The AMY2B gene encodes pancreatic amylase, a critical enzyme for starch digestion. While previous studies have examined AMY2B copy number variation (CNV) in domestic and some wild animals, less is known about wild carnivores inhabiting regions with limited anthropogenic starch exposure. We analyzed blood samples for serum amylase activity and copy number variation in AMY2B gene from 8 wolves (Canis lupus), 11 brown bears (Ursus arctos), and 3 red foxes (Vulpes vulpes) from Bosnia and Herzegovina. AMY2B gene copy number was assessed using droplet digital PCR (ddPCR), and serum amylase activity and glucose levels were quantified. Although the number of fox samples was limited, foxes and wolves consistently harbored two copies of AMY2B, while brown bears exhibited higher CNV (3.67–8.40, mean 5.88). Serum amylase activity was highest in foxes, moderate in wolves, and variable but lower in bears. Despite differences in AMY2B copy number and serum amylase activity, circulating glucose concentrations did not differ significantly among species. Our findings suggest that variation in AMY2B copy number among wild carnivores may be associated with species-specific evolutionary histories and dietary adaptations, providing insight into genomic mechanisms underlying carbohydrate utilization in natural populations.

Phasianus chaphamaparvovirus 1 is a newly recognized pathogen associated with high mortality in young pheasant chicks with significant losses for pheasant breeders. Here we present an outbreak of necrotizing hepatitis with high mortality in a flock of young pheasant chicks in Bosnia and Herzegovina associated with Phasianus chaphamaparvovirus 1 and coccidiosis coinfection. Severe necrotizing hepatitis with characteristic intranuclear inclusion bodies in affected hepatocytes was observed on microscopic examination. In addition, multifocal areas of intestinal mucosal necrosis with numerous developing stages (meronts) of coccidia were present. Phasianus chaphamaparvovirus 1 was detected with qPCR from the livers of diseased birds. Our report represents the first data on the presence of this new virus in pheasant flocks in the Balkan region and suggests that the virus is widespread across Europe. It further supports the inclusion of Chaphamaparvovirus as a differential diagnosis of pheasant mortality outbreaks both in breeding facilities and in the wild.

Giardia duodenalis is a protozoan parasite with worldwide distribution and recognized zoonotic potential. Data on its molecular epidemiology in Bosnia and Herzegovina (BiH) are scarce, particularly in wild mesocarnivores. This study aimed to investigate the occurrence and genetic characterization of G. duodenalis in domestic and wild mesocarnivores across BiH. A total of 520 fecal samples were collected between 2023 and 2025, including dogs (Canis lupus familiaris, n = 433), cats (Felis catus, n = 21), red foxes (Vulpes vulpes, n = 39), golden jackals (Canis aureus, n = 17), European pine martens (Martes martes, n = 5), grey wolves (Canis lupus, n = 1), European badgers (Meles meles, n = 2), and European wildcats (Felis silvestris, n = 1). Screening was performed using fecal flotation and immunofluorescence assay (IFAT), with selected samples further analyzed by high-resolution melting (HRM) real-time PCR (qPCR-HRM) and targeted next-generation sequencing (NGS). Overall, G. duodenalis was detected in 20.96 % (109/520) of samples by flotation and IFAT. Cats showed the highest positivity rate (71.43 %), followed by dogs (21.02 %), whereas wild mesocarnivores exhibited substantially lower detection rates (5.13 % in red foxes and 5.88 % in golden jackals). Among dog subpopulations, hunting dogs showed the highest positivity (49.52 %) compared with shelter dogs (6.72 %). Molecular typing revealed assemblage D as predominant (65.91 %), followed by assemblages B (18.18 %), C (6.82 %), and F (4.55 %), with occasional mixed profiles. Assemblage D occurred across multiple hosts, while the zoonotic assemblage B was detected exclusively in wild canids. This study provides the first molecular epidemiological evidence of G. duodenalis assemblage circulation among domestic and wild mesocarnivores in Bosnia and Herzegovina. The findings identify cats and hunting dogs as key hosts contributing to parasite circulation and demonstrate limited but epidemiologically meaningful involvement of wild mesocarnivores, underscoring the importance of integrated One Health surveillance to assess transmission risks at the domestic-wildlife-human interface.

