Avian Parasites Research Articles

Diversity and Host Specificity of Avian Haemosporidians in an Afrotropical Conservation Region.

Afrotropical regions have high bird diversity, yet few studies have attempted to unravel the prevalence of avian haemosporidia in conservation areas. The diversity and host specificity of parasites in biodiversity hotspots is crucial to understanding parasite distribution and potential disease emergence. We test the hypothesis that biodiverse regions are associated with highly diverse parasites. By targeting the cytochrome b (Cytb) gene, we molecularly screened 1035 blood samples from 55 bird species for avian haemosporidia infections to determine its prevalence and diversity on sites inside and adjacent to the Kruger National Park. Overall infection prevalence was 28.41%. Haemoproteus, Leucocytozoon, and Plasmodium presented prevalences of 17.39%, 9.24%, and 4.64%, respectively. One hundred distinct parasite lineages were detected, of which 56 were new lineages. Haemoproteus also presented the highest diversity compared to Leucocytozoon and Plasmodium with varying levels of specificity. Haemoproteus lineages were found to be specialists while Plasmodium and Leucocytozoon lineages were generalists. We also found a positive relationship between avian host diversity and parasite diversity, supporting an amplification effect. These findings provide insight data for host-parasite and co-evolutionary relationship models.

Culicoides biting midges feeding behaviour as a key for understanding avian Haemoproteus transmission in Lithuania.

Investigations of host feeding behaviour in haematophagous insects are critical to assess transmission routes of vector-borne diseases. Understanding if a certain species has ornithophilic or mammalophilic feeding behaviour can facilitate future studies focused on pathogens transmission to and from certain host species. Culicoides Latreille (Diptera: Ceratopogonidae) are vectors of several pathogens, which include arboviruses, bacteria and parasites to a considerable diversity of vertebrate hosts. However, most of the studies focused on feeding habits target Culicoides species that could transmit the Bluetongue virus, consequently with a mammalophilic feeding behaviour, leaving aside the Culicoides species that are involved in the transmission of vector-borne parasites to birds, such as Haemoproteus Kruse (Haemosporida: Haemoproteidae). This study aimed to investigate the source of blood meals of wild-caught Culicoides using molecular-based methods and to correlate our findings with the reports of Haemoproteus parasites in Culicoides species. Engorged Culicoides females were collected using ultraviolet (UV)-light traps at seven different localities in Lithuania in 2021-2023. Biting midges were dissected, and the abdomens of engorged females were used for molecular investigation of the blood meal source. A polymerase chain reaction (PCR) protocol that amplifies a fragment of the Cytochrome B gene of vertebrates was used. Obtained sequences were compared to available information in GenBank database to confirm the source of the blood meal. In total, 258 engorged Culicoides females, representing nine different species, were analysed. The source of blood meal was identified in 29.1% of them with most of the insects having fed on birds (74.7%) and the remaining on mammals (25.3%). Culicoides segnis Campbell, Pelham-Clinton was the only species to feed exclusively on birds; Culicoides from the Obsoletus group, C. pallidicornis Kieffer and C. punctatus Latreille were found to feed exclusively on mammals; C. festivipennis Kieffer, C. kibunensis Tokunaga and C. pictipennis Staeger had an opportunistic feeding behaviour, with the first two preferably feeding on birds. Due to their feeding behaviour and the presence of Haemoproteus parasites reported in the literature, C. festivipennis, C. kibunensis, C. pictipennis, and C. segnis play an important role in the transmission of those avian vector-borne parasite in the wild. These Culicoides species were already confirmed as being able to support the development of several Haemoproteus species and lineages. Future studies focused on understanding the epidemiology of avian pathogens transmitted by Culicoides should target these species.

