First molecular confirmation of cyprinid herpesvirus 1 (CyHV1) in diseased carp in Serbia.

Mass mortality of farmed 1 yr old common carp Cyprinus carpio occurred at a carp farm in April 2022. In addition to high mortality, diseased fish exhibited papillomatous growths on the skin and fins, characteristic of carp pox. To investigate a possible viral cause, tissue samples were collected and nucleic acid was extracted using standard procedures. In a pooled sample from the gills and kidneys, carp edema virus (CEV) was detected by real-time PCR. In a skin tissue sample with papillomatous growths, cyprinid herpesvirus 1 (CyHV1) was detected by a conventional PCR targeting a conserved region of the DNA polymerase of cyprinid herpesviruses. PCR products were visualized through agarose gel electrophoresis, and the presence of CyHV1 DNA was confirmed by Sanger sequencing. This represents the first molecular confirmation of CyHV1 in common carp in Serbia.

Detection and characterisation of porcine circoviruses in wild boars in northeastern Serbia.

The objective was to expand and update the knowledge on the presence and genotype diversity of porcine circoviruses 2 and 3 (PCV2 and PCV3) in the wild boar populations from the hunting grounds in northeastern Serbia. The presence of PCV3 was not determined, and PCV2 was confirmed in 40.32% of the organ samples from 124 wild boars hunted from 2018 to 2019, indicating their significance in virus circulation since traditional pig farms with irregular PCV2 vaccination strategies are widespread in this region. The most prevalent genotype was PCV2d, followed by PCV2b and PCV2a in 55.6%, 38.9%, and 5.5% of the examined samples, respectively. Nucleotide sequences of the detected strains were homogenous within the genotype and clustered within the subgroups PCV2d-2, PCV2b-1A/B, and PCV2a-2D with high identity to European, Chinese, and Serbian domestic pig sequences suggesting their origin. Wild boars presented with no clinical or pathological signs of infection, implying that these animals might be less susceptible to disease, particularly since the cofactors present in pig farming systems that support the disease development are absent in the wild. The high PCV2 detection frequency demonstrates the importance of wildlife monitoring to track virus population dynamics, especially in regions with free-range pig farming in order to plan adequate disease control strategies.

Protoparvovirus carnivoran 1 infection of golden jackals Canis aureus in Serbia.

Parvoviruses are among the major animal pathogens that can cause considerable health disorders ranging from subclinical to lethal in domestic and wild animals. Golden jackal (Canis aureus), an expanding European species, is a reservoir of many pathogens, including vector-borne diseases and zoonoses. Given the importance of parvovirus infections in dogs and cats, this study aimed to unfold the virus prevalence and molecular characterisation in the golden jackal population in Serbia. The spleen samples from 68 hunted jackals during 2022/2023 were tested for the VP2-specific genome region of Protoparvovirus carnivoran 1 by PCR. BLAST analysis of partial VP2 sequences obtained from three animals (4.4%) revealed the highest similarity to Protoparvovirus carnivoran 1, genogroup Feline panleukopenia virus, which is the second report on FPV infection in jackals. Based on specific amino acid residues within partial VP2, the jackals' Protoparvovirus carnivoran 1 was also classified as FPV. One jackal's strain showed two synonymous mutations at positions 699 and 1167. Although species cross-transmission could not be established, jackals' health should be maintained by preventing the transmission of viruses to native species and vice versa. Although jackals are considered pests, their role as natural cleaners is of greater importance. Therefore, further monitoring of their health is needed to understand the influence of infectious diseases on population dynamics and to determine the relationship between domestic predators and jackals and the direction of cross-species transmission.

Performance of 5 commercial ELISA kits for the detection of antibody to bovine leukemia virus.

