In vitro amplification of the 16S rRNA genes from Mycoplasma bovis and Mycoplasma agalactiae by PCR.

Mycoplasma bovis and Mycoplasma agalactiae are two very closely related species which cause mastitis in cows and goats, respectively. M. bovis can also cause arthritis and respiratory disease in cattle. It has recently been shown that the 16S rRNA sequences differ only in 8 nucleotide positions between the two species [J.G. Mattsson, B. Guss and K.-E. Johansson (1994) FEMS Microbiol. Lett., 115: 325-328]. These nucleotide differences are distributed over the molecule in such a way that it is difficult to design specific identification systems, based on PCR only, for M. bovis and M. agalactiae. Two different PCR systems based on 16S rRNA sequence data have, however, been designed for these two species. The forward primers were identical in the two systems and complementary to a segment of the evolutionarily variable region V2. The reverse primers were complementary to the variable region V6, in which there are two nucleotide differences between M. bovis and M. agalactiae. The size of the PCR products, generated with these primers, was 360 bp. Cross-amplification was obtained with the two species in the heterologous PCR systems, but with approximately a 100-fold lower efficiency. Cross-amplification was not obtained with any other bovine or caprine mycoplasma except for Mycoplasma sp. strain A1343 of the caprine group 7. The detection limit of the PCR system for M. bovis with a reference culture was 4 x 10(2) CFU/ml and of the PCR system for M. agalactiae 2 x 10(2) CFU/ml. The M. bovis-PCR system was used to analyze nasal samples of calves from a herd where an outbreak of pneumonia had occured and it proved possible to detect M. bovis in these samples.