Avian nematode parasites of some wild birds of Bangladesh

Avian nematode parasites of some wild birds of Bangladesh

Avian haemosporidian parasites from wild-caught mosquitoes with new evidence on vectors of Plasmodium matutinum

Avian haemosporidian parasites are spread worldwide and pose a threat to their hosts occasionally. A complete life cycle of these parasites requires two hosts: vertebrate and invertebrate (a blood-sucking insect that acts as a vector). In this study, we tested wild-caught mosquitoes for haemosporidian infections. Mosquitoes were collected (2021–2023) in several localities in Lithuania using a sweeping net and a CDC trap baited with CO2, morphologically identified, and preparations of salivary glands were prepared (from females collected in 2022–2023). 2093 DNA samples from either individual after dissection (1675) or pools (418 pools/1145 individuals) of female mosquito's abdomens were screened using PCR for the detection of haemosporidian parasite DNA. Salivary gland preparations were analyzed microscopically from each PCR-positive mosquito caught in 2022 and 2023. The average prevalence of haemosporidian parasites for all analyzed samples was 2.0 % and varied between 0.6 % (2021) and 3.5 % (2022). DNA of Plasmodium ashfordi (cytochrome b genetic lineage pGRW02), P. circumflexum (pTURDUS1), P. homonucleophilum (pSW2), P. matutinum (pLINN1), P. vaughani (pSYAT05), Haemoproteus brachiatus (hLK03), H. majoris (hWW2), and H. minutus (hTUPHI01) were detected in mosquitoes. Coquilletidia richiardii (3.5 %) and Culex pipiens (2.9 %) were mosquito species with the highest prevalence of haemosporidian parasite DNA detected. Mixed infections were detected in 16 mosquitoes. In one of the samples, sporozoites of P. matutinum (pLINN1) were found in the salivary gland preparation of Culex pipiens, confirming this mosquito species as a competent vector of Plasmodium matutinum and adding it to the list of the natural vectors of this avian parasite.

Finding a needle in a haystack: DNA Haemoproteus columbae enrichment using percoll density gradient and flow cytometry

Isolation of genomic DNA of blood parasites in birds, reptiles, amphibians, and fishes is a challenging task, given that their red blood cells are nucleated; for that reason, parasite genomic DNA is only a fraction of the total extracted DNA, and it is challenging to obtain concentrated high-quality genetic material. Percoll Density Gradient (PDG) and flow cytometry are tools for separating and analyzing cell populations or even a single cell, and both represent potent approaches for isolating avian haemosporidians parasites. Our experimental design included several steps seeking to concentrate the parasite´s DNA. We used blood samples from a Rock pigeon infected with Haemoproteus columbae. After inducing parasite exflagellation and gametogenesis in vitro, we subjected the samples to a Percoll Density Gradient to separate the parasites from the rest of the blood cells. Following centrifugation, the layer containing extracellular parasites underwent a flow cytometry and cell sorting process, during which we selected two different subpopulations of cells for analysis. Based on qPCR analyses, we demonstrate parasite DNA enrichment in Percoll Density Gradient and flow cytometry samples; simultaneously, these samples showed the lowest concentration of Columba livia DNA. However, the concentration of parasite DNA was higher in the PDG than in the cell sorting sample. This study reports the concentration of the Haemoproteus parasite by flow cytometry without DNA-intercalating dyes, and this methodology can serve as a technique for DNA enrichment of blood parasites infecting nucleated red blood cells to improve techniques that allow obtaining complete genomes.

First insights on the susceptibility of native coccidicidal fungi Mucor circinelloides and Mucor lusitanicus to different avian antiparasitic drugs

BackgroundThe combined application of predatory fungi and antiparasitic drugs is a sustainable approach for the integrated control of animal gastrointestinal (GI) parasites. However, literature addressing the possible interference of antiparasitic drugs on the performance of these fungi is still scarce. This research aimed to assess the in vitro susceptibility of six native coccidicidal fungi isolates of the species Mucor circinelloides and one Mucor lusitanicus isolate to several antiparasitic drugs commonly used to treat GI parasites’ infections in birds, namely anthelminthics such as Albendazole, Fenbendazole, Levamisole and Ivermectin, and anticoccidials such as Lasalocid, Amprolium and Toltrazuril (drug concentrations of 0.0078–4 µg/mL), using 96-well microplates filled with RPMI 1640 medium, and also on Sabouraud Agar (SA).ResultsThis research revealed that the exposition of all Mucor isolates to the tested anthelminthic and anticoccidial drug concentrations did not inhibit their growth. Fungal growth was recorded in RPMI medium, after 48 h of drug exposure, as well as on SA medium after exposure to the maximum drug concentration.ConclusionsPreliminary findings from this research suggest the potential compatibility of these Mucor isolates with antiparasitic drugs for the integrated control of avian intestinal parasites. However, further in vitro and in vivo studies are needed to confirm this hypothesis.

