First Record of Mosquito-Borne Kyzylagach Virus in Central Europe.

RNA of Kyzylagach virus (KYZV), a Sindbis-like mosquito-borne alphavirus from Western equine encephalitis virus complex, was detected in four pools (out of 221 pools examined), encompassing 10,784 female Culex modestus mosquitoes collected at a fishpond in south Moravia, Czech Republic, with a minimum infection rate of 0.04%. This alphavirus was never detected in Central Europe before.

Bartonella species in medically important mosquitoes, Central Europe.

Here, we provide the first mass molecular screening of medically important mosquitoes for Bartonella species using multiple genetic markers. We examined a total of 72,115 mosquito specimens, morphologically attributed to Aedes vexans (61,050 individuals), Culex pipiens (10,484 individuals) and species of the Anopheles maculipennis complex (581 individuals) for Bartonella spp. The initial screening yielded 63 Bartonella-positive A. vexans mosquitoes (mean prevalence 0.1%), 34 Bartonella-positive C. pipiens mosquitoes (mean prevalence 0.3%) and 158 Bartonella-positive A. maculipennis group mosquitoes (mean prevalence 27.2%). Several different Bartonella ITS sequences were recovered. This study highlights the need for molecular screening of mosquitoes, the most important vectors of arthropod-borne pathogens, for potential bacterial agents.

The invasive Asian tiger mosquito Aedes albopictus (Diptera: Culicidae) in the Czech Republic: Repetitive introduction events highlight the need for extended entomological surveillance.

In the framework of a regional collaborative project between authorities and scientists, evidence was found of repeated introduction of Aedes (Stegomyia) albopictus (Skuse) alongside the main road entrances (E461 and E65) connecting Austria and the Slovak Republic with the Czech Republic. In comparison to data from 2012 (17 specimens collected on three occasions), the seasons 2016 (66 specimens on ten occasions) and 2017 (90 specimens on eight occasions) show an apparent increase of introduction events as well as of mosquito numbers and underline the need for more intense surveillance activities.

Urbanization impact on mosquito community and the transmission potential of filarial infection in central Europe.

BACKGROUND: Despite long-term research on dirofilariosis in Slovakia, little attention has thus far been paid to Dirofilaria vectors. The particular aim of the present study was molecular screening for filarioid parasites in two different habitats of Bratislava, the capital city of Slovakia. In addition, the effect of urbanisation on mosquito species abundance and composition, associated with the risk of mosquito-borne infections, was studied and discussed. METHODS: Mosquitoes were identified by morphological features, and molecular methods were also used for determination of selected individuals belonging to cryptic species from the Anopheles maculipennis and Culex pipiens complexes. The presence of filarioid DNA (Dirofilaria repens, Dirofilaria immitis and Setaria spp.) was detected using standard PCR approaches and sequencing. RESULTS: A total of 6957 female mosquitoes were collected for the study. Overall, the most abundant mosquito species was Aedes vexans, closely followed by unidentified members of the Cx. pipiens complex and the less numerous but still plentiful Ochlerotatus sticticus species. Further investigation of mosquito material revealed 4.26% relative prevalence of Dirofilaria spp., whereby both species, D. repens and D. immitis, were identified. The majority of positive mosquito pools had their origin in a floodplain area on the outskirts of the city, with a relative prevalence of 5.32%; only two mosquito pools (1.26%) were shown to be positive in the residential zone of Bratislava. Setaria spp. DNA was not detected in mosquitoes within this study. CONCLUSIONS: The study presented herein represents initial research focused on molecular mosquito screening for filarioid parasites in urban and urban-fringe habitats of Bratislava, Slovakia. Molecular analyses within the Cx. pipiens complex identified two biotypes: Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus. To our knowledge, Dirofilaria spp. were detected for the first time in Slovakia in mosquitoes other than Ae. vexans, i.e. D. repens in Anopheles messeae and unidentified members of An. maculipennis and Cx. pipiens complexes, and D. immitis in Coquillettidia richiardii and Cx. pipiens biotype pipiens. Both dirofilarial species were found in Och. sticticus. The suitable conditions for the vectors' biology would represent the main risk factor for dirofilariosis transmission.

Cryptic species Anopheles daciae (Diptera: Culicidae) found in the Czech Republic and Slovakia.

We report the distribution of mosquitoes of the maculipennis complex in two distinct areas of the Czech Republic (Bohemia and South Moravia) and in one locality of neighbouring Slovakia with emphasis on the detection of the newly described cryptic species Anopheles daciae (Linton, Nicolescu & Harbach, 2004). A total of 691 mosquitoes were analysed using a species-specific multiplex PCR assay to differentiate between the members of the maculipennis complex. In the Czech Republic, we found Anopheles maculipennis (with a prevalence rate of 1.4%), Anopheles messeae (49.0%) and Anopheles daciae (49.6%). In Slovakia, only An. messeae (52.1%) and An. daciae (47.9%) were detected. In this study, An. daciae was documented for the first time in the two countries where it represented a markedly higher proportion of maculipennis complex species (with an overall prevalence almost reaching 50%) in comparison to previous reports from Germany, Romania and Poland. The determination of the differential distribution of maculipennis complex species will contribute to assessing risks of mosquito-borne diseases such as malaria or dirofilariasis.

