Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species.

BACKGROUND: Identification of mosquitoes greatly relies on morphological specification. Since some species cannot be distinguished reliably by morphological methods, it is important to incorporate molecular techniques into the diagnostic pipeline. DNA barcoding using Sanger sequencing is currently widely used for identification of mosquito species. However, this method does not allow detection of multiple species in one sample, which would be important when analysing mosquito eggs. Detection of container breeding Aedes is typically performed by collecting eggs using ovitraps. These traps consist of a black container filled with water and a wooden spatula inserted for oviposition support. Aedes mosquitoes of different species might lay single or multiple eggs on the spatula. In contrast to Sanger sequencing of specific polymerase chain reaction (PCR) products, multiplex PCR protocols targeting specific species of interest can be of advantage for detection of multiple species in the same sample. METHODS: For this purpose, we adapted a previously published PCR protocol for simultaneous detection of four different Aedes species that are relevant for Austrian monitoring programmes, as they can be found in ovitraps: Aedes albopictus, Aedes japonicus, Aedes koreicus, and Aedes geniculatus. For evaluation of the multiplex PCR protocol, we analysed 2271 ovitrap mosquito samples from the years 2021 and 2022, which were collected within the scope of an Austrian nationwide monitoring programme. We compared the results of the multiplex PCR to the results of DNA barcoding. RESULTS: Of 2271 samples, the multiplex PCR could identify 1990 samples, while species determination using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I gene was possible in 1722 samples. The multiplex PCR showed a mixture of different species in 47 samples, which could not be detected with DNA barcoding. CONCLUSIONS: In conclusion, identification of Aedes species in ovitrap samples was more successful when using the multiplex PCR protocol as opposed to the DNA barcoding protocol. Additionally, the multiplex PCR allowed us to detect multiple species in the same sample, while those species might have been missed when using DNA barcoding with Sanger sequencing alone. Therefore, we propose that the multiplex PCR protocol is highly suitable and of great advantage when analysing mosquito eggs from ovitraps.

A citizen science report-Tiger mosquitoes (Aedes albopictus) in allotment gardens in Graz, Styria, Austria.

Aedes albopictus, the Asian tiger mosquito, is an invasive species not native to Europe. Due to its ability to transmit pathogens, such as dengue, chikungunya and Zika viruses, Ae. albopictus is considered a major health threat. In Austria, it was first reported in 2012 in the Western province of Tyrol and was documented in the metropolitan area of Vienna in 2020, demonstrating its ability to colonize urban areas. In July 2021, a garden owner from Graz, Styria, Austria, contacted experts because of the possible presence of tiger mosquitoes in an allotment garden complex. Accordingly, citizen scientists collected adult mosquitoes and set up ovitraps. Adults and eggs were sent to the laboratory for morphological examination and molecular DNA barcoding within the mitochondrial cytochrome c oxidase subunit I gene. In total, 217 eggs of Ae. albopictus were found at the allotment garden as well as at a second location in the city of Graz. In addition, 14 adult Ae. albopictus specimens, of which 7 were molecularly identified as an identical haplotype, were collected at the allotment garden. With its mild climate and numerous parks and gardens, Graz provides the perfect environment for reproduction of tropical/subtropical alien Aedes mosquitoes. The presence of eggs and adult specimens in the current study period indicates that Ae. albopictus is already breeding in Graz. However, monitoring efforts need to be continued to determine whether stable populations of Ae. albopictus can survive there.

Predicting Culex pipiens/restuans Population Dynamics Using a Weather-Driven Dynamic Compartmental Population Model.

