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British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Scanning and transmission electron microscopic observations of bacteria adhering to ileal epithelial cells in growing broiler and white leghorn chickens a
a
a
K. Yamauchi , Y. Isshiki , Z.‐X. Zhou & Y. Nakahiro
a
a
Laboratory of Animal Science, Faculty of Agriculture , Kagawa University , Miki‐cho, Kagawa‐ken, 761–07, Japan Published online: 08 Nov 2007.
To cite this article: K. Yamauchi , Y. Isshiki , Z.‐X. Zhou & Y. Nakahiro (1990) Scanning and transmission electron microscopic observations of bacteria adhering to ileal epithelial cells in growing broiler and white leghorn chickens, British Poultry Science, 31:1, 129-137, DOI: 10.1080/00071669008417238 To link to this article: http://dx.doi.org/10.1080/00071669008417238
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
Downloaded by [UQ Library] at 10:44 13 March 2015
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British Poultry Science (1990) 31: 129-137
SCANNING AND TRANSMISSION ELECTRON MICROSCOPIC OBSERVATIONS OF BACTERIA ADHERING TO ILEAL EPITHELIAL CELLS IN GROWING BROILER AND WHITE LEGHORN CHICKENS K. YAMAUCHI, Y. ISSHIKI, Z.-X. ZHOU AND Y. NAKAHIRO Laboratory of Animal Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa-ken, 761-07 Japan
Downloaded by [UQ Library] at 10:44 13 March 2015
Received for publication 17th July 1989
Abstract 1. Bacteria adhering to the ileal epithelial cells in broiler and White Leghorn (WL) chickens aged 1 to 60 d were observed with scanning (SEM) and transmission (TEM) electron microscopes. 2. In SEM observations, bacteria were not found on day 1 after hatching in either breed. In 10-d-old broilers many bacteria were observed around the apical area of villi. The number decreased with age and disappeared by 50 d. In WL chicks, the bacteria were first observed at 20 d. Numbers were much fewer than in broilers and none were seen after 30 d. 3. TEM investigations showed that bacteria had a cytoplasmic membrane, cell wall and nucleus but no nuclear membrane and organella, were compartmentalised and resembled Streptobacillus moniliformis. At the attachment zone to the epithelium, many mitochondria were observed in the epithelial cells; the bacterial membrane did not fuse to the epithelial cell membrane except at the apex of the attachment end where the bacterial membrane seemed to undergo lysis, suggesting a possibility that some bacterial components were transferred to the epithelial cells. 4. It is possible that the bacteria aid in the functioning of ileal epithelial cells. Possible functions are discussed in relation to the morphological features. INTRODUCTION
In mammals, the intestinal microflora is widely recognised to affect excretion of amino acids (Mason and Palmer, 1973; Mason et al., 1976). In contrast, the flora in poultry alimentary tracts appears to have little or no significant influence on the digestion of dietary proteins (Salter and Fulford, 1974) not on the excretion of amino acids (Parsons et al., 1982) but has an important role in producing ammonia from uric acid (Karasawa et al., 1988). Barnes et al. (1972) and Mead and Adams (1975) have described the intestinal anaerobic bacteria of chickens. However, in comparison with many studies on 129
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K. YAMAUCHI, Y. ISSHIKI, Z.-X. ZHOU AND Y. NAKAHIRO
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the bacteria floating in the intestinal lumen, there appears to be little information on bacteria associated with the intestinal epithelial cells, except for studies in mice and rats (Dubos et al., 1965; Hampton and Rosario, 1965). In chickens, bacteria associated with ileal epithelial cells were first detected by Fuller and Turvey (1971) and McNab (1973) reviewed their possible functions. However, the role of such bacteria in nutritional functions is not yet understood. We have observed that the number of bacteria adhering to epithelial cells differed with age as well as between meat and layer type chickens. We speculated that these bacteria may have a role in avian nutrition. The objectives of this study were to compare the distribution patterns of bacteria between broiler and WL chickens, to obtain more detailed morphological observations on bacteria and epithelial cells to which they adhered and to discover whether they caused cytopathologic damage in ultrastructural phases to the epithelial cells. MATERIALS AND METHODS
