Introduction West Nile Virus, an emerging zoonotic pathogen, has been circulating in Serbia for over a decade, with its first detection in mosquitoes in 2010. Since then, the virus has led to increasing cases in both animals and humans, peaking in 2018 with 415 human cases and 36 fatalities. This study aimed to explore the phylogenetic relationships between previously sequenced West Nile virus strains from Serbia and those sequenced in this study, while also identifying possible virulence factors. Materials and methods Whole genome sequencing was conducted using a targeted approach on the MinION Mk1C platform, following a two-step process involving cDNA synthesis and amplification. Bioinformatics analysis included demultiplexing, primer trimming, and sequence mapping using tools such as iVar, Minimap2, and Samtools. Phylogenetic analysis was performed using MAFFT alignment and the Maximum Likelihood method with the Tamura Nei model in MEGA X software. Virulence factors were assessed in both structural and nonstructural proteins, focusing on key glycosylation motifs and specific mutations. Homology modeling of the E protein was also performed to evaluate potential structural changes due to mutations. Results Phylogenetic analysis revealed two major sublineages within the E subclade, representing the majority of strains from Western and Central Europe. These sublineages likely originated from Austria, Serbia, and Hungary between 2008 and 2012. The study also identified three distinct sublineages within the D subclade, which includes more diverse strains from Southern Europe. The E protein exhibited significant variations, particularly at the E159 site, which is crucial for virulence. The EI159T aa change has become dominant in recent years, replacing the previously prevalent EI159M. Additionally, changes in the NS1 glycoprotein and NS3 protein, both of which are involved in immune modulation and viral replication, were identified, with potential implications for the virus’s virulence. Conclusion The study’s findings highlight the Western Balkans and Central Europe as key regions for the mixing and dissemination of West Nile virus strains from both Western-Central and Southern Europe. These results underscore the importance of continuous surveillance and phylogenetic analysis to monitor the evolution and spread of West Nile virus, particularly in light of the frequent mutations observed in virulence-associated sites.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which led to the COVID-19 pandemic, has significantly impacted global public health. The proper diagnosis of SARS-CoV-2 infection is essential for the effective control and management of the disease. This study investigated the SARS-CoV-2 infection using RT-qPCR tests from laboratories in Bosnia and Herzegovina. We performed a retrospective study of demographic data and Ct values from 170,828 RT-qPCR tests from April 2020 to April 2023, representing 9.3% of total national testing. Samples were collected from 83,413 individuals across different age groups. Of all tests, 33.4% were positive for SARS-CoV-2, with Ct values and positivity rates varying across demographics and epidemic waves. The distribution was skewed towards older age groups, although lower positivity rates were observed in younger age groups. Ct values, indicative of viral load, ranged from 12.5 to 38. Lower Ct values correlated with higher positive case numbers, while higher Ct values signaled outbreak resolution. Additionally, Ct values decreased during epidemic waves but increased with the dominance of certain variants. Ct value-distribution has changed over time, particularly after the introduction of SARS-CoV-2 variants of interest/concern. Established Ct value trends might, therefore, be used as an early indicator and additional tool for informed decisions by public health authorities in SARS-CoV-2 and future prospective pandemics. Moreover, they should not be overlooked in future epidemiological events.

