virology, bacteriology and molecular tests for avian influenza, avian paramyxovirus-1, avipoxvirus, Chlamydia psittaci, Plasmodium spp., Babesia spp., Leucocytozoon spp. and Toxoplasma gondii were negative. Tissue concentrations of a range of heavy metals (n?=?4 birds) were consistent with low level exposure, while examination of proventricular contents and mucus failed to detect any marine biotoxins or Clostridium botulinum toxin. Hepatic concentrations of total polycyclic aromatic hydrocarbons (PAHs) (n?=?5 birds) were similar to background concentrations of polycyclic aromatic hydrocarbons previously found in yellow-eyed penguins from the South Island of New Zealand, but there were significantly higher concentrations of 1-methylnapthelene and 2-methylnapthelene in the birds found dead in this mortality cluster. The biological significance of this finding is unclear. A temporal investigation of the epidemic did not indicate either a common source or propagative epidemic pattern. Although our investigation did not definitively implicate a toxic or infectious agent, we could not rule out causes such as toxic marine organisms or mycoplasmosis. Further investigations should therefore by carried out in the event of future mortality clusters.     

Role of TLRs/MyD88 in host resistance and pathogenesis during protozoan infection: lessons from malaria

Toll-like receptors (TLRs) are important to initiate the innate immune response to a wide variety of pathogens. The protective role of TLRs during infection with protozoan parasites has been established. In this regard, malaria represents an exception where activation of TLRs seems to be deleterious to the host. In this article, we review the recent findings indicating the contrasting role of Myeloid Differentiation Primary-Response gene 88 (MyD88) and TLRs during malaria and infection with other protozoa. These findings suggest that MyD88 may represent an Achilles’ heel during Plasmodium infection.     

Malaria parasites (Plasmodium spp.) infecting introduced, native and endemic New Zealand birds

Avian malaria is caused by intracellular mosquito-transmitted protist parasites in the order Haemosporida, genus Plasmodium. Although Plasmodium species have been diagnosed as causing death in several threatened species in New Zealand, little is known about their ecology and epidemiology. In this study, we examined the presence, microscopic characterization and sequence homology of Plasmodium spp. isolates collected from a small number of New Zealand introduced, native and endemic bird species. We identified 14 Plasmodium spp. isolates from 90 blood or tissue samples. The host range included four species of passerines (two endemic, one native, one introduced), one species of endemic pigeon and two species of endemic kiwi. The isolates were associated into at least four distinct clusters including Plasmodium (Huffia) elongatum, a subgroup of Plasmodium elongatum, Plasmodium relictum and Plasmodium (Noyvella) spp. The infected birds presented a low level of peripheral parasitemia consistent with chronic infection (11/15 blood smears examined). In addition, we report death due to overwhelming parasitemia in a blackbird, a great spotted kiwi and a hihi. These deaths were attributed to infections with either Plasmodium spp. lineage LINN1 or P. relictum lineage GRW4. To the authors’ knowledge, this is the first published report of Plasmodium spp. infection in great spotted and brown kiwi, kereru and kokako. Currently, we are only able to speculate on the origin of these 14 isolates but consideration must be made as to the impact they may have on threatened endemic species, particularly due to the examples of mortality.     

Description and molecular characterization of Plasmodium (Novyella) unalis sp. nov. from the Great Thrush (Turdus fuscater) in highland of Colombia

Plasmodium (Novyella) unalis sp. nov. was found in the Great Thrush, Turdus fuscater (Passeriformes, Turdidae) in Bogotá, Colombia, at 2,560 m above sea level where the active transmission occurs. This parasite is described based on the morphology of its blood stages and a fragment of the mitochondrial cytochrome b gene (lineage UN227). Illustrations of blood stages of new species are given, and the phylogenetic analysis identifies closely related species and lineages of avian malaria parasites. The new species is most similar to Plasmodium (Novyella) vaughani (lineage SYAT05), a cosmopolitan avian malaria parasite; these parasites are also closely related genetically, with a genetic difference of 3.2 % between them. P. unalis can be readily distinguished from the latter species morphologically, primarily due to the (1) presence of a single large, circular shaped pigment granule in the erythrocytic trophozoites and meronts; (2) presence of prominent vacuoles in trophozoites and growing meronts; and (3) presence of predominantly fan-like shaped erythrocytic meronts. Cytochrome b lineages with high similarity to the new species have been reported in Costa Rica, Brazil, Chile, and USA. It is probable that the new species of malaria parasite is widely distributed in the New World. This parasite has been reported only in the Great Thrush at the study site and might have a narrow range of avian hosts. Records of P. unalis are of particular theoretical interest due to its active transmission at highlands in Andes. Possible influence of urbanization on transmission of this malaria parasite in Bogotá is discussed.     