Fifty newly hatched male broilers and 50 WL chicks (Gallus gallus domesticus) were obtained from a commercial hatchery on the same day. They were kept in a battery-type brooder during the first week and then transferred to 9 larger cages, of 11 to 12 birds each. Birds were maintained in an environmentally-controlled room with a 06.00 to 20.00 h photoperiod where the temperature was reduced gradually from 32°C to 17°C. Birds were given ad libitum access to water and standard proprietary diets. Five chicks selected at random from each breed were killed at 1-d-old and at 10, 20, 30, 40 and 60 d of age. Samples of the ileum for SEM were taken immediately caudal to Meckel's diverticulum (MD), at the middle part between the MD and ileo-cecal-colonic junction (IC) and immediately rostral to the IC. Tissue samples were opened and washed with 0-1 M phosphate buffered saline at pH 7-4. Tissue samples were pinned out within a fixative containing paraformaldehyde (40 g/1) in 0-1 M phosphate buffer (pH 7-4), kept at room temperature for 30 min and cut into 10X10 mm squares for SEM and 2 x 1 0 mm for TEM. All segments were fixed in glutaraldehyde (30 ml/1) and paraformaldehyde (40 g/1) in 0-1 M cacodylate buffer (pH 7'4) for 2 h at room temperature. After washing in the same buffer all specimens were postfixed in osmium tetroxide (10 g/1) in 0-1 M ice-cold cacodylate buffer (pH 7-4) for 1 h. Specimens were washed in distilled deionised water and dehydrated in graded ethanol solutions (from 500 to 1000 ml/1 for 1 h each). The specimens for SEM were kept in isoamyl acetate and dried in a critical point drying apparatus (Hitachi HCP-1) using liquid carbon dioxide as the medium. The dried specimens were mounted on aluminum stubs with electrically conducting cement (silver paste), sputter coated with platinum (RMC-Eiko RE vacuum coater) at 100 millitorr, 7 milliamperes for 15 min and examined with a Hitachi S-800 SEM at 8 kV. Specimens for TEM were stained with ice-cold uranyl acetate (5 g/1) overnight after washing in distilled, deionised water, dehydrated and embedded in Spurr's plastic mixture (Spurr, 1969). Ultrathin sections were stained with lead citrate.
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RESULTS
At 1-d-old bacteria were not observed by SEM on the villi of small intestine in either breed. However, in 10-d-old broilers, many bacteria were found around the apical surface area of villi (Fig. la and b). Villi to which bacteria adhered occurred mainly between the MD and the IC (middle part of the ileum) and decreased in number at the rostral and caudal parts of the ileum. These dense distributions of bacteria decreased with increase in age; intestinal villi of 20-d-old broilers showed diminished tufts of bacteria (Fig. lc and e), while in 30-d-old broilers tufts had disappeared and each bacterium was distributed at random (Fig. lg). Bacteria had disappeared by 50 d. Compared with broilers, in WL chicks the appearance of bacteria was later and they were first observed sparsely distributed distribution at 20 d (Fig. Id and f). Numbers were also much fewer than in broilers (Fig. lc-f). Furthermore, the bacteria at the middle part of the ileum had decreased greatly and disappeared after 30 d. The bacteria were not observed in adult chickens of either breed except for one case in which two bacteria were found. Using the TEM, no cytopathologic changes and no acute inflammatory reactions could be detected in ileal epithelial cells to which bacteria had adhered (Figs. 2 and 3). In addition, in ultrastructural features