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In 1956, Robinson and colleagues (1) reported the first isolation of a mycoplasma from a contaminated cell culture. Subsequently, mycoplasmas have been shown to be common and bothersome contaminants capable of altering the activity of cells and affecting the results of study. Because many of the vaccines prepared for human use are produced in cell cultures and are subject to mycoplasma contamination, the Bureau of Biologics has maintained a continuing study for the past 18 years to examine various aspects of mycoplasma contamination. This report will review some of our findings and present a brief, updated status report on the incidence, prevalence and sources of mycoplasma contamination. The subject has been reviewed in detail elsewhere (2–5).

Attachment of Mycoplasma pneumoniae to host cells initiates disease, and the attachment components may represent important protective immunogens for preventing disease. We have studied the mechanisms of attachment using in vitro cell culture systems and selected pathogenic and nonpathogenic strains of M. pneumoniae. Attachment of the pathogenic strains M129 and PI-1428 was several fold greater than attachment of the nonpathogenic strain, and attachment of strains M129 and PI-1428 was reduced by 21 to 63% when human WiDr cell monolayers were exposed to neuraminidase, supporting the concept that M. pneumoniae attaches to mammalian cells by a neuraminidase-sensitive glycoconjugate. While attachment of the two pathogenic strains was markedly reduced by treating the WiDr cells with glutaraldehyde, glutaraldehyde treatment produced minimal effects on the attachment of the nonpathogenic strain B176. Glutaraldehyde treatment also altered the temperature dependence of attachment by the pathogenic strains. Because glutaraldehyde-treated WiDr cell monolayers showed little difference in attachment between pathogenic and nonpathogenic strains, glutaraldehyde-treated cells are not appropriate cell substrates for studying M. pneumoniae attachment mechanisms or identifying immunogens for vaccine development.

Lipoglycans (previously designated lipopolysaccharides) from several species of Acholeplasma and from Thermoplasma acidophilum were examined for endotoxin-like activities as measured by the standard rabbit fever test and the Limulus amoebocyte lysate assay. The lipoglycans from Acholeplasma granularum, Achloplasma laidlawii, Acholeplasma modicum , and Acholeplasma oculi caused a febrile response at concentrations of 1 ng/ml per kg or greater, whereas with control Escherichia coli EC-2 lipopolysaccharides, 6.25 ng/ml per kg was required. Similar results were obtained in the Limulus amoebocyte lysate test. The minimum concentrations in nanograms per milliliter required to stimulate formation of a solid clot were: Acholeplasma axanthum , 0.22; A. granularum , 0.85; A. modicum , 0.51; A. laidlawii , 1.05; A. oculi , 0.74. Standard E. coli 1B lipopolysaccharide required a concentration of 0.125 ng/ml. Thermoplasma lipoglycan was least active, requiring 4.25 ng/ml. Clotting of the Limulus lysate proceeds by the activation by lipopolysaccharide plus Ca 2+ of a proenzyme which cleaves an arginine-lysine peptide bond of the coagulogen. The clotting and amidase activities are inactivated by deoxycholate and can be reactivated by addition of lipopolysaccharide and Ca 2+ . As with E. coli 1B lipopolysaccharide, acholeplasmal lipoglycans were shown to restore both clotting and amidase activities of the deoxycholate-inactivated Limulus clotting enzyme. The degree of restoration of amidase activity by mycoplasmal lipoglycans relative to E. coli lipopolysaccharide (1.00) were: A. axanthum , 1.71; A. modicum , 1.22; A. granularum , 0.61; and Thermoplasma , 0.37. The coagulating enzyme, restored with either E. coli lipopolysaccharide or mycoplasmal lipoglycans, was able to react with the synthetic peptide benzoyl-Ile-Glu-(γ-OCH 3 )-Gly-p-nitroaniline (an analog of the coagulogen) or with the purified coagulogen itself to form the clot. The mycoplasmal lipoglycans alone were incapable of promoting these reactions when incubated with the synthetic peptide or with the purified coagulogen, thereby ruling out the contamination of these lipoglycans with proteases capable of cleaving the same Arg-Lys peptide bond of the coagulogen. These results show that acholeplasmal lipoglycans possess endotoxin-like activities. Their passive or active role in disease remains to be established.