Mosquitoes of the genus Culex are important vectors of a variety of arthropod-borne viral infections. In most of the northern parts of the USA, Cx. pipiens/restuans is the predominant representative of this genus. As vectors, they play a key role in the spreading of arboviruses and thus, knowledge of the population dynamic of mosquitoes is important to understand the disease ecology of these viruses. As poikilotherm animals, the vital rates of mosquitoes are highly dependent on ambient temperature, and also on precipitation. We present a compartmental model for the population dynamics of Cx. pipiens/restuans. The model is driven by temperature, precipitation, and daytime length (which can be calculated from the geographic latitude). For model evaluation, we used long-term mosquito capture data, which were averaged from multiple sites in Cook County, Illinois. The model fitted the observation data and was able to reproduce between-year differences in the abundance of the Cx. pipiens/restuans mosquitoes, as well as the different seasonal trends. Using this model, we evaluated the effectiveness of targeting different vital rates for mosquito control strategies. The final model is able to reproduce the weekly mean Cx. pipiens/restuans abundance for Cook County with a high accuracy, and over a long time period of 20 years.

Canine babesiosis in Austria in the 21st century - A review of cases.

A retrospective study on 699 cases of canine babesiosis presented to veterinary clinics in eastern Austria were evaluated for the location where infection had presumably taken place. Of these, 542 (77.54%) had acquired the infection in Austria, while the majority of non-autochthonous cases came from neighboring countries, most notable Hungary. Both groups were recorded primarily in Vienna, eastern Lower Austria and Burgenland, but cases from the southern (Styria, Carinthia) and western (Upper Austria, Tyrol, Salzburg) provinces of the country were also recorded. Records were made all year round, with most cases in spring (46.6%) and fall (48.4%). The annual cases ranged from four to 58 (mean: 31.8) with large fluctuations and no visible trend for an in- or decrease. The tick vector of Babesia canis, Dermacentor reticulatus, is present in Austria but displays a very patchy distribution, and its occurrence and activity are not readily foretold, which might be a reason why its presumably increasing density in Europe is not reflected by increased incidences of canine babesiosis. Another factor that may influence the numbers of cases per year could be the application (or non-application) of acaricidal or repellent compounds. A limitation of this study is that bias is exerted by the location of the participating clinics, and by the unknown rate of infections that does not induce clinical symptoms and is likely not presented in veterinary practices and clinics. The data, however, clearly show that at least the lowlands of Austria are endemic for B. canis, and appropriate tick control must be advised all year round.

Dermatitis linearis outbreak associated with Paederus balcanicus in Austria.

BACKGROUND: Dermatitis linearis is a toxic skin lesion caused by contact with certain beetles of the genus Paederus (Coleoptera: Staphylinidae). Dermatitis linearis outbreaks have been described mainly in tropical and subtropical regions, but so far not in Central Europe, and are considered an emerging public health concern potentially associated with climate change. MATERIAL AND METHODS: Following diagnosis of dermatitis linearis in a cluster of six adults and one child with reported exposure to beetles with morphological characteristics of Paederus species at a recreational public open-air bath at Lake Neusiedl (Illmitz, Burgenland, Austria), we performed on-site inspection and installed light and pitfall traps. Collected beetle specimens of the genus Paederus were classified using morphological characteristics and DNA barcoding. RESULTS: A total of 32 Paederus beetles were collected using an aspirator (n = 2) and light traps (n = 30). No individuals of the genus Paederus were captured with the pitfall traps. Morphological analyses identified them as members of the Paederus balcanicus species, which was confirmed by genetic specification of four arbitrarily chosen individuals. Dermatitis linearis lesions were treated with topical steroids and healed but partly leaving scars and hyperpigmentation, over the course of a few weeks in all affected persons. CONCLUSION: We report for the first time (a) an outbreak of dermatitis linearis associated with exposure to autochthonous Paederus species in Austria, and (b) that contact to the species Paederus balcanicus may cause dermatitis linearis in humans. Adequate measures should be taken to prevent dermatitis linearis outbreaks in areas with resident Paederus occurrence.

First Nationwide Monitoring Program for the Detection of Potentially Invasive Mosquito Species in Austria.