of organelles, specific morphological alterations were not observed in the cytoplasm except for the following modifications: at the surface of epithelial cells in contact with bacteria, microvilli were displaced and the plasmic membrane surrounding the attachment areas was characterised an increase in thickness by density (small arrows in Figs. 2c, d and 3b). The rest of the epithelial cell cytoplasm beneath the attachment zone was normal in morphological appearance. At a second nearer to the centre of the longitudinal axis of bacteria, its top showed a budlike protrusion which penetrated deeper into the cytoplasm (Figs. 2d and 3b). The thickened epithelial cell plasmic membrane conformed to the shape of the tip of the bacteria. At the cytoplasmic area immediately beneath the attachment zone, many mitochondria aggregated (Fig. 4a, d). In addition, as shown in Fig. 4b, the plasmic membrane of microvilli surrounding a transverse section of a bacterium, terminal web and rootlet of bundles of microvillar filaments in the cytoplasm beneath the microvilli did not show morphological changes. The morphology of bacteria showed characteristic features. The bacteria had a cytoplasmic membrane, periplasmic space, cell wall and nucleus but no nuclear membrane, flagella, pili and no organelles such as mitochondria, endoplasmic reticulum and lysosomes (Figs. 3b and 4c). Almost homogenous cytoplasm was bounded by a continuous cell wall but compartmentalised by cytoplasmic divisions (Figs. 2c and 3b). Mesosomes (arrow in Fig. 4c) were sometimes observed as a conspicuous structure. At the attachment zone of bacteria to the ileal epithelial cells, the plasmic membrane, periplasmic space and cell wall of bacteria showed a distinct lamellar structure parallel to the epithelial plasmic membrane (small arrows in Fig. 3b). Fusion between the plasmic membranes of epithelial cells and bacteria were not observed (small arrows in Fig. 3b) except for the apex of a bud-like protrusion of the attachment end where the cytoplasmic membrane and cell wall of bacteria
Downloaded by [UQ Library] at 10:44 13 March 2015
K. YAMAUCHI, Y. ISSHIKI, Z.-X. ZHOU AND Y. NAKAHIRO
FIG. 1.—Scanning electron micrographs of the intestinal villi from the middle portion of the chick ileum for comparing the appearance of bacteria by age and in number between broiler and White Leghorn (WL) chicks, a. Bacteria in 10-d-old broilers: many bacteria distributed on the apical surface of the villi have already appeared and covered the intestinal lumen: b. Higher magnification of the tuft of bacteria in 10-d-old broilers, c. Diminished tuft of bacteria (arrows) in 20-d-old broilers, d. The first appearance of bacteria (arrows) in 20-d-old WL; numbers of bacteria were much fewer than in broilers, e. and f. Higher magnifications of the bacteria in 20d-old broilers (e) and WL (0; villi of broiler showed a tuft and had more bacteria than WL. g. Sparsely distributed bacteria on villi in 30-d-old broilers; the tuft of bacteria disappeared and each bacterium was distributed at random. Scale bars: a, c, d, g, 33 fim; b, e, f, 8 fim.
seemed to be undergoing lysis (large arrow in Fig. 3b). Fig. 4 shows examples of transverse, oblique and tangential sections of bacteria. DISCUSSION
Based on ultrastructural features, the bacteria association with ileal epithelial cells were indistinguishable from prokaryotic microorganisms and, accord-
Downloaded by [UQ Library] at 10:44 13 March 2015
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FIG. 2.—a. and b. Transmission electron micrographs showing longitudinal sections of bacteria adhering to the ileal epithelial cells in 10-d-old broilers. No cytopathologic changes are visible in epithelial cells attached by a bacterium. G: Goblet cell. Scale bars: 1-5 fim. c. and d. Higher magnifications of bacteria in a. and b. respectively. At the attachment area, epithelial cell plasmic membranes show a characteristic thickening and increase of density (arrows). V: Microvilli. Scale bars: c. 0-3 pan; d. 0-2 fim.
