Evaluation of longitudinal passive immunity transfer against lumpy skin disease virus in calves by different serological methods.

To implement effective lumpy skin disease (LSD) control measures, such as timely vaccination, particularly in calves and serological monitoring, it is necessary to evaluate immune response after vaccination, both in adult cattle and in their calves. The aim of this study was to evaluate passive immunity transfer and duration of maternal antibodies against lumpy skin disease virus (LSDV) in calves born to vaccinated cows by two different serological methods. The longitudinal study was carried out on two farms in Serbia where no cases were reported during LSD outbreak in 2016. Fifteen cows on each farm were vaccinated and revaccinated with attenuated vaccine - Neethling strain. A total of 30 cows and 30 calves on both farms were included in the study. Serum samples from cows were collected on calving day and serum samples from their respective calves on days 10, 20, 30, 45, 60, 75, 90, 105 and 120 after birth. Colostrum samples were collected only from 15 cows on one farm. In order to determine the presence of antibodies against LSDV a total of 30 cow sera samples, 15 colostrum samples and 270 calf sera samples were examined by commercial enzyme-linked immunosorbent assay (ELISA) and modified virus neutralization test (VNT). Overall, the performance of both serological tests was very satisfactory. The results of this longitudinal study showed that persistence of passive immunity in calves is less than 4 months, and that most calves are not protected against LSDV at that age. Since the vaccination is the most important control measure against LSDV, the recommended age of six months for vaccination of calves born to vaccinated cows should be reassessed to achieve the most optimal protection against LSD.

Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus.

IMPORTANCE: Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015-2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

Serological Examinations of Significant Viral Infections in Domestic Donkeys at the Special Nature Reserve "Zasavica", Serbia.

The paper presents the findings of specific antibodies in the blood sera of donkeys against the following viruses: equine infectious anemia virus (EIAV), African horse sickness virus (AHSV), equine herpesvirus type 1 (EHV-1), equine influenza virus subtype H3N8 (EIV) and equine arteritis virus (EAV). The analyses were conducted during the year 2022. From a total of 199 donkeys bred in "Zasavica", blood was sampled from 53 animals (2 male donkeys and 51 female donkeys), aged 3 to 10 years. Specific antibodies against EIAV were not detected in any of the tested animals using the agar-gel immunodiffusion (AGID) assay. No specific antibodies against AHSV, tested by enzyme-linked immunosorbent assay (ELISA), or antibodies against EAV, tested by the virus neutralization test (VNT) and ELISA were detected in any of these animals. A positive serological result for EHV-1 was determined by the VNT in all animals, with antibody titer values ranging from 1:2 to 1:128, while a very low antibody titer value for EIV (subtype H3N8) of 1:16 was determined in 18 donkeys using the hemagglutination inhibition test (HI test).

African Swine Fever Outbreak in an Enclosed Wild Boar Hunting Ground in Serbia.

African swine fever (ASF) has been detected in many European countries since its introduction in Georgia in 2007. Serbia suffered its first case of ASF in the domestic pig population in 2019. At the beginning of 2020, ASF was detected in wild boars in open hunting grounds in the southeastern region of the country in districts along the country's borders with Romania and Bulgaria. Since then, all ASF outbreaks in wild boar were clustered in the population located in the same bordering areas. Despite the newly implemented biosecurity protocols for hunters in 2019, ASF was detected for the first time in June 2021 in the wild boar population located in an enclosed hunting ground in the northeast region of the country. In this study, we reported the first ASF outbreak in a wild boar population located in an enclosed hunting ground in close proximity to the Serbian-Romanian border. The epizootiological data on the field investigation of the ASF outbreak, with descriptions of the clinical signs and gross pathological lesions detected, including the total number as well as the estimated age, sex, and postmortem interval, were analyzed. Clinical signs were detected only in nine diseased wild boars, while in total, 149 carcasses were found in the open and enclosed part of the hunting ground. In addition, 99 carcasses from which samples (parts of spleen or long bones) were collected for molecular diagnostics (RT-PCR) were confirmed as ASF-positive. The results of the epidemiological investigations indicate the central role of wild boar movements as well as the constant risk of human-related activities in the countries bordering area.

