Ticks are key vectors of zoonotic pathogens, and their expanding distribution in Europe heightens public health concerns. In Bosnia and Herzegovina, while tick distribution is well documented, molecular data on tick-borne pathogens remain limited. This study aimed to illustrate the presence and diversity of these pathogens, focusing on areas with high human activity. Ticks (n = 556) were collected in April 2022 from eight diverse locations, including urban parks, private properties, and rural sites. PCR-based screening was employed to detect Anaplasmataceae, Borrelia, Francisella, Piroplasmida, Rickettsia, and tick-borne encephalitis virus (TBEV), with subsequent sequencing to confirm results. Further characterization of Borrelia burgdorferi sensu lato was achieved via reverse line blotting (RLB) hybridization and sequencing. Ixodes ricinus was the most prevalent species, followed by Dermacentor marginatus and D. reticulatus. Our analysis revealed an overall infection rate of 22.1% in questing ticks, with Rickettsia spp. and Borrelia spp. predominating. Notably, seven Borrelia species were identified in I. ricinus, alongside Anaplasma phagocytophilum, Rickettsia helvetica, and R. monacensis, with co-infections mainly observed in peri-urban areas. This study provides the first molecular evidence of multiple tick-borne pathogens in the region, underscoring the need for further surveillance and risk assessment of tick-borne diseases in the region.

The etiology of transmissible viral proventriculitis (TVP) of broiler chickens has been discussed since its initial recognition 40 years ago. Regardless of its low direct impact on mortality rate, it leads to high economic losses in the broiler industry through reduction of food conversion, weakening of birds, and their increased susceptibility to pathogens. The aim of the present study was to examine the potential presence of TVP on the broiler chicken farms in Bosnia and Herzegovina, to characterize microscopic lesions, and to investigate the viruses implicated in etiology of TVP by PCR-based methods. In total, 143 diseased broiler chickens from 16 farms in Bosnia and Herzegovina were euthanized and subjected to necropsy and subsequent histopathology of proventriculi. A representative number of proventriculi samples (n = 50) that exhibited histopathologic changes were processed for molecular detection of chicken proventricular necrosis virus (CPNV), girovirus (GyV3), chicken anemia virus (CAV), and infectious bursal disease virus (IBDV) by PCR-based methods. In addition, samples of bursa of Fabricius (n = 39) and spleen (n = 50) were tested for IBDV. Histopathology revealed changes consistent with TVP in 39.8% (57/143) and LP (lymphocytic proventriculitis) in 2.1% (3/143) of samples. All 50 proventricular samples showed positivity to CPNV with Ct values ranging between 18 and 26. GyV3 was detected in eight samples (16%), with Ct values ranging from 11.1 to 27.5. The presence of CAV was more prominent (38%), with 19 positive broiler chickens (Ct ranging from 9.6 to 35.6). Pooled samples of spleen, bursa, and proventriculi from three farms were positive for IBDV. The obtained results represent the first documented data on TVP and the first record of CPNV and GyV3 presence in broiler farms from Bosnia and Herzegovina.

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 (BcEnhancin) protein significantly reduces B. afzelii burden in the guts of Ixodes ricinus ticks. This effect is attributed to the ability of BcEnhancin 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.

Case summary A 2-year-old domestic longhair crossbred female cat was referred for a second opinion on a non-healing surgical wound after left eye enucleation. In addition to the left orbital lesion, ulcerative granular masses protruding from the left nostril and on the base of the left ear were noted. A diagnosis of cryptococcosis was established using histopathological examination and a latex cryptococcal antigen agglutination test. The cat was successfully treated with itraconazole. Relevance and novel information Cryptococcosis, commonly reported in Australia, western Canada and the western USA, is rarely reported in companion animals in Europe. This marks the first report of cryptococcosis in cats in Bosnia and Herzegovina, emphasising the need to raise awareness within the veterinary community, both local and regional, about this disease.

An integrated approach using phylogeographic data, climate modelling, and network analysis unlocks insights into the post-glacial expansion of the sand fly Phlebotomus mascittii in Europe and suggests two potential (parallel) dispersal routes. Phlebotomine sand flies (Diptera: Phlebotominae) are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). In Central Europe, Phlebotomus mascittii is the predominant species, but largely understudied. To better understand factors driving its current distribution, we infer patterns of genetic diversity by testing for signals of population expansion based on two mitochondrial genes and model current and past climate and habitat suitability for seven post-glacial maximum periods, taking 19 climatic variables into account. Consequently, we elucidate their connections by environmental-geographical network analysis. Most analyzed populations share a main haplotype tracing back to a single glacial maximum refuge area on the Mediterranean coasts of South France, which is supported by network analysis. The rapid range expansion of Ph. mascittii likely started in the early mid-Holocene epoch until today and its spread possibly followed two routes. The first one was through northern France to Germany and then Belgium, and the second across the Ligurian coast through present-day Slovenia to Austria, toward the northern Balkans. Here we present a combined approach to reveal glacial refugia and post-glacial spread of Ph. mascittii and observed discrepancies between the modelled and the current known distribution might reveal yet overlooked populations and potential further spread.

