Detection of metabolic inhibition antibody to Mycoplasma pneumoniae by new method with guinea-pig complement

Improvement of the fermentation-inhibition (FI) test for Mycoplasma pneumoniae was attempted. The sensitivity of detecting the FI antibody to M. pneumoniae in the homologous immune rabbit serum was notably elevated, when such FI medium containing gamma globulin-free horse serum and guinea-pig complement in substitution for unheated horse serum was used. The M. pneumoniae suspension was filtered to remove aggregates of the organisms and used as the antigen for this new FI test. In detection of the serum antibody of patients with M. pneumoniae infections, the new FI test showed much higher sensitivity than conventional FI test and strongly correlated (r = 0.84) with high density particle agglutination test known to detect both IgG and IgM antibodies.     

Comparison of the efficacy of egg yolk extract and horse serum for growth of Mycoplasma pneumoniae

The usefulness of chicken egg yolk extract as a substitute for horse serum in culture media for Mycoplasma pneumoniae was investigated. As a growth-supporting factor in the growth medium for M. pneumoniae and some other mycoplasmal species, the primary isolation medium for M. pneumoniae, and the metabolism-inhibition test medium for diagnosis of M. pneumoniae infection, egg yolk extract may be an excellent substitute for horse serum. The particular superiority of egg yolk extract to horse serum is that egg yolk extract, unlike horse serum, did not show any inhibitory effect on the growth of M. pneumoniae.     

A sensitive method for detecting the fermentation-inhibition antibody to Mycoplasma pneumoniae.

The fermentation-inhibition (FI) test for Mycoplasma pneumoniae was improved by using a combination of guinea pig complement and gamma globulin-depleted horse serum in place of unheated whole horse serum employed in the conventional assay system. As the test antigen for the new FI assay system, M. pneumoniae filtrated through a 3.0 microns membrane filter was used. Owing to the strong augmenting effect of guinea pig complement, the FI activity of rabbit immune serum was increased 32-fold in the new system compared with the conventional system. Furthermore, IgM antibody, which is barely detectable by the conventional system, could easily be titrated by the new system. With this sensitive method, rapid rise of FI titer was clearly demonstrable in most children with acute M. pneumoniae infections, and a prevalence of FI or growth-inhibitory antibody among healthy adults in Japan (82%) was revealed.     

Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion

Taylor-Robinson, David (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Otakar Sobeslavsky, and Robert M. Chanock. Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion. J. Bacteriol. 90:1432-1437. 1965.-Conditions are presented for the production of four lines of precipitate between Mycoplasma pneumoniae antigen and homologous hyperimmune rabbit serum in double diffusion in agar. The specificity of the reaction was shown by the fact that M. pneumoniae antigen did not react with antisera to the other human mycoplasma species, nor did M. pneumoniae antiserum produce lines with antigens prepared from the other human mycoplasmas. In addition, there was no reduction in the number or intensity of precipitation lines after absorption of M. pneumoniae antiserum with heterotypic mycoplasma antigens, or after absorption of heterotypic mycoplasma antisera with M. pneumoniae antigen. These findings indicate that, of the human mycoplasma species so far studied, M. pneumoniae is antigenically the most distinct.     

Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins

The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.     

Mycoplasma genitalium and Mycoplasma pneumoniae share a 67 kilodalton protein as a main cross-reactive antigen

It was demonstrated that a 67 kilodalton (kDa) protein of Mycoplasma pneumoniae is a main cross-reactive antigen with similar molecular weight protein of Mycoplasma genitalium by Western blot analysis using monoclonal antibody to 67 kDa protein of M. pneumoniae and hyperimmune rabbit sera directed against each mycoplasma strain.     

Medium components adsorbed to mycoplasmal cells

Washed cells of Mycoplasma pneumoniae mixed with complete Freund's adjuvant were capable of producing lethal anaphylactic shock in mice when injected repeatedly into the foot pad. The causative agent is associated with horse serum contained in the culture medium but cannot be removed from mycoplasmal cells by repeated washing with phosphate buffered saline. Mycoplasmas grown in medium containing rabbit serum had a similar effect. No lethal anaphylaxis occurs when M. pneumoniae is injected without adjuvant.     

Circulating immune complexes in the diagnosis of Mycoplasma pneumoniae infection

The possibility of detecting M. pneumoniae antigen and antibodies to it, incorporated into immune complexes, in the sera of patients with acute pneumonia by means of erythrocyte diagnosticums was studied, and the immunological characterization of these complexes was made. In patients with mycoplasmal pneumonia M. pneumoniae antigen and specific antibodies, both free and incorporated into immune complexes, were found to circulate in the blood. In children, antigenemia was detected twice as frequently as in adults. Dissociated M. pneumoniae antigens had different molecular weight, their location on the gel chromatogram of the serum being in fractions 7S and 19S. The dissociation of immune complexes permits the detection of M. pneumoniae antigen and antibodies to it in a bound state by means of the passive hemagglutination test, thus increasing the frequency of positive results in the diagnosis of M. pneumoniae infection.     

