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A Scanning Electron Microscope Study of the Caecal Tonsil: The Identification of a Bacterial Attachment to the Villi of the Caecal Tonsil and the Possible Presence of Lymphatics in the Caecal Tonsil1
A Scanning Electron Microscope Study of the Caecal Tonsil: The Identification of a Bacterial Attachment to the Villi of the Caecal Tonsil and the Possible Presence of Lymphatics in the Caecal Tonsil1 BRUCE GLICK 2 , KAREN A. HOLBROOK 3 , IMRE OLAH 2 , WILLIAM D. PERKINS 3 , and ROBERT STINSON2 2
Poultry Science Department, Mississippi State University, Mississippi State, Mississippi 39762 3 Department of Biological Structure, University of Washington School of Medicine,
ABSTRACT A scanning electron microscope (SEM) was used to compare the proximal region (PR) and distal region (DR) of the caecum. The caecal tonsil (CT) occupied the initial 4-10 mm of the PR. Villi were present in the PR and absent from the DR. Segmented structures were attached to the surface of PR. Transmission electron microscopy (TEM) revealed these structures to be bacteria. No difference in surface morphology could be discerned between the CT and the remainder of the PR. Lymphatic vessels were observed in the CT by employing TEM. INTRODUCTION T h e caecal tonsil is a l y m p h o m y e l o i d tissue in t h e chicken t h a t appears as an enlargement at t h e beginning of each c a e c u m ( M u t h m a n n , 1 9 1 3 ; Calhoun, 1 9 3 2 ; and Payne, 1971). Germinal centers are k n o w n t o be present in b o t h t h e caecal tonsil ( L o o p e r and Looper, 1 9 2 9 ; Jankovic et al., 1 9 6 6 ; and Jankovic and Mitrovic, 1 9 6 7 ) and in t h e distal p o r t i o n of t h e caecum, t h e caecal p o u c h ( L o o p e r and Looper, 1 9 2 9 ; and Calhoun, 1 9 3 2 ) . In t h e caecal tonsils these germinal centers a p p e a r t o be of t w o morphologically distinct types and t o include a specialized secretory cell (Olah and Glick, 1978). T h e present s t u d y was u n d e r t a k e n t o investigate t h e surface m o r p h o l o g y of t h e caecal tonsil by scanning electron microscopy and to c o m p a r e it with t h e remainder of t h e caecum, including t h e caecal p o u c h .
MATERIALS AND METHODS Animals and Management. T h e chickens were o b t a i n e d from a closed flock of New Hampshires m a i n t a i n e d b y Professor L. J. Dreesen of t h e P o u l t r y Science D e p a r t m e n t at Mississippi State University. All of t h e birds
'Journal article number 3751 from the Mississippi Agricultural and Forestry Experiment Station. 1978 Poultry Sci 57:1408-1416
received a basal ration (Morgan and Glick, 1 9 7 2 ) ad libitum. Birds were reared in batteries or o n litter. A coccidiostat was added t o t h e diet of litter-reared birds. Samples. Chickens were sampled at 3, 6, 19, 38, 52, 7 6 , and 8 4 days of age. Battery-reared birds were sampled at all ages while chickens raised o n litter were selected at 52 and 8 4 days of age. Samples of t h e caeca were prepared from p r o x i m a l (PR) and distal ( D R ) regions. The PR included tissue of t h e caecal tonsil (CT). Specimen Preparation for Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). T h e caeca were removed, slit open, a n d rinsed in Millonig's buffer ( p H 7.4) t o r e m o v e fecal material. T h e y were transferred t o dental wax and flooded with glutaraldehyde (2%) for dissection into t h e desired segments. Tissues were fixed in glutaraldehyde (1 h r ) , post-fixed in 2% buffered OSO4 (2 h r ) at r o o m t e m p e r a t u r e , and deh y d r a t e d t h r o u g h a graded series of e t h y l alcohol for 15 min each (50%, 70%, 90%, a n d 100% X 2). All samples t h e n were critical p o i n t dried in t h e pressure chamber of a Bomar S P C - 9 0 0 0 / Ex critical-point drying a p p a r a t u s a t t a c h e d t o a CO2 supply. T h e dried specimens were affixed t o a l u m i n u m stubs by double-stick scoth t a p e and coated with either gold palladium or gold in either a D e n t o n DV-502
1408
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
(Received for publication November 9, 1977)
SCANNING ELECTRON MICROSCOPE STUDY OF TONSIL
reared birds was approximately constant up to 12 weeks of age. The TEM revealed the segmented structures to be bacilli (Fig. 6). The bacilli were prominent around the distal portion of the villi and were absent at the entrance of the lieberkuhn crypts and on the surface of the mound which is filled with diffuse lymphatic tissue (Fig. 7). In the distal half of the villi's stroma many small irregular cavities can be observed which may correspond to the lymphatic space of the villi (Fig. 7 and 8). Among the basal parts of the epithelial cells covering the distal portion of the villi we observed what appeared to be intercellular spaces (Fig. 9). The distal region or caecal pouch lacked villi (Fig. 10).
