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Ureaplasma strains isolated from dogs (Canis familiaris) were characterized and compared with the type strains of five previously described species of the genus Ureaplasma, Ureaplasma urealyticum (isolated from humans), Ureaplasma diversum (isolated from cattle), Ureaplasma gallorale (isolated from chickens), Ureaplasma cati (isolated from cats), and Ureaplasma felinum (isolated from cats). The canine strains hydrolyzed urea but not arginine or glucose, were membrane bound, lacked a cell wall, passed through 450-nm-pore-size membrane filters, required cholesterol for growth, and formed minute colonies (diameter, 20 to 140 microns) on agar medium. These canine ureaplasma strains have been reported to be members of four serovars. The four serovars of canine strains fell into a single group on the basis of their genomic properties, as determined by DNA-DNA hybridization. On the basis of these findings, we propose that ureaplasmas with these characteristics belong to a new species, Ureaplasma canigenitalium, with strain D6P-C (= ATCC 51252) as the type strain.

Eight chimpanzees were examined. Two served as negative control and six inoculated with Mycoplasma pneumoniae became colonized. Colonization persisted for 28-68, 16-50 and 21 days with an average duration of 47, 32.5 and 21 days in the oropharyngeal, tracheal and lung tissues, respectively. Mycoplasma titers ranged from 10 8 to 10 1 color-changing units per specimen during the course of the infections. Seroconversion occurred within 12-15 days and peak antibody titers ranged from 1.256 to 1.1024 and developed between days 28 and 48 post-inoculation. Positive cold agglutinin titers were detected between 12 to 15 days and peak titers ranged from 1:80 to 1:640. Significant increases in sIgA and IgG immunoglobulin antibody levels were detected in lung lavage fluids. Unlike the many other experimentally infected animals examined, chimpanzees infected with M. pneumoniae had positive X-ray findings, developed cold agglutinins and showed overt signs of disease. These signs include persistent cough, low grade fever, rhinitis, oropharyngitis, diarrhea, and loss of appetite. Peak severity of disease corresponded with peak lung colonization, and the detection of cold agglutinins and positive X-ray findings. The microbiological, serological and clinical aspects of pneumonia induced in chimpanzees was similar to naturally occurring primary atypical pneumonia in humans.

Mycoplasma infection of cell cultures is widespread and has major detrimental effects on cellular physiology and metabolism. Since cell culture is used extensively, both in research and in industrial production processes, questions of primary concern arise, such as: how can mycoplasma contamination be detected; what are the effects of such contamination on cellular functions; what methods are available for eliminating contamination?

Using 12 different and elaborate broth, agar, and cell culture procedures, we failed to isolate mycoplasmas, ureaplasmas, spiroplasmas, or chlamydiae from the synovial fluid of 10 patients with rheumatoid arthritis (RA) and from six patients with non-rheumatoid arthritis (NRA). In addition, sera from 35 patients with RA and 12 patients with NRA also were examined. Although some of the sera had moderately high titers of metabolism-inhibiting antibody to some of the 10 human Mycoplasma species, especially to the common respiratory pathogen Mycoplasma pneumoniae, and to some of the eight Ureaplasma urealyticum serovars, especially serovars V and VII, there were no significant differences between titers of these antibodies in the two groups of patients. Among RA patients serum antibody titers to M. pneumoniae were 1:32 in five and 1:16 in eight; two patients had higher synovial fluid titers (1:16) than serum titers (1:4). The geometric mean titer (GMT) of antibody to serovar V in synovial fluid was higher in RA patients than in NRA patients, but the difference did not reach significance (P = .056). Reports on the possible role of infectious agents in the pathogenesis of rheumatoid arthritis are reviewed.

