Characterization of Mycoplasma pulmonis Variants Isolated from Rabbits I. Identification and Properties of Isolates

Mycoplasma showing at least two colony types were isolated from the nares and oropharynx of New Zealand white rabbits. Two strains were purified by single-colony passages and characterized. Morphology by phase-contrast and electron microscopy was typical of Mycoplasmataceae. Both grew anaerobically as well as aerobically, caused hemolysis of guinea pig, sheep, and horse red blood cells, and fermented glucose. These characteristics are shared by members of the species M. pulmonis, commonly isolated from the respiratory tracts of laboratory rats and mice. By use of the growth-inhibition test and agar-gel double-diffusion tests, the two strains were found to be serologically related to each other and to M. pulmonis ATCC 14267 but not to other representative Mycoplasma species from man and animals.     

Deoxyribonucleic Acid Homology and Relative Genome Size in Mycoplasma

The deoxyribonucleic acid homologies of Mycoplasma laidlawii type A and type B, M. pulmonis (#47 and #63), and M. hominis were determined by membrane methodology. The homology data revealed a difference in genome size between M. laidlawii type A and type B. This difference also held with stringent conditions of annealing (high temperature). Little or negligible homology was shown to exist between the M. laidlawii strains type A and type B and M. pulmonis strains 47 and 63 and M. hominis. M. hominis showed less than 10% homology to the M. pulmonis and M. laidlawii strains. Neither of the M. laidlawii strains showed more than 2% annealing to the M. pulmonis strains. Reaction rate studies are suggested as a means of demonstrating the phylogenetic relationship between the Mycoplasma and other microorganisms.     

The reactivity of antigens of Mycoplasma pulmonis derived from mice and rats to naturally infected rat sera

The reactivity of antigens of 4 mouse and 3 rat derived Mycoplasma pulmonis strains to 20 naturally infected rat sera was studied. The optical density values of the same serum by enzyme-linked immunosorbent assay using the 7 strains as the antigen revealed no marked difference among the strains. M. pulmonis antigens recognized by the antibodies were analyzed by the Western immunoblot method. The antigens with molecular weights of 92 K, 66 K, and 58 K were recognized in the 7 strains at a high frequency.     

Serological Studies on Actinobacillus actinomycetem-comitans

One hundred strains of Actinobacillus actinomycetem-comitans were examined serologically by determining their agglutination reactions with six absorbed antisera. Twenty-four reaction patterns were found, and the results were reproducible. The agglutinating antigens were heat-stable. Precipitation reactions were also studied by means of the Ouchterlony agar-gel diffusion technique with Fuller and acetone extracts. The agglutination test was considered preferable for epidemiological and ecological investigations on A. actinomycetem-comitans.     

Identification of Actinomyces israelii and Actinomyces naeslundii by Fluorescent-Antibody and Agar-Gel Diffusion Techniques

This study was an attempt to develop a fluorescent-antibody (FA) test to differentiate Actinomyces israelii and A. naeslundii as an aid in their laboratory identification. Two strains of A. israelii (X522 and A601) and two strains of A. naeslundii (X454 and X600), which had received intensive study by several investigators, were used for the immunization of rabbits. Working titers, based on tests with antigens prepared from the homologous strains and from well-established heterologous strains, were determined for each labeled antibody preparation. These conjugates and their normal serum control conjugates were used separately to stain 85 cultures of Actinomyes species and 23 strains of other species that might be confused with them. Acetone-precipitated soluble antigens from these same strains were tested with different antisera in the agar-gel diffusion test. Results showed that A. israelii (X522 and A601) and A. naeslundii (X454 and X600) labeled antiglobulins, when used at their working titers, stained most strains of their homologous species. Agar-gel diffusion results showed general agreement with those of the FA tests. The two tests appear to be equal in sensitivity, but the FA test is more specific, since several cross-reactions were noted with the agar-gel diffusion test whereas no cross-reactions were obtained with the FA reagents. Agar-gel and FA studies suggest that at least two serotypes of A. israelii may be associated with human disease. Although the majority of strains tested in this study appear to belong to a common serotype, "serotype 1," two strains of an apparent second serotype, "serotype 2," were encountered. FA staining of tissue impression smears from experimentally infected mice was successful when a counterstain, Evans Blue dye, was used.     

