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.

Molecular Detection of Porcine Cytomegalovirus, Porcine Parvovirus, Aujeszky Disease Virus and Porcine Reproductive and Respiratory Syndrome Virus in Wild Boars Hunted in Serbia during 2023.

Porcine cytomegalovirus (PCMV) infection is widespread worldwide and has a high prevalence in swine herds, especially in countries with intensive swine production. PCMV is zoonotic and can impact xenotransplants. It is the third swine virus known to be zoonotic, following swine influenza virus (influenza A) and hepatitis E virus genotype 3 (HEVgt3 or HEV-3). Wild boars, serving as reservoirs for various pathogens, including PCMV, pose a risk to both the pig industry and public health. This study aimed to investigate PCMV infection in Serbian wild boars using real-time PCR and assess other viral infections. We also tested samples for the presence of other viral infections: Aujeszky disease virus (ADV), Porcine parvovirus (PPV) and Porcine reproductive respiratory syndrome (PRRSV). Samples from 50 wild boars across 3 districts were tested. Results showed 8% positivity for PCMV DNA, with females showing higher infection rates. Porcine parvovirus (PPV) was detected in 56% of samples, while Porcine reproductive respiratory syndrome virus (PRRSV) was absent. ADV was found in 18% of samples, primarily in younger animals. This research contributes to understanding PCMV prevalence in Serbian wild boars and emphasizes the importance of monitoring viral infections in wild populations, considering the potential zoonotic and economic implications.

Bovine Parainfluenza Virus 3 and Bovine Respiratory Syncytial Virus: Dominant Viral Players in Bovine Respiratory Disease Complex among Serbian Cattle.

Bovine respiratory disease complex, a complex respiratory ailment in cattle, results from a combination of viral and bacterial factors, compounded by environmental stressors such as overcrowding, transportation, and adverse weather conditions. Its impact extends beyond mere health concerns, posing significant economic threats to the cattle industry. This study presents an extensive investigation into viral pathogens associated with BRDC in Serbian cattle, utilizing serum samples and nasal swabs. A cross-sectional study was conducted in 2024 across 65 randomly selected dairy farms in Serbia, excluding farms with vaccinated cattle. The farms were categorized by their livestock count: small (≤50 animals), medium (51-200 animals), and large (>200 animals). Serum samples from adult cattle older than 24 months were tested for antibodies against BVDV, BHV-1, BRSV, and BPIV3. Nasal swab samples from the animals with respiratory signs were tested using PCR for viral genome detection. The results showed seropositivity for all four viruses across all of the farms, with BPIV3 exhibiting universal seropositivity. Medium-sized and large farms demonstrated higher levels of seropositivity for BRSV and BHV-1 compared to small farms (p < 0.05). Our true seroprevalence estimates at the animal level were 84.29% for BRSV, 54.08% for BVDV, 90.61% for BHV-1, and 84.59% for BPIV3. A PCR analysis of the nasal swabs revealed positive detections for BRSV (20%), BHV-1 (1.7%), BVDV (8%), and BPIV3 (10.9%). Influenza D virus was not found in any of the samples. This study provides critical insights into the prevalence and circulation of viral pathogens associated with BRDC in Serbian cattle, emphasizing the importance of surveillance and control measures to mitigate the impact of respiratory diseases in cattle populations.

Simultaneous Detection of Antigen and Antibodies of African Swine Fever in a Novel Combo Lateral Flow Assay.

African swine fever (ASF) is a contagious disease of wild boar and domestic pigs notifiable to the World Organisation for Animal Health due to its high socio-economic impact. ASF is caused by the complex ASF virus (ASFV), and it can present different clinical manifestations that can be confused with other diseases; for this reason, laboratory testing is necessary for the proper diagnosis of clinically suspected animals. Despite the efforts put into it over decades, no treatment or safe vaccine is globally available, and disease control is based on early diagnosis and the implementation of strict biosecurity measures. In this context, rapid tests have the potential to accelerate and facilitate the identification of infected animals by giving fast on-site results. In this work, we improved the available point-of-care assays for the diagnosis of the disease by the development of a more specific antigen test and a more sensitive antibody test. This antibody detection test allowed for the earlier detection of infected animals than two commercial indirect ELISAs (statistically significant). Moreover, we developed a combined dual rapid test, unifying, in the same cassette, an antigen detection strip and an antibody detection strip. In this study, we confirmed that this combo approach is a useful tool for implementing rapid tests in the field since it increases the percentage of positive samples detected, even when PCR turns negative, while maintaining a good specificity.