Gastrointestinal nematodes, particularly Haemonchus contortus, represent a major threat to ruminant health and productivity worldwide, largely due to the widespread emergence of anthelmintic resistance. In Bosnia and Herzegovina, benzimidazole resistance has previously been confirmed in domestic ruminants; however, data on wildlife remain lacking. Given the frequent spatial and temporal overlap between domestic and wild ruminants on shared pastures, this study aimed to investigate the occurrence of benzimidazole-resistant H. contortus genotypes within a multi-host system. During the 2024/2025 season, a total of 111 abomasal samples were collected from sheep (n = 20), lambs (n = 12), goats (n = 17), roe deer (n = 40) and chamois (n = 22) across four localities in Bosnia and Herzegovina (Laktaši, Banja Luka, Modriča and Višegrad). Adult H. contortus specimens were morphologically identified and confirmed using real-time quantitative PCR (rt-qPCR). Benzimidazole resistance was assessed by allele-specific rt-qPCR targeting the F200Y mutation in the β-tubulin isotype 1 gene. Statistically significant interspecies differences in β-tubulin genotype distribution were observed (p < 0.05), primarily driven by variation in the homozygous resistant (RR) genotype. High RR prevalence was detected in sheep (60%), lambs (50%) and roe deer (52.5%), whereas lower proportions were observed in chamois (27.3%) and goats (23.5%). Overall, 44.1% of all analyzed H. contortus isolates carried homozygous resistant alleles, indicating an advanced stage of benzimidazole resistance within this multi-host system. These findings demonstrate that benzimidazole resistance in H. contortus is not confined to domestic livestock but is also present in wild ruminants sharing the same grazing areas, consistent with circulation of resistant parasites within shared grazing systems.

Simple Summary African swine fever is a deadly viral disease of pigs and wild boar that causes major losses for farmers and threatens food security. The disease does not affect people, but its rapid spread and high fatality in pigs make it one of the most serious challenges for animal health in Europe. Since 2019, the disease has been present in Serbia, and in 2023, it was first reported in Bosnia and Herzegovina. In this study, we examined virus samples collected from pigs and wild boar during outbreaks between 2023 and 2025 to better understand how the virus is spreading in the region. By looking at several important parts of the virus genome, we found that all the samples belonged to the same group, known as cluster 19. This shows that the same type of virus has been circulating for several years without major changes. The results suggest that the disease is being maintained locally, mainly through contact between wild boar and pigs kept on small farms with little or no protection. The discovery of the same virus type in Bosnia and Herzegovina highlights that the disease crosses borders, making regional cooperation and continued monitoring essential for controlling its spread.

Introduction With their remarkable flight capabilities, wild and captive birds play a pivotal role in the global dissemination of zoonotic pathogens including Chlamydia psittaci, Avian Influenza viruses (AIV), Chikungunya virus (CHIKV), Usutu virus (USUV), and West Nile virus (WNV). They function both as hosts and reservoirs responsible for transporting the mentioned infectious agents across vast geographic regions. Additionally, captive birds and birds inhabiting urban environments, particularly in tourist destinations, present significant public health concerns due to facilitated close interactions with humans. Methods A total of 358 samples originating from fifteen bird species were collected across 21 locations in Sarajevo Canton, over three consecutive years (2022–2024). Upon collection, the samples were subjected to molecular analysis to detect the presence of zoonotic pathogens. For detection of Chlamydia spp., and C. psittaci, real-time PCRs (qPCR) were used following established protocols. Additionally, reverse transcriptase real-time PCR (RT-qPCR) were utilized for the detection of emergent viral pathogens including avian influenza viruses, Chikungunya, Usutu, and West Nile virus. Results Chlamydia spp. was detected in 29.9% (95% CI: 25.2–34.9) of samples. Further, C. psittaci was identified in 10.3% (95% CI: 5.2–17.7) of positive samples originating from captive birds and birds inhabiting urban environments. One sample (0.3%) originating from a wild bird was positive to West Nile Virus. None of the samples tested positive for Avian Influenza viruses, Chikungunya and Usutu virus. Discussion The identification of C. psittaci and West Nile virus highlights the increased likelihood of zoonotic transmission. This underscores the imperative for bolstered biosecurity measures and public health strategies aimed at mitigating the risk associated with both environmental exposure and direct contact, especially in areas characterized by substantial tourist activity.