Seven ureaplasma strains isolated from the oral cavities of domestic cats (Felis domestica) were characterized and compared with the type strains of the three previously established species of this genus, Ureaplasma urealyticum (humans), Ureaplasma diversum (cattle), and Ureaplasma gallorale (chickens). The feline strains hydrolyzed urea but not arginine or glucose, were membrane bound, lacked cell walls, passed through 0.45-micron membrane filters, required cholesterol for growth, and formed minute (15- to 140-microns) colonies on agar medium. The seven feline strains fell into two distinct groups based on (i) their antigenic properties (determined by using the metabolism and growth inhibition and indirect immunoperoxidase procedures), (ii) their genomic properties (determined by using DNA-DNA hybridization and DNA cleavage pattern procedures), and (iii) their polypeptide profiles (determined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses). Based on these properties, the two feline groups were unrelated to each other or to the three previously established species, and each group represents a distinct Ureaplasma species. Thus, we propose that ureaplasmas with these phylogenetic and genomic properties be given taxonomic status as Ureaplasma felinum and Ureaplasma cati, with strain FT2-B (= ATCC 49229 = NCTC 11709) and strain F2 (= ATCC 49228 = NCTC 11710) as the type strains, respectively.

Following immunization, peak geometric mean serum metabolism inhibition antibody (MIT) titres were 1:13 and 1:16 for groups of three chimpanzees each that received either the formalin-inactivated OSU-1A or experimental acellular extract vaccine, respectively. Following challenge, the mean titres for chimpanzees given the acellular vaccine peaked at 1:256 in 4 weeks and was 1:48 at 10 weeks. Chimpanzees given the OSU-1A vaccine peaked at 1:80 in 4 weeks and remained at 1:80 at 10 weeks. There was no direct correlation between the serum MIT response and the severity of disease or colonization, and thus the MIT response was not a reliable measurement of protection. The two non-immunized chimpanzees showed significant signs of disease, including cough, pharyngitis, rhinitis, fever and abnormal X-ray findings, for about 5 weeks. The chimpanzees immunized with either vaccine were less colonized and showed far less disease than non-immunized controls. Protection afforded the chimpanzees was similar to that of vaccinees in the human clinical trial given the same OSU-1A vaccine (Wenzel et al., 1977). The two previously infected chimpanzees were most protected against colonization and disease on challenge.

Mycoplasma hominis was isolated in pure culture from septic synovial aspirates from an individual (patient A) during 16 different bouts of exacerbation over a 70-month period of observation. Two isolates, 10(7) and also 10(6) color-changing units (CCU) of the 1620 isolate and 5 x 10(4) CCU of the 1628 isolate, caused inflammation in chimpanzees inoculated intraarticularly. Inflammation was also induced with 10(7) CCU of the 2010B isolate, serovar VII of Ureaplasma urealyticum, recovered from an agammaglobulinemic individual (patient B) with septic polyarthritis and with 3 x 10(6) CCU of the PI-1428 isolate of Mycoplasma pneumoniae. Inflammation persisted for up to 36 days and was self-limiting. The aspirates contained up to 220,000 white blood cells/mm3 and up to 10(7) CCU/mL. There was good correlation between the severity of inflammation and the numbers of organisms, but antibody was not detected in aspirates during the peak severity of disease. As the numbers of organisms, decreased, detectable levels of antibody increased, thus suggesting that antibody may have been bound to antigen. Chimpanzees previously infected with either the 1628 isolate of M. hominis or the 2010B isolate of U. urealyticum were protected on challenge with > 100 times the minimal dose causing arthritis. Chimpanzees showed little or no inflammation when inoculated intraarticularly with 5 x 10(8) CCU of the type strain PG-21 of M. hominis or with the type strain CO of U. urealyticum or when inoculated intravenously with 3 x 10(8) CCU of the arthrogenic 1620 isolate of M. hominis.

This report will briefly review our current knowledge of: 1) the incidence, prevalence and sources of mycoplasma contamination in cell cultures; 2) the procedures for isolation, detection and identification of mycoplasmas, including procedures recommended for testing biological products produced for human use; 3) the effects of mycoplasma contamination and /or infection on the function and activities of various infected cell cultures; and 4) the recommended procedures for prevention and elimination of mycoplasma contamination. Extensive reviews on mycoplasma contamination in cell culture have been reported earlier (Barile, 1979; DelGiudice and Hopps, 1978; McGarrity and Kotani, 1985).

