Genomic analysis of Brucella Ovis in Italy reveals high genetic diversity and a novel lineage

Abstract

Background

Brucella ovis, the causative agent of ovine brucellosis, poses an underestimated threat to sheep farming worldwide due to its impact on reproductive health and flock productivity. Although B. ovis is non-zoonotic, the disease is globally distributed and often excluded from mandatory control programs, especially in Europe, resulting in scarce prevalence data and limited genomic information that hinder effective management strategies.

This study investigated the global and regional phylogenetic structure of B. ovis using Multilocus Variable-Number Tandem Repeat (MLVA) analysis, core genome Multilocus Sequence Typing (cgMLST), and Single Nucleotide Polymorphism (SNP) analysis. Strains from the Americas, Europe, Oceania, and a novel Italian dataset were analyzed to better understand the evolutionary dynamics and distribution of this pathogen.

Results

MLVA revealed limited continental clustering and the presence of shared genotypes across geographically distant regions. cgMLST analysis identified two dominant sequence types: the globally distributed ST-13 and a novel, Italy-specific ST-97. While ST-13 showed significant allelic diversity, ST-97 was found exclusively in two geographically distant Italian isolates. Italian strains demonstrated higher genomic diversity overall, forming multiple distinct clusters. SNP analysis confirmed these observations, highlighting clear geographic segregation within Italy and close genetic relationships between some Italian strains and those from Spain, Croatia, and the USA. Notably, ST-97 strains lacked the virulence-associated btpA gene and exhibited a 34-gene deletion, including putative prophage-related genes.

Conclusion

The findings reveal novel aspects of B. ovis phylogeography, emphasizing the unexpectedly high genetic diversity within Italy and identifying a distinct lineage (ST-97) with potential differences in virulence and adaptability. These insights enhance our understanding of the global epidemiology of B. ovis and may inform the development of targeted control strategies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-025-05124-w.

Background

Ovine brucellosis, caused by the Gram-negative bacterium Brucella ovis, is a significant yet often under-recognized threat to global sheep production. While considered non-zoonotic, its impact is substantial, primarily inducing epididymitis and orchitis in rams, and placentitis and abortions in ewes, leading to infertility, decreased lamb production, increased culling rates, and consequently, significant economic hardship for sheep farmers [1, 2]⁠. The economic impact stems not only from direct production losses, such as reduced lambing rates and increased culling of infertile rams, but also from trade restrictions and the costs associated with control programs where they exist [3]. First identified in Australia and New Zealand in the early 1950 s [4]⁠, B. ovis has since been reported across South and North America, Europe, and Oceania, highlighting its widespread distribution and substantial economic impact.

Despite its global distribution, significant knowledge gaps persist regarding the epidemiology and genomic diversity of B. ovis, particularly in Europe. There is a lack of comprehensive genomic datasets from European outbreaks, insufficient phylogenetic resolution to trace strain circulation between flocks or countries, and scarce information on possible genomic markers associated with virulence or host specificity. In many European countries, unlike infections with B. melitensis, ovine brucellosis caused by B. ovis is not subject to mandatory surveillance or eradication programs. This has led to a scarcity of prevalence data, with our understanding often limited to localized reports rather than a harmonized, continent-wide picture. Consequently, the amount of publicly available genomic data from European isolates is also limited, hindering high-resolution epidemiological analyses and a deeper understanding of the pathogen’s population structure and transmission dynamics across the region [5, 6]⁠. The absence of disease control programs allows the disease to spread, further exacerbating economic losses and animal welfare concerns [7]⁠. Therefore, understanding the genetic diversity and evolutionary history of B. ovis is crucial for developing effective control and eradication programs.

