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Mycoplasmas recovered recently from dogs were found unrelated to three classified canine Mycoplasma serotypes but were similar in biological and serological properties to a Mycoplasma strain (C21, PG-24) isolated 18 years earlier. It is proposed that strains with the characteristics described be designated Mycoplasma edwardii sp. n.
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.
Mycoplasma contaminants of animal and human cell cultures were rapidly detected and identified by an indirect immunofluorescent technique. Cells suspected of being contaminated by mycoplasmas were grown as monolayers on chamber slides in a culture medium selected to promote mycoplasmal growth. Before fixation by acetone, the monolayers were subjected to a hypotonic treatment to cause swelling of the mycoplasmas. Detection and identification were then performed by indirect immunofluorescence using rabbit antisera to various mycoplasma species. The correlation between results obtained by the standard isolation procedure and those obtained by this method was very close.
The genomes of Mycoplasma flocculare and Mycoplasma hyopneumoniae, two mycoplasmas of the porcine respiratory system, were studied. Based upon antigenic cross-reactivity and DNA-DNA hybridization, these species have given indication of a close genetic relationship. By using field-inversion gel electrophoresis and employing the restriction digest fragments obtained from the gels as the probes, physical maps of the genomes of the two species were constructed. Mycoplasma hyopneumoniae is similar to M. flocculare in having a single set of rRNA genes and the 5S-rRNA gene is separated from the 16S and 23S rRNA genes. Based upon the location of the rRNA genes on the physical maps in both species, the distance between the 5S and the 16S and 23S rRNA genes is at least 150 kbp. Thus, there is further evidence for the close relationship between these organisms.
83 continuous cell lines were screened for mycoplasma contamination by three methods: microbiological seeding for enriched media, staining with Hoechst-33258 stain, and autoradiography with 3H-thymidine incorporation. It has been shown that combination of different methods is necessary for the strict control of mycoplasma-contamination in cell cultures. Simultaneous manipulation with mycoplasma infected and pure cell lines leads to cross-contamination in 2-3 passages. Precautions are described to preclude mycoplasma-contamination during prolonged cell cultivation.
Cloned DNA segments of the Mycoplasma hyorhinis genome have been identified by specific immunological detection of gene products expressed in Escherichia coli. Individual, distinct segments collectively representing greater than 10% of the genome each synthesize multiple proteins, some of which are immunogenic. Molecular genetic and immunologic tools have identified mycoplasma genomic fragments useful in 1) generating and analyzing specific, isolated mycoplasma gene products, 2) studying structure and expression of specific mycoplasma genes, and 3) generating specific gene probes. Immunological detection provides the potential for preselection of specific genes of interest.
Thirty young adult mice, of strain BALB/c, treated previously with progesterone, were inoculated intravaginally (10 mice) or directly into the uterus (10 mice) with Mycoplasma pulmonis and 10 mice remained uninoculated. Ten mice not treated with the hormone were also inoculated intrauterinely with M. pulmonis. The same numbers of mice treated with oestradiol were inoculated in the same ways with M. hominis. Vaginal swab specimens were obtained from all mice 7, 14 and 28 days after inoculation and samples of genital tract tissue were collected from pairs of mice at the same time intervals. Large numbers of M. pulmonis and M. hominis organisms were isolated from the vagina throughout the course of the experiments and they were cultured also from the cervix and uterine horns. Mycoplasma-like bodies were demonstrated by scanning electron microscopy in the cervix and in the uterus, but neither mycoplasmal species was found attached to vaginal epithelium. The results of silver-enhanced immunogold labelling in conjunction with scanning microscopy provided assurance that the mycoplasma-like bodies were, in fact, mycoplasmas. The importance of hormone treatment was indicated by the diminished susceptibility of untreated mice to M. pulmonis and the almost complete insusceptibility to M. hominis, shown by culture and scanning electron microscopy.