RESULTS
The caecum was divided into a proximal region (PR) and distal region (DR). The PR appeared flesh colored, generally lacked fecal material, and had villi along its entire length. The DR was dark, contained fecal material, and lacked villi. The length of the PR varied with age; it was generally less than 35mm in birds, under 5 weeks of age and was increased in length to 35—50mm in birds up to 12 weeks of age. On the inner wall at the origin of the PR, the caecal tonsil was seen as an enlarged patch of tissue. Depending upon the age of the bird, the CT occupied the initial 4—10mm of the PR. After glutaraldehyde fixation other occasional patches of enlarged tissues were observed in the PR beyond the CT. Villi were present in the CT of 3-day-old chickens and were similar in size to the villi of 6-day-old chickens (Fig. 1). Threadlike structures appeared in the villi surface by 19 days of age (Fig. 2). Removal of the tips of villi revealed the columnar epithelial cells of the villi and numerous distinct cavities in the central core (Fig. 3). Occasionally, by 19 days of age a lymphocyte-like cell was observed in the lumen of the caecum among villi of the CT (Fig. 4). We observed segmented structures in association with the epithelial surface of the PR of the caecum. The concentration of these structures in the caecum depend upon the mode in which the bird was reared and, in the case of the battery-reared birds, upon the age. In batteryreared birds the segmented structures were observed first in birds 19 days of age and were increased markedly in birds older than 38 days of age (Figs. 5 and 5a). The numbers of these structures associated with the CT of litter-
DISCUSSION
The surface morphology of the CT does not markedly differ from the remainder of the PR of the caecum. However, in accord with the observations of other investigators (Calhoun, 1932, and Whitlock et ai, 1975), the villi of the PR may be shorter and broader than the villi of the CT. Thick sections have revealed a greater concentration of germinal centers (GC) in the CT than in the remainder of the PR. A more detailed study of the CT's basic unit which contains GC will be the subject of a subsequent paper. The morphology and fine structure of the CT's GC has been presented (Olah and Glick, 1978). This discussion will be restricted to structures initially revealed by SEM. Indigenous microorganisms of various types are known to be intimately associated with or attached to the epithelial surfaces of the gastrointestinal tracts of rats (Davis and Savage, 1976; Savage, 1969), mice (Savage, 1972; Savage and Blumershine, 1974), swine (Dubos et ah, 1965), and monkeys (Takeuchi and Zeller, 1972). Epithelial cell-associated bacteria have been described in the chicken (Fuller, 1973; and Fuller and Turvey, 1971). They observed bacteria associated with the intestinal wall of the crop, ileum, and caecum. From our observations the segmented filamentous bacteria were more numerous and appeared in most litter-reared birds while their presence in battery-reared birds was less consistent. It was not possible to differentiate the CT from the remaining PR of the caecum by the presence of these bacteria. While the microbial-epithelial associations are not well understood (Savage and Blumershine, 1974), bacteria attached to the epithelial
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
vacuum evaporator with an omni-rotary (Cherry Hill, NJ) or a Hummer (Model II) sputtering device. The Hitachi-2R scanning electron microscope was used to examine the specimens at an accelerating voltage of 20 kv. After viewing the SEM preparations, a selected segment of the CT was removed from the stub, placed in propylene oxide, and then embedded in araldite (Durcupan ACM). Thick sections (1 /zm) were cut and stained with toluidine blue for light microsopic examination. For transmission electron microscopy (TEM) the thin sections were contrasted with uranyl acetate and lead citrate and viewed with a Siemens 101 Elmiskop.