Donkey Milk: An Overview of its Chemical Composition and Main Nutritional Properties or Human Health Benefit Properties.

The donkey milk has a remarkable similarity to human milk, in addition to its valuable nutritional composition and content of numerous immune factors. The donkey milk is the subject of research worldwide, and data from the literature suggest significant differences with respect to the contents of individual components. However, some basic characteristics of donkey milk have been established: low contents of fat and cholesterol, total proteins and casein and high contents of lactose, whey proteins, calcium, selenium, and Vitamin D3. The donkey milk is rich in various protective proteins (α-lactalbumin, lysozyme, lactoferrin, lactoperoxidase, and immunoglobulins), and shows strong antioxidant, antibacterial, antiviral, antifungal, hypoglycemic, antiparasitic, and antitumor activity. Donkey milk can be considered functional food, having in mind that its fatty-acid profile and content of essential fatty acids are beneficial for cardiac health. The total fat content in donkey milk is low compared to human milk, and for this reason it is necessary to enrich donkey milk with other fat in order to provide enough calories in a diet for children. Commercialization of donkey milk and dairy products is still limited due to low production levels, that is lack of products on the market, and lack of product information. Considering the research data from the literature, there is a need for human clinical trials in order to obtain a stronger evidence of the therapeutic properties of donkey milk.

Diagnosis of Infectious Laryngotracheitis Outbreaks on Layer Hen and Broiler Breeder Farms in Vojvodina, Serbia.

Infectious laryngotracheitis (ILT) is a respiratory disease of poultry characterized by high morbidity and variable mortality. ILT is caused by Gallid alpha herpesvirus-1 (GaHV-1), which is transmitted horizontally and most susceptible are chickens older than 4 weeks. After almost two decades since last appearance of this disease in Vojvodina, an outbreak occurred from April 2020 to August 2021 on five laying hen farms and one broiler breeder flock farm. Clinical signs were mild to severe respiratory symptoms, unilateral or bilateral head swelling, serous nasal discharge, conjunctivitis and increased tearing. There was a decrease in feed consumption (2.1-40.0%) and egg production (2.7-42.0%), weight loss and mortality increased (0.8-31.5%). Pathomorphological changes were localized in the upper respiratory tract. Total of 200 carcasses were examined; 40 pooled samples were analyzed by PCR, and 40 by bacteriological analysis. ILT virus was confirmed in tracheal tissue samples. Infected flocks were not vaccinated against this disease. Five flocks had coinfection with Avibacterium paragallinarum. Three-to-four weeks after the first reported case in the flock, clinical symptoms had ceased. Future control and prevention strategies will involve the procurement of flocks vaccinated by recombinant vaccine or the registration of live attenuated vaccines and their use during the rearing period.

Development and Optimization of Indirect ELISAs for the Detection of Anti-Capripoxvirus Antibodies in Cattle, Sheep, and Goat Sera.

Sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD) are economically significant pox diseases of ruminants, caused by sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively. SPPV and GTPV can infect both sheep and goats, while LSDV mainly affects cattle. The recent emergence of LSD in Asia and Europe and the repeated incursions of SPP in Greece, Bulgaria, and Russia highlight how these diseases can spread outside their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. We expressed and tested two recombinant truncated proteins, the capripoxvirus homologs of the vaccinia virus C-type lectin-like protein A34 and the EEV glycoprotein A36, as antigens for an indirect ELISA (iELISA) to detect anti-capripoxvirus antibodies. Since A34 outperformed A36 by showing no cross-reactivity to anti-parapoxvirus antibodies, we optimized an A34 iELISA using two different working conditions, one for LSD in cattle and one for SPP/GTP in sheep and goats. Both displayed sound sensitivities and specificities: 98.81% and 98.72%, respectively, for the LSD iELISA, and 97.68% and 95.35%, respectively, for the SPP/GTP iELISA, and did not cross-react with anti-parapoxvirus antibodies of cattle, sheep, and goats. These assays could facilitate the implementation of capripox control programs through serosurveillance and the screening of animals for trade.

Intensive West Nile Virus Circulation in Serbia in 2018-Results of Integrated Surveillance Program.