Historically, molecular typing methods like Multilocus Variable Number of Tandem Repeats Analysis (MLVA) have provided valuable insights into B. ovis epidemiology [8]⁠. However, the advent of WGS has revolutionized the study of bacterial pathogens, offering higher resolution through approaches such as core genome Multi-Locus Sequence Typing (cgMLST) and Single Nucleotide Polymorphism (SNP) analysis [6, 8, 9]⁠⁠. These techniques enable the detection of subtle genetic variations, providing a more detailed picture of pathogen evolution and transmission. Notably, while WGS-based methods are now commonly employed and considered standard tools in pathogen surveillance, still there is a significant lesser number of publicly available B. ovis genomic sequence data, with previous analyses relying heavily on MLVA [10–12]⁠⁠. This paucity of data constrains our understanding of the global population structure, evolutionary trajectories, and adaptive mechanisms of this pathogen. Specifically, the limited genomic information currently available impedes accurate reconstruction of the origin and dissemination of B. ovis lineages, the identification of putative virulence and host-adaptation determinants, and a comprehensive understanding of the evolutionary processes underlying its persistence across diverse environments and hostsThis study aims to fill these knowledge gaps by applying a suite of genomic tools to a novel collection of Italian B. ovis isolates, placing them within the current global genomic context. By elucidating the fine-scale genetic relationships between these strains, we provide new insights into the regional and international circulation patterns of B. ovis, which may facilitate the future development of targeted control strategies.

Methods

Bacterial strains

Eleven historical isolates of B. ovis collected from sheep between 2015 and 2021 in Italy and stored by the National Reference Centre for Brucellosis in Italy were used in the study (Supplementary Table 1). Cultures were grown on Modified Thayer-Martin agar at 37 °C ± 1 °C (5–10% CO₂) for 72 h. The species was confirmed using PCR, traditional biochemical testing, and serotyping, as previously described [13]⁠. Genomic DNA was extracted from 11 strains using the Maxwell 16 tissue DNA purification kit (Promega Corporation, Madison, WI) according to the manufacturer’s instructions.

Multi-Locus Variable-Number tandem repeat analysis (MLVA)

Isolates were genotyped using the MLVA-16 panel described by Le Flèche et al. (2006) (3). Loci were amplified in multiplex PCR with the published primer set as described in Garofolo et al., 2013 [14]⁠. Amplicons were separated by capillary electrophoresis on an ABI 3500 instrument with POP 7 polymer, and allele types were assigned using GeneMapper 4.1 (Applied Biosystems, Carlsbad, CA). MLVA-16 allelic profiles were analyzed using the goeBURST algorithm implemented in PHYLOViZ v2.0 [15]⁠. MLVA profiles of additional 55 B. ovis strains were obtained from https://microbesgenotyping.i2bc.paris-saclay.fr/databases/public (Supplementary Table 2).

Whole genome sequencing (WGS)

Genomic DNA was quantified using the Qubit DNA HS assay (Thermo Fisher Scientific Inc., Waltham, MA, USA). Sequencing libraries were prepared using the Nextera XT Library Preparation Kit (Illumina, San Diego, CA, USA) and sequenced using a NextSeq 500/550 Mid Output Reagent Cartridge v2 (300 cycles) according to the manufacturer’s instructions, generating 150 bp paired-end reads. Following demultiplexing and adapter removal, read quality was assessed using FastQC v0.11.5 [16]⁠. Reads were trimmed for quality with Trimmomatic v0.36 using the following quality parameters - Leading: 25; Trailing: 25; Slidingwindow: 20:25 [17]. ⁠. De novo assembly was performed using SPAdes v3.11.1 [18]⁠ with the --careful option. Assembly quality was assessed using QUAST v4.3 [19]⁠. The sequence reads were deposited in NCBI Bioproject PRJNA1244283 and the reads accession numbers are shown in Supplementary Table 1. Additional 19 genome assemblies from public repositories were included in the analysis and these contained strains collected in Argentina, Spain, New Zealand, Australia, USA and Croatia (Supplementary Table 3).

MLST and CgMLST analysis

Multilocus sequence typing (MLST) was performed using Ridom SeqSphere + software version 6.0.2, employing the nine-locus MLST (MLST-9) scheme available at the PubMLST database (https://pubmlst.org/brucella/, accessed May 18, 2023). Core genome MLST (cgMLST) profiles were subsequently assigned using the same software, utilizing a Brucella spp. cgMLST template comprising 1764 core genes [8]⁠. Minimum spanning tree (MST) was generated using Ridom SeqSphere + with default software settings. MSTs were constructed by pairwise comparison of cgMLST target genes, with missing values ignored during distance calculations.

