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Introduction: The region of Serbia is characterised by a high prevalence and diversity of Borrelia species, with Borrelia lusitaniae dominating, followed by Borrelia afzelii. Before this report, there were no data on Borrelia species causing Lyme borreliosis (LB) in Serbia. Case presentation: We report the case of a 10-year-old boy with a clinical presentation of disseminated erythema migrans (EM). His results showed IgM antibodies at 6.27 (negative ˂ 0.20; positive ˃ 0.32) against Borrelia burgdorferi sensu lato, which was confirmed via ELFA. Except for skin lesions, the patient did not show any other clinical signs of systemic infection. His blood was taken to isolate and cultivate spirochetes and for molecular analysis. Antimicrobial therapy was prescribed according to the recommended treatment for patients with LB. A follow-up examination was conducted after nine days. The EMs on the skin had disappeared, and antibiotic therapy was continued for 14 days. A second follow-up was conducted one month after the end of therapy. The boy's health condition was normal. After 16 days of incubation in BSK-H medium, viable, motile, and spiral-shaped spirochetes were observed in the culture tube, and cultivation was prolonged for 29 days. PCR and sequencing were successful in both the blood sample and the culture and confirmed the presence of B. lusitaniae. Conclusions: The results presented here is the first Borrelia isolate from the blood of a patient with the clinical manifestation of LB-disseminated EM. The presented results confirm the potential of B. lusitaniae for dissemination via the hematogenous route.

Ixodid ticks are distributed across all countries of the Western Balkans, with a high diversity of species. Many of these species serve as vectors of pathogens of veterinary and medical importance. Given the scattered data from Western Balkan countries, we have conducted a comprehensive review of available literature, including some historical data, with the aim to compile information about all recorded tick species and associated zoonotic pathogens in this region. Based on the collected data, the tick fauna of the Western Balkans encompasses 32 tick species belonging to five genera: Ixodes , Haemaphysalis , Dermacentor , Rhipicephalus and Hyalomma . A range of pathogens responsible for human diseases has also been documented, including viruses, bacteria and parasites. In this review, we emphasize the necessity for integrated surveillance and reporting, urging authorities to foster research by providing financial support. Additionally, international and interdisciplinary collaborations should be encouraged that include the exchange of expertise, experiences and resources. The present collaborative effort can effectively address gaps in our knowledge of ticks and tick-borne diseases. Graphical Abstract

In order to determine whether conserved tick salivary protein AV422 is immunogenic, the goal of our study was to detect specific IgG response within at-risk populations. Study groups included 76 individuals, differing in occurrence of recently recorded tick bites and health status. Western blotting with recombinant (r) protein derived from Ixodes ricinus (Ir) was performed. IgG response to Borrelia/Rickettsia, as indicators of previous tick infestations, was also assessed. Additionally, a detailed in silico AV422 protein sequence analysis was performed, followed by modelling of the interactions between peptides and corresponding MHC II molecules by molecular docking. Anti-rIrAV422 seroprevalences among individuals exposed to ticks were high (62.5, 57.9 and 66.7%) and anti-Borrelia/Rickettsia seroprevalences were 54.2, 15.8 and 44.4% among individuals with/without recent tick bite and patients suspected of tick-borne disease, respectively. In silico analysis of AV422 protein sequence showed a high level of conservation across tick genera, including also the predicted antigenic determinants specific for T and B cells. Docking to the restricted MHC II molecules was performed for all predicted AV422 T cell epitopes, and the most potent (highly immunogenic) epitope determinants were suggested. The epitope prediction reveals that tick salivary protein AV422 may elicit humoral immune response in humans, which is consistent with the high anti-rIrAV422 seroprevalence in tested at-risk subjects. Tick-borne diseases are a growing public health concern worldwide, and AV422 is potentially useful in clinical practice and epidemiological studies.

