Vol. 60, No. 5
INFECrION AND IMMUNITY, May 1992, p. 2121-2124 0019-9567/92/052121-04$02.00/0 Copyright © 1992, American Society for Microbiology
Ultrastructural Study of Helicobacterpylori Adherence Properties in Gnotobiotic Piglets DANIEL G. RUDMANN,t KATHRYN A. EATON,* AND STEVEN KRAKOWKA
Department of Veterinary Pathobiology, School of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210 Received 19 December 1991/Accepted 24 February 1992
Ultrastructural examination of gastric mucosa from Helicobacterpylori-infected gnotobiotic piglets identified four general adherence patterns comparable to those observed in human patients. Intimate associations between the bacterial and mucosal cell membranes, including cuplike invaginations and adherence pedestals, were present and were accompanied by alterations to microvilli and cell membrane morphology.
patterns and ultrastructure confirmed that the recovered strain was identical to the challenge strain (8).
The spiral-shaped, gram-negative bacterium Helicobacter pylon is the primary cause of human type B gastritis (1, 3) and is thought to be an important part of duodenal and peptic ulcer pathogenesis in humans (12). Ethical considerations limit H. pylon research with human volunteers. As a result, a concentrated search for an appropriate animal model has been undertaken. The gnotobiotic piglet stomach can be colonized by H. pylon and produces gastric lesions similar to those observed in humans (14). This model has been useful in determining the pathogenesis of H. pylon infection and identifying various bacterial virulence factors (6, 7, 9). The present study was initiated to determine the ultrastructural morphology of H. pylon adherence patterns in gnotobiotic piglets and to compare the morphology with that of human infection. Bacterial strains and animal model. A total of 14 3-day-old gnotobiotic domestic Yorkshire piglets were randomly selected from two litters of date-mated pregnant sows derived by Caesarian section. The infected group (n = 5) was challenged with 109 CFU of laboratory H. pylon 26695 (7). Infected (n = 5) and control (n = 9) groups were separated and maintained in sterile isolation units as previously described (10). Transmission electron microscopy. At sacrifice, 21 to 25 days after infection, 1- to 2-mm biopsy samples from the antrum, fundus, and cardia of the stomach were obtained by utilizing an aseptic surgical technique (7). Infected and control group specimens were fixed for 24 h in 3% glutaraldehyde (pH 7.4), rinsed in cacodylate, postfixed in Millonig's 1% osmium tetroxide, dehydrated in graded ethanol solutions, and embedded in Medcast epoxy resin (Ted Pella, Inc., Redding, Calif.). One-micrometer-thick toluidine blue sections were screened for orientation and microbes. Selected areas were identified, and the blocks were trimmed, stained with uranyl acetate and Reynold's lead citrate, and mounted on copper grids. Twenty-five grids from three infected piglets were examined ultrastructurally with a Philips 300 electron microscope. Restriction endonuclease and sodium dodecyl sulfate-polyacrylamide gel electrophoresis
H. pylon organisms were observed in approximately 35 fields from prepared tissue grids of three infected piglets. In less than 25% of the fields, bacteria were located on the luminal surface of gastric epithelial cells. It was more common to find H. pylon on the surface of gastric pit epithelial cells and in gastric intraepithelial spaces. In fundic segments, the organisms sometimes were identified adjacent to parietal cells within gastric pits. The darkly stained, flagellated organisms were 0.5 to 0.7 ,um wide and 2 to 4.5 p,m long and possessed a double unit membrane encircling a granular protoplasm. No organisms in any of the control sections were identified. A close association between the surface of the epithelial cells and the bacteria was observed. Four adherence patterns were noted. In most cases, the bacterial membrane was closely apposed to the epithelial cell membrane, with some loss of microvilli and mild alterations in membrane morphology (Fig. 1A). Less often, bacteria were adhered to epithelial cell microvilli. The bacterial membranes either were directly apposed to the microvilli or appeared fused with them. Some bacteria were adhered to indented sites on the epithelial cell surface, with subsequent loss of microvilli (Fig. 1B). In three instances, the epithelial cell surface extended outward and fused with the bacterial membrane, possibly representative of an adherence pedestal (Fig. 1C). Twice, the epithelial cell membrane appeared to engulf the bacterium (Fig. 1D). In situ morphologic evidence of replication (e.g., binary fission) was seen frequently. Ultrastructural studies with humans indicate that there is a close association between H. pylon and the gastric epithelium in chronic gastritis (2, 5, 9, 11, 13, 14). Patterns of physical contact that are thought to represent different evolutive stages of H. pylon infection have been recognized previously (4). Several of the bacterial adherence patterns which have been observed include the formation of cuplike projections (11), indentation sites (9), and pedestals (4, 9). The appearance of pedestals is believed to be an important virulence factor and indication of the pathogenicity of the organism (9). A property of adherence present in many of the patterns is partial or complete bacterial and epithelial membrane fusion, often resulting in no change in the contour of the epithelial cell (9, 14). A few bacteria are covered by or resting on an epithelial microvillus, sometimes resulting in loss of normal
* Corresponding author. t Present address: Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47906.