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.

Cross-sectional serosurvey of selected infectious diseases in wild ruminants in Serbia.

The role of wildlife in maintaining infectious diseases in veterinary medicine is often neglected, although the disease eradication process in domestic animals is continuously affected by the risk of pathogens transmission from wildlife as a primary source. The main aim of this paper was to estimate the prevalence and distribution of selected infectious diseases in wild ruminants in Serbia. In total, 259 sera from wild ruminants were tested for specific antibodies to bluetongue virus, Schmallenberg virus, Bovine viral diarrhea/border disease virus, Capripox virus, West Nile fever virus, Bovine herpes virus-1, Coxiella burnetii, Brucella spp., and Leptospira spp. Specific Capripox virus and Leptospira spp. antibodies were not detected in any of the 259 wild ruminant samples. Although one animal was detected positive for BVDV/BDV specific antibodies, with 99.8% confidence, the prevalence of BVD within this population could be very low i.e. essentially free from BVD infection. One and three positive animals were detected for Brucella spp. and Coxiella burnetii antibodies, respectively. Bovine herpes virus-1 specific antibodies were detected in 20.85% of the samples. The estimated seroprevalence of vector-borne diseases was 20.5% for Schmallenberg disease, 34.3% for West Nile fever, and 38.6% for Bluetongue. Considering the reported results, wildlife health status is a result of different factors in complex relation, such as the presence of disease in domestic animals, disease nature, pathogen characteristics, environmental factors, presence, and vector competence. Wildlife should be considered not only as a risk but as a source of important information on disease distribution and its indicators.

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.

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.

Seroprevalence of Viral Enzootic Diseases in Swine Backyard Farms in Serbia.

Contrary to pig farming in developed Western countries, in a large part of the world, pigs are still traditionally kept in small backyard farms, usually for family needs. Their main characteristics are low biosecurity, swill feeding, natural breeding and uncontrolled trade. Given the high number of backyard farms in Serbia and the risk they are thought to pose to intensive pig farming, the main aim of this study was to evaluate the prevalence of major viral diseases of swine among traditionally kept pigs in small holdings with low biosecurity. For this investigation, 222 serum samples from 69 backyard holdings were randomly selected and tested for antibodies to Porcine Reproductive and Respiratory Syndrome virus (PRRSV), Aujeszky's disease virus (ADV), Porcine Parvovirus (PPV) and Swine influenza Virus (SIV) by enzyme-linked immunosorbent assay (ELISA). The herd-level seroprevalence of PRRS, Aujeszky's disease and PPV was 2.9%, 27.5% and 37.7%, respectively. Swine influenza seroconversion was not confirmed in any of the tested holdings. Despite widely distributed PPV and AD in backyard farms in Serbia, almost 50% of them are still negative for all the tested diseases. The backyard farms must be monitored, and owners must be educated as their role in eradication programs and obtaining country-free status may be crucial.

Phylogenetic analysis of spring viraemia of carp virus isolated in Serbia.