Avian Plasmodium in invasive and native mosquitoes from southern Spain

BackgroundThe emergence of diseases of public health concern is enhanced by factors associated with global change, such as the introduction of invasive species. The Asian tiger mosquito (Aedes albopictus), considered a competent vector of different viruses and parasites, has been successfully introduced into Europe in recent decades. Molecular screening of parasites in mosquitoes (i.e. molecular xenomonitoring) is essential to understand the potential role of different native and invasive mosquito species in the local circulation of vector-borne parasites affecting both humans and wildlife.MethodsThe presence of avian Plasmodium parasites was molecularly tested in mosquitoes trapped in five localities with different environmental characteristics in southern Spain from May to November 2022. The species analyzed included the native Culex pipiens and Culiseta longiareolata and the invasive Ae. albopictus.ResultsAvian Plasmodium DNA was only found in Cx. pipiens with 31 positive out of 165 mosquito pools tested. None of the Ae. albopictus or Cs. longiareolata pools were positive for avian malaria parasites. Overall, eight Plasmodium lineages were identified, including a new lineage described here. No significant differences in parasite prevalence were found between localities or sampling sessions.ConclusionsUnlike the invasive Ae. albopictus, Cx. pipiens plays a key role in the transmission of avian Plasmodium in southern Spain. However, due to the recent establishment of Ae. albopictus in the area, further research on the role of this species in the local transmission of vector-borne pathogens with different reservoirs is required.Graphical

Natural Vector of Avian Haemoproteus asymmetricus Parasite and Factors Altering the Spread of Infection.

Avian haemosporidians (Apicomplexa, Haemosporida) are widespread blood protists, often causing severe haemosporidiosis, pathology, or even mortality in their hosts. Migrant birds regularly bring various haemosporidian parasites from wintering grounds to European breeding areas. Some haemosporidian parasites are prevalent in breeding sites and complete their life cycles in temperate climate zones and can be transmitted, but others do not. The factors altering the spread of these haemosporidians are not fully understood. Culicoides biting midges (Diptera: Ceratopogonidae) play an important role in the transmission of worldwide distributed avian haemosporidian parasites belonging to the genus Haemoproteus, but this information is particularly scarce and insufficient. The key factors limiting the spread of these pathogens in temperate climate zones, which we suspect and aim to study, are the absence of susceptible vectors and the ecological isolation of birds from vectors during the breeding period when transmission occurs. The primary objective of this study was to evaluate how the habitats of biting midges and bird breeding sites influence parasite transmission while also seeking to expand our understanding of the natural vectors for these parasites. Biting midges were collected using UV traps on the Curonian Spit, Lithuania, in different habitats, such as woodland and reeds, from May to September. Parous Culicoides females were identified, dissected, and investigated for the presence of Haemoproteus parasites using both microscopy and PCR-based tools. Among the dissected 1135 parous Culicoides females, the sporozoites of Haemoproteus asymmetricus (genetic lineage hTUPHI01) have been detected for the first time in the salivary glands of Culicoides festivipennis. The sporozoites of four Haemoproteus lineages were detected in Culicoides segnis, C. festivipennis, and Culicoides kibunensis biting midges. PCR-based screening showed that the females of seven Culicoides species were naturally infected with Haemoproteus parasites. The DNA of the parasite of owls, Haemoproteus syrnii (hSTAL2), was detected for the first time in Culicoides punctatus. The highest abundance of collected Culicoides females was in June, but the highest prevalence of Haemoproteus parasites in biting midges was in July. The abundance of Culicoides was higher in the woodland compared with reeds during the season. The acquired findings indicate the varied abundance and diversity of biting midges throughout the season and across distinct habitats. This variability could potentially impact the transmission of Haemoproteus parasites among birds with diverse breeding site ecologies. These outcomes hold the potential to enhance our understanding of the epizootiology of Haemoproteus infections within temperate climatic zones.