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FIG. 2. Bacteria (B) embedded in the epithelium of villi (V) from the CT of a 19-day-old battery-reared bird. X266.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 1. High villi appear in the CT of a 6-day-old chick. X40.
SCANNING ELECTRON MICROSCOPE STUDY OF TONSIL
1411
FIG. 4. A lymphocyte-like cell appeared (L) among the villi of a CT from a 19-day-old battery-reared bird. On the surface of the villi are bacteria (B). The smaller globulin structures may be mucin droplets. X1460.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 3. The cut tip of a villus with a striated epithelial border and central openings, some of which may be lymphatics. X534.
1412
GL1CK ET AL.
FIG. 5a. The highly segmented chains of bacteria (B) project from the surface of CT-villi of 76-day-old litterreared birds. X6000.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 5. Individual and multiple bacteria (B) attached to the CT-villi of 38-day-old birds. X 300.
SCANNING ELECTRON MICROSCOPE STUDY OF TONSIL
1413
*
*
FIG. 7. A thick section (ljuM) of the CT illustrating the distal half of a villus with apparent lymphatics (Ly), bacteria (B), and intercellular spaces (Ic) between epithelial cells. The area of the mound (M) contain lymphocytes. X 560.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 6. The bacteria (B) are attached to the cell membrane. Around the attached surface the cell produced a dark substance which may be similar to that occurring at cell junctions. X 31,500.
GLICK ET AL.
•Mi
,
I
«m
0
FIG. 9. Intercellular (Ic) spaces among the epithelial cells of the distal portion of the CT's villi. In several spaces a fine precipitate may be ovserved. X 1,400.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 8. Lymphatic (Ly) spaces located in the distal portion of the CT's villi. X 10,150.
SCANNING ELECTRON MICROSCOPE STUDY OF TONSIL
1415
surface would have a distinct survival advantage over those not capable of attachment. It has been theorized that some bacteria may gain an important early foothold in the host by an ability to adhere to mucosal surfaces. This may be an important determinant of the organism's virulence. Adherence has been described in several species of pathogenic bacteria, including Streptococcus pyogenes, Escherichia coli, Streptococcus mutatis, Vibrio cholera, Salmonella typhimurium, Neisseria gonorrhea, Clostridium perfringens, and Mycoplasma sp. (Smith, 1977). The ecological significance of end-to-end attachment and end-on attachment to the CT by the bacteria is unknown. Occasionally, the bacteria seem to be almost embedded into the cells. While they appear to be firmly attached, they do not penetrate the cells. This is in agreement with observation of Fuller and Turvey (1971) in chicken ilea. It would be interesting to determine the influence of these bacteria on the cellular development of the CT, especially since their presence is influenced by the environment. Fuller (1973) suggested that the lactobacilli associated with the crop epithelium of the chicken plays a role in the regulation of the
chicken's intestinal flora. It would seem plausible that among the non-pathogenic bacteria of the gut, those which adhere to the intestinal epithelium, are more likely to have an important influence on the host than those which do not (Fuller and Turvey, 1971). Although the method of attachment has not been ascertained by SEM, the attachment of the filaments to the proximal end of the caeca and not the distal might reflect important functional differences between the surface epithelium of these two regions. We have not studied the SEM and TEM of the CT in agammaglobulinemic birds. Agammaglobulinemia might lead to a modification in the attachment of these microorganisms. The lymphatic system of the chicken offers a challenge to the micro-anatomist (Kampmeier, 1969; and Dransfield, 1945). Gut-associated villi would be expected to possess lymph vessels engaged in lipid transport. Also, lymphatics associated with the villi of the CT might be important in communication with a portion of the lymphomyeloid complex. Unpublished observations in our laboratory have revealed lymphatic-nodules in the tibia-tarsal region. Perhaps there are similar structures draining the gut-associated tissue of the chicken. The cavi-
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 10. The distal portion of the caecum lacks villi but contains numerous bacteria (B). X 300.
GLICK ET AL.