SNP analysis and phylogenetic reconstruction

Single nucleotide polymorphism (SNP) identification was carried out using In Silico Genotyper (ISG) version 0.16.10-3.10 [20]⁠. The analysis employed default filtering parameters to exclude SNPs located within duplicated genomic regions. Quality parameters included minimum Phred quality score of 30 and a minimum allele frequency of 90% across all samples. The ISG pipeline utilized BWA-MEM version 0.7.12-r1039 [21]⁠ for sequence alignment and GATK version 3.9 for SNP calling [22]⁠, with the Brucella ovis ATCC 25,840 genome (GenBank accession numbers NC_009504.1 and NC_009505.1) as the reference.

Phylogenetic reconstruction was performed using IQ-TREE multicore version 1.6.9 [23]⁠⁠. A multiple sequence alignment of 30 clean unique variant sequences obtained from the ISG tool, each 1694 base pairs in length, was used as input. The optimal nucleotide substitution model was determined using ModelFinder implemented through the -m MFP option within IQ-TREE [24]⁠. The K3P + ASC model was selected as the best-fit model based on the Bayesian Information Criterion (BIC). Ultrafast bootstrap support values were generated using 1000 replicates Maximum-likelihood tree was midpoint-rooted and visualized in iTOL [25]⁠.

In silico genome characterization

In silico detection of virulence factors and antimicrobial resistance genes was performed using ABRicate version 1.0.1 (https://github.com/tseemann/abricate/), employing default settings and the Virulence Factor Database (VFDB, updated February 21, 2023, containing 2,597 sequences; NCBI, updated February 21, 2023, containing 5,386 sequences). Genome annotation of all 30 B. ovis genomes was conducted using Prokka [26]⁠, and GFF files were generated. Pangenome analysis was performed using Panaroo [27]⁠, and the resulting pangenome was visualized using Phandango [28]⁠.

Results

Multilocus Variable-Number Tandem Repeat Analysis (MLVA) of B. ovis Global Distribution

To investigate the global distribution of B. ovis, we analyzed MLVA profiles of publicly available strains from South America, North America, Europe, Oceania, and our Italian dataset (Fig. 1). All strains were located within a maximum distance of 3 MLVA alleles from their closest neighbor. Although genotypes generally segregated according to the country of origin, some exceptions were observed. Notably, French, Spanish, and Italian strains were found in several distantly related branches of the minimum spanning tree (MST). Furthermore, no clear cluster division based on the continent of B. ovis isolation was apparent. The largest distance among Italian isolates was 4 MLVA alleles, with their closest neighboring strains including B. ovis from Argentina and Australia.

Fig. 1.

Minimum spanning tree (MST) based on B. ovis MLVA-16 typing results. The tree was generated using 62 Italian (n = 11) and publicly available (n = 55) MLVA profiles. The nodes are colored according to the country of strain isolation

Multilocus Sequence Typing (MLST) and cgMLST Reveals Distinct B. ovis Lineages

In order to characterize Italian strains further, we sequenced their genomes and performed cgMLST and SNP analysis. Additional 19 genome assemblies from public repositories were included in the analysis and these contained strains collected in Argentina, Spain, New Zealand, Australia, USA and Croatia. The cgMLST analysis, utilizing core genome profiles of 30 B. ovis genomes (11 Italian and 19 publicly available), revealed distinct clustering patterns associated with sequence types and geographic origins (Fig. 2). MLST grouped majority of B. ovis strains into ST-13 and a novel ST-97 was assigned to two of the strains originating in Italy, based on a point mutation in trpE gene. ST-13 encompassed genomes from diverse countries, including Argentina, Australia, and the United States, indicating a broad global distribution. Within ST-13, allelic distances ranged from 0 to 144, while a maximum distance of 223 alleles was observed between any two genomes in the entire dataset. Conversely, ST-97 was exclusively identified in two Italian genomes, suggesting regional persistence or localized transmission. Isolates from other regions, such as Australia, Croatia, and New Zealand, formed separate clusters, with some exhibiting close genetic relationships despite geographic distance, implying potential intercontinental transmission or shared ancestry. The largest subset of strains originated from Argentina, with pairwise allelic distances ranging from 3 to 66 alleles. Notably, Italian B. ovis strains displayed greater genomic diversity, forming three distinct clusters separated by over 134 alleles, along with a singleton genome (2021-TE-319475-1-2) that was 134 alleles distant from its closest Italian counterpart (2018-AZ-4603-1-10). Overall, the Italian genomes were dissimilar to those from other countries, with the closest neighboring genomes originating from Spain, Croatia, and the USA.

