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Aedes koreicus is an invasive mosquito species originating from East Asia. It has recently been introduced into several countries in Southern, Central and Eastern Europe as well as Central Asia in many of which it has successfully established populations. The biology and ecological requirements of the species are largely unknown, but it is considered as a potential vector of pathogens that requires careful monitoring. We report here the first detection of Ae. koreicus in the Czech Republic, based on a citizen report.

Bats are an important reservoir for many viral pathogens in humans. However, their role in the transmission of bacterial pathogens is neglected, as is that of their ectoparasites. This study focuses on the molecular detection of Bartonella spp. in bat bugs Cimex pipistrelli using partial sequences of gltA (citrate synthase), ssrA (transfer messenger RNA, tmRNA), and the 16S-23S rDNA internal transcribed spacer (ITS) region as targets. Bartonella DNA was detected in 2/112 (1.79% prevalence) samples from bat bugs. Due to the fact that bat bugs can sporadically bite humans, more extensive surveillance and vector competence studies are needed to ascertain zoonotic risk of bat-associated Bartonella spp.

Cile: Invazni druhy komarů představuji pro clověka významnou zdravotni hrozbu, protože jsou vektory řady zavažných patogenů, jako jsou viry dengue, chikungunya ci zika, a je tedy nezbytne provadět rutinni surveillance těchto vektorů. Metodika: V letech 2016 až 2020 byl proveden výzkum zaměřený na sledovani výskytu invaznich druhů komarů na uzemi jižni Moravy (Břeclavsko a Znojemsko) s využitim pasti typu ovitrap (odchyt vajec) doplněných o pasti BG-Sentinel (odchyt dospělců). Výsledky: Invazni druh Aedes albopictus byl prokazan ve vsech sledovaných lokalitach. Zavěr: Výsledky monitoringu naznacuji riziko možneho usidleni a nasledneho siřeni tohoto druhu v důsledku globalnich klimatických změn.

Kidney samples from 300 bat cadavers from the Czech and Slovak Republics were tested for

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.

Rejection (nomen rejiciendum) of the name Borreliella and all new combinations therein is being requested on grounds of risk to human health and patient safety (Principle 1, subprinciple 2 and Rule 56a) and violation to aim for stability of names, to avoid useless creation of names (Principle 1, subprinciple 1 and 3) and that names should not be changed without sufficient reason (Principle 9 of the International Code of Nomenclature of Prokaryotes).

Background Aedes japonicus is a mosquito species native to North-East Asia that was first found established outside its original geographic distribution range in 1998 and has since spread massively through North America and Europe. In the Czech Republic, the species was not reported before 2021. Methods Aedes invasive mosquitoes (AIM) are routinely surveyed in the Czech Republic by ovitrapping at potential entry ports. This surveillance is supported by appeals to the population to report uncommon mosquitoes. The submission of an Ae. japonicus specimen by a citizen in 2021 was followed by local search for aquatic mosquito stages in the submitter’s garden and short-term adult monitoring with encephalitis virus surveillance (EVS) traps in its surroundings. Collected Ae. japonicus specimens were subjected to nad4 haplotype and microsatellite analyses. Results Aedes japonicus was detected for the first time in the Czech Republic in 2021. Aquatic stages and adults were collected in Prachatice, close to the Czech-German border, and eggs in Mikulov, on the Czech-Austrian border. Morphological identification was confirmed by molecular taxonomy. Genetic analysis of specimens and comparison of genetic data with those of other European populations, particularly from Germany, showed the Prachatice specimens to be most closely related to a German population. The Mikulov specimens were more distantly related to those, with no close relatives identifiable. Conclusions Aedes japonicus is already widely distributed in Germany and Austria, two countries neighbouring the Czech Republic, and continues to spread rapidly in Central Europe. It must therefore be assumed that the species is already present at more than the two described localities in the Czech Republic and will further spread in this country. These findings highlight the need for more comprehensive AIM surveillance in the Czech Republic. Graphical Abstract

West Nile virus lineage 2 (WNV-2) was detected in the brain of 17 goshawks (Accipiter gentilis) that succumbed to neuroinvasive disease in the Czech Republic during 2018: twelve birds were captive and five wild. Furthermore, two wild sparrowhawks (Accipiter nisus) and three other captive birds of prey (golden eagle Aquila chrysaetos, hybrid saker falcon Falco cherrug × F. rusticolus and Harris's hawk Parabuteo unicinctus) also died due to WNV encephalitis. The 2018 outbreak in Czech raptors clearly reflects a new epidemiological situation and indicates an increasing risk of both raptor and human infection with WNV-2 in the country.

