Kohler, 1969) described desquamation of

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1 Br. J. exp. Path. (1978) 59, 1 78 THE RATE OF DEVELOPMENT OF POSTMORTEM ARTEFACT IN THE SMALL INTESTINE OF NEONATAL CALVES From the G. R. PEARSON AND E. F. LOGAN Veterinary Research Laboratories, Stormont, Belfast BT4 3SD Received for publication November, Summary. The rate of development of postmortem artefacts was investigated in the mucosa of the small intestine of a calf dually infected with an enteropathogenic strain of Esch. coli and rotavirus, and an uninfected control calf. Samples were removed under general anaesthesia from the proximal, middle and distal small intestine and compared with those taken from adjacent sites 1-3 min after severing the major blood vessels of the neck. In the challenged calf, changes occurred in the villous mucosa by 3 min after severance, whilst in the control calf good fixation was obtained until 1 min after severance. IT has been recognized for many years that intestinal mucosa undergoes rapid autolytic change shortly after the death of an animal (Badawy et al., 1957; Fell, 1961). Fell (1961) obtained good fixation of intestine of sheep and rats within 1 min of death and Thorpe and Thomlinson (1967) described a similar finding in pigs. These latter authors, however, demonstrated that by 1- h post mortem, changes were well advanced. Other workers (Cross and Kohler, 1969) described desquamation of villous epithelium in the duodenum and jejunum within 24 min after death in conventionally reared pigs. In a recent study of intestinal disease in neonatal calves (Pearson, McNulty and Logan, 1978b) desquamation of epithelium was not observed in samples of intestine removed under general anaesthesia, but, when a calf infected with rotavirus and an enteropathogenic strain of E8cherichia coli was exsanguinated and re-sampled several minutes later, desquamation of epithelium from the tips of some villi was a notable feature. Conversely, a similarly treated, uninfected calf did not show the same degree of change. The purpose of this paper was to investigate and compare the rate of onset of postmortem changes in the small intestine of a normal and a diseased calf. MATERIALS ANI) METHODS Experimental procedure.-two calves less than 1 week old were kept in isolation. One calf was orally inoculated on arrival with cultures of an enteropathogenic strain of Esch. coli known to cause pathological lesions (Pearson, McNulty and Logan, 1978a), and rotavirus recovered from the faeces of a diarrhoeic calf (McNulty, Allan and McFerran, 1976) whilst the other was an uninfected control. On the day after arrival each calf was anaesthetized with pentobarbitone and, with a minimum of handling, samples of the proximal, middle and distal small intestine were removed and each site marked with a small numbered clip. In order to follow the method of sampling adopted by other workers (Mebus et al., 1971; Morin et al., 1974) each calf was then stunned with a captive-bolt humane killer and the major vessels of the neck were severed. At 1, 3, 5, 7, 1, 15, 2 and 3 min after severance further samples were removed from sites adjacent to those previously sampled, the intestine remaining in situ throughout the experiment. Thus the sample removed under anaesthesia from each calf served as a control for the remainder of the experiment. As each portion of intestine was removed the time was noted. The time taken to open the sample and place it in formalin was also recorded. At the end of the experiment the position of the clips in relation to the length of the small intestine was noted, allowance being made for the amount of tissue removed. Histopathology.-Each portion of intestine was opened immediately after removal and placed in 1% neutral buffered formalin. Four hours later the tissue was trimmed and processed by the paraffin method. Sections were cut at

2 POSTMORTEM ARTEFACTS IN SMALL INTESTINE 179 6,um and stained with haematoxylin and eosin. In order to assess the postmortem changes, all properly orientated villi in 2 sections from each site at each time were counted. RESULTS At time of death, both calves were clinically normal. In both calves exsanguination took approximately 3 min. Because of the length of the intestine an inevitable delay occurred in locating the numbered sites at 1 and 3 min following severance of the carotid arteries -(Table). At all times samples were placed in formalin within 2 s of removal from the abdomen. In the control calf the samples relating to the proximal small intestine were removed from a point 5-1% of the length of the small intestine from the pylorus, the middle small intestine 53-58%, and the distal small intestine 85-9%. In the challenged calf the proximal small intestine samples were removed from a point 2-7%, the middle 29-34%, and the distal ileum from a point 91-96% of the length of the small intestine from the pylorus. The postmortem changes are summarized in the Table. Samples removed from both calves under general anaesthesia showed good preservation of the epithelium which was contiguous with the lamina propria (Fig. 1). The villi of the challenged TABLE.-The Relationship of Epithelial Cell Shedding in the Small Intestine of Calves to the Time of Fixation Following Severance of the Carotid Arteries Challenged calf Control calf - Actualt Number Epithelium time of separation Site in small Time* (min, villi ratiot intestine (min) sec) counted (%) Proximal Middle Distal Percentage of denuded villi * 3* * Actual time (min, sec) Number Epithelium of separation villi ratio counted (%) 1 ioo * Predetermined time in minutes for removing samples after severing the carotid arteries. t Time at which the sample was removed from the small intestine. : Number of villi with sub-epithelial space: number of villi counted. Sample removed under general anaesthesia. Percentage of denuded villi *

