Abstract
The Paenibacillus larvae is known as the causative agent of American foulbrood, which is widespread throughout the world. It is a highly contagious, fatal disease of honeybees. The aim of this study was to investigate the phylogenomic relationships of ERIC II genotype P. larvae isolates from Slovakia and to compare them with ERIC II genotype isolates originating from other countries. Phylogenomic analyses of P. larvae D3, 5S, and M1 isolates from Slovakia revealed a very low level of genetic diversity among our isolates, with low genome size variability (3.56 ± 0.04 Mbp) and average nucleotide and amino acid identity levels above 99%. Genomes showed similar gene counts (3,700 ± 59 genes per genome), with the highest variability in the mobile elements, especially bacteriophage-related genes (from 220 to 281 genes per genome). Comparative genomic analyses of several ERIC II and ERIC I types of P. larvae genomes available in the GenBank showed a clear geographical pattern, indicating that P. larvae strains spreading in the Slovak region differ from strains found in other parts of Europe and the rest of the world, thus indicating the possible existence of regionalism in the P. larvae species distribution.
1 Introduction
Accurate identification and typing of microorganisms are important steps not only for understanding microbial biogeography and heterogeneity but also for gaining knowledge about the pathogenicity of infectious agents and the epidemiology of microbial diseases. American Foulbrood Disease (AFB) is currently the most invasive bacterial disease affecting bee colonies worldwide. The disease is caused by the Gram-positive spore-forming bacterium Paenibacillus larvae, which causes major economic losses to the beekeeping industry worldwide each year by reducing bee populations, honey production, and other bee products. Because honeybees represent both an agricultural asset and an essential component of ecosystem health, AFB is of major veterinary and economic concern and has become one of the best-studied diseases of honeybees (1, 2). Even though this disease is called American Foulbrood, the origin of the disease is unknown. Outbreaks of AFB are most often caused by clonally related bacteria that share inherited biochemical and genetic properties, but sufficient diversity remains at the species level, leading to variations in the virulence of P. larvae. Despite its global distribution, the evolutionary origin and population structure of P. larvae remain incompletely resolved. Several molecular typing approaches have been applied to characterize P. larvae diversity. Enterobacterial repetitive intergenic consensus (ERIC)-PCR has been widely used for genotyping. ERIC-PCR generates genomic fingerprints based on amplification of repetitive intergenic elements but provides limited genome-wide resolution. Recently, five enterobacterial repetitive intergenic consensus (ERIC) genotypes (I-V) have been described (3). Differences in virulence between ERIC genotypes are associated with variation in disease progression, larval mortality rates, and outbreak severity (3, 4) highlighting the need for precise genotyping in veterinary diagnostics and surveillance. Genotype II is considered the most virulent (6). In recent years, the Multi Locus Sequence Typing (MLST) approach has been used for typing P. larvae isolates (7). It provides improved resolution but still interrogates only a small fraction of the genome: seven housekeeping genes. Proteomic approaches such as Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) of total cellular proteins, which are combined with 16S rRNA sequencing, have been increasingly used as an alternative and fast tool for identifying, typing, and differentiating closely related strains (8, 9).
Our results so far indicate that MALDI-TOF MS is effective in differentiating P. larvae, and that 16S rRNA analysis provides a very low level of differentiation between P. larvae isolates and is unable to explain the observed phenotypic variability (6, 10). Understanding the intraspecies diversity of P. larvae is crucial for research into the epidemiology of the disease. Whole-genome sequencing (WGS) provides a powerful tool for veterinary epidemiology, enabling precise strain discrimination, identification of virulence-associated genes, and tracking of transmission pathways (11). Understanding genome-level variation is essential for assessing outbreak sources, monitoring pathogen spread, and improving disease control strategies.
In this study, we obtained drafts of whole-genome sequences of Slovak isolates and performed phylogenomic and comparative genomic analyses to evaluate genome-wide similarity, gene content variation, and the contribution of mobile genetic elements to strain diversification of closely related strains. By resolving intraspecific diversity at high genomic resolution, this study aims to improve understanding of AFB epidemiology in Slovakia and to provide genomic data relevant for veterinary surveillance and disease control programs.