Evolutionary conservation of Trichomonas-mycoplasma symbiosis across the host species barrier.

The protozoan parasite Trichomonas vaginalis is the most common cellular sexually transmitted disease in humans, and the closely related species Trichomonas gallinae is an avian parasite of ecological and economic importance. Phylogenetic evidence suggests T. vaginalis arose during bird to human transmission of a T. gallinae-like ancestor. Trichomonas vaginalis shares a strong clinical association with the independent sexually transmitted pathogen Metamycoplasma (formerly Mycoplasma) hominis, and the uncultured bacterium "Candidatus Malacoplasma (formerly Mycoplasma) girerdii," with the latter association being an order of magnitude stronger. Both bacterial species have been shown to profoundly influence T. vaginalis growth, energy production and virulence-associated mechanisms. Evidence for a novel Malacoplasma sp. was discovered by in vivo Illumina metatranscriptomics sequencing of the T. gallinae-infected pigeon mouth. We leveraged published 16S rDNA profiling data from digestive tract of 12 healthy and 24 T. gallinae-infected pigeons to investigate association between the novel Malacoplasma sp. and T. gallinae. We utilised Illumina metagenomics sequencing targeted to pigeon oral and crop samples infected with the novel Malacoplasma sp. to generate its full-length genome sequence. Sequence similarity network analysis was used to compare annotated proteins from the novel Malacoplasma sp. with a range of other related species. Here we present evidence for a novel Malacoplasma species, related to "Ca. M. girerdii," that is strongly associated with T. gallinae in the upper digestive tract of domestic pigeons. Analysis of the genome sequence revealed gene features apparently specific to a Trichomonas-symbiotic Malacoplasma lineage. These data support a model of long-term association between Trichomonas and Malacoplasma spp. that has been conserved across diversification of the Trichomonas lineage and the host species barrier from birds to human.

Bird community effects on avian malaria infections

In community assembly processes, interspecific interactions play an important role in shaping community diversity, especially at the local scale. Changes in species richness or abundance can modify local infectious disease dynamics, either reducing or increasing the risk of transmission within the community. This study evaluates the effects of bird community on avian haemosporidians infections in a Neotropical region. Bird samples were collected from areas surrounding three dams, and molecular analysis were performed to identify blood-parasitic haemosporidia infecting the birds. Generalized linear models were used to analyze the relationships between the bird community and the prevalence, number of infections, and richness of avian haemosporidian lineages. Non-significant effects of bird community dominance and richness on the prevalence of avian parasites and the number of infections of Haemoproteus were found. However, there was evidence of an amplification effect. Host dominance was associated with the total number of infections, the number Plasmodium infections and the expected richness of Plasmodium lineages, while the expected richness of Haemoproteus lineages was associated with the richness of bird species. These findings highlight the role of host community dominance and richness in the dynamics of parasite infections, potentially influenced by the availability of competent hosts. This study contributes significantly to our understanding of blood parasite diversity in tropical birds within a relatively understudied region of South America.

Introducing the land snail Bradybaena pellucida increased infection risk of the avian parasite Postharmostomum commutatum in the Kanto region of Japan.

The trematode Postharmostomum commutatum is a parasite of the chicken Gallus gallus domesticus. Its heavy infection can cause inflammation and hemorrhage in the cecum of host birds. We found a severe infection of metacercariae of P. commutatum, which was identified based on DNA barcodes with morphology, in the introduced land snail Bradybaena pellucida and its related species in the Kanto region of Japan. Our field survey revealed that metacercariae were detected in 14 of 69 sampling locations in this region. B. pellucida was thought to be the major second intermediate host of metacercariae of the trematode because this snail was most frequently found in the study area and the prevalence and infection intensity were higher than those of the other snail species. The observed increase in metacercariae in introduced populations of B. pellucida can enhance the infection risk of chickens and wild host birds, probably owing to the spillback effect. Our seasonal field study showed that the prevalence and infection intensity of metacercaria seemed to be high in populations of B. pellucida during the summer and early autumn. Therefore, chickens should not be bred outdoors during these seasons to prevent severe infection. Our molecular analysis, based on cytochrome c oxidase subunit I sequences, showed a significantly negative value for Tajima's D in P. commutatum, suggesting an increase in its population size. Thus, P. commutatum distributed in the Kanto region may have increased its population size with the introduction of the host snail.