Background Despite the known circulation of West Nile virus (WNV) and Usutu virus (USUV) in Slovakia, no formal entomological surveillance programme has been established there thus far. Aim To conduct contemporaneous surveillance of WNV and USUV in different areas of Slovakia and to assess the geographical spread of these viruses through mosquito vectors. The first autochthonous human WNV infection in the country is also described. Methods Mosquitoes were trapped in four Slovak territorial units in 2018 and 2019. Species were characterised morphologically and mosquito pools screened for WNV and USUV by real-time reverse-transcription PCRs. In pools with any of the two viruses detected, presence of pipiens complex group mosquitoes was verified using molecular approaches. Results Altogether, 421 pools containing in total 4,508 mosquitoes were screened. Three pools tested positive for WNV and 16 for USUV. USUV was more prevalent than WNV, with a broader spectrum of vectors and was detected over a longer period (June–October vs August for WNV). The main vectors of both viruses were Culex pipiens sensu lato. Importantly, WNV and USUV were identified in a highly urbanised area of Bratislava city, Slovakias’ capital city. Moreover, in early September 2019, a patient, who had been bitten by mosquitoes in south-western Slovakia and who had not travelled abroad, was laboratory-confirmed with WNV infection. Conclusion The entomological survey results and case report increase current understanding of the WNV and USUV situation in Slovakia. They underline the importance of vector surveillance to assess public health risks posed by these viruses.

Hyalomma spp. ticks (Acari: Ixodidae) represent a public health threat because of their prominent role in the transmission of Crimean- Congo haemorrhagic fever (CCHF) virus. Moreover, these ticks can transmit other medically important arboviruses such as Thogoto, Dhori, West Nile, and Bhanja viruses; human pathogenic bacteria like Rickettsia conorii, R. aeschlimannii, and Anaplasma marginale; and protozoans like Theileria annulata and Babesia caballi (Huba ´lek and Rudolf, 2011). Many records of larval and nymphal H. marginatum ticks on birds migrating from North Africa and Southern Europe into Central and Northern Europe have been published.

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.

Rejection (

Spirochetal bacteria were successfully isolated from mosquitoes (Culex pipiens, Aedes cinereus) in the Czech Republic between 1999 and 2002. Preliminary 16S rRNA phylogenetic sequence analysis showed that these strains differed significantly from other spirochetal genera within the family Spirochaetaceae and suggested a novel bacterial genus in this family. To obtain more comprehensive genomic information of these isolates, we used Illumina MiSeq and Oxford Nanopore technologies to sequence four genomes of these spirochetes (BR151, BR149, BR193, BR208). The overall size of the genomes varied between 1.68 and 1.78 Mb; the GC content ranged from 38.5 to 45.8%. Draft genomes were compared to 36 publicly available genomes encompassing eight genera from the class Spirochaetes. A phylogeny generated from orthologous genes across all taxa and the percentage of conserved proteins (POCP) confirmed the genus status of these novel spirochetes. The genus Entomospira gen. nov. is proposed with BR151 selected as type species of the genus. For this isolate and the closest related isolate, BR149, we propose the species name Entomospira culicis sp. nov. The two other isolates BR208 and BR193 are named Entomospira nematocera sp. nov. (BR208) and Entomospira entomophilus sp. nov. (BR193). Finally, we discuss their interesting phylogenetic positioning.

We found a male Hyalomma rufipes Koch, 1844 tick feeding on a horse grazing near Valtice, south Moravia, Czech Republic on October 24, 2019. The horse was born in Czechland and did not leave the country at least during the last five years. Relevant findings of Hyalomma ticks in other parts of central Europe are reviewed, including also records of pre-imaginal Hyalomma marginatum complex ticks on migrating birds all over Europe.

To determine whether hepatitis E virus (HEV) was distributed in the population of wild boars in South Moravia between 1990 and 2008, a total of 366 samples of archived sera from wild boars were investigated using serological (commercial ELISA) and molecular (RT-qPCR) methods. A total of 31 (8.5%) wild boars were seropositive, and from two of them, RNA sequences were recovered by nested RT-PCR. The presented results, with one of the oldest animal's HEV-positive serum (collected in 1990), suggest that wild boars may be a reservoir of HEV in the Czech Republic and that this virus has been circulating in studied areas for more than 20 years.

