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

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

Bartonella species in medically important mosquitoes, Central Europe.

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

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

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

West Nile virus in overwintering mosquitoes, central Europe.

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

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

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

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

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

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

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

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

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

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

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

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

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

Results of the Screening of Tick-Borne Encephalitis Virus Antibodies in Human Sera from Eight Districts Collected Two Decades Apart.

The sera from healthy individuals aged 10-59 years randomly selected from the general population during repeated cross-sectional surveys were stored at -20°C at the serum bank of the National Institute of Public Health in Prague. The sera included in the present study were collected in the 1980s and in 2001 in eight districts of the Czech Republic. The proportional representation of the study localities was similar in both periods. The sera were uniformly distributed in 5-year age groups for 10- to 19-year-olds and in 10-year age groups for 20- to 59-year-olds. Males and females were nearly equally represented in both periods. Altogether 704 sera, 434 from the period 1978-1989 and 270 from 2001, were screened for antibodies against tick-borne encephalitis virus (TBEV) by the virus neutralization test. The seroprevalence rates were 11.5% in the 1980s and 26.3% in 2001. From the logistic regression model, it follows that the chance of detecting anti-TBEV antibodies was more than twice higher in 2001 than in 1978-1989 (odds ratio [OR]=2.22). The differences between males and females were not statistically significant, nor was sex-period interaction. The time trends in the seropositivity rates were similar in all age groups, with the exception of the 10- to 14-year-olds (p=0.914). The rate of seropositives in the age group 15-59 years increased 1.9 times, whereas that in the age group 10-14 years rose 5.1 times. In areas comparable to those where the study sera were collected, the average incidence rates of tick-borne encephalitis (TBE) per 100,000 population aged 10-59 increased significantly from 3.35 in 1978-1989 to 8.96 in 2001 (p<0.001). The age-specific antibody trends in adult age groups in both periods suggest that clinically manifest or inapparent TBE cases do not induce lifelong immunity, but they are likely to reflect the previous epidemiological situation.

First evidence of Babesia venatorum and Babesia capreoli in questing Ixodes ricinus ticks in the Czech Republic.

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 host-seeking I. ricinus ticks in the Czech Republic.

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

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

Human pathogenic borreliae in Ixodes ricinus ticks in natural and urban ecosystem (Czech Republic).

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 Belský 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.

Rickettsiae in questing Ixodes ricinus ticks in the Czech Republic.

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.

West Nile Virus equine serosurvey in the Czech and Slovak republics.

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.

Dirofilaria repens microfilariae in Aedes vexans mosquitoes in Slovakia.

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 Kosické Olsany 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.