Demonstration of membrane association and surface location of Mycoplasma pneumoniae antigens using monoclonal antibodies

We examined 10 monoclonal antibodies (mAbs) directed against Mycoplasma pneumoniae proteins of 200, 170, 67, 46 and 42 kDa, and one mAb directed against a glycolipid component. The membrane association of the antigens reacting with our mAbs was investigated, in particular by phase-fractionation involving use of the detergent Triton X-114. The 170 kDa protein was shown to be membrane-associated, and surface exposure of this antigen was demonstrated by its disappearance from SDS-PAGE patterns after treatment of intact mycoplasmas with proteolytic enzymes. Cross-reactions with protein antigens of Mycoplasma genitalium were also shown. A mAb directed against a component of a lipid extract, prepared by the method used for preparation of the antigen used in the complement fixation (CF) test for serological diagnosis of M. pneumoniae infection, reacted with one major and a few minor bands in thin-layer chromatography (TLC) of the crude extract. The glycolipid character of this major antigen was demonstrated by treatment of the extract with sodium periodate, and by development of the TLC with orcinol/ferric chloride. These reactive bands were the same as those detected by the use of polyclonal mouse antiserum and a human convalescent serum, a result showing that the CF antigen contains a glycolipid moiety reacting with our mAb. The surface exposure of this antigen was demonstrated by binding of mAbs to intact cells.     

Identification of two new hemagglutinins of Escherichia coli, N-acetyl-D-glucosamine-specific fimbriae and a blood group M-specific agglutinin, by cloning the corresponding genes in Escherichia coli K-12.

Genes encoding the Escherichia coli IH11165 hemagglutinins with specificity for terminal N-acetyl-D-glucosamine and blood group M antigen, respectively, were cloned by a cosmid cloning procedure. A 22-kilobase-pair subclone expressed both hemagglutination specificities in the nonhemagglutinating E. coli HB101 recipient strain. Derivatives obtained by insertion and deletion mutagenesis expressed either one of the two hemagglutination specificities. Both agglutinins were purified; the agglutinin recognizing terminal N-acetyl-D-glucosamine was associated with a new type of fimbria (G fimbria) with an apparent subunit molecular mass of 19.5 kilodaltons, whereas the blood group M agglutinin (M agglutinin) was nonfimbrial and had an apparent subunit mass of 21 kilodaltons.     

Lobar pneumonia caused by Mycoplasma pneumoniae.

Seven patients with Mycoplasma pneumoniae pneumonia presented with moderate to dense consolidation in one (in five patients) or more lobes. The diagnosis was suspected in five patients after failure to respond to 1 to 6 (average 2.6) antibiotics administered for 2 to 12 (average 7) days, and in one patient upon the development of hemolytic anemia. Clues to the diagnosis of nonbacterial pneumonia included a nonrespiratory viral-like prodromal period (in five), a nonproductive cough (in five), lack of rigors (in seven), recent "pneumonia" in family members (in ;three), normal total leukocyte and neutrophil counts (in six) and the absence of bacterial pathogens in smears and cultures of sputum (in all seven). The diagnosis of M. pneumoniae infection was supported by the presence of cold agglutinins (in a titre of 1.64 or greater) in ;the serum of five or six patients and was confirmed by diagnostic levels or increases in the titre of M. pneumoniae complement fixing antibodies. Awareness of the fact that M. pneumoniae can present as lobar consolidation and close attention to the clinical and laboratory data can usually suggest a nonbacterial cause and thus prevent delay in appropriate antibiotic treatment.     

Improved sensitivity and specificity of enzyme immunoassays with P1-adhesin enriched antigen to detect acute Mycoplasma pneumoniae infection

An in-house P1-enriched (168-kDA protein) Mycoplasma pneumoniae antigen preparation was compared in IgG, IgA and IgM enzyme immunoassays (EIAs) to the respective EIAs employing crude antigen lysate, antigen prepared by Triton X-114 partition and two commercial antigens, one of which was an ether-extracted antigen and the other a P1-enriched antigen. In addition, three commercial kits from Sanofi Pasteur, Novum Diagnostica and Savyon Diagnostics were also assessed for comparison. Diagnostic sensitivity was studied with paired samples from adults (n=37) with acute respiratory illness interpreted as acute, recent or past infection to M. pneumoniae on the basis of the results of complement fixation test (CFT). If the consensus of at least two methods is taken as the true positive for acute infection, the diagnostic sensitivities of combined IgG and IgM EIAs were 100% for the Platelia(R), Sero MP and in-house EIAs whereas for the Novum EIAs and CFT- 97% and 74%, respectively. Moreover, the sensitivity of the P1-enriched antigen was proven superior on the basis of systematically highest OD(405 nm) ratios between convalescent and acute serum samples.Analytical specificity was studied by screening serum samples from 92 Finnish blood donors and 111 serum samples from cord blood. Diagnostic specificity was studied in a blind testing of 30 paired serum samples from infants with pneumonia of variable etiology. No single misinterpretation of acute infection from the group of samples with other respiratory diseases did occur.The present study confirmed and extended the earlier observations of the usefulness of P1-enriched antigen for reliable serologic diagnosis of acute M. pneumoniae infection.     