Specific antigens of Lactobacillus acidophilus.

Antigens specific for Lactobacillus acidophilus were investigated by double immunodiffusion in agar-gel. Antigenic materials were extracted from whole bacteria and some walls with cold trichloroacetic acid. Antisera were prepared by intravenous injection into rabbits of suspensions of whole organisms in solutions of bovine serum albumin, which had been heated and then washed. Four specific antigens were found as precipitinogens and denoted as antigens 11, 12, 13 and 14. Of 43 strains of L. acidophilus studied, 33 strains possessed antigen 11, six strains antigen 12, two strains antigen 13 and two strains antigen 14. Sugar compositions of wall preparations were analysed in an attempt to characterize the determinants of antigens 11 and 12. The walls contained glucose, galactose, hexosamine and sometimes glycerol, but no rhamnose was found. It was considered that alpha-glucopyranose was the major component of the determinant of antigen 11 since trehalose and maltose significantly inhibited the reaction between antibody 11 and its antigen; the determinant of antigen 12 was not clarified.     

Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.     

Expression of Mycoplasma pulmonis antigens in Escherichia coli

The expression of Mycoplasma pulmonis antigen in Escherichia coli was investigated by cloning genomic DNA derived from M. pulmonis m 53, and the DNA fragment participating in antigen expression was identified. When the DNA library of M. pulmonis was screened by colony immunoassay using anti-M. pulmonis serum, 10 recombinant clones expressing seroreactive antigens were obtained. The recombinant plasmids isolated from these clones included 3.7-6.5 kilobase pair (kbp) DNA inserts, while all clones contained a common 2.3-kbp DNA fragment. Subcloning of initial DNA inserts showed that the common 2.3-kbp fragment is essential for antigen expression. Moreover, antiserum against the recombinant antigen generated from the 2.3-kbp DNA fragment recognized a native M. pulmonis antigen. The reactivity of this antiserum was absorbed specifically with M. pulmonis. These results suggest that the cloned 2.3-kbp DNA fragment codes an antigen specific to M. pulmonis.     

Alfalfa mosaic virus isolates from lucerne in South Australia: biological variability and antigenic similarity

Alfalfa mosaic virus (AMV) was isolated from lucerne (Medicago sativa) plants with a variety of disease symptoms in eight of 13 sites in South Australia indicating that the virus is widespread in the state. The host ranges and symptomatology of the virus isolates varied considerably. Twelve selected local lesion isolates were shown to be distinct when mechanically inoculated to a range of plant species and cultivars. However, agar-gel diffusion and enzyme-linked immunoassay tests with polyclonal antisera prepared against glutaraldehyde-fixed virus preparations of the five most readily distinguishable AMV isolates, failed to reveal significant antigenic differences between the 12 virus isolates. This indicates that serological tests with polyclonal antisera can detect a wide range of AMV variants but would be unlikely to differentiate between strains. The wide host range and variability of AMV precluded the grouping of isolates into strains of the virus.     

