Scanning Electron Microscopy of the Small Intestine of a Normal Unsuckled Calf and a Calf with Enteric Colibacillosis

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1 Vet. Pathol. 15; (1978) Scanning Electron Microscopy of the Small Intestine of a Normal Unsuckled Calf and a Calf with Enteric Colibacillosis G. R. PEARSON. E. F. LOGAN and G. P. BRENNAN Departmcnt of 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 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 10 sites (I to 10) of 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 of 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

2 Scanning Electron Microscopy of Calf Intestine 40 1 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. la) and the length and shape were confirmed by light microscopy (fig. lb, 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

3 402 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 to 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.

4 Scanning Electron Microscopy of Calf Intestine 403 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 of villus tip; columnar epithclial cclls (arrow). narrow lamina propria, horizontal furrow (F) and promincnt microvillous brush border (B). Fig. 6: Cross section of 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 mucus and neutrophil leukocytes on 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).

5 404 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

6 Scanning Electron Microscopy of Calf Intestine 405 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 and have been described in the small intestine of adult persons (71. In an earlier study in the pig [4] the term bridging was used to describe any confluent villi. In our study confluent villi were divided into bifurcated, trifurcated and 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 be an artifact of the plane of section.

7 406 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 1 MARSH. M.N.; SWIFT, J.A.; WILLIAMS. E.D.: Studies of small intestinal mucosa with the scanning electron microscope. Br Med J 4:95-Y MEBUS, C.A.; NEWMAN, L.E.; STAIR, E.L.: Scanning electron. light, and transmission clectron microscopy of intestine of gnotobiotic calf. Am J Vet Res , MOON, H.W.; JOEL. D.D.: Epithelial cell migration in the small intestine of sheep and calves. Am J Vet Res 36: I975 4 MOUWEN. J.M.V.M.: White scours in piglets. I. Stereomicroscopy of the mucosa of the small intestine. Vet Pathol 8: , PEARSON, G.R.; MCNULTY, M.S.; LOGAN, E.F.: Pathological changes in the smali intestine of neonatal calves with enteric colibacillosis. Vet Pathol 15:92-101, 1Y78 6 SMITH. T.: Hydropic stages in the intestinal epithelium of newborn calves. J Exp Med 41: TONER, P.G.; CARR, K.E.: The use of scanning electron microscopy in the study of the intestinal villi. J Pathol 97: , TONER, P.G.; CARR, K.E.; WYBURN. G.M.: The Digestive System: an Ultrastructural Atlas and Review. p. 64; Butterworth's, London, 1971 Y WAXLER, G.L.: Lesions of transmissible gastroenteritis in the pig as determined by scanning electron microscopy. Am J Vet Res 33: Request reprints from G. R. Pearson. Department of Agriculture, Veterinary Research Laboratories. Stormont. Belfast BT4 3SD.