Mycoplasma pulmonis and Mycoplasma arthritidis are both significant causes of infection in colonies of rodents, which are common experimental animals for biomedical research. Since differential diagnosis has proven difficult due to similar homology between these two murine Mycoplasma species, the development of a reliable identification system is of importance. In this study DNA probes specific for M. pulmonis and M. arthritidis were generated after cross-hybridization between two reference strains and cloning of the specific DNA fragments into the pGEM-3 blue vector. Two clones, harboring plasmids pGEM-89 (specific for M. pulmonis) and pGEM-31 (specific for M. arthritidis) were selected based on their specificity upon colony hybridization and were used as species-specific probes. These probes could detect in dot blots 150 pg of M. pulmonis DNA or 300 pg of M. arthritidis DNA with no visible hybridization with up to 100 ng of heterologous DNA. When hybridized with dot blots of culture grown organisms, the pGEM-89 probe produced a positive signal with all 12 isolates of the M. pulmonis strains tested, but none of the four M. arthritidis, and vice versa for the pGEM-31 probe. We thus anticipate that these probes will be useful for routine detection and identification of mycoplasmal infections of rodents.

The current classification of the Kingdom, Prokaryotae, includes four divisions, three of which encompass the Gram-negative bacteria, the Gram-positive bacteria and the archaeobacteria. The remaining division, Tenericutes, (soft skin) covers the prokaryotes which lack a cell wall. It contains only one class, Mollicutes, three orders, four families and six genera (Subcomm. Tax. Mollicutes, 1979; Freundt and Edward, 1979; Razin and Freundt, 1984; Freundt and Razin, 1984; Murray, 1984 (Table 3.1). Mollicute(s) is used as the trivial term to denote all organisms belonging to the class, and the trivial names; mycoplasmas, ureaplasmas, acholeplasmas, etc., denote members of the corresponding genus.

A prospective analysis of bronchoalveolar lavage (BAL) in 13 patients with Wegener's granulomatosis (WG), 20 disease control subjects with idiopathic pulmonary fibrosis (IPF), and 24 normal control subjects was conducted to (1) evaluate the quality of the alveolar inflammatory response associated with active WG; (2) determine whether antineutrophil cytoplasmic antibody (ANCA) is present in alveolar fluid and produced in the lungs of patients with WG; and (3) determine whether inhaled particles or infectious agents may play an etiologic role in WG. BAL in untreated active WG had a marked increase in neutrophils (mean = 42% of total WBC count), and usually in eosinophils (mean = 4%) compared with that in normal control subjects (1.6% neutrophils, 0% eosinophils), and untreated WG in remission (5.9% neutrophils, 0% eosinophils). Disease control subjects with IPF, a process known to be associated with neutrophilic alveolitis, had an increased population of neutrophils (15.4%) and eosinophils (2.7%) in BAL. Leukocyte remnants, as well as intact leukocytes, could be identified within BAL macrophages in the patients with WG and IPF, and rarely in the normal control subjects. Normal subjects and control patients with IPF were all negative for ANCA in serum, whereas ANCA was found in serum and BAL in all patients with active WG who had generalized disease. Protein analysis of BAL revealed a disproportionate increase in the IgG to albumin ration compared with serum values (IgG index) in patients with active untreated disease. The increase in the IgG index suggests that IgG with ANCA reactivity is produced by pulmonary lymphoid tissue. An infectious agent in BAL was not identified by any of the techniques applied in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

The levels of DNA relatedness among two unclassified feline ureaplasma serogroups, four unclassified canine ureaplasma serogroups, and the three previously established Ureaplasma species were examined and compared. The strains examined included five feline strains representing two feline serogroups, four canine strains representing four canine serogroups, and the type strains of the three established species. Each strain representing each species or serogroup exhibited 78% or more actual DNA homology with its homologous DNA, but less than 10% DNA homology with DNAs from the heterologous strains. These findings indicate that each of these human, bovine, avian, feline, and canine strains is genomically distinct. In addition, the three previously recognized species (Ureaplasma urealyticum [human], Ureaplasma diversum [bovine], and Ureaplasma gallorale [avian]), which were established on the basis of phenotypic properties, were also shown to be genomically distinct. The three feline serogroup SI strains were genomically related (from 89 to 100% DNA homology) to each other but were unrelated (less than 10% DNA homology) to the feline serogroup SII strains, indicating that these two feline serogroups are also genomically distinct. Conversely, the two feline serogroup SII strains were genomically very similar (from 83 to 100% DNA homology) to each other but were unrelated (less than 10% DNA homology) to the three feline serogroup SI strains. However, canine serogroup SI strain D1M-C exhibited 73% DNA homology with serologically distinct canine serogroup SII strain D29M, indicating that these strains representing two separate serogroups belong to the same genomic species.(ABSTRACT TRUNCATED AT 250 WORDS)

