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.