Background In Bosnia and Herzegovina, domestic and wild carnivores represent a significant driver for the transmission and ecology of zoonotic pathogens, especially those of parasitic aetiology. Nevertheless, there is no systematic research of Trichinella species in animals that have been conducted in Bosnia and Herzegovina, even though trichinellosis is considered the most important parasitic zoonosis. The available results of the few studies carried out in Bosnia and Herzegovina are mainly related to the confirmation of parasitic larvae in the musculature of domestic pigs and wild boars or data related to trichinellosis in humans. The objective of our study was to present the findings of a comprehensive investigation into the species composition of Trichinella among 11 carnivorous species within the territory of Bosnia and Herzegovina, as follows: red fox ( Vulpes vulpes ), grey wolf ( Canis lupus ), brown bear ( Ursus arctos ), wildcat ( Felis silvestris ), pine marten ( Martes martes ), European badger ( Meles meles ), weasel ( Mustela nivalis ), European polecat ( Mustela putorius ), Eurasian lynx ( Lynx lynx ), but also dog ( Canis lupus familiaris ) and cat ( Felis catus ). Results In the period 2013–2023, carnivore musculature samples ( n  = 629), each consisting of 10 g of muscle tissue, were taken post-mortem and individually examined using the artificial digestion method. In the positive samples ( n  = 128), molecular genotyping and identification of parasitic larvae of Trichinella spp. were performed using a PCR-based technique up to the species/genotype level. Positive samples were used for basic PCR detection of the genus Trichinella (rrnS rt-PCR technique) and genotyping (rrnl-EVS rt-PCR technique). The Trichinella infection was documented for the first time in Bosnia and Herzegovina among red foxes, grey wolves, brown bears, dogs, badgers and Eurasian lynx, with a frequency rate of 20.3%. Additionally, the presence of T. britovi infection was newly confirmed in Bosnia and Herzegovina, marking the initial documented cases. Furthermore, both T. britovi and T. pseudospiralis infections were observed in the wildcat population, whereas T. britovi and T. spiralis infections were detected in pine martens. Consistent with previous research, our findings align particularly regarding carnivores, with data from other countries such as Germany, Finland, Romania, Poland and Spain, where T. britovi exhibits a wider distribution (62.5–100%) compared to T. spiralis (0.0–37.5%). T. britovi is more common among sylvatic carnivores (89.0%), while T. spiralis prevails in wild boars (62.0%), domestic swine (82.0%) and rodents (75.0%). Conclusion The results of our study represent the first molecular identification of species of the genus Trichinella in Bosnia and Herzegovina. Additionally, our findings underscore the necessity for targeted epidemiological studies to thoroughly assess trichinellosis prevalence across diverse animal populations. Considering the relatively high frequency of trichinellosis infection in investigated animal species and its public health implications, there is an evident need for establishing an effective trichinellosis surveillance system 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.

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

Echinococcus multilocularis has been spreading through Central Eastern Europe but has not yet been reported in Bosnia and Herzegovina (B&H). Recently, this parasite is confirmed in Croatia suggesting the movement of the parasite's distribution limit further south. Given that there is no surveillance or monitoring system for echinococcosis in B&H, our study was designed as a pilot study of E. multilocularis. A total of 57 red foxes originating from 24 localities all over the country were collected during the routine rabies monitoring, autopsied and examined for the presence of echinococcosis. Based on intestinal scraping technique and microscopy, E. multilocularis adult worms have been detected in one (1/57, 1.75%) red fox. To verify this finding and to differentiate Echinococcus spp., DNA extracted from adult worms was subjected to species-specific PCR targeting part of the mitochondrial 12S ribosomal RNA gene. E. multilocularis PCR-positive samples were further confirmed by NGS sequencing of a 203 bp amplified fragment of 12S rRNA, which has been deposited in GenBank (Accession no.: OP047920). This finding represents the first detection of E. multilocularis in B&H, strongly suggesting its presence in the country. The confirmation of the parasite in the same locality where migrants/refugees temporarily stay on their route to Western Europe highlights the need for a One Health approach in addressing all future questions. Moreover, the first detection of E. multilocularis in B&H warrants the need for the implementation of an appropriate state surveillance program.

Crimean-Congo haemorrhagic fever virus is a tick-borne zoonotic agent of the genus Orthonairovirus, and the causal agent of Crimean-Congo haemorrhagic fever in humans. The study is aimed at investigating the presence of CCHFV RNA in ticks and cattle in Bosnia and Herzegovina (B&H), as well as the exposure of cattle to CCHFV in this country. Only one pool of three male Hyalomma marginatum ticks tested positive for viral RNA (3/760, 0.39%). No viral RNA was detected in 206 bovine blood samples using real-time reverse-transcriptase PCR (RT-qPCR). The overall seroprevalence of CCHFV in the cattle population was estimated as 14.97% (72/481). The results indicate the presence and the established circulation of CCHFV in B&H. Considering the endemic character of CCHFV in the Balkans as well as our results, the possibility that CCHFV is endemic in the territory of Bosnia and Herzegovina is considered to be very high.