The objective of the study was to assess the genetic diversity of the Tornjak shepherd dog by analyzing 10 micro- satellite loci. The dogs were divided into three main groups: Tornjaks from Bosnia and Herzegovina (TBA), Tornjaks from Croatia (THR), and a control group containing four subgroups: German Shepherd (GS), Belgian Shepherd (BS), crossbreeds of Tornjaks and other breeds (MIXT), and various other dog breeds (MIXA). The average number of genotypes (GN) and alleles (AN) was 19.2 and 8.4 (TBA), 8.2 and 5.4 (THR), 7.9 and 5.1 (BS), 4.4 and 3.6 (GS), 7.6 and 5.5 (MIXT), and 8.6 and 6.0 (MIXA), respectively. The average values of observed (HO) and expected heterozy- gosity (HE) were 0.7261 and 0.7392 (TBA); 0.7625 and 0.7139 (THR); 0.6857 and 0.6837 (BS); 0.4900 and 0.4640 (GS); 0.6786 and 0.6760 (MIXT); and 0.7067 and 0.7160 (MIXA), respectively. In the TBA population, “private” alleles were observed at all ten loci. The average overall inbreeding coefficient (F) value between the Tornjak and the control group was 0.0768. The AMOVA test revealed the highest degree of variation within the TBA group (55.43%), while no significant variations were observed in the control subgroup GS. The smallest differentiation for TBA was found with THR at 1.86% used the pairwise FST (pFST). The constructed Neighbor-Joining (NJ) dendrogram shows clear grouping of TBA and THR in comparison to the breeds of the control group. In relation to the data presented, a high level heterogeneity of has been established in the studied Tornjak population (TBA).

Anthelmintic resistance in livestock is a growing concern worldwide, with significant implications for animal health and agricultural productivity. This study explores the perceptions of veterinarians and farmers in Bosnia and Herzegovina regarding the factors contributing to anthelmintic resistance in Haemonchus contortus nematodes. Data were collected through structured questionnaires completed by 106 veterinarians and 188 farmers in 2022 and 2023. The analysis focused on self-reported therapeutic practices, farm management and environmental variables. Logistic regression, including Firth’s penalized approach, was used to assess associations between these perceived factors and the reported occurrence of resistance. Notably, combination anthelmintic treatments were perceived as a significant risk factor (OR > 49.3), while higher altitude was seen as potentially protective (OR = 0.10). Routine prophylactic deworming was associated with an increased likelihood of perceived resistance (OR = 173.7), whereas staying informed about newly registered products was perceived as protective (OR = 0.34). Although the findings are based on the self-reported perceptions and practices of veterinarians and farmers, they align with globally recognized trends and offer the first structured insights into factors perceived to contribute to anthelmintic resistance in Bosnia and Herzegovina. This study underscores the importance of awareness and responsible anthelmintic use and the need for improved diagnostics and ongoing education to combat anthelmintic resistance.

Echinococcus multilocularis, the causative agent of alveolar echinococcosis (AE), is a parasitic zoonosis of increasing public health significance in Europe. Although previously confirmed in red foxes in Bosnia and Herzegovina, this study provides the first report of E. multilocularis in golden jackals (Canis aureus) in the country. Between December 2024 and March 2025, a total of 44 golden jackals were examined across 15 localities in Bosnia and Herzegovina, with E. multilocularis detected in 6.8 % of the samples from three sites in Western and Central Bosnia and Herzegovina. Adult Echinococcus spp. worms were first detected using the intestinal scraping technique and identified by microscopy. To confirm these findings and differentiate between Echinococcus species, DNA extracted from adult worms was then subjected to species-specific PCR targeting a fragment of the mitochondrial 12S ribosomal RNA gene. PCR-positive samples for E. multilocularis were further validated by next-generation sequencing (NGS) of a 203 bp amplicon of the 12S rRNA gene. These findings indicate an expanding distribution of E. multilocularis in Bosnia and Herzegovina, highlighting the role of golden jackals as definitive hosts for the parasite. Given the growing jackal population in the country and the increasing public health concerns, enhanced surveillance and further research are warranted, particularly regarding human cases of E. multilocularis infection, to better understand the associated epidemiological risks.

This study aims to estimate the number of AMY2B gene copies and measure serum amylase activity in several Balkan dog breeds. Additionally, it explores the relationship between these genetic and biochemical parameters. Blood samples from 85 dogs representing eight breeds were collected, DNA was extracted, and AMY2B copy numbers were determined using droplet digital PCR. AMY2B gene copies ranged from 7.7 to 18.4, with a mean of 12.4 ± 2.2. Significant breed-related differences were observed (p = 0.025), with Istrian Wire-Haired Hounds showing the highest mean copy number (13.9 ± 1.5) and Posavatz Hounds the lowest (10.8 ± 1.5). Serum amylase activity ranged from 3.3 to 17.8 µkat/L, with a mean of 8.7 ± 2.6, and showed significant interbreed differences (p = 0.004), with Barak breed displaying the highest activity. Serum glucose levels varied widely, but no significant interbreed differences were detected (p = 0.340). No significant correlation was found between AMY2B copy numbers and serum amylase activity or glucose levels. The study concludes that Balkan dogs have AMY2B copy numbers similar to other European breeds, likely reflecting historical agricultural practices in the region, thereby facilitating better starch digestion. While significant variations exist among breeds, the lack of correlation between gene copy number and amylase activity suggests that other factors influence enzyme levels.