Inflammatory toxin from Mycoplasma bovis: isolation and characterization.

An inflammatory toxin was extracted from Mycoplasma bovis with 75 percent aqueous ethanol. The toxin is a complex polysaccharide composed of glucose, glucosamine or galactosamine, and a heptose, is heat-stable, devoid of protein and lipid, and has a molecular weight of 73,000. The holotoxin in the cell membrane is a glycoprotein; however, it is the polysaccharide portion that is toxic. This inflammatory toxin increases vascular permeability and is capable of activating complement. Infusion of 0.9 milligram of toxin into the bovine udder resulted in the characteristic eosinophilic mastitis produced by Mycoplasma bovine.

Simultaneous detection of reverse transcriptase and high molecular weight RNA unique to oncogenic RNA viruses.

The simultaneous assay of the reverse transcriptase and 60S to 70S RNA of oncogenic RNA viruses is described. The virus can be detected at low concentrations in biological fluids containing enzymatically active cell fragments or other contaminants. The fact that two features diagnostic of the oncornaviruses are used in the tests increases the certainty with which a positive outcome can be interpreted.

Induction of adenine salvage in mouse cell lines deficient in adenine phosphoribosyltransferase.

Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) pseudorevertant cell lines were isolated under selective conditions requiring adenine salvage for survival; yet they were found to be deficient in measurable APRT activity and resistant to the purine analog 2'6'-diaminopurine (DAP) (M.S. Turker, J. A. Tischfield, P. Rabinovitch, P.J. Stambrook, J.J. Trill, A.C. Smith, C.E. Ogburn, and G.M. Martin, manuscript in preparation). Adenine salvage was examined in two APRT pseudorevertant cell lines, their two APRT homozygous deficient parental cell lines, and a genotypic APRT revertant cell line (i.e., one with measurable APRT activity and DAP sensitivity). Adenine accumulation was observed in both revertant phenotypes and was demonstrated by high-performance liquid chromatography to be linked with adenine metabolism. The ability to salvage adenine declined substantially in the pseudorevertant cell lines when they were removed from selective media containing inhibitors of de novo 5'-AMP synthesis (alanosine and azaserine); for one pseudorevertant cell line this decline was accelerated by the addition of DAP to the medium. The readdition of alanosine or azaserine to the growth medium of the pseudorevertant lines induced adenine salvage to its previous levels. An APRT-like cross-reacting material was found in the pseudorevertant cell lines, although its relationship to adenine salvage is unknown. A low level of constitutive adenine salvage was found in the parental APRT-deficient lines, and it was also possible to induce adenine salvage in these cell lines. These findings suggest a novel regulatory mechanism for adenine salvage.

Histone-like protein in the prokaryote Thermoplasma acidophilum.

The DNA of the prokaryote Thermoplasma acidophilum is associated with a histone-like protein that has the following properties: it has a high content (23%) of basic amino acids, is positively charged at neutral pH, is soluble in acid, and can stabilize DNA against thermal denaturation. In polyacrylamide gel electrophoresis, in the presence of either sodium dodecylsulfate or urea, it migrates at the same rate as histone IV (F2a1) of calf thymus. The amino acid composition, however, it unusually rich in the amides of acidic amino acids (16-20%), and it does not appear to be closely homologous to any of the classes of eukaryotic histones. Escherichia coli DNA, on the other hand, was associated with no detectable acid-soluble proteins, and the nucleoprotein thermally denatured at a lower temperature than pure DNA.

Analysis of membrane fractions from Mycoplasma gallisepticum.

Membrane fractions have been isolated from Mycoplasma gallisepticum following a procedure derived from that described by Maniloff, J. and Quinlan, D.C. (J. Bacteriol. (1974) 120, 495-501). A light fraction F1 was obtained which contained structures resembling the bleb-infrableb apparatus characteristic of M. gallisepticum. It was enriched in DNA and had an electrophoretic profile different from that of unfractionated membranes. Cholesterol-to-phospholipid ratios higher than two and elevated values of the ratio of saturated to unsaturated fatty acids were other characteristics of this fraction. The two other fractions isolated (FII and FIV) also differed from intact membranes by their cholesterol and phospholipid content as well as by their saturation ratios. The membrane fluidity of FII and FIV, estimated by fluorescence polarization, was similar to that of unfractionated membranes while a slight but significant difference was recorded for the light fraction. Possible relationships between the lateral heterogeneity of the M. gallisepticum membrane and the obtainment of fractions are discussed.

Studies on the phospholipid composition of pathogenic cell membranes of Mycoplasma hyopneumoniae.

The membrane phospholipid and fatty acid compositions of Mycoplasma hyopneumoniae, a pathogen of porcine enzootic pneumoniae isolated in China, was studied by thin-layer chromatography and gas chromatography. The results showed that membrane phospholipids consisted predominantly of diphosphatidylglycerol. The percentage of C16 - C18 fatty acids comprised 79% of the total fatty acids, of which oleic acid as well as palmitic acid are the major fatty acids. Some differences were shown in fatty acid composition as compared with membranes of other species of Mycoplasma.

