Acta path. microbiol. scand. Sect. B, 85: 462-465, 1977
BRIEF REPORTS EXPERIMENTAL MYCOPLASMAL PNEUMONIA IN DOGS : ELECTRON MICROSCOPY OF INFECTED TISSUE S. Rosendal and 0. Vinther The Institute of Medical Microbiology, University of Aarhus, Denmark
Rosendal, S. & Vinther, 0.Experimental mycoplasmal, pneumonia i n dogs: Electron microscopy of infected tissue. Acta path. microbiol. scand. Sect. B, 85: 462-465, 1977. Thin sections of lung tissue from dogs with pneumonia induced by endobronchial inoculation of Mycoplasma cynos were examined by electron microscopy. Mycoplasmas were observed extracellularly in lumen of bronchus and in alveoli in the earlier stages of the infection. The infection also resulted i n degenerative changes in the bronchial epithelial cells, including in particular destruction and loss of cilia. Key words: Mycoplasma cynos; mycoplasmal pneumonia; electron microscopy.
S. Rosemdal, Institute of Medlica4 Microbiology, Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark.
Received 9.vii.77
Accepted 14.x.77
Pneumonia was induced by endobronchial inoculation of Mycoplatma cynos, strain D19, !into 1-week-old dogs (4). The present communication gives the results of electron microscopic examination of infected tissue from three dogs killed a t regular intervals after inoculation. Histologically, the lesions induced were characterized by severe inflammation of bronchi and adjacent respiratory tissue. At post-mortem, large numbers of mycoplasmas were isolated from affected tissue of the two dogs killed first, whereas none were recovered from the last one. Material and Methods The dogs, D23, D24 and D25, were killed with an overdose of barbiturate 2, 3 and 4 weeks after inoculation, and tissue was removed immediately and cut into blocks about 1 x 1 x 2 mm: These blocks were fixed by immersion in 2 per cent glutaraldehyde in 0.1 M d i u m cacodylate buffer, p H 7.2, for 4 h at mom temperature. After a brief wash in cacodylate buff= containing 3 per cent sucrose, the blocks were further fixed in 1 per cent OsO, in Michaelis buffer, p H 7.2, for 1 h at room temperature, and subsequently stained for 15 min
462
in 2 per cent uranyl acetate in 70 per cent ethanol. After acetone dehydration and embedding in Vestopal-W, u1trathi.n seotions were obtained with an LKB Ultrotome-I11 microtome, post-stained with magnesium uranyl acetate and lead citrate, and examined in a JEOL JEM 100 B electron m i c m scope. Based on microscopical examination of toluidine blue (0.1 per cent) stained sections, representative areas of bronchial and alveolar inflammatory lesions were chosen for electron m~croscopy. For
Fig. 1. D23. Macrophage in lumen of bronchus. Several mycoplasmas ( M ) are present extracelluLrly. One mycoplasma ( M p ) seems to be in the process of being phagocytosed. Lysosomes (L). Possible phagosome ( P ) . x 41,000. Bar represents 250 nm. Fig. 2. D23. Macrophage containing phagosome (P) i,n the lumen of a bronchus. Mycoplasma structures ( M ) are seen in the phagosome and extracellularly. In addition, there is membrane debris (Me) in the phagosome. The macrophage surface is irregular with many pseudopods (Ps). x 61,000. Bar represents 250 nm.
463
464
comparison purposes, lung tissue from areas without histologically visible lesions was also included in the study.