2 Materials and methods
2.1 Origin of samples and identification of bacterial isolates
Bacterial isolates of P. larvae D3, 5S, and M1 were obtained from the collection of microorganisms of the Centre of Biosciences of the Slovak Academy of Sciences, Institute of Animal Physiology in Kosice. The isolates were isolated from different localities in Eastern Slovakia from infected brood combs (D3 – Velka Lodina; 5S – Kosice and M1 – Lorincik) and identified and typed by using a combination of 16S rRNA sequencing, MALDI-TOF MS spectrometry, and ERIC-PCR (see publication (6)).
2.2 Isolation of DNA, sequencing, and phylogenetic analysis
The DNA was extracted from bacterial cultures grown in MYPGP medium (1.5% yeast extract, 0.3% K2PO4, 0.2% glucose, 0.1% sodium pyruvate, and 1.0% Mueller–Hinton broth) using the GenElute™ Bacterial Genomic DNA Kit according to the manufacturer’s protocol (Sigma Aldrich, St. Louis, United States). The quality and quantity of the extracted DNA were confirmed by agarose electrophoresis in 0.8% agarose gel in a TAE buffer and by using NanoDrop 2000c Spectrophotometer (Thermo Scientific, United States) and subsequently subjected to whole-genome sequencing by Eurofins Genomics Europe Pharma and Diagnostics (Constance, Germany), using Illumina technology, mode NovaSeq PE 150. The quality of raw sequences was checked using FastQC v 0.11.9 (12). Trimmomatic v0.39 (13) was used to remove sequences with a quality score lower than Q20. The high-quality sequences were de novo assembled using the SPAdes tool (14). Finally, the contigs shorter than 200 bp were excluded from the obtained draft genomes. Draft genome sequences were deposited in the GenBank database1 under the following accession numbers: JBLOBJ000000000 and GCA_047786425.1 for the D3 isolate, JBLOBH000000000 and GCA_047786405.1 for the 5S isolate, and JBLOBI000000000 and GCA_047786385.1 for the M1 isolate. All quality metrics and basic genome descriptions are shown in Table 1.
Table 1
| The features of genomes | D3 isolate | 5S isolate | M1 isolate |
|---|---|---|---|
| JBLOBJ000000000 | JBLOBH000000000 | JBLOBI000000000 | |
| Genome size (bp) | 3,714,613 | 3,708,420 | 3,679,277 |
| Sequenced reads | 6,396,195 | 7,601,786 | 8,891,576 |
| Trimmed reads | 6,395,799 | 7,601,503 | 8,891,243 |
| Genome coverage | 436x | 518x | 606x |
| GC content (%) | 44.6 | 44.6 | 44.6 |
| Largest contig (bp) | 100,864 | 100,865 | 101,339 |
| N50 (bp) | 25,137 | 23,599 | 25,134 |
| L50 | 45 | 47 | 45 |
| Number of contigs | 359 | 381 | 358 |
| Completeness (%)* | 99.19 | 99.19 | 99.19 |
| Contamination (%) | 0.02 | 0 | 0 |
The features of P. larvae genomes originating from Slovakia according to the RAST server.
* Genome completeness and contamination calculated by the CheckM tool using the NCBI database.
Phylogenetic position of the studied isolates within P. larvae strains of known ERIC genotype (genome assemblies are available in the GenBank database with accession numbers GCA_000511405.1, GCA_002951895.1, GCA_002082155.1, GCA_032710345.1, GCA_002003265.1, GCA_002951875.1, and known genotype) was performed using digital DNA–DNA hybridization (dDDH) and construction of a Genome BLAST Distance Phylogeny (GBDP) phylogenetic tree using the Type (Strain) Genome Server (TYGS) tool (15). The analysis was supplemented by calculating the Average Amino Acid Identity (AAI) and Average Nucleotide Identity (ANI) values using the EzAAI tool (16) and the JspeciesWS server (17).
Annotation of the studied genomes was performed using the Rapid Annotation using Subsystem Technology (RAST) (18). The PhageBoost server2 was used to detect prophage regions (at least 7 bacteriophage related genes in a row) (19). The presence of plasmids in draft sequences was analyzed using an online search against the PLSDB database (available at https://ccb-microbe.cs.uni-saarland.de/plsdb2025/) (20).
