May 17, 1972 - The loss of viability and ultrastructural ... *Department of Veterinary Pathology, University of ... tubes, and for ultrastructural studies, 10 ml.
Viability and Ultrastructural Changes in Group E Streptococci After in vitro Phagocytosis by Swine Macrophages C. F. Cargill and L. D. Olson*
ABSTRACT
RESUME
The loss of viability and ultrastructural changes were studied in group E Streptococci (GES) after in vitro phagocytosis in immune swine macrophages. There was a 50% reduction in the viability of intracellular GES during the first 60 minutes of incubation and a total loss of viability by 300 minutes as compared to the control tubes where the GES increased. Loss of viability in phagocytized group E Streptococci was associated with the appearance of degenerative changes in the bacterial cytoplasm. This was followed by disruption of the bacterial cell membrane and its separation from the bacterial cell wall. No definite evidence of cell wall degeneration could be found. Unphagocytized organisms incubated for similar periods and fixed in the same manner did not lose viability nor have any degenerative changes.
Cette etude visait a determiner la perte de viabilite et les changements ultrastructuraux des streptocoques du groupe E (SGE), suite a leur phagocytose in vitro par des macrophages de porcs immuns. On nota une reduction de la viabilite des SGE intracellulaires, de l'ordre de 50%, au cours des 60 premieres minutes d'incubation, et une perte totale de leur viabilite, au bout de 300 minutes, comparativement a une augmentation du nombre des SGE, dans les tubes temoins. La perte de viabilite chez les SGE phagocytes s'accompagnait de changements degeneratifs de leur cytoplasme. On nota ensuite une dislocation de leur membrane cellulaire, et sa separation de la capsule. Celle-ci ne manifestait cependant aucune evidence de degenerescence. La viabilite des microbes non phagocytes, incube6s durant des periodes de temps semblables, et fixes de la meme maniere, demeura intacte; ils ne manifesterent pas non plus de changements dege'neratifs.
INTRODUCTION *Department of Veterinary Pathology, University of Missouri. Columbia, Missouri 65201. Portion of a dissertation presented by the senior author to the Graduate Faculty of the University of Missouri as partial fulfillment of the requirements for the degree, Doctor of Philosophy. Present address of senior author: Institute for Pathological Anatomy, University of Zurich, Switzerland. Supported by Cooperative Agreement No. 12-14-100-9741 (45) from the U.S. Department of Agriculture.
Submitted May 17, 1972.
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Group E Streptococci (GES) can induce a lymphadenitis in swine which is confined principally to the cervical region (3). It is thought that the GES penetrate the tonsillar tissue and quickly pass to the regional lymph nodes where the organisms either are destroyed or produce an abscess (5). Swine between three and 12 months old are most
Can. J. comp. Med.
ESTABLISHMENT OF MACROPHAGE CULTURES
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Blood leukocytes were collected and macrophage cultures were established using the methods as described by the authors in a previous report (5).
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The inoculum was prepared as previously described (5) with a final concentration of 2 to 5 x 107 colony forming units (CFU)/ ml serum. For viability studies, 1 ml of leukocyte suspension containing 2.5 x 105 monocytes/ml were placed in Leighton tubes, and for ultrastructural studies, 10 ml of leukocyte suspension were placed in 100 ml tissue culture bottles.
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60 120 300 MINUTES AFTER REMOVAL OF UNPHAGOCYTIZED GES
Fir. 1. The reduction of viable intracellular GES in macrophages (the number of viable intracellular organisms present at the end of 30 minutes of incubation was taken as l00c8c).
susceptible to infection (11). Immunity has been produced in such swine by exposing them intravenously to viable GES (12). It has been observed that macrophages from both susceptible and immune ten week old swine phagocytized and destroyed GES Xn vitro; however, the macrophages from immune swine phagocytized and reduced the viability of GES more rapidly (2). This study was conducted to determine the ultrain GES after structural changes that phagocytosis by macrophages and to compare them with the loss of viability.
VIABILITY
STUDIES
Each macrophage culture was infected with 2 x 106 CFU of GES and incubated at 37°C in an incubator gassed with 5% C02. After an incubation of 30, 60, 120 and 300 minutes, cultures were washed three times with Hanks balanced salt solution (HBSS) which removed essentially all of the unphagocytized GES. The number of viable intracellular organisms present at each of these intervals was determined by adding 1 ml of 10% saponin to a Leighton tube in each series and incubating the tubes for 15 minutes before making serial dilutions and plating a sample of each dilution on a blood agar plate. The number of viable intracellular GES present 15 minutes after the removal of unphagocytized GES was taken as 100l
occur
MATERIALS AND METHODS MACROPHAGES
AND IMMUNE SERUM
Macrophages and immune serum were collected from 14 week old specific-pathogen-free swine which had been administered intravenously 1 x 108 colony forming units (CFU) of GES four weeks previous-
Fig. 2. Unphagocytized group E Streptococcus after 30 minutes of incubation in Hanks basal salt solution and The cell envelope (arrows) is surrounded by flocculent electron dense material (f). X34,960.
serum.
