An approach to characterizing uncultivated prokaryotes: the Grey Lung agent and proposal of a Candidatus taxon for the organism, 'Candidatus Mycoplasma ravipulmonis'.

An approach to characterizing uncultivated bacteria which combines a PFGE procedure for obtaining purified full-length chromosomes with PCR amplification is described. Isolated chromosomes from uncultivated organisms provide a specifically identifiable source material for hybridization, amplification and cloning. The availability of purified chromosomes for DNA amplification should aid in examining the diversity of microbial populations and should also reduce the possibility of forming hybrid DNA artifact molecules. The approach is illustrated by isolating the chromosome of the uncultivated agent of rodent Grey Lung disease and using the purified chromosomes to amplify and directly sequence the evolutionarily conserved 16S rRNA gene. The Grey Lung agent (GLA) contains a 650 kb chromosome and is shown to be a Mycoplasma sp. located phylogenetically in the hominis group of mycoplasmas. If a simple genomic lesion(s) is responsible for the unculturability of GLA, it is conceivable that complementation with DNA from a close relative could permit growth on artificial media.

Mycoplasma auris sp. nov., Mycoplasma cottewii sp. nov., and Mycoplasma yeatsii sp. nov., new sterol-requiring mollicutes from the external ear canals of goats.

Three mycoplasma strains, designated GIHT (T = type strain), UIAT, and VIST, were isolated from the external ear canals of goats and were shown to be serologically distinct from each other and from previously described Acholeplasma, Entomoplasma, Mesoplasma, and Mycoplasma species. Using light and transmission electron microscopy, we showed that the cells of these organisms were small, pleomorphic, coccoid, nonmotile, and nonhelical and that each cell was surrounded by a single cytoplasmic membrane. There was no evidence of a cell wall, and the organisms grew freely in media containing penicillin at concentrations of 1,000 U/ml or more and thallous acetate (final concentration, 1:4,000) and produced the "fried-egg" morphology typical of most mollicutes. Growth occurred both aerobically and anaerobically (as determined by the GasPak method). The ability to catabolize glucose and mannose and the ability to hydrolyze arginine varied among the three strains. All three strains required sterol for growth, and none of the strains hydrolyzed urea. The guanine-plus-cytosine contents of the DNAs of strains UIAT, VIST, and GIHT were determined to be 26.9, 27.0, and 26.6 mol%, respectively. Our data indicate that the three strains represent new Mycoplasma species, for which we propose the names Mycoplasma auris, Mycoplasma cottewii, and Mycoplasma yeatsii. The type strain of M. auris is UIA (= ATCC 51348 = NCTC 11731), the type strain of M. cottewii is VIS (= ATCC 51347 = NCTC 11732), and the type strain of M. yeatsii is GIH (= ATCC 51346 = NCTC 11730).

Serological specificity of a monoclonal antibody to Mycoplasma capricolum strain F38, the agent of contagious caprine pleuropneumonia.

A previously described mouse monoclonal antibody (WM25), directed against the caprine pathogenic mycoplasma strain F38 (Mycoplasma capricolum subspecies capripneumoniae), was further examined for its diagnostic efficacy in serological tests against various isolates of this group and of each of the five other 'M mycoides cluster' groups (M mycoides subspecies mycoides, small colony (SC) and large colony (LC) biotypes, M mycoides subspecies capri, M capricolum subspecies capricolum and bovine group 7) and M agalactiae. The methods used were the conventional disc-growth inhibition and colony-immunofluorescence techniques. The same strains were also tested against polyclonal rabbit antisera for each mycoplasmal group. With the polyclonal antisera, many cross reactions occurred between members of the different groups. The two F38-polyclonals gave cross reactions only against various M capricolum subspecies capricolum and bovine group 7 strains, apart from one of M mycoides SC in the immunofluorescence test only. The F38 monoclonal antibody gave more specific results, in that only the F38-type strains (M capricolum subspecies capripneumoniae) and three of the four bovine group 7 strains reacted positively in growth inhibition tests; the only heterologous reaction by colony immunofluorescence was with one M capricolum subspecies capricolum strain. The F38 monoclonal antibody, WM25, may therefore be useful for the specific serological identification of caprine F38-type isolates by the disc-growth inhibition method, which will exclude M agalactiae, M capricolum subspecies capricolum and the other members of the 'M mycoides cluster' associated with goats, but not bovine group 7 (which is not found in goats), which can be excluded by colony immunofluorescence tests.

