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Mycoplasma virus Hr 1 is a short tailed bacteriophage with a polyhedral head about 34 nm across and a tail about 14 nm long. It produces plaques on some strains of Mycoplasma hyorhinis.
A procedure for transformation of the murine pathogen Mycoplasma pulmonis with plasmid pAM120 was developed. This plasmid replicates in Escherichia coli and contains the gram-positive transposon Tn916. The transformation protocol also proved effective for the swine pathogen Mycoplasma hyorhinis. The tetracycline resistance determinant of Tn916 was expressed in transformed myocoplasma cells, and Tn916 was found inserted into numerous sites in the recipient chromosomes of M. pulmonis and M. hyorhinis, indicating that transposition had occurred. Interestingly, some transformants of M. pulmonis and M. hyorhinis contained cointegrate structures which apparently had a complete copy of the entire donor plasmid (pAM120) inserted into the recipient chromosome. Subsequent transposition of inserted Tn916 was observed in passaged clones of transformed M. pulmonis.
A small but significant reduction in the number of Mycoplasma dispar colonising the respiratory tract after intratracheal challenge was observed in gnotobiotic-calves previously inoculated subcutaneously three times with formalin-killed organisms and oil adjuvant. Injection of M. dispar by the intramuscular route, with oil adjuvant, and 2 weeks later by the intratracheal route, without adjuvant, failed to induce immunity to subsequent intratracheal challenge. Following the subcutaneous injection of killed M. dispar, the amount of antibody detected by single radial haemolysis (SRH) increased markedly with increasing age in groups of calves with average ages of 16 to 155 days when first injected. Most calves aged less than 40 days failed to produce an antibody response to a single injection of M. dispar. With M. bovis a smaller difference was observed between antibody levels generated in calves of different ages; also, calves less than 40 days old produced a detectable SRH antibody response following a single injection of killed M. bovis. IgG1 and IgG2 antibody to M. dispar and M. bovis were measured by ELISA. IgG1 appeared before IgG2 antibody and this was particularly pronounced in younger calves. Also, for both mycoplasmas IgG2 antibody levels were lower in younger than older calves. The IgG1 response to M. dispar was compared in three groups of calves with average ages of 16, 55 and 155 days and was greatest in the oldest and least in the youngest animals. In contrast, the IgG1 response to M. bovis varied little in calves of different ages. It therefore appears that the immune response of young calves to M. dispar is impaired or defective.
The interaction of Mycoplasma mobile 163 K with epithelial cells was investigated in tracheal organ cultures of gnotobiotic rats by electron microscopy. Ultrathin sections showed that the epithelial cells were heavily colonized and damaged by the mycoplasmas 2 days after infection. Numerous organisms were attached to fragments of the cytoplasmic membranes; others entered the injured cells and adhered to cell structures. Organisms were interconnected by fibrillary material supporting their adherence to the cell surface. Non-infected tracheal rings remained viable, differentiated and well organized up to 28 days of cultivation. The present investigation characterizes M. mobile 163 K as a surface parasite.
Using enzyme-linked immunosorbent assays with Mycoplasma ovipneumoniae as antigen, the cross-reactivity of antigens between this species and 22 other mycoplasma species was examined using reference polyclonal antisera. Significant cross-reactivity with M. ovipneumoniae was demonstrated by five species, only, viz. M. bovoculi, M. dispar, M. flocculare, M. hyopneumoniae and M. hyorhinis. Using one-dimensional SDS-PAGE and immunoblotting techniques with homologous and heterologous antisera, cross-reacting antigens of M. dispar, M. flocculare, M. hyopneumoniae and M. ovipneumoniae were further investigated. Cross-reacting antigens with apparent molecular weights of 64, 44 and 32 kDa were common to all and a 184 kDa cross-reacting antigen occurred in all except M. ovipneumoniae. Further cross-reacting antigens (one-way and two-way) between two of the four species are reported. Four monoclonal antibodies against different antigens of M. ovipneumoniae did not recognise any antigen in the other three species examined.
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)
Several Mycoplasma and Acholeplasma species chosen at random and solubilized with sodium dodecyl sulphate showed a common periodic acid-Schiff positive band with an apparent molecular weight of about 64 000, when examined by polyacrylamide gel electrophoresis. Another more cathodic minor band was detected in M. hyopneumoniae and M. flocculare. The common periodic acid-Schiff positive band appeared when a precipitate of serum constituents of the uninoculated growth medium after incubation was examined. The minor band was identified as a serum glycoprotein contaminating mycoplasmas grown in the presence of swine serum. We draw attention to the compounds as a possible source of error in serological tests or in the lymphocyte stimulation response. After lithium diiodosalicylate solubilization and aqueous phenol extraction, polyacrylamide gel electrophoresis showed a periodic acid-Schiff positive band in membranes from M. hyopneumoniae (molecular weight 75 000) and M. hyorhinis (molecular weight 80 000), suggesting the presence of a membrane glycoprotein. Such a glycoprotein was absent from A. granularum. Since the common periodic acid-Schiff positive band was not extracted by aqueous phenol, this growth medium constituent did not contaminate the preparations of membrane glycoproteins. However, the minor band was present in glycoprotein preparations of M. hyopneumoniae grown in the presence of swine serum.