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ties observed in the CT's-villi by SEM and TEM appear to be lymphatics. There may be a relationship among the lymphatic spaces of the villi's stroma, the dilated intercellular-space among the epithelial cells, and the bacilli which occur in this portion of the villi.
ACKNOWLEDGEMENTS
REFERENCES Calhoun, M. Louis, 1932. The microscopic anatomy of the digestive tract of Gallus domesticus. Iowa State College J. Sci. 7:261-281. Davis, C. P., and C. D. Savage, 1976. Effect of penicillin on the succession, attachment, and morphology of segmented filamentous microbes in the m u r i n e small b o w e l . I n f e c t . Immunity 13:180-188. Dransfield, J. W., 1945. The lymphatic system of the domestic fowl. Br. Vet. J. 101:171-179. Dubos, R., R. W. Schaedler, R. Costello, and P. Hoet, 1965. Indigenous, normal, and autochthonous flora of the gastrointestinal tract. J. Exp. Med. 122:67-76. Fuller, R., 1973. Ecological studies on the lactobacillus flora associated with the crop epithelium of the fowl. J. Appl. Bact. 36:131-139. Fuller, R., and A. Turvey, 1971. Bacteria associated with the intestinal wall of the fowl (Gallus domesticus). J. Appl. Bact. 34:617-622. Jankovic, B. D., K. Mitrovic, L. Popeskovic, and M. Milosevic, 1966. Tonsilla caecalis: An immunologically active tissue in the chicken. Yugoslav. Physiol. Pharmacol. ACTA 2:71-75. Jankovic, B., and K. Mitrovic, 1967. Germinal centers
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
The a u t h o r s wish to express their appreciation t o t h e University Electron Microscope Center of Mississippi State University, Dr. Greta Tyson, Head, for t h e use of their facilities, Doris T h o m p s o n [BS, MT (ASCP)] for her l a b o r a t o r y assistance, and Karen A n d e r s o n for t y p i n g t h e manuscript.
in the tonsilla caecalis relationship to the thymus and the bursa of Fabricius. Page 34 in Germinal centers in the immune response. M. Cottier, N. Odartchenko, R. Schneller, and C. C. Congdon, ed. Springer, NY. Kampmeier, O. F., 1969. Evolution and comparative morphology of the lymphatic system. Charles C Thomas Publ. IL. Looper, J. B., and M. H. Looper, 1929. A histological study of the colic caeca in the bantam fowl. J. Morphol. and Physiol. 48:585-609. Morgan, W., and B. Glick, 1972. A quantitative study of serum proteins in bursectomized and irradiated chickens. Poultry Sci. 51:771—778. Muthmann, E., 1913. Beitrage fur vergluchende anatomie der blind darmes der lymphoiden organe des darmkanales ber saugetieren und vogeln. Anat. Hefte 48:67-114. Olah, Imre, and Bruce Glick, 1978. A description of germinal centers and a secretory cell in the chicken's caecal tonsil. Manuscript. Payne, L. N., 1971. Lymphoid system (A review) In. Physiology and biochemistry of the domestic fowl. Vol. 2. D. J. Bell and B. M. Freeman, ed. Academic Press, NY. Savage, D. C , 1969. Localization of certain indigenous microorganisms on the ileal villi of rats. J. Bacteriol. 97:1505-1506. Savage, D. C , 1972. Associations and physiological interactions of indigenous microorganisms and gastrointestinal epithelia. Am. J. Clin. Nutr. 25:1372-1379. Savage, D. C , and R. V. H. Blumershine, 1974. Surface-surface associations in microbial communities populating epithelial habitats in the murine gastrointestinal ecosystem: Scanning electron microscopy. Infect. Immunity 10:240—250. Smith, H., 1977. Microbial surfaces in relation to pathogenicity. Bacteriol. Rev. 41:475—500. Takeuchi, A., and J. A. Zeller, 1972. Ultrastructural identification of spirochetes and flagellated microbes at the brush border of the large intestinal epithelium of the rhesus monkey. Infect. Immunity 6:1008-1018. Whitlock, D. R., W. B. Lushbaugh, H. D. Danforth, and M. D. Ruff, 1975. Scanning electron microscopy of the caecal mucosa in Eimeria-tenella infected and uninfected chickens. Avian Dis. 19:293-304.