To prevent significant economic losses, some countries have successfully eradicated enzootic bovine leukosis (EBL), which is caused by bovine leukemia virus (BLV) infection. In Serbia, efforts to eliminate EBL commenced in the late 1990s. Recognizing the disparities in test selection among laboratories and variations in quality, we evaluated the diagnostic sensitivity and specificity of commercial ELISAs using field samples in Serbia. Using 5 commercial ELISA kits, we tested 138 cattle serum samples, submitted for confirmatory testing between 2020 and 2023, along with 100 serum samples from BLV-negative herds. We found 100% agreement of the ID Screen BLV Competition (IDvet), Svanovir BLV gp51-Ab (Svanova), and INgezim BLV Compac 2.0 (Ingenasa) ELISAs. We observed 93% agreement comparing these 3 kits to the Bovine Leukemia Virus Antibody test kit (VMRD). Agreements of 92% and 88.4% were determined between Idexx and IDvet, Svanova, and Ingenasa kits, and between Idexx and VMRD kits, respectively.

Evaluation of commercial ELISA kits' diagnostic specificity for FAST diseases in wild animals.

Wild animals, sharing pathogens with domestic animals, play a crucial role in the epidemiology of infectious diseases. Sampling from wild animals poses significant challenges, yet it is vital for inclusion in disease surveillance and monitoring programmes. Often, mass surveillance involves serological screenings using enzyme-linked immunosorbent assay (ELISA) tests, typically validated only for domestic animals. This study assessed the diagnostic specificity of commercially available ELISA tests on 342 wild ruminant serum samples and 100 from wild boars. We evaluated three tests for foot-and-mouth disease: two for Peste des petits ruminants, two for Rift Valley fever and one for Capripox virus. Diagnostic specificity was calculated using the formula True Negative/(False Positive + True Negative). Cohen's kappa coefficient measured agreement between tests. Results showed high specificity and agreement across all tests. Specificity for foot-and-mouth disease (FMD) ranged from 93.89% for Prionics to 100% for IDEXX, with IDvet showing 99.6%. The highest agreement was between FMD IDvet and IDEXX at 97.1%. Rift Valley fever (RVF) tests, Ingezim and IDvet, achieved specificities of 100% and 98.83%, respectively. The optimal specificity was attained by retesting single reactors and inactivating the complement.Contribution: Commercially available ELISA kits are specific for foot-and-mouth disease and similar transboundary animal diseases and can be used for highly specific wild animal testing.

Retrospective phylogenetic analysis of rabies virus G and N genes from Serbia.

Rabies is a viral disease of the central nervous system of all warm-blooded animals and one of the oldest and most important zoonoses. In the Republic of Serbia, rabies is controlled by compulsory vaccination of dogs and cats and oral vaccination of wild carnivores, which has been implemented since 2010. In the period 2009-2018, 367 rabies cases were recorded. The last rabies case in Serbia was recorded in 2018 in a red fox (Vulpes vulpes), while the last case in dogs was diagnosed in 2011. This study aimed to perform a retrospective phylogenetic analysis of G and N genes of the rabies virus from Serbia from 2009 to 2018 to understand sources and pathways of infection better and to enable molecular virus tracing in the future based on the association of rabies virus genetic lineages with the geographic distribution. For the phylogenetic analysis of the rabies virus, 14 historically positive brain samples of red foxes from 2009 to 2018, collected in central Serbia, were used. All field strains from Serbia were identified as classic rabies virus and grouped within the Cosmopolitan lineage. Phylogenetic analysis of N gene sequences revealed that 13 rabies virus strains (92.9%) from Serbia belonged to the EE sub-lineage of isolates, while one virus (7.1%) ON988027 from 2011 clustered together with isolates from the WE sub-lineage.

Molecular characterization of Canine parvovirus type 2 from diarrheic dogs in Serbia from 2008 to 2020.

Canine parvovirus 2 (CPV-2) is the causal agent of canine parvovirosis an infectious disease with the high fatality rate among dogs. However, in Serbia, it has never been investigated thoroughly. This study was conducted on samples collected from dogs with diarrhea in anamnesis, submitted for various reasons to the Institute of Veterinary Medicine of Serbia, and stored in the sample bank. In total, 50 rectal swab samples were collected from the period 2008 to 2020, and consequently tested. Out of 50 rectal swab samples, the CPV-2 genome was detected in 14 (28%). This retrospective study showed the presence of three different subtypes of CPV-2 in diarrheic dogs during the last 12 years in Serbia. CPV-2a was the most prevalent subtype (60%) followed by CPV-2b (30%), and CPV-2c (10%). Interestingly, CPV-2a had been the predominantly detected subtype up until 2018. Nevertheless in 2019, there was the first detected occurrence of the CPV-2b, followed by the first detection of the CPV-2c in 2020. This study reports the evidence and distribution of CPV-2 from 2008 to 2020, providing new information about the presence of virus strains in Serbia.