The results of the Serbian national integrated West Nile virus (WNV) surveillance program conducted in 2018 and funded by the Serbian Veterinary Directorate are presented. The WNV surveillance program encompassed the entire territory of Serbia and was conducted by the veterinary service in collaboration with entomologists and ornithologists. The objective of the program was early detection of WNV circulation in the environment and timely reporting to the public health service and local authorities to increase clinical and mosquito control preparedness. The program was based on the detection of WNV presence in wild birds (natural hosts) and mosquitoes (virus vectors) and on serological testing of sentinel horses (WNV-specific IgM antibodies). The season 2018 was confirmed to be the season of the most intensive WNV circulation with the highest number and severity of human cases in Serbia ever reported. The most intense WNV circulation was observed in the northern and central parts of Serbia including Vojvodina Province, the Belgrade City area, and surrounding districts, where most positive samples were detected among sentinel animals, mosquitoes and wild birds. The majority of human cases were preceded by the detection of WNV circulation during the surveillance. The WNV surveillance program in 2018 showed satisfactory results in the capacity to indicate the spatial distribution of the risk for humans and sensitivity to early detection of WNV circulation in the environment.

Complete Genome Sequence of an Equine Arteritis Virus Strain Isolated from a Lipizzaner Stallion in 2015 in Serbia.

Here, we report the first whole-genome sequence of an equine arteritis virus (EAV) strain, RS1, isolated from the semen of a Lipizzaner stallion held in the Vojvodina region of Serbia.

Complete genome sequences of two strains of classical swine fever virus of subgenotype 2.3 detected during outbreaks in 2005 and 2006 in Serbia.

Two strains of classical swine fever virus (CSFV) (SRB/CSFV/1264/2005 and SRB/CSFV/6168/2006), producing serious clinical signs of disease during outbreaks in 2005 and 2006 in Serbia, were isolated on porcine kidney cells, and their complete genomes were determined by next-generation sequencing. This first complete genome characterization of Serbian CSFV strains provides new data about the evolution of CSFV in the Balkan region and enables further detailed phylogenetic studies of the various strains.

Methodology and results of integrated WNV surveillance programmes in Serbia.

Studies conducted during the past few years have confirmed active West Nile virus (WNV) circulation in Serbia. Based on these studies and the epidemiological situation, the Veterinary Directorate of the Ministry of Agriculture and Environmental Protection launched national WNV surveillance programmes in 2014 and 2015. The programmes encompassed the territory of Serbia and were conducted by the veterinary service in collaboration with entomologists and ornithologists. The objective of the programmes was early detection of WNV and timely reporting to the public health service and local authorities to increase both clinical and mosquito control preparedness. The WNV surveillance programmes were based on direct and indirect surveillance of the presence of WNV by the serological testing of initially seronegative sentinel horses and chickens as well as through viral detection in pooled mosquito and wild bird samples. The most intense WNV circulation was observed in all seven districts of Vojvodina Province (northern Serbia) and Belgrade City, where most of the positive samples were detected among sentinel animals, mosquitoes and wild birds. The West Nile virus surveillance programmes in 2014 and 2015 showed satisfactory results in their capacity to indicate the spatial distribution of the risk for humans and their sensitivity to early detect viral circulation at the enzootic level. Most of the human cases were preceded by the detection of WNV circulation as part of the surveillance programmes. According to the existing data, it can be reasonably assumed that WNV infection, now an endemic infection in Serbia, will continue to present a significant problem for the veterinary service and public health.

Serological evidence of equine arteritis virus infection and phylogenetic analysis of viral isolates in semen of stallions from Serbia.