3 Results
In this study, the draft whole-genome sequences were analyzed for three ERIC II genotype P. larvae isolates (D3, 5S, and M1) obtained from three localities in Eastern Slovakia for performing phylogenomic and comparative genomic analyses to evaluate genome-wide similarity, gene content variation, and the contribution of mobile genetic elements to strain diversification of closely related strains. The features of genomes are described in Table 1.
Phylogenomic analyses of three genomes indicated very low genome size variability (3.71 ± 0.03 Mbp) among isolates from Slovakia. The analysis by ANI, AAI, and dDDH showed very high genetic similarity values exceeding 99%. The lower AAI, ANI, and dDDH values were observed for the genomes from other areas of the world (see Table 2). The isolates from Slovakia were placed in a single clade in the GBDP tree constructed using the TYGS server (25–27), and together with isolates from Germany formed a well-separated clade (Figure 1).
Table 2
| Genomes of P. larvae | Country | Genotype | ANI in % | AAI in % | dDDH (d4 in %) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 5S | M1 | D3 | 5S | M1 | D3 | 5S | M1 | D3 | |||
| 5S GCA_047786405.1 | Slovakia | ERIC II | – | 99.87 | 99.96 | – | 99.95 | 99.93 | – | 99.9 | 99.9 |
| M1 GCA_047786385.1 | Slovakia | ERIC II | 99.87 | – | 99.89 | 99.95 | – | 99.98 | 99.9 | – | 100.0 |
| D3 GCA_047786425.1 | Slovakia | ERIC II | 99.96 | 99.89 | – | 99.93 | 99.98 | – | 99.9 | 100.0 | – |
| GCA_000511405.1 | Germany | ERIC II | 99.96 | 99.90 | 99.95 | 99.97 | 99.98 | 99.98 | 99.9 | 99.7 | 99.9 |
| GCA_002951895.1 | Germany | ERIC II | 99.96 | 99.89 | 99.96 | 99.97 | 99.98 | 99.98 | 99.9 | 99.7 | 99.9 |
| GCA_002082155.1 | Argentina | ERIC II | 98.22 | 98.13 | 98.20 | 98.63 | 98.63 | 98.62 | 90.6 | 90.4 | 90.5 |
| GCA_032710345.1 | Canada | ERIC I | 99.10 | 99.06 | 99.09 | 99.28 | 99.29 | 99.28 | 96.2 | 96.0 | 96.1 |
| GCA_002003265.1 | Argentina | ERIC I | 99.19 | 99.16 | 99.19 | 99.26 | 99.28 | 99.27 | 96.5 | 96.2 | 96.4 |
| GCA_002951875.1 | USA | ERIC I | 99.22 | 99.17 | 99.21 | 99.31 | 99.31 | 99.31 | 96.5 | 96.3 | 96.4 |
The phylogenomic analysis of P. larvae genomes originating from Slovakia and different countries worldwide according to ANI, AAI, and dDDH comparison.
Figure 1
The D3, 5S, and M1 genomes exhibited very small differences in gene counts (3,700 ± 59 genes per genome) predicted using the RAST server (18), with the highest variability observed in mobile genetic elements, particularly in the genes of putative bacteriophage origin (ranging from 220 to 281 genes per genome, Table 3). Similarly, the PhageBoost server predicted 194 to 258 genes per genome in our genomes, whereas in the genome DSM25430, it was 338 per genome (data not shown). Different sets of plasmids were detected in the genomes of D3, 5S, and M1 isolates during search against the PLSDB database (20) using a stringent search (minimal identity 0.99). In the genomes of D3 and M1 isolates, 3 plasmids were detected (the same set as in the genome of the geographically related DSM 25430 strain from Germany). In the 5S genome, a single plasmid (NZ_CP019653.1, P. larvae subsp. larvae DSM 25430 plasmid unnamed1, Table 3) was detected, indicating that mobile gene elements contribute to the observed genomic variability in P. larvae.