Isolation of saprophytic filamentous fungi from avian fecal samples and assessment of its predatory activity on coccidian oocysts

Fungal strains used in the biocontrol of animal gastrointestinal parasites have been mainly isolated from pasture soil, decaying organic matter, and feces from herbivores and carnivores. However, their isolation from birds and assessment of predatory activity against avian GI parasites has been scarce thus far. This research aimed to isolate filamentous fungi from avian fecal samples and evaluate their predatory activity against coccidia. A pool of 58 fecal samples from chickens, laying hens, and peacocks, previously collected between July 2020-April 2021, were used for isolation of filamentous fungi and assessment of their in vitro predatory activity against coccidian oocysts, using Water-Agar medium and coprocultures. The Willis-flotation technique was also performed to obtain concentrated suspensions of oocysts. A total of seven Mucor isolates was obtained, being the only fungal taxa identified, and all presented lytic activity against coccidia. Isolates FR3, QP2 and SJ1 had significant coccidiostatic efficacies (inhibition of sporulation) higher than 70%, while isolates FR1, QP2 and QP1 had coccidicidal efficacies (destruction of the oocysts) of 22%, 14% and 8%, respectively, after 14 days of incubation, being a gradual and time-dependent process. To our knowledge, this is the first report regarding the isolation of native predatory fungi from avian feces and demonstration of their lytic activity against coccidia.

Gradual changes in model shape affect egg-directed behaviours by parasitic shiny cowbirds Molothrus bonariensis in captivity.

Eggs are critically important for avian reproduction as all birds are oviparous. Accordingly, the recognition and care of own eggs represent the cornerstones of avian breeding, whereas the elimination of foreign objects, including brood-parasitic eggs and non-egg items from the nest are known to also increase fitness by refocusing incubation effort on the parents' own eggs. But egg recognition also plays a role in some avian obligate brood parasites' reproductive strategy through the pecking of already present eggs in the hosts' clutch to reduce nestmate competition with the parasite's own hatchling. Here we tested egg shape recognition in this parasitic egg-pecking context by exposing two different series of 3D printed models to captive obligate brood-parasitic shiny cowbirds (Molothrus bonariensis) in artificial nests. Natural egg-shaped models were pecked more often compared with increasingly thinner models, but there was no effect of increasing angularity on pecking rates, implying that a natural, rather than an artificial, range of variability elicited adaptive responses from parasitic cowbirds.

2-hydroxy-1,4-naphthoquinones with 3-alkyldiarylether groups: synthesis and Plasmodium falciparum inhibitory activity.

Background: In 1948, the synthesis and Plasmodium lophurae activity of 2-hydroxy-1,4-naphthoquinones containing 3-alkyldiarylether side chains was reported. Method/results: The synthesis of five related compounds, designed to be more metabolically stable, was pursued. The compounds were synthesized using a radical alkylation reaction with naphthoquinones. One compound had a lower IC50 value against various strains of Plasmodium falciparum and assay data indicate that it binds to the Qo site of cytochrome bc1. With a low yield for the radical alkylation of the most active compound, a reductive alkylation method with used to improve reaction yields. Conclusion: Further synthetic knowledge was obtained, and the assay data indicate that there are sensitivity differences between avian and human malarial parasites for these molecules.