Fig. 2.

Minimum spanning tree (MST) generated for 30 isolates of B. ovis using the gene-by-gene approach. MST was calculated by pairwise comparison of 2076 target genes with missing values ignored. Nodes correspond to unique profiles and are colored according to the country of strain isolation and the branches correspond to the number of discriminating loci. Strains grouped by MLST profiles into ST-13 and ST-97 are highlighted in gray and blue, respectively

Single Nucleotide Polymorphism (SNP) Analysis and Genomic Characterization of B. ovis Strains

Phylogenetic analysis, based on 1694 unique SNPs, revealed distinct clustering patterns among the Italian B. ovis strains (Fig. 3). Three genetically separate Italian clusters were observed, with pairwise SNP differences within the clusters ranging from 2 to 74. Two ST-97 strains formed the most basal branch, and were separated from one another by 18 SNPs, while strain 2021-TE-319475-1-2 occupied a unique branch. ST-97 was separated from the strains of ST-13 by a minimum of 592 SNPs, while a maximum distance observed within ST-13 was 364 SNPs. Five additional Italian strains clustered together while three other Italian strains grouped with Spanish, Croatian, and US strains.

Fig. 3.

Global phylogeny of B. ovis. The ML tree was generated from an alignment of 1694 unique SNPs from 30 B. ovis WGS sequences. The tree is mid-point rooted, and branches with bootstrap values between 0.95 and 1 are marked with a star. Metadata, including MLST profile, country of strain isolation, and region of isolation (Italian strains only) are shown on the right. Virulence factors detected are shown as grey boxes

Geographic analysis of the Italian strains revealed regional segregation. The five-strain cluster originated in Sicily, while the three-strain cluster was associated with Lazio and the Lazio-Abruzzo border. The two ST-97 strains, however, were isolated from geographically distant regions: Piedmont (northern Italy) and Abruzzo (Adriatic coast) (Supplementary Fig. 1).

In silico screening identified 41 Brucella virulence-associated genes (Fig. 3). All ST-13 strains exhibited a uniform virulence factor profile. Notably, the btpA TIR-domain effector gene was absent in both ST-97 strains. Pangenome analysis further revealed a 34-gene deletion in the ST-97 strains compared to the ST-13 strains (Supplementary Fig. 2). This deleted region, in addition to btpA, contained genes encoding a site-specific prophage integrase, an ImmA/IrrE family metalloendopeptidase, and AlpA, suggesting a possible prophage related origin.

No antimicrobial resistance genes were detected in the strains analysed in our study.

Discussion

This study provides valuable insights into the genetic diversity and distribution of B. ovis strains, particularly in Italy, using a combination of MLVA, cgMLST, and SNP analyses. Our findings reveal a complex population structure with evidence of both regional clustering and intercontinental transmission. MLVA analysis provided a broad overview of B. ovis global distribution, confirming previous observations of limited clustering by continent. It also highlighted the presence of shared genotypes across geographically distant regions, suggesting historical dissemination and underscoring the limitations of MLVA for high-resolution discrimination. However, the observation of shared genotypes among French, Spanish, and Italian strains demonstrated likely epidemiological links within Europe.

Our results are concordant with published MLVA work showing B. ovis structured into two main clusters with limited continent-level segregation. Notably, European profiles (France, Spain) have been placed adjacent to South American isolates in MSTs, consistent with historical livestock movements [10]⁠. The occurrence of shared French–Spanish–Italian MLVA types is readily explained by transmission routes (predominantly venereal, with documented semen shedding) and frequent cross-border movement of breeding rams within Europe [3, 29]⁠. In addition, VNTR homoplasy limits the phylogenetic specificity of identical MLVA profiles, reinforcing the need for genome-wide markers [29]⁠.