During 2008–2018, we collected samples from rodents and patients throughout the Czech Republic and characterized hantavirus isolates. We detected Dobrava-Belgrade and Puumala orthohantaviruses in patients and Dobrava-Belgrade, Tula, and Seewis orthohantaviruses in rodents. Increased knowledge of eco-epidemiology of hantaviruses will improve awareness among physicians and better outcomes of patients.

A total of 7778 host-seeking adult Dermacentor reticulatus (Ixodida: Ixodidae) ticks were examined for the prevalence of Francisella tularensis holarctica (Thiotrichales: Francisellaceae) in a natural focus of tularaemia in the floodplain forest-meadow ecosystem along the lower reaches of the Dyje (Thaya) river in South Moravia (Czech Republic) between 1995 and 2013. Ticks were pooled (10 specimens per pool) and their homogenates inoculated subcutaneously in 4-week-old specific pathogen-free mice. Dead mice were sectioned, their spleens cultivated on thioglycollate-glucose-blood agar and impression smears from the spleen, liver and heart blood were Giemsa-stained. Sixty-four pools were positive for F.tularensis: the overall minimum infection rate (MIR) was 0.82%. Overall MIRs for the 4714 female and 3064 male D.reticulatus examined were 0.89 and 0.72%, respectively; MIRs fluctuated across years between 0.0 and 2.43%. The estimated bacterial load in infected ticks varied from 0.84 to 5.34 log(10) infectious F.tularensis cells per tick (i.e. from about seven to 220000 cells). Ticks with low loads were more prevalent; more than 1000 infectious cells were detected in 24 ticks (0.3% of all ticks and 37.5% of infected ticks). Monitoring of D.reticulatus for the presence and cell numbers of F.tularensis may be a valuable tool in the surveillance of tularaemia.

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.

The way in which European genetic variants of Anaplasma phagocytophilum circulate in their natural foci and which variants cause disease in humans or livestock remains thus far unclear. Red deer and roe deer are suggested to be reservoirs for some European A. phagocytophilum strains, and Ixodes ricinus is their principal vector. Based on groEL gene sequences, five A. phagocytophilum ecotypes have been identified. Ecotype I is associated with the broadest host range, including strains that cause disease in domestic animals and humans. Ecotype II is associated with roe deer and does not include zoonotic strains. In the present study, questing I. ricinus were collected in urban, pasture, and natural habitats in the Czech Republic, Germany, and Slovakia. A fragment of the msp2 gene of A. phagocytophilum was amplified by real-time PCR in DNA isolated from ticks. Positive samples were further analyzed by nested PCRs targeting fragments of the 16S rRNA and groEL genes, followed by sequencing. Samples were stratified according to the presence/absence of roe deer at the sampling sites. Geographic origin, habitat, and tick stage were also considered. The probability that A. phagocytophilum is a particular ecotype was estimated by a generalized linear model. Anaplasma phagocytophilum was identified by genetic typing in 274 I. ricinus ticks. The majority belonged to ecotype I (63.9%), 28.5% were ecotype II, and both ecotypes were identified in 7.7% of ticks. Ecotype II was more frequently identified in ticks originating from a site with presence of roe deer, whereas ecotype I was more frequent in adult ticks than in nymphs. Models taking into account the country-specific, site-specific, and habitat-specific aspects did not improve the goodness of the fit. Thus, roe deer presence in a certain site and the tick developmental stage are suggested to be the two factors consistently influencing the occurrence of a particular A. phagocytophilum ecotype in a positive I. ricinus tick.