Results 0 2 3 . The epithelial cells of the i d d e d bronshi were swollen and vacuolated. Cilia were fractured and deformed and were less in number compared to (the normal bronchus. Membranous debris and desquamated epithelial cells were seen in the lumen together with neutrophils and macrophages. Mycoplannas were observed in different areas of the lumen. In Fig. 1, several mycoplasmas, characterized by their densely packed ribosomes and their triple-layered limiting membrane, are located near a macrophage which is swollen and surrounded by a damaged or broken plasma membrane. The c y t e plasmic matrix of the macrophage is lucent and the c y t o p l m i c organelles partly disorganized. One organism, possibly in an early stage of being phagocytosed, can be seen. Macrophages with phagosoma containing mycoplasma-like structures, as well as unidentifiable membrane debris, were also encountered (Fig. 2). I n sections of alveoli, the Jininmg celels were swollen and vacuolated. Alveolar spaces contained neutrophils, macrophages, and unidentifiable debris. Mycoplasmas were also observed in the alveoli. 0 2 4 . The epithelial cells of the bronchi were very swollen and appeared to be vacuolated due to pronounced distention of the endoplasmic reticulum. The mitochondria were also swollen. However, the most striking feature was the severe destruction of cilia which, in mast cases could only be identified by their remaining basal bodies. Membrane and cilia debris were observed in the
Fig. 3. D24. Section of bronchus. The epithelial cells are swollen and show distended endoplasmic reticulum (ER) and swollen mitochondria (Mi). Mycoplasmas ( M ) , cilia ( C ) and membrane debris (Me) are present in the lumen. Basal bodies (B) of cilia can be seen at the epithelial surface. x 12,000. Bar represents 1 pn. Fig. 4. D25. Alveolar macrophages Degeneration of these cells is evident from the marginated and clumped chromatin, the distended endoplasmic reticulum (ER), the swollen mitochondria (Mi), and the prominent myelin figures (My) present in their cytoplasm. x 10,000. Bar represents 1 pn. Fig. 5. D24. Alveolar epithelial cell from an area without histologically visible lesions. T h e ultrastructure of the cell is well preserved. Round and elongated mitochondria and flat endoplasmic reticulum are observed in the cytoplasm of the cell. x 33,000. Bar represents 250 run.
lumen together with a few mycoplasmas (Fig. 3 ) . Several plasma cells and macrophages were present in the peribronchial area. Degenerated neutrophils and macrophages were observed in the alveoli, together with unidentifiable debris. Mycoplasmas could not be demonstrated in the alveoli of this dog. Degenerative changes i n bronchial and alveolar epithelium were not observed in tissue without visible lesions (Fig. 5). 0 2 5 . Only very few basal bodies were left of the cilia of the epithelial cells, otherwise the same changes were observed as seen in D24. The alveoli were filled with more or less degenerated cells and membrane debris. Fig. 4 shows alveolar macrophages. The endoplasmic reticulum of these cells is distended and their mitochondria are swollen. In addition myelin-like structures are very prominent in the cytoplasm. Mycoplasmas were not seen, either in the bronchus or i n the alveoli.
Discussion This paper reports on the electron microscopical demonstration of mycoplasmas in dogs after endobronchial inoculation of the animals with M . cynos. The mycoplasmas were easily recognized on the basis of their characteristic morphology, as demonstrated previously in laboratory cultures of M . cynos (3). The organisms were mainly located extracellularly, but some could also be found in phagosomes of macrophages and neutrophaic granulocytes. The histopathologic characteristics (Rosendal, unpublished results (4) ) were, in general, confirmed by electron microscopy. One feature which deserves special attention is the degenerative changes in the bronchial epithebial cells, including the destruction and loss of cilia. These changes are probably caused by the rnycoplasmas and not by inadequate fixation, since the ultrastructure of lung tissue from areas without visible lesions was generally well preserved. The bronchial changes resemble closely those observed i n rats inoculated with Mycoplasma pulmonis (2) and in hamsters infected with Mycoplasma pneumonia (1). References: 1. Collier, A . M . , Clyde, W . A,, Ir. & Denny, F. W . : Proc. Soc. Exptl. Biol. Med. 136: 569-573, 1971.-2. Kohn, D . F.: Lab. Anim. Sci. 21: 856-861, 1971.-3. Rosendal, S.: Int. J. Syst. Bacteriol. 23: 49-54, 1 9 7 3 . 4 . Rosendal, S.: Unpublcished results.
