Pneumonia due to mycoplasma in gnotobiotic mice. I. Pathogenicity of Mycoplasma pneumoniae, Mycoplasma salivarium, and Mycoplasma pulmonis for the lungs of conventional and gnotobiotic mice

Lutsky, Irving I. (Marquette University School of Medicine, Milwaukee, Wis.), and Avrum B. Organick. Pneumonia due to mycoplasma in gnotobiotic mice. I. Pathogenicity of Mycoplasma pneumoniae, Mycoplasma salivarium, and Mycoplasma pulmonis for the lungs of conventional and gnotobiotic mice. J. Bacteriol. 92:1154-1163. 1966.-Two species of mycoplasma of human origin, Mycoplasma pneumoniae and M. salivarium, were tested for their ability to produce respiratory disease in the Ha/ICR mouse when inoculated by the intranasal route. The mouse pathogen M. pulmonis was studied as a positive control. Conventional and gnotobiotic Ha/ICR mice were employed, the latter to provide a system free from indigenous mycoplasma and bacteria. Pneumonia from which mycoplasma were isolated was produced in all groups of the conventional Ha/ICR mice, including those inoculated with sterile broth. Only M. pulmonis produced disease when inoculated intranasally into the gnotobiotic mice, and the gross and microscopic lesions resembled those described in conventional mice. The gnotobiotic mouse provided a tool to study the pathogenicity of different mycoplasma species, and indicated marked differences in host specificity that could not be clearly seen when conventional mice were used.     

Pneumonia due to mycoplasma in gnotobiotic mice. II. Localization of Mycoplasma pulmonis in the lungs of infected gnotobiotic mice by electron microscopy

Organick, Avrum B. (Marquette University School of Medicine, Milwaukee, Wis.), Kenneth A. Siegesmund, and Irving I. Lutsky. Pneumonia due to mycoplasma in gnotobiotic mice. II. Localization of Mycoplasma pulmonis in the lungs of infected gnotobiotic mice by electron microscopy. J. Bacteriol. 92:1164-1176. 1966.-Lesions in lungs of gnotobiotic mice inoculated intranasally with Mycoplasma pulmonis were examined by electron microscopy after osmic acid fixation. At 1 week after infection, mycoplasma cells were found in large numbers in the bronchi at the surface of bronchial epithelial cells and, in smaller numbers, in the alveoli where active phagocytosis by polymorphonuclear leukocytes (PMN) occurred. Cytopathic changes in underlying bronchial epithelial cells, not apparent by light microscopy, were observed. At 3 weeks after infection, mycoplasma cells were rarely seen in the bronchi, and were no longer seen free in the alveolar spaces or within PMN. Lungs examined after glutaraldehyde fixation 1 week after infection confirmed the presence of mycoplasma cells in the alveolar spaces and within phagocytic vacuoles of PMN, but also revealed numerous ring forms within granular pneumocytes. These forms seemed to represent intracytoplasmic developmental stages of M. pulmonis, in which elementary bodies appeared in large numbers.     

Pneumonia Due to Mycoplasma in Gnotobiotic Mice IV. Localization and Identification of Mycoplasma pulmonis in the Bronchi of Infected Gnotobiotic Mice by Immunofluorescence and by Light Microscopy

The model of pneumonia produced by intranasal inoculation of Mycoplasma pulmonis in gnotobiotic mice provided the opportunity to study the localization and identification of the infecting organisms in the tissues by immunofluorescence techniques. Frozen sections of pneumonic mouse lung were fixed in acetone, layered with rabbit anti-M. pulmonis serum, washed, layered again with fluorescein-isothiocyanate-labeled goat anti-rabbit globulin, washed again, and examined by fluorescence microscopy. A bright line of fluorescence was seen at the bronchial epithelial surface, usually in a continuous layer. Occasional masses of fluorescence were seen in the polymorphonuclear leukocytic exudate in the bronchial lumen. Sections of tissues fixed in Helle's or 10% Formalin fixatives and stained with hematoxylin and eosin were reviewed by light microscopy and revealed a zone of blue-staining material composed of tiny coccoid bodies in the same locations at the bronchial epithelial surface as in the immunofluorescent preparations and in previously reported electron microscope studies.     

