Background: Dermacentor reticulatus is an emerging tick species in Central Europe with growing significance as a vector of various pathogens. This study reports, for the first time, the presence of human-pathogenic microsporidia in D. reticulatus ticks collected from South Moravia, Czech Republic. Methods: Ticks trapped by flagging from vegetation at ten various sites in the Czech Republic were homogenized and tested for presence of microsporidial DNA using PCR and sequencing, and the microsporidial load was quantified by qPCR. Results: A total of 277 ticks were analyzed using molecular methods, revealing microsporidian DNA in 7.58 % of samples. Several zoonotic species and genotypes were identified, including Encephalitozoon cuniculi genotypes I and II, E. intestinalis, and Enterocytozoon bieneusi genotypes D, horse 1, and Peru 8. Dual infections were observed in two ticks. Spore burden ranged from 102 to 103 spores per tick. Conclusions: Although spore loads were relatively low, the presence of microsporidia in ticks indicates a potential role for D. reticulatus as a mechanical or biological vector. Our findings highlight the need for further investigation into the ecological and public health implications of microsporidia transmission by ticks.

Microsporidia, obligate intracellular parasitic fungi, are increasingly associated with asymptomatic infections. One of the most prevalent zoonotic species, Encephalitozoon cuniculi, has been detected in Ixodes ricinus ticks. Nevertheless, the capacity of microsporidia to replicate within tick hosts remains unresolved. We used tick cell lines IRE11, IRE/CTVM19 and IRE/CTVM20 along with mammalian line Vero E6 for in vitro testing of E. cuniculi propagation. We evaluated the spore number using quantitative RT-PCR and documented the presence of microsporidia in host cells microscopically. We reported successful growth of Encephalitozoon cuniculi in tested tick cell lines, with IRE/CTVM19 providing the most suitable conditions for E. cuniculi propagation, reaching up to 1459 % increase of the inoculum. Moreover, the parasitophorous vacuoles containing developmental stages of microsporidia were observed in all cell lines. Our data support the plausibility of tick-mediated transmission, providing novel insights into the epidemiology of microsporidia.

Lyme disease, caused by Borrelia burgdorferi sensu lato (s.l.), is the most common vector-borne disease in the Northern Hemisphere, with Ixodes ticks as its primary vectors. However, many patients do not recall tick bites, fueling speculation about alternative transmission routes, particularly via mosquito bites. This belief is reinforced by studies reporting Borrelia presence in mosquitoes. This study evaluates whether three mosquito species can acquire, maintain, and transmit Borrelia spirochetes. Mosquitoes (Aedes aegypti, Culex quinquefasciatus, and Culex pipiens biotype molestus) were fed on Borrelia-infected mice to assess pathogen acquisition. Additional experiments involved ex vivo feeding on Borrelia-enriched blood to examine spirochete persistence in the mosquito gut. The potential for mechanical transmission was tested by simulating interrupted feeding between infected and naive hosts. The role of trypsin in Borrelia survival and infectivity was also investigated. Mosquitoes exhibited low efficiency in acquiring Borrelia from infected hosts. Spirochetes artificially introduced through ex vivo blood meals were rapidly eliminated during digestion, primarily due to trypsin activity. Inhibition of trypsin prolonged spirochete persistence and infectivity in the mosquito gut. Mechanical transmission experiments revealed no evidence of Borrelia transmission from infected to naive hosts. Our findings demonstrate that mosquitoes lack the biological capacity to efficiently acquire and maintain B. burgdorferi s.l. spirochetes and are unable to transmit them through natural or mechanical means. This study provides compelling evidence against mosquito-borne transmission of Lyme disease and reinforces Ixodes ticks as the sole competent vectors, which is crucial for targeted public health interventions and accurate risk communication.