Virulent strains of Mycoplasma pneumoniae, PI-1428 and M129, were radiolabeled wtih [3H]palmitic acid or [3H]thymidine and examined for attachment to hamster tracheal organ cultures, tracheal outgrowth monolayers, human O-positive erythrocytes, and human WiDr carcinoma cell cultures. Although attachment to each cell substrate was readily detected, the WiDr cell culture monolayers provided the most satisfactory substrate for quantitating mycoplasma attachment. Serious technical limitations were encountered with each of the other substrates that we examined; these limitations interfered with reproducibility or sensitivity and rendered tracheal organ cultures and erythrocyte suspensions unsuitable for routine attachment and attachment inhibition assays. Moreover, the WiDr cell monolayer was the most sensitive substrate for determining attachment inhibition activity in protein-containing extracts prepared from M. pneumoniae. The significance of these findings is discussed.

Attachment of Mycoplasma pneumoniae to human WiDr cell culture monolayers was examined by using radiolabeled M. pneumoniae. The amount of attachment was proportional to the density of the WiDr cells and to the concentration of M. pneumoniae in the assay. Saturation of the monolayers was achieved with 40 micrograms of virulent strain M129 per assay, whereas binding of avirulent strain B176 was 70% less than that of strain M129. A competitive attachment inhibition assay was used to measure specific binding component activity. Attachment was inhibited when WiDr cells were pretreated with unlabeled virulent strain M129, whereas avirulent noncytadsorbing strain B176 did not inhibit attachment as well as the virulent strain. A protein-rich extract prepared from virulent, cytadsorbing strains of M. pneumoniae also inhibited attachment. The amount of inhibition was dependent on the amount of extract used, and units for binding component activity in the extract were calculated from the competitive attachment inhibition assays. The competitive attachment inhibition assay was also used to investigate the nature of the receptor site on the WiDr cells. Attachment was inhibited when the radiolabeled M. pneumoniae suspensions were pretreated with human sialoglycoproteins, such as orosomucoid and ceruloplasmin, and bovine gangliosides. These findings support the present concept that the mammalian receptor site for M. pneumoniae is a sialic acid-containing glycoprotein.

Mycoplasma pneumoniae strains PI-1428, M129, B176, FH, and MAC were analyzed for homology by DNA-DNA hybridization, thermal elution midpoints of the DNA-DNA heteroduplexes, DNA cleavage patterns with restriction endonucleases, and protein gel electrophoretic profiles. These properties were compared with biological assays, such as antigenic reactivity with reference antisera in the growth inhibition and metabolic inhibition tests, as well as the ability to attach to human WiDr cell cultures. The avirulent, nonhemadsorbing strain B176 could be differentiated from the others by diminished attachment capacity to WiDr cells and by slightly reduced DNA homology. The other strains appeared to be identical when examined by these procedures. No significant differences were detected among any of the strains by the metabolic inhibition or growth inhibition tests. The DNA cleavage patterns of the five strains were also similar, although strains FH and MAC differed from the other three strains in a few bands. These results demonstrate that these five strains are similar and that the species M. pneumoniae is remarkably homogeneous.

SUMMARY: This study compares the intraspecies genetic relatedness among strains of two established species of Acholeplasma. Radiolabeled DNA probes were prepared from three strains of Acholeplasma laidlawii and two strains of Acholeplasma axanthum, by using the nick translation method. The labelled DNA probes of these two strains were hybridized to an excess of unlabelled DNA from 12 strains of Acholeplasma laidlawii and from six strains of Acholeplasma axanthum, respectively. Nucleic acid hybridization analyses showed a wide variation among strains within each of the two established species, ranging from 48 to 100% homology. The results demonstrate that strains isolated from diverse hosts and habitats within a given species of Acholeplasma exhibit extensive genotypic variations.

Anderson, Douglas R. (National Cancer Institute, Bethesda, Md.), and Michael F. Barile . Ultrastructure of Mycoplasma hominis . J. Bacteriol. 90: 180–192. 1965.—Both thin-sectioning and negative staining were used in an electron microscopic study of the morphology of pleuropneumonia-like organism (PPLO) strain HEp-2 ( Mycoplasma hominis , type I) grown in an artificial liquid medium. The morphology is quite variable and seems to depend, in part, on the age of the culture. The smallest form observed (“elementary body”) is 80 to 100 mμ in diameter. The internal components of the larger PPLO cells (0.5 to 1 μ) are variable—some have ribosomelike granules and nuclear areas of netlike strands, and others have only irregular dense areas in a pale groundplasm. Some of the forms have dense cytoplasmic bodies which look much like elementary bodies. Others have vacuoles which may contain structures which look like smaller organisms. Especially in older cultures, very large (10 μ) vacuolated organisms are seen, probably corresponding to the “large bodies” described by light microscopists. Filamented forms are also seen. These observations suggest several possible modes of reproduction, each perhaps operating under different cultural conditions or at different ages of the culture.