West Nile virus in overwintering mosquitoes, central Europe.

BACKGROUND: West Nile virus (WNV) is currently the most important mosquito-borne pathogen spreading in Europe. Data on overwintering of WNV in mosquitoes are crucial for understanding WNV circulation in Europe; nonetheless, such data were not available so far. RESULTS: A total of 28,287 hibernating mosquitoes [27,872 Culex pipiens, 73 Anopheles maculipennis (sensu lato), and 342 Culiseta annulata], caught in February or March between 2011 and 2017 in a WNV-endemic region of South Moravia, Czech Republic, were screened for the presence of WNV RNA. No WNV positive pools were found from 2011 to 2016, while lineage 2 WNV RNA was detected in three pools of Culex pipens mosquitoes collected in 2017 at two study sites. CONCLUSIONS: To the best of our knowledge, this is the first record of WNV RNA in overwintering mosquitoes in Europe. The data support the hypothesis of WNV persistence in mosquitoes throughout the winter season in Europe.

Serological Survey for West Nile Virus in Wild Artiodactyls, Southern Moravia (Czech Republic).

Antibodies to West Nile virus (WNV) were examined by plaque-reduction neutralization test in the blood sera of 1023 wild artiodactyls: 105 roe deer (Capreolus capreolus), 148 red deer (Cervus elaphus), 287 fallow deer (Dama dama), 71 mouflons (Ovis musimon), and 412 wild boars (Sus scrofa), sampled in South Moravian district of Breclav (Czech Republic) in the years 1990-2008. Neutralizing antibodies were detected in 5.9% of wild ruminants (4.8% roe deer, 4.1% red deer, 6.3% fallow deer, 9.9% mouflons) and 4.1% of wild boars, with titers ranging between 1:20 and 1:320. The results indicate that WNV has circulated in wild artiodactyls at a variable frequency during the years in the area.

First report of Rickettsia raoultii and Rickettsia helvetica in Dermacentor reticulatus ticks from the Czech Republic.

Tick-borne rickettsioses nowadays present a significant health threat among emerging tick-borne infections. In the study, we screened a total of 997 questing adult Dermacentor reticulatus ticks (576 females and 421 males) collected in Southern Moravia (Czech Republic) from April to September of 2010 and 2011 for the presence of human pathogenic rickettsiae. Here we provide the first evidence on the occurrence of human pathogenic Rickettsia raoultii in the local D. reticulatus population indicating possible future emergence of Dermacentor-borne necrotic erythema and lymphadenopathy (DEBONEL) in the region.

Molecular detection and phylogenetic analysis of Hepatozoon spp. in questing Ixodes ricinus ticks and rodents from Slovakia and Czech Republic.

By amplification and sequencing of 18S rRNA gene fragments, Hepatozoon spp. DNA was detected in 0.08 % (4/5057) and 0.04 % (1/2473) of questing Ixodes ricinus ticks from Slovakia and Czech Republic, respectively. Hepatozoon spp. DNA was also detected in spleen and/or lungs of 4.45 % (27/606) of rodents from Slovakia. Prevalence of infection was significantly higher in Myodes glareolus (11.45 %) than in Apodemus spp. (0.28 %) (P < 0.001). Sequencing of 18S rRNA Hepatozoon spp. gene amplicons from I. ricinus showed 100 % identity with Hepatozoon canis isolates from red foxes or dogs in Europe. Phylogenetic analysis showed that at least two H. canis 18S rRNA genotypes exist in Slovakia of which one was identified also in the Czech Republic. The finding of H. canis in questing I. ricinus suggests the geographical spread of the parasite and a potential role of other ticks as its vectors in areas where Rhipicephalus sanguineus is not endemic. Sequencing of 18S rRNA gene amplicons from M. glareolus revealed the presence of two closely related genetic variants, Hepatozoon sp. SK1 and Hepatozoon sp. SK2, showing 99-100 % identity with isolates from M. glareolus from other European countries. Phylogenetic analysis demonstrates that 18S rRNA variants SK1 and SK2 correspond to previously described genotypes UR1 and UR2 of H. erhardovae, respectively. The isolate from Apodemus flavicollis (Hepatozoon sp. SK3b) was 99 % identical with isolates from reptiles in Africa and Asia. Further studies are necessary to identify the taxonomic status of Hepatozoon spp. parasitizing rodents in Europe and the host-parasite interactions in natural foci.

Molecular survey of arthropod-borne pathogens in sheep keds (Melophagus ovinus), Central Europe.