2121
2122
NOTES
INFECT. IMMUN.
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'i
;.,, .-
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," 's
"
:,
A
F VL
5,
-,
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B: 4
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-.
--I
FIG. 1. (A) Several H. pylon organisms closely associated with gastric epithelium. Some bacterial membranes appear fused with epithelial cell membrane (arrows), while others are only intimately apposed (star). Magnification, x 15,000. (B) H. pylon adherence associated with loss of underlying gastric epithelial microvilli (arrows). Magnification, x43,000. (C) Pedestal extending from gastric epithelial cell to adherence site on H. pylori organism (arrow). Magnification, x 32,000. (D) An epithelial cell membrane appears to engulf an H. pylon organism, with subsequent fusion of membranes. Magnification, x32,000.
microvillus structure. In general, these adhered bacteria are present both at intercellular junctions and at the surfaces of epithelial cells, but more often in the former (4). The patterns of adherence seen in this study were similar to those seen in ultrastructural studies of human gastric mucosa colonized by H. pyloni.
How the adherence patterns seen in these piglets directly affect the host gastric epithelial cells remains unanswered.
Probably, the bacteria utilize substances that the adjacent cells require for survival. Some believe that H. pyloni may produce an exotoxin that damages the underlying adhered cells (3). It may be that direct bacterial adherence blocks the
wr
FIG.
1-Continued. 2123
2124
NOTES
normal function of the gastric epithelial cell membrane by interfering with ion transport channels and/or blocking and destroying membrane receptors. When adherence between the bacteria and cell was intimate, we observed a loss of architecture of the adhering surfaces. It became impossible to distinguish the bacterial cell wall from the epithelial cell membrane. What exactly occurs at this point of fusion is unknown. Whether the fusion is a virulence factor of H. pylon or a protective response of the epithelial cell requires further study. As seen in humans (5), bacteria were frequently observed near resting-phase parietal cells. There exists the possibility that the bacteria, through some mechanism, inhibit normal functioning of parietal cells, resulting in a local achlorhydria. As a result, the immediate gastric environment is made less acidic and more favorable for H. pylon colonization. H. pylon in humans and Helicobacter mustelae in ferrets are proposed to be the only Helicobacter species to attach to gastric mucosa. It is interesting to note that during our studies of H. pylon in gnotobiotic piglets, we investigated an outbreak of gastritis in a group of cheetahs at the zoo in Columbus, Ohio. An unpublished ultrastructural study of the cheetah gastric lesions showed evidence of similar adherence patterns by Gastrospillum spp. The role that this bacterium plays in the pathogenesis of cheetah gastric disease requires further investigation. The similarities in adherence patterns of H. pylon in humans and gnotobiotic piglets provide further evidence that supports the usefulness of the gnotobiotic piglet as an animal model of human H. pylon type B gastritis. The exact pathogenic effect of these adherence patterns in humans and in gnotobiotic piglets must now be elucidated to better understand the precise role of H. pylon in human type B gastritis and duodenal and gastric ulceration. We thank E. Handley for her assistance in preparation of electron microscopy samples and in operation of the microscope. This study was completed as part of an off-campus research externship offered by the Purdue University School of Veterinary Medicine and The Ohio State University College of Veterinary Medicine.
REFERENCES 1. Blaser, M. J. 1990. Helicobacterpylori and the pathogenesis of gastroduodenal inflammation. J. Infect. Dis. 161:626-633.