Poultry Science Department, Mississippi State University, Mississippi State, Mississippi 39762 3 Department of Biological Structure, University of Washington School of Medicine,
ABSTRACT A scanning electron microscope (SEM) was used to compare the proximal region (PR) and distal region (DR) of the caecum. The caecal tonsil (CT) occupied the initial 4-10 mm of the PR. Villi were present in the PR and absent from the DR. Segmented structures were attached to the surface of PR. Transmission electron microscopy (TEM) revealed these structures to be bacteria. No difference in surface morphology could be discerned between the CT and the remainder of the PR. Lymphatic vessels were observed in the CT by employing TEM. INTRODUCTION T h e caecal tonsil is a l y m p h o m y e l o i d tissue in t h e chicken t h a t appears as an enlargement at t h e beginning of each c a e c u m ( M u t h m a n n , 1 9 1 3 ; Calhoun, 1 9 3 2 ; and Payne, 1971). Germinal centers are k n o w n t o be present in b o t h t h e caecal tonsil ( L o o p e r and Looper, 1 9 2 9 ; Jankovic et al., 1 9 6 6 ; and Jankovic and Mitrovic, 1 9 6 7 ) and in t h e distal p o r t i o n of t h e caecum, t h e caecal p o u c h ( L o o p e r and Looper, 1 9 2 9 ; and Calhoun, 1 9 3 2 ) . In t h e caecal tonsils these germinal centers a p p e a r t o be of t w o morphologically distinct types and t o include a specialized secretory cell (Olah and Glick, 1978). T h e present s t u d y was u n d e r t a k e n t o investigate t h e surface m o r p h o l o g y of t h e caecal tonsil by scanning electron microscopy and to c o m p a r e it with t h e remainder of t h e caecum, including t h e caecal p o u c h .
MATERIALS AND METHODS Animals and Management. T h e chickens were o b t a i n e d from a closed flock of New Hampshires m a i n t a i n e d b y Professor L. J. Dreesen of t h e P o u l t r y Science D e p a r t m e n t at Mississippi State University. All of t h e birds
'Journal article number 3751 from the Mississippi Agricultural and Forestry Experiment Station. 1978 Poultry Sci 57:1408-1416
received a basal ration (Morgan and Glick, 1 9 7 2 ) ad libitum. Birds were reared in batteries or o n litter. A coccidiostat was added t o t h e diet of litter-reared birds. Samples. Chickens were sampled at 3, 6, 19, 38, 52, 7 6 , and 8 4 days of age. Battery-reared birds were sampled at all ages while chickens raised o n litter were selected at 52 and 8 4 days of age. Samples of t h e caeca were prepared from p r o x i m a l (PR) and distal ( D R ) regions. The PR included tissue of t h e caecal tonsil (CT). Specimen Preparation for Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). T h e caeca were removed, slit open, a n d rinsed in Millonig's buffer ( p H 7.4) t o r e m o v e fecal material. T h e y were transferred t o dental wax and flooded with glutaraldehyde (2%) for dissection into t h e desired segments. Tissues were fixed in glutaraldehyde (1 h r ) , post-fixed in 2% buffered OSO4 (2 h r ) at r o o m t e m p e r a t u r e , and deh y d r a t e d t h r o u g h a graded series of e t h y l alcohol for 15 min each (50%, 70%, 90%, a n d 100% X 2). All samples t h e n were critical p o i n t dried in t h e pressure chamber of a Bomar S P C - 9 0 0 0 / Ex critical-point drying a p p a r a t u s a t t a c h e d t o a CO2 supply. T h e dried specimens were affixed t o a l u m i n u m stubs by double-stick scoth t a p e and coated with either gold palladium or gold in either a D e n t o n DV-502
1408
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
(Received for publication November 9, 1977)
SCANNING ELECTRON MICROSCOPE STUDY OF TONSIL
reared birds was approximately constant up to 12 weeks of age. The TEM revealed the segmented structures to be bacilli (Fig. 6). The bacilli were prominent around the distal portion of the villi and were absent at the entrance of the lieberkuhn crypts and on the surface of the mound which is filled with diffuse lymphatic tissue (Fig. 7). In the distal half of the villi's stroma many small irregular cavities can be observed which may correspond to the lymphatic space of the villi (Fig. 7 and 8). Among the basal parts of the epithelial cells covering the distal portion of the villi we observed what appeared to be intercellular spaces (Fig. 9). The distal region or caecal pouch lacked villi (Fig. 10).
