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.

Brucellosis in pregnancy: case reports with different outcomes in an endemic region.

Different outcomes of brucellosis in pregnancy regarding the fetus/neonate and the mother are described. Medical records of five pregnant women with brucellosis were retrospectively analyzed. Patients were treated in several departments of infectious diseases in the Republic of Macedonia between 1995 and 2009. The diagnosis of brucellosis was based on clinical findings compatible with the disease supported by detection of specific antibodies. Pregnancy outcomes in patients were as follows: spontaneous abortion, intrauterine fetal death, premature delivery in two cases (one with twin pregnancy) and term delivery. One of the women experienced relapse. Follow-up results of neonates showed no infection and their normal growth and development. Brucellosis, especially if acquired in early pregnancy, can have an impact on pregnancy outcome. In endemic regions, in pregnant women with persisting fever and unspecific manifestations one should always have in mind brucellosis. In these areas, cases with unexplained spontaneous abortion, intrauterine fetal death and premature delivery should also be investigated for brucellosis.

Experimental lumpy skin disease virus infection of cattle: comparison of a field strain and a vaccine strain.

Lumpy skin disease virus (LSDV) infections can cause massive clinical signs in cattle and have great economic impact due to severe trade restrictions. For LSDV control, only live attenuated vaccines are commercially available, but they currently are not authorized in the European Union. Moreover, these vaccine virus strains can induce substantial side effects with clinical signs similar to infections with virulent LSDV. In our study, we compared clinical symptoms, viremia, and seroconversion of cattle inoculated either with a virulent field strain from North Macedonia isolated from diseased cattle in 2016 or with the attenuated LSDV vaccine strain "Neethling". Using specimens from the field and from experimental inoculation, different diagnostic tools, including a pan-capripox real-time qPCR, newly developed duplex real-time qPCR assays for differentiation between virulent and attenuated LSDV strains, and several serological methods (ELISA, indirect immunofluorescence test and serum neutralization test [SNT]) were evaluated. Our data show a high analytical sensitivity of both tested duplex real-time qPCR systems for the reliable distinction of LSDV field and vaccine strains. Moreover, the commercially available capripox double-antigen ELISA seems to be as specific as the SNT and therefore provides an excellent tool for rapid and simple serological examination of LSDV-vaccinated or infected cattle.

Molecular characterisation of rabies virus strains in the Republic of Macedonia.

Rabies, a worldwide zoonosis, remains a public-health concern despite oral wildlife vaccination in Europe. After a ten-year break, Macedonia reported eight rabies cases in 2011-2012. Two countries (Serbia and Bulgaria) bordering Macedonia are reporting cases in domestic and wild animals. This report describes the genetic characterisation of eight isolates from Macedonia compared with representative samples from neighbouring countries. All of the isolates tested belong to the Eastern European group, with a high degree of nucleotide sequence identity in the nucleoprotein gene. The close genetic relationship between isolates from the three bordering countries suggests that wildlife is responsible for rabies movements in the region.

Emergence and Persistent Circulation of Highly Pathogenic Avian Influenza Virus A (H5N8) in Kosovo, May 2021-May 2022.

In this study, we report the first outbreak of highly pathogenic avian influenza (HPAI) A H5N8, clade 2.3.4.4b in Kosovo on 19 May 2021. The outbreak consisted of three phases: May-June 2021, September-November 2021, and January-May 2022. In total, 32 backyards and 10 commercial holdings tested positive for the virus. Interestingly, the third and last phase of the outbreak coincided with the massive H5N1 clade 2.3.4.4b epidemic in Europe. Phylogenetic analyses of 28 viral strains from Kosovo revealed that they were closely related to the H5N8 clade 2.3.4.4.b viruses that had been circulating in Albania, Bulgaria, Croatia, Hungary, and Russia in early 2021. Whole genome sequencing of the 25 and partial sequencing of three H5N8 viruses from Kosovo showed high nucleotide identity, forming a distinctive cluster and suggesting a single introduction. The results of the network analysis were in accordance with the three epidemic waves and suggested that the viral diffusion could have been caused by secondary spreads among farms and/or different introductions of the same virus from wild birds. The persistent circulation of the same virus over a one-year period highlights the potential risk of the virus becoming endemic, especially in settings with non-adequate biosecurity.

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.

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.

Re-emergence of BTV serotype 4 in North Macedonia, July 2020.

Bluetongue virus serotype 4 (BTV-4) was confirmed in sheep in North Macedonia in July 2020. The full genome of this BTV-4 strain (MKD2020/06) was shown to be most closely related (99.74% nt identity) to the Greek GRE2014/08 and the Hungarian HUN1014 strains, indicating the re-emergence of this BTV serotype in the Balkan region since it was last reported in 2017.

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.

Gaps in brucellosis eradication campaign in sheep and goats in Republic of Macedonia: lessons learned.