Ureaplasmas isolated from avian sources were compared to the two established Ureaplasma species, U.urealyticum from humans and U.diversum from cattle, by the metabolic inhibition test, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell protein patterns, restriction endonuclease cleavage patterns of genomic DNA and by [3H] DNA-DNA hybridization. All seven unclassified avian ureaplasma strains tested were serologically related to each other but were different from the two established bovine and human Ureaplasma species. The antigenic differences were also evident in Western blot analysis. The [3H] DNA-DNA hybridization data further confirmed these results by showing that avian ureaplasmas were genetically related to each other but distinct from the established Ureaplasma species. The restriction endonuclease cleavage patterns revealed variations among the avian strains that were not apparent by the serological and biochemical techniques. Our results support the notion that the avian ureaplasmas are distinct, are unrelated to the two established Ureaplasma species and merit consideration as a new species.

Seven ureaplasma strains isolated from the oropharynx of chickens (Gallus gallus var. domesticus) were characterized and compared with the two previously described species of this genus, Ureaplasma urealyticum from humans and Ureaplasma diversum from cattle. The avian isolates were found to have similar biological, biochemical, serological, and genomic properties and were readily distinguished from the two previously described species. These avian ureaplasma strains have been reported to hydrolyze urea, but not to catabolize arginine or glucose; they lack a cell wall, pass through a 450-nm membrane filter, require cholesterol for growth, and form minute colonies (15 to 60 μm) on agar medium. The seven avian strains constitute a homogeneous group based on their antigenic properties as determined by the metabolism and growth inhibition tests and by protein composition analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the basis of these findings, we propose that ureaplasmas with these characteristics belong to a new species, Ureaplasma gallorale, with strain D6-1 (= ATCC 43346 = NCTC 11707) as the type strain.

Species of the Acholeplasmataceae differ from species of the Mycoplasmataceae and Spiroplasmataceae in many respects, including lack of a nutritional requirement for sterol, ability of most species to synthesize saturated fatty acids and polyterpenes from acetate, and several other properties related to lipid metabolism and to the incorporation and location of lipids in the cell membrane. Acholeplasma species have also been found to differ from Mycoplasma species in possessing a nicotinamide adenine dinucleotide-dependent lactate dehydrogenase that is specifically activated by fructose 1,6-diphosphate and in containing superoxide dismutase, as well as glucose-6-phosphate and 6-phosphogluconate dehydrogenases. In addition, reduced nicotinamide adenine dinucleotide oxidase activity is located in the cell membrane of Acholeplasma species and is associated with the soluble cytoplasmic fraction of Mycoplasma and Spiroplasma species. Finally, significant differences exist between the nucleic acids of the Acholeplasmataceae and the Mycoplasmataceae. The genome molecular weight for Acholeplasma species is about 1.0 × 109, compared with about 5.0 × 108 for species of the Mycoplasmataceae. Moreover, a recent comparison of ribosomal ribonucleic acid oligonucleotide catalogs has demonstrated that Acholeplasma species are more closely related phylogenetically to two clostridial species than to the Mycoplasma and Spiroplasma species tested. Because the characteristics of species of the Acholeplasmataceae differ in major respects from those of other families of the Mollicutes, we propose elevation of the family Acholeplasmataceae to the rank of a new order, Acholeplasmatales. We provide a description of the proposed taxon, the second order of the class Mollicutes.