BACKGROUND: Equine arteritis virus (EAV) is responsible for infections in equids. It can spread easily within the horse population and has a major impact on the horse breeding industry. No EAV outbreak has ever been reported in Serbia. To determine whether EAV is nonetheless circulating there, especially in the Vojvodina region, 340 horse serum samples were subjected to serology testing to detect EAV antibodies. In parallel, semen samples from three seropositive stallions were collected to evaluate their EAV status, using RT-qPCR and virus isolation on cell culture. RESULTS: Horse sera with EAV antibodies represented 15.88% (54/340) of the tested samples, 83.23% (283/340) being negative, and just three samples (0.89%) being uninterpretable due to cytotoxicity. Only 7.2% (10/138) of horses kept by private owners on their own property were seropositive for EAV, whereas 21.8% (44/202) of horses kept on stud farms had EAV antibodies. Phylogenetic analysis showed that the Serbian EAV isolate was most closely related to isolates from the neighbouring Hungary. CONCLUSIONS: EAV is circulating in the Serbian horse population, especially among the breeding population certainly due to the use of EAV shedder stallions since there is no surveillance programme in Serbia and only limited checks on racehorses. Moreover, phylogenetic analysis indicates that the EAV isolated from a Lipizzaner stallion in Serbia is closely related to isolates from Hungary, and together form a new cluster.

Complete Genome Sequence of Lumpy Skin Disease Virus Isolate SERBIA/Bujanovac/2016, Detected during an Outbreak in the Balkan Area.

The lumpy skin disease virus (LSDV) isolate SERBIA/Bujanovac/2016 consists of 150,661 nucleotides and has a 99.95% nucleotide identity with the Neethling Warmbaths LW strain isolated in South Africa in 1999. This is the first complete LSDV genome determined in Serbia and also in the Balkan area.

West Nile virus 'circulation' in Vojvodina, Serbia: Mosquito, bird, horse and human surveillance.

Efforts to detect West Nile virus (WNV) in the Vojvodina province, northern Serbia, commenced with human and mosquito surveillance in 2005, followed by horse (2009) and wild bird (2012) surveillance. The knowledge obtained regarding WNV circulation, combined with the need for timely detection of virus activity and risk assessment resulted in the implementation of a national surveillance programme integrating mosquito, horse and bird surveillance in 2014. From 2013, the system showed highly satisfactory results in terms of area specificity (the capacity to indicate the spatial distribution of the risk for human cases of West Nile neuroinvasive disease - WNND) and sensitivity to detect virus circulation even at the enzootic level. A small number (n = 50) of Culex pipiens (pipiens and molestus biotypes, and their hybrids) females analysed per trap/night, combined with a high number of specimens in the sample, provided variable results in the early detection capacity at different administrative levels (NUTS2 versus NUTS3). The clustering of infected mosquitoes, horses, birds and human cases of WNND in 2014-2015 was highly significant, following the south-west to north-east direction in Vojvodina (NUTS2 administrative level). Human WNND cases grouped closest with infected mosquitoes in 2014, and with wild birds/mosquitoes in 2015. In 2014, sentinel horses showed better spatial correspondence with human WNND cases than sentinel chickens. Strong correlations were observed between the vector index values and the incidence of human WNND cases recorded at the NUTS2 and NUTS3 levels. From 2010, West Nile virus was detected in mosquitoes sampled at 43 different trap stations across Vojvodina. At 14 stations (32.56%), WNV was detected in two different (consecutive or alternate) years, at 2 stations in 3 different years, and in 1 station during 5 different years. Based on these results, integrated surveillance will be progressively improved to allow evidence-based adoption of preventive public health and mosquito control measures.

West Nile virus serosurveillance in pigs, wild boars, and roe deer in Serbia.

West Nile virus (WNV) is maintained in nature in an enzootic transmission cycle between birds and mosquitoes, but it also infects many other vertebrates, including humans and horses, in which it can induce severe neurological diseases; however, data about virus circulation in other mammals is scarce. WNV has a history of recent outbreaks in Europe, including Serbia, where it was identified for the first time in 2010 in mosquitoes and in 2012 in birds and humans, being responsible for over 300 confirmed human cases and 35 deaths there along 2013. To assess WNV circulation among mammals in the country, 688 samples obtained from 279 farm pigs, 318 wild boars, and 91 roe deer were investigated for the presence of antibodies to WNV by enzyme-linked immunosorbent assay (ELISA) and viral neutralization test (VNT), and the specificity of their reactivity was assayed against Usutu virus (USUV). ELISA-reactive sera were identified in 43 (15.4%) pigs, 56 (17.6%) wild boars, and 17 (18.7%) roe deer. Of these, 6 (14%), 33 (59%), and 4 (23.5%) respectively, neutralized WNV. One out of the 45 ELISA negative sera tested, from a roe deer, neutralized WNV. Cross-reactivity neutralization test indicated that all deer and pigs neutralizing sera were WNV specific, while in 5 (15.2%) of the wild boar samples the specificity could not be established. Four wild boar sera showed USUV specificity. All these data confirm the circulation of both flaviviruses in Serbia, and highlight the need for the implementation of global coordinated surveillance programs in the region.

