Thirteen Acholeplasma strains representing three named species, Acholeplasma laidlawii, Acholeplasma granularum, and Acholeplasma oculi, were examined for the presence of 30 enzymes by horizontal starch gel electrophoresis. A total of 29 enzymes were demonstrated. Twelve of these enzymes have not been reported previously in acholeplasmas (alcohol dehydrogenase, aldolase, alkaline phosphatase, arginase, arginine deiminase, carbamyl phosphokinase, galactose dehydrogenase, galactose-6-phosphate dehydrogenase, glutamate dehydrogenase [nicotinamide adenine dinucleotide], glutamate dehydrogenase [nicotinamide adenine dinucleotide phosphate], a-glycerophosphate dehydrogenase, and xanthine dehydrogenase). An average group cluster analysis was carried out on the basis of estimates of dissimilarity coefficients. This analysis distinguished among the strains tested, was of some help in identification, and may also be useful in epidemiological investigations.In the past, serology has been a cornerstone of Acholeplasma taxonomy. Some of the membrane-associated lipoglycans from different Acholeplasma species share antigenic determinants (20). Consequently, methods based on surface antigens, like growth inhibition and immunofluorescence tests, may not be reliable for definitive identification of acholeplasmas . The growth precipitation test appears to be almost species specific within the genus Acholeplasma, as cross-reactions seldom occur between recognized species (8, 14). In an exploratory study, Lanham et al. (17) found that isoenzyme analysis by (thin-layer) electrophoresis is useful for distinguishing between Acholeplasma laidlawii and Acholeplasma equifetale, as well for demonstrating intraspecific variation. The value of isoenzyme analysis in mycoplasma taxonomy received further substantial support from a study by O'Brien et al. (24).The purpose of this study was to characterize and differentiate the type strains and a few field strains of three Acholeplasma species, A . laidlawii, Acholeplasma granularum, and Acholeplasma oculi, by analyzing soluble isoenzymes. These organisms have been reported to cross-react in growth inhibition and immunofluorescence tests and cannot be differentiated by any of the common biochemical tests.
MATERIAL AND METHODSAcholeplusma strains. The following 13 strains were included in this study: the type strains of A. laidlawii (strain PG8 [sewage strain A]), A . granularum (strain BTS-39), and A. oculi (strain 19-L), A . faidfawii strain PG9 (sewage strain B), and 9 strains isolated from bovine, caprine, ovine, equine, and avian sources. We attempted to classify these strains before isoenzyme analysis by the agar well modification of the growth inhibition test (4), the indirect epi-immunofluorescence technique (25), and the growth precipitation test (8). All cultures were filtered through a membrane filter (pore size, 450 nm; Millipore Corp., Bedford, Mass.) and cloned on solid media by picking single colonies. This procedure was repeated three times. Cultures were selected for this study b...