INFECT. IMMUN. 2. Bonvicini, F., P. Versura, S. Pretolani, G. Gasbarrini, and R. Laschi. 1989. Scanning electron microscopy in the study of Campylobacter pylori associated gastritis. Scanning Microsc. 3:355-368. 3. Buck, G. E. 1990. Campylobacter pylon and gastroduodenal disease. Clin. Microbiol. Rev. 3:1-12. 4. Caselli, M., N. Figura, L. Trevisani, P. P. Pazzi, P. Guglielmetti, M. R. Bovolenta, and G. Stabellini. 1989. Patterns of physical modes of contact between Campylobacter pylori and gastric epithelium: implications about the bacterial pathogenicity. Am. J. Gastroenterol. 84:511-513. 5. Chen, X. G., P. Correa, J. Offerhaus, E. Rodriguez, F. Janney, E. Hoffmann, J. Fox, F. Hunter, and S. Diavolitsis. 1986. Ultrastructure of the gastric mucosa harboring Campylobacterlike organisms. Am. J. Clin. Pathol. 86:575-582. 6. Eaton, K. A., C. L. Brooks, D. R. Morgan, and S. Krakowka. 1991. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylon in gnotobiotic piglets. Infect. Immun. 59:2470-2475. 7. Eaton, K. A., D. R. Morgan, and S. Krakowka. 1989. Campylobacter pylori virulence factors in gnotobiotic piglets. Infect. Immun. 57:1119-1125. 8. Eaton, K. A., D. R. Morgan, and S. Krakowka. 1990. Persistence of Helicobacter pylori in conventionalized piglets. J. Infect. Dis. 161:1299-1301. 9. Hessey, S. J., J. Spencer, J. I. Wyatt, G. Sobala, B. J. Rathbone, A. T. R. Axon, and M. F. Dixon. 1990. Bacterial adhesion and disease activity in Helicobacter associated chronic gastritis. Gut 31:134-138. 10. Krakowka, S., D. R. Morgan, W. G. Kraft, and R. D. Leunk. 1987. Establishment of gastric Campylobacter pylon infection in the neonatal gnotobiotic piglet. Infect. Immun. 55:27892796. 11. Neman-Simha, V., and F. Megraud. 1988. In vitro model for Campylobacter pylori adherence properties. Infect. Immun. 56:3329-3333. 12. Peterson, W. L. 1991. Helicobacter pylon and peptic ulcer disease. N. Engl. J. Med. 324:1043-1047. 13. Price, A. B., J. Levi, J. M. Dolby, P. L. Dunscombe, A. Smith, J. Clark, and M. L. Stephenson. 1985. Campylobacterpylonidis in peptic ulcer disease: microbiology, pathology, and scanning electron microscopy. Gut 26:1183-1188. 14. Tricottet, V., P. Bruneval, 0. Vire, and J. P. Camilleri. 1986. Campylobacter-like organism and surface epithelium abnormalities in active, chronic gastritis in humans: an ultrastructural study. Ultrastruct. Pathol. 10:113-122.
INFECrION AND IMMUNITY, May 1992, p. 2121-2124 0019-9567/92/052121-04$02.00/0 Copyright © 1992, American Society for Microbiology
Ultrastructural Study of Helicobacterpylori Adherence Properties in Gnotobiotic Piglets DANIEL G. RUDMANN,t KATHRYN A. EATON,* AND STEVEN KRAKOWKA
Department of Veterinary Pathobiology, School of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210 Received 19 December 1991/Accepted 24 February 1992
Ultrastructural examination of gastric mucosa from Helicobacterpylori-infected gnotobiotic piglets identified four general adherence patterns comparable to those observed in human patients. Intimate associations between the bacterial and mucosal cell membranes, including cuplike invaginations and adherence pedestals, were present and were accompanied by alterations to microvilli and cell membrane morphology.
patterns and ultrastructure confirmed that the recovered strain was identical to the challenge strain (8).
The spiral-shaped, gram-negative bacterium Helicobacter pylon is the primary cause of human type B gastritis (1, 3) and is thought to be an important part of duodenal and peptic ulcer pathogenesis in humans (12). Ethical considerations limit H. pylon research with human volunteers. As a result, a concentrated search for an appropriate animal model has been undertaken. The gnotobiotic piglet stomach can be colonized by H. pylon and produces gastric lesions similar to those observed in humans (14). This model has been useful in determining the pathogenesis of H. pylon infection and identifying various bacterial virulence factors (6, 7, 9). The present study was initiated to determine the ultrastructural morphology of H. pylon adherence patterns in gnotobiotic piglets and to compare the morphology with that of human infection. Bacterial strains and animal model. A total of 14 3-day-old gnotobiotic domestic Yorkshire piglets were randomly selected from two litters of date-mated pregnant sows derived by Caesarian section. The infected group (n = 5) was challenged with 109 CFU of laboratory H. pylon 26695 (7). Infected (n = 5) and control (n = 9) groups were separated and maintained in sterile isolation units as previously described (10). Transmission electron microscopy. At sacrifice, 21 to 25 days after infection, 1- to 2-mm biopsy samples from the antrum, fundus, and cardia of the stomach were obtained by utilizing