Download A Scanning Electron Microscope Study of the Caecal

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
(Received for publication November 9, 1977)
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
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3
Department of Biological Structure,
University of Washington School of Medicine,
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
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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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1410
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.
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FIG. 1. High villi appear in the CT of a 6-day-old chick. X40.
SCANNING ELECTRON MICROSCOPE STUDY OF TONSIL
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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.
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FIG. 3. The cut tip of a villus with a striated epithelial border and central openings, some of which may be
lymphatics. X534.
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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.
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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
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*
*
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.
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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.
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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-
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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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