Table 3
| DSM25430 strain | D3 isolate | 5S isolate | M1 isolate | |
|---|---|---|---|---|
| NZ_CP019652 | JBLOBJ000000000 | JBLOBH000000000 | JBLOBI000000000 | |
| Germany | Slovakia | Slovakia | Slovakia | |
| Genome size (bp) | 4,056,006 | 3,714,613 | 3,708,420 | 3,679,277 |
| Number of genes in category | ||||
| Coding sequences | 3,796 | 4,401 | 4,393 | 4,369 |
| RNAs | 102 | 77 | 76 | 77 |
| Putative prophage regions | 20 | 11 | 10 | 8 |
| Phage-related genes | 422 | 255 | 220 | 281 |
| The presence of a plasmid | ||||
| pPLA2_10 (NC_023147.1) | + | + | − | + |
| Unnamed 1 (NZ_CP019653.1) | + | + | − | + |
| Unnamed 2 (NZ_CP019654.1) | + | + | + | + |
| Virulence, disease and defense | 63 | 67 | 66 | 62 |
| Stress response | 77 | 80 | 79 | 79 |
| Regulation and cell signaling | 53 | 58 | 58 | 46 |
| DNA metabolism | 343 | 124 | 122 | 121 |
| Motility and chemotaxis | 38 | 73 | 73 | 73 |
The comparative genomics of P. larvae isolates from Slovakia and the closest ERICII type relative from Germany.
Genomic comparison with the DSM25430 genome showed that genomes of P. larvae isolates from Slovakia show a similar number of genes in most categories, including the Virulence, Disease and Defense, Stress Response, and Regulation and Cell Signaling categories identified by RAST annotation (18), but show a significantly lower number of genes for RNA and in the DNA Metabolism and Motility and Chemotaxis categories (Table 3). In the DNA Metabolism category, all Slovak isolates lack multiple genes participating in DNA repair.
4 Discussion
AFB represents one of the most destructive bacterial diseases affecting honeybee (Apis mellifera) colonies worldwide, with significant ecological and economic impacts. The etiological agent of the disease, P. larvae, exhibits considerable genetic diversity, which has been linked to differences in virulence, epidemiology, and transmission dynamics. In Slovakia and other countries worldwide, despite long-term monitoring and control measures, AFB remains a persistent threat, particularly in regions with high colony density and frequent movement of beekeeping materials. Understanding the routes of AFB spread and the population structure of P. larvae is therefore essential for improving disease management strategies. Traditional genotyping methods, such as ERIC-PCR, have identified several genotypes, among which ERIC II is often associated with increased virulence and rapid disease progression. However, these approaches provide only limited resolution for elucidating fine-scale genetic relationships and transmission pathways. In this context, the WGS combined with phylogenomic and comparative genomic analyses offers a powerful framework for high-resolution characterization of pathogen populations (5).
For a better understanding of the American foulbrood spread routes and variability of P. larvae, the etiological agent of AFB in Slovakia, a phylogenomic approach and comparative genomics were used in this study. Draft whole-genome sequences were obtained for three ERIC II genotype P. larvae isolates (D3, 5S, and M1) obtained from three localities in Eastern Slovakia separated by more than 15 km, thereby minimizing the probability of direct epidemiological linkage. This spatial distribution provides an opportunity to assess genomic variability within the ERIC II lineage at a regional scale and to explore potential patterns of dissemination independent of immediate local transmission. Phylogenomic analyses predicted a very low genome size variability (3.71 ± 0.03 Mbp) in isolates from Slovakia, which is the lowest among P. larvae genomes (of about 4 Mbp for the isolates in the GenBank database) available in the GenBank database. Three Slovak origin genomes showed very high genetic similarity, with ANI, AAI, and dDDH values exceeding 99%. The distance between the localities is greater than the usual flight range of worker bees, so robbing or bee drifting cannot explain the similarities observed. Similar low genetic variability was observed in P. larvae outbreaks in Slovenia (21), where the ERIC II genotype was detected, or in Australia, where the ERIC I genotype was confirmed (22). On the other hand, lower AAI, ANI, and dDDH values (see Table 2) were observed for the genomes from other areas of the world (see Figure 1), and there is a correlation between genomic and geographic distances between