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Because immunoglobulin A (IgA) is the predominant immunoglobulin at mucosal surfaces, IgA proteases produced by pathogenic bacteria are considered potential virulence factors for organisms that cause disease or gain entry at mucous membranes. To determine the role of IgA protease in the pathogenicity of mycoplasmal disease, a variety of human and animal mycoplasma and ureaplasma species were examined for IgA protease activity with human, murine, porcine, and canine IgA. None of the mycoplasma species examined showed detectable IgA protease activity with any of the IgAs tested. Twenty-eight strains of Ureaplasma urealyticum isolated from human urogenital tissues cleaved human IgA1, but no cleavage of human IgA2 or murine, porcine, or canine IgA was observed. Ureaplasmas isolated from nonhuman hosts (feline, canine, avian, and bovine [Ureaplasma diversum]) did not cleave human IgA1. Two strains of canine ureaplasmas were able to cleave canine IgA, but not murine IgA. Thus, ureaplasmas from other species can produce IgA protease, but the specificity of the enzyme was restricted to the IgA of the appropriate host. This finding suggests that IgA proteases could play a role in the selective host specificity of mucosal pathogens.

Crude extracts of triple-cloned, purified cultures of 22 species of Mycoplasma and Acholeplasma were examined for expression of 21 isozyme systems routinely used to type mammalian cells. Nine previously described enzymes (purine nucleoside phosphorylase, adenylate kinase, dipeptidase, esterase, glyceraldehyde-3-phosphate dehydrogenase, glucose phosphate isomerase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and superoxide dismutase) and three enzymes not previously reported in mycoplasma (triose phosphate isomerase, inorganic pyrophosphatase, and acid phosphatase) were detected in some or all of the species examined. These findings provide new information on the enzymatic expressions of these organisms. Three of the isozyme systems (superoxide dismutase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) were present in Acholeplasma species but not in any Mycoplasma species. The characteristic pattern of electrophoretic mobility of the 12 isozyme systems also provides a useful biochemical property for identification, characterization, and classification of these mycoplasmas. Mycoplasma isozyme expression for seven of the enzymes were readily detected in various infected-cell culture lines by using either cell extracts or concentrated cell culture fluids. Mycoplasma-specific enzymes found in infected-cell extracts had the same electrophoretic mobility patterns as enzymes obtained from broth-grown mycoplasmas of the same species. Expression of homologous mammalian enzymes was not detectably altered by infection with mycoplasmas.

3H-labeled Acholeplasma DNA probes were generated in vitro by the nick-translation method and used to determine the nucleotide sequence homology among the type strains of the eight currently recognized species of Acholeplasma. Very little nucleotide sequence homology (less than or equal to 18%) was found among the eight species, with heteroduplexes showing at least 12% or more mismatching as determined by thermal elution midpoints. The small amount of nucleotide sequence homology among the eight species indicates that these species are quite distinct and are not closely related to each other genomically.

Mycoplasmas recovered recently from dogs were found unrelated to three classified canine Mycoplasma serotypes but were similar in biological and serological properties to a Mycoplasma strain (C21, PG-24) isolated 18 years earlier. It is proposed that strains with the characteristics described be designated Mycoplasma edwardii sp. n.

Anderson, Douglas R. (National Cancer Institute, Bethesda, Md.), Hope E. Hopps, Michael F. Barile, and Barbara C. Bernheim . Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culture. J. Bacteriol. 90: 1387–1404. 1965.—In an effort to make a valid comparison of the ultrastructure of several intracellular parasites, selected agents were propagated under identical conditions in a single type of tissue culture cell; such infected preparations were processed for examination by electron microscopy by use of a standardized procedure for fixation and embedding. The organisms studied were: the Breinl and E strains of epidemic typhus, Rickettsia prowazeki ; the Bitterroot strain of R. rickettsii ; the Karp strain of R. tsutsugamushi ( R. orientalis ); R. sennetsu; the P-4 strain of ornithosis virus; and the HEp-2 strain of Mycoplasma hominis type I. Each of the rickettsial species examined had a cell wall and a plasma membrane, and contained ribosomes and deoxyribonucleic acid (DNA) in a ground substance. However, certain differences were noted. Both strains of R. prowazeki contained numerous intracytoplasmic electron-lucent spherical structures (4 to 10 mμ), not previously described. R. sennetsu , unlike the other rickettsiae, was not free in the host cytoplasm but was always enclosed in a vacuole. R. rickettsii was observed intranuclearly and in digestive organelles of the host cell as well as in the cytoplasm. Cells infected with ornithosis virus contained several forms representing the stages in its life cycle. The “initial bodies,” made up of ribosomes and DNA strands, were morphologically similar to the rickettsiae. In cultures infected with M. hominis , most of the cells became large and multinucleate. Although the Mycoplasma organisms were readily cultivated from these cultures, only a few could be found in the electron microscope preparations. These organisms were extracellular and lacked a cell wall, being bound only by a unit membrane. Again, the internal components were ribosomes and DNA strands. Under the uniform preparative conditions employed here, the three groups of organisms were morphologically distinguishable from one another.

Barile, Michael F. (National Institutes of Health, Bethesda, Md.), Walter F. Malizia, and Donald B. Riggs . Incidence and detection of pleuropneumonia-like organisms in cell cultures by fluorescent antibody and cultural procedures. J. Bacteriol. 84: 130–136. 1962—A total of 102 tissue-cell cultures from 17 separate laboratories was examined for pleuropneumonia-like organisms (PPLO) by the fluorescent antibody and cultural procedures. PPLO were isolated from 48 of the 49 tissue-cell cultures found positive for PPLO by the fluorescent antibody procedure, and results of the two procedures agreed in 101 of the 102 (99%) cases. PPLO were isolated from none of 10 primary-cell cultures prepared from six animal species and from 48 of 92 (52%) continuous-cell cultures prepared from eight animal species. Cells grown in media containing antibiotics were more frequently contaminated with PPLO (72%) than cells grown in antibiotic-free media (7%). Cultures (91%) from tissue-culture-producing laboratories and cultures (76%) used for propagation of microorganisms were contaminated with PPLO, although none used for tissue-culture metabolic studies was contaminated. In addition, our findings support the view that PPLO contamination of cell cultures is probably owing to bacterial contaminants which revert to L forms in the presence of antibiotics.

Barile, Michael F. (Division of Biologics Standards, National Institutes of Health, Bethesda, Md.), Robert T. Schimke, and Donald B. Riggs . Presence of the arginine dihydrolase pathway in Mycoplasma . J. Bacteriol. 91 :189–192. 1966.—The presence of the arginine dihydrolase pathway was examined in 61 Mycoplasma strains representing at least 18 Mycoplasma species isolated from nine different sources: human, bovine, avian, murine, swine, goat, canine, sewage, and tissue cell culture origin. Some species were represented by only one or two strains. Different strains of the same species gave the same results. Ten species (56%) were positive. Many nonpathogenic Mycoplasma species ( M. hominis , type 1 and 2, M. fermentans, M. salivarium , and M. gallinarum ) were positive, whereas most pathogenic species ( M. pneumoniae, M. gallisepticum, M. neurolyticum , and M. hyorhinis ) were negative. The presence of arginine dihydrolase activity among Mycoplasma species may prove to be useful for purposes of identification and classification.