Non-invasive elevation of circulating corticosterone increases the rejection of foreign eggs in female American robins (Turdus migratorius)

Avian obligate brood parasites rely on other species to raise their offspring. In turn, many brood parasite hosts have evolved defensive behaviors to reduce the costs of brood parasitism, yet the proximate bases underlying these defenses remain poorly understood. Recent studies regarding the potential endocrine mechanisms of foreign-egg rejection have implicated corticosterone as a physiological mediator of anti-parasitic defenses. For example, corticosterone is elevated in response to non-mimetic eggs in an egg rejecter thrush, the Eurasian blackbird (Turdus merula) and this hormone's suppression reduces egg rejection rates in the congeneric American robin (T. migratorius). American robins are also among the few host species of obligate brood parasitic brown-headed cowbirds (Molothrus ater) that readily reject foreign eggs from their nests. We non-invasively elevated corticosterone levels in incubating female robins by dissolving it in DMSO gel which was then applied onto eggs already in the clutch. Relative to controls treated with pure DMSO gel, corticosterone-treated female robins were more likely to reject a non-mimetic, cowbird-sized foreign egg (72 %) than control females (50 %) when accounting for the known effect of lower clutch sizes on greater egg rejection. Future studies are needed to assess the sensory and cognitive impact(s) of corticosterone, as well as other hormones essential for parental care, in this and other hosts' defense behaviors against avian brood parasitism.

Exploring the thermal limits of malaria transmission in the western Himalaya.

Environmental temperature is a key driver of malaria transmission dynamics. Using detailed temperature records from four sites: low elevation (1800), mid elevation (2200 m), and high elevation (2600–3200 m) in the western Himalaya, we model how temperature regulates parasite development rate (the inverse of the extrinsic incubation period, EIP) in the wild. Using a Briére parametrization of the EIP, combined with Bayesian parameter inference, we study the thermal limits of transmission for avian (Plasmodium relictum) and human Plasmodium parasites (P. vivax and P. falciparum) as well as for two malaria‐like avian parasites, Haemoproteus and Leucocytozoon. We demonstrate that temperature conditions can substantially alter the incubation period of parasites at high elevation sites (2600–3200 m) leading to restricted parasite development or long transmission windows. The thermal limits (optimal temperature) for Plasmodium parasites were 15.62–34.92°C (30.04°C) for P. falciparum, 13.51–34.08°C (29.02°C) for P. vivax, 12.56–34.46°C (29.16°C) for P. relictum and for two malaria‐like parasites, 12.01–29.48°C (25.16°C) for Haemoproteus spp. and 11.92–29.95°C (25.51°C) for Leucocytozoon spp. We then compare estimates of EIP based on measures of mean temperature versus hourly temperatures to show that EIP days vary in cold versus warm environments. We found that human Plasmodium parasites experience a limited transmission window at 2600 m. In contrast, for avian Plasmodium transmission was not possible between September and March at 2600 m. In addition, temperature conditions suitable for both Haemoproteus and Leucocytozoon transmission were obtained from June to August and in April, at 2600 m. Finally, we use temperature projections from a suite of climate models to predict that by 2040, high elevation sites (~2600 m) will have a temperature range conducive for malaria transmission, albeit with a limited transmission window. Our study highlights the importance of accounting for fine‐scale thermal effects in the expansion of the range of the malaria parasite with global climate change.

Did extreme nest predation favor the evolution of obligate brood parasitism in a duck?

Obligate brood parasites depend entirely on other species to raise their offspring. Most avian obligate brood parasites have altricial offspring that require enormous amounts of posthatching parental care, and the large fecundity boost that comes with complete emancipation from parental care likely played a role in the independent evolution of obligate parasitism in several altricial lineages. The evolution of obligate parasitism in the black‐headed duck, however, is puzzling because its self‐feeding precocial offspring should not constrain parental fecundity of a potential brood parasite in the way that altricial offspring do. We used an experimental nest predation study to test the idea that high nest predation rates played a role in the evolution of brood parasitism in this enigmatic duck. Experimental duck eggs in untended nests suffered massive rapid predation, while eggs in tended nests of the three main hosts, all aggressive nest defenders, had very high success, illustrating the benefits of parasitizing these ‘bodyguard’ hosts.

A new duplex real-time PCR for simultaneous detection and differentiation of Tetratrichomonas gallinarum and Trichomonas gallinae.