isolates. Isolates from Slovakia were placed in a well-separated branch in the GBDP tree, indicating that the locally differentiated genomic lineage ERIC II of P. larvae spreads on the territory of Eastern Slovakia, distinct from ERIC II and ERIC I genomic lineages from Germany or North and South America (Figure 1). The D3, 5S, and M1 genomes exhibited very small differences in gene counts (3,700 ± 59 genes per genome) detected using the RAST server (18), with the highest variability observed in mobile genetic elements, particularly in the genes of putative bacteriophage origin (ranging from 220 to 281 genes per genome, Table 3). Confirming the presence of prophages in our genomes was realized by adding analysis using the PhageBoost server (see footnote 2). Analysis predicted 194 to 258 genes per genome, and in the genome DSM25430, it was 338 per genome (data not shown). In P. larvae, a large part of the genome size variability is due to integrated bacteriophages (prophages). Comparing our genomes with the German genome DSM25430 showed a difference in the number of phage regions /genes. Our genomes had approximately half the number of phage regions / genes compared to the DSM25430 genome (see Table 3). This means that if our strains have lost or never acquired prophages, their genomes may be up to hundreds of kilobases smaller. Different sets of plasmids were detected in the genomes of D3, 5S, and M1 isolates, too, using the PLSDB database (20), which indicates that mobile gene elements contribute to the observed genomic variability in P. larvae. Mobile genetic elements are DNA segments capable of moving between genomes or within a genome. In the disease of American foulbrood caused by the bacterium P. larvae, they play a very important role. They influence the virulence of the bacterium, its ability to adapt, horizontal gene transfer, and, last but not least, the epidemiology of the disease in bee populations. Plasmids and transposons can carry resistance genes and promote the selection of resistant strains. Although antibiotic therapy is mostly banned in Europe, selection pressure may have affected P. larvae populations in the past. Mobile gene elements accelerate recombination, genome rearrangement, and the creation of new variants. These factors, therefore, significantly complicate disease control, diagnosis, and, last but not least, virulence prediction (23, 24).
Next, genomic comparison with the German origin DSM25430 strain genome showed that genomes of P. larvae isolates from Slovakia show a similar number of genes in most categories, including the Virulence, Disease and Defense, Stress Response, and Regulation and Cell Signaling categories identified by RAST annotation, but show a significantly lower number of genes for RNA and in the DNA Metabolism and Motility and Chemotaxis categories (Table 3). In the DNA Metabolism category, all Slovak isolates lack multiple genes participating in DNA repair. On the other hand, in the Motility and Chemotaxis category, the German strain completely lacks genes in the Flagellum subcategory, where genes for flagellar biosynthesis, flagellar motor stator proteins, and flagellar motility belong. The biological importance of observed differences must be confirmed experimentally, but these findings point to distinct genomic adaptations in Slovak isolates and the reference strain, particularly in functions related to genome stability and motility, which may have implications for their pathogenicity, environmental persistence, and evolutionary relationships. Variability in metabolic functions may reflect differences in the ability of individual strains to utilize available nutrients or adaptation to specific environmental conditions. Differences in genes related to motility and chemotaxis may indicate differences in mechanisms of colonization and interaction with the host. The lower representation of genes classified into DNA and RNA categories may be related to variability in regulatory mechanisms, DNA repair processes, or genes involved in nucleic acid metabolism. Since the analyzed strains belonged to the same ERIC II genotype, it is likely that the observed differences represent a variable component of the genome and reflect microevolutionary processes occurring within this genetic lineage, while genes involved in virulence and basic cellular functions remain evolutionarily conserved.
