Immune response of chimpanzees infected with human mycoplasmas.

Seven chimpanzees were inoculated intra-articularly with one of three Mycoplasma species. Three chimpanzees were inoculated with clinical synovial isolate 1620, and one was inoculated with type strain PG-21 of M. hominis. The fifth animal was inoculated with clinical synovial isolate 2010B, and the sixth was inoculated with the type strain CO of Ureaplasma urealyticum. The seventh animal was inoculated with clinical isolate PI-1428 of M. pneumoniae. The synovial isolates induced intense antibody responses, antibodies recognized more antigens, and the reactive antigens were distinct from the corresponding type strains. Chimpanzees infected with synovial isolate 1620, but not type strain PG-21, recognized antigens of molecular mass 223, 208, 168, 165, 155, 150, 142, 134, 115, 105, 52, 50, 45, 38, and 36 kDa. The chimpanzee infected with synovial isolate 2010B, but not type strain CO, recognized antigens at about 277, 237, 62, 57, and 43 kDa. The major antibody reactive antigens of M. pneumoniae isolate PI-1428 migrated at about 170, 90, 56, 40, 32, and 30 kDa. The reported biologic activities of antigenic proteins derived from these Mycoplasma species are reviewed.

Protection of immunized and previously infected chimpanzees challenged with Mycoplasma pneumoniae.

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.

Experimentally induced septic arthritis in chimpanzees infected with Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma urealyticum.

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.

Immunoblot analyses of chimpanzee sera after infection and after immunization and challenge with Mycoplasma pneumoniae.

Consecutive weekly or biweekly serum specimens obtained during a 3- or 4-month study from 16 chimpanzees were examined by immunoblot analyses to identify the immunogenic components of Mycoplasma pneumoniae. Six experimentally infected chimpanzees showed significant signs of overt disease, including cough, pharyngitis, rhinitis, fever, and loss of appetite. The sera of these infected chimpanzees recognized from 17 to 20 protein bands. Two control chimpanzees that were not inoculated were included in the study. Three chimpanzees immunized with a formalin-inactivated OSU-1A vaccine and three chimpanzees immunized with an experimental acellular vaccine showed minimal signs of disease on challenge. After challenge, the serum immunoblot responses of the immunized chimpanzees were similar to those of the infected chimpanzees. Before challenge, the sera of two previously infected chimpanzees recognized protein bands of 169 (which comigrated with the P1 adhesin), 148, 130, 117, 86, 61, 44, 35, 30, and 29 kDa. After challenge, the previously infected chimpanzees showed the most intense serum immunoblot responses and were most protected against colonization and disease. The sera from each of the 16 chimpanzees examined recognized a large number of immunogenic components, and the serum immunoblot responses were virtually identical to those of patients. Sera from each chimpanzee and patient recognized 169-, 148-, 130-, 117-, 86-, 44-, and 35-kDa bands and many of them recognized 67-, 63-, 61-, 56-, 32-, 30-, and 29-kDa protein bands.

Ureaplasma canigenitalium sp. nov., isolated from dogs.

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.

Experimentally induced Mycoplasma pneumoniae pneumonia in chimpanzees.

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 fermentans (incognitus strain) cells are able to fuse with T lymphocytes.

Characteristics of the fusion of Mycoplasma fermentans (incognitus strain) with cultured lymphocytes were investigated. The rate and extent of fusion were monitored continuously in an assay that measured lipid mixing on the basis of dequenching of a fluorescent probe, octadecylrhodamine (R18), incorporated into mycoplasmas. Fusion of M. fermentans was detected with CD4+ (Molt-3) cells, CD4- (12E1) cells, and primary peripheral-blood lymphocytes. The level of fusion was relatively low (8%-12%). Detection of a similarly low level of fusion by fluorescence microscopy suggested the involvement of a specific lymphocyte subpopulation. After a short lag period, fusion at 37 degrees C proceeded exponentially for approximately 30 minutes and was virtually complete at 60 minutes. Throughout the process, lymphocytes remained intact. Fusion was stimulated by CaCl2 but not by MgCl2; its inhibition by antisera to M. fermentans and by pretreatment of M. fermentans with proteolytic enzymes implied that the mycoplasmas possessed a proteinase-sensitive receptor involved in fusion. Mycoplasmas were rendered nonfusogenic by treatment with the uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone; 5 microM) but were unaffected by treatment with chlorpromazine (10 microM) or DCCD (dicyclohexylcarbodiimide; 50 microM); these findings suggested that a proton gradient across the cell membrane is required for fusion.