The infection with SARS-CoV-2 virus in cats and dogs raised issue of human-to-animal transmission of SARS-CoV-2 in domestic pets in close contacts with their owners. Our study was designed to research this in the framework of Bosnia and Herzegovina. Using ELISA, AFIAS fluorescent immunoassay, RT-qPCR and WGS on Nanopore MinION platform with ARTIC Network Amplicon sequencing protocol for SARS-CoV-2, we showed that three out of thirteen dogs and one out of five cats from the households with confirmed human cases of COVID-19 in Bosnia-Herzegovina were infected with SARS-CoV-2. The high viral RNA load was detected in samples collected from a 4-year-old male Havanese (Ct = 12.52), a 6-year-old German Shepherd (Ct = 21.36) and a 9-year-old female American Staffordshire terrier (Ct = 25.74). The antibody response in dogs and one cat was observed. The viral genetic sequences from dogs were identical to the sequences detected in the owners suggesting the human-to-animal transmission of the virus. These findings, especially the low initial Ct values detected, from the public health perspective additionally stress the need for precautionary measures to protect both humans and animals.

Abstract Background Crimean–Congo haemorrhagic fever (CCHF) is a widespread tick‐borne zoonosis with reported detection of virus and/or virus‐specific antibodies from over 57 countries across Africa, Asia, Europe and the Middle East and is endemic in the Balkans. Detection of Crimean–Congo Haemorrhagic Fever Virus (CCHFV) antibodies in domestic ruminants has been important in providing initial evidence of virus circulation and in localising CCHFV high‐risk spots for human infection. Objectives The present study investigated the possible exposure of sheep to CCHFV in Bosnia and Herzegovina (B&H). Methods To investigate the presence of anti‐CCHFV antibodies in sheep, all sera (n = 176) were tested using multi‐species double antigen enzyme‐linked immunosorbent assay (ELISA). Reactive sera were further complementary tested by adapted commercial indirect immunofluorescence assay (IFA) using FITC‐conjugated protein G instead of anti‐human immunoglobulins. Results CCHFV specific antibodies were detected in 17 (9.66%) animals using ELISA test. All negative sera were determined as negative by both tests, while 13 out of 17 ELISA‐positive reactors were also determined as unambiguously positive by IFA test. The age group with the highest proportion of seropositive rectors were the oldest animals. Conclusions This is the first report of anti‐CCHFV antibodies in sheep from B&H providing the evidence of CCHFV circulation in the country's sheep population. So far, these findings indicate the circulation of the virus in the westernmost region of the Balkans and point to the potential CCHFV spread further out of this endemic area.

Red foxes are the most abundant wild carnivore species in Europe commonly exposed to pathogenic Leptospira and Hepatozoon canis. Despite high seroprevalence, the clinical disease caused by these pathogens in red foxes has never been reported. Herein, we report the first-ever case of a fatal Leptospira spp. and H. canis coinfection in a two-month-old red fox cub with acute haemolytic anaemia, mild bronchopneumonia, intraalveolar haemorrhage, and tubulonephrosis. The presence of pathogenic Leptospira spp. DNA was detected in the kidney and lung tissues of the infected animal. In contrast to our previous knowledge, we believe that such fatal cases due to concomitant infection by Leptospira spp. and H. canis, especially in young animals, may commonly occur in nature. However, further studies are required to identify other factors that possibly contribute to the severity and the pathogenic effect of Leptospira spp. and H. canis infections in red foxes.

The International Organisation for Animal Health (OIE), from the onset of COVID-19 pandemic, promoted One Health in global and national responses. The OIE accentuated the role of the veterinary profession due to testing capacity of animal health laboratories and expertise. Veterinary Faculty Sarajevo through its Veterinary Institute participates in the national veterinary service with diagnostic and advisory roles. It has proactively enhanced the scope and quality of laboratories, including strengthening the interdisciplinarity and internationality. Development achieved through earlier pandemic threats resulted in having laboratory and technical facilities for molecular SARS-CoV-2 detection in the wake of the unveiling COVID-19 pandemic (early 2020). From confirmation of the first COVID-19 cases in Bosnia and Herzegovina (BiH), our staff participated in crisis response teams and, so far, held over sixty media addresses promoting public awareness and science based information. Our laboratories were included in the official detection system and were the first to sequence SARS-CoV-2, then to establish the Alpha COVID-19 variant in BiH human samples and to substantiate one-way virus transmission from humans to pets. The aim of this paper is to describe our activities as a participant in the response to the COVID-19 pandemic, alongside faced challenges and gained experiences.