DNA relatedness between field isolates of Mycoplasma F38 group, the agent of contagious caprine pleuropneumonia, and strains of Mycoplasma capricolum.

DNA-DNA hybridization experiments were carried out in order to clarify the taxonomic relationships between the F38 group of caprine mycoplasmas, the established etiologic agents of classical contagious caprine pleuropneumonia, and Mycoplasma capricolum, an organism associated with septicemia, arthritis, and mastitis in goats and sheep. The taxonomic status of the F38 group has been uncertain, principally because of the serological, genomic, and other properties which it shares with M. capricolum. Tritium-labeled DNAs from the M. capricolum type strain (California kid) and from prototype strain F38 were hybridized with unlabeled DNAs from these two strains and from four other isolates belonging to each group. The results showed consistent DNA relatedness values of about 70% between the F38 and M. capricolum groups, compared with levels of relatedness of about 90 and 85%, respectively, for the strains within each group. In addition, the results of comparisons of these 10 strains in which growth inhibition and immunofluorescence tests were used confirmed the previously reported serological relationships between the two groups and reinforced other observations concerning their shared genomic and cell membrane characteristics, indicating that there is a close taxonomic relationship. However, as the 70% DNA relatedness values between the M. capricolum and F38 groups also indicate a degree of genomic difference inconsistent with a relationship at the species level, we conclude that our findings support previous proposals for classification of the F38 group as a subspecies of M. capricolum.(ABSTRACT TRUNCATED AT 250 WORDS)

Proposal for designation of F38-type caprine mycoplasmas as Mycoplasma capricolum subsp. capripneumoniae subsp. nov. and consequent obligatory relegation of strains currently classified as M. capricolum (Tully, Barile, Edward, Theodore, and Ernø 1974) to an additional new subspecies, M. capricolum subsp. capricolum subsp. nov.

A subspecies relationship with the existing species Mycoplasma capricolum is appropriate for the F38 group of mycoplasmas, the causative agent of classical contagious caprine pleuropneumonia. We believe that this classification is justified on the basis of the close DNA-DNA relationship recently reported for isolates belonging to the two groups and the other known serological and biological similarities and differences of these organisms. Strain F38T (T = type strain) and taxonomically indistinguishable strains are therefore proposed as members of a new subspecies of M. capricolum, M. capricolum subsp. capripneumoniae. Strain F38 (= NCTC 10192) is the type strain of M. capricolum subsp. capripneumoniae subsp. nov. As a consequence of this subdivision of the species M. capricolum, strains previously classified as M. capricolum are now necessarily relegated to subspecies status, as M. capricolum subsp. capricolum subsp. nov. Strain California kid (= ATCC 27343 = NCTC 10154) is the type strain of M. capricolum, as well as of M. capricolum subsp. capricolum. A taxonomic description of M. capricolum subsp. capripneumoniae and a brief amended description of M. capricolum subsp. capricolum are presented.

Relationship between Mycoplasma mycoides subsp. mycoides ('large-colony' strains) and M. mycoides subsp. capri, as indicated by numerical analysis of one-dimensional SDS-PAGE protein patterns.

Twenty-five strains classified as Mycoplasma mycoides subsp. mycoides LC or subsp. capri have been compared by one-dimensional SDS-PAGE of their cellular proteins. A computerized numerical analysis revealed that the protein patterns of all but two aberrant strains formed one large phenon that separated clearly from representatives of the four other members of the 'M. mycoides cluster' at a similarity level (S) of 66% and which remained undivided at up to 78% S. At higher similarity levels, these strains fell heterogeneously into mixed sub-phenons containing strains of both subspecies. Serological comparisons by immunofluorescence largely confirmed the subspecies designations of the test strains, but also showed that some were serologically intermediate between subsp. mycoides and subsp. capri, being cross-reactive with both. These results confirm and enlarge upon those of our earlier studies indicating the protein-pattern inseparability of subsp. capri and subsp. mycoides LC strains and their distinctiveness from the classical M. mycoides subsp. mycoides SC strains and other members of the 'M. mycoides cluster'. As also recognized by other workers, subsp. mycoides LC and subsp. capri strains appear to comprise one large group, wherein those most readily identifiable as either type lie at either end of a serological spectrum that also contains serologically cross-reactive strains. Our observations therefore suggest the lines along which the three groups classified at present within the species M. mycoides (SC and LC strains of subsp. mycoides; subsp. capri) might eventually be reclassified, subject to direct genomic comparisons.

