Background: Vector-borne pathogens (VBPs) are increasing in significance in veterinary medicine and public health settings, with wildlife playing a potentially crucial role in their transmission. Eurasian badgers (Meles meles) are widely distributed across Europe. However, information currently available on the prevalence of VBPs in badgers is limited. The objective of the current study was to investigate the occurrence of Anaplasmataceae, Bartonella spp., Mycoplasma spp., Rickettsia spp., Piroplasmida, Trypanosomatida and Filarioidea in badgers and subsequently, based on the results, assess the potential risk to domestic animals, other wildlife and humans. Methods: Between 2017 and 2021, blood or spleen samples from 220 badgers were collected in nine continental European countries: Austria (n = 7), Bosnia and Herzegovina (n = 2), Croatia (n = 22), France (n = 44), Germany (n = 16), Hungary (n = 7), Italy (n = 16), Romania (n = 80) and Serbia (n = 26). VBPs were identified by performing PCR analysis on the samples, followed by Sanger sequencing. Additionally, to distinguish between different Babesia lineages we performed restriction fragment length polymorphism (RFLP) analysis on piroplasm-positive samples, using HinfI as restriction enzyme. A phylogenetic analysis was performed on Mycoplasma spp. Results: The pathogens identified were Babesia sp. badger type A (54%), B (23%), and C (37%); Trypanosoma pestanai (56%); Mycoplasma sp. (34%); Candidatus Mycoplasma haematomelis (8%); Candidatus Mycoplasma haematominutum (0.5%); and Ehrlichia spp. (2%). Rickettsia spp., Bartonella spp. and filarioid nematodes were not detected among the tested samples. Conclusions: The large sample size and diverse study populations in this study provide valuable insights into the distribution and epidemiology of the analyzed pathogens. Some of the VBPs identified in our study show high similarity to those found in domestic animals, such as dogs. This finding suggests that badgers, as potential reservoirs for these pathogens, may pose a threat not only to other wildlife but also to domestic animals in close vicinity. Continuous surveillance is essential to monitor VBPs in wildlife as a means to enable the assessment of their impact on other wildlife species, domestic animals and human health. Graphical

The European wildcat ( Felis silvestris silvestris ) is a rare species in Croatia, yet data on its vector-borne pathogens (VBPs) have been entirely lacking. This study provides the first molecular survey of VBPs in twelve Croatian wildcats collected between 2018 and 2025. Using broad-range PCR assays and sequencing, we found pathogen DNA in all analyzed animals (12/12). The most common pathogen was Hepatozoon felis (10/12), followed by Cytauxzoon europaeus (4/12), Mycoplasma spp. (2/12), and Hepatozoon silvestris (1/12). Dual infections were identified in 5/12 animals. Notably, phylogenetic analysis of hemotropic mycoplasmas revealed a Mycoplasma suis -like genotype, typically associated with swine, suggesting potential cross-species transmission at the sylvatic-domestic interface. The high prevalence of pathogens highlights a potential health risk for this protected population as well as to domestic cats ( Felis catus ) in the region. These findings fill a critical knowledge gap in the studied region and emphasize the need for integrated conservation strategies that consider the domestic-sylvatic interface in pathogen dynamics.

Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis, is considered endemic in Mediterranean Europe and is primarily associated with the brown dog tick, Rhipicephalus sanguineus sensu lato. In Croatia, previous molecular investigations in dogs failed to detect the pathogen, despite sporadic serological findings and the confirmed presence of competent tick vectors. The aim of this study was to investigate the presence of E. canis DNA in ticks collected in Croatia. A total of 583 ticks from five genera were collected from the environment, domestic animals, wildlife, and humans across approximately 130 locations. All ticks were screened by PCR targeting the 16S rRNA gene, and positive samples were further characterised by sequencing of the full-length 16S rRNA gene. Six ticks tested positive for Ehrlichia sp. DNA, and sequencing confirmed all isolates as E. canis. All positive ticks were identified as Rhipicephalus bursa and originated from coastal Mediterranean regions of Croatia. Positive specimens were collected from goats, sheep, and chamois (Rupicapra rupicapra). These findings represent the first molecular evidence of E. canis in Croatia and indicate its circulation in ruminant-associated tick populations within the Mediterranean part of the country. Further studies are needed to clarify transmission dynamics and the potential epidemiological relevance for domestic animals.

