Mária Kazimírová

Herein we report the occurrences of two exotic tick species, Rhipicephalus sanguineus and Hyalomma marginatum and of the endophilic Ixodes kaiseri, and a new regional record of Ixodes frontalis in Slovakia. Ticks were identified to species and stage according to taxonomic keys. Morphological identification of all species was confirmed molecularly by PCR targeting specific fragments of the cytochrome C oxidase subunit 1 (cox1) and 16S rRNA genes Rhipicephalus sanguineus (Latreille, 1806) were collected from the walls in an apartment and from two mixed- breed dogs in Bratislava in 2017-2018 (Didyk et al. 2022). Based on molecular analyses ticks clustered in the Rhipicephalus sp. IIa mitochondrial “temperate” lineage, which contains geographically similar ticks from the mid- and western Mediterranean regions. Ixodes kaiseri Arthur, 1957 were collected from the skin of hunted Vulpes vulpes and Meles meles in 2021. Sequence analyses showed that the I. kaiseri isolate from Slovakia was identical to isolates from other European countries (Krumpálová et al. 2023). In July 2022, one questing Hyalomma female, climbing cloths, was collected in district Púchov (western Slovakia). The tick was morphologically and molecular identified as H. marginatum Koch, 1844 Ixodes frontalis (Panzer, 1798) ticks were collected from vegetation in the Botanical Garden of Comenius University in Bratislava in 2023-2025. Molecular analyses showed 100% identity of the I. frontalis DNA sequences with other European isolates of the species. The current findings represent the second record of H. marginatum and the third record of I. frontalis in Slovakia. Although the current risk of introduction of pathogens carried by these ticks to Slovakia is relatively low, continuous monitoring of their occurrence is necessary Acknowledgements: The research was funded by grant APVV-22-0372, Vega2/0083/25, Vega2/0092/26.

Questing larvae and nymphs of Ixodes frontalis were collected for the first time from vegetation in the Botanical Garden in Bratislava, south-western Slovakia, in the autumn, winter and spring from 2023 to 2025. Previous reports on the occurrence of the species in the country were from the Slovak Karst (eastern Slovakia) and the Zvolen basin (central Slovakia). The identification of ticks is based on morphological and molecular (the cytochrome C oxidase subunit 1 ( cox 1) and 16S rRNA genes) characteristics. Phylogenetic analysis confirmed the classification of the ticks to a specific gene group together with other European isolates of I. frontalis. Infections of the studied ticks with bacterial ( Borrelia burgdorferi sensu lato complex, Anaplasma phagocytophilum and Rickettsia spp.) and protozoan ( Babesia/Theileria spp.) tick-borne pathogens have not been confirmed.

Among the 24 tick species documented in Slovakia, Ixodes ricinus is the most widespread and abundant. In some sites, 2–5 epidemiologically important tick species co-occur. Such sites represent hotspots for studying the co-circulation of tick-borne pathogens. Sympatric occurrence of five exophilic species (I. ricinus, Dermacentor reticulatus, D. marginatus, Haemaphysalis concinna, and H. inermis) was confirmed in the environs of the Žemberovce village (Levice region, south-western Slovakia). Here, the seasonal activity and abundance of questing ticks and the tick infestation of game and sheep were investigated. Questing ticks, spleens of game, and ticks removed from game and sheep were examined for the presence of tick-borne pathogens (Babesia spp., Theileria spp., Anaplasma phagocytophilum, Rickettsia spp., Borrelia burgdorferi s.l., and Borrelia miyamotoi) by molecular methods. Among the questing ticks, I. ricinus prevailed. Presence of Babesia crassa, B. microti, Rickettsia helvetica, R. raoultii, A. phagocytophilum, Borrelia afzelii, B. garinii, B. valaisiana, B. burgdorferi sensu stricto and B. miyamotoi was detected. Dermacentor marginatus, I. ricinus and H. concinna were collected from sheep. They were infected with A. phagocytophilum, A. ovis, R. slovaca, and R. raoultii. Anaplasma phagocytophilum was detected in all examined red deer and roe deer and in 55.6% of wild boar. All cervids were positive for Theileria spp. Infestation of game with all five tick species, with a predominance of I. ricinus, was confirmed. In these ticks, A. phagocytophilum, R. helvetica, R. raoultii, Babesia sp. hc-hlj212, B. crassa, B. microti, Babesia spp. and B. miyamotoi were detected. This study confirmed the presence of B. crassa in Slovakia for the first time. The investigated area, with the co-occurrence of five exophilic tick species and a wide spectrum of tick-borne pathogens, represents an epidemiologically important hotspot with the risk of infections of humans and domestic animals.