Eighteen male and eight female primates, representing five subhuman species, were inoculated urogenitally with Mycoplasma genitalium, a microorganism recovered from men with nongonococcal urethritis. Male rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) monkeys apparently were resistant. Female squirrel monkeys (Saimiri sciureus) and female tamarins (Saguinus mystax) exhibited low-level, genital-tract infections. Male chimpanzees (Pan troglodytes) developed an obvious genital-tract infection, with some shedding organisms for 21 weeks. M. genitalium was recovered from the blood of two of the male chimpanzees, usually when large numbers of organisms were in the urethra. Female chimpanzees generally shed organisms for 12-15 weeks. Most chimpanzees colonized with the organism exhibited increased numbers of polymorphonuclear leukocytes in the genital tract and developed a significant antibody response. The results offer substantial evidence for the pathogenicity of M. genitalium for the urogenital tract of higher primates and suggest the microorganism may have a role in human genital-tract infections.

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.

The present study compared the genetic characteristics of three previously unclassified acholeplasmas of plant origin with those of the eight established species ofAcholeplasma. A radiolabeled DNA probe was derived from the lemon (L1) strain acholeplasma by using the nick translation method and hybridized to the excess unlabeled DNA isolated from the eight established species ofAcholeplasma and three new isolates from plants. Nucleic acid hydridization and serological tests confirmed that the L1 acholeplasma was distinct from all eight established species ofAcholeplasma but closely related to strains isolated from the flowers of grapefruit trees (GF1) and powder puff plants (PP2). The results suggest that these new acholeplasmas isolated exclusively from plants may represent a new species ofAcholeplasma.

Purified deoxyribonucleic acids (DNAs) of representative strains of nine established serovars of the human mycoplasma Ureaplasma urealyticum and of five strains of Mycoplasma pneumoniae were digested with a variety of restriction endonucleases. The cleavage patterns obtained by electrophoresis of the digestion products separated the nine U. urealyticum serovars into two definite clusters, one consisting of serovars 1, 3, and 6 and the other consisting of serovars 2, 4, 5, 7, 8, and 9, in agreement with previous results obtained by electrophoresis of cell proteins and DNA-DNA hybridization. Although the five M. pneumoniae strains differed in virulence and adherence capacity, they exhibited similar DNA cleavage patterns, indicating a high degree of genetic homogeneity. The enzymes KpnI, XhoI, BamHI, and PstI, which possess guanine-plus-cytosine-rich recognition sequences, cleaved the DNA of U. urealyticum (guanine-plus-cytosine content, about 28 mol%) into a small number of fragments, whereas SmaI, with the recognition sequence CCC/GGG, failed to cleave U. urealyticum DNA into visibly distinct fragments. The same restriction enzymes produced multiband cleavage patterns with M. pneumoniae DNA, which has a guanine-plus-cytosine content of about 40 mol%. Restriction endonucleases EcoRI, HindIII, HpaI, and XbaI, which have recognition sequences rich in adenine plus thymine, produced multiband patterns with the DNAs of both U. urealyticum and M. pneumoniae. We concluded that the cleavage patterns of mycoplasmal DNAs digested with restriction endonucleases provide a means for determining genetic relatedness among mycoplasmas.

SUMMARY: Electrophoretic patterns of digestion products of Acholeplasma and Mycoplasma DNA by restriction endonucleases were compared. The patterns of Acholeplasma axanthum strains isolated from a variety of hosts and habitats differed markedly from each other, indicating considerable genotypic heterogeneity among strains included in this species. Heterogeneity was less marked among the Acholeplasma oculi strains tested, and was minimal among strains of the avian pathogen Mycoplasma gallisepticum. Strains of Mycoplasma genitalium isolated from the urethra of patients with non-gonococcal urethritis and from the urethra of an experimentally infected chimpanzee yielded identical cleavage patterns, indicating a high degree of genetic homogeneity of these strains. The data support the notion that mycoplasma species of strict host and tissue specificity exhibit marked genetic homogeneity. The advantages and deficiencies of the use of DNA cleavage patterns for classification purposes are discussed.