Spring viraemia of carp (SVC) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (Cyprinus carpio carpio). The causative agent is the Rhabdovirus carpio or SVC virus (SVCV), a member of the Sprivivirus genus, within the Rhabdoviridae family. Phylogenetically, SVCV is divided into four genogroups (SVCV a, SVCV b, SVCV c and SVCV d), which have a reasonable correlation with the geographical distribution of the virus. In the late twentieth century, the disease was widespread in Serbian aquaculture and caused massive deaths in common carp. This study aimed to molecularly characterize the circulating SVCV isolates in Serbia over a 17-year period. The genetic relationships between 21 SVCV isolates from common carp and rainbow trout in Serbia between 1992 and 2009 were determined based on the partial nucleotide sequence of the glycoprotein gene (G gene). The phylogenetic analysis showed that the dominant SVCV isolates in Serbia belong to the SVCV d genogroup, with only one isolate belonging to genogroup SVCV b. The SVCV strains circulating in Serbia exhibited high homogeneity, as several isolates shared 100% similarity within these genogroups. Most Serbian isolates belonged to SVCV d1 and d2 subgroups, with one isolate notably different and included in a new subgroup SVCV d5. Understanding the SVCV genetic variants circulating in Serbia would be helpful in future epizootic investigations.

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.

Genetic analysis reveals multiple intergenic region and central variable region in the African swine fever virus variants circulating in Serbia.

This study provides the first comprehensive report on the molecular characteristics of African swine fever virus (ASFV) variants in Serbia between 2019 and 2022. Since its first observation in July 2019, the disease has been found in wild boar and domestic swine. The study involved the analysis of 95 ASFV-positive samples collected from 12 infected administrative districts in Serbia. Partial four genomic regions were genetically characterized, including B646L, E183L, B602L, and the intergenic region (IGR) between the I73R-I329L genes. The results of the study suggest that multiple ASFV strains belonging to genotype II are circulating in Serbia, as evidenced by the analysis of the IGR between I73R-I329L genes that showed the most differences. Furthermore, the phylogenetic analysis of the B602L gene showed three different clades within the CVR I group of ASFV strains. Regarding the IGR, 98.4% were grouped into IGR II, with only one positive sample grouped into the IGR III group. These findings provide essential insights into the molecular characteristics of ASFV variants in Serbia and contribute to the knowledge of circulating strains of ASFV in Europe. However, further research is necessary to gain a better understanding of ASFV spread and evolution.

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.

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.

First Molecular Detection of Pneumocystis spp. in the Golden Jackal (Canis aureus).

Forty-six golden jackals (Canis aureus) were collected between November 2020 and February 2021 in five counties of Serbia. Lung samples were screened for the presence of Pneumocystis DNA by pan-Pneumocystis PCR on the mtLSU rRNA gene obtaining PCR products of 370 bp in length. Pneumocystis DNA was detected in the lungs from 6/46 (13.04%) golden jackals. Four were females and two were males; four were classified as adults and two as subadults. Positive samples were confirmed in 4/5 investigated counties. No gross pathologic lung lesions were observed in this study. The sequences of Pneumocystis spp. from golden jackals were identical to one another and showed the highest similarity with Pneumocystis spp. sequences of dogs (98% nucleotide identity). The genetic variation was comparable to Pneumocystis spp. of raccoon dogs (95-97% nucleotide identity), red foxes (91-95% nucleotide identity), ferrets (86% nucleotide identity), and another Pneumocystis type in dogs (P. canis Ck2, 81% nucleotide identity) was higher. Golden jackals may be carriers and may play a nonnegligible role in the spread of Pneumocystis spp. Although this finding cannot be directly related to any clinical manifestation or pathologic lesions, a possible role in the exacerbation of different pulmonary disorders should be considered.

First confirmation of salmonid alphavirus infection in rainbow trout in Bosnia and Herzegovina and Serbia.

Sleeping disease is a highly infectious viral disease caused by salmonid alphavirus subtype 2 (SAV2 FW), affecting mainly rainbow trout Oncorhynchus mykiss farmed in freshwater. During March to May 2014, disease episodes with clinical signs of sleeping disease in rainbow trout fingerlings occurred almost simultaneously in 2 trout farms located in Bosnia and Herzegovina (BiH) and Serbia. The infection of rainbow trout with SAV2 FW in 2 farms was confirmed by virus isolation and molecular methods. This is the first isolation and molecular characterization of SAV2 FW in BiH and Serbia.