In Austria, only fragmented information on the occurrence of alien and potentially invasive mosquito species exists. The aim of this study is a nationwide overview on the situation of those mosquitoes in Austria. Using a nationwide uniform protocol for the first time, mosquito eggs were sampled with ovitraps at 45 locations in Austria at weekly intervals from May to October 2020. The sampled eggs were counted and the species were identified by genetic analysis. The Asian tiger mosquito Aedes albopictus was found at two sites, once in Tyrol, where this species has been reported before, and for the first time in the province of Lower Austria, at a motorway rest stop. The Asian bush mosquito Aedes japonicus was widespread in Austria. It was found in all provinces and was the most abundant species in the ovitraps by far. Aedes japonicus was more abundant in the South than in the North and more eggs were found in habitats with artificial surfaces than in (semi-) natural areas. Further, the number of Ae. japonicus eggs increased with higher ambient temperature and decreased with higher wind speed. The results of this study will contribute to a better estimation of the risk of mosquito-borne disease in Austria and will be a useful baseline for a future documentation of changes in the distribution of those species.

First records of Aedes pulcritarsis (Rondani, 1872) (Diptera: Culicidae) in Austria.

Aedes pulcritarsis is a tree-hole breeding species with its main distribution in the Mediterranean area. Within the scope of two independent monitoring programmes, this mosquito species was detected for the first time in Austria, in the province of Lower Austria (2018, districts Mistelbach and Gaenserndorf; 2020, district Bruck an der Leitha). As the climatic and habitat situation in Central Europe seems to be generally suitable for this species, the most likely explanation for the species not being recorded previously is that it might have been overlooked in the past due to its specialized breeding habitat. However, further research on the distribution of Ae. pulcritarsis in Austria would be needed to support this hypothesis. The results from this study will contribute to the investigation of the northern distribution limit of Ae. pulcritarsis in Europe and possible changes thereof.

AIMSurv: First pan-European harmonized surveillance of Aedes invasive mosquito species of relevance for human vector-borne diseases.

Human and animal vector-borne diseases, particularly mosquito-borne diseases, are emerging or re-emerging worldwide. Six Aedes invasive mosquito (AIM) species were introduced to Europe since the 1970s: Aedes aegypti, Ae. albopictus, Ae. japonicus, Ae. koreicus, Ae. atropalpus and Ae. triseriatus. Here, we report the results of AIMSurv2020, the first pan-European surveillance effort for AIMs. Implemented by 42 volunteer teams from 24 countries. And presented in the form of a dataset named "AIMSurv Aedes Invasive Mosquito species harmonized surveillance in Europe. AIM-COST Action. Project ID: CA17108". AIMSurv2020 harmonizes field surveillance methodologies for sampling different AIMs life stages, frequency and minimum length of sampling period, and data reporting. Data include minimum requirements for sample types and recommended requirements for those teams with more resources. Data are published as a Darwin Core archive in the Global Biodiversity Information Facility- Spain, comprising a core file with 19,130 records (EventID) and an occurrences file with 19,743 records (OccurrenceID). AIM species recorded in AIMSurv2020 were Ae. albopictus, Ae. japonicus and Ae. koreicus, as well as native mosquito species.

Diversity of West Nile and Usutu virus strains in mosquitoes at an international airport in Austria.

Increased globalization and international transportation have resulted in the inadvertent introduction of exotic mosquitoes and new mosquito-borne diseases. International airports are among the possible points of entry for mosquitoes and their pathogens. We established a mosquito and mosquito-borne diseases monitoring programme at the largest international airport in Austria and report the results for the first two years, 2018 and 2019. This included weekly monitoring and sampling of adult mosquitoes, and screening them for the presence of viral nucleic acids by standard molecular diagnostic techniques. Additionally, we surveyed the avian community at the airport, as birds are potentially amplifying hosts. In 2018, West Nile virus (WNV) was detected in 14 pools and Usutu virus (USUV) was detected in another 14 pools of mosquitoes (minimum infection rate [MIR] of 6.8 for each virus). Of these 28 pools, 26 consisted of female Culex pipiens/torrentium, and two contained male Culex sp. mosquitoes. Cx. pipiens/torrentium mosquitoes were the most frequently captured mosquito species at the airport. The detected WNV strains belonged to five sub-clusters within the sub-lineage 2d-1, and all detected USUV strains were grouped to at least seven sub-clusters among the cluster Europe 2; all strains were previously shown to be endemic in Austria. In 2019, all mosquito pools were negative for any viral nucleic acids tested. Our study suggests that airports may serve as foci of arbovirus activity, particularly during epidemic years, and should be considered when designing mosquito control and arbovirus monitoring programmes.