Tetratrichomonas gallinarum and Trichomonas gallinae are pathogenic avian parasites that infect a wide range of bird species. The pathologic potential of T. gallinarum is controversial, whereas T. gallinae causes disease in many avian species. Infections are often asymptomatic in doves and pigeons; thus, columbids are presumed to represent the natural hosts for trichomonads. The detection of T. gallinarum and T. gallinae is based on direct microscopic observation or a conventional PCR assay. Microscopy is not very sensitive, and identification of the trichomonads at the genus or species level is not possible. Conventional PCR assays have been developed primarily for phylogenetic studies, which detect a wide range of Trichomonas spp. but do not allow their differentiation. We developed a duplex real-time PCR (rtPCR) assay for the simultaneous detection and differentiation of T. gallinarum and T. gallinae. We found that the rtPCR assay detected 102 plasmid DNA copies of T. gallinarum and as few as 101 plasmid DNA copies of T. gallinae.

Culicoides segnis and Culicoides pictipennis Biting Midges (Diptera, Ceratopogonidae), New Reported Vectors of Haemoproteus Parasites.

As bloodsuckers of birds, Culicoides biting midges (Diptera, Ceratopogonidae) play an important role in the transmission of avian haemosporidian (Haemoproteus) parasites, which are prevalent in many bird populations and cause disease, pathology, or even mortality in their hosts. Information about the role of the various Culicoides species in the transmission of Haemoproteus parasites remains insufficient. This presents an obstacle for the better understanding of the epizootiology of haemoproteosis. The aim of this study was to determine new Culicoides species involved in the transmission of Haemoproteus parasites in the wild. Biting midges were collected using UV traps on the Curonian Spit, Lithuania. Only parous Culicoides females were investigated: they were identified and were diagnosed for the presence of Haemoproteus parasites using both microscopy and PCR-based methods. We collected and dissected 420 parous Culicoides females. PCR-based screening showed that 28 parous Culicoides biting midges were infected with avian Haemoproteus parasites. Haemoproteid DNA was detected in Culicoides kibunensis, Culicoides pictipennis, Culicoides festivipennis, Culicoides segnis, Culicoides pallidicornis, and Culicoides obsoletus biting midges. The DNA of Haemoproteus palloris, genetic lineage hWW1, was found for the first time in C. pallidicornis. Haemoproteus sporozoites were detected in the salivary glands of two Culicoides segnis biting midges. According to the PCR results, one female contained Haemoproteus tartakovskyi (genetic lineage hHAWF1) DNA and another Haemoproteus majoris (genetic lineage hCCF5) DNA. The sporozoites of Haemoproteus parasites were also detected in the salivary glands of four C. pictipennis biting midges using microscopy, and this finding was confirmed by PCR as Haemoproteus parabelopolskyi DNA (genetic lineage hSYAT02) was detected in three out of the four biting midges. The obtained results supplement existing information about Culicoides biting midges as natural vectors of Haemoproteus spp. and add two new Culicoides species to the vector list, showing the low specificity of these parasites for the invertebrate hosts.

Prevalence of filarial parasites in field-caught mosquitoes in northwestern California.

Mosquitoes were collected in Lake County, CA, in 2014 and tested using standard polymerase chain reaction for filarial parasite DNA. Filarial parasites were detected in 23 out of 1,008 total pools. DNA from Dirofilaria immitis, the parasite causing dog heartworm, was detected in Aedes increpitus (MIR=4.62), Aedes sierrensis (MIR=6.72), Anopheles freeborni (MIR=1.08), and Culex tarsalis (MIR=0.10). Setaria yehi, deer body worm, was detected in Ae. sierrensis (MIR=13.42), Anopheles franciscanus (MIR=0.55), An. freeborni (MIR=2.69), and Culex stigmatosoma (MIR=0.41). The avian parasite Splendidofilaria could not be identified to species but was detected in Cx. tarsalis (MIR=0.20). DNA was also detected for three unidentified filarial parasites in Culex. Filarial-positive pools spanned May-August, with Splendidofilaria earlier in the season and S. yehi later. For D. immitis, MIR tended to be highest in June, when the 130 HDU development threshold was reached. Interestingly, D. immitis was also detected prior to the HDU threshold, and D. immitis was not detected August-September, though HDU remained high enough for development. This suggests that there are other factors influencing dog heartworm transmission in the area.