Background The epidemiological status concerning many canine tick‐borne diseases (TBDs) in Serbia is still insufficiently known. Objectives Our study aimed to investigate the presence of tick‐borne pathogens of the family Anaplasmataceae and Hepatozoon spp., as a cause of illnesses accompanied by clinical signs that can occur in dogs with anaplasmosis, ehrlichiosis and hepatozoonosis. Methods Dogs are included in the study based on the presence of a minimum of three clinical and/or pathological findings that could be associated with anaplasmosis, ehrlichiosis and hepatozoonosis. During the study (April–October 2018), 11 dogs met the conditions to be included in the survey. Identification of the causative agent in the blood of diseased dogs was performed by conventional PCR followed by sequencing. Results The presence of the pathogens was confirmed in three animals (3/11, 27.3%). The presence of Ehrlichia canis was confirmed in 3‐month‐old female Rottweiler puppy, an 8‐year old Miniature Schnauzer female was positive for Hepatozoon canis infection, while 4‐year‐old mixed breed male dog was co‐infected with both mentioned pathogens. These are the first cases of autochthonous infection with E. canis and H. canis in dogs from Serbia confirmed by molecular methods. Conclusions The results of our study indicate the importance of molecular methods to establish a reliable diagnosis of TBDs. Also, the confirmed presence of causative agents of canine monocytic ehrlichiosis and hepatozoonosis in Serbia appeals to veterinary practitioners that it is necessary to exclude the presence of those diseases in suspicious dogs. Dogs are included in the study based on the presence of a minimum of three clinical and/or pathological findings that could be associated with canine ehrlichiosis and hepatozoonosis. The presence of the pathogens was confirmed in three animals (3/11, 27.3%), Ehrlichia canis was confirmed in one dog, as well as Hepatozoon canis, while one dog was co‐infected with both mentioned pathogens. The confirmed presence of causative agents of canine monocytic ehrlichiosis and hepatozoonosis appeals to veterinary practitioners that is necessary to exclude the presence of those diseases in suspicious dogs

Past studies in Serbia have reported concurrent infections of Ixodes ricinus ticks with Borrelia burgdorferi sensu lato genospecies, Anaplasma phagocytophilum and Francisella tularensis . As a step forward, this investigation included a broader range of microorganisms and five most common and abundant tick species in Serbia. Five tick species were identified ( Dermacentor marginatus , D. reticulatus , Haemaphysalis punctata , H. concinna and I. ricinus ) and analyzed for the presence of seven pathogens. Anaplasma ovis , A. phagocytophilum , Babesia canis , B. burgdorferi s.l., Coxiella burnetii , Rickettsia helvetica and R. monacensis were detected. Sequencing of samples positive for F. tularensis revealed the presence of Francisella -like endosymbionts. No Bartonella spp. DNA was amplified. Concurrent infections were present in three tick species ( D. reticulatus , H. concinna and I. ricinus ). The rate of co-infections was highest in I. ricinus (20/27), while this tick species harbored the broadest range of co-infection combinations, with dual, triple and a quadruple infection(s) being detected.

Human activities such as deforestation, urbanization, and environmental pollution lead to a reduction in the spatial boundary between wild animals, domestic animals and humans. These activities increase the risk for the emergence of pathogens from the sylvatic cycle in the population of domestic animals and humans. Foxes are recognized as potential reservoirs for a number of bacterial pathogens of medical and public health concern. The aim of the present study was to investigate the prevalence and spatial distribution of bacterial tick-borne pathogens from the Anaplasmataceae family, sensu lato (s.l.), spp., , , spp., in the red fox population from Serbia and to discuss the obtained results from the epidemiological point of view. Legally hunted red foxes ( ) from 14 localities in Serbia were included in the study and spleen samples from 129 animals were tested with conventional PCR assays for the presence of bacterial tick-borne pathogens. DNA of Neoehrlichia sp. (FU98), sensu stricto, and was detected in 6 (4.7%), 1 (0.8%), 2 (1.6%) and 1 (0.8%) animals, respectively. Co-infection by Neoehrlichia sp. (FU98) and was detected in one animal. All samples were negative for other tested bacterial tick-borne pathogens. The results of the present study indicate the potential role of foxes in natural cycles of Neoehrlichia sp. (FU98) and causative agents of Lyme borreliosis in the investigated areas. Further research is required to elucidate the role of foxes in the epidemiology of these and other tick-borne zoonotic pathogens in the Republic of Serbia.

The majority of ixodid ticks display host-specificity to varying extents. Feeding on different hosts affects their development and reproduction. Consequences can be analyzed at the level of the egg, as it is the initial stage of tick development. Tick egg proteins are abundant and diverse, providing nutrients for embryonic development. However, studies on tick egg profiles are scarce. In this study, we aimed to analyze whether feeding Haemaphysalis qinghaiensis ticks on the yaks ( Bos grunniens ) and domestic sheep ( Ovis aries ) has an impact on the variety and variability of the egg proteome. Detached engorged females were used to lay eggs, which were then collected, dewaxed, and subjected to protein extraction. The extracted egg proteins were enzymatically digested using Filter-Aided Sample Preparation (FASP), and the unique peptides were separated and detected by Liquid Chromatography-tandem Mass Spectrometry (LC–MS/MS). The MS data were searched against the previously constructed whole tick transcriptome library of H. qinghaiensis, and the UniProt database for the identification of tick-derived egg proteins. The analysis revealed 49 and 53 high-confidence proteins identified in eggs collected from B. grunniens (EggBg) and O. aries (EggOa), respectively. Of these, 46 high-confidence proteins were common to both egg types, while three were unique to EggBg and seven to EggOa. All the identified proteins mainly belonged to enzymes, enzyme inhibitors, transporters, and proteins with unknown functions. The differential abundance analysis showed that nine proteins were significantly more present in EggBg, while six were significantly more present in EggOa. Overall, enzymes were the most diverse group, while vitellogenin (Vg) was the most abundant. Blood meal uptake on different hosts has a certain effect on the egg proteome composition and the abundance of some proteins, but it may also lead to compensation of protein roles.