Mycoplasma (Pleuropneumonia-Like Organisms) and Blood Group I; Associations with Neoplastic Disease

A diphtheria toxin shown to have high toxicity and avidity and to combine with antitoxin in multiple proportions was selected for the U.S. standard diphtheria toxin for the Schick test and freeze-dried. Assayed values per vial were 1.09 L f , 1.09 L r , 1,090 Schick test doses (STD), 33 LD 80 , 38 LD 50 , and 43,000 minimum skin reactive doses. One STD (L r /1000) is slightly more toxic than one unit of the international standard (L f /1,000). Experiments showed that the potency assay of Schick test toxin by guinea pig erythema toxicity, determined relative to the toxicity of the standard, was highly reproducible and significantly more reproducible than the lethal (minimum lethal dose) test and that the STD, defined as one L r /1000, was equivalent to approximately 1/50 minimum lethal dose. The erythema potency assay was prescribed in the U.S. standards for Schick test toxin effective in 1969.

Schimke, Robert T. (National Institutes of Health, Bethesda, Md.) and Michael F. Barile . Arginine metabolism in pleuropneumonia-like organisms isolated from mammalian cell culture. J. Bacteriol. 86: 195–206. 1963.—Arginine degradation is a significant metabolic process for pleuropneumonia-like organisms (PPLO; Mycoplasma ) isolated from cell culture. The conversion of arginine to ornithine in PPLO-contaminated cell culture was rapid, and occurred by the arginine dihydrolase pathway involving arginine deiminase, ornithine transcarbamylase, and carbamyl phosphokinase. In the absence of PPLO contamination, arginine conversion to ornithine was minimal and took place by an arginase activity present in the cell culture, but not in the PPLO. All five PPLO strains isolated from cell culture accomplished the conversion of arginine to ornithine, and contained the requisite enzyme of the arginine dihydrolase system, whereas PPLO-free cell cultures did not. Supplementation of PPLO culture broth with arginine increased the extent of PPLO growth. When the arginine content of the culture limited growth, arginine was completely converted to ornithine. When growth was limited in the presence of excess arginine, citrulline was the major breakdown product. It is suggested that the conversion of arginine to ornithing constitutes a significant, and possibly major, source of adenosine triphosphate for this class of organisms.

SummaryAn assay using the L-929 mouse fibroblast cell culture line was described to evaluate the toxicity of Arlacel A. The toxic lots of AA produced a characteristic cytotoxic effect consisting of intracytoplasmic refractile bodies. The results obtained by the cell culture and mouse assays were similar for 18 of 19 lots tested; one lot was moderately toxic by the cell culture assay only. A correlation was seen between the amount of FFA and the toxicity of AA. The cell culture assay provides a sensitive and reproducible assay to evaluate AA toxicity.We thank Dr. B. S. Berlin for his critical review of the manuscript, the Cell Biology Section (DBS) for providing the cells used in these studies, and acknowledge the excellent assistance of Mr. Donald Riggs, Mrs. Norma Duffin, Mrs. Marion Grabowski, Mr. Ethan Summers, and Mrs. Hazel Young.

Mycoplasmas isolated from simian ( Cercopithecus aethiops ) tissues were shown to have biological, biochemical, and serological properties and electrophoretic cell protein patterns similar to strain Navel isolated from man 15 years ago. The simian and human Navel strains comprised a single serogroup, distinct from the established Mycoplasma and Acholeplasma species of the class Mollicutes . It is proposed that strains with the properties described be named Mycoplasma primatum .

Publisher Summary This chapter discusses incidence, source, prevention, and problems of the elimination of mycoplasma contamination of cell cultures. A test for mycoplasmas is required for viral vaccines produced in cell cultures. Several procedures are used, including a standard culture procedure for isolating mycoplasmas from contaminated cell cultures; a more sensitive large specimen-broth culture procedure for isolating small numbers of mycoplasmas from minimally infected or contaminated specimens; and a semisolid broth culture procedure for screening cell cultures for microbial contamination. Because mycoplasmas are frequently present in oral and genital tissues of man and animals, and in neoplastic tissues, cell cultures prepared from these tissues would theoretically have a greater contamination risk. The 1063 contaminants isolated in the study were identified as either human, bovine, swine, murine, avian, or canine species of mycoplasmas. Human, bovine, and swine species of mycoplasmas caused 1046 of the contaminations observed. Contamination can be spread from cell to cell either by contaminated virus pools, antisera, or by other reagents commonly used in cell culture studies. The measures used are designed to control the sources and the spread of contamination.

