Effects of dietary change and rotavirus infection on small intestinal structure and function in gnotobiotic piglets

Research in Veterinary Science /989, 47, 2/9-224

Effects of dietary change and rotavirus infection on small intestinal structure and function in gnotobiotic piglets G. A. HALL, K. R. PARSONS, Agricultural and Food Research Council, Institute for Animal Health, Compton Laboratory, Compton, near Newbury, Berkshire, RG160NN, G. L. WAXLER, Department of Pathology, Michigan State University, East Lansing, Michigan, USA, K. J. BUNCH, Agricultural and Food Research Council, Institute for Animal Health, Compton Laboratory, Compton, near Newbury, Berkshire, RG160NN, R. M. BATT, Department of Veterinary Pathology, University of Liverpool, PO Box 147, Liverpool intestinal damage and malfunction at weaning, there was no evidence of it five weeks later (Hall et al 1983). The purpose of this study was to examine the combined effects of weaning and rotavirus infection on the structure and function of the small intestinal mucosa, to ascertain whether damage might persist and affect growth rate. Germ-free pigs were used to exclude possible effects of unidentified infectious agents.

The combined effects of weaning and rotavirus infection on small intestinal structure and function and on growth rate were studied in 28 gnotobiotic piglets. There was little damage by rotavirus to the proximal small intestine, some damage to the mid small intestine and relatively severe damage to the distal small intestine; villi were stunted, crypts lengthened and activities of all brush border enzymes decreased. The damage was short-lived despite the synchronisation of rotavirus infection with simulated weaning. There was no evidence of persistent damage to the small intestine and growth rate was unaffected.

Materials and methods

WEANING of piglets is often followed by diarrhoea (Wilson 1986) and there appear to be a number of contributory icauses. These include proliferation of Escherichia coli (Kenworthy and Crabb 1963, Tzipori et al 1980, Leece et al 1983), the action of rotavirus (Woo de et at'1976, Tzipori et a11980, Leece et a11983) and the effects of dietary change (Tzipori et al 1980, Leece et al 1983, Miller et al 1984). There is evidence for an interaction between E coli infection and a change of diet from milk to solids in the pathogenesis of postweaning diarrhoea (Tzipori et al 1984), and also for an interaction between dietary change and infection with rotavirus (Tzipori et al 1980, Leece et al 1983). Weaning also causes marked alterations in the structure of the small intestine (Gay et al 1976, Kenworthy 1976, Hampson I986a) and these changes may occur in the presence (Kenworthy 1976) and absence (Hampson 1986a) of diarrhoea. A check in growth rate is another consequence of weaning (Leece and King 1978); often this follows postweaning diarrhoea, but may occur in the absence of diarrhoea. The postweaning check in growth rate may be the result of the structural changes that occur in the small intestine at weaning. However, the structure and function of the small intestine appeared normal in a study of eightweek-old pigs that had grown slowly since weaning (Hall et al 1983); if the poor growth was caused by

Animals Twenty-eight gnotobiotic piglets were derived from three sows by the method of Tavernor et al (1971). They were housed in plastic isolators and fed a milk diet from birth to 21 days when they were weaned on to a solid diet. On the day of weaning, 15 pigs were inoculated with rotavirus by mouth, while the remaining I3 were not inoculated and served as controls. Six inoculated pigs were killed three days after weaning, five at seven days and four at 22 days. Five controls were killed three days after weaning, five at seven days and three at 22 days.

Diets The milk diet was evaporated milk (Carnation Foods) diluted with an equal volume of mineral solution (Dennis et al 1976). The solid diet was commercially prepared for three-week-weaned piglets (Piglets I; Colborn Nutrition); it contained a supplement of copper (14 g t-') but not of antibiotics or growth promoters. This diet was sterilised by exposure to 5 Mrad ionising radiation before it was passed into the isolators.

Weighing

219

Piglets were weighed each day in a sling attached to

G. A. Hall, K. R Parsons, G. L. Waxler, K. J. Bunch, R. M. Ball

220

TABLE 1: Mean villus height, crypt depth. ratio of villus height:crypt depth. E mm -1 and A mm -1 in proximal. mid and distal small intestines in rotavirus·inoculated and control pigs

Villus height (I'm)

Site

Treatment

3

Proximal

Inoculated Control Inoculated Control Inoculated Control

267411 202372

Inoculated Control Inoculated Control Inoculated Control

159 127 114 90 95 80

Mid Distal Crypt depth (I'm)

Proximal Mid Distal

Ratio villus height: crypt depth

Proximal Mid Distal

E mm-' (rnm)

