Free-living animals frequently play a key role in the circulation of various zoonotic vector-borne pathogens. Bacteria of the genus Bartonella are transmitted by blood-feeding arthropods and infect a large range of mammals. Although only several species have been identified as causative agents of human disease, it has been proposed that any Bartonella species found in animals may be capable of infecting humans. Within a wide-ranging survey in various geographical regions of the Czech Republic, cadavers of accidentally killed synurbic mammalian species, namely Eurasian red squirrel (Sciurus vulgaris), European hedgehog (Erinaceus europaeus) and Northern white-breasted hedgehog (Erinaceus roumanicus), were sampled and tested for Bartonella presence using multiple PCR reaction approach targeting several DNA loci. We demonstrate that cadavers constitute an available and highly useful source of biological material for pathogen screening. High infection rates of Bartonella spp., ranging from 24% to 76%, were confirmed for all three tested mammalian species, and spleen, ear, lung and liver tissues were demonstrated as the most suitable for Bartonella DNA detection. The wide spectrum of Bartonella spp. that were identified includes three species with previously validated zoonotic potential, B. grahamii, B. melophagi and B. washoensis, accompanied by ‘Candidatus B. rudakovii’ and two putative novel species, Bartonella sp. ERIN and Bartonella sp. SCIER.

Background: Vector-borne pathogens (VBPs) are a major threat to humans, livestock and companion animals worldwide. The combined effect of climatic, socioeconomic and host composition changes favours the spread of the vectors, together with the expansion of invasive carnivores contributing to the spread of the pathogens. In Europe, the most widespread invasive species of carnivores are raccoons (Procyon lotor) and raccoon dogs (Nyctereutes procyonoides). This study focused on the detection of four major groups of VBPs namely Babesia, Hepatozoon, Anaplasma phagocytophilum and Bartonella in invasive and native carnivores in the Czech Republic, with the emphasis on the role of invasive carnivores in the eco-epidemiology of said VBPs. Methods: Spleen samples of 84 carnivores of eight species (Canis aureus, Canis lupus, Lynx lynx, P. lotor, Martes foina, Lutra lutra, Mustela erminea and N. procyonoides) were screened by combined nested PCR and sequencing for the above-mentioned VBPs targeting 18S rRNA and cytB in hemoprotozoa, groEL in A. phagocytophilum, and using multilocus genotyping in Bartonella spp. The species determination is supported by phylogenetic analysis inferred by the maximum likelihood method. Results: Out of 84 samples, 44% tested positive for at least one pathogen. Five different species of VBPs were detected in P. lotor, namely Bartonella canis, Hepatozoon canis, Hepatozoon martis, A. phagocytophilum and Bartonella sp. related to Bartonella washoensis. All C. lupus tested positive for H. canis and one for B. canis. Three VBPs (Hepatozoon silvestris, A. phagocytophilum and Bartonella taylorii) were detected in L. lynx for the first time. Babesia vulpes and yet undescribed species of Babesia, not previously detected in Europe, were found in N. procyonoides. Conclusions: Wild carnivores in the Czech Republic are hosts of several VBPs with potential veterinary and public health risks. Among the studied carnivore species, the invasive raccoon is the most competent host. Raccoons are the only species in our study where all the major groups of studied pathogens were detected. None of the detected pathogen species were previously detected in these carnivores in North America, suggesting that raccoons adapted to local VBPs rather than introduced new ones. Babesia vulpes and one new, probably imported species of Babesia, were found in raccoon dogs. Graphical

