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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.

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.

Emerging flaviviruses are causative agents of severe and life-threatening diseases, against which no approved therapies are available. Among the nucleoside analogues, which represent a promising group of potentially therapeutic compounds, fluorine-substituted nucleosides are characterized by unique structural and functional properties. Despite having first been synthesized almost 5 decades ago, they still offer new therapeutic opportunities as inhibitors of essential viral or cellular enzymes active in nucleic acid replication/transcription or nucleoside/nucleotide metabolism. Here, we report evaluation of the antiflaviviral activity of 28 nucleoside analogues, each modified with a fluoro substituent at different positions of the ribose ring and/or heterocyclic nucleobase. Our antiviral screening revealed that 3'deoxy-3'-fluoroadenosine exerted a low-micromolar antiviral effect against tick-borne encephalitis virus (TBEV), Zika virus, and West Nile virus (WNV) (EC50 values from 1.1 +/- 0.1 mu M to 4.7 +/- 1.5 mu M), which was manifested in host cell lines of neural and extraneural origin. The compound did not display any measurable cytotoxicity up to concentrations of 25 mu M but had an observable cytostatic effect, resulting in suppression of cell proliferation at concentrations of > 12.5 mu M. Novel approaches based on quantitative phase imaging using holographic microscopy were developed for advanced characterization of antiviral and cytotoxic profiles of 3'-deoxy-3'-fluoroadenosine in vitro. In addition to its antiviral activity in cell cultures, 3'-deoxy-3'-fluoroadenosine was active in vivo in mouse models of TBEV and WNV infection. Our results demonstrate that fluoro-modified nucleosides represent a group of bioactive molecules with excellent potential to serve as prospective broad-spectrum antivirals in antiviral research and drug development.

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.

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.

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.

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.

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.

West Nile virus (WNV) is a medically important emerging arbovirus causing serious neuroinfections in humans 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 ofC2′-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 post-infection, 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 post-infection (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 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.

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.

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.

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.

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.

In this study, we screened field-caught mosquitoes for presence of Dirofilaria spp. by using a polymerase chain reaction (PCR) assay. Potential occurrence of Dirofilaria repens and Dirofilaria immitis microfilariae was examined in 3,600 mosquitoes of eight species (Aedes vexans, Aedes cinereus, Aedes rossicus, Culex pipiens, Culiseta annulata, Ochlerotatus sticticus, Ochlerotatus cantans and Ochlerotatus caspius) collected from five locations in two districts (Kosice and Trebisov) of Eastern Slovakia, endemic region of canine dirofilariasis. Collection of mosquitoes was performed between May and August 2012 in premises known to be inhabited by Dirofilaria-infected dogs. PCR assays were performed on 72 pools, each pool containing 50 mosquitoes of the same species, collected on the same location. Each pool was examined separately for the presence of D. immitis and D. repens, respectively. A positive finding of D. repens was recorded in one pool of A. vexans mosquitoes collected in Košické Olšany village. Minimum infection rate in A. vexans was 1:1,750, i.e. 0.57 per 1,000 mosquitoes. The identity of D. repens was confirmed by direct sequencing of PCR product which has shown 100 % homology with sequence attributed to D. repens (GenBank accession number AJ271614). This study represents the first molecular evidence of D. repens microfilariae in mosquitoes in Slovakia and highlights a need for better surveillance of zoonotic dirofilariasis in central Europe.

International audience; 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. (C) 2016 Elsevier GmbH. All rights reserved.

