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Wild canids are globally recognised as hosts and reservoirs of a large number of ecto- and endoparasites. Data that reveal the importance of the grey wolf (Canis lupus L.1758) in the spread of hepatozoonosis are very scarce. There are a large number of different potential host species that can be infected by Hepatozoon canis, but the most common are domestic and wild carnivores, such as dogs, jackals, foxes, and wolves. In this study, the epidemiological significance of the grey wolf as a host for the pathogen was analysed for the first time in Serbia, as well as the genetic variability of H. canis. The presence of H. canis in wolf spleens has been demonstrated using molecular methods. A total of 107 wolf spleen samples from 30 localities in Serbia were analysed. The presence of H. canis was confirmed in 62 (57.94%) individuals from 26 out of 30 localities. According to the analysis, the sampled H. canis sequences were found to be characterised by a certain heterogeneity. Based on five mutated nucleotide sites in the sequences, H. canis could be divided into five sequence types, S1 to S5. The five sequence types can potentially circulate in grey wolf populations as well as among other domestic and wild canids. This study is the first confirmation of the presence of H. canis in grey wolf populations in Serbia. Considering that the role of this vector-borne disease is poorly researched in wild carnivores, it is very important to indicate the role of this species in the circulation of this pathogen in natural ecosystems.

Hepatozoon canis is a protozoan tick-borne parasite infecting domestic and wild canids, including foxes, wolves, and jackals. It is mainly found in dogs but has also been detected in several wild carnivores, including foxes, wolves, and jackals. Host transmission primarily occurs through the ingestion of infected ticks, typically Rhipicephalus sanguineus, with documented instances of transplacental transmission from infected females to cubs. In Serbia, the golden jackal is common throughout the country, and its population has increased in recent years. Previous research has documented the presence of several vector-borne pathogens in the jackal population in Serbia, so we conducted this study to determine the presence, prevalence, and genetic variability of H. canis. Over eleven years (2010–2020), 114 animal samples were collected from 23 localities in Serbia. A total of 90/114 (78.95%) jackals were positive for H. canis, and they came from 22 localities. Among 15 juveniles, almost half (6/15 (40%)) tested positive for H. canis. In addition to the high prevalence, high genetic variability of the pathogen was also found. According to the mutated positions, four sequence types (S4–S7) of H. canis were determined. Based on our earlier research on the grey wolf and on this study, it can be observed that various sequence types of H. canis circulate within wild canid populations in Serbia. The prevalence of H. canis infection in wild carnivores raises significant concerns for wildlife conservation and animal health. Infected animals may act as reservoirs for the disease, posing a potential risk to domestic animals by acting as a source of infection.

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.

Canine circovirus (CanineCV) is an emerging virus of interest in both domestic and wild carnivores that is scarcely reported in southeastern Europe. This study examined the presence, genetic diversity, and evolutionary characteristics of CanineCV in red foxes (Vulpes vulpes) and golden jackals (Canis aureus) from northwestern Serbia, a region marked by expanding mesopredator populations overlapping with human habitats. Out of 98 sampled animals, circoviral DNA was detected in 31.6%. Jackals were mostly positive for CanineCV genotype 4, while genotype 5, associated with wild carnivores, was dominant in foxes. Mixed genotype 4/genotype 5 infections were only found in jackals. Phylogenetic and haplotype analyses indicated that most jackal-derived CanineCV strains clustered along sequences from Europe, Africa, and the Americas, while genotype 5 sequences grouped separately from other genotype representatives. A recombinant strain was identified as a divergent lineage, and several sequences showed evidence of recombination between Rep and Cap genes. Despite Cap protein amino acid differences, purifying selection dominated, suggesting functional constraints on viral evolution. The results indicate that jackals may act as recombination hotspots and bridging hosts between viral lineages. This study provides insight into the molecular epidemiology of CanineCV in the Balkans, highlighting the importance of ongoing surveillance.