Diagnostic differentiation between the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis is known to be problematic when only conventional serological and biochemical tests are used. The main reason for this is that both agents share a considerable number of related proteins and common epitopes. DNA-based detection methods offer advantages in terms of specificity and sensitivity. However, there is an urgent need to compare currently used PCR assays because they target different genomic regions and, therefore, may perform differently. In the present work, five laboratories, which use PCR routinely, evaluated the specificity of four different PCR systems for M. agalactiae and three systems for M. bovis on a total of 41 strains of the two Mycoplasma species including six previously unidentified strains. As the vast majority of PCR examinations (97.1% of all tests) correctly identified the strains the specificity of all seven detection systems appears to be high. In four cases, incorrect identification by conventional diagnostic methods was rectified by PCR. Isolates from non-typical hosts, i.e. three M. bovis strains from small ruminants and two M. agalactiae strains from cattle, were characterised by sequencing the 16S and part of the 23S ribosomal RNA genes.
Maternal antibodies can protect avian embryos against vertically transmitted pathogens during embryogenesis and also young birds after hatching. In contrast to the well-known transfer of maternal immunoglobulin (Ig) G (also termed IgY) from the yolk to embryonic blood, information about the transfer of IgA, IgG and IgM from the egg albumen to the extra-embryonic fluids is very limited. In our study, IgA, IgG and IgM to Mycoplasma gallisepticum and Mycoplasma synoviae were detected in oviduct washings of naturally infected hens and in the corresponding egg albumen samples. In their progeny embryos, IgA, IgG and IgM antibodies to these Mycoplasma species were detected in the allantoic fluid (ALF) and amniotic fluid (AMF) on day 14 of embryonic development (ED). Examination of embryos from chickens immunized with antigens of M. synoviae revealed that the appearance of IgA and IgG and of antibodies to M. synoviae in ALF could vary even among embryos of the same dam. However, IgA, IgG and IgM were detected as early as day 7 of ED in ALF and AMF in certain embryos from hens infected with M. synoviae. In five groups of embryos examined on day 7, IgG to M.synoviae was found in 51% of ALF and 33% of AMF samples. M. synoviae was isolated from 2.3% of ALF samples. IgA to M. synoviae appeared in ALF and AMF on day 12 of ED, and could be found in the majority of AMF samples examined from day 14 onwards. IgM to M. synoviae appeared in AMF on day 13 and in ALF on day 14, but was detected in those fluids less frequently than IgA or IgG.
The susceptibilities of 40 strains of various Mycoplasma species to 10 classes of antimicrobial agents were compared in vitro by a broth microdilution method. The strains tested comprised 20 strains of four AIDS-associated species--M. penetrans (1 strain), M. fermentas (5 strains), M. pirum (6 strains) and M. genitalium (8 strains)--nine strains of the urogenital tract species M. hominis and 11 strains of M. pneumoniae. The results demonstrated wide variation in the susceptibilities of the different Mycoplasma spp. to different classes of antimicrobial agent. All the mycoplasmas were susceptible or highly susceptible to the fluoroquinolones, with sparfloxacin the most active, and to the diterpine antibiotic tiamulin. M. pneumoniae and M. genitalium strains were also highly susceptible to the macrolides, particularly azithromycin and had similar antibiotic susceptibility patterns to most other antimicrobial agents. However, all strains of M. genitalium were resistant to streptomycin (MIC 250->500 mg/L) whereas all M. pneumoniae isolates, except the MAC strain, were susceptible (MICs 1.25-12.5 mg/L). M. pirum isolates varied considerably in their susceptibility to macrolides (MIC range versus azithromycin 0.0025->100 mg/L). M. fermentans strains were susceptible to the tetracyclines, lincosamides and mupirocin, but varied in susceptibility to aminoglycosides. Most M. hominis strains were susceptible to the tetracyclines and all were susceptible to clindamycin and mupirocin. M. penetrans GTU 54 was susceptible to azithromycin, the tetracyclines and lincosamides as well as to the fluoroquinolones and tiamulin.