AIM: To identify why "test and slaughter policy" for eradication of brucellosis did not significantly reduce the prevalence in sheep and goats in Macedonia. METHOD: Coverage of sampled vs expected number of sheep and goats, absolute number of positive animals, prevalence, frequency distribution, and classes of disease prevalence were retrospectively analyzed at the village level for 2004-2006. A comparative analysis of the disease prevalence in the investigated villages was also performed. The percentage of slaughtered animals was analyzed for 2000-2006. RESULTS: We found differences between the expected and actual number of sampled animals, which were related to the type of livestock breeding. Traditionally maintained flocks and migratory flocks were considered to be responsible for the transmission of the disease. The absolute number of positive animals and the number of infected vs non-infected holdings did not decrease over the study period. Most of the villages had between 1 and 10 positive animals. Between 2000 and 2006, 55% of the positive animals were slaughtered, 41% in 2001 and up to 79% in 2002. Moreover, in 2005 and 2006, 34% and 53% of sheep and goats were found to be positive at the slaughter line, respectively, demonstrating that only 21%-23% of the infected animals were correctly removed from the herds. CONCLUSION: Based on the findings of this study, Macedonia changed its control strategy from "test and slaughter" to a vaccination policy for sheep and goats in 2008.

Minimum Infective Dose of a Lumpy Skin Disease Virus Field Strain from North Macedonia.

Infection with Lumpy Skin Disease virus (LSDV), as well as infections with other Capripox virus species, are described as the most severe pox diseases of production animals and are therefore listed as notifiable diseases under the guidelines of the World Organization for Animal Health (OIE). To our knowledge there is only a single study examining dose dependency, clinical course, viremia, virus shedding, as well as serological response following experimental LSDV "Neethling" inoculation. Here, we inoculated cattle with four different doses of LSDV strain "Macedonia2016", a recently characterized virulent LSDV field strain, and examined clinical symptoms, viremia, viral shedding, and seroconversion. Interestingly, around 400 cell culture infectious dose50 (CCID50) of LSDV-"Macedonia2016" were sufficient to induce generalized Lumpy Skin Disease (LSD) in two out of six cattle but with a different incubation time, whereas the other animals of this group showed only a mild course of LSD. However, differences in incubation time, viral loads, serology, and in the clinical scoring could not be observed in the other three groups. In summary, we concluded that experimental LSDV infection of cattle with an infectious virus titer of 105 to 106 CCID50/mL of "Macedonia2016" provides a robust and sufficient challenge model for future studies.

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.

Impact of measures to control brucellosis on disease characteristics in humans: experience from an endemic region in the Balkans.

AIM: To analyze the impact of measures undertaken to control brucellosis in the Republic of Macedonia on demographic, epidemiological and clinical characteristics of the disease in humans. METHODS: Demographic, epidemiological and clinical characteristics of 340 patients with brucellosis were retrospectively analyzed. Patients were treated at the University Clinic of Infectious Diseases in Skopje during three time periods: 1989-1990 when no national strategy for disease control existed, 2000-2001 when some activities for control were performed and 2011-2014 when an organized and financially supported strategy was accomplished. RESULTS: A drastic decrease of the number of cases of human brucellosis during the last period when compared to the first two ones (3.9 and 3.3 times lower, respectively) was evident. Patients from the third period were significantly older, with median age 41 years (range 4-73) vs. 31 years (3-77) and 34 years (1-74), respectively (p = .013). There were more males (86.6 vs. 73.6% and 63.2%, respectively, p = .002) and the disease was more often acquired professionally (80.6 vs. 60.1% and 59.2%, respectively, p = .006). Fewer patients from the third period had elevated body temperature either as a symptom (50.7 vs. 73.6% and 69.6%, respectively, p = .003), or as a sign (35.8 vs. 55.4% and 56.8%, respectively, p = .011) and less often exhibited focal forms (40.3 vs. 56.8% and 64.0%, respectively, p = .007). CONCLUSION: The national strategy for brucellosis control, which included vaccination of small ruminants, resulted in a decrease of human brucellosis cases in the Republic of Macedonia and there was a tendency to milder disease manifestations.

Circulation of Crimean-Congo Hemorrhagic Fever Virus in the former Yugoslav Republic of Macedonia revealed by screening of cattle sera using a novel enzyme-linked immunosorbent assay.

BACKGROUND: There are only few assays available for the detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-specific antibodies in animals, and data about diagnostic sensitivity and specificity are incompletely documented for most of these tests. This is unfortunate since CCHFV antibodies in animals can be used as indicator for virus circulation in a geographic area and therewith potential risk of human exposure. This paper therefore reports on a novel ELISA for the detection of CCHFV-specific antibodies in cattle and on its application for testing ruminant sera from the Former Yugoslav Republic of Macedonia. PRINCIPAL FINDINGS: A highly sensitive and specific ELISA was developed to detect CCHFV-specific IgG antibodies in cattle. The assay was validated by using 503 negative serum samples from a country where CCHFV has never been detected until now, and by using 54 positive serum samples. The positive sera were verified by using two commercially available assays (for testing human serum) which we have adapted for use in animals. The sensitivity of the novel ELISA was 98% and its specificity 99%. The presence of Hyalomma ticks was demonstrated in the Former Yugoslav Republic of Macedonia and depending on the region antibody prevalence rates up to 80% were detected in the cattle population. CONCLUSION: This article describes a fully validated, highly sensitive and specific ELISA for the detection of CCHFV-specific IgG antibodies in cattle. Using this assay, CCHFV-specific antibodies were detected for the first time in cattle in the Former Yugoslav Republic of Macedonia, giving evidence for an active circulation of this virus in the country. Supporting this conclusion, the occurrence of the main vector of CCHFV was demonstrated in the present work for the first time in Former Yugoslav Republic of Macedonia.