Mycoplasma pulmonis infection in gnotobiotic and conventional mice: aspects of pathogenicity including microbial enumeration and studies of tracheal involvement.

Intranasal inoculation of Mycoplasma pulmonis into gnotobiotic mice resulted in positive cultures from the anterior nares, nasopharynx, trachea, and lungs persisting for 21 da. Similar inoculation of conventional, mycoplasma-free mice resulted in an increase in colony-forming units reaching a maximum by the 8th post-inoculation day and remaining constant in trachea and lungs through the 22nd day by an organ homogenate serial dilution method. All mice appeared ill with loss of total body weight, an increase in lung weight, and a peak of mortality between the 6th and 8th days. Between the 8th and 22nd, day, the number of deaths declined and survivors improved in appearance despite persistence of large numbers of colony-forming units in the trachea and lungs. M pulmonis was demonstrated in the trachea by immunofluorescence and electron microscopy.

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.

Pneumonia due to Mycoplasma in gnotobiotic mice. 3. Lesions in the lungs of gnotobiotic mice after multiple intranasal inoculations of broth cultures of Mycoplasma pneumoniae.

Lung lesions characterized by extensive peribronchial and perivascular round cell infiltration and intrabronchial plugging with polymorphonuclear leukocytes were produced in 5 of 44 Ha/ICR gnotobiotic mice sacrificed 3 to 10 days after three intranasal inoculations of broth cultures of Mycoplasma pneumoniae. After 10 days, no significant lesions were seen, and the proportion of lungs positive for M. pneumoniae dropped off sharply. M. pneumoniae persisted for longer periods (up to 24 days) in the trachea, nasopharynx, and anterior nares. These findings would seem to represent a self-limited respiratory infection due to M. pneumoniae in gnotobiotic mice. Ring forms within granular alveolar pneumocytes were seen by electron microscopy in the lungs of triply inoculated gnotobiotic controls receiving sterile horse-serum broth as well as in the lungs of mice receiving broth cultures of M. pneumoniae. Ring forms must now be considered to be part of the nonspecific cellular reactions of pneumocytes to foreign substances in the lungs of mice rather than intracytoplasmic developmental forms of mycoplasma as previously proposed.

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.