Numerical analysis of PAGE protein patterns and the taxonomic relationships within the 'Mycoplasma mycoides cluster'.

Twenty-six isolates belonging to the 'Mycoplasma mycoides cluster' have been characterized by one-dimensional SDS-PAGE of their cellular proteins. A numerical classification based on the resulting patterns and using a correlation coefficient revealed four distinct phenons at a similarity (S) level of 70%, comprising: (a) bovine group 7 strains; (b) M. capricolum and F38-like strains; (c) M. mycoides subsp. capri and LC strains ('subsp. mycoides'); (d) M. mycoides subsp. mycoides (SC). At the 75% S level, they could be divided further to give eight phenons. The composition of the clusters at both levels was in good agreement with their previous classification, except for M. mycoides subsp. mycoides LC and M. mycoides subsp. capri, which were clustered in a single phenon at 70% S and could not be clearly separated at 75% S. We conclude that high-resolution SDS-PAGE, combined with computerized analysis of protein patterns, provides an extremely effective approach to the investigation of taxonomic relationships within this group of mycoplasmas.

Taxonomy of the Mycoplasma mycoides cluster.

Some of the mycoplasmas found in diseases of ruminants, Mycoplasma capricolum, M. mycoides subsp. capri (PG3), bovine group 7 (PG50), strain F38 and strain Y goat show various degrees of relationship to M. mycoides subsp. mycoides (PG1) and to one another, such that they are referred to as the M. mycoides cluster. Studies using serology, DNA homology, isoenzyme analysis and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of proteins have been carried out on the type and reference strains of these organisms and some representative strains in an effort to resolve this cluster into well-defined taxa. An ad hoc committee set up by the Subcommittee on the Taxonomy of the Mollicutes examined the known data, but found that it led to conflicting opinions on the classification of the unspeciated strains, partly because of the lack of guidelines on the relative weight to be given to each criterion, and partly because insufficient strains of each member of the cluster had been examined. Although classifications were proposed, total agreement was not achieved on any one proposal, and the committee believes that decisions on the taxonomy would have to await the results of repeat tests, in which several representative strains of each of the six groups would be included with the type strain.

Construction of the mycoplasma evolutionary tree from 5S rRNA sequence data.

The 5S rRNA sequences of eubacteria and mycoplasmas have been analyzed and a phylogenetic tree constructed. We determined the sequences of 5S rRNA from Clostridium innocuum, Acholeplasma laidlawii, Acholeplasma modicum, Anaeroplasma bactoclasticum, Anaeroplasma abactoclasticum, Ureaplasma urealyticum, Mycoplasma mycoides mycoides, Mycoplasma pneumoniae, and Mycoplasma gallisepticum. Analysis of these and published sequences shows that mycoplasmas form a coherent phylogenetic group that, with C. innocuum, arose as a branch of the low G+C Gram-positive tree, near the lactobacilli and streptococci. The initial event in mycoplasma phylogeny was formation of the Acholeplasma branch; hence, loss of cell wall probably occurred at the time of genome reduction to approximately to 1000 MDa. A subsequent branch produced the Spiroplasma. This branch appears to have been the origin of sterol-requiring mycoplasmas. During development of the Spiroplasma branch there were several independent genome reductions, each to approximately 500 MDa, resulting in Mycoplasma and Ureaplasma species. Mycoplasmas, particularly species with the smallest genomes, have high mutation rates, suggesting that they are in a state of rapid evolution.

Isolation of mycoplasma spp. from racing pigeons (Columba livia).

