A comparison of Mycobacterium tuberculosis complex isolates from seals (pinnipeds) in Australia, Argentina, Uruguay, Great Britain and New Zealand was undertaken to determine their relationships to each other and their taxonomic position within the complex. Isolates from 30 cases of tuberculosis in six species of pinniped and seven related isolates were compared to representative and standard strains of the M. tuberculosis complex. The seal isolates could be distinguished from other members of the M. tuberculosis complex, including the recently defined 'Mycobacterium canettii ' and 'Mycobacterium caprae', on the basis of host preference and phenotypic and genetic tests. Pinnipeds appear to be the natural host for this 'seal bacillus', although the organism is also pathogenic in guinea pigs, rabbits, humans, Brazilian tapir (Tapirus terrestris) and, possibly, cattle. Infection caused by the seal bacillus is predominantly associated with granulomatous lesions in the peripheral lymph nodes, lungs, pleura, spleen and peritoneum. Cases of disseminated disease have been found. As with other members of the M. tuberculosis complex, aerosols are the most likely route of transmission. The name Mycobacterium pinnipedii sp. nov. is proposed for this novel member of the M. tuberculosis complex (the type strain is 6482 T =ATCC BAA-688 T =NCTC 13288 T ).Abbreviations: BCG, Bacille Calmette-Gué rin; FAFLP, fluorescent amplified fragment length polymorphism; PZA, pyrazinamide; SS, seal spoligotype; TCH, thiophen-2-carboxylic acid hydrazide.

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DNA preparations from two reference (H37Ra and H37Rv) and two wild strains of Mycobacterium tuberculosis and one re-isolated strain of Mycobacterium bovis BCG were analysed using 17 restriction endonucleases. The enzyme BstEII revealed the greatest differences between strains. Electrophoretic DNA patterns from the wild M. tuberculosis strains differed from each other and from the reference strains at relatively few positions. At the highest resolution attained, patterns from the two reference strains remained indistinguishable from each other. The pattern of the M. bovis BCG strain was substantially different from, but had many bands in common with, the M. tuberculosis patterns.

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The effects of electroporation temperature, biochemical pretreatment of cells and stage of culture on electroporation efficiency for slow-growing mycobacteria were investigated. The efficiency of transformation into Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium intracellulare increased markedly with temperature. In contrast, the efficiency of transformation into Mycobacterium smegmatis, a fast-growing species, was higher at 0 degree C and decreased with temperature. While stage of culture had little effect, a further increase in efficiency of 2-4-fold was obtained following glycine or ethionamide pretreatment. Electroporation at 37 degrees C has been chosen as a standard condition for slow-growing species as it usually resulted in a transformation efficiency several orders of magnitude higher than that obtained at 0 degree C.

A better tuberculosis vaccine is urgently required to control the continuing epidemic. Molecular techniques are now available to produce a better live vaccine than BCG by producing avirulent strains of the Mycobacterium tuberculosis complex with known gene deletions. In this study, 1000 illegitimate recombinants of Mycobacterium bovis were produced by illegitimate recombination with fragments of mycobacterial DNA containing a kanamycin resistance gene. Eight recombinant strains were selected on the basis of their inability to grow when stationary-phase cultures were inoculated into minimal medium. Five of these recombinants were found to be avirulent when inoculated into guinea pigs. Two of the avirulent recombinants produced vaccine efficacy comparable to BCG against an aerosol challenge in guinea pigs with M. bovis. One of these recombinants had an inactivated glnA2 gene encoding a putative glutamine synthetase. Transcriptional analysis showed that inactivation of glnA2 did not affect expression of the downstream glnE gene. The other recombinant had a block of 12 genes deleted, including the sigma factor gene sigG. Two avirulent recombinants with an inactivated pckA gene, encoding phosphoenolpyruvate carboxykinase which catalyses the first step of gluconeogenesis, induced poor protection against tuberculosis. It is clear that live avirulent strains of the M. tuberculosis complex vary widely in their ability as vaccines to protect against tuberculosis. Improved models may be required to more clearly determine the difference in protective effect between BCG and potential new tuberculosis vaccines.

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