Fourteen conjunctival strains of mycoplasma were isolated from infected sheep and goats in two separate naturally occurring outbreaks of keratoconjunctivitis (pink-eye). The biological and serological properties and cell protein patterns of these strains are presented in this report. These strains were related to each other and were unrelated to 40 recognized Mycoplasma and Acholeplasma species and serotypes. We are proposing that this group of mycoplasmas be named M. conjunctivae. The role of M. conjunctivae in the etiology of keratoconjunctivitis in sheep and goats remains to be determined.

Mycoplasma pneumoniae stimulates transformation and mitosis in cultures of lymphocytes obtained from sensitized individuals1, as do other antigens2. In contrast, M. hominis3 and M. arthritidis4 inhibit lymphocyte transformation induced by phytohaemagglutinin (PHA). We have examined the effects of different species of Mycoplasma on lymphocyte cultures in an attempt to elucidate the mechanism of mycoplasma inhibition of PHA-induced transformation.

Ten strains of Mycoplasma were isolated from brain tissues of a scrapie infected sheep and mouse, joint exudate of an arthritic goat, and cell cultures established from human, chimpanzee, and dog tissues. These strains were related to each other, but were unrelated to known species of Mycoplasma. The present report characterizes the new strains, proposes that they be classified as Mycoplasma ariginini and discusses their ecological significance.Materials and Methods. Media for isolation. Hayflick (1), BYE (2), and BBL (3) broth and agar media were used under aerobic and anaerobic (5% CO2 in nitrogen) conditions (2). See Table I for media used for isolation of individual strains.Media for metabolic reactions. Arginine utilization reaction: Hayflick and BYE broth media were supplemented with 10 mM arginine, 10 mM glutamine, vitamins (4), 0.002% phenol red, and adjusted to pH 7.1.Glucose fermentation reaction: Hayflick broth medium was supplemented with 0.5% glucose, 10 mM glutamine, vitamins, 0.004% cresol ...

The Bureau of Biologics (formerly the Division of Biologics Standards) requires that a test for mycoplasmas be performed on viral vaccines produced in tissue cultures. Consequently, we have maintained a continuing study of the contamination of cell cultures by Mycoplasma, the presence of Mycoplasma in animal tissues used in cell-culture preparations for production of vaccine, and the mycoplasmal flora of oral and genital issues of various animals, including the African green monkey, Cercopithecus aethiops. The monkeys examined were obtained from Kenya and were quarantined in the United States for at least six weeks prior to study. The study was supported by our contracts program and was supported by four participating laboratories [1]. Two laboratories were responsible for maintaining the monkeys and for collecting the tissues, while two others, and in particular Mr. R. A. DelGiudice of Huntington Research Center, were responsible for the isolation and identification of species of Mycoplasma and Acholeplasma. To minimize gross bacterial contamination, tissues were approached through the thoracic and peritoneal cavities. A midline incision was made and the entire larynx and cervix or prostratic urethral tissues were removed aseptically. The mucosal linings of these tissues were cultured for Mycoplasma. Duplicate broth and agar cultures were incubated aerobically or in a C02-N2 environment [2]. Positive broth cultures were subcultured to a series of agar plates. The agar subcultures and the original agar cultures were stained directly with a series of fluorecein-conjugated antisera, and species of Mycoplasma were identified by epiimmunofluorescence [3] (table 1). The microscope used was the Carl Zeiss epiilluminator, which directs ultraviolet light to the surface of the

AT certain times, unexpectedly high yields of vesicular stomatitis virus (VSV) have been obtained from infected primary hamster embryo fibroblast (HEF) cell cultures,, which were later found to have been contaminated with mycoplasma. The work reported here indicates that this contamination can enhance VSV replication.

IT has been established that continuous tissue cell cultures are commonly contaminated with pleuropneumonia-like organisms and/or L-forms of bacteria1–5. These results have encouraged investigators to search for reasonably simple methods for the detection of these agents, especially in relation to studies for their elimination. Of methods proposed for their detection, cultivation and isolation have proved most successful6. With the advent of the fluorescent antibody technique as a rapid method for the identification of micro-organisms, it seemed to us that this procedure might be useful as a means of detecting contamination of pleuropneumonia-like organisms in tissue cell cultures. The feasibility of this technique is presented here.

FOUR laboratories have collaborated to isolate and identify mycoplasmas from the pharyngeal and lower urogenital tissues of the African green monkey. Seventy mycoplasmas were isolated from fifty-two of 142 specimens obtained from seventy-one monkeys. Most of the simian mycoplasmas isolated were similar or identical to Mycoplasma species previously isolated only from man.

The purpose of the Round Table is to summarize the current status of information on selected topics in mycoplasmology. Invited speakers will introduce the subject and summarize salient data followed by an open discussion.