In the study, we screened a total of 399 adult sheep keds (Melophagus ovinus) for the presence of RNA and DNA specific for arboviral, bacterial, and protozoan vector-borne pathogens. All investigated keds were negative for flaviviruses, phleboviruses, bunyaviruses, Borrelia burgdorferi, Rickettsia spp., Anaplasma phagocytophilum, "Candidatus Neoehrlichia mikurensis," and Babesia spp. All ked pools were positive for Bartonella DNA. The sequencing of the amplified fragments of the gltA and 16S-23S rRNA demonstrated a 100 % homology with Bartonella melophagi previously isolated from a sheep ked and from human blood in the USA. The identification of B. melophagi in sheep keds in Central Europe highlights needs extending a list of hematophagous arthropods beyond ticks and mosquitoes for a search of emerging arthropod-borne pathogens.

Neglected tick-borne pathogens in the Czech Republic, 2011-2014.

In this study, we screened a total of 2473 questing (years 2011-2014) and 199 engorged (years 2013 and 2014) Ixodes ricinus ticks for the presence of Rickettsia spp., "Candidatus Neoehrlichia mikurensis", Anaplasma phagocytophilum, and Babesia spp. Host-seeking ticks were collected at three study sites corresponding to natural woodland, urban park and pastureland ecosystem, and analyzed using molecular techniques. All pathogens tested were present at all study sites. The prevalence rates for Rickettsia spp., 'Candidatus Neoehrlichia mikurensis', Anaplasma phagocytophilum, and Babesia spp. ranged from 2.6% to 9.2%, 0.8% to 11.6%, 0% to 12.1%, and 0% to 5.2%, respectively. Engorged I. ricinus ticks collected from sheep on pastureland in the years 2013 and 2014 yielded prevalence rates 7.4% and 6.3%, respectively, for Rickettsia spp., 38.5% and 14.1% for 'Candidatus N. mikurensis', 18.5% and 12.5% for A. phagocytophilum, and 4.4% and 0.0% for Babesia spp. Monitoring of neglected tick-borne pathogens within the scope of epidemiological surveillance is an important tool for prevention and control of human tick-borne infections.

Overwintering of Uranotaenia Unguiculata Adult Females in Central Europe: A Possible Way of Persistence of the Putative New Lineage of West Nile Virus?

We report the overwintering of Uranotaenia unguiculata adult females in Central Europe (Czech Republic, Hungary, Austria). This finding suggests a potential mode of winter persistence of putative novel lineage of West Nile virus in the temperate regions of Europe.

Co-circulation of Usutu virus and West Nile virus in a reed bed ecosystem.

BACKGROUND: Mosquito-borne flaviviruses are a major public health threat in many countries worldwide. In Central Europe, West Nile virus (WNV) and Usutu virus (USUV), both belonging to the Japanese encephalitis virus group (Flaviviridae) have emerged in the last decennium. Surveillance of mosquito vectors for arboviruses is a sensitive tool to evaluate virus circulation and consequently to estimate the public health risk. METHODS: Mosquitoes (Culicidae) were collected at South-Moravian (Czech Republic) fishponds between 2010 and 2014. A total of 61,770 female Culex modestus Ficalbi mosquitoes, pooled to 1,243 samples, were examined for flaviviruses by RT-PCR. RESULTS: One pool proved positive for USUV RNA. Phylogenetic analysis demonstrated that this Czech USUV strain is closely related to Austrian and other Central European strains of the virus. In addition, nine strains of WNV lineage 2 were detected in Cx. modestus collected in the same reed bed ecosystem. CONCLUSIONS: This is the first detection of USUV in Cx. modestus. The results indicate that USUV and WNV may co-circulate in a sylvatic cycle in the same habitat, characterised by the presence of water birds and Cx. modestus mosquitoes, serving as hosts and vectors, respectively, for both viruses.

Zoonotic Dirofilaria repens (Nematoda: Filarioidea) in Aedes vexans mosquitoes, Czech Republic.

The surveillance of vectors for arthropod-borne pathogens is nowadays an important tool in surveillance programmes throughout Europe. Whereas many studies have been performed to screen arthropods for viruses or bacterial pathogens, only limited information is available concerning the geographical distribution and vector range of pathogenic filariae in Central Europe. To consider the prevalence of filarial parasites in mosquito vectors, we performed a molecular survey of mosquitoes for filarial DNA. Mosquito collection was conducted at six study sites in the South Moravian region (Czech Republic) close to the borders with Slovakia and Austria from 2009 to 2011. Molecular screening of mosquitoes was conducted using conventional PCR with primers designed to amplify the mitochondrial cytochromoxidase subunit I gene as well as the partial 5.8S ribosomal RNA gene. A total of 13,222 mosquitoes belonging to six species were captured and distributed into 237 pools with different numbers of individuals. Overall, four pools were positive for Dirofilaria repens (a minimum infection rate 0.03%) at two study sites (both natural and urban). Another filarial parasite detected during a study into Aedes vexans mosquitoes revealed the closest homology to Setaria spp. We detected specific D. repens DNA in Ae. vexans mosquitoes for the first time in the Czech Republic and confirmed the circulation of Dirofilaria spp. in a natural focus of infection providing an epidemiological link between autochthonous canine cases and mosquito vectors in the area studied.