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

The aim of this review is to present briefly background information on 27 tick-borne viruses (“tiboviruses”) that have been detected in Europe, viz flaviviruses tick-borne encephalitis (TBEV), louping-ill (LIV), Tyuleniy (TYUV), and Meaban (MEAV); orthobunyaviruses Bahig (BAHV) and Matruh (MTRV); phleboviruses Grand Arbaud (GAV), Ponteves (PTVV), Uukuniemi (UUKV), Zaliv Terpeniya (ZTV), and St. Abb's Head (SAHV); nairoviruses Soldado (SOLV), Puffin Island (PIV), Avalon (AVAV), Clo Mor (CMV), Crimean-Congo hemorrhagic fever (CCHFV); bunyavirus Bhanja (BHAV); coltivirus Eyach (EYAV); orbiviruses Tribec (TRBV), Okhotskiy (OKHV), Cape Wrath (CWV), Mykines (MYKV), Tindholmur (TDMV), and Bauline (BAUV); two thogotoviruses (Thogoto THOV, Dhori DHOV); and one asfivirus (African swine fever virus ASFV). Emphasis is laid on the taxonomic status of these viruses, range of their ixodid or argasid vectors and vertebrate hosts, pathogenicity for vertebrates including humans, and relevance to public health. In general, three groups of tibovirus diseases can be recognized according to main clinical symptoms produced: (i) febrile illness—usually with a rapid onset, fever, sweating, headache, nausea, weakness, myalgia, arthralgia, sometimes polyarthritis and rash; (ii) the CNS affection—meningitis, meningoencephalitis or encephalomyelitis with pareses, paralysis and other sequelae; (iii) hemorrhagic disease. Several “European” tiboviruses cause very serious human (TBEV, CCHFV) or animal (LIV, ASFV) diseases. Other arboviruses play definite role in human or animal pathology though the disease is usually either less serious or infrequently reported (TYUV, BHAV, AVAV, EYAV, TRBV, DHOV, THOV). The other European arboviruses are “orphans” without a proven medical or veterinary significance (BAHV, MTRV, MEAV, GAV, PTVV, ZTV, SAHV, UUKV, SOLV, PIV, AVAV, CMV, OKHV, CWV, MYKV, TDMV, BAUV). However, certain arbovirus diseases of free-living vertebrates (but also those of domestic animals and even man) may often pass unnoticed or misdiagnosed and eventually, they might potentially appear as emerging diseases. Active search for new tiboviruses or for new, pathogenic variants of the known tiboviruses in Europe should therefore continue.

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.

In vitro effect of salivary gland extract from fed Ixodes ricinus, the competent vector of Lyme borreliosis in Europe, on the growth of Borrelia burgdorferi sensu lato (B. garinii, B. afzelii and B. burgdorferi sensu stricto) was examined in BSK-H medium. Motility rate, concentration of motile spirochetes and their morphology were estimated at intervals of 0, 2, 4, 6 and 8 days using darkfield microscopy. Salivary gland extract derived from I. ricinus stimulated markedly the growth of three genomic species of borreliae. The results confirm a substantial role of salivary glands in the mechanism of pathogen transmission to vertebrate host.

Six kea (Nestor notabilis) in human care, naturally infected with West Nile virus (WNV) lineage 2 in Vienna, Austria, in 2008, developed mild to fatal neurological signs. WNV RNA persisted and the virus evolved in the birds' brains, as demonstrated by (phylo)genetic analyses of the complete viral genomes detected in kea euthanized between 2009 and 2014. WNV antibodies persisted in the birds, too. Chronic WNV infection in the brain might contribute to the circulation of the virus through oral transmission to predatory birds.

The detection of West Nile virus (WNV) nucleic acid in a blood donation from Vienna, Austria, as well as in Culex pipiens pupae and egg rafts, sampled close to the donor’s residence, is reported. Complete genomic sequences of the human- and mosquito-derived viruses were established, genetically compared and phylogenetically analyzed. The viruses were not identical, but closely related to each other and to recent Czech and Italian isolates, indicating co-circulation of related WNV strains within a confined geographic area. The detection of WNV in a blood donation originating from an area with low WNV prevalence in humans (only three serologically diagnosed cases between 2008 and 2014) is surprising and emphasizes the importance of WNV nucleic acid testing of blood donations even in such areas, along with active mosquito surveillance programs.

Questing Ixodes ricinus L. (Acari: Ixodidae) ticks were collected on a forest trail that had been completely cleared of shrubs and ground vegetation in winter 2002 and on a nearby control uncleared forest transect in South Moravia (Czech Republic). Samples were collected each May in 2003, 2004 and 2005. Nymphal ticks were 3.4 times, 1.9 times and 1.2 times less frequent on cleared forest than on uncleared forest trails in the three respective years, whereas adult tick abundance was 27.2 times, 4.0 times and 2.2 times lower, respectively. The ticks were examined for borreliae by dark-field microscopy: prevalence of nymphal ticks infected with Borrelia burgdorferi sensu lato (12.6% to 20.0%) did not differ significantly between the cleared and uncleared trail during the 3 years. In conclusion, the habitat modification appeared to result in a decreased abundance of I. ricinus as well as a reduced frequency of infected ticks (and thus indirectly a lower potential risk of Lyme borreliosis), which lasted, however, for only 2 years. Eight cultures of borreliae isolated from the ticks were all identified as the ‘ornithophilic’ genomic species Borrelia garinii, possibly indicating a greater role of forest birds than that of forest rodents as the hosts of immature I. ricinus in the tick (and borrelial) colonization of the cleared part of the forest.

