Tick-borne diseases are a serious threat to both public and veterinary health. In this study, we used high-throughput sequencing to characterize the virome of three tick species implicated in the spread of vector-borne disease throughout Croatia. Ten viruses were identified, including seven potential novel species within the viral families Flaviviridae, Nyamiviridae, Rhabdoviridae, Peribunyaviridae, Phenuiviridae, and Nairoviridae.

Tick-borne encephalitis (TBE) represents an important public health problem in Europe. We analyzed the epidemiology of TBE based on data from humans, animals, and Ixodes ricinus ticks in endemic regions of continental Croatia. In the period from 2017 to 2023, cerebrospinal fluid (CSF) and serum samples of 684 patients with neuroinvasive diseases, 2240 horse serum samples, and 300 sheep serum samples were tested for TBEV. In addition, 8751 I. ricinus ticks were collected. CSF samples were tested using RT-PCR. Serological tests (serum, CSF) were performed using commercial ELISA, with confirmation of cross-reactive samples by a virus neutralization test. Eighty-four autochthonous human TBEV cases were confirmed. The majority of patients were in the age group of 40–69 years (58.3%) with a male predominance (70.2%). TBE showed a bimodal seasonality with a large peak in April–August and a small one in October–November. In addition to humans, TBEV IgG antibodies were found in 12.2% of horses and 9.7% of sheep. Seasonal tick abundance corresponds to the reported number of human infections. Continental Croatia is still an active natural focus of TBE. Continuous monitoring of infections in humans, sentinel animals, and ticks is needed for the implementation of preventive measures.

Since the role of wild rodents/small mammals in hepatitis E virus (HEV) epidemiology has been a subject of considerable debate, this study was conducted to investigate the potential presence of HEV RNA in small rodents collected within their natural habitats and to detect if they can be potential reservoirs of the virus. A total of 483 small rodents were captured using snap traps placed at 11 regions in Croatia. Sampling was undertaken in 2008 and repeated from 2010 to 2014. Liver samples were tested for the presence of HEV RNA. HEV RNA was detected in only one liver sample (0.21%) originated from Apodemus flavicollis from the location Medvednica, nearby Zagreb collected in 2014. According to the sequence analysis, the isolate has shown to be a member of Orthohepevirus A species, genotype HEV-3. The genotyping results confirmed grouping into subtype 3a, general cluster 3abchij.The detected HEV strain showed to be genetically highly related to strains found in humans and/or domestic pigs and wild boars from Croatia. Our finding indicates that wild small mammals could play a role in the epidemiology of HEV-3 infection and therefore should be taken under consideration as potential reservoirs or/and transmitters of the disease. However, further investigation is needed to recognize their potential for maintaining the infection in natural conditions.

As tick-borne disease incidence and distribution is increasing worldwide, there is a true need for better understanding of the distributional ecology of their vectors. The aim of this study was to determine the diversity of hard ticks fauna (Acari: Ixodidae) and their seasonal dynamics in different habitats at three urban recreational sites in Croatia’s capital, the City of Zagreb, known as the natural foci of Lyme borreliosis and tick borne encephalitis. Within a three-year period (2016 - 2018), the only species detected was Ixodes ricinus Linnaeus, 1758. Using flag dragging method 506 ticks were sampled; 273 (54%) in their nymphal stage, 166 (33%) as larvae and 64 (13%) as adults. The highest abundance of ticks was recorded at forest habitat. Seasonal activity showed their peaks at midspring and midsummer. Continuous monitoring of hard tick population in urban areas should and could become a standard method of tick-borne diseases prevention.

Neuroinvasive arboviruses such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Usutu virus (USUV), and Toscana virus (TOSV) have (re-)emerged with increasing incidence and geographic range. We analyzed the epidemiology of arboviral infections in Croatia during the 2024 transmission season. A total of 154 patients with neuroinvasive diseases (NID), 1596 horses, 69 dead birds, and 7726 mosquitoes were tested. Viral RNA was detected using RT-qPCR. IgM/IgG-specific antibodies were detected using commercial ELISA or IFA, with confirmation of cross-reactive samples by virus neutralization test. RT-qPCR-positive samples were Sanger sequenced. Arboviral etiology was confirmed in 33/21.42% of patients with NID. WNV was most frequently detected (17/11.03%), followed by TBEV (10/6.49%), USUV (5/3.24%), and TOSV (1/0.64%). WNV infections were reported in regions previously known as endemic, while in one continental county, WNV was recorded for the first time. USUV infections re-emerged after a six-year absence. In addition to human cases, acute WNV infections were recorded in 11/395 (2.78%) of horses and two dead crows. WNV IgG seropositivity was detected in 276/1168 (23.63%) and TBEV IgG seropositivity in 68/428 (15.88%) horses. None of the tested mosquito pools were positive for WNV and USUV RNA. Phylogenetic analysis showed the circulation of WNV lineage 2 and Usutu Europe 2 lineage. Climate conditions in 2024 in Croatia were classified as extremely warm, which could, at least in part, impact the quite intense arboviral season. The spreading of flaviviruses in Croatia highlights the need for continuous surveillance in humans, animals, and vectors (“One Health”).

Altitudinal distributions, population structures and seasonal dynamics of tick fauna at three localities in Continental Croatia (Medvednica and Papuk) and an alpine biogeographic region (Gorski Kotar) were studied. Sampling of questing ticks was performed twice a year (spring and autumn, from 2019 to 2021) at different altitudes (200, 400, 600, 800 and 1000 m above sea level) using the flagging method. In total, 2942 ticks (53.9% larvae, 40.1% nymphs, 6.0% adults) were sampled and 2937 (99.83%) were determined as Ixodes ricinus, 4 (0.14%) as Haemaphysalis concinna and 1 (0.03%) as Ixodes frontalis. Ixodes ricinus was the only species found at all altitudes and sampling sites. The highest tick abundance was recorded at higher altitudes (800–1000 m asl.) on Medvednica and in Gorski Kotar within mixed forests of European beech and European silver fir, while on Papuk most of the ticks were sampled at lower altitudes (200 m asl.) in Sessile oak forest. From 27 pools containing 305 ticks, 1 (3.7%) was positive for Borrelia burgdorferi sensu lato infection. Borrelia burgdorferi s.l. was detected in 20% (1/5) of the pools containing nymphs and adults collected in Gorski Kotar (600 and 800 m asl.). Sequencing of the ospA gene and phylogenetic analysis revealed the presence of the Borrelia burgdorferi sensu stricto genotype.