Background Anaplasma phagocytophilum is currently regarded as a single species. However, molecular studies indicate that it can be subdivided into ecotypes, each with distinct but overlapping transmission cycle. Here, we evaluate the interactions between and within clusters of haplotypes of the bacterium isolated from vertebrates and ticks, using phylogenetic and network-based methods. Methods The presence of A. phagocytophilum DNA was determined in ticks and vertebrate tissue samples. A fragment of the groEl gene was amplified and sequenced from qPCR-positive lysates. Additional groEl sequences from ticks and vertebrate reservoirs were obtained from GenBank and through literature searches, resulting in a dataset consisting of 1623 A. phagocytophilum field isolates. Phylogenetic analyses were used to infer clusters of haplotypes and to assess phylogenetic clustering of A. phagocytophilum in vertebrates or ticks. Network-based methods were used to resolve host-vector interactions and their relative importance in the segregating communities of haplotypes. Results Phylogenetic analyses resulted in 199 haplotypes within eight network-derived clusters, which were allocated to four ecotypes. The interactions of haplotypes between ticks, vertebrates and geographical origin, were visualized and quantified from networks. A high number of haplotypes were recorded in the tick Ixodes ricinus. Communities of A. phagocytophilum recorded from Korea, Japan, Far Eastern Russia, as well as those associated with rodents had no links with the larger set of isolates associated with I. ricinus, suggesting different evolutionary pressures. Rodents appeared to have a range of haplotypes associated with either Ixodes trianguliceps or Ixodes persulcatus and Ixodes pavlovskyi. Haplotypes found in rodents in Russia had low similarities with those recorded in rodents in other regions and shaped separate communities. Conclusions The groEl gene fragment of A. phagocytophilum provides information about spatial segregation and associations of haplotypes to particular vector-host interactions. Further research is needed to understand the circulation of this bacterium in the gap between Europe and Asia before the overview of the speciation features of this bacterium is complete. Environmental traits may also play a role in the evolution of A. phagocytophilum in ecotypes through yet unknown relationships.

West Nile virus (WNV) is a medically important emerging arbovirus causing serious neuroinfections in humans and against which no approved antiviral therapy is currently available. In this study, we demonstrate that 2=-C-methyl- or 4=-azido-modified nucleosides are highly effective inhibitors of WNV replication, showing nanomolar or low micromolar anti-WNV activity and negligible cytotoxicity in cell culture. One representative of C2=-methylated nucleosides, 7-deaza-2=-Cmethyladenosine, significantly protected WNV-infected mice from disease progression and mortality. Twice daily treatment at 25 mg/kg starting at the time of infection resulted in 100% survival of the mice. This compound was highly effective, even if the treatment was initiated 3 days postinfection, at the time of a peak of viremia, which resulted in a 90% survival rate. However, the antiviral effect of 7-deaza-2=-Cmethyladenosine was absent or negligible when the treatment was started 8 days postinfection (i.e., at the time of extensive brain infection). The 4=-azido moiety appears to be another important determinant for highly efficient inhibition of WNV replication in vitro. However, the strong anti-WNV effect of 4=-azidocytidine and 4=- azido-aracytidine was cell type dependent and observed predominantly in porcine kidney stable (PS) cells. The effect was much less pronounced in Vero cells. Our results indicate that 2=-C-methylated or 4=-azidated nucleosides merit further investigation as potential therapeutic agents for treating WNV infections as well as infections caused by other medically important flaviviruses.

The risks of depletion of energy reserves and encountering lethally low temperatures are considered as two important mortality factors that may limit winter survival of mosquito, Culex pipiens f. pipiens populations. Here we show that the autumn females carry lipid reserves, which are safely sufficient for at least two overwintering periods, provided the females diapausing at temperatures typical for underground spaces (0 °C - 8 °C) would continuously rest at a standard metabolic rate (SMR). The overwintering females, however, switch from SMR to much higher metabolic rate during flight, either seeking for optimal microhabitat within the shelter or in response to disturbances by air current or predator attack. These behaviors result in fast oxidation of lipid reserves and, therefore, the autumn load of energy reserves may actually limit winter survival under specific circumstances. Next, we show that the level of females' cold hardiness is physiologically set relatively weak for overwintering in open field, above-ground habitats, but is ecologically entirely sufficient for overwintering in most underground spaces. The characteristics of suitable overwintering shelters are: no or limited risk of contact with ice crystals, no or limited air movements, winter temperatures relatively stable between +2 and + 6 °C, winter minimum does not drop below −4 °C for longer than one week, or below −8 °C for longer than 1 day.

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.

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.