Proximal Mid Distal Proximal Mid Distal

Days after inoculation 7 22

476

4n

505 501 367

589 582

509

63

399 319

293

427 364 397

31

200156 143 116 11588

292243 175 183 173 159

Inoculated Control Inoculated Control Inoculated Control

3·09 3·79 2·344·56 2·074·61

2·63 3·35 2·60 3·40 2·82 3·56

2·25 2·55 2·88 2·31 2·37 2·73

Inoculated Control Inoculated Control Inoculated Control

4·21 4·00 3·064·41 2·443'82

4·31 4·22 3-97 4·63 2·85 3·22

4·87 3·86 4·65 4·23 3·24 3·65

Inoculated Control Inoculated Control Inoculated Control

331 324 221 254 168237

SEot

(19 dfl

390 347

296 293

209 196

565 490

388 375 312

295

63

19 14 10 0·45 0·44 0·44 0·69 0·55 0·44 45 40 22

- Significantly different from control (P
t Average standard error of difference between means of inoculated and control groups at a given time

a spring balance. An analysis of variance model with parameters for treatment and litter was fitted to data from day 22 onwards. The mean weight of each piglet at days 20 and 21, immediately before the treatment period, was taken as a covariate. The model was used to test for differences in mean weight between the two groups allowing for any initial pretreatment differences in weight.

Necropsy procedures Tissues for microscopy and enzymology were removed under pentobarbitone sodium (Sagatal; May & Baker) anaesthesia. First, short lengths (approximately 10 ern) of small intestine were ligated and filled with mercuric formol at three sites; proximally, at the ligament of Treitz (proximal small intestine) distally, adjacent to the i1eocaecal junction (distal small intestine) and at a point approximately mid-way between the above sites (mid small intestine). Secondly, a further 2 cm length of intestine was removed from the proximal and distal sites placed in a

glass vial and snap frozen in liquid nitrogen at - 20°C for subsequent enzyme assay. Tissues for enzyme assay were not collected from piglets killed at 22 days after weaning. Thirdly, the ligated loops of intestine were removed and immersed in fixative.

Morphometries Histological sections of intestinal tissue were prepared and stained with haematoxylin and eosin or by the immunoperoxidase method for rotavirus antigen (Parsons et al 1984) and villus height, crypt depth, number of villi mm -I of muscularis mucosa, number of crypts mm - I of muscularis mucosa, length of epithelial surface mm - 1 of muscularis mucosa (E mm - I) and mucosal area mm - I of muscularis mucosa (A mm -I) were measured (Hall et al 1983). Analysis of variance models with parameters for treatment, litter and day of slaughter were used to examine the effects of infection on these variates. This analysis took the form of a split-plot model with site as the subplot for two of the variates which were the ratio of

Effects of diet and rotavirus on pig intestine

221

TABLE 2: Geometric meen enzyme ectivities ImU mg- 1 proteinl in mucosel scrapes from the proximal and distal small intestines of rotavirus-inoculated and control pigs Days after infection Enzyme

Site

Treatment

3

7

Alkaline phosphatase

Proximal

Inoculated Control Inoculated Control

18·3" 30·5 16·3 24·2

10·9 15·4 17·7 21·3

Inoculated Control Inoculated Control

4·4 4·2 2·8" 5·2

4·5" 8·4 12·6 10·2

Inoculated Control Inoculated Control

8·5 9·0 13·9" 53·6

4·2 6·0 19·9 13·8

Inoculated Control Inoculated Control

2·0 2·1 2·1" 7·9

3·8 6·0 5·3 4·3

Inoculated Control Inoculated Control

107·4 104·7 109·4" 181·1

121·6 124·2 259·4 187·1

Inoculated Control Inoculated Control

75·3 116·9 4·0" 102·8

85·5 86·1 22·7 8·6

Inoculated Control Inoculated Control

43·0 45·1 42·1 93·8

32·1 42·7 82·4 73·1

Distal Aminopeptidase N

Proximal Distal

a-glucosidase

Proximal Distal

y-glutamyl transferase

Proximal Distal

Maltase

Proximal Distal

Lactase

Proximal Distal

Sucrase

Proximal Distal

" Significantly different from control (P
villus height to crypt depth and E mm - I. This splitplot model was considered inappropriate for villus height, crypt depth and A mm -I for which error variances were different for different sites. Therefore, data from each site were analysed separately for these variates.

Enzymology Samples of intestine stored at - 20°C were thawed and assayed for the brush border enzymes alkaline phosphatase, aminopeptidase N, a-glucosidase and yglutarnyl transferase (Hall et al 1983)and for maltase, sucrase and lactase (Peters et al 1976). Protein was determined (Shacterle and Pollack 1973) with bovine serum albumin (Armour Pharmaceutical) as a standard. Enzymology data were analysed by statistical methods similar to those described above. Results

Weights There was no significant difference in weight

between inoculated and control animals. After adjusting for initial weight differences, the mean values for the two groups were 6' 32 kg and 6' 55 kg, respectively.

