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Spoligotyping and exact tandem repeat (ETR) analysis ofAs a result, the official TB-free status was achieved starting from 1970 in some Italian areas such as the Autonomous Region of Trentino Alto Adige, Friuli Venezia Giulia Region (northeast Italy), and some provinces of northern and central Italy. In the rest of the national territory, TB still persists with different prevalence rates: 6.6% in Sicilia, 0.77% in Puglia, and 0.59% in Lazio and Campania. In northern Italy, where about the 70% of the cattle population is reared, TB is still present in the Piedmont (0.4%), Lombardy (0.15%), Veneto (0.1%), and Emilia-Romagna (0.08%) regions. In order to accelerate the achievement of a TB-free status, the authorities in these areas are implementing specific TB control measures, taking into account local situations and problems such as mean distance among herds, trade of animals from other territories, and common pasturing.In this respect, the origin of TB infection often remains undetermined despite its importance. Molecular typing of isolates has become a valuable tool in the study of M. bovis epidemiology, allowing investigators to better identify the sources of infection and achieve a wider knowledge of TB transmission routes. A genetic profile database collection of M. bovis isolates may help to confirm or reject hypotheses outlined by traditional epidemiological investigations.Genotyping of M. bovis probably lacks sufficiently informative methods. IS6110 restriction fragment length polymorphism (RFLP) typing has been considered a gold standard method for differentiation of M. tuberculosis strains for a long time; this has provided only limited discrimination among M. bovis populations where the majority of the isolates harbor only one or few IS copies (11). PCR-based spoligotyping (16) has been widely used to genotype M. bovis isolates (11); it is highly reproducible and rapid and represents the first universally recognized typing system for M. bovis populations. However, studies performed on M. bovis isolates in Northern Ireland (32,33), France (12), Australia, Canada, the Republic of Ireland, and Iran (7) showed a limited discrimination power of this method.Over the last 10 years several PCR-based genotyping methods have become available for rapid molecular epidemiology

We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda, Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2) clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general, four or more copies of the insertion sequence IS6110, in contrast to the majority of M. bovis strains isolated from cattle, which are thought to carry only one or a few copies.

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.