3 18 G. R. PEARSON AND E. F. LOGAN FIG. 1. Proximal small intestine, control calf. Sample removed under anaesthesia shows intact villous epithelium contiguous with the lamina propria. H. and E. x 7. FIG. 2. Proximal small intestine, challenged calf. Sample rernoved 3 min after severance shows development of a sub-epithelial space at the tips of two villi (arrows). A third, normal villus (N), is also present. H. and E. x 7. FIG. 3. Proximal small intestine, challenged calf. Sample removed 7 min after severance shows denudation of villous epithelium (arrows). H. and E. x 7. FIG. 4.-Proximal small intestine, challenged calf. Sample removed 3 min after severance shows dissociation of villous epithelium. Individual epithelial cells (E), and short ribbons of epithelial cells (arrow) are present in the lumen. H. and E. x 7. FIG. 5.-Distal small intestine, challenged calf. Sample removed 1 min after severance shows development of a space (arrow) between the lamina propria and the epithelium of a villus. A central lacteal (L) in a villus with intact epithelium can also be seen. H. and E. x 7. FIG. 6. Proximal small intestine, control calf. Sample removed 3 min after severance shows detachment of the epithelium from the villus cores with resulting sub-epithelial space (arrow). The ribbons of detached epithelium are intact. H. and E. x 7.

4 POSTMORTEM ARTEFACTS IN SMALL INTESTINE 181 calf were generally shorter than those.of the control calf and the villous lamina propria was slightly thickened due to an increased number of reticular cells, which was indicative of infection (Pearson et al., 1978b). In the proximal small intestine of the challenged calf the development of small sub-epithelial spaces towards the tips of the villi was first noted at 3 min (Fig. 2). The presence of this sub-epithelial space was used in the assessment of the postmortem changes in the mucosa. The number of villi with a space (whether small or involving the whole villus) relative to the number of villi counted in the 2 sections was designated the "epithelium separation ratio" (Table). By 5 min 28% of the villi were affected, and by 7 min more than half of the villi had a subepithelial space and there was a large number of denuded villi (villi with partial loss of epithelium leaving exposed lamina propria) (Fig. 3). By 15 min, almost all villi had sub-epithelial spaces and twothirds of them showed denudation. By 2 min all the villi were denuded, leaving the lamina propria exposed, and there was complete dissociation of epithelial cells which were seen lying free in the lumen (Fig. 4). In the middle small intestine a similar progression was observed but the changes started earlier. Complete dissociation of epithelium leaving denuded villi did not occur until 3 min after bleeding. In the distal small intestine the changes were later in onset than those in the proximal and middle small intestine. The formation of sub-epithelial spaces was first observed at 1 min (Fig. 5) but total dissociation as seen in the proximal and middle small intestine did not occur. In the control calf there were variations between different areas of intestine in time of onset and severity of postmortem changes. Changes started slightly earlier in the proximal small intestine but they progressed more rapidly in the middle small intestine, so that by 15 min the appearance of the mncosa was approximately the same in both regions. Between 5 and 1 min a small sub-epithelial space was observed at the tips of occasional villi. By 15 min, approximately one-third of the villi showed the development of a sub-epithelial space with some contraction of the connective tissue lamina propria, but there were only a few denuded villi. By 2 and 3 min the epithelium was separated from the lamina propria of the majority of villi but remained intact and adjacent to it (Fig. 6). The temporal sequence of changes in the terminal ileum was slightly slower than in the proximal and middle intestine, and 1 min after the severance of the carotid arteries resembled the sample (Fig. 1) removed under anaesthesia. Thereafter, the changes were mild and by 3 min approximately one-quarter of the villi showed the development of a sub-epithelial space, but only occasional villi were denuded. DISCUSSION In the control calf, good fixation of the small intestine was obtained under general anaesthesia and until 1 min after severing the carotid arteries, a finding similar to that of earlier workers in other species (Badawy et al., 1957; Fell, 1961; Thorpe and Thomlinson, 1967). The development of a sub-epithelial space was the earliest detectable change and in the control calf was minimal until 15 min, when approximately one-third of the villi were affected. By 2 min all the villi had pronounced sub-epithelial spaces at their lateral borders, with the epithelium forming sheets of cells lying adjacent to the lamina propria. In contrast, in the challenged calf subepithelial spaces were seen as early as 3 min after severance and were severe in the proximal small intestine by 7 min. In both calves exsanguination took approximately 3 min by which time postmortem changes were observed in the challenged calf but not in the control calf. This earlier start in a challenged calf was observed previously (Pearson et al., 1978b); therefore only