The inactivation by ultraviolet (UV) light irradiation of mycoplasma cells of five human strains was monitored by investigating the colony-forming ability. The survival curves of five strains tested indicated that the cells of Mycoplasma buccale only are single and homogenously susceptible to UV light. The effect of the repair inhibitor, caffeine, on the colony-forming ability of UV-irradiated cells was investigated with M. buccale because of its homogenous susceptibility to UV light. The colony formation of irradiated cells was markedly depressed by post-irradiation treatment with caffeine at concentrations that had little or no effect on the colony formation of unirradiated cells. The colony-forming units (CFU) of UV-irradiated cells which were kept in broth without caffeine in the dark increased without a lag as the time in the dark increased. The colony-forming ability of the irradiated cells completely recovered after 3 hr in the dark. However, when irradiated cells were kept in the presence of caffeine, no increase in their CFU was observed. The mode of action of caffeine on UV-irradiated cells closely resembles that described for other organisms which possess dark reactivation systems for UV-induced damage in deoxyribonucleic acid (DNA). Thus, the results obtained provide evidence for the existence of a dark repair function in M. buccale.
Multiple restriction fragments, homologous to the previously described Mycoplasma bovis vspA gene, were identified in the chromosome of Mycoplasma agalactiae. The vspA, a representative variable surface lipoprotein gene of the vsp gene family, and four synthetic oligonucleotides, representing sequences complementary to selected regions of the vsp genes, were used as probes against digested chromosomal DNAs of several M. agalactiae clinical isolates. The resulting Southern blot analysis demonstrated a marked DNA polymorphism of multiple vspA-related fragments among the isolates. An oligonucleotide representing a conserved 5'-region common to all known vsp genes, was found to hybridize to multiple M. agalactiae genomic fragments while the other three oligonucleotides, representing distinct repetitive structures within the coding region of three known vsp genes (vspA, vspE, and vspF), failed to react. These results argue for the possible existence of a gene family in M. agalactiae analogous to the vsp system of M. bovis but comprised of diverse genes.
The 16S rRNA gene sequences of Mycoplasma cavipharyngis and Mycoplasma fastidiosum have been determined. Phylogenetic analysis showed that these species formed a new cluster within the so-called pneumoniae group of the mollicutes (class Mollicutes). This cluster will be referred to as the M. fastidiosum cluster. Interestingly, the M. fastidiosum cluster formed a sister lineage to the haemotrophic bacteria. Eperythrozoon spp. and Haemobartonella spp. The two latter genera, formerly classified as rickettsias, formed a stable phylogenetic entity in the tree as judged from branch lengths, bootstrap values and sequence signatures. Thus, the members of the M. fastidiosum cluster are the closest known relatives to the haemotrophic bacteria. Our data strongly support that the haemotrophic bacteria should be reclassified to reflect their actual phylogenetic affiliation.
The in vitro activity of trovafloxacin against 125 strains of Mycoplasma species and Ureaplasma urealyticum, including fluoroquinolone-susceptible and fluoroquinolone-resistant species, was compared to those of other fluoroquinolones, doxycycline, and erythromycin. The MIC at which 90% of isolates are inhibited for all fluoroquinolone-susceptible strains was 0.25 microg/ml. Whatever the associated mutations, trovafloxacin exhibited greater activity than the other fluoroquinolones tested against fluoroquinolone-resistant Mycoplasma hominis and U. urealyticum isolates.
Mycoplasma canadense and Mycoplasma verecundum were cultured in a serum-free medium containing bovine serum albumin, cholesterol, oleic acid, and palmitic acid in order to avoid the addition of horse serum. Growth was detected by measurement of A640 and by colony formation. The level of growth attained in this medium was less than that obtained in the horse serum-supplemented media, but colonies retained their distinctive morphology.
Lung lesions characterized by extensive peribronchial and perivascular round cell infiltration and intrabronchial plugging with polymorphonuclear leukocytes were produced in 5 of 44 Ha/ICR gnotobiotic mice sacrificed 3 to 10 days after three intranasal inoculations of broth cultures of Mycoplasma pneumoniae. After 10 days, no significant lesions were seen, and the proportion of lungs positive for M. pneumoniae dropped off sharply. M. pneumoniae persisted for longer periods (up to 24 days) in the trachea, nasopharynx, and anterior nares. These findings would seem to represent a self-limited respiratory infection due to M. pneumoniae in gnotobiotic mice. Ring forms within granular alveolar pneumocytes were seen by electron microscopy in the lungs of triply inoculated gnotobiotic controls receiving sterile horse-serum broth as well as in the lungs of mice receiving broth cultures of M. pneumoniae. Ring forms must now be considered to be part of the nonspecific cellular reactions of pneumocytes to foreign substances in the lungs of mice rather than intracytoplasmic developmental forms of mycoplasma as previously proposed.