West Nile virus (WNV) is a medically important emerging arbovirus causing serious neuroinfections in humans and against which no approved antiviral therapy is currently available. In this study, we demonstrate that 2′-C-methyl- or 4′-azido-modified nucleosides are highly effective inhibitors of WNV replication, showing nanomolar or low micromolar anti-WNV activity and negligible cytotoxicity in cell culture. One representative of C2′-methylated nucleosides, 7-deaza-2′-C-methyladenosine, significantly protected WNV-infected mice from disease progression and mortality. Twice daily treatment at 25 mg/kg starting at the time of infection resulted in 100% survival of the mice. This compound was highly effective, even if the treatment was initiated 3 days postinfection, at the time of a peak of viremia, which resulted in a 90% survival rate. However, the antiviral effect of 7-deaza-2′-C-methyladenosine was absent or negligible when the treatment was started 8 days postinfection (i.e., at the time of extensive brain infection). The 4′-azido moiety appears to be another important determinant for highly efficient inhibition of WNV replication in vitro. However, the strong anti-WNV effect of 4′-azidocytidine and 4′-azido-aracytidine was cell type dependent and observed predominantly in porcine kidney stable (PS) cells. The effect was much less pronounced in Vero cells. Our results indicate that 2′-C-methylated or 4′-azidated nucleosides merit further investigation as potential therapeutic agents for treating WNV infections as well as infections caused by other medically important flaviviruses.

In 2017, we isolated and identified West Nile virus (WNV) lineage 2 from two dead captive goshawks (Accipiter gentilis), for the first time in the Czech Republic. Goshawk might serve as an early indicator species for the ongoing WNV emergence in several European countries.

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.

The incidence of tick-borne diseases caused by Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp. has been rising in Europe in recent decades. Early pre-assessment of acarological hazard still represents a complex challenge. The aim of this study was to model Ixodes ricinus questing nymph density and its infection rate with B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in five European countries (Italy, Germany, Czech Republic, Slovakia, Hungary) in various land cover types differing in use and anthropisation (agricultural, urban and natural) with climatic and environmental factors (Normalized Difference Vegetation Index (NDVI), Normalized Difference Water Index (NDWI), Land Surface Temperature (LST) and precipitation). We show that the relative abundance of questing nymphs was significantly associated with climatic conditions, such as higher values of NDVI recorded in the sampling period, while no differences were observed among land use categories. However, the density of infected nymphs (DIN) also depended on the pathogen considered and land use. These results contribute to a better understanding of the variation in acarological hazard for Ixodes ricinus transmitted pathogens in Central Europe and provide the basis for more focused ecological studies aimed at assessing the effect of land use in different sites on tick–host pathogens interaction.

West Nile virus (WNV), the most widespread flavivirus, is distributed throughout Africa, Asia, Europe, and Australia, and since 1999, WNV has also been present in the Americas (1). Within the last 2 decades, WNV infection has caused an increasing number of cases of neuroinvasive disease in humans and become a substantial public health problem (1). Up to 8 lineages of WNV, based on genetic differences, have been proposed (1,2) (Table 1). Lineage 1 is widely distributed and further divided into lineage 1a, which includes the American strains; lineage 1b, which is also referred to as Kunjin virus and mainly described in Australia; and lineage 1c, which is also referred to as lineage 5 and comprises isolates from India. Lineage 2 has been detected in Africa and several parts of Europe, lineage 3 (Rabensburg virus) has been isolated only in the Czech Republic, and lineage 4 has been reported from Russia (3). A putative sixth lineage, based on a small genome fragment, has been described from Spain (4), and putative lineages 7 (Koutango virus) and 8 have been reported from Senegal (2). Table 1 Overview of West Nile virus lineages WNV is maintained in an enzootic cycle between mosquitoes and wild birds (1). In 2013, ≈100 Uranotaenia unguiculata Edwards, 1913, mosquitoes were trapped during mosquito-monitoring projects at Lake Neusiedl-Seewinkel National Park in Austria and near Sedlec in the Czech Republic. In Russia, Ur. unguiculata mosquitoes have been described as hosting lineage 4 WNV strains (A. Platonov, unpub. data) (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"FJ154906","term_id":"204324973","term_text":"FJ154906"}}FJ154906–49 and {"type":"entrez-nucleotide","attrs":{"text":"FJ159129","term_id":"204305343","term_text":"FJ159129"}}FJ159129–31). To determine whether Ur. unguiculata mosquitoes in Austria and the Czech Republic also host WNV, we investigated the mosquitoes collected in 2013 for the presence of WNV, focusing on lineage 4 viruses.

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.

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.

Two strains of Tahyna virus (TAHV; Orthobunyavirus, Bunyaviridae) were isolated from 4,568 (92 pools) female Anopheles hyrcanus Pallas (Diptera, Culicidae) mosquitoes collected on the fishponds in South Moravia (Czechland, central Europe) during July-August 2013. This is the first isolation of TAHV from An. hyrcanus in Europe. An. hyrcanus is a species new to Czechland since 2007; its population density was very high in the year 2013 at these ponds. The virus isolation procedure was based on intracerebral inoculation of newborn mice; moreover, the positive pools were also tested by polymerase chain reaction and found to contain TAHV RNA. An. hyrcanus, feeding preferentially on mammals including humans, may be a new potential vector for TAHV in Europe.