Background: Anaplasma phagocytophilum is characterized by a worldwide distribution and distinguished from other Anaplasmataceae by the broadest range of mammalian hosts and high genetic diversity. The role carnivores play in the life cycle of A. phagocytophilum in Europe is uncertain. Currently, only the red fox is considered a suitable reservoir host. In this study, we focused on native and invasive medium-sized carnivore species that live in sympatry and represent the most abundant species of wild carnivores in Poland. Methods: A total of 275 individual spleen samples from six carnivore species (Vulpes vulpes, Meles meles, Procyon lotor, Nyctereutes procyonoides and Martes spp.) were screened combining nested PCR and sequencing for A. phagocytophilum targeting a partial groEL gene with subsequent phylogenetic analysis inferred by the maximum likelihood method. Results: The DNA of A. phagocytophilum was detected in 16 of 275 individuals (5.8%). Eight unique genetic variants of A. phagocytophilum were obtained. All detected haplotypes clustered in the clade representing European ecotype I. Three variants belonged to the subclade with European human cases together with strains from dogs, foxes, cats, and wild boars. Conclusions: While carnivores might have a restricted role in the dissemination of A. phagocytophilum due to their relatively low to moderate infection rates, they hold significance as hosts for ticks. Consequently, they could contribute to the transmission of tick-borne infections to humans indirectly, primarily through tick infection. This underscores the potential risk of urbanization for the A. phagocytophilum life cycle, further emphasizing the need for comprehensive understanding of its ecological dynamics. Graphical

Ixodes arboricola and Ixodes lividus are ornithophilic tick species. The former is typically associated with tree-hole dwelling birds, while the latter is a host-specific parasite of sand martins (Riparia riparia). These two tick species share important morphological characters that make them difficult to identify when they are collected from atypical hosts, such as birds of prey. Despite this, high resolution digital pictures have not been reported to compare I. arboricola and I. lividus, nor was their complete mitogenome reported. The aim of this study was to compensate for this lack of illustrations and sequence data. Nymphs and females of I. arboricola and I. lividus were used for morphological comparison, and one specimen of each species to generate mitogenome sequences. The results showed that females of these two species are different in the shape of their scutum, porose areas, the length of basis capituli, palps, coxae, genital pore, anal groove and tarsus I. On the other hand, nymphs of I. arboricola and I. lividus can be distinguished according to their cervical grooves, cornuae, auricular ridges and spiracular plates. The mitochondrial genome size was 14,539 and 14,536 bp, for I. arboricola and I. lividus, respectively. The mitogenome sequences of I. arboricola and I. lividus were 91.1% identical to each other. Phylogenetic analysis of Ixodes species showed that I. arboricola and I. lividus are sister species, and cluster together with Ixodes crenulatus/canisuga under strong support. In conclusion, results of this study confirmed that the front of the basis capituli is crucial in distinguishing Ixodes species, especially in the subgenus Pholeoixodes where these two species are phylogenetically closest related to I. canisuga. Another phylogenetically relevant morphological character is the scutal surface which is wrinkled (rugose) as a common feature of Pholeoixodes species in the clade of I. arboricola and I. lividus (including I. canisuga, I. rugicollis and I. ariadnae). Although the host ranges of I. arboricola and I. lividus do not substantially overlap, they may transmit some of the shared pathogens. Relevant data indicate that the eco-epidemiological significance of the two ornithophilic tick species studied here may in part be similar and they may play a role in the transmission of rickettsiae, borreliae and viruses of which birds act as reservoirs.

Ticks are vectors of pathogens affecting wildlife, livestock, and humans. Knowledge of their abundance, species composition, and ecological drivers is essential for an understanding of tick-borne disease ecology. In this study, four natural sites in Sicily (southern Italy) were selected, and 39 sampling events from April 2024 to August 2025 yielded 1200 ticks: 407 larvae, 474 nymphs, and 319 adults. Five genera were identified (Dermacentor, Haemaphysalis, Rhipicephalus, Ixodes, and Hyalomma); Rhipicephalus bursa was the most abundant species, followed by Haemaphysalis punctata. Most ticks were collected in spring–summer, except at one site where abundance peaked in autumn–winter. A significant association was found between site and season (p < 0.0001), but tick abundance per sampling event did not differ among sites (p = 0.431) or seasons (p = 0.769). No significant correlations emerged between tick abundance and environmental variants. However, significant associations were detected between site and sex (p < 0.0001), site and developmental stage (p < 0.0001), and species and developmental stage (p < 0.0001). Sites exclusively hosting wild boar showed higher species richness. These findings underscore the ecological complexity of tick populations in Mediterranean protected areas and provide baseline data useful for integrated tick surveillance and wildlife health management.