The immunodominant 90-kilodalton protein is localized on the terminal tip structure of Mycoplasma pneumoniae.

Immunoblot analysis of convalescent-phase sera of experimentally infected chimpanzees or monoclonal antibodies (MAbs) specific to the 90- and 40-kDa proteins of Mycoplasma pneumoniae indicated that both proteins were present in cytadsorbing, pathogenic strains PI-1428, M129, and FH but absent in noncytadsorbing, nonpathogenic strain M129-B176. Adsorption of convalescent-phase chimpanzee sera with virulent strain PI-1428 removed reactivity, whereas adsorption with avirulent strain M129-B176 did not remove reactivity to these two proteins. By using proteolysis and specific MAbs, we demonstrated that the 90- and 40-kDa proteins were surface exposed. Immunoelectron microscopy employing specific MAbs showed that the 90-kDa protein is localized on the terminal tip attachment apparatus. However, the MAb specific for the 40-kDa protein failed to indicate a similar localization. Nevertheless, these data, taken together, indicate that the immunodominant 90- and 40-kDa proteins are surface exposed, are localized on the terminal tip apparatus, and might be involved in the attachment mechanism.

Beware of mycoplasmas.

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?

Differential detection of Mycoplasma pulmonis and Mycoplasma arthritidis with species-specific DNA probes.

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.

Fusion of Mycoplasma fermentans strain incognitus with T-lymphocytes.

The ability of Mycoplasma fermentans (strain incognitus) to fuse with cultured lymphocytes was investigated and the fusion process was characterized. Fusion was measured using an assay to determine lipid mixing based on the dequenching of the fluorescent probe, octadecylrhodamine (R18), that was incorporated into the mycoplasma cells. Fusion of M. fermentans was detected with both CD4+ (Molt 3) and CD4- (12-E1) cells. The amount of fusion induced was relatively low and ranged from 5-10% with either cell culture. When primary peripheral blood lymphocytes were used the fusion yield was somewhat higher, reaching 12% of the cell population. Similar findings were obtained with fluorescent microscopy analysis suggesting that a predetermined, but unidentified subpopulation of cultured lymphocytes, were being fused. The rate of fusion was temperature dependent. Following a short lag period fusion at 37 degrees C was virtually completed in 60 min. The lymphocytes remained intact throughout the fusion process, as determined by the Trypan blue staining procedure. Fusion was almost completely inhibited by anti-M. fermentans antisera and by pretreatment of M. fermentans cells with proteolytic enzymes, suggesting that a surface-exposed proteinaceous component is involved in the fusion process.

Successive synovial Mycoplasma hominis isolates exhibit apparent antigenic variation.

The expression of surface proteins by 14 successive Mycoplasma hominis isolates obtained from the synovial fluid of a chronically infected septic arthritis patient was examined. Marked differences in the expression of surface proteins, as determined by monoclonal antibodies raised against the first isolate, were observed. However, identical restriction patterns and virtually identical hybridization patterns with probes containing the conserved genes of the Mycoplasma capricolum rRNA operon and the Escherichia coli elongation factor Tu suggest that the protein differences might reflect antigenic variation by M. hominis during infection.

Rheumatoid arthritis: new findings on the failure to isolate or detect mycoplasmas by multiple cultivation or serologic procedures and a review of the literature.

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.