Mycoplasma contamination of cell cultures was first reported in 1956 and continues today to be a major concern to the investigator. Although awareness has increased over the years, mycoplasma infection of cell cultures remains a common, troublesome problem that affects many cell functions and can markedly influence proper interpretation of test results. Up-dated, sensitive mycoplasma test procedures are described. These include isolation of mycoplasmas by agar and large volume broth media procedures, and detection in an indicator cell culture system with a DNA-binding fluorochrome stain. Although prevention is the first line of defense, occasionally the considerable effort required to eliminate the contaminant from an irreplaceable cell line can be justified.

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.

Four spiroplasma strains and eleven isolates tentatively identified as acholeplasmas were obtained from fall flowers in Colorado, Nebraska, Illinois, and Maryland. Although the acholeplasma isolates were heterogeneous, all showed antigenic sharing with a group of unnamed organisms (L1 and related strains) isolated in othe studies from flowers in Florida. The W20 and W24 isolates from Nebraska were partially related to the L1 group by DNA-DNA homology and polyacrylamide gel electrophoresis (PAGE) analyses. A Colorado spiroplasma (W13) was identifed as a new strain of group IV complex. Three spiroplasma strains from flowers in Maryland “old fields” represent a new serovar with closest affinity to subgroup I-4 and to the LB12 and N525 serovars of group I. Widespread occurrence of acholeplasmas on flowers in this study, and on plant surfaces in general, suggests that, like spiroplasmas they probably will be found to reside in arthropods.

Similarities in the biochemical and serological characteristics of sterol-nonrequiring acholeplasmas (mycoplasmas) have created problems in the taxonomy and classification. The present study was designed to compare the genetic characteristics of two unclassified acholeplasma strains with those of the established speciesAcholeplasma. Radiolabeled DNA probes were prepared toA. laidlawii, A. granularum, and to the two unclassified acholeplasmas (S2 and 72-043 strains) by the nick-translation method. Nucleic acid hybridization results presented here confirm and extend previously reported genetic interrelationships observed betweenA. laidlawii (PG8) andA. granularum (BTS39). The DNA of theseAcholeplasma species failed to hybridize with DNA prepared from aMycoplasma species (M. capricolum, California kid strain) or from duck cells in control studies. The two unclassified acholeplasmas were found to be related to each other but distinct from all of the established species ofAcholeplasma and may represent a new species within the genusAcholeplasma. In light of difficulties in making species separation in the acholeplasmas, data from nucleic acid hybridization tests can offer very important information useful to the classification of these organisms.

31P-NMR studies of Mycoplasma gallisepticum cells have been carried out using a continuous perfusion technique; these are the first such studies with this organism. Using this technique, glucose metabolism was monitored in the intact organisms, and cell extracts were prepared to identify the intermediates. Under glycolytic conditions, high levels of fructose-1,6-diphosphate were observed, indicating that this sugar may play a key role in the regulation of metabolism. The level of phosphoenolpyruvate was low under normal glycolytic conditions, and did not increase during starvation. From the position of the internal inorganic phosphate peak, the intracellular pH was estimated. The cells were found to maintain an intracellular pH of approximately 7.1 over an investigated external pH range of 6.6-8.6.

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.

The association between transitional L-forms of bacteria and aphthous lesions was tested by searching for these microorganisms in histopathologic sections stained by the Giemsa method. Microorganisms were found in the majority of aphthous lesions (93.3 per cent) and less frequently in nonaphthous lesions (40 per cent) and oral mucosa obtained at autopsy (47 per cent). The high incidence of these microorganisms in aphthous lesions tends to support the thesis that there may be an association between the transitional L-forms of bacteria (probably a streptococcus) and the pathogenesis of aphthous conditions. The presence of microorganisms in histopathologic sections may be a useful adjunct to clinical and microbiologic findings for the diagnosis of aphthous conditions. Systemic use of antibiotics did not seem to affect the incidence of these organisms in the oral tissues.

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.

SummaryThe effects of mycoplasma infection on interferon production were studied in hamster embryo fibroblasts. Both mycoplasmas chosen for investigation, M. arginini and M. kyorhinis, were able to replicate and establish a chronic infection in these cells. Neither mycoplasma was able to induce interferon by itself. However, both caused a pronounced decrease in the amount of interferon induced by a virus (Semliki Forest virus) and in the amount of viral resistance induced by a synthetic complexed RNA (polyinosinic and polycytidilic acid). In addition to depressing interferon production, mycoplasma infection rendered the cells less sensitive to exogenously supplied interferon. Viral transcription, however, was not impaired. Rather, under the appropriate conditions, enhanced SFV yields could be obtained from cultures previously infected with mycoplasma.