AIM: To use polymerase chain reaction (PCR)-microtiter plate hybridization assays to detect Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum (biovar 1) and Ureaplasma urealyticum (biovar 2) in first-voided urine specimens from patients with non-gonococcal urethritis (NGU). METHODS: A total of 153 male patients with NGU, who visited one of 24 clinics in Japan, were recruited for this study. All were examined using PCR-microtiter plate hybridization assays for the presence of M. genitalium, M. hominis, U. parvum (biovar 1) and U. urealyticum (biovar 2) in first-voided urine specimens. They were also examined for the presence of Chlamydia trachomatis. RESULTS: Of these 153 patients, 73 (47.7%) were positive for C. trachomatis. Overall, the prevalence was 17.0% for M. genitalium, 16.3% for U. urealyticum (biovar 2), 7.8% for U. parvum (biovar 1) and 2.6% for M. hominis. In the 80 patients with non-chlamydial NGU, the prevalence of M. genitalium, U. urealyticum (biovar 2), U. parvum (biovar 1) and M. hominis was 23.8%, 18.8%, 8.8% and 2.6%, respectively. CONCLUSIONS: This study shows the prevalence of mycoplasmas and ureaplasmas in NGU in Japan. M. genitalium and U. urealyticum (biovar 2) might be pathogens of NGU and could be associated with persistent and recurrent urethritis. When patients with NGU are treated, such pathogens should be taken into account. This PCR-microtiter plate hybridization assay provides a useful method for diagnosing NGU caused by M. genitalium and U. urealyticum (biovar 2).
In previous studies with hyperimmune rabbit sera and monoclonal antibodies against the P1 protein of Mycoplasma pneumoniae, we obtained evidence of a shared antigenic determinant with a single protein of Mycoplasma genitalium. Because of biologic and morphologic similarities between these two human Mycoplasma species, attempts were made to characterize this cross-reacting protein of M. genitalium (designated MgPa). The protein was surface exposed and had an estimated molecular size of 140 kilodaltons. Electron microscopy with monoclonal antibodies produced against either MgPa or P1 demonstrated that MgPa is located over the surface of the terminal structure of M. genitalium which is covered by a nap layer. These immunologic and morphologic findings suggest that the MgPa protein of M. genitalium could be the counterpart of the P1 protein of M. pneumoniae.
VanDemark, P. J. (University of South Dakota, Vermillion), and P. F. Smith. Respiratory pathways in the Mycoplasma. II. Pathway of electron transport during oxidation of reduced nicotinamide adenine dinucleotide by Mycoplasma hominis. J. Bacteriol. 88:122-129. 1964.-Unlike the flavin-terminated respiratory pathway of the fermentative Mycoplasma, the respiratory chain of the nonfermentative M. hominis strain 07 appears to be more complex, involving quinones and cytochromes in addition to flavins. In addition to reduction by reduced nicotine adenine dinucleotide (NADH) and reduced nicotine adenine dinucleotide phosphate, nonpyridine nucleotide-linked reduction of the respiratory chain of this organism occurred with succinate, lactate, and short-chained acyl coenzyme A derivatives as electron donors. Enzymes catalyzing the oxidation of NADH included an NADH oxidase, a diaphorase, a quinone reductase, and a cytochrome c reductase. The oxidation of NADH was sensitive to a variety of inhibitors, including 10(-4)m Atabrine, 10(-3)m sodium amytal, 10(-5)mp-chloromercuribenzoate, 10(-4)m antimycin A, and 10(-4)m potassium cyanide. The oxidase was resolved by the addition of 5% trichloroacetic acid and reactivated by the addition of flavin adenine dinucleotide but not flavin mononucleotide. The M. hominis sonic extract contained an NADH-coenzyme Q reductase. The oxidation of NADH was stimulated by the addition of either menadione or vitamin K(2) (C(35)). The oxidase was inactivated by extraction with ether or irradiation at 360 mmu. The ether-inactivated enzyme was partially reactivated by the addition of "lipid" extract of the enzyme and coenzyme Q(6). Difference spectra of the cell extracts revealed the presence of "b" and "a" type cytochromes. These cell extracts were found to contain a cyanide-and azide-sensitive cytochrome oxidase and catalase.