Subsequent WGS-based analysis, offering higher discriminatory power, revealed two dominant STs, the globally distributed ST-13 and the novel, Italy-specific ST-97. The wide range of cgMLST allelic distances within ST-13 likely reflects its long evolutionary history, while the restricted presence of ST-97 in Italy suggests either recent emergence or limited dispersal. Finally, SNP-based phylogenetic analysis corroborated the cgMLST findings, confirming the unique position of the ST-97 strains and revealing distinct Italian clusters with evidence of geographic segregation.

Whole-genome phylogenies consistently place B. ovis as a compact species-level lineage within the genus, reflecting comparatively low internal diversity across available genomes [30]⁠. Genomic analyses also document extensive pseudogenization in B. ovis, a hallmark of host restriction that is concordant with shallow intra-species divergence on WGS trees [5]⁠. PMC Where sampling has been dense, country-level groupings are evident: the largest study that incorporated publicly available B. ovis genomes reported that Argentinian isolates clustered together and were most closely related to genomes from New Zealand and Australia, indicating long-range affinities rather than strict continent-specific segregation [6]⁠. Notably, that analysis also emphasized how few B. ovis genomes were publicly available at the time, which helps explain why no Europe-exclusive lineage has been defined in published WGS datasets to date [6]⁠. Taken together, current whole-genome trees support a picture of a small number of deep lineages with membership spanning multiple regions, alongside localized clusters that emerge in areas with intensive sampling [30, 31]⁠.

This genomic diversity observed among Italian B. ovis strains, characterized by distinct clusters and regional segregation, might be attributed to a combination of factors, including prolonged endemic circulation and multiple introduction events. This pattern is similar to the evolutionary dynamics observed in B. melitensis within the same geographic area, where high genetic diversity has been attributed to multiple introduction events, potentially dating back to Neolithic times, and sustained by historical trade routes, particularly those connecting the Italian peninsula with other Mediterranean regions [20]⁠⁠. The observed connections between some Italian B. ovis strains and those from other Mediterranean countries like Spain and Croatia may also reflect historical trade and the movement of livestock across the region. In particular, connections between Italian genomes and those from Spain and Croatia can be interpreted in light of documented livestock movements across the Mediterranean and the Adriatic [32]⁠. Historical records show sustained transfer of Spanish Merino rams into southern Italy from the fifteenth century onward, underpinning Italian Merino-derived breeds and establishing long-standing breeding links between the two countries [32]⁠. In the Single Market era, intra-Community trade in ovine and caprine animals under Council Directive 91/68/EEC has enabled high volumes of live-animal movements within the EU, with Spain-Italy exchanges among the most active (https://eur-lex.europa.eu/eli/dir/1991/68/oj/eng, accessed 26-08-2025). EUR-Lex European Parliament Consistent with these pathways, recent trade statistics report large annual imports of live sheep into Italy, including from Spain, and ongoing bilateral flows with Croatia (https://wits.worldbank.org/trade/comtrade/en/country/ITA/year/2023/tradeflow/Imports/partner/ALL/product/010410, accessed 26-08-2025). Given the venereal transmission of B. ovis and documented semen shedding in rams, such movements provide plausible routes for the cross-border proximity observed in our phylogeny [33]⁠.

This underscores the complex influence of historical events, trade, and regional evolution on shaping Brucella population structure in the Mediterranean. The high level of diversity of B. ovis in Italy has also implications for disease management. The presence of multiple, distinct genetic clusters suggests that control strategies may need to be tailored to specific regions within Italy to be most effective [34]⁠. At flock level, systematic serological screening of rams with removal of reactors, pre-breeding examinations, and sourcing from accredited or repeatedly tested flocks have been described as effective control measures [7, 35]⁠. These measures target the main transmission route because infected rams shed B. ovis in semen [35]⁠.