Immunostructure of school-age children inoculated against measles

The mass serological survey of school children immunized against measles was carried out by means of the hemagglutination inhibition test. As a result, 20.4% of these school children were found to be seronegative, and in 9.5% of them even the minimum concentration of measles antihemagglutinins (when titrated with 1 hemagglutinating unit of the antigen) was not detected. The accumulation of a considerable seronegative (measles-susceptible) stratum among children of school age occurred due to the low immunogenic potency of some batches of live measles vaccine, used for immunization in 1973, as well as due to the formerly practiced immunization of children under 1 year of age. A direct and close dependence of focal measles morbidity among immunized children having had contacts with the source of infection on the number of children among them, found to be seronegative after titration with 1 hemagglutination unit of measles antigen, was established.     

A chimeric human-bovine parainfluenza virus type 3 expressing measles virus hemagglutinin is attenuated for replication but is still immunogenic in rhesus monkeys

The chimeric recombinant virus rHPIV3-N(B), a version of human parainfluenza virus type 3 (HPIV3) that is attenuated due to the presence of the bovine PIV3 nucleocapsid (N) protein open reading frame (ORF) in place of the HPIV3 ORF, was modified to encode the measles virus hemagglutinin (HA) inserted as an additional, supernumerary gene between the HPIV3 P and M genes. This recombinant, designated rHPIV3-N(B)HA, replicated like its attenuated rHPIV3-N(B) parent virus in vitro and in the upper and lower respiratory tracts of rhesus monkeys, indicating that the insertion of the measles virus HA did not further attenuate rHPIV3-N(B) in vitro or in vivo. Monkeys immunized with rHPIV3-N(B)HA developed a vigorous immune response to both measles virus and HPIV3, with serum antibody titers to both measles virus (neutralizing antibody) and HPIV3 (hemagglutination inhibiting antibody) of over 1:500. An attenuated HPIV3 expressing a major protective antigen of measles virus provides a method for immunization against measles by the intranasal route, a route that has been shown with HPIV3 and respiratory syncytial virus vaccines to be relatively refractory to the neutralizing and immunosuppressive effects of maternally derived virus-specific serum antibodies. It should now be possible to induce a protective immune response against measles virus in 6-month-old infants, an age group that in developing areas of the world is not responsive to the current measles virus vaccine.     

Trypsinized Human Group O Erythrocytes as an Alternative Hemagglutinating Agent for Japanese Encephalitis Virus

Trypsinized human group O erythrocytes were found to be a suitable alternative to gander cells in hemagglutination (HA) and hemagglutination inhibition (HAI) tests for Japanese encephalitis (JE) virus. In the HAI test, no cross-reactions against JE virus were observed with immune sera containing antibody to taxonomically related or unrelated viruses, with mouse brain antigen, or with nonantibody serum inhibitors; specific antibody rise could be detected in an immunized rabbit. Gander and trypsinized human group O cells gave comparable titers in the HAI test, but the latter were preferable since (i) they required less challenging HA antigen, being more sensitive to agglutination by JE virus, and (ii) all human and some animal sera investigated were devoid of natural agglutinins for these cells, thereby eliminating or reducing the need for prior adsorption with packed cells.     

Susceptibility of newly established mouse strain MPS to Mycoplasma pneumoniae infection

A newly established mouse strain, MPS, which is more sensitive to Mycoplasma pulmonis than ICR, ddY and other mouse strains was examined for its susceptibility to Mycoplasma pneumoniae. In experimental infections with M. pneumoniae, it was observed that M. pneumoniae attached to tracheas of MPS mice, and M. pneumoniae cells were isolated from tracheas and lungs of MPS mice even after four weeks of infection, while no mycoplasmas were isolated from ICR and ddY mice after one week of infection. Specific antibodies against M. pneumoniae were also observed by the Western blotting in the sera of MPS mice infected with M. pneumoniae. Although any lung lesion could not be observed in this work, this newly established mouse strain MPS may be useful for experiments of M. pneumoniae infection, especially for the analysis of strain differences in susceptibility to M. pneumoniae infection.     

