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.

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.

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.

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.

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.

Genotyping of Leptospira spp. in wild rats leads to first time detection of L. kirshneri serovar Mozdok in Serbia.

Introduction: This study aimed to investigate the prevalence and molecular characterization of Leptospira species in Belgrade, Serbia, an area where this disease is underexplored. Specifically, the study sought to employ molecular and multilocus sequence typing analyses to fill the gap in understanding the diversity and distribution of Leptospira species within the region. Methods: A comprehensive molecular analysis was conducted on kidney samples obtained from Norway rats (Rattus norvegicus) in the urban environment. The study utilized molecular diagnostic techniques including real-time PCR targeting the lipL32 gene and performing sequence-based typing schemes utilizing adk, icdA, lipL32, lipL41, rrs2, and secY genes. These methodologies were applied to ascertain the presence and characterize different Leptospira species and serovars, respectively. Results: The findings revealed the presence of two Leptospira species and three separate serovars in the Belgrade area. This study identified the presence of L. kirschneri serovar Mozdok in Serbia for the first time, a significant discovery previously undocumented in the region. This pioneering investigation sheds light on the molecular diversity and prevalence of Leptospira species in Serbia. Discussion: The study underscores the importance of employing molecular typing methods to gain insights into the epidemiology and characterization of Leptospira species. These findings significantly contribute to both local and global perspectives on leptospirosis epidemiology, providing vital insights for the development of effective control strategies and interventions. Summary: In our recent study, we explored the presence and performed molecular typing of the Leptospira species, the bacteria responsible for leptospirosis, in wild rats in Serbia. This was the first time such a study was conducted in the region. Leptospirosis is a serious disease that affects both animals and humans, often transmitted through contact with water contaminated by infected animals. Our focus was on understanding which types of Leptospira were present in these animals. Excitingly, we discovered a particular strain of Leptospira, known as L. kirshneri serovar Mozdok, for the first time in Serbia. This finding is significant because it sheds light on the presence and spread of different Leptospira serovars in Serbia. It also raises awareness about the potential health risks associated with this serovar, which was previously unknown in the area. Our work fits into a broader context of disease surveillance and public health. By identifying the types of Leptospira present in a specific region, we can better understand the risks to public health and take steps to prevent and control the spread of leptospirosis. This discovery is not just important for scientists studying infectious diseases; it has real implications for public health officials, veterinarians, and anyone concerned with preventing and treating leptospirosis. Our findings highlight the need for ongoing monitoring of Leptospira in wildlife and synanthropic fauna, to protect both animal and human health.

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).

Highly Pathogenic Avian Influenza H5N8 Outbreak in Backyard Chickens in Serbia.

In winter 2016/2017, the highly pathogenic avian influenza virus H5N8 was detected in backyard poultry in Serbia for the first time. The second HPAI outbreak case in backyard poultry was reported in 2022, caused by subtype H5N1. This is the first study that documents the laboratory identification and pathology associated with highly pathogenic avian influenza in poultry in Serbia during the first and second introduction waves. In both cases, the diagnosis was based on real-time reverse transcriptase PCR. The most common observed lesions included subepicardial hemorrhages, congestion and hemorrhages in the lungs, and petechial hemorrhages in coelomic and epicardial adipose tissue. Histologically, the observed lesions were mostly nonpurulent encephalitis accompanied by encephalomalacia, multifocal necrosis in the spleen, pancreas, and kidneys, pulmonary congestion, and myocardial and pulmonary hemorrhages. In H5N8-infected chickens, immunohistochemical examination revealed strong positive IHC staining in the brain and lungs. Following these outbreaks, strict control measures were implemented on farms and backyard holdings to prevent the occurrence and spread of the disease. Extensive surveillance of birds for avian influenza virus did not detect any additional cases in poultry. These outbreaks highlight the importance of a rapid detection and response system in order to quickly suppress outbreaks.

Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform.

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

E6/E7 mRNA Expression of the Most Prevalent High-Risk HPV Genotypes in Cervical Samples from Serbian Women.