Cerebral Plasmodium falciparum malaria: The role of PfEMP1 in its pathogenesis and immunity,and PfEMP1-based vaccines to prevent it

Malaria, a mosquito-borne infectious disease caused by parasites of the genus Plasmodium continues to be a major health problem worldwide. The unicellular Plasmodium-parasites have the unique capacity to infect and replicate within host erythrocytes. By expressing variant surface antigens Plasmodium falciparum has evolved to avoid protective immune responses; as a result in endemic areas anti-malaria immunity develops gradually over many years of multiple and repeated infections. We are studying the role of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed by asexual stages of P. falciparum responsible for the pathogenicity of severe malaria. The immunopathology of falciparum malaria has been linked to cyto-adhesion of infected erythrocytes to specific host receptors. A greater appreciation of the PfEMP1 molecules important for the development of protective immunity and immunopathology is a prerequisite for the rational discovery and development of a safe and protective anti-disease malaria vaccine. Here we review the role of ICAM-1 and EPCR receptor adhering falciparum-parasites in the development of severe malaria; we discuss our current research to understand the factors involved in the pathogenesis of cerebral malaria and the feasibility of developing a vaccine targeted specifically to prevent this disease.     

Hemoprotozoa in free-ranging birds from rural areas of Mazandaran Province, northern Iran

A range of protozoa infect the blood of birds including Plasmodium, Aegyptianella, Haemoproteus, and Leukocytozoon and are transmitted by known vectors. The parasites occur worldwide and can cause reduced production, anemia, and even death. The aim of present study was to undertake the first survey of hemoprotozoa in the native birds of the Central zone of Mazandaran province. A total of 213 blood samples were collected from five different species of birds, 70 (32.9%) were infected by at least one parasite. The prevalence of infection by Aegyptianella pullorum, Plasmodium spp., and Haemoproteus spp. was 23.9%, 2.3%, and 6.6%, respectively. Our results showed a high prevalence of A. pullorum (38.3%) among ducks, as well as a high prevalence of Haemoproteus spp. (87.5%) in pigeons. Moreover, only ducks and turkeys were found to be infected with Plasmodium spp. Infections with multiple genera were found in 30.04% (64/213) of birds examined which included 91.4% of the 70 infected birds. It also seems that birds which migrate to the area annually might play an important role in infecting domestic birds in the province.     

Avian haemosporidians in haematophagous insects in the Czech Republic

The degree to which avian haemosporidian parasites can exploit different vectors as a definitive host has ecological implications for their transmission and biogeography. Studies targeting haemosporidian parasites using precise molecular detection methods are almost lacking in Central Europe, however. Here, we utilized PCR-based molecular methods to detect avian haemosporidians in insect vectors in the Czech Republic. Nine lineages of parasites belonging to three genera, Haemoproteus, Plasmodium, and Leucocytozoon, were detected in pooled samples of insect individuals, of which three lineages had not yet been discovered in previous studies. All three Leucocytozoon lineages were found exclusively in black flies, while five Haemoproteus lineages were found in biting midges. The most abundant insect species Culicoides kibunensis harbored three Haemoproteus lineages, and the second-most numerous species Culicoides segnis even four. The positive mosquitoes of Culex pipiens complex hosted two parasite lineages, one Plasmodium and one Haemoproteus, the latter of which, however, could suggest the aberrant development of this parasite in an unusual invertebrate host. The co-occurrence of Haemoproteus ROFI1 and TURDUS2 lineages in both insects and birds at the same study plot suggests a transmission of these lineages during breeding season of birds.