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Fig. 3. Group E Streptococci (s) within a cytoplasmic vesicle (cv) of a macrophage 30 minutes after engulfment. Vesicle membrane (vm). X32,230.
Fig. 4. Group E Streptococci approximately 90 minutes after engulfment. Cytoplasmic degenerative changes (r) have extended to the bacterial cell wall with disruption of the cell membrane (arrows). Mesosome (m). X67,685.
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and the percentage loss in viability of the intracellular GES was calculated as a function of time. Control tubes containing either medium and saponin or medium alone were inoculated with GES and the number of viable organisms determined at the same intervals. ULTRASTRUCTURAL STUDIES
Macrophages were prepared for electron microscopy after zero, 60 and 240 minutes of incubation and the removal of unphagocytized GES. A technique developed for platelet studies was used without modification (13). The same technique was used to prepare unphagocytized GES for electron microscopy after zero, 30 and 240 minutes of incubation in serum and HBSS. The agar cubes were embedded in fresh epon (6) and sections cut with a Sorvall MT-2 Porter-Blurcn ultra-microtome. Sections were stained with Reynold's lead citrate (8) for five minutes. A Radio Corporation of America EMU-3 electron microscope was used to view the sections.
RESU LTS
VIABILITY STUDIES
There was a 50% reduction in the viability of intracellular GES during the first 60 minutes of incubation and a total loss of viability by 300 minutes (Fig. 1). In the control tubes, the number of organisms increased from 6 x 106 CFU/ml to 9 x 106 CFU/ml during the 120 minutes of incubation. ULTRASTRUCTURAL STUDIES
Ultrastructural changes were not observed in the unphagocytized GES fixed after suspension in the medium for varying periods. A flocculent material was attached to the external surface of the bacterial cell wall which appeared as a less dense layer (Fig. 2).
Fig. 5. Group E Streptococcus 90 minutes after engulfm ent. The bacterial cell wall (cw) and cell membrane (cm) have separated. X78,400.
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Organisms were observed in cytoplasmic vesicles of macrophages fixed 30 minutes after the addition of GES to the culture medium (Fig. 3). The majority of these organisms did not differ morphologically from the unphagocytized GES. However, in some organisms a reticular pattern, associated with a decreased density, was observed in the cytoplasm of the bacterial cell. In many organisms within macrophages fixed 60 minutes after incubation, the reticular pattern of the cytoplasm extended almost to the bacterial cell wall and evidence of a disruption of the bacterial cell membrane was observed (Fig. 4). In some organisms, the cell wall was separated from the bacterial cell membrane (Fig. 5), but mesosomes could still be observed (Fig. 4). After 240 minutes of incubation, in a majority of the unphagocytized organisms, the cell membrane had begun to separate from the cell wall.
changes were observed in phagocytized GAS (4, 7) as well as in GAS subjected to phage-associated lysin and Streptomyces albus enzyme (1). Neither the method of fixation nor the medium appeared to induce any degenerative changes in the unphagocytized GES. The flocculent material attached to the bacterial cell wall was considered to be proteinaceous material from the medium as was also concluded in a different study where similar material surrounded GAS (4).
ACKNOWLEDGMENTS We gratefully acknowledge the College of Agriculture, University of Missouri for the use of the Electron Microscopy Facility and Drs. Merton Brown (Department of Plant Pathology) and Edward Adelstein (Department of Medical Pathology) for their generous advice.
DISCUSSION
REFERENCES
The degenerative changes in the phagocytized GES observed ultrastructurally correlated with the loss in viability. These changes also correlated with the results of a different study where, at the same time after the unphagocytized GES were removed, there was marked reduction in the number of phagocytized GES per macrophage staining positive with fluorescein conjugated anti-GES serum (9). As in this study, the initial ultrastructural change in group A Streptococci (GAS) after ingestion by phagocytes has been reported to be a dissolution of the bacterial cytoplasm (1, 4). It has been suggested that phagocytes killed bacteria through their hydrogen peroxide activity and that lysosomal enzymes were responsible for the degradation (10). In this study, degenerative changes developed in the cytoplasm of the bacteria before changes were observed in the bacterial cell membrane. If the degradation was due to lysosomal enzymes, the cell wall of the organism must have become permeable to the enzymes responsible for degradation even though definite cell wall degeneration was not observed. The same sequence of
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