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.     

Evaluation of the Pathogenicity of the Bacteroides fragilis Toxin Gene Subtypes in Gnotobiotic Mice

Enterotoxigenic Bacteroides fragilis (ETBF) strains produce a metalloprotease toxin (BFT) related to diarrheal disease in animals, young children, and adults. Three different isoforms of the enterotoxin, designated BFT-1, BFT-2, and BFT-3, have been identified and sequenced. In the present study, the pathogenicity of the ETBF strains carrying bft-1 or bft-2 was evaluated. Each toxin gene subtype of ETBF (bft-1 or bft-2) was intragastrically monoassociated to germ-free mice during 10 days and histopathological data from intestines and liver compared with those from mice monoassociated to a non-enterotoxigenic B. fragilis. Histopathological alterations were observed in all groups of animals related to ETBF. These alterations were characterized mainly by ulceration, edema, and inflammatory infiltration in intestine. However, these lesions were slightly more severe in mice monoassociated with bft-2 subtype. No alteration or lesion was observed in animals associated with the non-enterotoxigenic B. fragilis. In conclusion, strains harboring bft-1 or bft-2 gene subtypes were able to induce histopathological alterations in intestine of a gnotobiotic mice model and it could explain the effect produced for the enterotoxin.     

悉生小鼠体内大肠杆菌和青春型双歧杆菌对艰难梭菌的拮抗作用

本文应用悉生小鼠做模型,研究了大肠杆菌(E.coli)和青春型双歧杆菌(Bifidobacterium adolescentis)对艰难梭菌(Clostridium diffi-cile)的拮抗作用。E.coli和B.adolescentis预先接种无菌SSB小鼠,再用C.difficile攻击。结果表明,E.coli和E.coli B.adolescentis对小鼠均有保护作用,保护平分别为87.5%(7/8)和100%(8/8)。B.adolescentis定值后数量达10~(10.28)CFU/g,且对E.coli数量和小鼠本身无影响。E.coli和B.adolescentis联合比E.coli单独抑制C.difficile在肠道中繁殖的作用更强(0.02>P>0.01),但对其毒素产生和粘附力的作用无明显差异。C.difficile攻击后的1～14天,小鼠粪便中C.difficile菌数在10~4至10~8CFU/g内变化,细胞毒素为10~3CFU/g,A毒素滴度为10~2/g,B.adolescentis也一度下降10~2CFU/g。接种C.difficile后,小鼠虽无明显的腹泻症状,但组织学仍可观察到肠粘膜有充血和分泌增加等轻度损害。扫描电镜和普通光镜均发现E.coli单独或与B.adolescentis共同吸附在肠粘膜微绒毛表面,未见有C.difficile吸附。     

Clinical complications of Mycoplasma pneumoniae disease--central nervous system

The mechanism of the neurologic complications associated with primary atypical pneumonia is unknown. To examine the ability of Mycoplasma pneumoniae to enter the brain of experimental animals, the organism was inoculated into adult and suckling mice by various routes. After intranasal infection, M. pneumoniae was isolated from brains and lungs of both groups of mice. After intracerebral inoculation, the high levels of the mycoplasma persisted for two months or more in the brains of suckling mice. In addition, after intravenous infection, the systemic spread of infection occurred in the mice treated with high doses of cyclophosphamide. Our results suggest that M. pneumoniae may be able to reach the brain via blood and it may occur with relative ease in compromised hosts.     