RESULTS
The caecum was divided into a proximal region (PR) and distal region (DR). The PR appeared flesh colored, generally lacked fecal material, and had villi along its entire length. The DR was dark, contained fecal material, and lacked villi. The length of the PR varied with age; it was generally less than 35mm in birds, under 5 weeks of age and was increased in length to 35—50mm in birds up to 12 weeks of age. On the inner wall at the origin of the PR, the caecal tonsil was seen as an enlarged patch of tissue. Depending upon the age of the bird, the CT occupied the initial 4—10mm of the PR. After glutaraldehyde fixation other occasional patches of enlarged tissues were observed in the PR beyond the CT. Villi were present in the CT of 3-day-old chickens and were similar in size to the villi of 6-day-old chickens (Fig. 1). Threadlike structures appeared in the villi surface by 19 days of age (Fig. 2). Removal of the tips of villi revealed the columnar epithelial cells of the villi and numerous distinct cavities in the central core (Fig. 3). Occasionally, by 19 days of age a lymphocyte-like cell was observed in the lumen of the caecum among villi of the CT (Fig. 4). We observed segmented structures in association with the epithelial surface of the PR of the caecum. The concentration of these structures in the caecum depend upon the mode in which the bird was reared and, in the case of the battery-reared birds, upon the age. In batteryreared birds the segmented structures were observed first in birds 19 days of age and were increased markedly in birds older than 38 days of age (Figs. 5 and 5a). The numbers of these structures associated with the CT of litter-
DISCUSSION
The surface morphology of the CT does not markedly differ from the remainder of the PR of the caecum. However, in accord with the observations of other investigators (Calhoun, 1932, and Whitlock et ai, 1975), the villi of the PR may be shorter and broader than the villi of the CT. Thick sections have revealed a greater concentration of germinal centers (GC) in the CT than in the remainder of the PR. A more detailed study of the CT's basic unit which contains GC will be the subject of a subsequent paper. The morphology and fine structure of the CT's GC has been presented (Olah and Glick, 1978). This discussion will be restricted to structures initially revealed by SEM. Indigenous microorganisms of various types are known to be intimately associated with or attached to the epithelial surfaces of the gastrointestinal tracts of rats (Davis and Savage, 1976; Savage, 1969), mice (Savage, 1972; Savage and Blumershine, 1974), swine (Dubos et ah, 1965), and monkeys (Takeuchi and Zeller, 1972). Epithelial cell-associated bacteria have been described in the chicken (Fuller, 1973; and Fuller and Turvey, 1971). They observed bacteria associated with the intestinal wall of the crop, ileum, and caecum. From our observations the segmented filamentous bacteria were more numerous and appeared in most litter-reared birds while their presence in battery-reared birds was less consistent. It was not possible to differentiate the CT from the remaining PR of the caecum by the presence of these bacteria. While the microbial-epithelial associations are not well understood (Savage and Blumershine, 1974), bacteria attached to the epithelial
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
vacuum evaporator with an omni-rotary (Cherry Hill, NJ) or a Hummer (Model II) sputtering device. The Hitachi-2R scanning electron microscope was used to examine the specimens at an accelerating voltage of 20 kv. After viewing the SEM preparations, a selected segment of the CT was removed from the stub, placed in propylene oxide, and then embedded in araldite (Durcupan ACM). Thick sections (1 /zm) were cut and stained with toluidine blue for light microsopic examination. For transmission electron microscopy (TEM) the thin sections were contrasted with uranyl acetate and lead citrate and viewed with a Siemens 101 Elmiskop.
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GLICK ET AL.
FIG. 2. Bacteria (B) embedded in the epithelium of villi (V) from the CT of a 19-day-old battery-reared bird. X266.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 1. High villi appear in the CT of a 6-day-old chick. X40.
