Bacillus (B.) anthracis, the causal agent of anthrax, is effectively controlled by the Sterne live spore vaccine (34F2) in animals. However, live spore vaccines are not suitable for simultaneous vaccination and antibiotic treatment of animals being at risk of infection in an outbreak situation. Non-living vaccines could close this gap. In this study a combination of recombinant protective antigen and recombinant Bacillus collagen-like antigen (rBclA) with or without formalin inactivated spores (FIS), targeted at raising an immune response against both the toxins and the spore of B. anthracis, was tested for immunogenicity and protectiveness in goats. Two groups of goats received from local farmers of the Kars region of Turkey were immunized thrice in three weeks intervals and challenged together with non-vaccinated controls with virulent B. anthracis, four weeks after last immunization. In spite of low or none measurable toxin neutralizing antibodies and a surprisingly low immune response to the rBclA, 80% of the goats receiving the complete vaccine were protected against a lethal challenge. Moreover, the course of antibody responses indicates that a two-step vaccination schedule could be sufficient for protection. The combination of recombinant protein antigens and FIS induces a protective immune response in goats. The non-living nature of this vaccine would allow for a concomitant antibiotic treatment and vaccination procedure. Further studies should clarify how this vaccine candidate performs in a post infection scenario controlled by antibiotics.

Brucellosis represents a significant global health concern that is endemic in many regions of the world, especially in Maghreb (Tunisia, Morocco and Algeria). In Tunisia the diagnosis of human brucellosis is primarily based on serological tests and cultivation of the causative pathogen, without the knowledge of the underlying Brucella species or biovar. In addition, the scarcity of laboratories with adequate biosecurity measures to handle suspected specimens constitutes a significant challenge. Furthermore, the absence of full genome data limits our understanding of the genetic diversity of Brucella in Tunisia in comparison to the strains circulating in the North African region and the world. In the present study, a total of 36 bacterial isolates derived from human patients diagnosed with brucellosis in Tunisia were subjected to examination. Real-time PCR confirmed all isolates as B. melitensis. In the subsequent core genome-based MLST analysis (cgMLST) based on 2706 target genes, the isolates formed two separate but closely related clusters with a distance of 140 alleles. The intra-cluster diversity was one to six alleles. In the larger geographical context and in comparison to almost 1000 other Brucella genomes, the isolates showed the highest genetic relationship to B. melitensis isolates from Italy and Egypt with distances of 130 and 150 alleles, respectively. All the isolates were most similar to the biovar 3 genotype. Markedly, strains from a reported brucellosis outbreak in Austria were grouped closely (26 and 27 alleles, respectively) together with strains from Tunisia, suggesting that this country may represent their geographical origin. This research represents a significant advancement in our understanding of B. melitensis strains circulating in the Maghreb region, as it is the first study to elucidate the molecular characterization of strains isolated from humans in Tunisia. The cgMLST analysis of the strains provided information on the regional distribution of the strains and the association with neighboring countries and significant outbreaks in the region. The data will form the basis of a future reference framework for strains circulating in the Mediterranean region.

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 represents a significant global health concern that is endemic in many regions of the world. It is most prevalent in the Mediterranean region, especially in Maghreb (Tunisia, Morocco and Algeria). In Tunisia the diagnosis of human brucellosis is primarily based on serological tests and cultivation of the causative pathogen, without the knowledge of the underlying Brucella species or biovar. In addition, the scarcity of laboratories with adequate biosecurity measures to handle suspected specimen constitutes a significant challenge. Furthermore, the absence of full genome data limits our understanding of the genetic diversity of Brucella in Tunisia in comparison to the strains circulating in the North African region and the world. In the present study, a total of 36 bacterial isolates derived from human patients diagnosed with brucellosis in Tunisia were subjected to examination. Real-time PCR confirmed all isolates as B. melitensis. In subsequent core genome-based MLST analysis (cgMLST) based on 2706 target genes, the isolates formed two separate but closely related clusters with a distance of 140 alleles. Intra-cluster diversity was 1 to 6 alleles. In the larger geographical context and in comparison to almost 1000 other Brucella genomes, the isolates showed the highest genetic relationship to B. melitensis isolates from Italy and Egypt with distances of 130 and 150 alleles, respectively. All isolates were most similar to biovar 3 genotype. Markedly, strains from a reported brucellosis outbreak in Austria were grouped closely (26 and 27 alleles respectively) together with strains from Tunisia, suggesting that this country may represent their geographical origin. This research represents a significant advancement in our understanding of B. melitensis strains circulating in the Maghreb region, as it is the first study to elucidate the molecular characterization of strains isolated from humans in Tunisia. The cgMLST analysis of the strains provided information on the regional distribution of the strains and the association with neighboring countries and significant outbreaks in the region. The data will form the basis of a future reference framework for strains circulating in the Mediterranean region.