Simple Summary Ticks are ectoparasites with medical significance. They inhabit diverse environments and maintain close interactions with numerous vertebrate hosts. Ixodes ticks can transmit various pathogens to animals and humans. The aim here was to examine Ixodes ticks from Bosnia and Herzegovina to check for specific pathogens. This study found Rickettsia spp., Babesia spp., Anaplasma phagocytophilum, and Borrelia burgdorferi sensu lato in ticks from domestic animals. These findings highlight the need for the ongoing monitoring of ticks and tick-borne pathogens to protect animal and public health. Additionally, this study provides valuable insights into the occurrence and spread of these pathogens, emphasizing the importance of broader surveillance and control measures. Effective prevention, surveillance, and control of tick-borne diseases require urgent regional and international collaboration. Abstract Limited information is available regarding the presence of tick-borne pathogens and their distribution within Ixodes species in Bosnia and Herzegovina. This study aimed to identify Rickettsia spp., Babesia spp., Anaplasma phagocytophilum, and Borrelia burgdorferi sensu lato (s.l.) in Ixodes ticks collected from domestic and wild animals and vegetation in different regions across Bosnia and Herzegovina. A total of 7438 adult ticks, including 4526 Ixodes ricinus, Ixodes canisuga, and Ixodes hexagonus, were collected. Real-time PCR screening of 450 pooled I. ricinus samples revealed a 22.1% infection rate with at least one pathogen. Rickettsia spp. (6.3%) were found in ticks from dogs, cats, and goats, Babesia spp. (3.1%) in ticks from dogs and cattle, A. phagocytophilum (8.8%) in ticks from dogs, goats, and cattle, and B. burgdorferi s.l. (3.4%) in ticks from dogs and cats. Mixed infections with B. burgdorferi s.l. and A. phagocytophilum, as well as B. burgdorferi s.l. and Rickettsia spp., were found in two pools of I. ricinus from dogs and cats, respectively. Additionally, co-infection with Rickettsia spp. and A. phagocytophilum was confirmed in three tick pools from dogs and goats. Each tick from these pooled samples was individually retested to confirm the presence of pathogens. In the examined pooled samples of I. canisuga (1) and I. hexagonus (6), none of the tested pathogens were detected. Our findings represent the first detection of Rickettsia spp., Babesia spp., A. phagocytophilum, and B. burgdorferi s.l. in I. ricinus collected from domestic animals and vegetation in Bosnia and Herzegovina. Considering the established infection rates, the detection of tick-borne pathogens in adult ticks collected from domestic animals and vegetation enriches the current knowledge of the presence of tick-borne pathogens at the local, regional, national, and broader levels.

Dermacentor (D.) reticulatus ticks carry and transmit a wide range of pathogens to vertebrate hosts. Limited information is available about the existence of emerging tick-borne pathogens and the distribution of D. reticulatus in Bosnia and Herzegovina. The study aimed to investigate the occurrence and distribution of D. reticulatus and to detect the presence of Anaplasma spp., Borrelia (B.) burgdorferi s.l., Rickettsia spp., and Babesia spp. in samples originating from questing ticks and ticks collected from domestic animals in various regions of Bosnia and Herzegovina. A total of 402 collected D. reticulatus ticks were widely distributed throughout the country. Of the 41 pools consisting of 205 individual D. reticulatus ticks, 21 (51.2%) indicated the presence of Rickettsia spp., 17 (41.4%) of Babesia spp., 2 (4.8%) of Anaplasma spp., and 1 (2.4%) of B. burgdorferi s.l. after real-time PCR screening. Our study indicates that D. reticulatus has significantly expanded its distribution and host range in Bosnia and Herzegovina. Moreover, our results represent the first detection of Babesia spp. in D. reticulatus in Bosnia and Herzegovina. Given the demonstrated presence of emerging pathogens in questing and feeding ticks, there is an urge to establish a surveillance system for ticks and tick-borne pathogens in Bosnia and Herzegovina.

Abstract Parasitic diseases of wild animals represent an important area of research. In addition to the significant impact on wildlife health and fitness, many parasitic diseases have zoonotic implications. Due to limited scientific information, this research aimed to investigate parasitic diseases in wildlife in Bosnia and Herzegovina (B&H), focusing on the Federation of Bosnia and Herzegovina (FB&H), emphasizing zoonotic species. In the period from April 2020 to November 2022, we conducted research on 9 wildlife species. We analyzed fecal samples to detect and identify diagnostic stages (eggs, larvae, cysts, and oocysts) of various animal endoparasites using coprological methods, such as sedimentation, flotation, and the Baermann technique. The MERIFLUOR® Cryptosporidium/Giardia test was also used for the detection of Cryptosporidium oocysts and Giardia cysts. In the case of red foxes, intestinal samples were examined using the intestinal scraping technique to detect adult helminths. All collected muscle samples were subjected to the artificial digestion method for Trichinella detection. From 1,278 samples, 70.9% were positive. Parasitic infections were confirmed in 15.9% (11/69) of bears; 83.7% (262/313) of red foxes; 67.6% (44/65) of wolves; 25% (1/4) of wildcats; 20% (1/5) of badger; 43.7% (7/16) of martens; 39.7% (76/191) of wild boars; 84.5% (350/414) of deer, and 77.1% (155/201) of hares. The finding of zoonotic parasites (Toxocara canis, Uncinaria spp., Trichinella spp., Echinococcus spp. etc.) is particularly important due to their potential detrimental effects on human health, which highlights the need for further investigations.

