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.

The molecular fingerprints of 1,349 isolates of Mycobacterium bovis received between 1979 and August 2000 at Agence Française de Sécurité Sanitaire des Aliments (Afssa) have been obtained by spoligotyping. The majority of the isolates (1,266) were obtained from cattle living in France. An apparently high level of heterogeneity was observed between isolates. One hundred sixty-one spoligotypes were observed in total, of which 153 were from French isolates. The two predominant spoligotypes, designated BCG-like and GB54, accounted for 26 and 12% of the isolates, respectively. In addition, 84% of the spoligotypes were found fewer than 10 times. Analysis of the results by clustering and parsimony-based algorithms revealed that the majority of the spoligotypes were closely related. The predominant spoligotype was identical to that of the vaccine strain Mycobacterium bovis BCG, which was isolated in France at the end of the 19th century. Some spoligotypes were closely associated with restricted geographical areas. Interestingly, some spoligotypes, which were frequently observed in France, were also observed in neighboring countries. Conversely, few spoligotypes were common to France and England, and those that were shared were observed at very different frequencies. This last point illustrates the potential role for an international data bank, which could help trace the spread of M. bovis across national borders.Bovine tuberculosis (TB) was endemic in France until the 1960s, with herd prevalence rates of 25% in 1955 (9). From this time onwards, a national program for TB control based on tuberculin skin testing with control of animal movements and total slaughter of infected herds was implemented. This control strategy resulted in a dramatic decrease in bovine tuberculosis leading to a herd prevalence rate of 0.09% in 1998 (2), suggesting that cattle are the most important reservoir, or even the sole reservoir, for Mycobacterium bovis in France. Due to the success of this control strategy, France was declared "officially free of bovine TB" by the European Commission (3).The very low level of TB in cattle has resulted in the introduction of new control strategies. Consequently, there has been a progressive reduction in the use of skin testing, with an increasing emphasis on systematic sampling of suspect lesions identified at slaughterhouses for M. bovis isolate identification and molecular typing. New laboratory tools were therefore required in order to improve the traceability of the infections and identification of the origin of the outbreak (i.e

Letters to the Editor Mycobacterium microti: More Widespread than Previously Thought Mycobacterium microti typically causes disease in voles, wood mice, and shrews (6). Only rarely has this bacterium been isolated from other animals, among which are a llama, cats, pigs, a rock hyrax, and a ferret (2, 4, 5). Recently, M. microti isolates from different sources were characterized by using novel genetic markers (5), and this study for the first time disclosed four cases of M. microti infections in humans in The Netherlands. Here we describe the finding of additional M. microti infections in animals and a human in the United Kingdom (UK) that were identified by spoligotyping (3). In the framework of a European Union project on the development of novel standardized methodology for the identification of and nomenclature for Mycobacterium bovis, spoli

Osteomyelitis is a major cause of morbidity worldwide but there are few data investigating pathogenesis of infection and no investigations into local secretion of proinflammatory cytokines in patients. Tumour necrosis factor-alpha (TNF), interleukin (IL)-6 and IL-8 concentrations were measured in pus of infected bone from 30 Zambian patients with chronic osteomyelitis (principally caused by Staphylococcus aureus), in plasma, and after lipopolysaccharide stimulation of whole-blood leucocytes. Patients had reduced body mass index compared to controls (P = 0.025) and an acute-phase response. Elevated concentrations of proinflammatory cytokines were detected in bone compared to plasma (all P < 0.0002). Bone IL-8 concentrations were greater than IL-8 levels after lipopolysaccharide stimulation of whole blood (P < 0.01). In contrast, systemic and ex-vivo-stimulated concentrations of proinflammatory cytokine were similar in patients and controls, despite differences in body mass index and an acute-phase response. In summary, we observed marked local TNF, IL-6 and IL-8 secretion in established bacterial osteomyelitis without systemic cytokine release.