Polymerase chain reaction and restriction endonuclease digestion for selected members of the 'Mycoplasma mycoides cluster' and Mycoplasma putrefaciens

A specific diagnostic method using the polymerase chain reaction, together with restriction en- donuclease digestion patterns, was developed for members of the " Mycoplasma mycoides cluster" that normally occur in the United States (i.e., Mycoplasma mycoides subsp. mycoides Large Colony and Mycoplasma capri- colum subsp. capricolum in addition to "cluster" mycoplasma, bovine serogroup 7, and Mycoplasma putrefaciens. The digestion of "cluster" polymerase chain reaction DNA (1,225 bp) amplification products with restriction enzymes AseI and SspI gave mycoplasma species-specific patterns for all strains of M. mycoides subsp. mycoides Large Colony, M. capricolum subsp. capricolum, and bovine group 7 tested. Moreover, we found a nonspecific amplification product for M. putrefaciens that occurred with the oligonucleotide primers used for the " M. mycoides cluster" reaction. However, the restriction endonuclease digestion patterns observed with the restriction enzymes AluI, AseI, and SspI for M. putrefaciens were different than the digestion patterns obtained for the other "cluster" mycoplasmas. This report confirms the usefulness of polymerase chain reaction DNA ampli- fication allied with restriction enzyme digestion profile analysis for the rapid and specific identification of mycoplasmas belonging to the "M. mycoides cluster" and M. putrefaciens. Mycoplasma infections in ruminants are frequently one serious outbreak/ For the purpose of this report, observed, particularly in goats. However, it is extreme- the "M. mycoides cluster" shall infer only MmLC, MC, ly important to distinguish between mycoplasmas and IM. sp. serogroup 7. Other members of the "clus- causing severe and contagious diseases with serious ter" were not analyzed due to their "exotic" status in economic consequences and mycoplasmas that may the United States. cause sporadic disease with minor economic impact. In addition to the basic questions of taxonomy with- For example, worldwide, the most important myco- in the "cluster," differential diagnostic tests are often plasmas economically are the related ruminant patho- difficult to develop due to shared common antigens gens classified as the " Mycoplasma mycoides cluster." among these mycoplasma. 10 Cross-reactive antibodies This "cluster" 4 consists of six mycoplasma species: can impede the identification of the pathogenic agents Mycoplasma mycoides subsp. mycoides Small Colony thereby complicating the interpretation of serological (MmSC), Mycoplasma mycoides subsp. mycoides Large tests. 6 Moreover, biochemical differentiation may be Colony (MmLC), Mycoplasma mycoides subsp. capri time-consuming and ambiguous. Recently, DNA (Mmc), Mycoplasma capricolum subsp. capricolum probes have been used to develop more specific di- (MC), Mycoplasma capricolum subsp. capripneumon- agnostic procedures .