Use of PCR methods for detection of selected genes of Mycoplasma pneumoniae

The aim of this study was standardization of PCR for the detection of gene encoding the P1 protein, 16S rRNA and elongation factor Tu of M. pneumoniae. A total of 13 strains of M. pneumoniae, 28 strains of other mycoplasmas and 14 strains of different bacteria causing respiratory tract infections were tested. In all of tested M. pneumoniae strains the presence of the sought genes was confirmed. The specificity of DNA was confirmed by the restriction endonuclease analysis with enzymes Hind III, Alu I and Hha I. With none of primers specific for the M. pneumoniae genes amplification of DNA from other bacteria was noted. The PCR method with the selected primers allowed to detect from 10(2) to 10(4) cfu M. pneumoniae/ml suspended in broth. The obtained results indicate that the PCR method can be used for detection of M. pneumoniae genes. A very good sensitivity and specificity predestine++ PCR as a potential quick diagnostic method for identification of M. pneumoniae in clinical specimens.     

Protective effect of colostrum in Mycoplasma pneumoniae infection induced in infant mice

The immunological responses and mechanism of maternal immunity in Mycoplasma pneumoniae infection of mice were investigated. ICR female mice, 4 weeks old, and infant mice, 2 to 4 days old, were infected with M. pneumoniae. Anti-M. pneumoniae antibodies in serum and colostrum were determined by enzyme-linked immunosorbent assay. The specific IgG antibody production persisted for 9 months or longer in both the young and infant mice. These infected mice were protected from rechallenge with M. pneumoniae. In addition, the infected dams conferred passive immunity on their offspring. The infant mice born to uninfected normal dams were protected from the challenge with M. pneumoniae when fed by infected foster dams. Conversely, the infant mice born to infected dams were not protected from the challenge with M. pneumoniae when the infants were fed by uninfected dams. The specific IgG antibody appeared in serum of infant mice inoculated orally with M. pneumoniae-infected mouse serum and the infants were protected from challenge with M. pneumoniae, while the infants given protein A-absorbed serum were not protected from the challenge. These results suggest that one of the factors involved in the resistance of infant mice to M. pneumoniae infection is the specific IgG antibody present in the colostrum rather than the result of transplacental transfer.     

Protective efficacy of a Mycoplasma pneumoniae P1C DNA vaccine fused with the B subunit of Escherichia coli heat-labile enterotoxin

In the present study, we investigated the immunomodulatory responses of a DNA vaccine constructed by fusing Mycoplasma pneumoniae P1 protein carboxy terminal region (P1C) with the Escherichia coli heat-labile toxin B subunit (LTB). BALB/c mice were immunized by intranasal inoculation with control DNAs, the P1C DNA vaccine or the LTB-P1C fusion DNA vaccine. Levels of the anti-M. pneumoniae antibodies and levels of interferon-γ and IL-4 in mice were increased significantly upon inoculation of the LTB-P1C fusion DNA vaccine when compared with the inoculation with P1C DNA vaccine. The LTB-P1C fusion DNA vaccine efficiently enhanced the M. pneumoniae-specific IgA and IgG levels. The IgG2a/IgG1 ratio was significantly higher in bronchoalveolar lavages fluid and sera from mice fusion with LTB and P1C than mice receiving P1C alone. When the mice were challenged intranasally with 10(7) CFU M. pneumoniae strain (M129), the LTB-P1C fusion DNA vaccine conferred significantly better protection than P1C DNA vaccine (P < 0.05), as suggested by the results, such as less inflammation, lower histopathological score values, lower detectable number of M. pneumoniae strain, and lower mortality of challenging from 5 × 10(8) CFU M. pneumoniae. These results indicated that the LTB-P1C fusion DNA vaccine efficiently improved protective efficacy against M. pneumoniae infection and effectively attenuated development of M. pneumoniae in mice.     

Experimental Dental Caries on Gnotobiotic Inbred Mice

The purpose of this study in mono-infected gnotobiotic BALB/cA and C3H/HeN mice was to evaluate the cariogenicity of Enterococcus faecalis. The caries incidence and mean caries score in the BALB/cA mice were significantly higher than those in the C3H/HeN. In both of the mouse strains, the mean number of E. faecalis isolated from the cecum content was almost the same, however, the mean number of E. faecalis from the maxilla of BALB/cA was significantly higher than that of C3H/HeN. These results indicate that C3H/HeN has some factors that prevent E. faecalis from attaching to the tooth surfaces.     