Dirofilaria spp. and Angiostrongylus vasorum: Current Risk of Spreading in Central and Northern Europe.

In the past few decades, the relevance of Dirofilaria immitis and Dirofilaria repens, causing cardiopulmonary and subcutaneous dirofilariosis in dogs and cats, and of Angiostrongylus vasorum, causing canine angiostrongylosis, has steadily increased in Central and Northern Europe. In this review, a summary of published articles and additional reports dealing with imported or autochthonous cases of these parasites is provided for Central (Austria, Czechia, Germany, Hungary, Luxemburg, Poland, Slovakia, Slovenia, and Switzerland) and Northern (Denmark, Finland, Iceland, Norway, and Sweden) Europe. Research efforts focusing on Dirofilaria spp. and A. vasorum have varied by country, and cross-border studies are few. The housing conditions of dogs, pet movements, the spread of competent vectors, and climate change are important factors in the spread of these nematodes. Dogs kept outside overnight are a major factor for the establishment of Dirofilaria spp. However, the establishment of invasive, diurnal, synanthropic, competent mosquito vectors such as Aedes albopictus may also influence the establishment of Dirofilaria spp. The drivers of the spread of A. vasorum remain not fully understood, but it seems to be influenced by habitats shared with wild canids, dog relocation, and possibly climatic changes; its pattern of spreading appears to be similar in different countries. Both Dirofilaria spp. and A. vasorum merit further monitoring and research focus in Europe.

Arrival of the Asian tiger mosquito, Aedes albopictus (Skuse, 1895) in Vienna, Austria and initial monitoring activities.

Aedes albopictus was recorded in Vienna, Austria, in August 2020 for the first time. The species was found to occur in three sites within the city; morphology-based monitoring was followed by DNA-barcoding. Mitochondrial COI barcode sequences recovered three different haplotypes, however this data does not reveal whether single or multiple introduction events have occurred. The vicinity of Viennese Ae. albopictus sites to major traffic routes highlights the importance of passive transport for range expansion of this species.

Integrative Approach to Phlebotomus mascittii Grassi, 1908: First Record in Vienna with New Morphological and Molecular Insights.

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood-feeding insects that transmit the protozoan parasites Leishmania spp. and various arthropod-borne (arbo) viruses. While in Mediterranean parts of Europe the sand fly fauna is diverse, in Central European countries including Austria mainly Phlebotomus mascittii is found, an assumed but unproven vector of Leishmania infantum. To update the currently understudied sand fly distribution in Austria, a sand fly survey was performed and other entomological catches were screened for sand flies. Seven new trapping locations of Ph. mascittii are reported including the first record in Vienna, representing also one of the first findings of this species in a city. Morphological identification, supported by fluorescence microscopy, was confirmed by two molecular approaches, including sequencing and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) protein profiling. Sand fly occurrence and activity were evaluated based on surveyed locations, habitat requirements and climatic parameters. Moreover, a first comparison of European Ph. mascittii populations was made by two marker genes, cytochrome c oxidase subunit 1 (COI), and cytochrome b (cytb), as well as MALDI-TOF mass spectra. Our study provides new important records of Ph. mascittii in Austria and valuable data for prospective entomological surveys. MALDI-TOF MS protein profiling was shown to be a reliable tool for differentiation between sand fly species. Rising temperatures and globalization demand for regular entomological surveys to monitor changes in species distribution and composition. This is also important with respect to the possible vector competence of Ph. mascittii.

