Tick-Borne Pathogens in Dermacentor reticulatus Ticks from Bosnia and Herzegovina.

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.

Diversity of Trichinella species in carnivores from Bosnia and Herzegovina.

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.

First Report of Benzimidazole Resistance in Field Population of Haemonchus contortus from Sheep, Goats and Cattle in Bosnia and Herzegovina.

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 ß-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 ß-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.

Ixodid ticks and zoonotic tick-borne pathogens of the Western Balkans.

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.

Metagenomic Sequencing of Lloviu Virus from Dead Schreiber's Bats in Bosnia and Herzegovina.

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.

Molecular characterization and phylogenetic analysis of highly pathogenic H5N1 clade 2.3.4.4b virus in Bosnia and Herzegovina.

Influenza A virus continues to represent a growing problem affecting mainly birds but with an increasing number of mammal transmission events reported each year. Nevertheless, molecular characterization and phylogenetic analysis of influenza A viruses originating from all confirmed cases have not been systematically performed in all parts of the world. In this study, we investigated a sample originating from a mute swan that died in November 2021 in the northern part of Bosnia and Herzegovina with RT-qPCR and whole genome sequencing using ONT MinION. It was diagnosed as a highly pathogenic Influenza A virus, subtype H5N1 of 2.3.4.4b clade, and phylogenetic analysis revealed high sequence homology with other European HPAI H5N1 sequences at the time. The notable detected mutations in HA (N110S and T139P) and NA genes (H155Y), that facilitate the host specificity shift and enable the resistance to some antiviral drugs respectively, underscore the necessity of virus evolution surveillance. Therefore, the rapid dissemination of information, including virological and molecular data, is essential for the introduction of tailored prevention measures for infected animals, providing clearer insight and better awareness of a potential public health threat.

First detection of Echinococcus multilocularis in Bosnia and Herzegovina.

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.

Distribution of ticks in Bosnia and Herzegovina.

Ticks are one of the important groups of parasites, whose populations have increased in Europe in the last 20 years and, as vectors of many emerging diseases, are a major threat to human and animal health. Bosnia and Herzegovina has both climate and environmental conditions favorable for ticks. Given this risk, the aim of this study was to identify tick species currently present in this country and to compare the findings with previous studies, in order to update information on their spatial distribution and other parameters of occurrence. From March 2017 to April 2020, ticks were collected in 42 sampling areas (municipalities) throughout the country. Prespecified identification keys were used for identifying the ticks to the species level. A total of 6193 tick specimens from the Ixodidae family were collected from animal hosts or from vegetation. Ixodes ricinus was the most dominant tick species found. However, our results indicate a decrease in the relative contribution of this tick species to the overall abundance of tick populations. Dermacentor marginatus, which almost doubled in abundance compared to earlier studies, is the second most common tick species. We further confirmed the presence of Dermacentor reticulatus, Rhipicephalus bursa, Rhipicephalus sanguineus sensu lato, Hyalomma marginatum and Haemaphysalis punctata. Ixodes hexagonus, Ixodes canisuga and Haemaphysalis concinna, which were previously reported at low abundance were not found amongst our specimens.

Contamination of Soil and Vegetation with Developmental Forms of Parasites in the Area of the Federation of 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.