Development of lung tumors in Syrian hamsters with a mixed Mycoplasma pneumoniae and influenza virus infection

The authors studied morphological alterations in the lungs of Syrian hamsters infected intranasally with Mycoplasma pneumoniae and influenza virus. The animals were first infected with M. pneumoniae and after 7 days with influenza A/PR8 virus. On days 1-3 after infection with influenza virus (days 8-11 following infection with M. pneumoniae) the animals showed the appearance of multiple foci of bronchiolar epithelium proliferation. At the later stages of experiment the size of the foci of proliferation increased. On days 14-21 after infection with influenza virus (days 21-28 of experiment) the animals developed lung tumors.     

Mycoplasma pulmonis Arthritis in Congenitally Athymic (Nude) Mice

Histopathological examinations were performed on arthritic joints and other organs of strain BALB/cA nu/nu and nu/+ mice intravenously injected with Mycoplasma pulmonis strain m53. In both groups of mice suffering from polyarthritis, acute inflammatory lesions with infiltration of polymorphonuclear leukocytes in the synovia and periarticular tissues were observed one to two weeks after injection. In nu/nu mice, the acute inflammation appeared repeatedly up to 20 weeks after inoculation, when the experiment was terminated, and furthermore, extensive synovial and periarticular necrosis were characteristically present after the 4th week. Only a small number of lymphocytes and plasma cells were in the lesions. In nu/+ mice, after the early acute inflammation of arthritis, relapses of the infiltration of polymorphonuclear leukocytes were also observed in some mice in and after the 10th week. In addition, infiltration of lymphocytes and plasma cells was substantial after the 15th week. Focal necrosis was sometimes found in the liver of nu/nu mice. Perivascular infiltration of small lymphocytes and plasma cells was found in the lungs, liver and kidney of nu/+ mice in and after the 15th week. Repair mechanisms of injured articular tissues in nu/nu mice were histopathologically poor, while those in nu/+ mice seemed to be progressive and quite similar to those reported by many investigators for mice with the thymus intact. The histopathological differences are discussed in respect to the thymus-dependent immune responses.     

Morphology and Ultrastructure of Mycoplasma pneumoniae Spherules

During growth in fluid medium, most strains of Mycoplasma pneumoniae produce free-floating granules which become larger with time. We have called these granules "spherules." This study describes the morphological and ultrastructural features of M. pneumoniae strain AP-164 spherules, both free and in association with HeLa cells in cell culture. In thin section, spherules were composed of lobulated cells, connected together by membranes, and ring-shaped cells. The two-dimensional morphology observed varied according to the plane of section and to the age of the culture. In HeLa cell cultures, mycoplasmata were found attached to plasma membranes of cells; in sections, individual mycoplasmata were often aligned in radial apposition to the membranes. Mycoplasmata were not found intracellularly. The three-dimensional morphology of spherules was examined by the critical point method and by scanning electron microscopy. Both methods revealed lobulated forms, ring-shaped forms, and star-shaped forms. Treatment of the spherules with crude porcine pancreatic lipase effectively released large numbers of free organisms. Phosphotungstic acid preparations of these uncentrifuged forms revealed a morphology in agreement with the other methods used. Lobulated ring forms with "beaded" filaments were prominent. In respect to morphology, M. pneumoniae under our conditions resembles that of the type species M. mycoides.     

Ultrastructural Features of Mycoplasma pneumoniae

The ultrastructure of Mycoplasma pneumoniae cultivated in broth on glass and plastic surfaces was studied by scanning and transmission electron microscopy. The organisms grew as filaments, which by over-crossing eventually formed a dense network on the surface and in colonies composed mainly of rounded and elongated forms. The filaments were usually thinner at the ends and terminated with a knob-like structure. Some filaments possessed short ramifications which also ended with a knob, and others showed constrictions. Sectioned organisms were seen to contain ribosome-like structures. Many organisms had a specialized structure at their thinner end, which consisted of a dense rod surrounded by electron-lucent cytoplasm and ending with a platelike thickening.     

