Snapshot of Mycobacterium bovis and Mycobacterium caprae Infections in Livestock in an Area with a Low Incidence of Bovine Tuberculosis

Zoonotic bovine tuberculosis (bTB), caused by Mycobacterium bovis and, more rarely, by Mycobacterium caprae, is an important disease of livestock and of public health concern (1). A low incidence of bTB has been observed until now in Portugal, where a comprehensive eradication scheme has been implemented. Epidemiological surveys carried out in several countries suggest that test-and-slaughter policies may reduce strain diversity, favoring clonal expansion as a result of a bovine population bottleneck (2, 6). A remarkable exception to this observation was the first epidemiological analysis carried out in Portugal (3), which highlighted the apparent high genotype diversity of Mycobacterium bovis, possibly due to the low prevalence rates and the absence of major epidemics favoring ongoing transmission of the same strain(s). In the present work, we reassess and update the molecular epidemiology of bTB in Portugal. The previous analysis, concerning the period 2002 to 2007 (3), was extended to 2009 by including an additional 183 M. bovis and 10 Mycobacterium caprae isolates from cattle, goats, and sheep. Spoligotyping, based on the direct repeat (DR) region (5), identified 30 M. bovis patterns, including 11 profiles previously unrecognized in Portugal and seven new profiles that were deposited in the international database (www.mbovis.org). Spoligotype SB0157 remains the single M. caprae pattern acknowledged in the territory. The epidemiological significance of the most relevant genotypes in relation to the temporal and geographical distribution was investigated globally for the period 2002 to 2009 (results from this study and from reference 3). Consistent with previous findings, the high prevalence of SB0121 (25%) and SB0119 (13%) is confirmed. Remarkably, SB0119 and seven other profiles identified in Portugal differ from SB0121 in the absence of a single spacer (Table ​(Table1). 1). Altogether, their cumulative prevalence in cattle is superior to 53%. Furthermore, they have also been detected in wildlife (3). Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis based on eight previously validated loci (4) distinguished 90 SB0121-related strains into 50 closely associated allelic profiles (data not shown), further suggesting their genetic relatedness. Taking this into consideration and the trend of the DR region to evolve primarily by the loss of single or multiple contiguous direct variable repeats (DVRs) (7), our observations suggest the presence of a clonal complex. Supporting this hypothesis is also the recent detection of SB0121-like strains in the national territory in comparison to SB0121, the sole genotype identified until 2004. To our knowledge, three SB0121-related spoligotypes, SB1090, SB1093, and SB1095, have been reported only in Portugal. Geographical distribution analysis also shows that SB0121 and SB0119 have remarkably expanded throughout the territory, being isolated in roughly all bTB-affected districts, while most SB0121-related genotypes are geographically localized (Fig. ​(Fig.11). FIG. 1. Geographical distribution across the districts of the six bTB-affected regions of mainland Portugal during the period 2002 to 2009 (joint results from this study and from reference 3) of M. bovis spoligotype SB0121 (•) and of strains with spoligotyping . TABLE 1. Patterns, hosts, prevalence, and geographical distribution of M. bovis and M. caprae spoligotypes isolated in Portugal from 2007 to 2009 (this study) and 2002 to 2009 (joint results from this study and from reference 3) Our results support the notion that a country tends to have a dominant and locally evolved clone. Moreover, clonally related strains may emerge, exhibiting biogeographical specificities, possibly in response to barriers for expansion or adaptation to new ecological niches and new host species. Further studies are needed to improve our understanding of the epidemiological significance of geographically important M. bovis strains which may define specific lineages with increased virulence and the ability to escape tuberculin test and, consequently, slaughter of their hosts.