SummaryA rapid, simple and routine chemical method for detection of PPLO contamination in cell cultures is reported. The method is based on the presence of arginine deiminase activity in all tissue culture PPLO strains, but absence from all mammalian tissue and PPLO-free cell cultures. In a total of 73 cell cultures examined, the detection of PPLO determined by presence of arginine deiminase activity agreed entirely with the most sensitive cultural procedure which requires 14 days.

SummaryVaccinia virus yields were decreased from cells previously chronically infected with M. arginini but not M. hyorhinis. This decrease was found to be a result of utilization of arginine from the medium by this mycoplasma. When added arginine was incorporated, vaccinia yields were the same as in cells not infected by mycoplasma. Thus, vaccinia virus was found to be arginine dependent.

A case of acute serous pulpitis from which a pure culture of PPLO was isolated is presented, and its possible effect on our present concepts of dental pathology and therapy is discussed. Although no general conclusions are attempted on the basis of one case report, we hope that this report will generate and stimulate further interest in the possible role of filterable forms in dental pathology.

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.

SummaryMycoplasmas were isolated from 73 of 379 (19%) commercial bovine serum lots examined, including 55 of 239 (23%) unprocessed fetal bovine and calf serum sublots received from 4 suppliers and 18 of 140 (13%) final lots obtained from 2 of 5 manufacturers. Of 31 mycoplasma strains examined for identity, 28 were identified as M. arginini and 3 strains were unrelated to 39 well established Mycoplasma species and serotypes and may represent new species. Mycoplasma arginini was isolated from 10 uninoculated cell cultures and one virus pool also. Data presented indicate that M. arginini is a common contaminant of commercial bovine sera and of cell cultures and that bovine serum is a major source of bovine mycoplasma contamination of cell cultures.Addendum. Since Acholeplasma laidlawii is a common cell culture contaminant (1) of bovine origin, attempts to isolate this agent from commercial bovine serum were continued. The culture procedure was modified as follows: the horse serum component was deleted from t...

Pure cultures of a transitional L form isolated from numerous lesions in three aphthous stomatitis patients examined suggest that a relationship exists between the L form of bacteria and the pathogenesis of this disease and that some cases of recurrent aphthous stomatitis may be infectious in nature. The “L form theory” presented proposes that the stable L form variant of pathogenic bacteria is the dormant-carrier agent in recurrent aphthous ulcerations.

SummaryTwo hundred and forty-nine mycoplasmas were isolated from 87 of 185 laryngeal and urogenital tissues of 93 normal purebred beagle and mongrel dogs and were identified by the plate immunofluorescence procedure. There were 68 M. spumans, 66 M. canis, 55 M. maculosum, and 59 mycoplasmas related to strain PG-24 (recently named Mycoplasma edwardii) and one unidentified strain HRC 689 which may represent a new Mycoplasma species. Mixed mycoplasmal flora were found in cultures of 57 laryngeal, 7 cervical, and 6 prostatic-urethral tissues. The findgings indicate that four species of Mycoplasma are commensal inhabitants of dogs.

Mycoplasma arginini Barile et al. 1968 and Mycoplasma leonis Heyward et al. 1969 are regarded as synonyms on the basis of comparisons of the original species descriptions and of the serological and biochemical characteristics of authentic strains, including the type strains, of each of the organisms. According to the rules of nomenclature, the correct name of the species formed by the union of these two taxa is M. arginini Barile et al., which name has priority over M. leonis Heyward et al.