Comparative Studies of Antigens from Mycoplasma mycoides and Escherichia coli

Extracts of sonically disrupted Mycoplasma mycoides and Escherichia coli were fractionated by sucrose density gradient centrifugation. The presence of antigen in each of the fractions was determined by complement-fixation and agar-gel diffusion precipitin tests, in which cow, pig, and rabbit anti-M. mycoides sera and rabbit anti-E. coli serum were used. Fractions of M. mycoides, with a buoyant density of 1.225 or lower, fixed complement with cow and pig anti-M. mycoides sera. These fractions also formed precipitin lines with pig antiserum. Fractions in the buoyant density range of 1.10 to 1.20 fixed complement with rabbit anti-E. coli serum, but precipitin lines were not formed. All E. coli fractions fixed complement and gave precipitin lines with homologous serum. But fractions in the buoyant density range of 1.10 to 1.20 had minimal complement fixation with heterologous M. mycoides sera. The cross-reacting antigens in M. mycoides and E. coli had a buoyant density of 1.10 to 1.20; the specific antigens were isolated from M. mycoides at a buoyant density of 1.08 to 1.02.     

Identification of Isolates of Clostridium perfringens Types C and D by Agglutination and Fluorescent-Antibody Methods

Agglutination and fluorescent-antibody methods were employed for screening Clostridium perfringens types C and D from 393 isolates of this organism. All of 50 strains which were isolated in Japan and were agglutinable with an antiserum prepared against a stock strain of type C no. 3182 toxigenically belonged to type C, but the antiserum showed no cross-agglutination with any of type C strains isolated in Denmark. All of the latter strains, however, were agglutinated by an antiserum prepared against a Danish strain, CWC11. Of 64 strains, showing heat-labile agglutinability by type D antiserum L9, 22 strains were toxigenically identified as type D strains which can be divided into three groups by the heat-stable antigens; no strains which were L-agglutination-positive but O-agglutination-negative were epislon-toxigenic. All of 13 strains, the heat-stable antigen of which was agglutinable by a type D antiserum VX81, were toxigenically type D strains. The results of fluorescent-antibody tests were almost in agreement with those of agglutination test with type C strains and completely with those of the O-agglutination test with type D strains. No beta-, epsilon- or delta-toxigenicity could be demonstrated in strains which were not agglutinated by our test sera for types C and D strains. Further examination of cultural properties of Japanese and Danish type C strains revealed that the two groups were considerably different in urease production, capsule formation, and delta- and alpha-toxigenicities.     

Cross-reactive antibodies to mycoplasmas found in human sera by the enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycoplasma pneumoniae in patients' sera with M. pneumoniae infection were measured by the complement fixation (CF) test and enzyme-linked immunosorbent assay (ELISA). Many patients' sera cross-reacted with heterologous mycoplasmal ELISA antigens such as M. hominis, M. hyorhinis, M. orale, M. pulmonis and M. salivarium. The sera with high CF (CF greater than or equal to 40) titers gave significantly higher ELISA values to M. hyorhinis (P less than 0.001) and M. pulmonis (P less than 0.001), which are not parasitic for humans, than those with low CF (CF less than 20) titer. Human normal immunoglobulin G (human normal IgG) containing 98% or more IgG, prepared from pooled plasma of at least 500 normal human donors, showed ELISA reactions with all mycoplasmal strains used. The nonspecific adsorption of human normal IgG on the surface of plate wells and on medium components which might contaminate mycoplasmal ELISA antigens could be disregarded. These results suggest that cross-reactive antibodies to mycoplasmas exist in human sera, and they affect the results of ELISA for serodiagnosis of M. pneumoniae infection.     

Serological comparison of ten glycolytic Mycoplasma species

Seventeen strains of mycoplasmata representing 11 named species were compared serologically by three parameters: growth inhibition on agar, double immunodiffusion, and complement fixation. In growth-inhibition studies, a strain labeled Mycoplasma histotropicum was found related to and perhaps best classified as M. pulmonis, a relationship confirmed by double immuno-diffusion studies. A comparison of the remaining 10 species demonstrated that two pairs of species could be shown to be closely related by complement fixation and double immunodiffusion but not by growth inhibition; these were: M. granularum-M. laidlawii and M. felis-M. canis. M. pneumoniae and M. gallisepticum were the most serologically unique organisms in this study, showing very few cross-reactions with each other or other species. Overall, taxonomic groupings obtained by comparative serology appeared to correlate with the groupings obtained when the guanine plus cytosine contents of the deoxyribonucleic acid of mycoplasmata were employed as classification criteria. The group of organisms having a guanine plus cytosine content of 23 to 28% (M. canis, M. fermentans, M. hyorhinis, M. neurolyticum, and M. pulmonis) appeared to be generally serologically related. Thus the remarkable heterogeneity observed in the base composition of the deoxyribonucleic acid of order Mycoplasmatales is also reflected and apparently paralleled by a corresponding serological heterogeneity.     