Growth temperature range, resistance to selective antibiotics, activities of 23 enzymes, protein fingerprints and fatty acids composition of the spirochaetal strain BR91, isolated from the Culex pipiens mosquito, were tested. The spirochaetes were grown in BSK-H Complete liquid medium. The optimal in vitro growth temperature of the strain was 33 °C. Strain BR91 was sensitive to trimethoprim, nalidixic acid, 5-fluorouracil, and tolerated phosphomycin. The strain produced acid and alkaline phosphatase, esterase (C4), esterase–lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and α-fucosidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay revealed several major proteins in the size range of 13–16 kDa, 22–30 kDa and 37–131 kDa. Fatty acid methyl ester (FAME) analysis showed that C14:0, C16:0, C18:1 ω9c and summed feature 5 (C18:2 ω6,9c and/or C18:0 anteiso) are major fatty acids. This study highlights certain phenotypic differences between strain BR91 and the Lyme disease spirochaete Borrelia burgdorferi, and supports the hypothesis that strain BR91 represents a unique taxonomical entity in a system of spirochaetal species.

Tick-borne rickettsiae are an important topic in the field of emerging infectious diseases. In the study, we screened a total of 1473 field-collected Ixodes ricinus ticks (1294 nymphs, 99 males, and 80 females) for the presence of human pathogenic rickettsiae (Rickettsia helvetica, R. monacensis, 'Candidatus Neoehrlichia mikurensis', and Anaplasma phagocytophilum) in natural and urban ecosystems using molecular techniques. The minimum infection rate (MIR) for Rickettsia spp. was found to be 2.9% in an urban park and 3.4% in a natural forest ecosystem; for 'Candidatus Neoehrlichia mikurensis', we observed MIRs of 0.4% in the city park and 4.4% in the natural habitat, while for A. phagocytophilum the MIR was 9.4% and 1.9%, respectively. Our study provides the first data on the occurrence of human pathogenic rickettsiae in questing I. ricinus ticks in the Czech Republic.

The way in which European genetic variants of Anaplasma phagocytophilum circulate in their natural foci and which variants cause disease in humans or livestock remains thus far unclear. Red deer and roe deer are suggested to be reservoirs for some European A. phagocytophilum strains, and Ixodes ricinus is their principal vector. Based on groEL gene sequences, five A. phagocytophilum ecotypes have been identified. Ecotype I is associated with the broadest host range, including strains that cause disease in domestic animals and humans. Ecotype II is associated with roe deer and does not include zoonotic strains. In the present study, questing I. ricinus were collected in urban, pasture, and natural habitats in the Czech Republic, Germany, and Slovakia. A fragment of the msp2 gene of A. phagocytophilum was amplified by real-time PCR in DNA isolated from ticks. Positive samples were further analyzed by nested PCRs targeting fragments of the 16S rRNA and groEL genes, followed by sequencing. Samples were stratified according to the presence/absence of roe deer at the sampling sites. Geographic origin, habitat, and tick stage were also considered. The probability that A. phagocytophilum is a particular ecotype was estimated by a generalized linear model. Anaplasma phagocytophilum was identified by genetic typing in 274 I. ricinus ticks. The majority belonged to ecotype I (63.9%), 28.5% were ecotype II, and both ecotypes were identified in 7.7% of ticks. Ecotype II was more frequently identified in ticks originating from a site with presence of roe deer, whereas ecotype I was more frequent in adult ticks than in nymphs. Models taking into account the country-specific, site-specific, and habitat-specific aspects did not improve the goodness of the fit. Thus, roe deer presence in a certain site and the tick developmental stage are suggested to be the two factors consistently influencing the occurrence of a particular A. phagocytophilum ecotype in a positive I. ricinus tick.