465
BRIEF REPORTS EXPERIMENTAL MYCOPLASMAL PNEUMONIA IN DOGS : ELECTRON MICROSCOPY OF INFECTED TISSUE S. Rosendal and 0. Vinther The Institute of Medical Microbiology, University of Aarhus, Denmark
Rosendal, S. & Vinther, 0.Experimental mycoplasmal, pneumonia i n dogs: Electron microscopy of infected tissue. Acta path. microbiol. scand. Sect. B, 85: 462-465, 1977. Thin sections of lung tissue from dogs with pneumonia induced by endobronchial inoculation of Mycoplasma cynos were examined by electron microscopy. Mycoplasmas were observed extracellularly in lumen of bronchus and in alveoli in the earlier stages of the infection. The infection also resulted i n degenerative changes in the bronchial epithelial cells, including in particular destruction and loss of cilia. Key words: Mycoplasma cynos; mycoplasmal pneumonia; electron microscopy.
S. Rosemdal, Institute of Medlica4 Microbiology, Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark.
Received 9.vii.77
Accepted 14.x.77
Pneumonia was induced by endobronchial inoculation of Mycoplatma cynos, strain D19, !into 1-week-old dogs (4). The present communication gives the results of electron microscopic examination of infected tissue from three dogs killed a t regular intervals after inoculation. Histologically, the lesions induced were characterized by severe inflammation of bronchi and adjacent respiratory tissue. At post-mortem, large numbers of mycoplasmas were isolated from affected tissue of the two dogs killed first, whereas none were recovered from the last one. Material and Methods The dogs, D23, D24 and D25, were killed with an overdose of barbiturate 2, 3 and 4 weeks after inoculation, and tissue was removed immediately and cut into blocks about 1 x 1 x 2 mm: These blocks were fixed by immersion in 2 per cent glutaraldehyde in 0.1 M d i u m cacodylate buffer, p H 7.2, for 4 h at mom temperature. After a brief wash in cacodylate buff= containing 3 per cent sucrose, the blocks were further fixed in 1 per cent OsO, in Michaelis buffer, p H 7.2, for 1 h at room temperature, and subsequently stained for 15 min
462
in 2 per cent uranyl acetate in 70 per cent ethanol. After acetone dehydration and embedding in Vestopal-W, u1trathi.n seotions were obtained with an LKB Ultrotome-I11 microtome, post-stained with magnesium uranyl acetate and lead citrate, and examined in a JEOL JEM 100 B electron m i c m scope. Based on microscopical examination of toluidine blue (0.1 per cent) stained sections, representative areas of bronchial and alveolar inflammatory lesions were chosen for electron m~croscopy. For
Fig. 1. D23. Macrophage in lumen of bronchus. Several mycoplasmas ( M ) are present extracelluLrly. One mycoplasma ( M p ) seems to be in the process of being phagocytosed. Lysosomes (L). Possible phagosome ( P ) . x 41,000. Bar represents 250 nm. Fig. 2. D23. Macrophage containing phagosome (P) i,n the lumen of a bronchus. Mycoplasma structures ( M ) are seen in the phagosome and extracellularly. In addition, there is membrane debris (Me) in the phagosome. The macrophage surface is irregular with many pseudopods (Ps). x 61,000. Bar represents 250 nm.
463
464
comparison purposes, lung tissue from areas without histologically visible lesions was also included in the study.