ing to Bergey's classification (Krieg and Holt, 1984), corresponded to Streptobacillus moniliformis as facultatively anaerobic Gram-negative rods. In mice, Hampton and Rosario (1965) have already described the ileal epitheliumassociated bacteria as Streptobacillus moniliformis in an ultrastructural study. In chickens, such bacteria associated with ileal wall have also been investigated by light microscopy by Fuller and Turvey (1971); they seem not to be a typical inhabitant of the small intestine and may have come from the caecum. In our electron microscopic observations, these bacteria do not appear to cause intestinal disease but are normal flora. The physiological functions of epithelium-associated bacteria (reviewed by McNab, 1973) are not clear but their
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K. YAMAUCHI, Y. ISSHIKI, Z.-X. ZHOU AND Y. NAKAHIRO
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a-*"!' "uwr^-vr
b "
FIG. 3.—a. Ultrastructure of a section nearer to the centre of the longitudinal axis of a bacterium associated with ileal epithelium in 10-d-old broilers. G: Goblet cell. Scale bar: 1 flm b. Higher magnification of a bacterium in a. The bacterium having a plasmic membrane, cell wall and nucleus shows no nuclear membrane and organelles and is compartmentalised by cytoplasmic divisions. Note that at the attachment area bacterial membranes show no fusion to the epithelial cell membrane (small arrows) except for the apex of a bud-like protrusion of attachment end where the bacterial membrane seems to be undergoing lysis (large arrow). M: Mitochondria. N: Nucleus. R: Rootlet of bundles of microvillar filaments. T: Terminal web. V: Microvilli. Scale bar: 0-2 fim.
attachment to epithelial cells suggests a possible role in nutrient absorption. Transport of bacteria from the intestinal lumen to other tissues has been shown in chickens (Fuller and Jayne-Williams, 1968; 1970). It is unlikely that the adhesion of bacteria to the epithelial surface represents an early stage of such a translocation because most of bacterial membranes remained intact. As shown in TEM images (Fig. 1), the bacteria were almost as long as epithelial cells were high and so more advanced stages of translocation should surely have been detected. Hampton and Rosario (1965) speculated that the intestinal epithelium is an effective barrier to bacterial passage from the gut lumen to the lamina propria and that epithelial cells aid in controlling the number of species of intestinal flora through digestion of some components of some microorganisms by enzymatic properties in the brush border region. Morphological evidence that the cytoplasmic membrane of epithelial cells surrounding the attachment zone showed thickening and increased density has parallels to the neural post-synaptic membrane which shows increased thickness and density. Such a synapse with an asymmetric membrane is known as an
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a
•.•A«r«.«.-.
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Scanning and transmission electron microscopic observations of bacteria adhering to ileal epithelial cells in growing broiler and white leghorn chickens a
a
a
K. Yamauchi , Y. Isshiki , Z.‐X. Zhou & Y. Nakahiro
a
a
Laboratory of Animal Science, Faculty of Agriculture , Kagawa University , Miki‐cho, Kagawa‐ken, 761–07, Japan Published online: 08 Nov 2007.
To cite this article: K. Yamauchi , Y. Isshiki , Z.‐X. Zhou & Y. Nakahiro (1990) Scanning and transmission electron microscopic observations of bacteria adhering to ileal epithelial cells in growing broiler and white leghorn chickens, British Poultry Science, 31:1, 129-137, DOI: 10.1080/00071669008417238 To link to this article: http://dx.doi.org/10.1080/00071669008417238
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
Downloaded by [UQ Library] at 10:44 13 March 2015
Terms & Conditions of access and use can be found at http://www.tandfonline.com/ page/terms-and-conditions
British Poultry Science (1990) 31: 129-137
SCANNING AND TRANSMISSION ELECTRON MICROSCOPIC OBSERVATIONS OF BACTERIA ADHERING TO ILEAL EPITHELIAL CELLS IN GROWING BROILER AND WHITE LEGHORN CHICKENS K. YAMAUCHI, Y. ISSHIKI, Z.-X. ZHOU AND Y. NAKAHIRO Laboratory of Animal Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa-ken, 761-07 Japan
Downloaded by [UQ Library] at 10:44 13 March 2015
Received for publication 17th July 1989