Usutu virus (USUV) is a flavivirus (Flaviviridae: Flavivirus) of an African origin transmitted among its natural hosts (diverse species of birds) by mosquitoes. The virus was introduced multiple times to Europe where it caused mortality of blackbirds (Turdus merula) and certain other susceptible species of birds. In this study, we report detection of USUV RNA in blackbirds, Culex pipiens and Cx. modestus mosquitoes in the Czech Republic, and isolation of 10 new Czech USUV strains from carcasses of blackbirds in cell culture. Multiple lineages (Europe 1, 2 and Africa 3) of USUV were found in blackbirds and mosquitoes in the southeastern part of the country. A single USUV lineage (Europe 3) was found in Prague and was likely associated with increased mortalities in the local blackbird population seen in this area in 2018. USUV genomic RNA (lineage Europe 2) was detected in a pool of Cx. pipiens mosquitoes from South Bohemia (southern part of the country), where no major mortality of birds has been reported so far, and no flavivirus RNA has been found in randomly sampled cadavers of blackbirds. The obtained data contributes to our knowledge about USUV genetic variability, distribution and spread in Central Europe.

Monitoring West Nile virus (WNV) and Usutu virus (USUV) activity now has the highest priority among mosquito-borne pathogenic viruses circulating in the European Union. This study documents a first time detection and the co-circulation of WNV lineage-2 (with the minimal prevalence of 0.46%) and USUV clade Europe 2 (with the minimal prevalence of 0.25%) in mosquitoes from the same habitat of south-western Slovakia and underlines necessity to perform rigorous surveillance in birds, mosquitoes, horses and humans in that country.

We examined 146 common coots (Fulica atra) on fishponds in central Moravia, Czech Republic, for antibodies to West Nile (WNV) and Usutu (USUV) flaviviruses. Eighteen birds reacted in the plaque-reduction neutralization test against WNV; these WNV seropositive samples were then titrated in parallel against USUV and tick-borne encephalitis virus (TBEV) to exclude flavivirus cross-reactivity. Two birds (1.4% overall) had the highest titers against WNV while 9 birds (6.2% overall) were seropositive for USUV, and in 7 birds the infecting flavivirus could not be differentiated with certainty. Our results indicate that both WNV and USUV infections occur in common coots; these birds might serve as a 'sentinel' species indicating the presence of these viruses at fishpond and wetland habitats in Central Europe.

Introduction and objective. Ixodes ricinus is the most common tick species occurring in Central Europe and it serves as a principal vector of emerging human pathogens. The aim of this study was to determine the prevalence of Babesia spp. in host-seeking I. ricinus in urban and natural habitats. Materials and methods. PCR was applied on samples to assess prevalence of Babesia spp. in questing ixodid ticks. Sequencing was used for Babesia species determination. Results. 1,473 I. ricinus ticks (1,294 nymphs, 99 males and 80 females) were examined for the presence of Babesia spp. at the two study sites. Minimum infection rate for Babesia spp. was found to be 0.5% (infected I. ricinus nymphs were only detected in the natural ecosystem). Two Babesia species were identified by sequencing: B. venatorum (formerly called Babesia sp. EU1) and B. capreoli. Conclusions. The results obtained represent the first evidence of the occurrence of B. venatorum and B. capreoli in hostseeking I. ricinus ticks in the Czech Republic.

A serological survey for West Nile virus (WNV) infection involved 395 horses from 43 administrative districts of the Czech Republic (163 animals) and 29 districts of Slovakia (232 animals), sampled between 2008 and 2011. Using a plaque-reduction neutralization microtest, antibodies to WNV were not detected in any horse from the Czech Republic, whereas 19 nonvaccinated horses from Slovakia had specific antibodies to WNV (no cross-reactions were observed with tick-borne encephalitis and Usutu flaviviruses in those animals). The seropositivity rate of nonvaccinated horses in Slovakia was 8.3% (95% confidence interval [CI] 4.7-11.9%), and autochthonous local infection with WNV occurred at least in 11, i.e., 4.8% (95% CI 2.0-7.6%) of the animals. All seropositive horses lived in six lowland districts of southern Slovakia; overall, 15.1% (95% CI 8.8-21.4%) of 126 nonvaccinated horses were seropositive in those districts, situated relatively closely to the border with Hungary, i.e., the country where WNV disease cases have been reported in birds, horses and humans since 2003.