SCANNING ELECTRON MICROSCOPE STUDY OF TONSIL
1411
FIG. 4. A lymphocyte-like cell appeared (L) among the villi of a CT from a 19-day-old battery-reared bird. On the surface of the villi are bacteria (B). The smaller globulin structures may be mucin droplets. X1460.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 3. The cut tip of a villus with a striated epithelial border and central openings, some of which may be lymphatics. X534.
1412
GL1CK ET AL.
FIG. 5a. The highly segmented chains of bacteria (B) project from the surface of CT-villi of 76-day-old litterreared birds. X6000.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 5. Individual and multiple bacteria (B) attached to the CT-villi of 38-day-old birds. X 300.
SCANNING ELECTRON MICROSCOPE STUDY OF TONSIL
1413
*
*
FIG. 7. A thick section (ljuM) of the CT illustrating the distal half of a villus with apparent lymphatics (Ly), bacteria (B), and intercellular spaces (Ic) between epithelial cells. The area of the mound (M) contain lymphocytes. X 560.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 6. The bacteria (B) are attached to the cell membrane. Around the attached surface the cell produced a dark substance which may be similar to that occurring at cell junctions. X 31,500.
GLICK ET AL.
•Mi
,
I
«m
0
FIG. 9. Intercellular (Ic) spaces among the epithelial cells of the distal portion of the CT's villi. In several spaces a fine precipitate may be ovserved. X 1,400.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 8. Lymphatic (Ly) spaces located in the distal portion of the CT's villi. X 10,150.
SCANNING ELECTRON MICROSCOPE STUDY OF TONSIL
1415
surface would have a distinct survival advantage over those not capable of attachment. It has been theorized that some bacteria may gain an important early foothold in the host by an ability to adhere to mucosal surfaces. This may be an important determinant of the organism's virulence. Adherence has been described in several species of pathogenic bacteria, including Streptococcus pyogenes, Escherichia coli, Streptococcus mutatis, Vibrio cholera, Salmonella typhimurium, Neisseria gonorrhea, Clostridium perfringens, and Mycoplasma sp. (Smith, 1977). The ecological significance of end-to-end attachment and end-on attachment to the CT by the bacteria is unknown. Occasionally, the bacteria seem to be almost embedded into the cells. While they appear to be firmly attached, they do not penetrate the cells. This is in agreement with observation of Fuller and Turvey (1971) in chicken ilea. It would be interesting to determine the influence of these bacteria on the cellular development of the CT, especially since their presence is influenced by the environment. Fuller (1973) suggested that the lactobacilli associated with the crop epithelium of the chicken plays a role in the regulation of the
chicken's intestinal flora. It would seem plausible that among the non-pathogenic bacteria of the gut, those which adhere to the intestinal epithelium, are more likely to have an important influence on the host than those which do not (Fuller and Turvey, 1971). Although the method of attachment has not been ascertained by SEM, the attachment of the filaments to the proximal end of the caeca and not the distal might reflect important functional differences between the surface epithelium of these two regions. We have not studied the SEM and TEM of the CT in agammaglobulinemic birds. Agammaglobulinemia might lead to a modification in the attachment of these microorganisms. The lymphatic system of the chicken offers a challenge to the micro-anatomist (Kampmeier, 1969; and Dransfield, 1945). Gut-associated villi would be expected to possess lymph vessels engaged in lipid transport. Also, lymphatics associated with the villi of the CT might be important in communication with a portion of the lymphomyeloid complex. Unpublished observations in our laboratory have revealed lymphatic-nodules in the tibia-tarsal region. Perhaps there are similar structures draining the gut-associated tissue of the chicken. The cavi-
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 18, 2015
FIG. 10. The distal portion of the caecum lacks villi but contains numerous bacteria (B). X 300.
GLICK ET AL.
1416
ties observed in the CT's-villi by SEM and TEM appear to be lymphatics. There may be a relationship among the lymphatic spaces of the villi's stroma, the dilated intercellular-space among the epithelial cells, and the bacilli which occur in this portion of the villi.
ACKNOWLEDGEMENTS
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The a u t h o r s wish to express their appreciation t o t h e University Electron Microscope Center of Mississippi State University, Dr. Greta Tyson, Head, for t h e use of their facilities, Doris T h o m p s o n [BS, MT (ASCP)] for her l a b o r a t o r y assistance, and Karen A n d e r s o n for t y p i n g t h e manuscript.
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