Ticks are obligate hematophagous ectoparasites of public health importance, acting as vectors of numerous pathogens affecting wildlife, domestic animals and humans. Although birds play an important role in tick ecology and dispersal, information on tick infestations of pheasants in Europe remains limited. This study investigated the prevalence, species composition, developmental stage structure and haplotype diversity of ticks infesting pheasants in Croatia using ecological, morphological and molecular approaches. During the hunting seasons, 255 pheasants from continental and Mediterranean regions of Croatia were examined. Ticks were collected from harvested pheasants and road-killed individuals, morphologically identified, and representative specimens were molecularly confirmed. Overall, 213 pheasants were infested, resulting in a prevalence of 83.5%, and a total of 1103 ticks were collected. Six tick species were identified, with Ixodes ricinus dominating the tick community (80.1%). Molecular analyses confirmed the occurrence of several closely related Mediterranean Ixodes species, including Ixodes festai and Ixodes ventalloi. Nymphs predominated (78.2%), highlighting the importance of pheasants as hosts for immature tick stages. Significant geographical differences were observed. Continental localities were dominated by I. ricinus, whereas coastal and island regions showed significantly higher tick species diversity. Mean tick abundance per pheasant was significantly higher in Mediterranean regions. Male pheasants exhibited higher infestation prevalence and intensity than females. The marked regional differences highlight the influence of Croatia’s biogeographical heterogeneity on tick community composition. These findings demonstrate that pheasants serve as important hosts for diverse tick species and likely contribute to the maintenance of local tick populations, thereby providing a valuable baseline for future surveillance of tick-borne pathogens in Croatia.

Ticks are hematophagous ectoparasites of major veterinary and public health importance, acting as vectors of numerous pathogens affecting wildlife, domestic animals, and humans. Data on tick species infesting lagomorphs in Croatia remain scarce. This study investigated the species composition and genetic diversity of ticks collected from European brown hares (Lepus europaeus) in Croatia during 2024–2025 using morphological and molecular methods. A total of 158 hares from 18 localities were examined, and ticks were detected on 145 individuals, resulting in an infestation prevalence of 91.8%. Morphological identification and molecular analyses revealed nine tick species. The tick community was dominated by Ixodes ricinus (59.1%; n = 821), followed by Ixodes festai (26.8%; n = 372) and Rhipicephalus bursa (9.6%; n = 133), together accounting for more than 95% of all collected ticks. Molecular analyses confirmed species identification and revealed 10 haplotypes of I. ricinus, eight of I. festai, two of I. ventalloi, and one in I. inopinatus. The results demonstrate that European brown hares are important hosts for diverse tick species in Croatia and provide baseline data for future studies on tick ecology, tick-borne pathogens, and wildlife health management.

Ticks (Acari: Ixodidae) are important ectoparasites and vectors of numerous pathogens affecting wildlife, domestic animals, and humans. Although birds play a significant role in tick ecology, information on tick infestations in wild galliform birds remains limited. The aim of this study was to identify tick species associated with rock partridge (Alectoris graeca) populations in Mediterranean and sub-Mediterranean habitats of Croatia, and to evaluate their potential importance in tick ecology. A total of 39 rock partridges were examined from four localities in Croatia (Pag, Lozovac, Primorski Dolac and Svilaja). A total of 47 ticks were collected from 27 birds (69.2%). Molecular analysis based on sequencing of the mitochondrial 16S rRNA gene confirmed the morphological identification of all tick species. Four tick species belonging to the genus Ixodes were identified: Ixodes festai, Ixodes gibbosus, Ixodes ventalloi and Ixodes hexagonus. Ixodes festai was the dominant species (72.3%) and was recorded at all four localities. The remaining ticks belonged to I. gibbosus (10.6%), I. ventalloi (8.5%), and I. hexagonus (8.5%) and were found exclusively on Pag Island. Among the collected ticks, adult stages (55.3%) and nymphs (42.6%) predominated. The results indicate that rock partridges represent important hosts for several Mediterranean tick species of the genus Ixodes and may contribute to the maintenance of their local populations. The dominance and widespread occurrence of I. festai suggest a strong ecological association between this ornithophilic tick species and rock partridge populations. Furthermore, the exclusive occurrence of I. ventalloi, I. gibbosus and I. hexagonus on Pag Island indicates the influence of local ecological conditions and host community composition on tick population structure. The results of this study contribute to a better understanding of tick ecology and provide an important basis for future research on tick-borne pathogens in Mediterranean ecosystems.