Presence of Human and Animal Viruses in Surface Waters in Vojvodina Province of Serbia.

For the first time in Serbia, a small surveillance study was conducted in order to estimate the presence and frequency of occurrence of selected human [adenoviruses (HAdV), noroviruses, (NoV GI, NoV GII) and hepatitis A virus (HAV)], animal [porcine adenovirus (PAdV) and bovine polyomavirus (BPyV)] and zoonotic [hepatitis E virus (HEV)] viruses in selected surface waters. In total, 60 surface water samples were collected in two sampling occasions at 30 locations, with each sampling time being separated by 1-5 months. In addition, six sewage effluent samples were collected at one sampling site per each of the three tested town sewage systems, in two sampling occasions with 2 months intervals, before their discharge into the surface waters. The most prevalent virus found was HAdV which was detected in 43.33 % samples. NoV GII was found in 40 % samples. NoV GI was found in 10 % samples, and PAdV, BPyV and HEV were detected in 5 (8.33 %), 4 (6.67 %) and 2 (3.33 %) samples, respectively. HAV was not found in any of analysed surface waters or urban sewage samples. The obtained results confirm the presence of pathogenic enteric viruses of both human and animal origin in surface waters in Serbia indicating the existence of diverse contamination sources.

Quantitative farm-to-fork risk assessment model for norovirus and hepatitis A virus in European leafy green vegetable and berry fruit supply chains.

Fresh produce that is contaminated with viruses may lead to infection and viral gastroenteritis or hepatitis when consumed raw. It is thus important to reduce virus numbers on these foods. Prevention of virus contamination in fresh produce production and processing may be more effective than treatment, as sufficient virus removal or inactivation by post-harvest treatment requires high doses that may adversely affect food quality. To date knowledge of the contribution of various potential contamination routes is lacking. A risk assessment model was developed for human norovirus, hepatitis A virus and human adenovirus in raspberry and salad vegetable supply chains to quantify contributions of potential contamination sources to the contamination of produce at retail. These models were used to estimate public health risks. Model parameterization was based on monitoring data from European supply chains and literature data. No human pathogenic viruses were found in the soft fruit supply chains; human adenovirus (hAdV) was detected, which was additionally monitored as an indicator of fecal pollution to assess the contribution of potential contamination points. Estimated risks per serving of lettuce based on the models were 3×10(-4) (6×10(-6)-5×10(-3)) for NoV infection and 3×10(-8) (7×10(-10)-3×10(-6)) for hepatitis A jaundice. The contribution to virus contamination of hand-contact was larger as compared with the contribution of irrigation, the conveyor belt or the water used for produce rinsing. In conclusion, viral contamination in the lettuce and soft fruit supply chains occurred and estimated health risks were generally low. Nevertheless, the 97.5% upper limit for the estimated NoV contamination of lettuce suggested that infection risks up to 50% per serving might occur. Our study suggests that attention to full compliance for hand hygiene will improve fresh produce safety related to virus risks most as compared to the other examined sources, given the monitoring results. This effect will be further aided by compliance with other hygiene and water quality regulations in production and processing facilities.

Prevalence of hepatitis E virus (HEV) antibodies in Serbian blood donors.

