Vet. Pathol. 15; 400-406 (1978)
Scanning Electron Microscopy of the Small Intestine of a Normal
Unsuckled Calf and a Calf with Enteric Colibacillosis
G. R . PEARSON.
E. F. LOGANand G . P. BRENNAN
Departmcnt o f Agriculture. Veterinary Research Laboratories. Stormont. Belfast; and
Department of Zoology, Ouecn's University, Belfast
Abstract. Sections of the small intestine wcre taken under general anaesthesia from a
normal calf and from a calf with cnteric colibacillosis and examined by scanning electron
and light microscopy. In the normal calf villi were long and oval throughout the intestine
and in the challenged calf there was villous stunting and fusion in the distal small intestine.
The histological appearance of the small intestine of the neonatal calf has
received little attention. Hydropic change in the intestinal mucosa has been
reported [6] and recently the histological. ultrastructural and stereoscopic appearance of the small intestine of a gnotobiotic calf have been described [2].Other
workers described epithelial cell renewal in neonatal calves and gave details of
mucosal measurements, but did not describe the histological appearance (31. The
normal histological appearance of the small intestine of unsuckled neonatal calves
and of calves with experimental enteric colibacillosis has been reported recently
PI.
Materials and Methods
Two colostrum-deprived calves wcre used. One was a control and one was challenged
when less than 18 hours old with an cnteropathogenic strain of Escherichia c d i (type
0101 K?(A)). Samples of intestine wcre removed when both calves were 4 days old. Calves
were given general anaesthesia and samples removed immediately before slaughter.
For light microscopy 1 0 sites ( I t o 10) o f the small intestine (from the pylorus to the
ilcocolic junction) were placed immediately in 10% buffered formalin. Sections were cut at
6 micrometers and stained with haematoxylin and eosin (HE) and periodic acid-Schiff
(PAS) IS].
Samples for scanning electron microscopy were taken at the same time as those for light
microscopy from sites 2 , 5 and 10. Small blocks of tissue, 0.5 centimeter diameter, were
placed in a fresh solution o f two parts glutaraldehyde and one part osmium tctroxide at 4"C
for 2 hours. They were dehydrated in a graded series of ethanol from 70 to 100% and
stored at 4" C in amyl acetate until required. The tissues were dried in a critical-point drying
apparatus. With the villous surface uppermost the tissues were attached to aluminum stubs
with adhesive, coatcd with gold and examined.
400
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Fig. 1: Middle small intestine. control calf. a. Villi arc long. oval and have prominent
horizontal furrows. b. Longitudinal section. Villi have narrow lamina propria and central
lacteal (arrow).
Fig. 2: Cross section of villi from distal small
intestine, control calf. Villi are individually distinct, oval in outline with central lacteal (arrow)
and narrow lamina propria (L). A few erythrocytes (E) in lumen.
Results
There was no diarrhoea in the control calf, but it had been profuse for 42 hours
in the challenged calf at the time of sampling.
At low magnification control calf tissue had long oval villi with prominent
horizontal furrows at all sites by scanning electron microscopy (fig. l a ) and the
length and shape were confirmed by light microscopy (fig. l b , 2). Bridging villi
(fig. 3 ) were seen by light microscopy, but by scanning electron microscopy the
corresponding structure was a single villus with a longitudinal depression in the
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Pearson. Logan and Brennan
Fig. 3: Proximal small intestine. control calf. a . Bridging villus (B) with longitudinal
depression (arrow) in surface. b. Similar villus with central space lined with epithclium
(arrow) corresponding t o the depression.
Fig. 4: Middle small intestine. control calf. a . Bifurcation of tip of villus. b. Longitudinal
section of similar villus.
surface. Bifurcated villi (fig. 4) were seen by both techniques and trifurcated villi
were seen by scanning electron microscopy. At higher magnification the epithelial
cells had a flat luminal surface with a microvillous brush border (fig. 5 ) .
In the challenged calf the mucosa of the proximal and middle small intestine
resembled that of the control calf. In the distal small intestine, however, the villi
were thick, had become round in cross section (fig. 6) and were markedly stunted.