Our findings predict a possible regionalism in the occurrence of P. larvae species, since Slovak isolates differ from previously reported strains from other regions of Europe and the world. The isolates from Germany form a neighboring branch to isolates of Slovakia, even though they differ significantly in genomic characteristics (e.g., genome size 4.1 Mbp versus 3.7 Mbp for Slovak isolates). On the other hand, the plasmid occurrence in 2 Slovak isolates is identical to the profile of the German isolate. The cause of the possible observed regionalism is not yet clear, but it could be the transmission of P. larvae through human activity. Human-mediated transmission is one of the most important epidemiological factors for American foulbrood, as natural spread of the disease is possible but usually geographically limited (up to 10 km). In contrast, humans can inadvertently transfer P. larvae spores over long distances and between many colonies in a short time. A key problem is the extraordinary resistance of the spores. The spores can survive in honey, wax, propolis, wooden parts of hives, or on tools for decades without losing infectivity. As a result, even old or apparently clean material can become a source of infection. Epidemiologically, it is important that very small amounts of spores are sufficient to cause larval infection. One of the most important mechanisms of spreading is the movement of bee colonies. Commercial nomadic bees for pollination or trade in brood and queens create extensive contact networks between regions. If a colony is infected subclinically, i.e., without obvious visible symptoms, it can be relocated before the disease is diagnosed. Such “silent” spread significantly complicates disease control and promotes the geographical expansion of certain genotypes of P. larvae. Contaminated hive material is also of great importance. Frames, partitions, wax, feeders, or used hives can contain high concentrations of spores. Recycling wax is particularly risky, as spores can survive normal processing conditions. Similarly, honey used to feed bees can be a source of infection if it comes from contaminated colonies. All of the above factors are significant epidemiological mediators of this invasive bacterial disease.
5 Conclusion
This study provides new genomic data for P. larvae isolates from Europe and highlights the need for broader genomic sampling to clarify transmission routes and population structure. The aim of the future research is to obtain and analyze additional data (genomes) and confirm (or exclude) the cause of transmission and virulence of P. larvae associated with the presence of mobile genetic elements, including plasmids, transposons, and bacteriophages, which have been identified in Slovak P. larvae genomes. These elements may play a critical role in shaping bacterial virulence, adaptability, and evolutionary trajectories. However, their functional significance in the context of P. larvae pathogenicity and transmission remains to be conclusively determined. Future studies integrating genomic, functional, and epidemiological data will be essential to confirm or exclude their involvement in virulence modulation and disease spread.
A better understanding of these mechanisms could ultimately contribute to the development of more effective surveillance, prevention, and control strategies targeting AFB. Considering the significant economic and ecological impact of AFB on apiculture, such advancements are crucial not only for Slovakia but also for the global beekeeping community.
Statements
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/genbank/, JBLOBJ000000000; https://www.ncbi.nlm.nih.gov/genbank/, JBLOBH000000000; https://www.ncbi.nlm.nih.gov/genbank/, JBLOBI000000000.
Author contributions
AK: Methodology, Visualization, Conceptualization, Investigation, Writing – review & editing, Funding acquisition, Writing – original draft, Formal analysis. JK: Writing – review & editing, Software, Methodology. SI: Writing – review & editing. MPG-B: Funding acquisition, Writing – review & editing. PP: Formal analysis, Funding acquisition, Supervision, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was financially supported by the VEGA 2/0099/25 project to AK. Additional support was provided through the Bilateral Action CSIC-SAS (BISAS24004 to MPG-B and CSIC-SAS-2024-01 to PP).
Acknowledgments
We thank Zuzana Alexiova for her assistance during our experiments.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that Generative AI was not used in the creation of this manuscript.
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Summary
Keywords
American foulbrood, comparative genomic, diversity, honeybees, Paenibacillus larvae, phylogenomic analysis, sequencing, WGS
Citation
Kopcakova A, Kiskova J, Ivorova S, Garcillán-Barcia MP and Pristas P (2026) Phylogenomic analysis of Paenibacillus larvae isolates from Slovakia predicts regional genomic clustering. Front. Vet. Sci. 13:1872838. doi: 10.3389/fvets.2026.1872838
Received
05 May 2026
Revised
08 June 2026
Accepted
12 June 2026
Published
08 July 2026
Volume
13 - 2026
Edited by
Mohamed H. Maarouf, Suez Canal University, Egypt
Reviewed by
Joon Liang Tan, Multimedia University, Malaysia
Ahmed F. Roumia, Menoufia University, Egypt
Updates
Copyright
© 2026 Kopcakova, Kiskova, Ivorova, Garcillán-Barcia and Pristas.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Anna Kopcakova, kopcakova@saske.sk
Disclaimer
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