an aseptic surgical technique (7). Infected and control group specimens were fixed for 24 h in 3% glutaraldehyde (pH 7.4), rinsed in cacodylate, postfixed in Millonig's 1% osmium tetroxide, dehydrated in graded ethanol solutions, and embedded in Medcast epoxy resin (Ted Pella, Inc., Redding, Calif.). One-micrometer-thick toluidine blue sections were screened for orientation and microbes. Selected areas were identified, and the blocks were trimmed, stained with uranyl acetate and Reynold's lead citrate, and mounted on copper grids. Twenty-five grids from three infected piglets were examined ultrastructurally with a Philips 300 electron microscope. Restriction endonuclease and sodium dodecyl sulfate-polyacrylamide gel electrophoresis
H. pylon organisms were observed in approximately 35 fields from prepared tissue grids of three infected piglets. In less than 25% of the fields, bacteria were located on the luminal surface of gastric epithelial cells. It was more common to find H. pylon on the surface of gastric pit epithelial cells and in gastric intraepithelial spaces. In fundic segments, the organisms sometimes were identified adjacent to parietal cells within gastric pits. The darkly stained, flagellated organisms were 0.5 to 0.7 ,um wide and 2 to 4.5 p,m long and possessed a double unit membrane encircling a granular protoplasm. No organisms in any of the control sections were identified. A close association between the surface of the epithelial cells and the bacteria was observed. Four adherence patterns were noted. In most cases, the bacterial membrane was closely apposed to the epithelial cell membrane, with some loss of microvilli and mild alterations in membrane morphology (Fig. 1A). Less often, bacteria were adhered to epithelial cell microvilli. The bacterial membranes either were directly apposed to the microvilli or appeared fused with them. Some bacteria were adhered to indented sites on the epithelial cell surface, with subsequent loss of microvilli (Fig. 1B). In three instances, the epithelial cell surface extended outward and fused with the bacterial membrane, possibly representative of an adherence pedestal (Fig. 1C). Twice, the epithelial cell membrane appeared to engulf the bacterium (Fig. 1D). In situ morphologic evidence of replication (e.g., binary fission) was seen frequently. Ultrastructural studies with humans indicate that there is a close association between H. pylon and the gastric epithelium in chronic gastritis (2, 5, 9, 11, 13, 14). Patterns of physical contact that are thought to represent different evolutive stages of H. pylon infection have been recognized previously (4). Several of the bacterial adherence patterns which have been observed include the formation of cuplike projections (11), indentation sites (9), and pedestals (4, 9). The appearance of pedestals is believed to be an important virulence factor and indication of the pathogenicity of the organism (9). A property of adherence present in many of the patterns is partial or complete bacterial and epithelial membrane fusion, often resulting in no change in the contour of the epithelial cell (9, 14). A few bacteria are covered by or resting on an epithelial microvillus, sometimes resulting in loss of normal
* Corresponding author. t Present address: Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47906.
2121
2122
NOTES
INFECT. IMMUN.
..
..Amlbk I..
'i
;.,, .-
.1
," 's
"
:,
A
F VL
5,
-,
%
*.&
B: 4
4_
_7
F, i
'4*t'r
?"
-.
--I
FIG. 1. (A) Several H. pylon organisms closely associated with gastric epithelium. Some bacterial membranes appear fused with epithelial cell membrane (arrows), while others are only intimately apposed (star). Magnification, x 15,000. (B) H. pylon adherence associated with loss of underlying gastric epithelial microvilli (arrows). Magnification, x43,000. (C) Pedestal extending from gastric epithelial cell to adherence site on H. pylori organism (arrow). Magnification, x 32,000. (D) An epithelial cell membrane appears to engulf an H. pylon organism, with subsequent fusion of membranes. Magnification, x32,000.
microvillus structure. In general, these adhered bacteria are present both at intercellular junctions and at the surfaces of epithelial cells, but more often in the former (4). The patterns of adherence seen in this study were similar to those seen in ultrastructural studies of human gastric mucosa colonized by H. pyloni.
How the adherence patterns seen in these piglets directly affect the host gastric epithelial cells remains unanswered.
Probably, the bacteria utilize substances that the adjacent cells require for survival. Some believe that H. pyloni may produce an exotoxin that damages the underlying adhered cells (3). It may be that direct bacterial adherence blocks the
wr
FIG.