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Fig. 5: Proximal small intcstinc. control calf. a . Epithelial cells have raised polygonal
outline; microvilli are distinct, Small flecks. possibly niucus, on surface. b . Longitudinal
scction o f villus tip; columnar epithclial cclls (arrow). narrow lamina propria, horizontal
furrow (F) and promincnt microvillous brush border (B).
Fig. 6: Cross section o f villi, distal small intestine of challenged calf at same magnification as
figure 2 . Thick villi; increased number of cells in
lamina propria (L) and central lacteals are obliteratcd. Villi fused at epithelial surfaces (arrows).
Fusion of epithelial surfaces (fig. 6) caused the mucosa to look flat (fig. 7). There
was more exudate consisting of m u c u s and neutrophil leukocytes o n the mucosa
than in normal sites. Small accumulations of cellular debris attached to the tip of
occasional villi was seen by scanning electron microscopy. By light microscopy this
was shown to be epithelial cells and neutrophilic leukocytes becoming detached
from the tip of the villus, leaving intact cpithelium below (fig. 8).
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Pearson, Logan and Brennan
Fig. 7: Distal small intestine, challenged calf. a. Flat mucosa; stunting and fusion of villi.
Exudate on mucosa. b . Stunted villi cut tangentially and in cross section.
Fig. 8: Distal small intestine, challenged calf. a . Tip of round villus, covered by
epithelium with separation of some epithelial cells and excess debris (E). b . Separation of
epithelial cells (E) from underlying intact epithelium (arrows).
The epithelial cells covering some villi had a characteristic dome-shaped
appearance (fig. 9). Rod-shaped bacilli were adherent to the surface of epithelial
cells (fig. 10).
Discussion
The scanning electron microscopic appearance of the intestinal mucosa of
conventionally-reared calves has not been described previously and differs in some
respects from that reported for a gnotobiotic calf in which the villi are cylindrical in
profile [2], similar to the finger-shaped villi in human mucosa of adults [ 1, 71. In
contrast, in our study, in which the calves were histologically representative of
other calves [ 5 ] ,most villi were oval. Similar villi have been reported at some sites
in the pig [9].
Bifurcated and bridging villi have been seen by light microscopy [ 5 ] and scanning
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Fig. 9: Distal small intestine. challenged calf. a . Villus covered with dome-shaped
epithelial cells. b. Epithelial cells have domed profile (arrows) towards lumen.
Fig. 10: Distal small intestine. challenged calf. a . Bacteria on epithelial cell surface.
Microvillous brush-border (M). b. Rod-shaped bacilli (arrow) adherent to luminal surfacc
of epithelial cells.
electron microscopy. In addition, trifurcated villi, not seen by light microscopy
were detected by scanning electron microscopy. Bifurcated villi, however, are not
unique to the calf a n d have been described in the small intestine of adult persons
(71. In an earlier study in the pig [4] the term bridging was used t o describe any
confluent villi. In o u r study confluent villi were divided into bifurcated, trifurcated
a n d bridging villi. As seen by light microscopy bridged villi appear to be two villi
that are fused towards their apex with an intervening space lined by epithelium. By
scanning electron microscopy, however, the space appears to be only a depression
in the surface of a single villus. Thus the histological appearance of a bridge is
considered to b e an artifact of the plane of section.
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Pearson, Logan and Brennan
In the distal small intestine of the challenged calf the change in the threedimensional appearance of the mucosa as a result of lesions was pronounced,
confirming the histological findings IS]. The most noticeable feature was stunted
and thick villi with marked fusion between adjacent villi resulting in a flat mucosa.
On cross-section the villi were round. Some villi with dome-shaped epithelial cells
were detected. an appearance that has been described in coeliac disease in man
[ l ] . Although this is more commonly found in diseased specimens, there is the
possibility that it may be partly caused by artifact [8].
The adhesion of bacteria to the mucosa, previously shown by specific immunofluorescence to be the challenge strain of E . coli [ 5 ] , also was demonstrated by
scanning electron microscopy. Thus there was good correlation between light and
scanning electron microscopy in both calves and the latter technique was found to
be useful in confirming the histological findings.
Acknowledgements
The scanning electron microscope in the Department of Zoology. Queen's University.
was bought with a grant from the Science Research Council.
References
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Request reprints from G . R. Pearson. Department of Agriculture, Veterinary Research
Laboratories. Stormont. Belfast BT4 3SD.
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