Immunostaining Rotavirus antigen was detected only in pigs killed three days after weaning; in five of six inoculated pigs and not in controls.

Morphometries Control pigs. Villus height, crypt depth and A mm - 1 decreased with distance along the small intestine regardless of age (Table I). Crypt depth increased significantly at intervals after weaning throughout the small intestine (P
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G. A. Hall, K. R Parsons, G. L. Waxler, K. J. Bunch, R. M. Batt

the small intestine by approximately 25 per cent three and seven days after inoculation. This increase was maintained in the proximal small intestine until 22 days after inoculation (P
Enzymology

Control pigs. Table 2 shows the acnvines of enzymes in mucosal scrapes taken from the small intestine three and seven days after inoculation; 0'glucosidase and sucrase were significantly higher on average in the distal small intestine compared with the proximal (P<0'05); the activities of the remaining enzymes did not change significantly with site. The activity of y-glutamyl transferase in the proximal small intestine was significantly higher seven days after inoculation than at three days (P
Discussion In the control animals in this study, as in earlier studies of weaned and unweaned conventionally reared pigs (Hall et al1983, Hampson 1983), mucosal volume, villus height and crypt depth were lower in the distal than the proximal small intestine. Crypt depth increased significantly at intervals after weaning in control pigs throughout the small intestine, but villus height did not. Studies of conventionally reared pigs not infected with enteropathogens have shown that villi shorten and crypts lengthen markedly after weaning (Hampson 1986a); the cause of these changes is not understood but could be related to withdrawal of sows' milk (Hampson 1986a), the physical effects of diet (Tasman-Jones et al 1982) and the acquisition of a complex microbial flora (Kenworthy and Allen 1966). The diet of the control pigs in this study was changed from liquid to solid at weaning and could have been the cause of the changes to the crypts. Lengthening of crypts has been interpreted as evidence of an increased rate of production of crypt cells during repair processes following villus damage (AI-Mukhtar et aI1982). It is possible that dietary change caused villus damage and that repair processes were the cause of elongated crypts in the control pigs in this study. This is unlikely, however, because damaged villi were not seen and villus height increased rather than decreased, although this increase was not statistically significant. The reason for increased crypt depth is unclear, but it confirms previous observations of an underlying trend for crypt depth to increase with age reported in weaned and unweaned conventionally reared pigs (Smith 1984, Hampson 1986a). The morphological changes detected in the small intestines of inoculated pigs were consistent with those described previously in rotavirus-infected pigs (Crouch and Woode 1978, Theil et al1978) and were seen in association with rotavirus antigen revealed by immunoperoxidase staining. Typically, rotavirus infects and damages mature enterocytes in the small intestine, and these exfoliate resulting in a relatively immature population of enterocytes on villi of reduced height; these changes are accompanied by crypt hyperplasia. The mucosa is not infected throughout its length; earlier studies of rotavirus infections in'pigs less than one week old found that the mucosa of the mid and distal small intestine was infected most severely (Hall et al 1976, Pearson and McNulty 1977, Crouch and Woode 1978, TorresMedina and Underdahl 1980, Pospischil et al 1981) and a similar distal distribution of infection was reported in 10- to 14-day-old pigs (Theil et al 1978). Mucosal infection can be patchy within infected regions (Pearson and McNulty 1977). A similar, well defined restriction of infection and damage to the mid and distal small intestine was seen in this present study

Effects of diet and rotavirus on pig intestine of older gnotobiotic pigs. There was little morphological and only slight biochemical evidence for rotavirus-induced damage to the proximal small intestine, although an increase in crypt depth seven and 22 days after inoculation suggested that rotavirus infection had stimulated crypt cell production rate either by a local or systemically mediated response. There was evidence of damage in the mid-intestine at three days; villus height, the ratio of villus height to crypt depth and length of epithelial surface were reduced significantly. There was morphological and biochemical evidence of severe damage to the distal intestine three days after rotavirus inoculation. Villus height, villus height to crypt depth ratio, length of epithelial surface and mucosal area were all reduced significantly and the activities of all brush border enzymes were reduced, although the decrease in alkaline phosphatase was not significant. The overall function of the small intestine may not be affected by regional and patchy interference with structure and function, and this may have been the case in the present study. For example, reduced lactase activity was restricted to the mid small intestine of gnotobiotic lambs infected with rotavirus, but oral tolerance tests demonstrated no difference in the ability to digest lactose and absorb glucose compared with the controls (Ferguson et al 1981). Similarly, diarrhoea was not seen in calves where severe rotavirus infection and damage was located mainly in the proximal small intestine (Reynolds et al 1985). A sequence of events which results in postweaning diarrhoea can be postulated from these and previous studies. Weaning causes a reduction in small intestinal absorptive area and, because of the appearance of a less mature enterocyte population, a reduction in digestive enzymes (Hampson 1986a); these changes may be exacerbated by rotavirus infection, as shown in this study. Excessive feeding of a high nutrient, dry diet after weaning overwhelms digestive and absorptive function and an environment is created in the small intestine which stimulates the proliferation of E coli and diarrhoea occurs (Leece et al 1982, 1983, Tzipori et al 1984, Hampson 1986b). The moderate lesions and mild clinical signs seen in this study, and the mild clinical signs seen in an earlier study where rotavirus infection was coincident with a change from a milk to a dry diet, but where intestinal structure and enzymology were not examined (Tzipori et al 1980), suggest that some other factor, possibly pathogenic E coli may need to be present if severe diarrhoea is to develop. The design of this experiment does not allow assessment of the relative contributions of weaning and rotavirus infection to the pathogenesis of the lesions. However, there was no evidence that inoculation of gnotobiotic pigs with rotavirus at the time of intro-