International audience; Tick-borne diseases represent major public and animal health issues worldwide. Ixodes ricinus, primarily associated with deciduous and mixed forests, is the principal vector of causative agents of viral, bacterial, and protozoan zoonotic diseases in Europe. Recently, abundant tick populations have been observed in European urban green areas, which are of public health relevance due to the exposure of humans and domesticated animals to potentially infected ticks. In urban habitats, small and medium-sized mammals, birds, companion animals (dogs and cats), and larger mammals (roe deer and wild boar) play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Presence of ticks infected with tick-borne encephalitis virus and high prevalence of ticks infected with Borrelia burgdorferi s.l., causing Lyme borreliosis, have been reported from urbanized areas in Europe. Emerging pathogens, including bacteria of the order Rickettsiales (Anaplasma phagocytophilum, "Candidatus Neoehrlichia mikurensis," Rickettsia helvetica, and R. monacensis), Borrelia miyamotoi, and protozoans (Babesia divergens, B. venatorum, and B. microti) have also been detected in urban tick populations. Understanding the ecology of ticks and their associations with hosts in a European urbanized environment is crucial to quantify parameters necessary for risk pre-assessment and identification of public health strategies for control and prevention of tick-borne diseases.

Rabensburg virus, isolated from Culex pipiens mosquitoes in central Europe, represents a new lineage of West Nile virus or a novel flavivirus of the Japanese encephalitis virus group., A flavivirus (strain 97-103) was isolated from Culex pipens mosquitoes in 1997 following floods in South Moravia, Czech Republic. The strain exhibited close antigenic relationship to West Nile virus (WNV) prototype strain Eg-101 in a cross-neutralization test. In this study, mouse pathogenicity characteristics and the complete nucleotide and putative amino acid sequences of isolate 97-103, named Rabensburg virus (RabV) after a nearby Austrian city, were determined. RabV shares only 75%–77% nucleotide identity and 89%–90% amino acid identity with representative strains of WNV lineages 1 and 2. Another RabV strain (99-222) was isolated in the same location 2 years later; it showed >99% nucleotide identity to strain 97-103. Phylogenetic analyses of RabV, WNV strains, and other members of the Japanese encephalitis virus (JEV) complex clearly demonstrated that RabV is either a new (third) lineage of WNV or a novel flavivirus of the JEV group.

We report the detection and isolation of four almost identical strains of West Nile virus (WNV) lineage 2 from Culex modestus mosquitoes collected at three fish ponds in South Moravia, Czech Republic, during August 2013. Phylogenetic analysis demonstrated that the Czech WNV strains isolated are closely related to Austrian, Italian and Serbian strains reported in 2008, 2011 and 2012, respectively. Our findings show the current northernmost range of lineage 2 WNV in Europe. In South Moravia in the Czech Republic, surveillance activities for mosquitoes and mosquito-borne pathogens have been carried out for several decades, but until our findings in 2013 presented here, WNV lineage 2 (WNV-2) had not been detected.

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.