Abstract 1. Bacteria adhering to the ileal epithelial cells in broiler and White Leghorn (WL) chickens aged 1 to 60 d were observed with scanning (SEM) and transmission (TEM) electron microscopes. 2. In SEM observations, bacteria were not found on day 1 after hatching in either breed. In 10-d-old broilers many bacteria were observed around the apical area of villi. The number decreased with age and disappeared by 50 d. In WL chicks, the bacteria were first observed at 20 d. Numbers were much fewer than in broilers and none were seen after 30 d. 3. TEM investigations showed that bacteria had a cytoplasmic membrane, cell wall and nucleus but no nuclear membrane and organella, were compartmentalised and resembled Streptobacillus moniliformis. At the attachment zone to the epithelium, many mitochondria were observed in the epithelial cells; the bacterial membrane did not fuse to the epithelial cell membrane except at the apex of the attachment end where the bacterial membrane seemed to undergo lysis, suggesting a possibility that some bacterial components were transferred to the epithelial cells. 4. It is possible that the bacteria aid in the functioning of ileal epithelial cells. Possible functions are discussed in relation to the morphological features. INTRODUCTION
In mammals, the intestinal microflora is widely recognised to affect excretion of amino acids (Mason and Palmer, 1973; Mason et al., 1976). In contrast, the flora in poultry alimentary tracts appears to have little or no significant influence on the digestion of dietary proteins (Salter and Fulford, 1974) not on the excretion of amino acids (Parsons et al., 1982) but has an important role in producing ammonia from uric acid (Karasawa et al., 1988). Barnes et al. (1972) and Mead and Adams (1975) have described the intestinal anaerobic bacteria of chickens. However, in comparison with many studies on 129
130
K. YAMAUCHI, Y. ISSHIKI, Z.-X. ZHOU AND Y. NAKAHIRO
Downloaded by [UQ Library] at 10:44 13 March 2015
the bacteria floating in the intestinal lumen, there appears to be little information on bacteria associated with the intestinal epithelial cells, except for studies in mice and rats (Dubos et al., 1965; Hampton and Rosario, 1965). In chickens, bacteria associated with ileal epithelial cells were first detected by Fuller and Turvey (1971) and McNab (1973) reviewed their possible functions. However, the role of such bacteria in nutritional functions is not yet understood. We have observed that the number of bacteria adhering to epithelial cells differed with age as well as between meat and layer type chickens. We speculated that these bacteria may have a role in avian nutrition. The objectives of this study were to compare the distribution patterns of bacteria between broiler and WL chickens, to obtain more detailed morphological observations on bacteria and epithelial cells to which they adhered and to discover whether they caused cytopathologic damage in ultrastructural phases to the epithelial cells. MATERIALS AND METHODS
Fifty newly hatched male broilers and 50 WL chicks (Gallus gallus domesticus) were obtained from a commercial hatchery on the same day. They were kept in a battery-type brooder during the first week and then transferred to 9 larger cages, of 11 to 12 birds each. Birds were maintained in an environmentally-controlled room with a 06.00 to 20.00 h photoperiod where the temperature was reduced gradually from 32°C to 17°C. Birds were given ad libitum access to water and standard proprietary diets. Five chicks selected at random from each breed were killed at 1-d-old and at 10, 20, 30, 40 and 60 d of age. Samples of the ileum for SEM were taken immediately caudal to Meckel's diverticulum (MD), at the middle part between the MD and ileo-cecal-colonic junction (IC) and immediately rostral to the IC. Tissue samples were opened and washed with 0-1 M phosphate buffered saline at pH 7-4. Tissue samples were pinned out within a fixative containing paraformaldehyde (40 g/1) in 0-1 M phosphate buffer (pH 7-4), kept at room temperature for 30 min and cut into 10X10 mm squares for SEM and 2 x 1 0 mm for TEM. All segments were fixed in glutaraldehyde (30 ml/1) and paraformaldehyde (40 g/1) in 0-1 M cacodylate buffer (pH 7'4) for 2 h at room temperature. After washing in the same buffer all specimens were postfixed in osmium tetroxide (10 g/1) in 0-1 M ice-cold cacodylate buffer (pH 7-4) for 1 h. Specimens were washed in distilled deionised water and dehydrated in graded ethanol solutions (from 500 to 1000 ml/1 for 1 h each). The specimens for SEM were kept in isoamyl acetate and dried in a critical point drying apparatus (Hitachi HCP-1) using liquid carbon dioxide as the medium. The dried specimens were mounted on aluminum stubs with electrically conducting cement (silver paste), sputter coated with platinum (RMC-Eiko RE vacuum coater) at 100 millitorr, 7 milliamperes for 15 min and examined with a Hitachi S-800 SEM at 8 kV. Specimens for TEM were stained with ice-cold uranyl acetate (5 g/1) overnight after washing in distilled, deionised water, dehydrated and embedded in Spurr's plastic mixture (Spurr, 1969). Ultrathin sections were stained with lead citrate.