Separation of Legionella pneumophila proteases and purification of a protease which produces lesions like those of Legionnaires' disease in guinea pig lung

Six discrete protease activities were recovered from the supernatant broth of Legionella pneumophila cultures by ion-exchange chromatography. One of these demonstrated in vitro activity against collagen, casein and gelatin. When administered into the lungs of guinea-pigs this protease elicited lesions which were pathologically similar to those seen in clinical and experimentally induced Legionnaires' disease.     

Gnotobiotic, athymic mice; a possible system for the study of the role of bacteria in human amoebiasis

Two groups of 12 + 14 gnotobiotic, athymic mice were intracaecally injected with Entamoeba histolytica strain HK9 and NIH:200, respectively. Two groups of 16 and 15 mice were given amoebae together with a pure strain of Escherichia coli and a further two groups of 16 and 27 were given amoebae with a pure strain of Clostridium perfringens. Batches of 3-7 mice from each group were killed at intervals of 1-4 weeks. All the mice given NIH:200 alone were found to be infected with trophozoites. Of those given HK9 alone, 20% of the first and 57% of the second group to be examined were infected. Groups of mice given either strain of amoeba monocontaminated with E. coli were all found to be infected at post-mortem examination with no apparent clinical signs and little histological change. The group given HK9 and C. perfringens, although all were infected, failed to produce clinical signs or histological lesions, though some died expectedly. In the group given NIH:200 with C. perfringens the amoebae showed a change of activity and there was evidence of both caecal and liver lesions after 120 days. The usefulness of the system in studying the effect of individual species of bacteria on invasive amoebae is discussed.     

Effect of oxytetracycline and erythromycin on Syrian hamsters after intratracheal infection with Mycoplasma pneumoniae

Male Syrian hamsters were infected intratracheally with Mycoplasma pneumoniae and treated here after for ten days with either OTC or erythromycin. The purpose of this study was to investigate whether there exists a relation between the persistence of M. pneumoniae in the lungs and the infection dose respectively the days passed after infection before starting treatment with antibiotics. The influence of antibiotic therapy on the immune response of the hamsters was also investigated.     

A deficient TLR2 signaling promotes airway mucin production in Mycoplasma pneumoniae-infected allergic mice

The original hygiene hypothesis suggests that early childhood respiratory infections preceding allergen exposure may decrease the prevalence of allergic diseases. We have recently demonstrated that Mycoplasma pneumoniae infection preceding allergen exposure reduced allergic responses in mice. However, the molecular mechanisms underlying the protective role of M. pneumoniae in allergic responses, particularly airway mucin production, remain unclear. Wild-type and Toll-like receptor 2 (TLR2)-deficient mice with a respiratory M. pneumoniae infection preceding allergen (ovalbumin) challenge were utilized to determine the regulatory role of TLR2-IFN-gamma signaling pathway in airway mucin expression. Furthermore, air-liquid interface cultures of mouse primary tracheal epithelial cells were performed to examine the effects of IFN-gamma on mucin expression. In wild-type mice, M. pneumoniae infection preceding allergen challenge significantly reduced airway mucins but increased IFN-gamma. In sharp contrast, in TLR2-deficient mice, M. pneumoniae preceding allergen challenge resulted in increased mucin protein without a noticeable change of IFN-gamma. In cultured mouse primary tracheal epithelial cells, IFN-gamma was shown to directly inhibit mucin expression in a dose-dependent manner. Our study demonstrates for the first time that a respiratory M. pneumoniae infection preceding allergen challenge reduces airway epithelial mucin expression in part through TLR2-IFN-gamma signaling pathway. A bacterial infection in asthmatic subjects with weakened TLR2-IFN-gamma signaling may result in an exaggerated airway mucin production.