The objective of this chapter is to provide an updated and concise systematic review on taxonomy, history, arthropod vectors, vertebrate hosts, animal disease, and geographic distribution of all arboviruses known to date to cause disease in homeotherm (endotherm) vertebrates, except those affecting exclusively man. Fifty arboviruses pathogenic for animals have been documented worldwide, belonging to seven families: Togaviridae (mosquito-borne Eastern, Western, and Venezuelan equine encephalilitis viruses; Sindbis, Middelburg, Getah, and Semliki Forest viruses), Flaviviridae (mosquito-borne yellow fever, Japanese encephalitis, Murray Valley encephalitis, West Nile, Usutu, Israel turkey meningoencephalitis, Tembusu and Wesselsbron viruses; tick-borne encephalitis, louping ill, Omsk hemorrhagic fever, Kyasanur Forest disease, and Tyuleniy viruses), Bunyaviridae (tick-borne Nairobi sheep disease, Soldado, and Bhanja viruses; mosquito-borne Rift Valley fever, La Crosse, Snowshoe hare, and Cache Valley viruses; biting midges-borne Main Drain, Akabane, Aino, Shuni, and Schmallenberg viruses), Reoviridae (biting midges-borne African horse sickness, Kasba, bluetongue, epizootic hemorrhagic disease of deer, Ibaraki, equine encephalosis, Peruvian horse sickness, and Yunnan viruses), Rhabdoviridae (sandfly/mosquito-borne bovine ephemeral fever, vesicular stomatitis-Indiana, vesicular stomatitis-New Jersey, vesicular stomatitis-Alagoas, and Coccal viruses), Orthomyxoviridae (tick-borne Thogoto virus), and Asfarviridae (tick-borne African swine fever virus). They are transmitted to animals by five groups of hematophagous arthropods of the subphyllum Chelicerata (order Acarina, families Ixodidae and Argasidae-ticks) or members of the class Insecta: mosquitoes (family Culicidae); biting midges (family Ceratopogonidae); sandflies (subfamily Phlebotominae); and cimicid bugs (family Cimicidae). Arboviral diseases in endotherm animals may therefore be classified as: tick-borne (louping ill and tick-borne encephalitis, Omsk hemorrhagic fever, Kyasanur Forest disease, Tyuleniy fever, Nairobi sheep disease, Soldado fever, Bhanja fever, Thogoto fever, African swine fever), mosquito-borne (Eastern, Western, and Venezuelan equine encephalomyelitides, Highlands J disease, Getah disease, Semliki Forest disease, yellow fever, Japanese encephalitis, Murray Valley encephalitis, West Nile encephalitis, Usutu disease, Israel turkey meningoencephalitis, Tembusu disease/duck egg-drop syndrome, Wesselsbron disease, La Crosse encephalitis, Snowshoe hare encephalitis, Cache Valley disease, Main Drain disease, Rift Valley fever, Peruvian horse sickness, Yunnan disease), sandfly-borne (vesicular stomatitis-Indiana, New Jersey, and Alagoas, Cocal disease), midge-borne (Akabane disease, Aino disease, Schmallenberg disease, Shuni disease, African horse sickness, Kasba disease, bluetongue, epizootic hemorrhagic disease of deer, Ibaraki disease, equine encephalosis, bovine ephemeral fever, Kotonkan disease), and cimicid-borne (Buggy Creek disease). Animals infected with these arboviruses regularly develop a febrile disease accompanied by various nonspecific symptoms; however, additional severe syndromes may occur: neurological diseases (meningitis, encephalitis, encephalomyelitis); hemorrhagic symptoms; abortions and congenital disorders; or vesicular stomatitis. Certain arboviral diseases cause significant economic losses in domestic animals-for example, Eastern, Western and Venezuelan equine encephalitides, West Nile encephalitis, Nairobi sheep disease, Rift Valley fever, Akabane fever, Schmallenberg disease (emerged recently in Europe), African horse sickness, bluetongue, vesicular stomatitis, and African swine fever; all of these (except for Akabane and Schmallenberg diseases) are notifiable to the World Organisation for Animal Health (OIE, 2012).

A total of 1279 field-collected Ixodes ricinus ticks were screened for Borrelia burgdorferi sensu lato (s.l.) in a natural and an urban ecosystem of Ostrava city (Czech Republic) by using molecular methods. Minimal prevalence rate for Borrelia burgdorferi s.l. in ticks for the urban park Bělský les was found to be 13.8% (17.6% in males, 17.8% in females and 11.7% in nymphs), similarly for the natural site Proskovice was minimal prevalence 15% (12.5% in males, 20% in females and 14.9% in nymphs). Six proven human pathogenic genomic species have been recorded in the study: B. afzelii, B. garinii, B. burgdorferi s.s., B. valaisiana, B. lusitaniae, and B. spielmanii. Emerging B. spielmanii was detected for the first time in Ixodes ricinus ticks in the region. Our results highlight the need for surveillance of zoonotic tick-borne pathogens even in urban areas.

Specimens from residents (N = 497) of an area affected by the 2002 flood were examined serologically for mosquitoborne viruses. Antibodies were detected against Tahyna (16%), Sindbis (1%), and Batai (0.2%) viruses, but not West Nile virus. An examination of paired serum samples showed 1 Tahyna bunyavirus (California group) infection.

Tick-borne viruses of the families Flaviviridae, Bunyaviridae, Reoviridae and Orthomyxoviridae are dealt with with in this chapter. Talking in general terms, we are note yet able to evaluate the fine effects of climate trends on the epidemiology of the most prominent tick-borne viruses. We should keep in mind that the these changes do not affect only the dynamics of tick vectors, but also the abundance of hosts for immature stages of the tick, or their migratory timings in the case of birds (which may be hosts for the immatures of the ticks) or even how climate may affect the densities of hosts in natural conditions. While available models might greatly contribute to understand the behaviour of the ticks under variable climate conditions, we need yet to build upon those models to reach the necessary level of complexity. Local processes are not captured yet by these models, adding "noise" to the general background picture of the fine scale distribution of a tick, their vectors and the pathogens it transmit. There is an implicit need of further research at both local and regional scales.