INTRODUCTION:Black flies (Simuliidae) are globally distributed blood-feeding arthropods and vectors of viral, bacterial, and parasitic pathogens to many animal species, including humans. We investigated the occurrence of selected vector-borne pathogens in black flies in South Moravia, Czech Republic, and evaluated their possible role in the circulation of vector-borne pathogens. METHODS:A total of 11,600 black flies comprising four species of the genus Simulium, namely Simulium (Boophthora) erythrocephalum (De Geer, 1776), Simulium (Wilhelmia) lineatum (Meigen, 1804), Simulium (Wilhelmia) balcanicum (Enderlein, 1924), and Simulium (Wilhelmia) turgaicum (Rubtsov, 1940) were pooled and screened for the following arthropod-borne pathogens and parasites endemic in Central Europe: viruses (alphaviruses, bunyaviruses and flaviviruses), bacteria (Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum, Neoehrlichia mikurensis, Bartonella spp., Rickettsia spp., Francisella tularensis, Coxiella burnetii, and Brucella spp.), protista (Babesia spp., Encephalitozoon spp. and Enterocytozoon spp.) and filaria (Dirofilaria spp., Setaria spp., and Onchocerca spp.). RESULTS:Almost all pools were negative for known arthropod-borne pathogens and parasites. However, four new Bartonella spp. variants were found that share similarity with other bartonellae reported from diverse arthropods and humans. The phylogenetic analysis of Bartonella sequences from Czech black flies provides further evidence about an expanding diversity of Bartonella lineages in arthropods globally, including hematophagous species (e.g., ticks, mosquitoes, and biting flies) and non-hematophagous species (e.g., bees and ants). These bartonellae have the potential to cause pathogenic infections in humans who are exposed to arthropods carrying these bacteria. CONCLUSIONS:Summing up, this study provides for the very first time valuable data for characterising the risk to public and veterinary health from black flies and the infections they may carry in Europe. Further testing, however, should include a wider geographic, seasonal, and taxonomic range of black flies.

We detected 24 Encephalitozoon cuniculi positive Ixodes ricinus ticks of 284 collected in the Czech Republic. Since the route of transmission of microsporidia is not fully understood, the presence of microsporidia in ticks raises the question of whether they may be involved in the transmission of these pathogens.

Spirochetal bacteria isolated from arthropods of the genera Culex and Aedes are termed BR149, BR151 ( Entomospira culicis), BR193 ( Entomospira entomophila), and BR208 ( Entomospira nematocerorum). Genome sizes assembled from Illumina MiSeq and Oxford Nanopore reads varied between 1.67 and 1.78 Mb containing three to six plasmids. GC content ranged from 38.5% to 45.76%.

Tularemia is a zoonosis caused by Francisella tularensis, a gram-negative aerobic bacterium belonging to the class of Gammaproteobacteria and the family Francisellaceae. Despite its undeniable importance for human health, there is little data on the current distribution of F. tularensis in various hematophagous arthropods. The aim of this study was to perform a mass molecular screening of different possible hematophagous vectors: ticks (4348 ticks of the species Ixodes ricinus, Dermacentor reticulatus, and Haemaphysalis concinna), mosquitoes (4100 specimens of Aedes vexans), and blackflies (6900 specimens of the Simulium spp.) for the presence of F. tularensis in the B & rcaron;eclav district in 2022. Only two specimens were positive for the specific DNA of Francisella tularensis subsp. holarctica. Both samples originated from D. reticulatus, one collected from infested roe deer and the other included in a pooled sample (n = 10). Both positive samples were sequenced, and the presence of F. tularensis subsp. holarctica was confirmed. In addition, the absence of F. tularensis in mosquitoes and black flies was documented.

Abstract In the frame of the entomological VectorNet network and its capacity building activities, we collected original mosquito distribution data in southern Poland and bordering areas of the Czech Republic, Germany and Slovakia, in June and September–November 2023. Because of the suspected occurrence of Aedes japonicus or Ae. koreicus in Poland, provided by a photo posted early 2022 on iNaturalist, we targeted the exotic Aedes species in our sampling strategy, but also collected data on other mosquito species. Besides some adult catches, we mainly collected mosquito immature stages from artificial and natural water containers but occasionally from other aquatic habitats. In addition, we collated citizen data and modelled the distribution of Ae. japonicus in Europe incorporating the newly collected data. During this snapshot field study, a total of 162 samples, including 139 yielding mosquitoes, were taken from 111 locations across 47 administrative units, resulting on the detection of 22 mosquito taxa. Our study provides the first substantiated records of Ae. japonicus and Anopheles petragnani in Poland (the second confirmed by molecular identification). While Ae. japonicus is clearly established over a large part of the country, no other exotic mosquito species was detected. The presence of Ae. japonicus was also confirmed at one location by four citizen records submitted to MosquitoAlert in 2023. Regarding native mosquitoes, we identified their presence in 127 species/NUTS3 combinations (113 for Poland, including a single record for An. petragnani). An updated modelling of the distribution of Ae. japonicus suggests higher environment suitability in Central and Eastern Europe than has been previously estimated. Aedes japonicus is probably widespread in the Czech Republic and Slovakia, and might soon colonise the bordering region of Ukraine. Its establishment extends the putative mosquito vector list for West Nile and Rift Valley fever viruses in Central Europe.