Free-ranging animals may serve as sentinels for the occurrence of ticks and tick-borne pathogens within a given habitat. Between 2020 and 2022, ticks were collected from domestic animals and vegetation on a family-operated agrotouristic farm in Western Slovakia. A total of 391 ticks were obtained (163 host-feeding and 228 questing). Ixodes ricinus predominated (94.9%), followed by Dermacentor reticulatus (4.9%) and Haemaphysalis concinna (one specimen). All ticks were screened for Borrelia burgdorferi sensu lato (s.l.), Borrelia miyamotoi, Anaplasma phagocytophilum, Babesia spp., and Rickettsia spp. by using PCR-based methods and subsequent sequencing. Borrelia burgdorferi s.l. was detected in 15.9% of I. ricinus. Six species were identified, with Borrelia afzelii dominating. Borrelia miyamotoi was detected in 1.62% of questing I. ricinus. Anaplasma phagocytophilum was identified in 20.49% of I. ricinus, mainly in host-feeding specimens. Babesia spp. were detected in I. ricinus (2.7%), represented by Babesia microti, Babesia venatorum, and Babesia capreoli. Rickettsia spp. were detected in 5.39% of I. ricinus and 31.58% of D. reticulatus. They comprised Rickettsia helvetica, Rickettsia monacensis, and Rickettsia raoultii. Co-infections involving two or three pathogens were observed in several ticks. Our results underscore the considerable diversity of tick-borne pathogens in a rural area where humans and domestic animals are in close contact to wildlife.

Specific pathogen-free ticks infected with tick-borne viruses are required in vector competence studies and studies on tick-virus-host interactions. Infection of ticks by feeding on infected laboratory animals is the only natural way of infection. However, one of the main rules set in ethical guidelines is reduction of the usage of laboratory animals in experiments. Here we describe an alternative method of tick infection with tick-borne encephalitis virus through parenteral inoculation into the tick hemocoel. Although the method bypasses some of the natural barriers in comparison with virus uptake through the tick blood meal, the efficiency of infection is high with over 90% of ticks containing living virus.

Introduction: In Europe , Borrelia burgdorferi sensu lato (s.l.), the causative agent of Lyme borreliosis is transmitted by the castor bean tick, Ixodes ricinus . In the last decades, global changes affect the spread of ticks and also their bionomics. The aim of this study was summarization of a large dataset obtained during 20 years of research. Methods: The research was carried out in 1999-2019 at 16 localities in Slovakia that were continuously monitored. In total, 17,249 questing I. ricinus ticks were tested for the presence of B. burgdorferi s.l. Results: The total prevalence of infected ticks was 18.8% (3,248/17,249), with 15.1% (1,557/10,302) infected nymphs and 24.3% (1,691/6,947) infected adults. Nine species of B. burgdorferi s.l. were identified. Borrelia afzelii (37.1%), B. garinii / bavariensis (24.7%), and B. valaisiana (15.4%) were the most frequent and were present at all study sites, followed by B. lusitaniae (12.6%), B. burgdorferi sensu stricto (4.1%) and B. spielmanii (1.6%). Borrelia bavariensis was confirmed only in four samples (0.1%), however, detection of this species has been performed only since 2017. Borrelia bissettii and B. kurtenbachii were both recorded in one case. The total prevalence differed significantly among four habitat types (urban, suburban, natural, agricultural). The highest infection prevalence was confirmed in natural habitat (22.0%), the lowest in urban habitat (13.2%). In addition, molecular analysis was carried out on part of the collected ticks previously morphologically identified as I. ricinus . The analysis did not confirm the occurrence of Ixodes inopinatus in Slovakia. Conclusion: Long-term monitoring of the abundance and spread of ticks as well as the prevalence and genetic variability of tick-borne pathogens can reveal the impact of global climatic and socio-economic changes on different habitats, including natural foci of tick-borne pathogens.