A screening of enzymes on cell-free extracts of various species of mycoplasmas revealed the presence of enolase (EC 4.2.1.11) in significative amount in M. pneumoniae and M. fermentans, in lower amounts in M. hominis, A. laidlawii and in trace only in U. urealyticum. The value of activity of the various mycoplasmas could be correlated with their metabolism. From 40 g of cell paste of M. hominis, 2.5 mg of enolase purified over 70 folds, was obtained with successive steps of salt fractionation and column chromatography. Kinetic studies gave the following constants: Km for 2-phospho-D-glicerate, 0.7 x 10(-4)M; optimum of Mg++ concentration, 1 x 10(-3)M; optimum of pH, 7.7 inhibition by fluoride and phosphate, I = 0.77 X 10(-12)M4. Molecular weight estimation indicated for the native enzyme a value of about 100,000 daltons. These data suggest closer similarities with the enolase of microorganisms like E. coli and yeast than with the genus Thermus or B. stearothermophilus.
The mean flock size was ten chickens per rural farmer. Antibodies to Mycoplasma gallisepticum and Mycoplasma synoviae were detected in 57.88% and 67.33% of the chicken sera respectively.
During an experimental reproduction of CBPP, 5 inoculated cows and 5 contacts cows were bled twice a week and antibodies research was performed using complement fixation test (CFT), passive haemagglutination test (PHAT) and slide agglutination test (SAT). In the same period, trachobronchial washings were performed weekly to detect and to count Mycoplasma mycoides subsp. mycoides SC. For four months these tests were used to compare antibodies kinetics with kinetics of mycoplasmas excretion. With titer over 40 as threshold of positivity, PHAT detects antibodies earlier than CFT and SAT at the beginning of infection, but fails to detect chronic carriers. In contacts cows no failure has been observed with CFT and SAT except during prodromic phase of infection. These results are valid for natural infections. However, for inoculated animals, most serious failures to detect infected cows occur.
Searching for potential virulence markers of Ureaplasma (U.) urealyticum and Mycoplasma (M.) hominis, simple laboratory methods were used to detect strain specific properties. Haemadsorption, sperm adsorption, haemolysis, and peroxide formation were tested on clinical isolates and type strains of U. urealyticum and M. hominis in comparison to reference strains of seven other Mycoplasma species. In contrast to M. bovis, M. gallisepticum and M. pulmonis, all strains of M. hominis and U. urealyticum failed to adsorb human spermatozoa and erythrocytes from five vertebrate species to their colonies. Unexpectedly, colonies of M. arthritidis PG 6 adsorbed both human erythrocytes and spermatozoa. Using various methods, the known haemolytic and peroxide activities of the reference strains could be confirmed. None of the U. urealyticum strains tested demonstrated lysis of human or ovine erythrocytes or peroxide production. On the other hand, 76 of 108 isolates of M. hominis showed an alpha'- or beta-haemolysis of different degree. However, this phenomenon could not be reproduced in all cases and it was not attributable to the activity of a peroxide. None of the methods used in this study were found to be suitable for detection of possible virulence factors of U. urealyticum or M. hominis.
Temporal antisera (TA) prepared in susceptible Leg-horn-type chickens against Mycoplasma gallisepticum and M synoviae were evaluated to determine the extent of cross-reactivity in ELISA and hemagglutination inhibition tests. Species-specific and interspecies-specific polypeptides were identified after electrophoretic separation and protein immunoblotting with reference antisera, TA, and a monoclonal antibody specific for M gallisepticum. Mycoplasma gallisepticum antiserum cross-reacted with M synoviae polypeptides in ELISA and TA immunoblots. Two major M synoviae polypeptides (88 and 53 kilodaltons [kD]) cross-reacted with M gallisepticum antisera in TA immunoblots. An M gallisepticum polypeptide of 70 kD cross-reacted with M synoviae in TA immunoblots. In contrast, M gallisepticum and M synoviae reference antisera cross-reacted when immunoblotted with heterologous antigens. A monoclonal antibody specific for M gallisepticum bound to a 69-kD polypeptide in lectin-purified and whole-cell M gallisepticum protein fractions in immunoblot assays. The lectin-purified fraction hemagglutinated chicken RBC. Seemingly, the 69-kD polypeptide may constitute all or part of the M gallisepticum hemagglutinin.