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RESULTS
At 1-d-old bacteria were not observed by SEM on the villi of small intestine in either breed. However, in 10-d-old broilers, many bacteria were found around the apical surface area of villi (Fig. la and b). Villi to which bacteria adhered occurred mainly between the MD and the IC (middle part of the ileum) and decreased in number at the rostral and caudal parts of the ileum. These dense distributions of bacteria decreased with increase in age; intestinal villi of 20-d-old broilers showed diminished tufts of bacteria (Fig. lc and e), while in 30-d-old broilers tufts had disappeared and each bacterium was distributed at random (Fig. lg). Bacteria had disappeared by 50 d. Compared with broilers, in WL chicks the appearance of bacteria was later and they were first observed sparsely distributed distribution at 20 d (Fig. Id and f). Numbers were also much fewer than in broilers (Fig. lc-f). Furthermore, the bacteria at the middle part of the ileum had decreased greatly and disappeared after 30 d. The bacteria were not observed in adult chickens of either breed except for one case in which two bacteria were found. Using the TEM, no cytopathologic changes and no acute inflammatory reactions could be detected in ileal epithelial cells to which bacteria had adhered (Figs. 2 and 3). In addition, in ultrastructural features of organelles, specific morphological alterations were not observed in the cytoplasm except for the following modifications: at the surface of epithelial cells in contact with bacteria, microvilli were displaced and the plasmic membrane surrounding the attachment areas was characterised an increase in thickness by density (small arrows in Figs. 2c, d and 3b). The rest of the epithelial cell cytoplasm beneath the attachment zone was normal in morphological appearance. At a second nearer to the centre of the longitudinal axis of bacteria, its top showed a budlike protrusion which penetrated deeper into the cytoplasm (Figs. 2d and 3b). The thickened epithelial cell plasmic membrane conformed to the shape of the tip of the bacteria. At the cytoplasmic area immediately beneath the attachment zone, many mitochondria aggregated (Fig. 4a, d). In addition, as shown in Fig. 4b, the plasmic membrane of microvilli surrounding a transverse section of a bacterium, terminal web and rootlet of bundles of microvillar filaments in the cytoplasm beneath the microvilli did not show morphological changes. The morphology of bacteria showed characteristic features. The bacteria had a cytoplasmic membrane, periplasmic space, cell wall and nucleus but no nuclear membrane, flagella, pili and no organelles such as mitochondria, endoplasmic reticulum and lysosomes (Figs. 3b and 4c). Almost homogenous cytoplasm was bounded by a continuous cell wall but compartmentalised by cytoplasmic divisions (Figs. 2c and 3b). Mesosomes (arrow in Fig. 4c) were sometimes observed as a conspicuous structure. At the attachment zone of bacteria to the ileal epithelial cells, the plasmic membrane, periplasmic space and cell wall of bacteria showed a distinct lamellar structure parallel to the epithelial plasmic membrane (small arrows in Fig. 3b). Fusion between the plasmic membranes of epithelial cells and bacteria were not observed (small arrows in Fig. 3b) except for the apex of a bud-like protrusion of the attachment end where the cytoplasmic membrane and cell wall of bacteria
Downloaded by [UQ Library] at 10:44 13 March 2015
K. YAMAUCHI, Y. ISSHIKI, Z.-X. ZHOU AND Y. NAKAHIRO
FIG. 1.—Scanning electron micrographs of the intestinal villi from the middle portion of the chick ileum for comparing the appearance of bacteria by age and in number between broiler and White Leghorn (WL) chicks, a. Bacteria in 10-d-old broilers: many bacteria distributed on the apical surface of the villi have already appeared and covered the intestinal lumen: b. Higher magnification of the tuft of bacteria in 10-d-old broilers, c. Diminished tuft of bacteria (arrows) in 20-d-old broilers, d. The first appearance of bacteria (arrows) in 20-d-old WL; numbers of bacteria were much fewer than in broilers, e. and f. Higher magnifications of the bacteria in 20d-old broilers (e) and WL (0; villi of broiler showed a tuft and had more bacteria than WL. g. Sparsely distributed bacteria on villi in 30-d-old broilers; the tuft of bacteria disappeared and each bacterium was distributed at random. Scale bars: a, c, d, g, 33 fim; b, e, f, 8 fim.
