The current state of research on the anti‑SARS‑CoV‑2 potential of artemisinin‑related compounds has identified arteannuin B as a potent inhibitor of the nCoV‑2019BetaCov/Wuhan/WiV04/2019 and BetaCov/Italy/CDG1/2020 strains of the virus. The aim of this work was to fractionate the targeted sesquiterpenoid compounds, arteannuin B and artemisinin, from the complex matrix of the crude ethanolic leaf extract of Artemisia annua L. using high‑speed countercurrent chromatography (HSCCC) and to test the simplified or purified fractions against the genomically characterized Alpha SARS‑CoV‑2 variant in vitro. This is the first detailed in vitro anti‑SARS‑CoV‑2 study using an analytically characterized supercritical fluid extract of A. annua L. The preparative HSCCC method enabled the isolation of purified arteannuin B in a single chromatographic step, which was confirmed by LC‑ESI‑QTOF‑MS/MS. The MS data confirmed the selectivity of the HSCCC method for the targeted fractionation of artemisinin from the complex matrix, as it was successfully separated from the EtOH crude extract without co‑elution with arteannuin B. Antiviral activity determined by quantitative real‑time PCR (qRT‑PCR) yielded half‑maximal effective concentrations (EC₅₀) of 93.7 µg/mL (SC‑CO₂ extract), 173.5 µg/mL (EtOH extract), 187.3 µg/mL (artemisinin knockout fraction), 38.1 µg/mL (arteannuin B fraction), and >100 µg/mL (artemisinin). The arteannuin B fraction was highly active at 50 µg/mL (p < 0.0001) and 100 µg/mL (p < 0.0001), and inhibited the amplification of the SARS‑CoV‑2 N and RdRp genes by 84% and 100%, respectively. An important contribution of this study is the demonstration of the antiviral activity of arteannuin B against the Alpha variant of SARS‑CoV‑2, which is known to have increased infectivity and transmissibility.

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.

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.

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.

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.