Results 0 2 3 . The epithelial cells of the i d d e d bronshi were swollen and vacuolated. Cilia were fractured and deformed and were less in number compared to (the normal bronchus. Membranous debris and desquamated epithelial cells were seen in the lumen together with neutrophils and macrophages. Mycoplannas were observed in different areas of the lumen. In Fig. 1, several mycoplasmas, characterized by their densely packed ribosomes and their triple-layered limiting membrane, are located near a macrophage which is swollen and surrounded by a damaged or broken plasma membrane. The c y t e plasmic matrix of the macrophage is lucent and the c y t o p l m i c organelles partly disorganized. One organism, possibly in an early stage of being phagocytosed, can be seen. Macrophages with phagosoma containing mycoplasma-like structures, as well as unidentifiable membrane debris, were also encountered (Fig. 2). I n sections of alveoli, the Jininmg celels were swollen and vacuolated. Alveolar spaces contained neutrophils, macrophages, and unidentifiable debris. Mycoplasmas were also observed in the alveoli. 0 2 4 . The epithelial cells of the bronchi were very swollen and appeared to be vacuolated due to pronounced distention of the endoplasmic reticulum. The mitochondria were also swollen. However, the most striking feature was the severe destruction of cilia which, in mast cases could only be identified by their remaining basal bodies. Membrane and cilia debris were observed in the
Fig. 3. D24. Section of bronchus. The epithelial cells are swollen and show distended endoplasmic reticulum (ER) and swollen mitochondria (Mi). Mycoplasmas ( M ) , cilia ( C ) and membrane debris (Me) are present in the lumen. Basal bodies (B) of cilia can be seen at the epithelial surface. x 12,000. Bar represents 1 pn. Fig. 4. D25. Alveolar macrophages Degeneration of these cells is evident from the marginated and clumped chromatin, the distended endoplasmic reticulum (ER), the swollen mitochondria (Mi), and the prominent myelin figures (My) present in their cytoplasm. x 10,000. Bar represents 1 pn. Fig. 5. D24. Alveolar epithelial cell from an area without histologically visible lesions. T h e ultrastructure of the cell is well preserved. Round and elongated mitochondria and flat endoplasmic reticulum are observed in the cytoplasm of the cell. x 33,000. Bar represents 250 run.
lumen together with a few mycoplasmas (Fig. 3 ) . Several plasma cells and macrophages were present in the peribronchial area. Degenerated neutrophils and macrophages were observed in the alveoli, together with unidentifiable debris. Mycoplasmas could not be demonstrated in the alveoli of this dog. Degenerative changes i n bronchial and alveolar epithelium were not observed in tissue without visible lesions (Fig. 5). 0 2 5 . Only very few basal bodies were left of the cilia of the epithelial cells, otherwise the same changes were observed as seen in D24. The alveoli were filled with more or less degenerated cells and membrane debris. Fig. 4 shows alveolar macrophages. The endoplasmic reticulum of these cells is distended and their mitochondria are swollen. In addition myelin-like structures are very prominent in the cytoplasm. Mycoplasmas were not seen, either in the bronchus or i n the alveoli.
Discussion This paper reports on the electron microscopical demonstration of mycoplasmas in dogs after endobronchial inoculation of the animals with M . cynos. The mycoplasmas were easily recognized on the basis of their characteristic morphology, as demonstrated previously in laboratory cultures of M . cynos (3). The organisms were mainly located extracellularly, but some could also be found in phagosomes of macrophages and neutrophaic granulocytes. The histopathologic characteristics (Rosendal, unpublished results (4) ) were, in general, confirmed by electron microscopy. One feature which deserves special attention is the degenerative changes in the bronchial epithebial cells, including the destruction and loss of cilia. These changes are probably caused by the rnycoplasmas and not by inadequate fixation, since the ultrastructure of lung tissue from areas without visible lesions was generally well preserved. The bronchial changes resemble closely those observed i n rats inoculated with Mycoplasma pulmonis (2) and in hamsters infected with Mycoplasma pneumonia (1). References: 1. Collier, A . M . , Clyde, W . A,, Ir. & Denny, F. W . : Proc. Soc. Exptl. Biol. Med. 136: 569-573, 1971.-2. Kohn, D . F.: Lab. Anim. Sci. 21: 856-861, 1971.-3. Rosendal, S.: Int. J. Syst. Bacteriol. 23: 49-54, 1 9 7 3 . 4 . Rosendal, S.: Unpublcished results.
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