Serological Studies of Clostridium botulinum Type E and Related Organisms II. Serology of Spores

Pure spore antigens for the immunization of rabbits were prepared by enzymic digestion of vegetative components and separation of the cleaned spores in polyethylene glycol. Spore antisera were prepared to strains representative of toxigenic Clostridium botulinum type E; nontoxigenic boticin E-producing variants; nontoxigenic nonproducers of boticin E; nontoxigenic "atypical" strains, which differ somewhat from C. botulinum type E in their physiology; C. botulinum types A and B; and C. bifermentans. They were tested against these and additional strains representative of the above groups, other types of C. botulinum, and other Clostridium species. There was no evidence of agglutination of flagellar or somatic antigens of vegetative cells by these antisera. Agglutination and agglutinin absorption tests showed common antigens among toxigenic type E strains and nontoxigenic variants, both producers and nonproducers of boticin E. Some nontoxigenic "atypical" strains varied in their ability to be agglutinated by type E antisera, and others did not agglutinate at all. Of those atypical strains that were not agglutinated, one was agglutinated by C. bifermentans antiserum. Antisera prepared against C. botulinum types A and B and C. bifermentans did not agglutinate the spores of type E or its variants nor share antigens common to each other. Similarly, antisera to type E, its nontoxigenic variants, and nontoxigenic atypical strains did not agglutinate other C. botulinum types or any other Clostridium species investigated.     

Immunochemical characterization of cell wall protein antigen purified from the cell wall autolysate of Clostridium botulinum type A.

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components in immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.     

Adsorption of mycoplasma virus P1 to host cells.

The adsorption of mycoplasma virus P1, a virus which infects some strains of Mycoplasma pulmonis, to host cells was examined. Mutants of M. pulmonis to which P1 virus did not adsorb were isolated. Proteins from the mutants and from wild-type cells were compared by two-dimensional polyacrylamide gel electrophoresis, and the only observed difference was in the surface antigen V-1. The electrophoretic properties of V-1 also correlated with the host range of the virus. These data strongly suggest that the V-1 antigen affects adsorption of P1 virus to host cells.     

Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamm-irradiated Treponema pallidum and Treponema reiteri

Miller, James N. (University of California School of Medicine, Los Angeles), J. H. De Bruijn, and J. H. Bekker. Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamma-irradiated Treponema pallidum and Treponema reiteri. J. Bacteriol. 91:583-587. 1966.-Ultrasonic lysate preparations extracted from virulent Treponema pallidum, Nichols strain, suspensions exposed to 652,800 R of gamma-irradiation exhibited a loss in the serological reactivity of their heat-labile antigens; the heat-stable components of both the lysate and residue antigens were unaffected. The activity of heat-stable, cardiolipin T. pallidum complement-fixing antigen obtained from similarly irradiated organisms was also unaltered. gamma-Irradiation of the cultivable Treponema reiteri with dosages as high as 6,500,000 R failed to alter serologically either the heat-labile or heat-stable component of its lipopolysaccharide-protein (Reiter protein) antigen. The reactivity of the lipopolysaccharide portion of the Reiter protein complex with an antiserum to T. pallidum Nichols indicates previously unsuspected antigenic differences between the rabbit-adapted Nichols strain of the organism and so-called "wild" human strains of T. pallidum in which this antigen is generally absent.     