Abstract Avian influenza, commonly known as bird flu, is a highly infectious viral disease that affects birds, including wild water birds and poultry. The emergence and spread of highly pathogenic avian influenza (HPAI) strains, such as H5N1, have raised concerns due to their potential to cause severe outbreaks and cross the species barrier, leading to human infections and global public health emergencies. In this study, we report the first case of HPAI H5N1 detection in Montenegro. Twenty-six carcasses of dalmatian pelicans were found in Skadar Lake, Montenegro, and the H5N1 subtype was confirmed through molecular testing in the samples from pelicans. The whole influenza genome was sequenced and belonging to clade 2.3.4.4b was determined.

Haemonchus contortus is a globally significant parasitic nematode in ruminants, with widespread resistance to benzimidazole due to its excessive and prolonged use. Given the extensive use of benzimidazole anthelmintics in Bosnia and Herzegovina, we hypothesized that resistance is prevalent. The aim of this study was to identify the presence of anthelmintic resistance to benzimidazole in H. contortus from naturally infected sheep, goats and cattle in Bosnia and Herzegovina through the detection of the Phe/Tyr polymorphism in the amino acid at position 200 of the β-tubulin protein. From 19 locations in Bosnia and Herzegovina, a total of 83 adult H. contortus were collected from the abomasum of ruminants. Among these, 45 H. contortus specimens were isolated from sheep, 19 from goats and 19 from cattle. Results showed that 77.8% of H. contortus in sheep exhibited homozygous resistant genotypes at position 200 of the β-tubulin gene, with 15.5% being heterozygous. In goats, all tested H. contortus (100%) were homozygous resistant, and no heterozygous resistant or homozygous sensitive genotypes were found. Cattle had 94.7% homozygous resistant H. contortus, with no heterozygous resistant genotypes detected. In H. contortus from sheep and cattle, 6.7% and 5.3%, respectively, displayed homozygous sensitive genotypes. This study, for the first time, highlights the presence of a resistant population of H. contortus in sheep, goats and cattle in Bosnia and Herzegovina, using the rt-qPCR method. The resistance likely spread from sheep or goats to cattle, facilitated by shared pastures and the practice of transhumance, indicating a widespread and growing issue of anthelmintic resistance.

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.

A growing issue on a global scale is the emergence of helminth species and populations that are resistant to one or more anthelmintics. The majority of currently available anthelmintics used to control parasitic nematodes of cattle and sheep belong to only three main groups, benzimidazoles, imidazothiazoles and avermectins/milbemycins. The availability of reliable and precise techniques for its identification and monitoring is a critical component of the success of helminth control programs intended to prevent the spread of resistance in nematode populations. In vivo method like fecal egg count reduction test and in vitro methods such as egg hatch assays, larval motility test, larval development test and polymerase chain reaction (PCR) can be used for the detection of anthelmintic resistance although each has some reliability, repeatability, sensitivity, and ease of interpretation issues. The genetic basis of resistance to the majority of anthelmintics are still not well understood. Thanks to recent developments in high-throughput sequencing, it is now possible to define features such as drug resistance using genome-wide techniques. Keywords: Anthelmintics; Helminths; Resistance; Detection assays; Molecular diagnostics; Parasite control