Bovine tuberculosis (bTB) is an emerging disease among wild animals in many parts of the world. Wildlife reservoir hosts may thus represent a potential source of infection for livestock and humans. We investigated the role played by the Sicilian black pig, an autochthonous free- or semi-free-ranging domestic pig breed, as a potential source of bTB infection in an area where bTB prevalence in cattle is high. We initially performed a preliminary field study to assess the occurrence of bTB in such animals. We sampled 119 pigs at abattoir and found 6.7% and 3.4% of them to be affected by gross tuberculous-like lesions (TBL) and Mycobacterium bovis culture positive, respectively. We then proceeded to investigate the dissemination and characteristics of lesions in a second field study performed on 100 animals sampled from infected herds. Here, tissues collected at the abattoir were examined macroscopically, microscopically, and by culture tests. Most pigs with TBL showed generalized lesions in both gross and histological examinations (53% and 65.5%, respectively). Head lymph nodes were the most frequently affected in both localized and generalized TB cases observed macroscopically and microscopically. M. bovis was the most frequently isolated etiologic agent. The molecular characterization of isolates from both field studies by spoligotyping and analysis of 12 mycobacterial interspersed repetitive-unit–variable number tandem repeat (MIRU-VNTR) loci, followed by their comparison to isolates of cattle origin, suggested a potential transmission of mycobacteria from domestic animals to black pigs and vice versa. Our findings, along with ethological, ecological, and management considerations, suggest that the black pig might act as a bTB reservoir in the ecosystem under study. However, additional studies will be necessary to establish the true epidemiological significance of the Sicilian black pig.

Brucellosis is a serious problem in Sicily. Brucella melitensis was identified as the species most frequently isolated in humans in Italy. No data, however, are available about the molecular epidemiological characterization of Brucella isolates from humans. We have conducted this study to molecularly characterize clinical isolates of Brucella spp. and to evaluate their antimicrobial susceptibilities. Twenty Brucella isolates were studied. Differential growth characteristics and DNA polymorphisms such as the restriction patterns of the PCR-amplified omp2a and omp2b genes, rpoB nucleotide sequencing, and multiple-locus variable-number tandem repeat analysis of 16 loci (MLVA-16) were used to characterize the strains. In vitro antibiotic susceptibility was determined by the E-test method on two different agar media, and the results were compared. All isolates were identified as B. melitensis biovar 3. rpoB nucleotide sequence analysis allowed the identification of two different genotypes of B. melitensis biovar 3. On the other hand, the MLVA-16 typing assay recognized 17 distinct genotypes. All isolates were sensitive to all tested antibiotics (rifampin, doxycycline, ciprofloxacin, ceftriaxone, and trimethoprim-sulfamethoxazole), and the Mueller-Hinton agar plate is recommended for antibiotic susceptibility testing by the E-test method. Our findings identify B. melitensis biovar 3 as the etiological agent isolated in Sicily and encourage the use of both molecular methods, and in particular of the MLVA-16 assay, in epidemiological trace-back analysis. This study represents the first epidemiological data from molecular typing of Brucella strains circulating in Italy and, in particular, in eastern Sicily.

Rifampin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens. Its bactericidal activity is due to its ability to bind to the β subunit of the DNA-dependent RNA polymerase encoded by therpoBgene. Mutations of therpoBgene have been characterized in rifampin-resistant (Rif^r) strains ofEscherichia coliandMycobacterium tuberculosis.The genetic bases of Rif^rinBrucellaspp. are still unknown. In the present study, the nucleotide sequences of therpoBgene of the Rif^rvaccine strainBrucella abortusRB51 and of 20 Rif^rclones derived in our laboratory from twoBrucella melitensisisolates were determined. These sequences were then compared to those of the respective rifampin-susceptible (Rif^s) parental strains and to the publishedB. melitensisstrain 16M. All Rif^rstrains carried one or more missense mutations mapping in two regions of therpoBgene. These two “hot” regions were investigated in eight additional Rif^rBrucellalaboratory mutants and in 20 reference Rif^sBrucellastrains.rpoBmutations were found in all Rif^rmutants. In contrast, no missense mutations were found in any analyzed Rif^sstrains. Our results represent the first from a study of the molecular characterization ofrpoBmutations in resistantBrucellastrains and provide an additional proof of the association of specificrpoBmutations with the development of the Rif^rphenotype in prokaryotes. In addition, because of the relationship between Rif^rand the attenuation of virulence inBrucellaspp., studies of virulence in these mutants may provide useful information about the genetic basis of pathogenesis inBrucella.