Movement-related measures include quarantine and repeat testing before transfers between holdings or regions, and certification of semen donors and artificial insemination centres with recent negative tests, as set out in international standards [7]. Voluntary accreditation programmes for ram breeders are also reported and can support disease control where implemented [7]⁠.

The distinct genomic features of the Italian strains, particularly the ST-97 lineage, should be considered within the broader context of B. ovis pathogenesis. Unlike smooth species such as B. melitensis, the virulence of B. ovis is shaped by a unique evolutionary trajectory characterized by genome degradation, which narrows its host and tissue tropism primarily to the ovine male genital tract. A key difference is its rough lipopolysaccharide (R-LPS), which lacks the O-polysaccharide and fundamentally alters its interaction with the host’s innate immune system compared to smooth strains [5, 36]⁠. Furthermore, the virulence of B. ovis is modulated by a distinct profile of outer membrane proteins (OMPs) and differences in the regulation of essential virulence systems. For instance, while the Type IV Secretion System (T4SS) is critical for intracellular survival in all pathogenic Brucella spp., its expression in B. ovis is regulated differently, notably lacking the induction under acidic conditions seen in B. melitensis [37, 38]⁠. These intrinsic characteristics contribute to its unique pathophysiology.

The absence of the btpA gene and the presence of a 34-gene deletion, including putative prophage-related genes, in the ST-97 strains compared to ST-13 strains is an interesting finding. In other Brucella spp., including B. melitensis and B. abortus, btpA encodes a TIR domain-containing protein with NAD+-consuming activity. BtpA, a T4SS effector protein (referred to also as TcpB and Btp1), along with BtpB, modulates dendritic cell activation and contributes to virulence in B. abortus [39, 40]⁠. While the precise mechanism of BtpA remains unclear, it was shown to interact with TIRAP and MyD88, and may modulate microtubule dynamics and induce the unfolded protein response [41]⁠⁠. Thus, the btpA deletion in ST-97 B. ovis could suggest potentially different host interaction and virulence mechanisms compared to ST-13 strains. The presence of prophage-related genes in the deleted region suggests a possible role of phage activity in the evolution of these strains and might have contributed to the observed genomic differences. While absence of the btpA gene in ST-97 strains could suggest a potential alteration in the host-pathogen interaction pathway, without associated clinical, pathological, and host data (e.g., breed, age, disease severity), it is not possible to correlate this genotype with a specific phenotype. Further investigation is needed to determine the functional significance of these deleted genes and their impact on B. ovis biology.

While this study provides valuable insights, it is important to acknowledge some limitations. The sample size, particularly for certain geographic regions, could be expanded to provide a more comprehensive global picture of B. ovis diversity. Furthermore, while MLVA, cgMLST, and SNP analyses provide high-resolution genotyping, functional studies are needed to validate the observed genetic differences and their phenotypic consequences.

Conclusion

This study highlights the complex evolutionary dynamics of B. ovis and reveals significant genetic diversity, particularly within Italian strains. The identification of the novel ST-97 lineage, characterized by specific gene deletions, warrants further investigation into its potential impact on virulence. Given the economic impact of ovine brucellosis, especially in high-density sheep farming regions, these findings underscore the need for enhanced surveillance and the development of targeted control strategies.

Authors’ contributions

TR and AJ conceived and supervised the study. KZ, AA, CV and MA performed laboratory work and data curation. AJ conducted genome analyses. GG, MT, and FDM contributed to data interpretation and manuscript drafting. NDA was responsible for administration and funding. AJ, TR and CV wrote the manuscript with input from all authors. All authors read and approved the final manuscript.

Funding

The work was partially funded by the Italian Ministry of Health, Project grant n. IZS AM 06/23 RC.

Data availability

The raw sequence reads generated and analyzed during this study are available in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1244283. Accession numbers for individual strains are provided in Supplementary Table 1.

Declarations

Ethics approval and consent to participate

Not applicable. This study did not involve experiments on live animals or human participants. The Brucella ovis strains analyzed were historical isolates obtained from diagnostic activities and stored at the National and WOAH Reference Laboratory for Brucellosis in Italy.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.