Measles Virus Antibody in Cerebrospinal Fluids from Patients with Multiple Sclerosis

A strong measles-specific gel precipitation reaction was found in the cerebrospinal fluid (C.S.F.) of two patients with multiple sclerosis (M.S.) (total of 15 tested). The serum and C.S.F. specimens from these two patients were tested for measles antibody by six assay methods. The results were compared with those obtained from serum and C.S.F. specimens of a patient with subacute sclerosing panencephalitis (S.S.P.E.). The gel precipitation line produced by the C.S.F. from the M.S. patients was identical with one of the three lines produced by the C.S.F. from the S.S.P.E. patient. The main antigenic component responsible for measles antibody appearing in the C.S.F. of the S.S.P.E. patient and the M.S. patients was also electrophoretically similar, and the corresponding antibody was associated with IgG. The serum/C.S.F. antibody titre ratios with the various assay methods used suggest that the C.S.F. antibodies are mainly to other than envelope components of measles virus. No complement-fixing antibody against 27 other viruses or Mycoplasma pneumoniae was found in the C.S.F. of the two M.S. patients.     

Effect of pH on the Immunogenicity of Mycoplasma pneumoniae

Mycoplasma pneumoniae harvested from media which had become acid lost the ability both to induce formation of tetrazolium reduction inhibition antibody and to act as antigens in immunodiffusion against human convalescent-phase sera. Incorporation of N-tris(hydroxymethyl)-2-aminoethane sulfonic acid and N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid buffers into a new medium containing PPLO Serum Fraction instead of horse serum delayed the pH decline. Tris(hydroxymethyl)aminomethane, triethanolamine, and 3,6-endomethylene-1,2,3,6-tetrahydrophthalic acid buffers inhibited growth. Mycoplasmas obtained from buffered cultures retained antigenicity as measured by immunodiffusion and could stimulate tetrazolium reduction inhibition antibody formation in animals.     

Trypanosoma gambiense: immunity with thymic cell transfer in mice.

This report deals with the enhanced agglutinin production and protection in thymectomized, lethally irradiated mice (TI-mice) with transferred thymic cells from mice immune to T. gambiense. Such mice, when sensitized with trypanosome antigen showed protection against experimental infection and also produced agglutinins. Thymic cells from cortisone-treated immune mice were able to induce the production of agglutinins in TI-mice subsequently injected with antigen. However, these agglutinin titers were very low. In bovine serum albumin gradient centrifugation experiments, agglutinin production could be efficiently induced by inoculation of TI-mice with a rather high density thymic cell subpopulation taken from immune mice. Fractionated by Sephadex G-200, the agglutinins displayed a division into two parts, a first and second peak. The main agglutination reaction was seen in the first or macroglobulin peak. In the fractionation of serum by DEAE-cellulose column chromatography, agglutinins were eluted in two parts, the 0.0175 M and 0.4 M effluents. The agglutination by the 0.4 M effluent was much stronger than that of the 0.0175 M effluent, in agreement with the gel filtration results. The sera containing agglutinins were able to enhance the phagocytosis of trypanosomes by cultured macrophages from the peritoneal cavity of normal and irradiated mice. Delay of parasitemia was evident in some of the TI-mice having detectable agglutinins. The delayed parasitemia resulted from antigenically altered trypanosomes which were able to withstand the lethal factors of TI-mice. Transplantation of thymic cells was considered to be responsible for agglutinins induced by the antigenic stimulation in TI-mice and for protection against experimental infection.     

Synthesis of Recombinant P48 of Mycoplasma agalactiae by Site Directed Mutagenesis and its Immunological Characterization

Contagious agalactia caused by Mycoplasma agalactiae is an economically important disease of sheep and goats and has been prevalent worldwide including India. The present study was undertaken to evaluate the membrane protein P48 of M. agalactiae for specific diagnosis of disease. For this, p48 gene of the organism was amplified by PCR and subjected to site directed mutagenesis to convert three TGA codons to TGG’s and, subsequently, cloned into prokaryotic expression vector pPRO EX HTb. Purified recombinant P48 protein reacted to anti-P48 serum in western blotting, which confirmed its immunogenic nature. Furthermore, the immune-blotting of the cell lysates from various Indian isolates of M. agalactiae against anti-P48 serum resulted in a single band at ～ 48 kDa among all isolates, indicating the conserved nature of P48 antigen in M. agalactiae. Also, the cross reactivity of P48 antigen among various Mycoplasma spp. was checked by western blotting which revealed reactivity only with M. agalactiae and M. bovis. Hence, this antigen could be exploited to differentiate M. agalactiae from other pathogenic Mycoplasma species except M. bovis. However, the inability of P48 to distinguish M. agalactiae from M. bovis does not downgrade the significance of P48 as the two species are usually host specific.