Sedlec virus (SEDV) was isolated from the blood of a reed warbler (Acrocephalus scirpaceus) in July 1984 in South Moravia, Czech Republic. In this study first genetic data of SEDV are presented which allow an estimate on its phylogenetic and taxonomic positioning within the genus Orthobunyavirus. The phylogenetic analysis of a 369 nt long stretch within the S segment (nucleocapsid protein gene and non-structural S protein gene) indicates genetic relatedness of SEDV to Leanyer virus and Simbu group viruses, while the phylogenetic tree based on 1796 nt long sequences of the L segment (RNA-dependent RNA polymerase gene) demonstrates genetic relationship of SEDV to two yet unclassified orthobunyaviruses: I612045 virus (isolated in India in 1961) and Oyo virus (isolated in Nigeria in 1964). Considering the genetic distances and the phylogenetic analyses, SEDV might represent a novel serogroup of the Orthobunyavirus genus.

A set of 25 urease-producing, yellow-pigmented enterococci was isolated from environmental sources. Phenotypic classification divided the isolates into two phena. Both phena were characterized using 16S rRNA gene sequence analysis, DNA base composition, rep-PCR fingerprinting and automated ribotyping. The obtained data distinguished the isolates from all members of the genus Enterococcus with validly published names and placed them in the Enterococcus faecalis species group. DNA–DNA hybridization experiments, pheS and rpoA sequencing and whole-cell protein electrophoresis provided conclusive evidence for the classification of each phenon as a novel species of the genus Enterococcus , for which the names Enterococcus ureilyticus sp. nov. (type strain CCM 4629T = LMG 26676T = CCUG 48799T), inhabiting water and plants, and Enterococcus rotai sp. nov. (type strain CCM 4630T = LMG 26678T = CCUG 61593T), inhabiting water, insects (mosquitoes) and plants, are proposed.

In a set of pooled field collected Dermacentor reticulatus ticks, Rickettsia raoultii, the causative agent of Tick-borne lymphadenopathy/Dermacentor-borne necrosis erythema and lymphadenopathy, was found for the first time in Austria. The coordinates of the positive locations for tick and pathogen abundance are given and shown in a map.

From March to November 2010, a total of 68 samples of blood from 41 hunting and working dogs that never left the Czech Republic were examined. Some dogs were sampled repeatedly. Blood samples were examined by polymerase chain reaction for the presence of DNA of piroplasms with negative results. Specific IgG antibodies against Babesia canis were detected by indirect immunofluorescence test, and five dogs (12.21%) were seropositive. Titres ranged from 50 to 200. One dog was positive in two samplings within 3 months. The highest number of positive samples was taken in June. The results of this study suggest a likely contact of the examined dogs with the parasite; although in 2005, a total of 340 adult unfed Dermacentor reticulatus ticks in 34 pools screened by PCR for babesiae were negative. Dogs, Babesia canis, antibodies, Dermacentor reticulatus Canine babesiosis, one of the most important emerging tick-borne diseases of dogs with worldwide distribution, is transmitted by intra-erythrocytic protozoan of the genus Babesia. Traditionally, identification of species is based on morphology and host specificity. According to these criteria, canine piroplasms are divided into two distinct species, the large (4–5 µm) Babesia canis and the small (2.5 µm) Babesia gibsoni. Based on the differences in geographical distribution, vector specificity, antigenic properties, pathogenicity and ss-ribosomal RNA gene three subspecies of B. canis are distinguished, namely B. canis canis transmitted by Dermacentor reticulatus in Europe, B. canis vogeli transmitted by Rhipicephalus sanguineus in tropical and subtropical regions, and highly pathogenic B. canis rossi transmissible by Haemaphysalis leachi in South Africa (Uilenberg 2006). B. canis canis is the most importcant agent of babesiosis in Europe. The incidence of Dermacentor reticulatus in the Czech Republic is limited to the basins of the Morava and Dyje rivers in the Břeclav and Hodonin regions and along the border with Slovakia (Kubelova and Siroký 2010) (Fig. 1). The activity of adults has two peaks, with the first being in the spring from early March (however, ticks can be observed as early as late February, depending on weather conditions – adults are sometimes found even on snow) to mid April. The second peak of adults’ activity starts in September. This tick species inhabits mainly lowland biotopes, waterlogged broadleaved forests, meadows, inundated areas of rivers and fringes of forests. Incidence of Dermacentor reticulatus is irregular and insular. In Central Europe, autochthonous canine babesiosis due to B. canis was recorded in several countries. Surprisingly, no autochthonous case of canine babesiosis was reported so far in the Czech Republic, although babesiosis is present in all the countries surrounding the Czech Republic and the competent vector of the disease frequently occurs (Svobodova and Svobodova 2004). In Slovakia, first cases of autochthonous babesiosis

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.