Theileria orientalis is a tick-borne intraerythrocytic protozoan parasite of cattle causing oriental or benign theileriosis. It has a worldwide distribution and is not considered a zoonotic agent, while the disease symptoms range from subclinical to severe. Theileria spp. are very common co-infecting agents with other tick borne pathogens such as Babesia spp., Anaplasma spp. including zoonotic Anaplasma phagocytophilum with rising prominence. This study is an exploratory investigation of the occurrence of Anaplasma spp. and other blood parasites in cattle from Gacko and Bileća, Herzegovina. Blood samples from 35 clinically healthy cattle were selected based on veterinary reports of tick infestation and examined microscopically, by PCR ( Babesia/Theileria ; Anaplasmataceae ) and qPCR ( A. phagocytophilum, A.platys, A.marginale ). Microscopy revealed intraerythrocytic bacterial forms in multiple samples. A. phagocytophilum DNA was detected in one and Babesia/Theileria DNA in 34 samples. Based on 16S rRNA gene sequencing, the A. phagocytophilum isolate was grouped with isolates from Europe, suggesting regional pathogen circulation. AnkA gene analysis placed the isolate in European group I, but outside the zoonotic subcluster, indicating a ruminant-associated lineage maintained primarily in wildlife– Ixodes ricinus cycles. The 18S rRNA gene sequencing showed subtle regional clustering of T.orientalis isolates. This is the first molecular confirmation of A. phagocytophilum in cattle from Eastern Herzegovina and identification of possible enzootic foci of T. orientalis infection, deeming further investigation into tick vectors and reservoir hosts in the region.

Although the importance of the microbiome in the context of tick biology and vector competence has recently come into a broader research focus, the field is still in its infancy and the complex ecological interactions between the tick residential bacteria and pathogens are obscure. Here, we show that an environmentally acquired gut bacterium has the potential to impair Borrelia afzelii colonization within the tick vector through a secreted metalloprotease. Oral introduction of either Bacillus cereus LTG-1 isolate or its purified enhancin ( Bc Enhancin) protein significantly reduces B. afzelii burden in the guts of Ixodes ricinus ticks. This effect is attributed to the ability of Bc Enhancin to degrade a glycan-rich peritrophic matrix (PM), which is a gut protective barrier essential for Borrelia survival. Our study highlights the importance of the gut microbiome in determining tick vector competence and provides a deeper mechanistic insight into the complex network of interactions between Borrelia , the tick, and the tick microbiome.

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

Ixodid ticks are present throughout the Western Balkan countries, including Albania, Bosnia and Herzegovina, Croatia, Montenegro, North Macedonia and Serbia, with many species serving as vectors for pathogens of both veterinary and medical importance. We have conducted a thorough examination of existing literature, encompassing historical documents, to collect information on all documented non-zoonotic tick-borne pathogens found in ticks, pets, farm animals and wild animals across the Western Balkan region. A comprehensive review was necessary due to the scarcity and scattered nature of available data from this area. The tick fauna in the Western Balkans consists of 32 species across five genera: Ixodes , Haemaphysalis , Dermacentor , Rhipicephalus and Hyalomma . Various pathogens responsible for diseases in animals, including bacteria and parasites, have also been documented, many of which can cause important diseases and significant reductions in animal productivity. Initial efforts were directed towards pathogen surveillance and the characterisation of non-zoonotic tick-borne pathogens, resulting in the identification of Theileria orientalis , Anaplasma bovis and Anaplasma marginale in cattle, although significant gaps remain in the current research. Studies on small ruminants have been sparse, with confirmed cases of Anaplasma ovis and Babesia ovis in sheep, but no comprehensive and systematic research on pathogens in goats. In contrast, research on canine piroplasms has identified several species, including Babesia canis and Babesia vulpes . Studies on wild animals, however, have predominantly focused on wild canines and carnivores, with limited attention given to non-zoonotic pathogens. Notably, only one study has reported non-zoonotic tick-borne pathogens in artiodactyl species and wild felids. This review is a much needed overview of existing research on non-zoonotic tick-borne diseases in the Western Balkans, including the historical context, current data and research gaps. Given the significant impact of these diseases on animal health and productivity, as well as their potential biodiversity, further comprehensive studies and the establishment of national surveillance systems for tick-borne diseases are essential for a better understanding and mitigation of their impact. Graphical abstract

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

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.