223

duction of a solid diet caused persistent damage to the small intestine. The damage was short-lived and there was no effect on growth rate. The findings suggest that rotavirus alone is unlikely to be responsible for a post weaning check in growth rate in the pig, but does not exclude the possibility that this could occur due to interaction between rota virus and other microorganisms present in conventionally reared pigs. Acknowledgements

The authors thank Dr J. C. Bridger for the rotavirus inocula, Mr M. J. Dennis for the production and care of gnotobiotic pigs and Mr B. Turfrey for the histological sections. References AL-MUKHTAR, M. Y. T., POLAK, J. M., BLOOM, S. R. & WRIGHT, N. A. (1982) Mechanisms in Intestinal Adaptation. Eds J. W. L. Robinson, R. H. Dowling and E. G. Riecken. Lancaster, Raven Press. pp 3-28 CROUCH, C. F. & WOODE, G. N. (1978) Journal 0/ Medical Microbiology II, 325-334 DENNIS, M. J., DAVIES, D. C. & HOARE, M. N. (1976) British Veterinary Journal 132, 642-646 FERGUSON, A., PAUL, G. & SNODGRASS, D. R. (1981) Gut 22, 114-119 GAY, C. c.. BARKER, I. K. & MOORE, P. (1976) Proceedings of the 4th International Pig Veterinary Society Congress, Ames, Iowa, VII HALL, G. A., BRIDGER, J. c.. CHANDLER, R. L. & WOODE, G. N. (1976) Veterinary Pathology 13, 197-210 HALL, G. A.,IPARSONS, K. R., BATT, R. M. & BUNCH, K. J. (1983) Research in Veterinary Science 34,167-172 HAMPSON, D. J. (1983) PhD thesis, University of Bristol HAMPSON, D. J. (1986a) Research in Veterinary Science 40, 32-40 HAMPSON, D. J. (I 986b) Research in Veterinary Science 41, 63-69 KENWORTHY, R. (1976) Research in Veterinary Science 21, 69-75 KENWORTHY, R. & ALLEN, W. D. (1966) Journal of Comparative Pathology 76, 291-296 KENWORTHY, R. & CRABB, W. E. (1963) Journal of Comparative Pathology 73, 215-228 LECCE, J. G., BALSBAUGH, R. K., CLARE, D. A. & KING, M. W. (1982) Journal of Clinical Microbiology 16, 715-723 LECCE, J. G., CLARE, D. A., BALSBAUGH, R. K. & COLLIER, D. N. (1983) Journal of Clinical Microbiology 17, 689-695 LEeCE, J. G. & KING, M. W. (1978) Journal 0/ Clinical Microbiology 8, 454-458 MILLER, B. G., NEWBY, T. J., STOKES, C. R. & BOURNE, F. J. (1984) Research in Veterinary Science 36,187-193 PARSONS, K. R., WILSON, A. M., HALL, G. A., BRIDGER, J. C, CHANTER, N. & REYNOLDS, D. J. (1984) Journal 0/ Clinical Pathology 37,645-650 PEARSON, G. R. & McNULTY, M. S. (1977) Journal of Comparative Pathology 87,363-375 PETERS, T. J., BATT, R. M., HEATH, J. R. & TlLLERAY, J. (1976) Biochemical Medicine 15,145-148 POSPISCHIL, A., HESS, R. G., BACHMANN, P. A. (1981) Zentralblatt fur Veterinarmedizin Series B 28, 564-577 REYNOLDS, D. J., HALL, G. A., DEBNEY, T. G. & PARSONS, K. R. (1985) Research in Veterinary Science 38,264-269 SCHACTERLE, G. R. & POLLACK, R. L. (1973) Analytical Biochemistry 51, 654-655

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Received July 12, 1988 Accepted December 5, 1988