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Abstract Background Bovine tuberculosis (BTB), caused by Mycobacterium bovis, has remained a major source of concern to public health officials in Zambia. Previous investigations have used traditional epidemiological methods that are unable to identify the causative agent and from which dynamics of disease dispersion is difficult to discern. The objective of this study was to isolate, characterize and determine the genetic diversity and relatedness of M. bovis from major cattle rearing districts in Zambia by spoligotyping. A total of 695 carcasses were examined and 98 tissues had gross post-mortem lesions compatible with BTB. Results Forty-two out of the ninety-eight suspected tissues examined had culture properties characteristic of mycobacteria from which 31 isolates yielded interpretable spoligotypes. This technique showed good discriminatory power (HGDI = 0.98), revealing 10 different spoligotype patterns. Twenty-seven isolates belonged to one cluster with more than 95% similarity and inside the cluster, one predominant spoligotype was found in 20 (64.5%) of the isolates tested. The highest number of spoligotypes was observed among samples from Namwala district. Spoligotypes from 26 (83.9%) of the isolates belonged to five spoligotypes that have been reported before while the remaining 5 (16.1%) isolates had unique spoligotypes that are being reported for the first time; these have been assigned numbers SB1763 to SB1767. Five of the 6 districts had the predominant spoligotype (SB0120). Conclusion The study has described the dispersion patterns of M. bovis in Zambian cattle for the first time and has identified 5 spoligotype patterns specific to Zambia. The observation of an overlap in the spoligotype pattern SB0120 in 5 of the 6 districts suggests the probability of sharing a common source of infection.

Abstract Background The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database. Results The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network. Conclusion Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.