Detection of Crenosoma spp., Angiostrongylus vasorum and Aelurostrongylus abstrusus in Gastropods in Eastern Austria.

Canine and feline cardiorespiratory parasites are of utmost relevance in veterinary medicine. Key epizootiological information on major pet metastrongyloids, i.e., Angiostrongylus vasorum and Crenosoma vulpis infecting dogs, and Aelurostrongylus abstrusus and Troglostrongylus brevior infecting cats, is missing from Austria. This study investigated their occurrence in 1320 gastropods collected in the Austrian provinces of Styria, Burgenland, Lower Austria, and in metropolitan Vienna. Metastrongyloid larvae were microscopically detected in 25 samples, and sequence analysis confirmed the presence of metastrongyloids in nine samples, i.e., A. vasorum in one slug (Arion vulgaris) (0.07%), C. vulpis in five slugs (one Limax maximus and four A. vulgaris) (0.4%), A. abstrusus in two A. vulgaris (0.17%), and the hedgehog lungworm Crenosoma striatum was detected in one A. vulgaris. The present study confirms the enzooticity of major cardiorespiratory nematodes in Austria and that canine and feline populations are at risk of infection.

Acanthocephalan parasites collected from Austrian fishes: molecular barcoding and pathological observations.

Acanthocephalan parasites were collected from the intestinal tracts of 137 predominantly wild fish (1 barbel Barbus barbus, 3 European chub Squalius cephalus, 13 rainbow trout Oncorhynchus mykiss and 120 brown trout Salmo trutta) from 12 localities. The condition factor, intensity of acanthocephalan infection and pathological lesions, if applicable, were documented. Routine bacteriology and virology were performed, and the brown trout were additionally tested for the presence of the myxozoan parasite Tetracapsolioides bryosalmonae by PCR. In total, 113 acanthocephalans were barcoded by sequencing a section of the mitochondrial cytochrome oxidase subunit I (COI) gene. Barcoding of the acanthocephalan tissues resulted in 77 sequences, of which 56 were assigned to Echinorhynchus truttae (3 genotypes), 11 to Pomphorhynchus tereticollis (9 genotypes), 9 to Acanthocephalus sp. (5 genotypes) and 1 to Neoechinorhynchida. Most of these genotypes were detected for the first time. Statistically, the acanthocephalan infection did not have an impact on the condition factor of the brown trout. Infection with P. tereticollis caused more severe pathological changes in the digestive tract than E. truttae. The present study provides new data regarding the distribution of acanthocephalan species in Austria and their impact on individual fish. In addition, new barcoding data from acanthocephalan parasites are presented, and the occurrence of P. tereticollis in European chub in Austria and in brown and rainbow trout in general was confirmed for the first time.

Barcoding of the Genus Culicoides (Diptera: Ceratopogonidae) in Austria-An Update of the Species Inventory Including the First Records of Three Species in Austria.

Ceratopogonidae are small nematoceran Diptera with a worldwide distribution, consisting of more than 5400 described species, divided into 125 genera. The genus Culicoides is known to comprise hematophagous vectors of medical and veterinary importance. Diseases transmitted by Culicoides spp. Such as African horse sickness virus, Bluetongue virus, equine encephalitis virus (Reoviridae) and Schmallenberg virus (Bunyaviridae) affect large parts of Europe and are strongly linked to the spread and abundance of its vectors. However, Culicoides surveillance measures are not implemented regularly nor in the whole of Austria. In this study, 142 morphologically identified individuals were chosen for molecular analyses (barcoding) of the mitochondrial cytochrome c oxidase subunit I gene (mt COI). Molecular analyses mostly supported previous morphologic identification. Mismatches between results of molecular and morphologic analysis revealed three new Culicoides species in Austria, Culicoides gornostaevae Mirzaeva, 1984, which is a member of the Obsoletus group, C. griseidorsum Kieffer, 1918 and C. pallidicornis Kieffer, 1919 as well as possible cryptic species. We present here the first Austrian barcodes of the mt COI region of 26 Culicoides species and conclude that barcoding is a reliable tool with which to support morphologic analysis, especially with regard to the difficult to identify females of the medically and economically important genus Culicoides.

