Characterizion of Mycoplasma pulmonis variants isolated from rabbits. II. Basis for differentiation of antigenic subtypes

The antigen composition of Mycoplasma pulmonis variants was studied by complement-fixation, agar-gel diffusion, and growth-inhibition tests. Two classes of complement-fixing antigens were demonstrated for M. pulmonis strains 47 and 63: (i) cross-related, heat-labile, water-soluble antigens, and (ii) high-titered, subtype-specific, heat-stable, water-soluble antigens. Lipid antigens prepared by organic solvent fractionation were low-titered antigens and showed little specificity. With the aid of agar-gel double-diffusion plates, the subtype-specific antigens were found to be precipitated by trichloroacetic acid and to be stable to periodate, but they were inactivated by pronase. Pronase-stable, periodate-labile precipitating antigens were observed as common components between the two variants. Antisera prepared with boiled antigens were found to be serologically active on gel diffusion but lacked neutralizing ability in growth-inhibition tests. Each of three strains of M. pulmonis (47, 63, ATCC 14267) could be identified as a variant because each strain possessed immunologically distinct heat-stable subtype-specific antigen(s).     

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.     

A DNA probe for specific detection of Mycoplasma pulmonis

Mycoplasma pulmonis was specifically detected by using a 2.3 kilobase pair (kbp) cloned DNA fragment derived from M. pulmonis m 53 as a probe. This probe recognized 2.3-kbp DNA fragments of three M. pulmonis strains in Southern hybridization, while it did not hybridize with the DNA of M. arthritidis or M. neurolyticum. Determination of the sensitivity of the probe by dot hybridization revealed that 10 ng of M. pulmonis DNA was detected by a biotinylated probe and 1 ng of M. pulmonis DNA was detected by a radioactive probe.     

鼠肺支原体单克隆抗体的研究

通过杂交瘤技术建立了３株抗鼠肺支原体单克隆抗体细胞株,它们分别是ＢＡ１１,ＢＤ７和Ｂ６１２．试验表明：３株细胞所分泌的抗体均属ＩｇＧ１亚类,都是鼠肺支原体的特异性抗体,与多种其它支原体无交叉反应。     

An alternative method for the decontamination of rats carrying Mycoplasma pulmonis without the use of germfree isolators

Two strains of Lewis rat were successfully freed from Mycoplasma pulmonis infection by using a combination of oral treatment with oxytetracycline hydrochloride and obtaining young by hysterectomy. Laminar flow cabinets were used to perform hysterectomies on donor animals and for rearing hysterectomy-derived animals. After thorough microbiological examination the rats were brought to the breeding colony of the Laboratory Animal Centre. Periodic laboratory tests using both cultural and enzyme-linked immunosorbent assay methods showed that the animals have remained free from M. pulmonis for the last 3 years.     

Relation between Mycoplasmas and Leukaemia and Related Diseases

Forty-five bone marrow specimens from leukaemia patients and 40 marrows from nonleukaemia patients were cultured for mycoplasmas. No mycoplasmas were isolated. Sera from patients with leukaemia or reticuloses and from non-leukaemic subjects were examined for antibodies to the Negroni and 880 strains of Mycoplasma pulmonis and also to the K7, K10, and prototype (PG18) strains of M. fermentans. No significant differences were observed between the two groups of patients with respect to antibodies to these mycoplasmas. These findings support those of the majority of other workers in failing to show any relationship between mycoplasmas and neoplastic disease in man.     

Ten years-long survey on pathogen status of mouse and rat breeding colonies

Eleven pathogens including P. aeruginosa, Salmonella spp., E. coli O115a, c: K(B), P. pneumotropica, B. bronchiseptica, C. kutscheri, Tyzzer's organism, M. pulmonis, Sendai virus, MHV and Syphacia spp. were surveyed in 217 mouse and rat breeding colonies during 1972-1981. In conventional animals, P. pneumotropica and/or Syphacia spp. were detected in nearly 90% of 89 mouse and 64 rat colonies. Sendai virus, M. pulmonis, P. aeruginosa and MHV were positive in 51.7 to 23.6% of the colonies, and Tyzzer's organism, B. bronchiseptica and probably SDA virus were also detected in more than 10% of the rat colonies. Salmonella spp., E. coli O115a, c: K(B) and C. kutscheri were found in a few colonies. In SPF animals, P. aeruginosa was isolated from about one third of 33 mouse and 31 rat colonies, and P. aeruginosa was isolated from about one third of 33 mouse and 31 rat colonies, and P. pneumotropica was also positive in 3 rat colonies. Infection rates of P. pneumotropica, M. pulmonis, Sendai virus and Syphacia spp. were usually higher than 40% of animals sampled from colonies contaminated with them. Accidental contaminations of SPF colonies were usually caused by P. pneumotropica and Syphacia spp.     

