First detection of Bartonella spp. in bat bugs Cimex pipistrelli (Hemiptera: Cimicidae), Central Europe.

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.

Detection of Leptospira species in bat cadavers, Czech and Slovak Republics.

Kidney samples from 300 bat cadavers from the Czech and Slovak Republics were tested for Leptospira DNA using PCR and sequencing of three genes (lipL32, flab, and 16S ribosomal RNA). Overall detection rate was 4.7% and two bat species (Myotis myotis and Nyctalus noctula) were PCR-positive for at least one gene. Detected Leptospira sequences were similar to L. interrogans and L. borgpetersenii, and included a potentially novel species related to L. weilii.

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.

In the foothill zone-Sabanejewia balcanica (Karaman 1922), in the lowland zone-Sabanejewia bulgarica (Drensky, 1928): Myth or reality?

The status of golden loaches (genus Sabanejewia) in the region of Central Europe and Balkans is still ambiguous. The greatest controversy is caused by species Sabanejewia balcanica and S. bulgarica. Both species are characterized by a wide spectrum of morphological variability and overlapping of distinguishing features, which then lead to difficulties in their determination. Previous phylogenetic studies aimed on the resolving of their taxonomic status did not include samples from their type localities and so led to a lack of their true distribution in this region. Therefore, the main aim of this study was to identify taxonomic status of golden loaches populations in the region of the middle Danube basin and adjacent areas on the model territory of Slovakia. For this purpose, we used novelty approach (morphological, molecular, and microhabitat) and we also included the missing samples from the type localities of both species. Based on mtDNA all the Slovakian samples reflected haplotype richness revealed on the type locality of S. bulgarica, although the genetic distances from other representatives of the genus Sabanejewia occurring are not significant. Within the morphology, we have revealed a great measure of variability in studied populations, which is largely caused by different habitat conditions and thus representing a phenotypic plasticity of these fish.

Field study indicating susceptibility differences between salmonid species and their lineages to proliferative kidney disease.

Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) is the causative agent of proliferative kidney disease (PKD), which affects both wild and farmed salmonid fish. The objective of this study was to outline differences in susceptibility to PKD in different salmonid species, hybrids and breeding lineages. Susceptibility to T. bryosalmonae infection was established based on cumulative mortality, pathological findings and detection of T. bryosalmonae in the kidney using immunohistochemistry and molecular methods. Determination of pure and hybrid individuals of different species in the genus Salvelinus, and dissimilarity of rainbow trout lineages, was performed using traditional polymerase chain reaction (PCR) and microsatellite analyses. Rainbow trout displayed higher disease severity compared with brook trout and Alsatian charr. Moreover, the results indicated differences in infection susceptibility, not only among different salmonid species but also among different lineages of charr and rainbow trout. Our study indicated that some salmonid species and even different lineages of the same species are more suitable for farming under PKD pressure.

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.

Overwintering of Culex modestus and Other Mosquito Species in a Reedbed Ecosystem, Including Arbovirus Findings.

The overwintering strategy of the mosquito Culex modestus, an important West Nile virus (WNV) vector in Europe, was explored under field conditions in reedbed (Phragmites australis) ecosystems in early 2019. A total of 30 Cx. modestus females were found in a BG-Sentinel trap placed in a plastic greenhouse as well as in a reference BG-Sentinel trap placed under the open sky, both set up within the reedbeds and inspected every 2-3 days from February 27 to April 10, 2019. Moreover, 186 females of Cx. pipiens, 3 females of Anopheles hyrcanus, and 3 females of Culiseta annulata were trapped in the monitored time span. While all Cx. modestus females tested negative for the presence of WNV and other arboviruses circulating in Central Europe, we confirmed WNV lineage 2 and Tahyna virus infection in several pools of the collected Cx. pipiens, demonstrating arbovirus overwintering. This pilot study highlights the need for large-scale monitoring activities covering different regions to identify the overwintering strategy of both mosquito-borne viruses and their vectors in Central Europe.

Co-Circulation of West Nile and Usutu Flaviviruses in Mosquitoes in Slovakia, 2018.

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.

The Legacy of Sexual Ancestors in Phenotypic Variability, Gene Expression, and Homoeolog Regulation of Asexual Hybrids and Polyploids.

Hybridization and polyploidization are important evolutionary processes whose impacts range from the alteration of gene expression and phenotypic variation to the triggering of asexual reproduction. We investigated fishes of the Cobitis taenia-elongatoides hybrid complex, which allowed us to disentangle the direct effects of both processes, due to the co-occurrence of parental species with their diploid and triploid hybrids. Employing morphological, ecological, and RNAseq approaches, we investigated the molecular determinants of hybrid and polyploid forms. In contrast with other studies, hybridization and polyploidy induced relatively very little transgressivity. Instead, Cobitis hybrids appeared intermediate with a clear effect of genomic dosing when triploids expressed higher similarity to the parent contributing two genome sets. This dosage effect was symmetric in the germline (oocyte gene expression), interestingly though, we observed an overall bias toward C. taenia in somatic tissues and traits. At the level of individual genes, expression-level dominance vastly prevailed over additivity or transgressivity. Also, trans-regulation of gene expression was less efficient in diploid hybrids than in triploids, where the expression modulation of homoeologs derived from the "haploid" parent was stronger than those derived from the "diploid" parent. Our findings suggest that the apparent intermediacy of hybrid phenotypes results from the combination of individual genes with dominant expression rather than from simple additivity. The efficiency of cross-talk between trans-regulatory elements further appears dosage dependent. Important effects of polyploidization may thus stem from changes in relative concentrations of trans-regulatory elements and their binding sites between hybridizing genomes. Links between gene regulation and asexuality are discussed.

Mortality of Goshawks (Accipiter gentilis) Due to West Nile Virus Lineage 2.

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.

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

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

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.

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.

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

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

A multilocus assessment of nuclear and mitochondrial sequence data elucidates phylogenetic relationships among European spirlins (Alburnoides, Cyprinidae).

The phylogenetic relationships and taxonomy of the spirlins in the genus Alburnoides are examined by comparative sequencing analysis of mitochondrial and nuclear markers. Molecular analyses revealed 17 Eurasian lineages divided into two main clades, termed the Ponto-Caspian and European in accordance with the lineage distribution. The indel diagnostics of ß-actin and S7 markers and translation of cyt b to the amino acid chain were evaluated as a reliable identifying tool for most of the recognised lineages. Lineage richness is closely connected with the existence of known glacial refugia in most cases. The underestimation of species richness in the genus Alburnoides is confirmed: the genetic analyses support the validity of 11 morphologically accepted species; apart from them, four phylogenetic lineages requiring descriptions as separate species were revealed. The distribution area of the nominotypical species A. bipunctatus s. stricto is newly defined. Two diverging phylogenetic lineages, A. ohridanus, and A. prespensis complex, were observed in the Southeast Adriatic Freshwater Ecoregion, confirmed as a hotspot of endemic biodiversity. A. ohridanus demonstrates high divergence from the A. prespensis complex, represented by three similar mitochondrial lineages with the same nuclear haplotypes and sympatric occurrence. The range restricted endemism was confirmed for at least seven species. The Albanian river systems, as well as the wider Ponto-Caspian basin exhibit complications among definite species delineations and gaps in understanding of microevolutionary processes; these areas require further investigations.

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.