Cervical cancer caused by persistent infection with HR HPV genotypes is the second leading cause of death in women aged 15 to 44 in Serbia. The expression of the E6 and E7 HPV oncogenes is considered as a promising biomarker in diagnosing high-grade squamous intraepithelial lesions (HSIL). This study aimed to evaluate HPV mRNA and DNA tests, compare the results according to the severity of the lesions, and assess the predictive potential for the diagnosis of HSIL. Cervical specimens were obtained at the Department of Gynecology, Community Health Centre Novi Sad, Serbia, and the Oncology Institute of Vojvodina, Serbia, during 2017-2021. The 365 samples were collected using the ThinPrep Pap test. The cytology slides were evaluated according to the Bethesda 2014 System. Using a real-time PCR test, HPV DNA was detected and genotyped, while the RT-PCR proved the presence of E6 and E7 mRNA. The most common genotypes in Serbian women are HPV 16, 31, 33, and 51. Oncogenic activity was demonstrated in 67% of HPV-positive women. A comparison of the HPV DNA and mRNA tests to assess the progression of cervical intraepithelial lesions indicated that higher specificity (89.1%) and positive predictive value (69.8-78.7%) were expressed by the E6/E7 mRNA test, while higher sensitivity was recorded when using the HPV DNA test (67.6-88%). The results determine the higher probability of detecting HPV infection by 7% provided by the mRNA test. The detected E6/E7 mRNA HR HPVs have a predictive potential in assessing the diagnosis of HSIL. The oncogenic activity of HPV 16 and age were the risk factors with the strongest predictive values for the development of HSIL.

Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

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.

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.

West Nile virus in the Republic of Serbia-Diagnostic performance of five serological tests in dog and horse sera.

West Nile virus (WNV) is a zoonotic mosquito-borne virus classified as family Flaviviridae and genus Flavivirus. The first WNV outbreak in humans in the Republic of Serbia was recorded in 2012. Equids and dogs can show clinical symptoms after WNV infection and are often used as sentinels. This study aimed to (i) give insight into seropositivity for WNV in clinically healthy dog and horse sera in different regions of Serbia and (ii) compare diagnostic value of 'in-house' and commercially available indirect immunofluorescence (IFA) and enzyme-linked immunoassay (ELISA) tests to 'gold standard' virus neutralization test (VNT). Due to cross-reactivity, sera were tested for Usutu virus and tick-borne encephalitis virus in VNT based on the epidemiological data of field presence. Blood sera of dogs (n = 184) and horses (n = 232) were collected from 2011 to 2013. The seropositivity was confirmed by VNT in 36.9 % tested dog sera and 34.9% tested horse sera with highest positivity in regions near two big rivers, while in four dog and seven horse sera, positivity resulted from Usutu virus infection. Comparative results of diagnostic tests in dogs ranged from 18.7 % seropositivity by 'in-house' ELISA to 31.9% by commercially available ELISA. In horses, seropositivity ranged from 36.2% by 'in-house' IFA to 32.5% by commercially available IFA and from 26.3% by 'in-house' IgG ELISA to 20.9% by commercially available ELISA. There were no statistically significant differences according to the McNemar test between 'in-house' and commercially available IFA and ELISA test in horse sera, while the same was not true for two ELISAs used in dog sera (χ2  = 8.647, p = .003). Established seropositivity in dogs and horses was in accordance with the epidemiological situation and WNV spread in the Republic of Serbia and proven Usutu virus co-circulation. 'In-house' tests remain a valuable tool in early diagnostics of WNV.

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.

Spatiotemporal Analysis of West Nile Virus Epidemic in South Banat District, Serbia, 2017-2019.

West Nile virus (WNV) is an arthropod-born pathogen, which is transmitted from wild birds through mosquitoes to humans and animals. At the end of the 20th century, the first West Nile fever (WNF) outbreaks among humans in urban environments in Eastern Europe and the United States were reported. The disease continued to spread to other parts of the continents. In Serbia, the largest number of WNV-infected people was recorded in 2018. This research used spatial statistics to identify clusters of WNV infection in humans and animals in South Banat County, Serbia. The occurrence of WNV infection and risk factors were analyzed using a negative binomial regression model. Our research indicated that climatic factors were the main determinant of WNV distribution and were predictors of endemicity. Precipitation and water levels of rivers had an important influence on mosquito abundance and affected the habitats of wild birds, which are important for maintaining the virus in nature. We found that the maximum temperature of the warmest part of the year and the annual temperature range; and hydrographic variables, e.g., the presence of rivers and water streams were the best environmental predictors of WNF outbreaks in South Banat County.

