N. Palmer

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non-functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes >/=300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.

In Great Britain an independent scientific review for the government has concluded that the development of a cattle vaccine against Mycobacterium bovis infection holds the best long-term prospect for tuberculosis control in British herds. A precondition for vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected with M. bovis so that testing and slaughter-based control strategies can continue alongside vaccination. To date bacillus Calmette-Guérin (BCG), an attenuated strain of M. bovis, is the only available vaccine for the prevention of tuberculosis. However, tests based on tuberculin purified protein derivative cannot distinguish between M. bovis infection and BCG vaccination. Therefore, specific antigens expressed by M. bovis but absent from BCG constitute prime candidates for differential diagnostic reagents. Recently, two such antigens, ESAT-6 and CFP-10, have been reported to be promising candidates as diagnostic reagents for the detection of M. bovis infection in cattle. Here we report the identification of promiscuous peptides of CFP-10 that were recognized by M. bovis-infected cattle. Five of these peptides were formulated into a peptide cocktail together with five peptides derived from ESAT-6. Using this peptide cocktail in T-cell assays, M. bovis-infected animals were detected, while BCG-vaccinated or Mycobacterium avium-sensitized animals did not respond. The sensitivity of the peptide cocktail as an antigen in a whole-blood gamma interferon assay was determined using naturally infected field reactor cattle, and the specificity was determined using blood from BCG-vaccinated and noninfected, nonvaccinated animals. The sensitivity of the assay in cattle with confirmed tuberculosis was found to be 77.9%, with a specificity of 100% in BCG-vaccinated or nonvaccinated animals. This compares favorably with the specificity of tuberculin when tested in noninfected or vaccinated animals. In summary, our results demonstrate that this peptide cocktail can discriminate between M. bovis infection and BCG vaccination with a high degree of sensitivity and specificity.

In Great Britain a recent independent scientific review for the government has concluded that the development of a cattle vaccine against Mycobacterium bovis holds the best long-term prospect for tuberculosis control in British herds. A sine qua non for vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected with M. bovis so that test-and-slaughter-based control strategies can continue alongside vaccination. In order to assess the feasibility of developing a differential diagnostic test for a live vaccine, we chose M. bovis BCG Pasteur as a model system. Recombinant forms of antigens which are expressed in M. bovis but not, or only at low levels, in BCG Pasteur (ESAT-6, MPB64, MPB70, and MPB83) were produced. These reagents were tested either alone or in combination by using peripheral blood mononuclear cells from M. bovis-infected, BCG-vaccinated, and Mycobacterium avium-sensitized calves. All four antigens induced in vitro proliferation and gamma interferon responses only in M. bovis-infected animals. A cocktail composed of ESAT-6, MPB64, and MPB83 identified infected animals but not those vaccinated with BCG. In addition, promiscuous T-cell epitopes of ESAT-6, MPB64, and MPB83 were formulated into a peptide cocktail. In T-cell assays with this peptide cocktail, infected animals were identified with frequencies similar to those obtained in assays with the protein cocktail, while BCG-vaccinated or M. avium-sensitized animals did not respond. In summary, our results suggest that peptide and protein cocktails can be designed to discriminate between M. bovis infection and BCG vaccination.

Sixteen European badgers (Meles meles) from three statutory removal operations were studied. Samples of tracheal aspirate, pooled lymph nodes and urine were cultured for mycobacteria. Seven of the badgers were infected with Mycobacterium bovis and had tuberculous pulmonary lesions which varied in severity from extensive granulomatous consolidation to microgranulomas which were not detectable grossly. Tuberculous lesions were also observed in the upper respiratory airways, intestines, kidneys, spleen, liver, thymus, pleura and lymph nodes. One badger had tuberculous bite wounds. The histopathological characteristics of the tuberculous reactions and the associated tissue damage in various organs, together with the gross pathology, indicate that both mildly and severely infected badgers have the potential to excrete M. bovis by several routes.