Attachment of Mycoplasma pulmonis to rat and mouse synovial cells cultured in vitro

The attachment of Mycoplasma pulmonis m53 organisms to mouse and rat synovial cells was examined by using the organisms and the synovial cells treated in various ways. M. pulmonis treated with trypsin attached to the synovial cells, but the organisms treated with pronase, formaldehyde, glutaraldehyde, or heat did not. These findings suggest that the sites for binding M. pulmonis to the mouse and rat synovial cells are of polypeptide nature. Treatment of M. pulmonis with sialic acid and treatment of the synovial cell sheets with neuraminidase did not affect the attachment. The synovial cell surface for receptors M. pulmonis organisms would be different from those on respiratory cells or erythrocytes for M. pneumoniae or M. gallisepticum. Even nonviable organisms and M. pulmonis membranes attached to the mouse or rat synovial cells. The nature of the receptor of mouse synovial cells would be different from that of rat cells, since rat cells were affected by treatment with formaldehyde or glutaraldehyde, but mouse cells were not.     

兰州地区实验动物肺支原体感染调查和分离鉴定

目的了解兰州地区啮齿类实验动物的肺支原体感染情况和感染菌株.方法用分离培养法对兰州地区640只啮齿类实验动物肺支原体的感染情况分春夏秋冬进行调查,并对分离株进行克隆纯化,从形态学,生化特性和血清学方面进行鉴定.结果肺支原体在普通级小鼠中的感染率为23%,普通级豚鼠、地鼠、大鼠和清洁级小鼠未发现有感染,且感染率与季节无明显相关性.分离株经鉴定均为支原体科支原体属肺支原体.     

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.     

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.     

Antigenic analysis of three strains of Mycoplasma arginini by two-dimensional immunoelectrophoresis.

The occurrence of species-specific and strain-specific antigens in three strains of Mycoplasma arginini (G-230, leonis and 23243) was studied by two-dimensional immunoelectrophoresis. Approximately 20 antigenic components could be detected in each strain. It was possible to analyze 6 to 7 major and distinct components from each strain by two techniques: "enhancement" where antigen to an additional strain is added to the first phase of the electrophoresis which increases the size of common peaks and "suppression" where antiserum to an additional strain is incorporated in the second phase whereby peak size of components to which both sera have antibody are decreased. A total of 10 distinct antigens were recognized. Electrophoretic mobilities relative to bovine albumin ranged from 0.2 to 1.08. Three components were common to all strains; two of these represented major amounts of material. Four components represented strain-specific components. Unique fast components were found both in strains 23243 and G-230. Three antigens were distributed into only two of the three strains. The electrophoretic mobilities of some common antigens were quite different between strains.     

Isolation of monoclonal antibodies to chromatin and preliminary characterization of target antigens

This paper describes the isolation of monoclonal antibodies to chromatin-associated protein antigens and their use in the characterization of such proteins by indirect immunofluorescence. Hybridomas were derived by fusion of the mouse myeloma Ag8653 with spleen cells from mice immunized with chromatin from human liver, rat liver or a human lymphoblastoid cell line. Hybrids were screened by solid-phase radioimmunoassay. The proportion of positive hybrids varied with the immunizing chromatin as follows: human liver 55/83, human lymphoblast 8/183 and rat liver 2/82. Fifteen antibodies derived from these fusions (7, 7 and 1 respectively) were subjected to further analysis. Most of these (11/13) were IgM and recognized both human and rat chromatin (12/15). Most of the target antigens were protease sensitive (8/13) and nuclease resistant. In fact the binding of five antibodies to lymphoblast chromatin was more than doubled by preincubation with DNAase I. The subcellular location of target antigens was examined by indirect immunofluorescence. Seven antibodies stained at least one of several cultured cell lines tested. Three gave staining patterns consistent with the in vivo association of the target antigen with chromatin recognizing, respectively, the interphase nucleus and metaphase chromosomes, the nuclear periphery and the mitotic spindle and other microtubule-containing structures. The remaining four all recognized antigens associated with the intermediate filament network.     

The effect of thiol-active compounds and sterols on the membrane-associated hemolysin of Mycoplasma pulmonis

Abstract Previous studies had shown that Mycoplasma pulmonis contained a bovine serum albumin-dependent, membrane-associated hemolysin. Biochemical analyses were performed to further characterize this activity. The membrane-associated hemolytic activity could be activated by dithiothreitol and β-mercaptoethanol, and inactivated by oxidizing compounds, a sulfhydryl inhibitor and heat treatment. Cholesterol and other sterols were inhibitory in a stereo-specific manner, but they did not interfere with adherence of M. pulmonis to red blood cells. These results indicated that once attached, the M. pulmonis hemolysin recognized cholesterol in the opposing membrane leading to red cell lysis. Because of the unique location of this toxin and its sensitivity to cholesterol, the mycoplasma membrane hemolysins may belong to a unique class of bacterial toxins.     

Distribution of ecto 5'-nucleotidase on Mycoplasma species associated with arthritis

The enzyme ecto 5'-nucleotidase (5'N) was found to be active on 8/14 strains of Mycoplasma fermentans, K(m) (+/-S.D.) 3.8+/-2.8 microM 5'-AMP, and on the type strain of Mycoplasma pulmonis, K(m) 0.63 microM 5'-AMP. The six M. fermentans strains lacking 5'N activity were related by restriction fragment length polymorphism typing. At pH 8.5, the type strains of Mycoplasma arthritidis, Mycoplasma buccale and Ureaplasma urealyticum showed a relatively non-specific phosphatase activity against 5'-AMP but no activity was shown by the type strains of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma salivarium at this pH. M. fermentans has been reported from rheumatoid joints, which show a raised 5'N activity on their synovial cells and in their fluid which may be associated directly or indirectly with the mycoplasma.     