By use of a very sensitive nested PCR method targeting part of the strongly conserved mycoplasmal 16S RNA genes, Mycoplasma pneumoniae was found in the synovial fluid of 19/24 (79%) of rheumatoid arthritis patients, 6/6 (100%) of patients with nonrheumatoid inflammatory arthritis, and 8/10 (80%) of osteoarthritis patients attending the rheumatology clinic for drainage of joint effusions. It was not found in the synovial exudates of 13 people attending the orthopedic clinic with traumatic knee injuries or undergoing surgery for knee replacement. However, M. pneumoniae was detected in 2/4 synovial biopsy specimens from orthopedic patients with traumatic knee injuries. M. pneumoniae was associated with the increased synovial fluids found in arthritic flares but was not found in the synovial fluids of trauma patients. Mycoplasma salivarium occurred sporadically. Mycoplasma fermentans had previously been isolated from patients with inflammatory cellular infiltrates, such as rheumatoid arthritis, but it was not detected for osteoarthritic patients from either clinic. It is possible that these organisms may contribute to chronic inflammation within the joints.
A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritis was identified initially as Mycoplasma pneumoniae. In retrospective studies, the culture was shown also to contain Mycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports of M. genitalium in human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for both M. genitalium and M. pneumoniae in the clinical laboratory.
We developed a duplex PCR assay targeting the hemagglutinin multigene families, vlhA and pMGA, of Mycoplasma synoviae and Mycoplasma gallisepticum, respectively. The assay proved to be specific and sensitive enough to justify its use for the simultaneous detection of the two major avian mycoplasma species from field isolates.
During an outbreak of pneumonia and arthritis in beef calves and young cattle on a large farm in north-west Germany, Mycoplasma bovis and Mycoplasma californicum were isolated from tracheobronchial lavage fluids and synovial fluids. The microbiological findings in dead and living animals and the immunohistochemical demonstration of M californicum antigen in lung and arthritic joint tissue, indicated that under poor housing conditions and possibly other predisposing factors, this mycoplasma, like M bovis, can colonise the respiratory tract and may be able to cross the respiratory mucosal barrier to spread through an infected animal and cause systemic infections that may contribute to severe arthritis.
The aim of this work was to examine in vivo whether infection with Mycoplasma hyopneumoniae (M. hyop) and/or Mycoplasma flocculare (M. floc) would interact and influence the severity of enzootic pneumonia in piglets. Specific pathogen-free, hysterectomy-derived piglets were allocated to six groups and experimentally inoculated with M. hyop. and/or M. floc at the age of 2 or 8 weeks. Clinical symptoms, frequency of coughing and temperature measurement were noted daily. Lung lesions were recorded by post-mortem examination and histological observations. The cross-inoculation with both mycoplasmas did not influence the clinical or the pathological picture of the disease. Evolution of specific and crossreacting antibodies was analyzed by ELISA and immunoblotting. Animals inoculated with M. floc did not develop any lesions but showed a weak antibody response 6-8 weeks post-infection (p.i.). No cross-reacting antibodies against M. hyop proteins were detected. In animals inoculated with M. hyop, the first antibody response was detectable 4-5 weeks p.i. and was stronger in piglets infected at the age of 2 weeks than at the age of 8 weeks. Three cross-reacting antibodies against M. floc proteins with molecular weights of 110, 47 and 33 kDa were detected by antibodies to M. hyop. Experimental infections with both mycoplasmas did not show differences in the pattern of species-specific proteins.