In order to assess the presence of tuberculosis in Pleistocene bison and the origin of tuberculosis in North America, 2 separate DNA extractions were performed by 2 separate laboratories on samples from the metacarpal of an extinct long-horned bison that was radiocarbon dated at 17,870+/-230 years before present and that had pathological changes suggestive of tuberculosis. Polymerase chain reaction amplification isolated fragments of tuberculosis DNA, which were sequenced, and on which spoligotyping was also performed to help determine its relationship to the various members of the Mycobacterium tuberculosis complex. Extensive precautions against contamination with modern M. tuberculosis complex DNA were employed, including analysis of paleontologic and modern specimens in 2 geographically separate laboratories.

The lack of a convenient high-resolution strain-typing method has hampered the application of molecular epidemiology to the surveillance of bacteria of the Mycobacterium tuberculosis complex, particularly the monitoring of strains of Mycobacterium bovis. With the recent availability of genome sequences for strains of the M. tuberculosis complex, novel PCR-based M. tuberculosis-typing methods have been developed, which target the variable-number tandem repeats (VNTRs) of minisatellite-like mycobacterial interspersed repetitive units (MIRUs), or exact tandem repeats (ETRs). This paper describes the identification of seven VNTR loci in M. tuberculosis H37Rv, the copy number of which varies in other strains of the M. tuberculosis complex. Six of these VNTRs were applied to a panel of 100 different M. bovis isolates, and their discrimination and correlation with spoligotyping and an established set of ETRs were assessed. The number of alleles varied from three to seven at the novel VNTR loci, which differed markedly in their discrimination index. There was positive correlation between spoligotyping, ETR-and VNTR-typing. VNTR-PCR discriminates well between M. bovis strains. Thirty-three allele profiles were identified by the novel VNTRs, 22 for the ETRs and 29 for spoligotyping. When VNTR-and ETR-typing results were combined, a total of 51 different profiles were identified. Digital nomenclature and databasing were intuitive. VNTRs were located both in intergenic regions and annotated ORFs, including PPE (novel glycine-asparigine-rich) proteins, a proposed source of antigenic variation, where VNTRs potentially code repeating amino acid motifs. VNTR-PCR is a valuable tool for strain typing and for the study of the global molecular epidemiology of the M. tuberculosis complex. The novel VNTR targets identified in this study should additionally increase the power of this approach.

Mycobacterium bovis, the causative agent of tuberculosis (TB) in farmed livestock such as cattle and deer, continues to affect detrimentally farming and agricultural economies of many countries, resulting from imposition of trade restrictions and the costs of implementing eradication programs (18). M. bovis is also a recognized zoonotic pathogen that infects many people, particularly in the developing world (35). It also infects a broad range of feral and wildlife animals, such as badgers, deer, goats, and possums; some of these species are reservoirs of infection for farmed livestock (19). An important component in bovine TB eradication programs is the application of epidemiological "traceback." By developing a better understanding of the source(s) and mode(s) of TB transmission in field outbreaks, more-effective control measures can be implemented (7,17).The advent of molecular typing techniques has greatly improved the epidemiological knowledge that can be gained from studying TB outbreaks (1,17,20). The three principal M. bovis strain typing techniques described to date have been restriction enzyme analysis (REA) (5), restriction fragment length polymorphism (RFLP) analysis (26), and spoligotyping (11,13,22). Standard operating procedures for both REA and RFLP typing of M. bovis have been devised and are generally accepted as providing good levels of differentiation (5,6,26,34). However, there are practical processing and analysis limitations in both REA and RFLP analysis, requiring both skilled personnel and image analysis software. Spoligotyping (spacer oligotyping) is advantageous as it is PCR based and is a more rapid and easier technique to perform and analyze. The main disadvantage of spoligotyping is that all genetic polymorphism is restricted to a single genomic locus, the DR cluster.The Mycobacterium tuberculosis complex (MTBC) genome sequencing projects at the Sanger Center and The Institute for Genomic Research have released valuable sequence data from the genomes of M. tuberculosis H37Rv, M. bovis AF2122/97, and M. tuberculosis CDC1551, respectively, enabling the identification of polymorphic loci that may be useful for molecular typing (4). Much of the polymorphism occurs within regions of tandemly repeated DNA. Polymorphism at a tandem repeat (TR) locus can occur either as a result of nucleotide sequence changes between individual repeat units or as a result of variation in the number of repeat units, hence creating allelic variants. Variable-number TR (VNTR) typing is based upon repeat number polymorphism within these tandemly arranged repetitive DNA sequences. Many of these TR loci display

Two insertion sequences, IS6110 and IS1081, specific to the tuberculosis complex mycobacteria and a highly reiterated DNA element (pTBN12) cloned from Mycobacterium tuberculosis were systematically used to identify restriction fragment length polymorphism (RFLP) types among bovine isolates of Mycobacterium bovis in Northern Ireland. In a sample of 109 isolates, probes IS6110, IS1081, and pTBN12 identified 10, 2, and 12 distinct patterns, respectively. By combining the patterns generated by the three probes it was possible to identify 28 distinct RFLP types. The standard protocol advocated for RFLP analysis ofM. tuberculosis was used and would facilitate computer-based gel documentation and image analysis to establish a database of M. bovis types for large-scale epidemiological studies. These procedures will facilitate interlaboratory comparisons of M. bovis isolates and will help to elucidate the precise epidemiology of bovine tuberculosis in diferent countries.

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