Diversity in prevalence and characteristics of ESBL/pAmpC producing E. coli in food in Germany.

The spread of extended-spectrum ß-lactamases (ESBLs) in Escherichia coli is a major public health issue and ESBL-producing bacteria are frequently reported in livestock. For the assessment of the role of the foodborne transmission pathway in Germany, detailed data on the prevalence and characteristics of isolates of food origin are necessary. The objective of this study was to describe the prevalence of cefotaxime resistant E. coli as well as ESBL/pAmpC-producing E. coli and their characteristics in foods in Germany. Out of 2256 food samples, the highest prevalence of cefotaxime resistant E. coli was observed in chicken meat (74.9%), followed by turkey meat (40.1%). Prevalence in beef, pork and minced meat was considerably lower (4.2-15.3%). Whereas 18.0% of the raw milk samples, collected at farm level were positive, this was true only for few cheese samples (1.3%). In one out of 399 vegetable samples a cefotaxime-resistant E. coli was isolated. ESBL resistance genes of the CTX-M-group (10.1% of all samples) were most frequently detected, followed by genes of the pAmpC (2.6%), SHV (2.0%) and TEM (0.8%) families. Distribution of ESBL/AmpC-encoding E. coli resistance genes and E. coli phylogroups was significantly different between the chicken related food samples and all other food items. Our study results reflect that consumers might get exposed to ESBL/pAmpC-producing E. coli through several food chains. These results together with those collected at primary production and in the human population in other studies will allow more detailed analysis of the foodborne pathways, considering transmission from livestock populations to food at retail and to consumers in Germany.

Xenomonitoring of Mosquitoes (Diptera: Culicidae) for the Presence of Filarioid Helminths in Eastern Austria.

Information on mosquito-borne filarioid helminths in Austria is scarce, but recent discoveries of Dirofilaria repens indicate autochthonous distribution of this parasite in Eastern Austria. In the current xenomonitoring study, more than 48,000 mosquitoes were collected in Eastern Austria between 2013 and 2015, using different sampling techniques and storage conditions, and were analysed in pools with molecular tools for the presence of filarioid helminth DNA. Overall, DNA of D. repens, Setaria tundra, and two unknown filarioid helminths were documented in twenty mosquito pools within the mitochondrial cox1 gene (barcode region). These results indicate that S. tundra, with roe deer as definite hosts, is common in Eastern Austria, with most occurrences in floodplain mosquitoes (e.g., Aedes vexans). Moreover, DNA of D. repens was found in an Anopheles plumbeus mosquito close to the Slovakian border, indicating that D. repens is endemic in low prevalence in Eastern Austria. This study shows that xenomonitoring is an adequate tool to analyse the presence of filarioid helminths, but results are influenced by mosquito sampling techniques, storage conditions, and molecular protocols.

Trypanosomatid parasites in Austrian mosquitoes.

Trypanosomatid flagellates have not been studied in Austria in any detail. In this study, specific nested PCR, targeted on the ribosomal small subunit, was used to determine the occurrence and diversity of trypanosomatids in wild-caught mosquitoes sampled across Eastern Austria in the years 2014-2015. We collected a total of 29,975 mosquitoes of 19 species divided in 1680 pools. Of these, 298 (17.7%), representing 12 different mosquito species, were positive for trypanosomatid DNA. In total, seven trypanosomatid spp. were identified (three Trypanosoma, three Crithidia and one Herpetomonas species), with the highest parasite species diversity found in the mosquito host Coquillettidia richiardii. The most frequent parasite species belonged to the mammalian Trypanosoma theileri/cervi species complex (found in 105 pools; 6.3%). The avian species T. culicavium (found in 69 pools; 4.1%) was only detected in mosquitoes of the genus Culex, which corresponds to their preference for avian hosts. Monoxenous trypanosomatids of the genus Crithidia and Herpetomonas were found in 20 (1.3%) mosquito pools. One third (n = 98) of the trypanosomatid positive mosquito pools carried more than one parasite species. This is the first large scale study of trypanosomatid parasites in Austrian mosquitoes and our results are valuable in providing an overview of the diversity of these parasites in Austria.