INTRODUCTION: Hepatitis E virus (HEV) infection is rarely reported in industrialized countries, but recent studies have revealed quite variable seroprevalence rates among European populations, including blood donors. In Serbia, very limited data about HEV seroprevalence are available. This study aimed to determine the prevalence of anti-HEV IgG antibodies and HEV RNA in the sera of volunteer blood donors in Serbia. METHODOLOGY: Serum samples from 200 volunteer blood donors were tested for the presence of anti-HEV IgG by enzyme-linked immunosorbent assay (ELISA) using ORF-2 HEV genotype 3 recombinant proteins as antigen, and for the presence of HEV RNA by nested reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: In total, 15% of the volunteer blood donors were seropositive. The prevalence increased with age; 21.5%, 14.2%, and 5.4% HEV seroprevalence rates were found in individuals older than 51 years, between 31 and 50 years, and in those younger than 30 years of age, respectively. However, no HEV RNA was detected in any of the individuals analyzed. CONCLUSIONS: The prevalence of anti-HEV IgG among blood donors as representatives of the general population is quite high in Serbia compared to data from many European countries. One of the reasons for this could be the high prevalence of HEV among Serbian pigs and the traditional consumption of piglet meat in the country. The relatively high HEV seroprevalence found among Serbian blood donors indicates the need for further investigation.

Tracing enteric viruses in the European berry fruit supply chain.

In recent years, numerous foodborne outbreaks due to consumption of berry fruit contaminated by human enteric viruses have been reported. This European multinational study investigated possible contamination routes by monitoring the entire food chain for a panel of human and animal enteric viruses. A total of 785 samples were collected throughout the food production chain of four European countries (Czech Republic, Finland, Poland and Serbia) during two growing seasons. Samples were taken during the production phase, the processing phase, and at point-of-sale. Samples included irrigation water, animal faeces, food handlers' hand swabs, swabs from toilets on farms, from conveyor belts at processing plants, and of raspberries or strawberries at points-of-sale; all were subjected to virus analysis. The samples were analysed by real-time (reverse transcription, RT)-PCR, primarily for human adenoviruses (hAdV) to demonstrate that a route of contamination existed from infected persons to the food supply chain. The analyses also included testing for the presence of selected human (norovirus, NoV GI, NoV GII and hepatitis A virus, HAV), animal (porcine adenovirus, pAdV and bovine polyomavirus, bPyV) and zoonotic (hepatitis E virus, HEV) viruses. At berry production, hAdV was found in 9.5%, 5.8% and 9.1% of samples of irrigation water, food handlers' hands and toilets, respectively. At the processing plants, hAdV was detected in one (2.0%) swab from a food handler's hand. At point-of-sale, the prevalence of hAdV in fresh raspberries, frozen raspberries and fresh strawberries, was 0.7%, 3.2% and 2.0%, respectively. Of the human pathogenic viruses, NoV GII was detected in two (3.6%) water samples at berry production, but no HAV was detected in any of the samples. HEV-contaminated frozen raspberries were found once (2.6%). Animal faecal contamination was evidenced by positive pAdV and bPyV assay results. At berry production, one water sample contained both viruses, and at point-of-sale 5.7% and 1.3% of fresh and frozen berries tested positive for pAdV. At berry production hAdV was found both in irrigation water and on food handler's hands, which indicated that these may be important vehicles by which human pathogenic viruses enter the berry fruit chain. Moreover, both zoonotic and animal enteric viruses could be detected on the end products. This study gives insight into viral sources and transmission routes and emphasizes the necessity for thorough compliance with good agricultural and hygienic practice at the farms to help protect the public from viral infections.

Study of the genetic variability of porcine circovirus type 2 detected in Serbia and Slovenia.

Recent variants of porcine circovirus type 2 (PCV2) were obtained from tissues of domestic pigs with porcine circovirus associated disease and from randomly selected wild boar samples from Serbia and Slovenia. A 450-base-pair nucleotide sequence was obtained by PCR from the ORF2. The derived nucleotide and amino acid sequences were aligned and compared to the corresponding region of closely related PCV2 sequences determined in previous years and retrieved from the GenBank. The 30 Serbian and 17 Slovenian PCV2 sequences clustered into three previously determined genotypes (PCV2a: 7), (PCV2b: 38) and (PCV2d: 2). Three major variable regions, concerning 29 amino acid position substitutions within the ORF2, were observed, which further supports the segregation of the detected strains into three separate genotypes. This study indicates that PCV2b is the predominant genotype in Serbia and Slovenia and the detected PCV2 strains are closely related to those previously described in Europe and in other parts of the world.