Validation of TaqMan-Based Assays for Specific Detection and Differentiation of Wild-Type and Neethling Vaccine Strains of LSDV.

Lumpy skin disease (LSD) is an important animal disease with significant health and economic impacts. It is considered a notifiable disease by the OIE. Attenuated strains of LSDV have been successfully used as vaccines (LAV) but can also produce mild or systemic reactions. Vaccination campaigns using LAVs are therefore only viable if accompanying DIVA assays are available. Two DIVA qPCR assays able to distinguish Neethling-based LAVs and wild-type LSDV were developed. Upon validation, both assays were shown to have high sensitivity and specificity with a diagnostic performance comparable to other published DIVA assays. This confirmed their potential as reliable tools to confirm infection in animals during vaccination campaigns based on Neethling vaccine strains.

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.

Distribution of porcine circovirus 2 cap antigen in the lymphoid tissue of pigs affected by postweaning multisystemic wasting syndrome.

The lymphatic organs of 50 pigs from a total of eight farms located at different sites in the epizootiological region of North Backa County were studied to obtain data on the prevalence of circoviral infections in Serbia. All of the pigs examined had clinical signs suggestive of postweaning multisystemic wasting syndrome (PMWS). All pigs underwent necropsy and tissue samples were taken for histopathological, immunohistochemical (IHC) and PCR analysis. The presence of porcine circovirus 2 (PCV2) was established by PCR analysis in the organs of the pigs tested. The most frequent histopathological lesions of lymphoid tissue linked with the presence of positive immunostaining for PCV2 Cap antigen confirmed the existence of PMWS in all farms tested in North Backa County. Using PCR, histopathological and IHC techniques, the presence of PMWS was proved in the Republic of Serbia. During necropsy, generalised enlargement of the lymph nodes was evident. The most common histopathological finding was lymphocyte depletion in the follicular and perifollicular areas of lymph nodes. Infiltration by macrophages was also recorded. By IHC analysis, the cytoplasm of macrophages was shown to contain a large amount of the ORF2-coded Cap antigen of PCV2. Lymphocyte depletion and large numbers of macrophages were recorded in the tonsils, spleen, intestinal lymphatic tissue, Peyer's patches and ileocaecal valve. The presence of typical granulomatous lesions with multinuclear giant cells (MGCs) was also recorded in the lymphatic tissue. Cap antigen was shown to be present in macrophages and less often in lymphocytes.

Biological properties of a naturally attenuated infectious bursal disease virus isolated from a backyard chicken flock.

The biological properties of an infectious bursal disease (IBD) virus isolated from bursas collected during an outbreak in a village chicken flock in Macedonia are described. The mortality rate was 50%. Two viruses coexisted in the bursas of infected chickens (IBDVwt and IBDVtc). The virus termed IBDVtc grows on chicken embryo fibroblast (CEF) cells from the first passage. Specific pathogen free chickens inoculated with IBDVtc at passage level 4 did not develop any clinical signs of disease. Some discrete bleeding on the leg muscles was seen and the bursa of Fabricius revealed pathological lesions similar to those caused by classical strains. However, the bursa recovered quickly (bursa lesion score 2) by 14 days post infection (PI). We also found evidence of bursal repopulation by means of perinuclear antigen staining. Strong CD3 influx was evident at 4 days PI, and at 33 days PI the CD3+ cell finding was comparable to the control. The mean antibody titre was 9.2 log 2 at 14 days PI. The amino acid composition of VP2 in IBDVwt (222 Ala, 242 Ile, 253 Gln, 256 Ile, 279 Asp, 284 Ala, 294 Ile and 299 Ser) is described. The same sequence was found in IBDVtc, except for two point mutations, at Gln253→His and Ala284→Thr. Such amino acid substitution is responsible for partial attenuation and the ability of the strain to replicate in cell culture. None of the commercial vaccine viruses has a similar arrangement of amino acids in the variable domain of IBDV. This strongly suggests that IBDVtc originates from a very virulent strain. To the best of our knowledge, this is the first report of a concomitant infection of chickens with highly pathogenic IBDV and its mutant counterpart.