Monoclonal antibodies to surface antigens of a pathogenic Mycoplasma hominis strain.

Three monoclonal antibodies (MAbs) were prepared against an arthritogenic strain of Mycoplasma hominis isolated from the joint aspirates of a patient with chronic septic arthritis. Immunoblots of polyacrylamide gel-electrophoresed proteins before and after surface proteolysis showed that the predominant antigenic determinants were on surface-exposed polypeptides. These polypeptides have extensive hydrophobic characteristics, as demonstrated by Triton X-114 phase partitioning. The electrophoresed proteins from cells grown in medium containing [14C]palmitate were blotted onto nitrocellulose which was both reacted with the MAbs and exposed to X-ray film. Superimposable bands on both the immunoblots and the exposed film suggested that the proteins might be acylated. The MAbs were further tested for reactivity with 16 other strains of M. hominis isolated from patients with septic arthritis (1 strain), septicemia (10 strains), or nongonococcal urethritis (1 strain); from the cervix (1 strain), rectum (1 strain), or surgical wound (1 strain) of patients; and from a contaminated cell culture. No single protein was consistently recognized from strain to strain, although a 94-kDa protein from 16 of the 17 strains tested was bound by at least one of the MAbs. The apparent antigenic heterogeneity among strains of M. hominis, including those isolated from the same tissue source and/or from patients with the same type of clinical disease, might be misleading in that all strains express epitopes associated with a discrete number of proteins to which one, two, or all three MAbs bind. The expression of the epitopes on multiple proteins from the same or different strains may reflect a mechanism for generating antigenic diversity.

Analysis of Mycoplasma hyorhinis DNA in the presence of host cells without growing the mycoplasma axenically.

Mycoplasma hyorhinis coisolates with the mitochondria of the cells in which it is carried as an infection. Since both mitochondria and mycoplasmas synthesize DNA by using the prokaryotic DNA polymerase gamma, the use of aphidicolin, which inhibits eukaryotic DNA polymerase alpha, allows for selective synthesis of mycoplasmal and mitochondrial DNA. The restriction patterns of mitochondria and mycoplasmas can easily be differentiated from each other in mixtures of both DNAs. Thus, it is possible to study the molecular biology of this noncultivable mycoplasma in situ rather than after growth in artificial media, with its potential genetic consequences during adjustment to axenic growth.

Transmembrane diffusion channels in Mycoplasma gallisepticum induced by tetanolysin.

The permeability properties of Mycoplasma gallisepticum cells treated with a purified preparation of tetanolysin were investigated by determining the initial swelling rates of cells suspended in an isoosmotic solution of electrolytes or nonelectrolytes. The swelling, initiated by the tetanolysin, depended on the tetanolysin concentration and was markedly affected by the molecular size of the various osmotic stabilizers utilized. Thus, the initial swelling rates in an isoosmotic solution of monosaccharides were much higher than those in isoosmotic solutions of di-, tri-, or tetrasaccharides. Cell swelling induced by tetanolysin was much lower with energy-depleted M. gallisepticum cells, with arsenate-treated cells, or when the membrane potential (delta psi) was collapsed by valinomycin (10 microM) plus KCl (100 mM). Swelling was not affected by the proton-conducting ionophore carbonyl cyanide-m-chlorophenylhydrazone (1 to 10 microM) or by nigericin (5 microM). These results support the concept that the damage induced by tetanolysin is due to the formation of water-filled pores within the membranes of energized M. gallisepticum cells. Such pores allow the diffusion of hydrophilic molecules into the cells and may vary in size, depending on the tetanolysin concentration utilized.

Ureaplasma felinum sp. nov. and Ureaplasma cati sp. nov. isolated from the oral cavities of cats.

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.

DNA relatedness among established Ureaplasma species and unidentified feline and canine serogroups.

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)

Nucleotide sequence of the MgPa (mgp) operon of Mycoplasma genitalium and comparison to the P1 (mpp) operon of Mycoplasma pneumoniae.

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.