Differences Between Brucella Antigens Involved in Indirect Hemagglutination Tests with Normal and Tanned Red Blood Cells

Brucella antigens capable of sensitizing normal and tanned sheep red blood cells for indirect hemagglutination were compared with antigens involved in agglutination, gel diffusion, and immunoelectrophoresis. Hyperimmune rabbit sera, before and after absorption with various antigenic preparations from smooth and rough B. abortus, were used in the tests. Normal erythrocytes could be sensitized with an NaOH-treated ether-water extract (EW-T) of smooth Brucella. Tanned erythrocytes could be sensitized with a water-soluble extract from ultrasonically disrupted smooth or rough Brucella. The EW-T produced a single precipitation band and the water-soluble antigens produce 6 to 23 bands in immunoelectrophoresis with unabsorbed sera. After absorption of antisera with water-soluble extracts from smooth or rough Brucella cells or from smooth or rough cell walls, the hemagglutinins for sensitized tanned erythrocytes and the precipitins for water-soluble antigens were removed. Absorption with living smooth or rough Brucella cells or with EW-T did not remove these antibodies. The precipitins and hemagglutinins for the antigen EW-T, and agglutinins for smooth cells, were absorbed by smooth antigens but not by rough antigens. It appears that the antigens which sensitize tanned erythrocytes and diffuse through agar gels are present in both smooth and rough forms and may be situated in the cytoplasm or in the internal part of the cell wall, whereas the agglutinogen and the antigen which attaches to normal erythrocytes are surface antigens found only on the smooth Brucella cell.     

Production and characterisation of monoclonal antibodies to the principle sonicate antigens of Porphyromonas gingivalis w50

Abstract Protein antigens from whole cell sonicates of Porphyromonas gingivalis W50, previously shown to be discriminatory antigens for patients with adult periodontitis, were purified using SDS-PAGE. Electroeluted proteins were used to immunize mice for the production of monoclonal antibodies (mAbs). A combination of enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen hybridoma supernatants for mAbs. MAbs were successfully raised against M _r 115 000, M _r 55 000 and M _r 47 000 antigens together with a second M _r 55 000 polypeptide which was a contaminant of the M _r 55 000 antigen. No immunological cross-reactivity was found between these four proteins. The mAbs were used to examine the distribution of these antigens among fifteen P. gingivalis strains together with related oral bacteria using immunostaining of dot blots and Western blots. The antigens were confined to P. gingivalis with the M _r 115 000 and M _r 47 000 antigens being present in all strains tested . The distribution of the M _r 55 000 antigens were slightly more restricted: one M _r 55 000 (outer membrane location) was present in nine of the fifteen P. gingivalis strains tested, while the other M _r 55 000 (location unknown) was only absent from one strain. Whole cell ELISA demonstrated that the M _r 115 000 and the outer membrane M _r 55 000 antigen possess epitopes which are located on the surface of the bacterium.     

Spiroplasmas: serological grouping of strains associated with plants and insects.

Spiroplasma strains from plant and arthropod hosts, and from surfaces of flowers, were classified into three serological groups (designated I, II, and III) based on results from growth-inhibition tests. No significant cross reactions were observed among groups. The groupings were confirmed by ring-interface precipitin and microprecipitin tests, using membrane preparations as test antigens, and by organism-deformation tests. Serogroup I contained three subgroups: subgroup A (Spiroplasma citri strains Maroc R8A2 and C189), subgroup B (strain AS 576 and closely related strains from honeybee or flowers), and subgroup C (corn stunt spiroplasma strains). Serogroup II contained strains 23-6 and 27-31 isolated from flowers of the tulip tree (Liriodendron tulipifera L.) growing in Maryland. Serogroup III contained strains SR 3 and SR 9 isolated from flowers of the tulip growing in Connecticut. The subgroups of serogroup I were based on organism deformation, microprecipitin, and ring-interface precipitin tests. The data are consistent with the hypothesis that the three serogroups represent no less than three distinct spiroplasma species.