Mapping immunodominant antigens and H-2-linked antibody responses in mice urogenitally infected with Chlamydia muridarum

To identify immunodominant antigens and MHC-restricted antibody responses, seven different strains of mice were intravaginally infected with Chlamydia muridarum and compared for antibody responses to 257 C. muridarum proteins. The 7 strains of mice recognized a total of 109 proteins as antigens, of which, 5 antigens (TC0660, TC0727, TC0828, TC0726 & TC0268) were each recognized by 60% or more mice from each mouse strain and thus designated as immunodominant antigens. Furthermore, antibody responses to 19 other antigens displayed strong associations with mouse H-2 haplotypes, including 6 antigens (TC0480, TC0912, TC0229, TCA04, TC0289 & TC0892) whose antibody responses were linked to H-2(b), 8 (TC0035, TC0387, TC0052, TC0781, TC0373, TC0117, TC0066 & TC0396) to H-2(d) and 5 (TC0512, TC0177, TC0589, TC0794 & TC0596) to H-2(k) haplotypes respectively. Interestingly, H-2(b) was negatively associated with antibody responses to most of the antigens that were positively linked to H-2(d) or H-2(k) haplotypes. These results by mapping Chlamydia trachomatis antigens commonly recognized by mice with different strain background and H-2 genes and revealing antigen association with H-2 haplotypes have provided important information for developing chlamydial subunit vaccines and understanding chlamydial pathogenesis.     

Variation in the virulence of strains of Mycoplasma pulmonis related to susceptibility to killing by macrophages in vivo.

The virulence of five strains of Mycoplasma pulmonis, as judged by their ability to survive in the respiratory tract and induce pneumonia in CBA mice, was related to the ability of viable organisms to persist in the peritoneal cavity. This appeared to be the result of differences in the ability of the strains to resist killing by peritoneal macrophages in vivo. It is suggested that resistance to phagocytosis by macrophages is an important determinant of virulence for M. pulmonis.     

Experimental Arthritis in Mice with Mycoplasma pulmonis

A Mycoplasma pulmonis strain, recovered from the arthritic joints of mice employed in the serial passage of a chemically induced tumor, was found to be arthritogenic for mice under experimental conditions. Some joint involvement occurred in all mice challenged intravenously with this strain, and M. pulmonis was recovered frequently from the enlarged joints. The arthritis was migratory, appearing first in the radiocarpal joints and later in the tibiotarsal joints. There was little evidence of a generalized mycoplasmal infection as a consequence of the experimental challenge. Histopathologically, the early stages of the infection in the joints was characterized by an inflammatory response in the synovium and periarticular tissues. Exudate in the joint space contained about equal numbers of polymorphonuclear and mononuclear cells. The polyarthritis resolved slowly, but some residual joint enlargement was noted for as long as 4 months. Two other M. pulmonis strains were also observed to be arthritogenic for mice. Rats were not susceptible to M. pulmonis challenge. Characteristics of the nonsuppurative M. pulmonis arthritis in mice were compared to M. arthritidis joint infections in rats.     

Genetic Mapping of the Antigenic Determinants of Two Polysaccharide K Antigens, K10 and K54, in Escherichia coli

The genes controlling synthesis of the Escherichia coli acidic polysaccharide capsular antigens K10 and K54 were transferred by conjugation to E. coli strains of other serotypes. The genes concerned with these K antigen determinants showed genetic linkage with the serA locus. We propose to name the K antigen-controlling gene kpsA. The genetic determinants of the two K antigens could also be transferred to enteropathogenic serotypes, even though such strains have never been found in nature with special acidic polysaccharide K antigens. A noncapsulated derivative, K(-), of the K10 strain can transfer the genetic determinant of the K antigen, demonstrating the probable existence of another chromosomal locus involved in the production of such acidic polysaccharide K antigens.     

Analysis of common epitopes on gag proteins of HIV-1, HIV-2 and SIV[AGM] using monoclonal antibodies against HIV-1.

Five monoclonal antibodies (MoAbs) to gag proteins of HIV-1 were prepared in mice. Western blot analyses showed that three clones recognized p24 and the other two p17. Among the three MoAbs recognizing p24, all recognized two of three strains of HIV-2. The spectra of reactions to SIV[AGM] of these MoAbs against p24 were different from one to another; K3-24 recognized all four strains of SIV[AGM], L6-24 three of them, and K5-24 none of them. Of the two MoAbs recognizing p17, K7-17 recognized two of the three strains of HIV-2 but not any SIV[AGM] strain, and the other clone, L14-17 recognized none of analogous proteins of HIV-2 nor of SIV[AGM]. These results demonstrate that the gag proteins of HIV-2 and SIV[AGM] share some common epitopes with those of HIV-1 which are heterogenic in some degree among the different isolates.