Protein sequence features of H1N1 swine influenza A viruses detected on commercial swine farms in Serbia.

Introduction: Swine influenza A viruses (swIAVs) are characterised by high mutation rates and zoonotic and pandemic potential. In order to draw conclusions about virulence in swine and pathogenicity to humans, we examined the existence of molecular markers and accessory proteins, cross-reactivity with vaccine strains, and resistance to antiviral drugs in five strains of H1N1 swIAVs. Material and Methods: Amino acid (AA) sequences of five previously genetically characterised swIAVs were analysed in MEGA 7.0 software and the Influenza Research Database. Results: Amino acid analysis revealed three virus strains with 590S/591R polymorphism and T271A substitution within basic polymerase 2 (PB2) AA chains, which cause enhanced virus replication in mammalian cells. The other two strains possessed D701N and R251K substitutions within PB2 and synthesised PB1-F2 protein, which are the factors of increased polymerase activity and virulence in swine. All strains synthesised PB1-N40, PA-N155, PA-N182, and PA-X proteins responsible for enhanced replication in mammalian cells and downregulation of the immune response of the host. Mutations detected within haemagglutinin antigenic sites imply the antigenic drift of the five analysed viruses in relation to the vaccine strains. All viruses show susceptibility to neuraminidase inhibitors and baloxavir marboxil, which is important in situations of incidental human infections. Conclusion: The detection of virulence markers and accessory proteins in the analysed viruses suggests their higher propensity for replication in mammalian cells, increased virulence, and potential for transmission to humans, and implies compromised efficacy of influenza vaccines.

Genetic analysis of influenza A viruses of swine from commercial farms in Serbia.

Swine influenza presents a very important health and economic issue in pig productions worldwide. Viruses that cause the disease are genetically very diverse but usually belong to the H1N1, H1N2 and H3N2 subtype of influenza A viruses. In this study, we sequenced and analyzed the full genome of viruses detected in swine from seven commercial farms. Through the analysis of the complete sequences of internal gene cassette together with previously characterized HA and NA genes we found three different genotypes amongst five completely sequenced viruses. Two viruses possessed a completely H1avN1 genotype (40%) and belonged to the H1avN1 lineage, which is prevalent in European swine populations. The other three viruses have arisen through the reassortment of the genes of H1avN1 and H1N1pdm09 lineages. In one sample we detected coinfection with viruses of H3N2 subtype with genes of H1avN1, H1N1pdm09 and A/swine/Gent/1/1984-like H3N2 lineages that presents a potential environment for the generation of a triple reassortant virus. The presence of the H1N1pdm09 origin M gene in this sample implies the potential risk of the introduction of these viruses into the human population. Phylogenetic analysis of internal gene cassette revealed slower evolution within genes of H1N1pdm09 lineage than those of H1avN1 lineage.

Patterns of ASFV Transmission in Domestic Pigs in Serbia.

The first case of African swine fever in domestic pigs in Serbia was in 2019. The following year, the disease was confirmed in wild boar. Thenceforth, ASF has been continuously reported in both wild and domestic pigs. The outbreaks in domestic pigs could not be linked directly to wild boars, even though wild boars were endemically infected, and reservoirs for ASF. This study aimed to investigate outbreaks and routes of transmission in domestic pigs in a region of central Serbia where no outbreaks in wild boar were reported. Fourteen outbreaks of ASF on backyard farms with low biosecurity were traced back, and no connection to wild boar was found. The epidemic investigation covered 2094 holdings, with 24,368 pigs, out of which 1882 were tested for ASF. In surrounding hunting grounds, field searches were conducted. Dead wild boars were found, and 138 hunted wild boars were negative for ASFV. It was concluded that outbreaks in 2021 were provoked by the illegal trade of live animals and pig products. Even though infective pressure from wild boars is assumed, no positive cases have been found, while the ASFV spreads within the domestic swine population evidenced in four recent outbreaks in 2022.

