The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs. The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions. Because B. burgdorferi and M. genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors. Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families. The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.

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.Bovine tuberculosis (BTB) is caused by Mycobacterium bovis. It is a zoonotic disease and was the cause of approximately 6% of total human deaths due to BTB in the 1930s and 1940s and of more than 50% of all cervical lymphadenitis cases in children (13, 23). The introduction of pasteurization of milk in the 1930s dramatically reduced the transmission from cattle to humans, and in 1995 only 1% of 3,200 isolates from patients with TB in Great Britain were identified as M. bovis (23). However, human TB caused by M. bovis is still a major health issue in many developing countries (12,14,15).BTB has severe implications for animal welfare in both developed and developing countries, since it causes reduced productivity and premature death in cattle and since affected farms also suffer severe economic losses. A compulsory eradication program based on the slaughter of infected animals detec...

We have analyzed a panel of independent North American isolates of the Lyme disease agent spirochete, Borrelia burgdorferi (sensu stricto), for the presence of linear plasmids with sequence similarities to the 12 linear plasmids present in the B. burgdorferi type strain, isolate B31. The frequency of similarities to probes from each of the 12 B31 plasmids varied from 13 to 100% in the strain panel examined, and these similarities usually reside on plasmids similar in size to the cognate B31 plasmid. Sequences similar to 5 of the 12 B31 plasmids were found in all of the isolates examined, and >66% of the panel members hybridized to probes from 4 other plasmids. Sequences similar to most of the B. burgdorferi B31 plasmid-derived DNA probes used were also found on linear plasmids in the related Eurasian Lyme agents Borrelia garinii and Borrelia afzelii; however, some of these plasmids had uniform but substantially different sizes from their B. burgdorferi counterparts.The spirochetes that cause Lyme disease, members of the Borrelia burgdorferi (sensu lato) group of species, are known to harbor numerous extrachromosomal DNA elements. For ease of discussion we will refer to these elements as plasmids, although some may be present in all natural isolates, and some may carry essential genes, so they should perhaps more correctly be called "mini-chromosomes" (2). All natural isolates examined carry multiple linear plasmids in the 5 to 110 kbp size range and multiple circular plasmids in the 9 to 70 kbp range. Different isolates have similar but nonidentical linear plasmid band patterns in electrophoresis gels (e.g., those seen in references 3, 4, 5, and 33). Circular plasmid contents are more difficult to display, but in the isolates that have been analyzed, multiple, related plasmid types are always present (e.g., those seen in references 9, 22, 27, 31, and 36). A number of studies have shown that plasmid loss correlates with loss of infectivity in mice (15,25,34), so it is of interest to understand whether these plasmids have uniform structures in the wild and to understand the distribution of these plasmids among natural isolates.Only one Borrelia isolate, B. burgdorferi B31, has been the subject of a comprehensive study that unequivocally identified all of its plasmids. The analyzed culture of this strain, B31 MI, carries 12 linear and 9 circular plasmids, and the nucleotide sequence of each is known (8, 12). Over 90% of the genes on the characterized Lyme agent plasmids have no known homologs outside of the Borrelia genus (8), and a number of these genes encode outer surface proteins that are antigenic during infection of mammals (10,13,17,21,23,29,32,35). We report here an analysis of plasmids that are related to the 12 known linear B31 plasmids in a panel of Lyme disease borreliae. MATERIALS AND METHODSThe B. burgdorferi strains used were previously described (7); Borrelia garinii and Borrelia afzelii strains and sources are listed (see Table 4). Contour-clamped homogeneous electric field (CHEF) electrophoresis a...

Our data indicate that insufficient help of CD4(+) T cells may cause loss of IFN- gamma -producing CD8(+) T cells and loss of control of CMV dissemination. Increasing CMV-infected cell counts in the face of high CMV-specific perforin- and granzyme B-expressing CD8(+) T cell counts may explain the immune pathological characteristics of CMV disease.