seemed to be undergoing lysis (large arrow in Fig. 3b). Fig. 4 shows examples of transverse, oblique and tangential sections of bacteria. DISCUSSION
Based on ultrastructural features, the bacteria association with ileal epithelial cells were indistinguishable from prokaryotic microorganisms and, accord-
Downloaded by [UQ Library] at 10:44 13 March 2015
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FIG. 2.—a. and b. Transmission electron micrographs showing longitudinal sections of bacteria adhering to the ileal epithelial cells in 10-d-old broilers. No cytopathologic changes are visible in epithelial cells attached by a bacterium. G: Goblet cell. Scale bars: 1-5 fim. c. and d. Higher magnifications of bacteria in a. and b. respectively. At the attachment area, epithelial cell plasmic membranes show a characteristic thickening and increase of density (arrows). V: Microvilli. Scale bars: c. 0-3 pan; d. 0-2 fim.
ing to Bergey's classification (Krieg and Holt, 1984), corresponded to Streptobacillus moniliformis as facultatively anaerobic Gram-negative rods. In mice, Hampton and Rosario (1965) have already described the ileal epitheliumassociated bacteria as Streptobacillus moniliformis in an ultrastructural study. In chickens, such bacteria associated with ileal wall have also been investigated by light microscopy by Fuller and Turvey (1971); they seem not to be a typical inhabitant of the small intestine and may have come from the caecum. In our electron microscopic observations, these bacteria do not appear to cause intestinal disease but are normal flora. The physiological functions of epithelium-associated bacteria (reviewed by McNab, 1973) are not clear but their
134
K. YAMAUCHI, Y. ISSHIKI, Z.-X. ZHOU AND Y. NAKAHIRO
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a-*"!' "uwr^-vr
b "
FIG. 3.—a. Ultrastructure of a section nearer to the centre of the longitudinal axis of a bacterium associated with ileal epithelium in 10-d-old broilers. G: Goblet cell. Scale bar: 1 flm b. Higher magnification of a bacterium in a. The bacterium having a plasmic membrane, cell wall and nucleus shows no nuclear membrane and organelles and is compartmentalised by cytoplasmic divisions. Note that at the attachment area bacterial membranes show no fusion to the epithelial cell membrane (small arrows) except for the apex of a bud-like protrusion of attachment end where the bacterial membrane seems to be undergoing lysis (large arrow). M: Mitochondria. N: Nucleus. R: Rootlet of bundles of microvillar filaments. T: Terminal web. V: Microvilli. Scale bar: 0-2 fim.
attachment to epithelial cells suggests a possible role in nutrient absorption. Transport of bacteria from the intestinal lumen to other tissues has been shown in chickens (Fuller and Jayne-Williams, 1968; 1970). It is unlikely that the adhesion of bacteria to the epithelial surface represents an early stage of such a translocation because most of bacterial membranes remained intact. As shown in TEM images (Fig. 1), the bacteria were almost as long as epithelial cells were high and so more advanced stages of translocation should surely have been detected. Hampton and Rosario (1965) speculated that the intestinal epithelium is an effective barrier to bacterial passage from the gut lumen to the lamina propria and that epithelial cells aid in controlling the number of species of intestinal flora through digestion of some components of some microorganisms by enzymatic properties in the brush border region. Morphological evidence that the cytoplasmic membrane of epithelial cells surrounding the attachment zone showed thickening and increased density has parallels to the neural post-synaptic membrane which shows increased thickness and density. Such a synapse with an asymmetric membrane is known as an
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a
•.•A«r«.«.-.
