Molecular epidemiology of Mycobacterium bovis and Mycobacterium caprae in Spain
PhD Thesis defense by Sabrina Rodríguez Campos at the Faculty of Veterinary Medicine of the Complutense University of Madrid
March 9th, 2012
The eradication of bovine tuberculosis is indispensable due to its negative impact on animal health and welfare, and national as well as international trade. Moreover, the infection threatens endangered animal species and humans. Mycobacterium (M.) bovis and M. caprae are the main causative agents of animal tuberculosis in Spain. Although the cattle herd prevalence has been drastically reduced in the last decade (1.15%, 2010), bovine tuberculosis remains a problem. The use of typing techniques contributes to a better control and understanding of the molecular epidemiology of the disease and thus backs up the national eradication programme. The doctoral thesis is structured in four independent chapters. In the first three chapters, the two molecular standard techniques direct variable spacer oligonucleotide typing (DVR-spoligotyping) and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) were applied to study the strain diversity, geographical distribution as well as the implication of wildlife in Spain.
Chapter I contains large population surveys of M. bovis and M. caprae in Spain by DVR-spoligotyping. The 6215 M. bovis isolates analysed yielded 252 different spoligotypes showing a high spoligotype diversity similar to other countries in continental Europe, but in contrast to the low diversity observed in the United Kingdom and the Republic of Ireland. The 791 M. caprae isolates clustered in only 15 spoligotypes. The most prevalent spoligotypes were M. bovis SB0121 and M. caprae SB0157. Both pathogens were widespread in Spain, with the most frequent strains being present in domestic animals and wildlife. Interestingly, an increase of cases of bovine tuberculosis due to M. caprae has been observed during the last years. These large population surveys hinted at two spoligotype signatures: the absence of spacer 21 from M. bovis spoligotyping profiles in 67% of the population, and the absence of spacers 30 to 33 from M. caprae spoligotyping patterns contrasting with the strains described in Central and Eastern Europe.
The MIRU-VNTR typing of three sets of selected isolates of M. bovis, which revealed an expansion of closely related clonal groups of the prevalent spoligotypes SB0121 and SB0295, are comprised in chapter II. The different sets included isolates with the spoligotype SB0121, a selection from a geographically limited area to track an outbreak on two alpaca farms, and a selection of isolates from bullfighting cattle and wildlife sharing their habitat. The fact that MIRU-VNTR types seem to evolve quickly in Spain might lead to difficulties with the identification of possible outbreak sources as experienced in the case of the first alpaca outbreak in Spain; hence, a less stringent comparison of MIRU-VNTR types might be employed for specific spoligotypes or geographical settings.
Chapter III describes the national database of animal tuberculosis, mycoDB.es, which has been designed as a tool within the Spanish programme of eradication of bovine tuberculosis. The access to the database is restricted to Official Veterinary Services and currently offers 17273 spoligotyping data out of which 410 are additionally MIRU-VNTR typed.
Chapter IV of this dissertation addresses the phylogeny of Spanish M. bovis isolates. For this purpose DNA microarrays, whole genome sequencing and single nucleotide polymorphism (SNP) typing were applied. A new clonal complex of M. bovis was defined based on the absence of spacer 21 from the spoligotyping pattern and a synonymous SNP in guaA. This clonal complex, named European 2 (Eu2), is prevalent in Spain and Portugal, less frequent in France and Italy and absent from the British Isles. The screening of Spanish M. bovis isolates with spoligotype signatures hinting at the African 2 (Af2) or the European 1 (Eu1) clonal complex revealed that no Af2 strains were present in the Spanish populations and that the proportion of Eu1 strains is estimated to be low.
In conclusion, the findings of this PhD thesis improved not only our understanding of the molecular epidemiology of M. bovis and M. caprae at national level and in the European context, but also the exploitation of molecular data and thus, contributed to a progress of the national eradication campaign.