During the years 1999–2002, a total of 4,898 individuals of 26 species of hematophagous insects (4,149 mosquitoes, 583 black flies, and 166 tabanid flies) was examined for the presence of spirochetes using dark-field microscopy. There was an overall recovery of spirochetes from the midguts of Culicidae and Simuliidae of 23.5% and 11.4%, respectively. Spirochetes were not detected in Tabanidae. Seven spirochetal strains have been successfully recovered from mosquitoes and black flies: BR149 (Culex pipiens), BR151 (Cx. pipiens), BR173 (Cx. pipiens), BR177 (Cx. pipiens), BR193 (Aedes cinereus), BR208 (Cx. pipiens), and BR231 (Simulium noelleri). The strains have been adapted to laboratory conditions (BSK-H Complete medium). Their preliminary determination based on 16S rRNA gene sequencing has shown that they differ from the Lyme disease spirochete Borrelia burgdorferi sensu lato as well as other members of the Order Spirochaetales indicating novel bacterial species in the Family Spirochaetaceae.

Six viral isolates were obtained from 23,243 female mosquitoes (examined in 513 pools) belonging to 16 species and collected along the lower reaches of the Dyje River in South Moravia (Czech Republic, central Europe) during 2006-2008: five isolates of Orthobunyavirus Tahyna (TAHV, California group, family Bunyaviridae: three isolations from Aedes vexans (Meigen), one from Ae. sticticus (Meigen), one from Culex modestus Ficalbi); and one isolation of Flavivirus West Nile (WNV, Japanese encephalitis group, family Flaviviridae)-strain Rabensburg (proposed lineage 3 of WNV) from Ae. rossicus (Dolbeshkin et al). All viral isolates were recovered from mosquitoes collected in 2006 (15,882 mosquitoes examined), while no virus was isolated from mosquitoes trapped in 2007 and 2008, when 1,555 and 5,806 mosquitoes were examined, respectively. The population density of local mosquitoes was very low in 2007 and 2008 because of warm and dry summer including a considerably low water table, compared with environmental conditions favorable for mosquito development in 2006. The virus isolation procedure was based on intracerebral inoculation of newborn mice. In parallel, more than one-third of the samples (183 pools consisting of 8,470 individual mosquitoes) were also examined by inoculating Vero cell cultures in Leighton tubes. However, the latter method detected only three of the six virus isolates (including WNV-Rabensburg). Ae. rossicus is a new potential vector for WNV-Rabensburg. This species feeds mostly on mammals including man; this raises the question whether this virus lineage is not adapted to an alternative mosquito-mammal cycle in the South-Moravian natural focus.

This chapter presents a survey of zoonotic microorganisms that have been isolated from vertebrates (Vertebrata), and are potentially transmissible to humans. It is intended as an aid for microbiologists, zoologists and epidemiologists, making possible better orientation among hosts (and reservoirs) of zoonoses. A great number of sources have been used in this compilation, e.g. Davis et al. (1970), Kucheruk (1979, 1989), Karabatsos (1985–1995), Hubalek (1994), etc.

The first terrestrial forms of arthropods appeared in the Palaeozoic, at the turn of the Silurian and Devonian ages some 400 million years ago. Today, arthropods form about 85% of all known animal species. They are characterized by segmentation of the body and limbs, reticulated paired appendages (antennae etc.), exoskeleton formed mainly of chitin, and bilateral symmetry.

This chapter could also be called eco-epidemiological basics or background information for zoonoses and sapronoses. Epidemiology is the study of the process of the origin and spread of transmissible (communicable) infectious diseases of man, and their control. The modern conception of epidemiology is broader in that it also includes non-infectious diseases (such as diabetes, heart attack, cardiovascular diseases and carcinomas) and variables that influence their distribution in the human population. In both the classical and modern conception of epidemiology, these factors include variables concerning the host (age, sex, nutrition, occupation), the agent (virulence, antigenic variability), and the environment (chemical factors, contamination, emissions, temperature, precipitation and humidity, illumination, ionising radiation, noise etc.). Epidemiological data are then analysed, and the results of these studies can then be used for the prevention and control of these human diseases.