Evaluation of West Nile Virus Diagnostic Capacities in Veterinary Laboratories of the Mediterranean and Black Sea Regions.

The increasing incidence of West Nile virus (WNV) in the Euro-Mediterranean area warrants the implementation of effective surveillance programs in animals. A crucial step in the fight against the disease is the evaluation of the capacity of the veterinary labs to accurately detect the infection in animal populations. In this context, the animal virology network of the MediLabSecure project organized an external quality assessment (EQA) to evaluate the WNV molecular and serological diagnostic capacities of beneficiary veterinary labs. Laboratories from 17 Mediterranean and Black Sea countries participated. The results of the triplex real time RT-PCR for simultaneous detection and differentiation of WNV lineage 1 (L1), lineage 2 (L2) and Usutu virus (USUV) were highly satisfactory, especially for L1 and L2, with detection rates of 97.9% and 100%, respectively. For USUV, 75% of the labs reported correct results. More limitations were observed for the generic detection of flaviviruses using conventional reverse-transcription polymerase chain reaction (RT-PCR), since only 46.1% reported correct results in the whole panel. As regards the serological panel, the results were excellent for the generic detection of WNV antibodies. More variability was observed for the specific detection of IgM antibodies with a higher percentage of incorrect results mainly in samples with low titers. This EQA provides a good overview of the WNV (and USUV) diagnostic performance of the involved veterinary labs and demonstrates that the implemented training program was successful in upgrading their diagnostic capacities.

Molecular detection of black queen cell virus and Kashmir bee virus in honey.

Considering the intensive trading nowadays, the honey from the local market was tested for the presence of the six most common bee viruses. To prove the suitability of honey as a sample for the bee viruses detection, the set of different sample types taken directly from the hives we comparatively tested. The study included 30 samples of domestic and 5 samples of imported honey. Additionally, we tested 40 sets of samples including live bees, dead bees, and the honey taken from four apiaries for the evaluation of honey suitability for the virus detection, Two out of the six most common bee viruses were detected in the samples of honey from the market. Black queen cell virus (BQCV) genome was found in 24 domestic honey samples and Kashmir bee virus (KBV) genome was detected in one sample of imported honey. The nucleotide sequences of 24 BQCV isolates showed the highest identity (86.4%) with strains from Europe at the polyprotein gene, whilst the Serbian isolates between each other showed 98.5% similarity. By comparative testing of the different type of samples, in three out of four apiaries BQCV genome was detected in both bees and honey. Evaluating the suitability of honey for the detection of the viral disease by simultaneous testing of live, dead bees, and honey from the same hive, it was shown that the honey can be successfully used for the detection of BQCV. Since, as of yet, there has been no evidence of KBV circulation in Serbia, after its detection in imported honey, there is a substantial risk of its introduction and consequently the need for its surveillance. Therefore, the programs of bee diseases screening should be included in the regular control procedures for the international trade. In addition to this benefit, honey gives an opportunity to beekeepers for continuous monitoring of bees' health status.

Bovine viral diarrhea virus infection in wild boar.

Bovine viral diarrhea (BVD) is one of the most economically important diseases of cattle. With its very high prevalence, cattle kept on pastures become a source of the virus for the wildlife which, due to their susceptibility, then easily can serve as a source for re-infections of cattle. Therefore, we investigated the BVDV infection in Serbian wild boar and assessed the role of wild boar in BVDV epidemiology including possible spreading to domestic species. This study was based on examination of 50 spleen samples which were collected from wild boars located in Eastern Serbia during the hunting season 2016/2017. BVDV genome was detected in 4 of 50 samples (8%). Phylogenetic analysis based on 5'UTR revealed that BVDV strains from wild boars shared 100% identity. Belonging to the BVDV 1f subgenotype, the most common in cattle, we showed that BVDV infections of wild boar occurred as a result of either direct or indirect contact with domestic animals. Therefore, the occurrence of infectious disease in wildlife emphasizes the need to study the pathogens shared by wildlife and domestic animals by investigating the incidence of pathogens and disease patterns of those populations.