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Lejla Šatrović, A. Softić, A. Zuko, A. Kustura, A. Koro, Šejla Goletić, E. Šatrović, F. Llorente et al.

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.

A. Alić, Jovana Šupić, T. Goletić, E. Rešidbegović, Ismar Lutvikadić, A. Hodžić

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.

E. Pérez-Ramírez, C. Cano-Gómez, F. Llorente, B. Adzic, Maisa S. Al Ameer, Igor Djadjovski, J. El Hage, F. El Mellouli et al.

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.

A. Softić, A. Martin, E. Skjerve, N. Fejzic, T. Goletić, A. Kustura, E. G. Granquist

Background The production of milk and dairy products and their placement on the market represent a constant profit for the farmers/producers in Bosnia and Herzegovina (BH). The profitable operation of the dairy farms is influenced by the reproductive performance of the lactating animals. This study assessed individual animal reproductive characteristics in selected dairy farms and described their reproductive performance indicators. Results The median age at first insemination was 493 days (5th–95th percentile range 429–840), while the age at first calving was 802 days (5th–95th percentile range 708–1168). The median pregnancy proportion at first insemination was 40% (5th–95th percentile range 17–62), while the cumulative pregnancy rate calculated at day-60, day-80, day-100, and day-120 showed that approximately 64% of all pregnancies happened before day-120. The calculated interservice intervals showed that approximately 69% of the repeat breeding animals came back to the oestrus in the period of 18 to 24 days. This is an indication of very good oestrus detection in selected dairy farms. The mean number of services per pregnancy was 2.61 (range 1–12). The median calving-to-first-insemination interval was 62.5 days (5th–95th percentile range 16–408). The calving-to-conception interval was 101 day (5th–95th percentile range 36–506). Finally, the calving interval was 385 days (5th–95th percentile range 329–773). Conclusions There is a need for an organised, regular, and more comprehensive recording system for the reproduction of dairy cattle among dairy farms in Una-Sana Canton. The calculated reproductive measures indicated an undulant trend in reproductive performance among selected dairy farms in Una-Sana Canton. Knowing the apparent reproductive indicators described in this study, the farmers and veterinary authorities may identify and correct areas in their management that contribute to the reproductive underperformance.

The objective of this study was to investigate the effects of test mixture or probiotic addition to drinking water on the growth performance of broiler chickens. A total of 240 one-day-old Cobb 500 chickens were distributed into three groups with eight replicates in each (10 chickens in each replicate). The control group of chickens (C) were without treatment. The chickens in experimental group E1 were treated with the commercial probiotic Probios® and the chickens in experimental group E2 were treated with the test mixture (Lactobacillus acidophilus culture, inactivated baker’s yeast, C vitamin, lactose and glucose) prepared using the authors’ own recipe. Treatments of chickens were conducted during the first three days of life and for three days using the chickens’ vaccination drinking water. The experiment lasted for 42 days. Feed and water were offered ad libitum during the experiment. Body weight, daily feed intake, body weight gain, feed conversion ratio (FCR), carcass weight, carcass yield and European production index (EPI) were studied in this experiment. The addition of the experimental probiotic significantly increased (P<0.05) body weight gain at 21, 35 and 42 days of age, however, the probiotic Probios® improved body weight gain over the same period without any significant difference compared to the control group. FCR was significantly improved at 21 and 35 days of age in both E1 and E2 groups, but at the end of fattening the FCR was not affected. Feed consumption was not influenced by the treatments. The results obtained indicate that carcass weight significantly increased (P<0.05) in the groups of chickens treated by the test mixture or probiotic. It was concluded that addition of test mixture or probiotic improved body weight gain, feed conversion ratio, carcass weight and EPI.