In this study, the cDNAs of heat shock cognate 90 protein 1 ( HfHspc1 ) and heat shock cognate 90 protein 4 ( HfHspc4 ) from Haemaphysalis flava (Acari: Ixodidae) were obtained using the rapid amplification of cDNA ends (RACE) approach, and the expression patterns of HfHspc1 and HfHspc4 in different developmental stages, engorgement stages and tick organs were analyzed by qPCR. The full length of HfHspc1 was 2411 bp, and its open reading frame (ORF) was 2196 bp, encoding a protein of 732 aa, containing five HSPC family signatures, with MEEVD motif at its extreme C-terminal. The full length of HfHspc4 was 2800 bp, and its ORF was 2364 bp, encoding a protein of 789 aa, containing a signal peptide and five family signatures, with HEEL motif at its extreme C-terminal. The expression of HfHspc1 and HfHspc4 was the highest in males, while it was significantly the highest in the ovaries of fully engorged females, potentially implying the roles of these proteins in the successful digestion of blood and development of eggs.

Tick salivary proteins are crucial for efficient and successful tick feeding. Most of them are still uncharacterized, especially those involved in the formation of tick cement. Tick salivary protein PA107 is a putative cement protein, which is transcribed in salivary glands during the initial phase of tick feeding. It is a tick-unique protein, with homologs described in several tick genera. In this study, a detailed in silico analysis of its primary and tertiary structure was performed, along with the immunogenicity assessment for the PA107 protein from Ixodes ricinus species. The screening of the primary structure placed it to the glycine-rich protein family, revealing in parallel an overlapping 15mer at the C-terminus and borderline homology to non-tick proteins with antimicrobial activity. The analysis of tertiary structure revealed a high degree of intrinsic disorder for monomeric PA107, in contrast to highly ordered structures for different oligomeric states that might correlate with the putative role in the tick cement formation process. Regarding in silico PA107 immunogenicity inference, obtained results were inconclusive, which aligns with the in vitro findings showing definitely the lack of humoral response induction in experimentally infested rats and persons bitten by the I. ricinus ticks. The results represent new pieces of a huge puzzle depicting a complex tick-host relationship, but also identify PA107 as a possible compound of novel formulations to be used in biomedicine as bioadhesives, and as a target for new anti-tick strategies, by interfering with the cement cone formation and stability, i.e. tick attachment and feeding.

This study assessed the level of knowledge, attitudes, and practices (KAP) regarding tick-borne encephalitis virus (TBEV) and tick-borne diseases (TBDs) among different groups of people in Serbia. Professionally tick-exposed persons (PTEPs), health care workers (HCWs), and the general population (GP) were subjected to an anonymous, voluntary, online questionnaire using Microsoft Forms. A total of 663 questionnaire responses were collected (February–March 2021), while 642 were included in the analysis. The significant difference in knowledge in TBDs existed between GP and PTEPs, and HCWs (p < 0.001). The perception of risk-to-tick exposure and TBDs was generally high (42.4 (95% CI: 33.6–51.2) within GP, 44.9 (95% CI: 35.8–53.9) within PTEPs and 46.2 (95% CI: 38.0–54.5) within HCWs), while fear was low (13.7 (95% CI: 7.9–19.5) within GP, 12.6 (95% CI: 7.3–19.9) within PTEPs, and 13.5 (95% CI: 7.4–19.5) within HCWs). Protective practices differed across groups (F (2639) = 12.920, p < 0.001, η2 = 0.039), with both PTEPs (t = 3.621, Cohen d = 0.332, p < 0.001) and HCWs (t = 4.644, Cohen d = 0.468, p < 0.001) adhering to more protective practices than the GP, without differences between PTEPs and HCWs (t = 1.256, Cohen d = 0.137, p = 0.421). Further education about TBDs in Serbia is required and critical points were identified in this study.