Many wild animal populations are considered endangered due to anthropogenic activities. Wildlife and nature habitat preservation requires holistic and science based approaches supported by adequate regulations. One of the means for wildlife preservation is undoubtedly heath monitoring and investigation of infectious diseases of the wild animal populations, particularly if spillover effects are considered. Even though the theoretical background is well researched, implementation of disease prevention and control measures in wildlife populations entails more challenges than in domestic animal populations. Immediate signs of health disorders in wildlife often become evident when the infectious agent is well established in an area. Additionally, due to unrestricted and often long-range movement of wildlife, diseases are easily spread across borders. Brown bears, indigenous in Europe, are classified by EU regulations as endangered, almost extinct and rare. The wild bear population in Bosnia and Herzegovina shares a genetic lineage with bear populations of neighbouring Croatia, Serbia and Montenegro and is one of the few remaining fragments of bear populations in Europe. The aim of this paper is to describe implemented activities for health and telemetric monitoring of wild bears in the Nature Park Skakavac, Canton Sarajevo, Bosnia and Herzegovina.

Background: According to the WHO (2019), more than 1.5 billion people worldwide are infected with soil-transmitted parasites. Previous research in the Federation of Bosnia and Herzegovina (FB&H) was mainly conducted in the area of the Sarajevo Canton. Therefore, the aim of the research was to explore contamination of soil and vegetation with developmental forms of parasites in the other cantons of FB&H. Methods: Between Apr and Oct 2018, a total of 1,618 soil and vegetation samples were taken from 386 different locations in the 9 cantons of the FB&H. Results: Positive samples were observed, 65/66 (98.48%) municipalities/cities and on 239/386 (61.92%) locations. Out of 1,618 samples taken in total (1,263 soil samples and 355 vegetation samples), 357 (22.06%) were positive, out of which 337 (26.68%) and 20 (5.63%) were soil and plant samples, respectively. In total, the following adult and developmental forms were identified: Taeniidae eggs (7.30%), Toxocara spp. eggs (62.08%), Ancylostomatidae eggs (25.00%), Trichuris spp. eggs (9.55%), Capillaria spp. eggs (3.37%), Toxascaris leonina eggs (1.40%), Nematodes larvae (19.38%), Giardia duodenalis cysts (5.06%), Cryptosporidium spp. oocysts (1.4%), oocysts and cysts of different species of Protozoa (3.93%). Conclusion: The identified developmental forms of parasites pose a permanent threat to human health. It is necessary to carry out measures to reduce the contamination of soil and vegetation in coordination with systematic solutions (legislation), paralelly with contribution of animal owners, veterinarians, physicians, ecologists, parents and all the others involved in this issue.

Rift Valley fever (RVF) is an arboviral zoonosis that primarily affects ruminants but can also cause illness in humans. The increasing impact of RVF in Africa and Middle East and the risk of expansion to other areas such as Europe, where competent mosquitos are already established, require the implementation of efficient surveillance programs in animal populations. For that, it is pivotal to regularly assess the performance of existing diagnostic tests and to evaluate the capacity of veterinary labs of endemic and non-endemic countries to detect the infection in an accurate and timely manner. In this context, the animal virology network of the MediLabSecure project organized between October 2016 and March 2017 an external quality assessment (EQA) to evaluate the RVF diagnostic capacities of beneficiary veterinary labs. This EQA was conceived as the last step of a training curriculum that included 2 diagnostic workshops that were organized by INIA-CISA (Spain) in 2015 and 2016. Seventeen veterinary diagnostic labs from 17 countries in the Mediterranean and Black Sea regions participated in this EQA. The exercise consisted of two panels of samples for molecular and serological detection of the virus. The laboratories were also provided with positive controls and all the kits and reagents necessary to perform the recommended diagnostic techniques. All the labs were able to apply the different protocols and to provide the results on time. The performance was good in the molecular panel with 70.6% of participants reporting 100% correct results, and excellent in the serological panel with 100% correct results reported by 94.1% of the labs. This EQA provided a good overview of the RVFV diagnostic capacities of the involved labs and demonstrated that most of them were able to correctly identify the virus genome and antibodies in different animal samples.

Whole Genome Sequence of four samples from COVID-19 outbreaks was done in two laboratories in Bosnia and Herzegovina (Veterinary Faculty Sarajevo and Alea Genetic Center). All four BiH sequences cluster mainly with European ones (Italy, Austria, France, Sweden, Cyprus, England). The constructed phylogenetic tree indicates probable multiple independent introduction events. The success of future containment measures concernig new introductions will be highly challenging for country due to the significant proportion of BH population living abroad.