Brucellosis is one of the most important zoonoses worldwide, primarily affecting livestock but also posing a serious threat to public health. The major Brucella species are known to cause a feverish disease in humans with various clinical signs. These classical Brucella species are (re-)emerging, but also novel strains and species, some of them transmitted from rodents, can be associated with human infections. As a result of our review on rodent-borne brucellosis, we emphasise the need for more comprehensive surveillance of Brucella and especially Brucella microti in rodent populations and call for further research targeting the ecological persistence of rodent-associated Brucella species in the environment, their epizootic role in wild rodents and their virulence and pathogenicity for wildlife.

The family Cimicidae comprises ectoparasites feeding exclusively on the blood of endothermic animals. Cimicid swallow bugs specifically target swallow birds (Hirundinidae) and their nestlings in infested nests. Bugs of the genus Oeciacus are commonly found in mud nests of swallows and martins, while they rarely visit the homes of humans. Although-unlike other cimicid species-the house martin bug Oeciacus hirundinis has never been reported as a vector of zoonotic pathogens, its possible role in arbovirus circulation in continental Europe is unclear. Samples of O. hirundinis were therefore collected from abandoned house martin (Delichon urbicum) nests in southern Moravia (Czech Republic) during the 2021/2022 winter season and checked for alpha-, flavi- and bunyaviruses by RT-PCR. Of a total of 96 pools consisting of three adult bugs each, one pool tested positive for Usutu virus (USUV)-RNA. Phylogenetic analysis showed that the virus strain was closely related to Italian and some Central European strains and corresponded to USUV lineage 5. The detection of USUV in O. hirundinis during wintertime in the absence of swallows raises the question for a possible role of this avian ectoparasite in virus overwintering in Europe.

Ťahyňa virus (TAHV) is an orthobunyavirus and was the first arbovirus isolated from mosquitoes in Europe and is associated with floodplain areas as a characteristic biotope, hares as reservoir hosts and the mammal-feeding mosquitoes Aedes vexans as the main vector. The disease caused by TAHV ("Valtice fever") was detected in people with acute flu-like illness in the 1960s, and later the medical significance of TAHV became the subject of many studies. Although TAHV infections are widespread, the prevalence and number of actual cases, clinical manifestations in humans and animals and the ecology of transmission by mosquitoes and their vertebrate hosts are rarely reported. Despite its association with meningitis in humans, TAHV is a neglected human pathogen with unknown public health importance in Central Europe, and a potential emerging disease threat elsewhere in Europe due to extreme summer flooding events.

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.

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.

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 nad 4 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

Usutu virus (USUV) je emergentní patogen přenášený komáry, taxonomicky náleží do rodu Flavivirus čeledi Flaviviridae. Tento původně africký arbovirus v roce 2001 způsobil hromadné hynutí ptactva v okolí Vídně a poté se rozšířil prakticky po celé střední, jižní a západní Evropě. USUV je primárně patogenní pro některé druhy pěvců, především kosy černé (Turdus merula), avšak patogenita byla prokázána i u řady jiných druhů, zejména z řádů dravců a sov. V posledních letech přibývá případů infekce u člověka. Cílem práce je podat ucelené informace o taxonomii viru, jeho genomu, životním cyklu včetně komářích vektorů a obratlovčích rezervoárů, endemickém výskytu v Africe i následném expanzivním šíření v Evropě, surveillanci v jednotlivých evropských státech, patogenitě viru pro obratlovce včetně popisu onemocnění, jež způsobuje, laboratorní diagnostice a dostupné léčbě.

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.

Hyalomma marginatum and Hyalomma rufipes are important vectors of Crimean-Congo Hemorrhagic Fever Virus (CCHFV) in North Africa and Southern Europe. They are occasionally also reported from Central and Western Europe where they are likely introduced from their natural range by migratory birds. In this study, we report findings and molecular identification of adults and one nymph of H. marginatum and H. rufipes, primarily from horses from different regions of the Czech Republic. While the number of the reported ticks is small, this is likely to be an underrepresentation of the actual number. Due to their vector competence for CCHFV and potential expansion into new areas with a changing climate, surveillance programs in Europe are warranted.