A chemiluminescent assay for mycoplasmas in cell cultures.

A chemiluminescent assay for the detection of mycoplasma contamination of cell cultures is described. Cells (and supernatant) derived from mycoplasma-contaminated cultures stimulate a burst of luminol-dependent chemiluminescence in cell suspensions containing phagocytic effector cell types. The assay conditions for spleen cells, human and bovine polymorphonuclear leucocytes as the responder or indicator cells have been optimized. The chemiluminescent assay can be utilized for both monolayer and suspension cell cultures and is more sensitive than colony formation on agar plates and electron microscopy. Results are obtained within 3-5 h including the time required for the preparation of the indicator cells. CL can be measured in the tritium window of standard liquid scintillation spectrometers after switching off the coincidence circuit.

An improved method for elimination of mycoplasmas from cell cultures.

Cell lines infected by different species of mycoplasma (Mycoplasma orale, Mycoplasma hominis) were decontaminated by co-culture with human blood monocyte (BM)-derived macrophages and pooled human immunoglobulin preparations. Co-cultures with BM-derived macrophages or murine peritoneal macrophages (PM) alone were not successful. The phenotype of infected cell lines did not differ from that of uninfected cell lines as revealed by morphological, enzymecytochemical, and immunocytochemical analysis.

Rapid and simple identification of mycoplasmas by immunobinding.

A simple and rapid method of species identification of mycoplasmas by immunobinding assay is described. Small amounts of antigen of supernatant from cell cultures, broth cultures or clinical specimens were spotted onto nitrocellulose paper. This was followed by application of specific anti-mycoplasma antisera. After incubation, an enzyme-conjugated antiserum against the first antiserum was applied. A positive reaction was indicated by the development of intense blue color reaction when substrate was added. This method identified mycoplasma species with monoclonal and polyclonal antibodies. It detected 9.3 X 10(3) - 7.5 X 10(4) CFU/ml of organisms depending on mycoplasma species. For identification of mycoplasma, ureaplasma, acholeplasma and spiroplasma species, this assay is useful and rapid compared with other serological methods. In limited studies, the method correlated with microbiological assay of clinical specimens for Mycoplasma pneumoniae.

A sensitive and quantitative microassay for the detection of mycoplasma contamination: inhibition of IL-2 dependent cell line proliferation.

A simple, rapid and sensitive microassay for mycoplasma detection in cell culture is reported. The assay is based on the fact that culture supernatants from contaminated cells inhibit [3H]thymidine incorporation by an IL-2 dependent mouse cytotoxic T cell line (CTLL). The mechanism of inhibition is related to the production by several mycoplasma strains of a pyrimidine-specific nucleoside phosphorylase which can degrade the radiolabelled thymidine used for the measurement of DNA synthesis. These strains were the commonest contaminants in cultures of 24 cell lines from 5 different sources. To establish the sensitivity of the test to detect mycoplasmas we have also used the inhibition assay to monitor the clearance of mycoplasma from 2 contaminated cell lines.

The elimination of mycoplasma from infected hybridomas by passaging in BALB/c mice.

Hybridomas, which were found to be infected with mycoplasma, were cleared of contamination by passaging in BALB/c mice. This procedure was successfully applied to four of five cultures examined. The procedure offers a simple and effective means of eliminating mycoplasma from valuable and sometimes irreplaceable hybridoma cell cultures.

Medium-dependent properties of mycoplasmas.

Without a cell wall, the morphology, growth rate, and composition of mycoplasmas are culture media-dependent with variable properties best described as environmentally related. The adaptation of mycoplasmas to either a tissue cell or cell-free culture media, with dependency upon specific animal or plant products for survival, has led to investigations of their human host-related properties. The influence of culture media on the antibiotic sensitivities of mycoplasmas was measured by use of three different broths in two different assay systems. The variable results indicate that the inhibition of mycoplasma protein synthesis or growth may also be host-tissue dependent. The addition of noninhibitory penicillins to different culture media was found to affect the composition and antigenicity of some mycoplasmas. Using the complement fixation test, we found some human sera that were more reactive than rabbit antisera to mycoplasmas cultured in human synovial broth or in myelin-enriched broth. Mycoplasmas cultured in human lung broth and pig lung broth had media-dependent antigenicity. The antigenicity and the growth of mycoplasmas were found to depend on the proteolytic enzymes used to provide the essential peptides in tissue broths. The media-affected mycoplasmas indicate the presence of species-, strain-, and tissue-specific antigen sites that may determine immunopathogenicity in the genetically susceptible host.

Comparison of seven isolates of Mycoplasma meleagridis.