Hepatozoon spp. are tick-borne apicomplexan parasites of terrestrial vertebrates that occur worldwide. Tissue samples from small rodents and their parasitizing fleas were sampled for molecular detection and phylogenetic analysis of Hepatozoon-specific 18S rRNA gene region. After alignment and tree inference the Hepatozoon-sequences retrieved from a yellow-necked mouse (Apodemus flavicollis) placed into a strongly supported single clade demonstrating the presence of a novel species, designated Hepatozoon sp. SK3. The mode of transmission of Hepatozoon sp. SK3 is yet unknown. It is important to note that this isolate may be identical with the previously morphologically described Hepatozoon sylvatici infecting Apodemus spp.; however, no sequences are available for comparison. Furthermore, the previously reported variants Hepatozoon sp. BV1/SK1 and BV2/SK2 were detected in bank voles (Clethrionomys glareolus). It has been suggested that these variants should be identified as Hepatozoon erhardovae leading to the assumption that BV1 and BV2 are paralogous 18S rRNA gene loci of this species. Evidence has also been presented that fleas are vectors of H. erhardovae. In this study, we show with high significance that only the Hepatozoon sp. BV1 variant, but not BV2, infects the studied flea species Ctenophthalmus agyrtes, Ctenophthalmus assimilis, and Megabothris turbidus (p < 0.001). This finding suggests that Hepatozoon sp. BV2 represents an additional species besides H. erhardovae (= Hepatozoon sp. BV1), for which alternative arthropod vectors or non-vectorial modes of transmission remain to be identified. Future studies using alternative molecular markers or genome sequencing are required to demonstrate that BV1/SK1 and BV2/SK2 are different Hepatozoon species.

Lyme borreliosis (LB) is the most prevalent tick-borne human infection in Europe, with increasing incidence during the latest decades. Abundant populations of Ixodes ricinus, the main vector of the causative agent, spirochetes from the Borrelia burgdorferi sensu lato (Bbsl) complex, have been observed in urban and suburban areas of Europe, in general, and Slovakia, particularly. Understanding the spread of infectious diseases is crucial for implementing effective control measures. Global changes affect contact rates of humans and animals with Borrelia-infected ticks and increase the risk of contracting LB. The aim of this study was to investigate spatial and temporal variation in prevalence of Bbsl and diversity of its species in questing I. ricinus from three sites representing urban/suburban, natural and agricultural habitat types in Slovakia. Ixodes ricinus nymphs and adults were collected by dragging the vegetation in green areas of Bratislava town (urban/suburban habitat), in the Small Carpathians Mountains (natural habitat) (south-western Slovakia) and in an agricultural habitat at Rozhanovce in eastern Slovakia. Borrelia presence in ticks was detected by PCR and Bbsl species were identified by restriction fragment length polymorphism (RFLP). Borrelia burgdorferi s.l. species in coinfected ticks were identified by reverse line blot. Significant spatial and temporal variability in prevalence of infected ticks was revealed in the explored habitats. The lowest total prevalence was detected in the urban/suburban habitat, whereas higher prevalence was found in the natural and agricultural habitat. Six Bbsl species were detected by RFLP in each habitat type —B. burgdorferi sensu stricto (s.s.), B. afzelii, B. garinii, B. valaisiana, B. lusitaniae and B. spielmanii. Coinfections accounted for 3% of the total infections, whereby B. kurtenbachii was identified by RLB and sequencing in mixed infection with B. burgdorferi s.s, B. garinii and B. valaisiana. This finding represents the first record of B. kurtenbachii in questing I. ricinus in Slovakia and Europe. Variations in the proportion of Bbsl species were found between nymphs and adults, between years and between habitat types. Spatial variations in prevalence patterns and proportion of Bbsl species were also confirmed between locations within a relatively short distance in the urban habitat. Habitat-related and spatial variations in Borrelia prevalence and distribution of Bbsl species are probably associated with the local environmental conditions and vertebrate host spectrum. Due to the presence of Borrelia species pathogenic to humans, all explored sites can be ranked as areas with high epidemiological risk.

A study of ticks on wildlife was carried out in the area of Levice, Bratislava, Stupava, and Vrbovce (south-western Slovakia) during 2021 and 2022. Overall, 512 ticks were collected from 51 individuals of six wild mammalian species. Eight tick species were identified, namely Dermacentor reticulatus, D. marginatus, Haemaphysalis inermis, H. concinna, Ixodes ricinus, I. hexagonus, and two Ixodes spp. Ixodes hexagonus were collected from northern white-breasted hedgehogs (Erinaceus roumanicus), females belonging to Ixodes spp. were collected from red fox (Vulpes vulpes) and nymphs from European badger (Meles meles). Ixodes hexagonus and the Ixodes spp. were identified morphologically and molecularly based on sequences of fragments of two mitochondrial genes, COI and 16S rRNA. Molecular analysis of Ixodes spp. confirmed the identity of Ixodes kaiseri Arthur, 1957 and I. canisuga (Johnston, 1849). Sequence analyses show that the I. kaiseri isolate from Slovakia is identical to I. kaiseri isolates from Romania, Poland, Germany, Turkey, and Croatia. We demonstrate for the first time the presence of I. kaiseri in Slovakia using both morphological and molecular methods.