Bats are a natural host for a number of viruses, many of which are zoonotic and thus present a threat to human health. RNA viruses of the family Filoviridae, many of which cause disease in humans, have been associated with specific bat hosts. Lloviu virus is a Filovirus which has been connected to mass mortality events in Miniopterus schreibersii colonies in Spain and Hungary, and some studies have indicated its immense zoonotic potential. A die-off has been recorded among Miniopterus schreibersii in eastern Bosnia and Herzegovina for the first time, prompting the investigation to determine the causative agent. Bat carcasses were collected and subjected to pathological examination, after which the lung samples with notable histopathological changes, lung samples with no changes and guano were analyzed using metagenomic sequencing and RT-PCR. A partial Lloviu virus genome was sequenced from lung samples with histopathological changes and found to be closely related to Hungarian and Italian virus sequences. Further accumulation of mutations on the GP gene, coding the glycoprotein responsible for cell tropism and host preference, enhances the need for further characterization and monitoring of this virus to prevent spillover events and protect human health.

The objective of this paper was to create an economical and abecedarian 3D printing method for the production of solid microneedles, as a more efficient transdermal drug delivery method, for day-to-day use in companion animals. The process of 3D printing was conducted using two types of 3D printers, utilizing the FDM and SLA printing techniques. Modulus of Elasticity was calculated for the determination of mechanical properties of the material, wherethe printed specimen was subjected to axial loading, and deformations were measured using an optical scanner. Post-processing was conducted by washing microneedles in isopropyl alcohol, followed by UV curing. The procedure of testing penetration capabilities was conducted at two sites of cat skin: Auris externa and the lateral part of the abdomen. The SLA printing method was more precise, resulting in higher quality microneedles for animal use compared to the FDM printing technique. Modulus of Elasticity was calculated and the value E=0.9 GPa can be used. Testing proved that the printed model was able to penetrate the skin at the tested sites. The use of microneedles is simple and economical, and therefore has wide applications in small animal practice. Veterinarians can access microneedle design repositories and print them for more effective transdermal drug delivery. The multifunctionality and transferability of the design in the present study ensure that it can be further modified to provide personalized therapy.

Background Cystic echinococcosis (CE) is recognized as one of the most prevalent zoonotic diseases in Bosnia and Herzegovina. However, no systemic investigation of the genetic diversity of Echinococcus granulosus sensu lato circulating among animals and humans in the country has been performed to date. Methods In this preliminary study, we analysed one cyst each from 36 sheep, 27 cattle, 27 pigs, 11 wild boars and 16 human patients for amplification and partial sequencing of the adenosine triphosphate 6 ( atp 6) and cytochrome c oxidase 1 ( cox 1) genes. The host species, fertility rate and organ cyst location were recorded for each subject involved in the study. Results Overall, the atp 6 gene was successfully amplified and sequenced from 110 samples, while 96 of the PCRs for cox 1 were positive. Three zoonotic genotypes of E. granulosus sensu stricto (G1 and G3) and Echinococcus canadensis (G7) were identified in our isolates based on analyses of the atp 6 gene. These genotypes were represented by 11 different genetic variants (haplotypes), six of which were identified for the first time in the present study. Conclusions This study demonstrates, for the first time, that CE in Bosnia and Herzegovina is predominantly caused by E. granulosus sensu stricto and E. canadensis clusters, which exhibited a lower genetic diversity compared to isolates from other European countries. Further molecular studies employing other mitochondrial and nuclear genes are required to better understand the transmission cycles of E. granulosus sensu stricto among intermediate and definitive hosts in the country. Graphical Abstract

Abstract A scarcity of information on the occurrence of zoonotic vector‐borne pathogens (VBPs), alongside a lack of human and animal health authorities’ awareness of pre‐existing data, augment the risk of VBP infection for local people and limit our ability to establish control programs. This holds especially true in low‐middle income countries such as Bosnia and Herzegovina (BiH). This dearth of information on zoonotic VBPs is bolstered by the inability of previously used diagnostic tests, including conventional molecular diagnostic methods, to detect the full spectrum of relevant pathogens. Considering this, we set out to apply a microfluidic qPCR assay capable of detecting 43 bacterial and protozoan pathogens from blood to accrue critical baseline data for VBPs occurrence in BiH. A total of 408 dogs were tested of which half were infected with at least one VBP of zoonotic or veterinary importance. Leishmania infantum was found in 18% of dogs, reaching a prevalence as high as 38% in urbanized areas of Sarajevo. These data highlight substantially higher levels of L. infantum prevalence when compared to that previously reported using conventional methods using the same samples. Additionally, this high‐throughput microfluidic qPCR assay was able to detect pathogens rarely or never reported in canines in BiH, including Anaplasma phagocytophilum (3%), Anaplasma platys (0.2%), haemotropic Mycoplasma (1%) and Hepatozoon canis (26%). Our report of the endemicity of important zoonotic pathogens and those of clinical significance to dogs emphasizes the need for urgent implementation of surveillance and control for VBPs in BiH, targeting both animal and human infections within the country.