Cost distribution of bluetongue surveillance and vaccination programmes in Austria and Switzerland (2007-2016).

Bluetongue virus (BTV) is an emerging transboundary disease in Europe, which can cause significant production losses among ruminants. The analysis presented here assessed the costs of BTV surveillance and vaccination programmes in Austria and Switzerland between 2007 and 2016. Costs were compared with respect to time, type of programme, geographical area and who was responsible for payment. The total costs of the BTV vaccination and surveillance programmes in Austria amounted to €23.6 million, whereas total costs in Switzerland were €18.3 million. Our analysis demonstrates that the costs differed between years and geographical areas, both within and between the two countries. Average surveillance costs per animal amounted to approximately €3.20 in Austria compared with €1.30 in Switzerland, whereas the average vaccination costs per animal were €6.20 in Austria and €7.40 in Switzerland. The comparability of the surveillance costs is somewhat limited, however, due to differences in each nation's surveillance (and sampling) strategy. Given the importance of the export market for cattle production, investments in such programmes are more justified for Austria than for Switzerland. The aim of the retrospective assessment presented here is to assist veterinary authorities in planning and implementing cost-effective and efficient control strategies for emerging livestock diseases.

Avian Plasmodium in Eastern Austrian mosquitoes.

BACKGROUND: Insect vectors, namely mosquitoes (Diptera: Culicidae), are compulsory for malaria parasites (Plasmodium spp.) to complete their life cycle. Despite this, little is known about vector competence of different mosquito species for the transmission of avian malaria parasites. METHODS: In this study, nested PCR was used to determine Plasmodium spp. occurrence in pools of whole individuals, as well as the diversity of mitochondrial cytochrome b gene sequences in wild-caught mosquitoes sampled across Eastern Austria in 2013-2015. RESULTS: A total of 45,749 mosquitoes in 2628 pools were collected, of which 169 pools (6.43%) comprising 9 mosquito species were positive for avian Plasmodium, with the majority of positives in mosquitoes of Culex pipiens s.l./Culex torrentium. Six different avian Plasmodium lineages were found, the most common were Plasmodium vaughani SYAT05, Plasmodium sp. Linn1 and Plasmodium relictum SGS1. In 2014, mosquitoes of the Culex pipiens complex were genetically identified and Culex pipiens f. pipiens presented with the highest number of avian Plasmodium positives (n = 37; 16.74%). Despite this, the minimum infection rate (MIR) was highest in Culex torrentium (5.36%) and Culex pipiens f. pipiens/f. molestus hybrids (5.26%). During 2014 and 2015, seasonal and annual changes in Plasmodium lineage distribution were also observed. In both years P. vaughani SYAT05 dominated at the beginning of the sampling period to be replaced later in the year by P. relictum SGS1 (2014) and Plasmodium sp. Linn1 (2015). CONCLUSIONS: This is the first large-scale study of avian Plasmodium parasites in Austrian mosquitoes. These results are of special interest, because molecular identification of the taxa of the Cx. pipiens complex and Cx. torrentium enabled the determination of Plasmodium prevalence in the different mosquito taxa and hybrids of this complex. Since pools of whole insects were used, it is not possible to assert any vector competence in any of the examined mosquitoes, but the results are nonetheless valuable in providing an overview of avian Plasmodium species and lineages present in Austria.