IN-VITRO AMPLIFICATION OF THE 16S RIBOSOMAL-RNA GENES FROM MYCOPLASMA-BOVIS AND MYCOPLASMA-AGALACTIAE BY PCR

Mycoplasma bovis and Mycoplasma agalactiae are two very closely relate d species which cause mastitis in cows and goats, respectively. M. bov is can also cause arthritis and respiratory disease in cattle. It has recently been shown that the 16S rRNA sequences differ only in 8 nucle otide positions between the two species [J.G. Mattsson, B. Guss and K. -E. Johansson (1994) FEMS Microbiol. Lett,, 115: 325-328]. These nucle otide differences are distributed over the molecule in such a way that it is difficult to design specific identification systems, based on P CR only, for M. bovis and M. agalactiae. Two different PCR systems bas ed on 16S rRNA sequence data have, however, been designed for these tw o species. The forward primers were identical in the two systems and c omplementary to a segment of the evolutionarily variable region V2. Th e reverse primers were complementary to the variable region V6, in whi ch there are two nucleotide differences between M. bovis and M. agalac tine. The size of the PCR products, generated with these primers, was 360 bp. Cross-amplification was obtained with the two species in the h eterologous PCR systems, but with approximately a 100-fold lower effic iency. Cross-amplification was not obtained with any other bovine or c aprine mycoplasma except for Mycoplasma sp. strain A1343 of the caprin e group 7. The detection limit of the PCR system for M. bovis with a r eference culture was 4 X 10(2) CFU/ml and of the PCR system for M, aga lactiae 2X10(2) CFU/ml. The M. bovis-PCR system was used to analyze na sal samples of calves from a herd where an outbreak of pneumonia had o ccured and it proved possible to detect M. bovis in these samples.