1-Continued. 2123
2124
NOTES
normal function of the gastric epithelial cell membrane by interfering with ion transport channels and/or blocking and destroying membrane receptors. When adherence between the bacteria and cell was intimate, we observed a loss of architecture of the adhering surfaces. It became impossible to distinguish the bacterial cell wall from the epithelial cell membrane. What exactly occurs at this point of fusion is unknown. Whether the fusion is a virulence factor of H. pylon or a protective response of the epithelial cell requires further study. As seen in humans (5), bacteria were frequently observed near resting-phase parietal cells. There exists the possibility that the bacteria, through some mechanism, inhibit normal functioning of parietal cells, resulting in a local achlorhydria. As a result, the immediate gastric environment is made less acidic and more favorable for H. pylon colonization. H. pylon in humans and Helicobacter mustelae in ferrets are proposed to be the only Helicobacter species to attach to gastric mucosa. It is interesting to note that during our studies of H. pylon in gnotobiotic piglets, we investigated an outbreak of gastritis in a group of cheetahs at the zoo in Columbus, Ohio. An unpublished ultrastructural study of the cheetah gastric lesions showed evidence of similar adherence patterns by Gastrospillum spp. The role that this bacterium plays in the pathogenesis of cheetah gastric disease requires further investigation. The similarities in adherence patterns of H. pylon in humans and gnotobiotic piglets provide further evidence that supports the usefulness of the gnotobiotic piglet as an animal model of human H. pylon type B gastritis. The exact pathogenic effect of these adherence patterns in humans and in gnotobiotic piglets must now be elucidated to better understand the precise role of H. pylon in human type B gastritis and duodenal and gastric ulceration. We thank E. Handley for her assistance in preparation of electron microscopy samples and in operation of the microscope. This study was completed as part of an off-campus research externship offered by the Purdue University School of Veterinary Medicine and The Ohio State University College of Veterinary Medicine.
REFERENCES 1. Blaser, M. J. 1990. Helicobacterpylori and the pathogenesis of gastroduodenal inflammation. J. Infect. Dis. 161:626-633.
INFECT. IMMUN. 2. Bonvicini, F., P. Versura, S. Pretolani, G. Gasbarrini, and R. Laschi. 1989. Scanning electron microscopy in the study of Campylobacter pylori associated gastritis. Scanning Microsc. 3:355-368. 3. Buck, G. E. 1990. Campylobacter pylon and gastroduodenal disease. Clin. Microbiol. Rev. 3:1-12. 4. Caselli, M., N. Figura, L. Trevisani, P. P. Pazzi, P. Guglielmetti, M. R. Bovolenta, and G. Stabellini. 1989. Patterns of physical modes of contact between Campylobacter pylori and gastric epithelium: implications about the bacterial pathogenicity. Am. J. Gastroenterol. 84:511-513. 5. Chen, X. G., P. Correa, J. Offerhaus, E. Rodriguez, F. Janney, E. Hoffmann, J. Fox, F. Hunter, and S. Diavolitsis. 1986. Ultrastructure of the gastric mucosa harboring Campylobacterlike organisms. Am. J. Clin. Pathol. 86:575-582. 6. Eaton, K. A., C. L. Brooks, D. R. Morgan, and S. Krakowka. 1991. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylon in gnotobiotic piglets. Infect. Immun. 59:2470-2475. 7. Eaton, K. A., D. R. Morgan, and S. Krakowka. 1989. Campylobacter pylori virulence factors in gnotobiotic piglets. Infect. Immun. 57:1119-1125. 8. Eaton, K. A., D. R. Morgan, and S. Krakowka. 1990. Persistence of Helicobacter pylori in conventionalized piglets. J. Infect. Dis. 161:1299-1301. 9. Hessey, S. J., J. Spencer, J. I. Wyatt, G. Sobala, B. J. Rathbone, A. T. R. Axon, and M. F. Dixon. 1990. Bacterial adhesion and disease activity in Helicobacter associated chronic gastritis. Gut 31:134-138. 10. Krakowka, S., D. R. Morgan, W. G. Kraft, and R. D. Leunk. 1987. Establishment of gastric Campylobacter pylon infection in the neonatal gnotobiotic piglet. Infect. Immun. 55:27892796. 11. Neman-Simha, V., and F. Megraud. 1988. In vitro model for Campylobacter pylori adherence properties. Infect. Immun. 56:3329-3333. 12. Peterson, W. L. 1991. Helicobacter pylon and peptic ulcer disease. N. Engl. J. Med. 324:1043-1047. 13. Price, A. B., J. Levi, J. M. Dolby, P. L. Dunscombe, A. Smith, J. Clark, and M. L. Stephenson. 1985. Campylobacterpylonidis in peptic ulcer disease: microbiology, pathology, and scanning electron microscopy. Gut 26:1183-1188. 14. Tricottet, V., P. Bruneval, 0. Vire, and J. P. Camilleri. 1986. Campylobacter-like organism and surface epithelium abnormalities in active, chronic gastritis in humans: an ultrastructural study. Ultrastruct. Pathol. 10:113-122.
