Brucellosis remains endemic in Tunisia, causing abortions in small ruminants, and represents a public health threat through occupational exposure and the consumption of contaminated animal products. The aims of this study are to assess the antibiotic susceptibility of two Brucella melitensis isolates (TATA and SBZ) from aborted sheep, to analyze their genomes using hybrid whole-genome sequencing, and to investigate their antimicrobial resistance (AMR), potential virulence factors (VFs), and phylogenetic relationships. Both isolates were phenotypically confirmed to be susceptible to doxycycline, gentamicin, rifampicin, streptomycin, and trimethoprim–sulfamethoxazole, and no corresponding classical AMR genes were identified. However, several potential AMR-related genes (mprF, bepCDEFG, qacG, and adeF) and a mutation in the parC gene were detected. The analysis of the genotypes revealed 74 potential virulence genes, primarily involved in lipopolysaccharide synthesis and type IV secretion systems. Genomic comparison showed over 99% nucleotide identity between the Tunisian strains, B. melitensis bv. 1 16M and B. melitensis bv. 3 Ether. Five gene clusters, including three hypothetical proteins with 100% identity, were detected exclusively in the TATA and SBZ strains. Additionally, two unique gene clusters were identified in SBZ: a rhodocoxin reductase and another hypothetical protein. Both isolates were assigned to sequence types ST11 and ST89. Core-genome-based phylogenetic analysis clustered both strains with biovar 3 and ordered the Tunisian strains into two distinct groups: TATA within Tunisian Cluster 1 is closely related to strains from Egypt and Italy, while SBZ near MST Cluster 4 is more related to isolates from Austria and two outliers from Italy and Tunisia. This study provides the first genomic characterization of B. melitensis from aborted sheep in Tunisia and offers valuable insights into AMR, virulence, and phylogenetic distribution.

Brucellosis is an endemic zoonotic disease in Africa and Tunisia, severely affecting both human and animal health, particularly ruminants. In livestock, brucellosis causes reproductive failure, including abortions, leading to substantial economic losses. Despite surveillance and vaccination efforts in Tunisia, brucellosis remains widespread. This study is aimed at assessing the presence of Brucella infection in aborted animals (sheep, goats, and cattle) using an IS711 ‐based real‐time PCR assay, determining the circulating species ( Brucella melitensis and Brucella abortus ) by differential qPCR, and identifying the most suitable sample type for detection between 2020 and 2022. Samples including vaginal swabs, blood, placenta, and fetal organs (liver, spleen, stomach, and cotyledons) were collected from farms selected based on abortion reports from farm owners. A total of 272 samples were analyzed, of which 24.26% tested positive for Brucella spp., with 25.71% in sheep, 13.33% in goats, and 8.33% in cattle. The detection rate of Brucella spp. was estimated at 24% (60/250) in aborted females and 27.27% (6/22) in aborted fetal materials. Vaginal swabs exhibited a notable positivity rate of 31.13%, 28.57% in organs and 5.71% in blood. B. melitensis and B. abortus were detected in 46.96% and 19.69% of positive samples, respectively. Interestingly, one sample showed a coinfection with both species; however, neither B. abortus nor B. melitensis was detected in 21 positive samples. This study highlights the presence of Brucella spp. in aborted ruminants and the circulation of B. melitensis and B. abortus , underlining the importance of molecular tools for the reliable diagnosis of brucellosis, and their usefulness in mitigating the spread of this infection on farms by applying appropriate control measures.