T. Goletić, A. Gagić, V. Savić, E. Rešidbegović, Aida Kavazović, E. Šatrović, T. Harder, S. Prašović et al.

ABSTRACT Background: Towards preparation for a possible influenza pandemic, investigation of the molecular characteristics of the circulating avian H5N1 influenza virus strains is of crucial importance. These H5N1 viruses continue to spread, to infect animals and humans and to evolve and diversify providing so an ever-looming pandemic threat.Aim: To identify genetic structure and molecular biological characteristics of BiH's isolates of H5N1 HPAI as well as to assess the level of pathogenicity, phylogenetic origin and host- specificity of the isolates.Material and Methods: SPF embryonated chicken eggs were used for virus isolation. Viral RNA extracted using QIAamp viral RNA kit and manufacturer’s protocol (QIAGEN®) was used for PCR amplification. cDNA synthesis and PCR amplification of the coding region, using gene specific primer sets (primer sequences available on request), were carried out for all eight viral RNA segments separately. The Prism Big Dye Terminator v1.1 cycle sequencing kit (Applied Biosystems) was used and products were analyzed on an automatic ABI PRISM 3130 genetic analyzer (Applied Biosystems). Nucleotide sequences were analyzed using Bioedit software (v. 7.0.9.0) with an engine based on the ClustalW 1.4 algorithm. MEGA software (v. 4,0), using the neighbor joining tree inference analysis with the Tamura-Nei γ-model, was used to estimate phylogenies and calculate bootstrap values from the nucleotide sequences.Results: Full-length nucleotide sequences of the A/Cygnus olor/BIH/1/2006 (H5N1) strain were deposited in EMBL Nucleotide Sequence Database under accession nos. FN186008 to FN186014 and FM20943. The pathogenicity and host specificity of this strain, as polygenic traits, are determined in silico by the structure of its proteins, especially surface glycoproteins, HA and NA. Multibasic amino acid stretch PQGERRRKKR/GLF, marker of strains highly pathogenic to poultry, was present at the HA cleavage site of BiH strain. The RBS was typical for avian influenza viruses and contained Gln and Gly at positions 238 and 240 (H5 numbering) that is,226 and 228 according to H3 numbering with seven potential glycosylated sites but with increased binding to alpha2-6 sialoglycans thanks to substitutions, as follows, 110N, 171N, 171N, 172A, 205R and 251P. NA structure assigned this strain to the Z genotype, characterized also by the deletion of the five amino acid residues of the NS1 protein (positions 80-84). Amino acid residues, typical for the avian influenza viruses, were revealed in 40 out of 43 positions of M1, M2, NP, PA, PB2 and HA, determining the host range specificity. Phylogenetic analysis of the HA gene revealed that BiH isolates belonged to genetic clade 2.2., and presence of aspartic acid at the position of 403 of HA locate BiH isolates in 2.2.2. sub-clade.Conclusions: The BiH’s isolates were determined as HPAI virus with genes sequences closely related to A/Cygnus olor/Astrakhan/Ast05-2-10/2005 (H5N1). Three residues (M2 - 28V and 78K, NP - 33I), typical of human influenza viruses, were found, indicating a certain degree of intercurrent evolutionary adaptive changes in BiH isolates. Sequence comparison of HA and NA segments with relevant sequences in GenBank revealed that the BiH isolates and the ones from the southern Russia (Astrakhan region) group together phylogenetically, forming a monophyleticcluster in both genes indicating that these isolates have evolved from the same origin. Sequence derived phenotype markers of NA protein (E99, V129, D131, R136, H255 and Y256) as well as of M2 protein (26L, 27V, 30A, S31 and G34) showed that the isolates have an oseltamivir and amantadine sensitive genotype. 

D. Pudar, D. Petrić, X. Allène, B. Alten, N. Ayhan, Aleksandar Cvetkovikj, C. Garros, T. Goletić et al.

The prime significance of species belonging to the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) is their ability to transmit viruses such as bluetongue virus (BTV) to wild and domestic ruminants. Prior to 1998, BTV was considered exotic in Europe, but according to recent history of its outbreaks, it has become endemic in southern and eastern European countries circulating beyond its expected historical limits, into the Balkan region. The wind-borne long-distance dispersal of Culicoides spp. over water bodies and local spreading between farms emphasize the necessity of filling in the information gaps regarding vector species distribution. In most Balkan countries, data on Culicoides fauna and species distribution are lacking, or information is old and scarce. During this study, 8586 specimens belonging to 41 species were collected. We present the first faunistic data on Culicoides species in the former Yugoslav Republic of Macedonia (FYROM), Kosovo, Montenegro and Serbia. For other countries (Bosnia and Herzegovina, Bulgaria and Croatia), all historical records were compiled for the first time and then expanded with our findings to various extents. In all countries, confirmed or suspected BTV vector species belonging to the subgenera Avaritia and Culicoides were collected. The total number of species sampled during our field collections was 20 in Bosnia and Herzegovina (15 new records), 10 in Bulgaria (2 new records), 10 in Croatia (5 new records), 13 in FYROM, 9 in Kosovo, 15 in Montenegro, and 28 in Serbia. Of these, 14 species were registered for the first time in this part of the Balkans. This paper provides the first data about Culicoides fauna in FYROM, Kosovo, Montenegro and Serbia, as well as new records and an update on the checklists for Bosnia and Herzegovina, Bulgaria and Croatia. These findings provide preliminary insights into the routes of BTV introduction and spreading within the Balkans, and present a valuable contribution to further research related to Culicoides-borne diseases in Europe.

Ádám Csikós, A. Hodžić, E. Pašić-Juhas, A. Jávor, A. Hrković-Porobija, T. Goletić, Gabriella Gulyás, L. Czeglédi

Species identification in food has become a prominent issue in recent years as the importance of consumer protection has increased. DNA-based species identification methods were developed by researchers in the last two decades, as these are reliable, accurate, and low-cost techniques for species identification in raw and processed food products as well. In our study, universal primers were designed to conserved regions of mitochondrial 12S rRNA. Amplicons were heat-denatured and a PCR single strand conformation polymorphism (SSCP) method was developed to identify cattle, buffalo, sheep, and goat DNA. Sensitivity of this technique was tested on DNA mixtures of cattle-sheep, cattle-goat, and cattle-buffalo and the threshold limit of cattle DNA was 5%, 5%, and 3%, respectively. One hundred and five cheeses were purchased and collected from Bosnian and Hungarian farmers, retails, and supermarkets to reveal fraud, 32 percent of them (34 cheeses) were found to be mislabelled by species.

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