5 182 G. R. PEARSON AND E. F. LOGUAN samples removed under anaesthesia should be directly compared, since postmortem artefacts probably occur earlier in animals with intestinal damage. Denudation of villus tips has been described as part of a pathological process by some authors (Mebus et al., 1971; Morin et al., 1974; Doughri and Storz, 1977). However, the present study demonstrates that sub-epithelial spaces and denuded villi can be produced as artefacts of fixation. Sprinz (1962) also regarded desquamation (denudation) of villous epithelium as an artefact, even at the height of severe bacterial disease in man. Differences in time of onset were observed at different sites in the small intestine, particularly in the control calf. For example, changes occurred first in the proximal small intestine and thereafter the proximal and middle small intestine progressed at about the same rate. The distal small intestine, however, appeared to be more resistant, a finding observed in normal animals of other species (Badawy et al., 1957; Thorpe and Thomlinson, 1967). Although the challenged calf was killed whilst clinically normal, shorter villi were observed in samples removed under anaesthesia. In an earlier study (Pearson et al., 1978b), diarrrhoea had been present for 72 h and pathological lesions were severe in the distal small intestine, where postmortem artefacts were prominent. Thus the pathological state of the mucosa may also influence the development of such artefacts. In conclusion, the present study demonstrates that the removal of samples of the small intestine under anaesthesia before exsanguination, as first suggested by Badawy et al. (1957) for the normal animal, is the method of choice and is possibly of greater importance when examining samples taken from animals with intestinal disease. REFERENCES BADAXNx-, A. Al., CAMPBELL, R. AM., CUTHuEITSON, 1). P. & FELL, B. F. (1957) Changes in the Intestinal Muicosa of the Sheep) Following Death bv Humatie Killer. Natuore (Lonld.), 18, 756. CROSS, R. F. & KoIlEIIz, E. M. (1969) Autolytic Changes in the I)igestive System of Germfree, Escherichi i coli AMono-contaminated, an(l Convenitional Baby Pigs. COnt. J. comp. Med., 33, 18. DoU(GHRI, A. M. & STORZ, J. (1977) Light andi Ultrastructtural Pathological Changes in Intestinal Coronavirtis Tnfection of Ntewborn Calves. Zbl. Vet. Med. B, 24, 367. FELL, B. F. (1961) Cell Shedding in the Epithelium of the Intestinal Muicosa: Fact andl Artefact. J. Path. Bact., 81, 251. MCNULTY, M. S., ALLAN, G. AM. & AMcFERRAN, J. B. (1976) IsolatioIn of a Cytopathic Calf Rotavirus. Res. Vet. Sci.,21, 114. MEnIs, C. A., STAIR, E. L., UNIDERDAHL, N. R. & TWIEHAVS, M. J. (1971) Pathology of Neoinatal Calf Diarrhoea Itidutce(I by a Reo-like V'irus. VSret. Plth., 8, 49. AIORIN, Al., LAMTOTHE, P., GA(GNON, A. & MALO, R. (1974) A Case of Viral Neonatal C'alf Diarrhoea in a Quebec Dairv Herd. (Can. J. cormio. Mec., 38, 236. PEARS;ON, G. R., MNI-LITY, M\. S. & LOGAN, E. F. (1978(t) Pathological Changes in the Small Intestine of Neonatal Calves with Einteric Colibacillosis. Vet. Path., 15, 92. PEARSON, C. R., MC'Nl LTY, AM. S. & LOGAN, E. F. (1 978b) Pathological Changes in the Small Intestine of Neonatal Calves Natuirally Infected with Reo-like Virus (Rotavirus). Vet. Fec. (in press). SPRINz, H. (1962) MIoirphological Response of Intestinal Mucosa to Enteric Bacteria an(d its Implication for Spruie and Asiatic Cholera. Fedn Proc., 21, 57. THOR"E, E. & THOi\INioN, J. R. (1967) Atutolysis an(l Post-mortem Bacteriological Changes in the Alimenitary Tract of the Pig. J. Path. Bact., 93, 61.