A comparative study of seven isolates of Mycoplasma meleagridis indicated that they were indistinguishable morphologically. Two isolates, E2 and 8M92, induced hemagglutination of red blood cells of several different species while the others did not. Metabolic inhibition, growth inhibition and growth precipitation tests revealed minor differences among the seven isolates. According to these differences, isolates were divided into three groups: antiserum-sensitive isolate 1466, less sensitive isolates N, 8M92, RY3, 529 and E2 and insensitive isolate 1940. One dimensional polyacrylamide gel electrophoresis of cell proteins revealed that all isolates of M. meleagridis had virtually identical patterns and that they were electrophoretically distinct from Mycoplasma gallisepticum and Mycoplasma synoviae. When nonhemagglutinating isolate N, and hemagglutinating isolate E2 were examined by simple immunoelectrophoresis, no differences were detected. However, minor antigenic differences were detected between the two strains by means of two dimensional immunoelectrophoresis.

Evaluation and practical aspects of the use of a commercial DNA probe for detection of mycoplasma infections in cell cultures.

Cell cultures have been analyzed for mycoplasma infections by using a commercial DNA-probe based on rRNA genes from mycoplasmas. Both the original version, Mycoplasma T.C. Detection Kit, and the improved version of the kit, Mycoplasma T.C. II Rapid Detection System, were used. The sensitivities of the two tests were found to be adequate in most cases and the improved version of the kit was 10-100 times more sensitive than the original one. A batch variation was observed with the improved version, which is not satisfactory. This batch variation can, however, be checked and the performance of the method with a properly working lot was found to be good.

Heterogeneity of Mycoplasma iowae determined by restriction enzyme analysis.

Strains of Mycoplasma iowae were homogeneous in some characteristics and heterogeneous in others. Thus, the biochemical tests, immunofluorescence, and protein profiling by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were group-specific tests. However, some minor differences in protein patterns were seen among strains. The growth inhibition test tended to be strain-specific. Hemagglutination titers were very low and unstable for the majority of strains. One strain (RY-65) with a stable high-titer hemagglutinin failed to react in the hemagglutination-inhibition test against immune sera to the reference strains. Restriction endonuclease DNA analyses was the most useful method to differentiate 1 strain from another.

Mycoplasma gallisepticum strain variations detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Various strains of Mycoplasma gallisepticum (MG) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Minor but distinct and reproducible differences in protein banding patterns were detected between strains, which included the vaccine F strain from various sources, an atypical (variant) strain, and the standard (A5969, S6) strains.

Status of the 1805 strain of Mycoplasma iowae (K component of serovar I,J,K, N,Q,R) and the SA strain of M. gallopavonis (serovar F).

Contrary to historical evidence, recent biochemical and serological studies have suggested that the 1805 strain of M. iowae (K component of serovar I,J,K,N, Q,R) and the SA strain of M. gallopavonis (serovar F) were members of the species M. gallinaceum. After examining three seeds of strain 1805 and an original seed of the SA strain, we present evidence to indicate that 1805 and SA are indeed valid members of the species M. iowae and M. gallopavonis, respectively. However, some seeds of strains 1805 and SA may contain M. gallinaceum. In the case of strain 1805, it is possible that the original seed may have contained M. gallinaceum.

Identification of Mycoplasma gallisepticum and M. synoviae by flow cytometry.

Immunofluorescence and flow cytometric methods were examined to detect and distinguish Mycoplasma gallisepticum and M. synoviae. The procedure employed 24-hr broth cultures of each organism, direct immunofluorescence staining with either homologous or heterologous antiserum, and analyses by flow cytometry. The organisms were distinguishable on the basis of fluorescent profiles when stained with the appropriate antiserum.

Glycoconjugate heterogeneity among five strains of Mycoplasma gallisepticum.

Five strains of Mycoplasma gallisepticum (MG) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for the presence of carbohydrate-containing components. Staining with periodic acid-Schiff (PAS) demonstrated carbohydrate components in three of the five strains studied. The PAS-reactive bands counterstained for protein, indicating a possible glycoprotein nature. Western blot analysis using three biotinylated lectin probes demonstrated the presence of additional glycoconjugates in the blot profiles of each MG strain. The carbohydrate specificity of lectin binding was demonstrated by competition experiments using specific sugars. Differences in the number, electrophoretic mobility and the morphology of PAS and lectin reactive bands were reproducible among separate preparations of each MG strain. These findings indicate substantial phenotypic diversity among the five MG strains in their ability to produce or acquire glycoconjugates.

Comparison of strains of Mycoplasma iowae.

Comparison of biochemical test results and protein electrophoretic patterns of 21 strains of Mycoplasma iowae indicated that all were similar. Comparison of agglutination test results indicated marked within-species antigenic variation. None of 21 antigens prepared from different strains were effective in demonstrating turkey antibody against five reference strains. Examination of sera from turkeys exposed by intra-air-sac inoculation to two pathogenic strains also indicated antigenic variation. Neither the M. iowae type-strain, Iowa 695, nor the other reference strains were effective in demonstrating antibody against both strains used to expose the turkeys. These findings suggest that antigenic variation may be a major problem in effective serodiagnosis of M. iowae infections.