Incubation of the soluble fraction derived from Mycoplasma gallisepticum cells with [gamma-32P]ATP results in the phosphorylation of several endogenous proteins. One protein with an apparent molecular mass of 55 kDa was the acceptor of more than 95% of the radioactive phosphate. This protein was also found to be radiolabeled in intact cells grown in the presence of [32P]orthophosphate. Acid hydrolysis of the phosphorylated 55-kDa protein followed by two-dimensional electrophoresis revealed that the 32P-labeled material co-migrated with phosphoserine. The in vitro phosphorylation of the 55-kDa protein has an optimum pH of 5.5-6.0 and is not affected by various metabolites of glycolysis, by cAMP or by calmodulin with or without Ca2+. The phosphorylation is dependent upon divalent cations, a dependency that is best fulfilled by the simultaneous addition of Ca2+ and Zn2+ that act in a specific and cooperative manner. Of a variety of possible exogenous protein acceptors tested, the endogenous protein kinase was capable to phosphorylate only phosvitin. The phosphorylation of the 55-kDa protein is reversible through the activity of a phosphoprotein phosphatase present in the soluble fraction of M. gallisepticum. The phosphoprotein phosphatase has an optimum pH of 7.5-8.0, is inhibited by NaF and stimulated to a large extent by inorganic phosphate and arsenate and to a lesser extent by pyrophosphate ATP and ADP. The possible association of the reversible protein phosphorylation to cell shape and gliding motility of M. gallisepticum are discussed.

Mycoplasma organisms are a major cause of respiratory, urogenital and joint infections in man and animals. The spectrum of the disease is wide and includes a number of complications.

The attachment of Mycoplasma genitalium and Mycoplasma pneumoniae to ciliated epithelium involves two surface proteins designated MgPa and P1, respectively. We have previously cloned and sequenced the P1 (mpp) operon of M. pneumoniae, and report here the use of P1-derived probes to clone and sequence a 10.4-kb region of M. genitalium DNA that, by analogy to the P1 operon, contains the MgPa (mgp) operon. The deduced amino acid sequences of the 29-kDa (ORF-1), MgPa (160-kDa) and 114-kDa (ORF-3) proteins of the MgPa operon show extensive homologies with those of the 28-kDa, P1 (170-kDa) and 130-kDa proteins, respectively, encoded by the P1 operon. The common features and homology of these operons are consistent with previous observations that the MgPa and P1 proteins share cross-reactive epitopes, as well as similar biological function. The gene order of the MgPa operon is ORF-1, MgPa, ORF-3, with intervening regions of 6 and 1 nt, respectively. A consensus ribosome-binding site (RBS) sequence is found before ORF-1 and a sequence indicative of a transcription terminator is located beyond ORF-3; the absence of such sequences adjacent to the MgPa gene suggests that the operon is transcribed as a polycistronic message. The RBS sequence is followed by sequences of dyad symmetry that have the potential to form two alternative stem-and-loop structures, which could be involved in controlling initiation of translation.

It has been established that certain mycoplasmas can alter the activity and function of contaminated cell cultures and thereby markedly affect the results of study. Some of these effects have been reviewed elsewhere (1, 2). This presentation will briefly discuss three general aspects of mycoplasma-cell-virus interaction including: a) the cytopathic effects (CPE) produced by the myco-plasma fermenters; b) the inhibitory effect produced by the arginine utilizing mycoplasmas on lymphocyte blastformation; and c) the effects of mycoplasmas on virus propagation.

Knowledge of the pathogenesis of pneumonia due to Mycoplasma pneumoniae has been derived primarily from experimental infection of rodents. As part of an effort to establish a model with a closer resemblance to man, three seronegative, young, adult rhesus monkeys (Macaca mulatta) were inoculated with M. pneumoniae (10(7.4) cfu per animal) by oropharyngeal administration of coarse-particle aerosol. Five to six days after exposure of the animals, cultures obtained from the upper respiratory tract were positive for M. pneumoniae. Each animal subsequently developed a serologic response, as determined by complement fixation, complement-mediated killing, and tetrazolium-reduction inhibition techniques. Infection was subclinical, and serial chest roentgenograms failed to disclose pneumonia throughout the course of infection. Blood cell counts and titers of cold agglutinins remained unchanged. Althought M. pneumoniae was recovered from the upper respiratory tract of two monkeys for 50 days, there was no evidence of transmission of infection to cage-mate controls inoculated with broth.