Live and dead racing pigeons (Columba livia) from five lofts in Norfolk and Suffolk were examined clinically and cultured for Mycoplasma spp. Both clinically healthy birds and those showing signs of mild respiratory disease were included. The oropharynx was the culture site for 130 live birds, the nasal sinuses and other tissues for 58 carcases. Mycoplasma columbinum, M. columborale and M. columbinasale were isolated from the oropharynges and nasal sinuses; M. columbinum and M. columbinasale from the brain and M. columbinum and M. columborale from lungs and air sacs. One or more of these three Mycoplasma spp. was isolated at necropsy from 28% of 58 pigeons. Only 11% of 37 pigeons reacted sero-logically by the metabolism inhibition test to M. columbinum and none to M. columborale. Twenty-five birds examined for M. gallisepticum antibody by the haemagglutination-inhibition test were negative. No sex or age predilection to infection with Mycoplasma was apparent. About 10% of pigeons in all five lofts showed clinical signs of the respiratory disease sometimes described as 'mycoplasmosis catarrh', but most dead birds from which Mycoplasma spp. were isolated also had concomitant infections of various kinds. Although suggestive, the results of these investigations provide no clear evidence that Mycoplasma spp. are aetiologically involved in natural respiratory disease of pigeons. No conclusive satisfactory treatment was found for the elimination of mycoplasmas.

Effect of Mycoplasma pulmonis infection on protein and glycosaminoglycan synthesis of cultured connective tissue cells.

Human synovial cells, fetal skin fibroblasts and rat granulation tissue fibroblasts were experimentally infected with Mycoplasma pulmonis, a species identified as a contamination of cell cultures, and studied for collagen, total protein and glycosaminoglycan synthesis. Hyaluronic acid and sulfated glycosaminoglycan synthesis were stimulated in cultures where the infection reduced cell density, while they were retarded in cultures which had proliferated into higher density than the controls. An extra polypeptide with molecular weight of 20 kD was seen in [35S]methionine-labelled cells. Media of rat granulation tissue cells showed a shift of a 39-42 kD polypeptide to 33-36 kD position in [35S]methionine and [3H]proline labellings. Other minor changes were also noticed. Collagen synthesis or procollagen conversion to collagen were, however, not altered.

Standard antisera produced in ponies for the identification of bovine mycoplasmas: comparative growth-inhibition results from six laboratories.

Antisera to 10 mycoplasma species of bovine origin were produced in 10 ponies and were distributed for evaluation in growth-inhibition tests at 6 laboratories in Australia, England, Denmark, France, and the United States. Except for a few failures with some antigens produced at the 6 laboratories, the antisera induced large zones of growth inhibition in homologous, but not heterologous, systems. These antisera may be useful as standard reagents for the identification of the bovine mycoplasmas.

Creutzfeldt-Jakob disease. Failure to detect spiroplasmas by cultivation and serological tests.

Specimens from confirmed cases of Creutzfeldt-Jakob disease (CJD) were examined for evidence of spiroplasma infection. No spiroplasmas or other mycoplasmas were cultivated from brain tissue of 18 cases and no antibodies to several recognised Spiroplasma spp. were detected in sera from 15 patients. These negative cultural and serological results provide no support for published suggestions that CJD brain contains structures morphologically resembling spiroplasmas.

An evaluation of a ward pharmacy service.

An evaluation of a ward pharmacy service showed: 1. Its introduction led to a 40-50% reduction in errors and ambiguities of prescribing. 2. It assisted in the safe and more effective use of medicines in hospitals. 3. Medical staff particularly valued the safety checking applied to their prescriptions and the patient's total medication profile, and the opportunity of seeking information about drugs. Nursing staff most appreciated the checking and clarifying elements which assisted during the administration of drugs, the promotion of the most effective use of medicines and the opportunity of discussing problems of drug therapy.

The plaque-forming factor for mink lung cells present in cytomegalovirus and herpes-zoster virus stocks identified as Mycoplasma hyorhinis.

Previous investigation of the ability of cytomegalovirus and varicella-zoster virus to replicate in a variety of cell lines suggested that both virus types plaqued with high efficiency in mink lung cells. However, many of the virus isolates used appeared to be contaminated with mycoplasma. We now report that the observed cytopathic effect is due to a mycoplasma which grows lytically to high titre in mink lung cells, but is difficult to cultivate in cell-free media. The mycoplasma was plaque-purified and shown to contain DNA with a buoyant density of 1.684 g/ml, with restriction endonuclease patterns identical to the porcine mycoplasma M. hyorhinis. This was confirmed by serological identification.