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Influence of creep feeding and dietary intake after weaning on malabsorption and occurrence of diarrhoea in the newly weaned pig
Research in Veterinary Science /986, 4/, 63-69
.Influence of creep feeding and dietary intake after weaning on malabsorption and occurrence of diarrhoea in the newly weaned pig D. J. HAMPSON, Department of Veterinary Pathology and Public Health, W. C. SMITH, Department of Animal Science, Massey University, Palmerston North, New Zealand
ships between the pattern of creep feeding, dietary intake after weaning, the proliferation of enteropathogens in the intestine and the occurrence of diarrhoea in the newly weaned pig.
The influence the pattern of creep feeding has on the ability of pigs to absorb xylose after weaning at three weeks old was investigated. All weaned pigs showed a reduction in their ability to absorb xylose one week after weaning, but this was not influenced by the pattern of creep feeding before weaning. Diarrhoea occurred in some animals after weaning, but did not occur in pigs which did not have access to creep food before weaning. This protective effect of withholding creep food was associated with a low dietary intake after weaning with this regimen. Pigs which developed diarrhoea tended to be those which consumed more meal after weaning than their contemporaries. Haemolytic enterotoxigenic Escherichia coli and rotaviruses were present in the faeces of most pigs after weaning, but, in those animals that ate too much and developed diarrhoea, excretion of the E coli continued for approximately twice as long as in animals that remained healthy.
Materials and methods
Animals Seventy crossbred piglets from five White-cross sows served by Hampshire boars, and three similar sows mated to Landrace boars, were obtained from the Pig Research Centre, Massey University. Two batches of 35 piglets, each divided into six groups, were used in separate but otherwise identical trials conducted three months apart. In each trial piglets of groups I, 2 and 3 were selected randomly from the progeny of two sows whose litters were allowed to mix from three days old; piglets of groups 4, 5 and 6 were selected from the litters of a further two similarly managed sows.
IT has been reported that diarrhoea following weaning in the pig may result from a transient intestinal hypersensitivity to antigenic components of the weaning diet (Miller et al 1984a). These authors demonstrated that piglets given a small amount of creep food as a 'priming' dose before weaning went on to show a significantly greater depression in their ability to absorb xylose eight days after weaning and suffered more diarrhoea than did other weaned pigs. It was suggested that while consumption of small amounts of creep food 'primes' hypersensitivity, consumption of large amounts before weaning causes the animal to become tolerant to the food antigens it meets later. Subsequently, in a commercial piggery, these authors (Miller et al 1984b) demonstrated that withholding creep food before three week weaning had a protective effect against diarrhoea after weaning; they claimed that this 'abrupt' weaning was beneficial because it limited intestinal hypersensitivity responses to the weaner diet. The purpose of the present investigation was to attempt to confirm these potentially important observations and to define more closely the relation-
Treatments and diets Piglets of group I were offered a commercial creep meal (19 per cent protein, no antibiotics) ad libitum from three days old and were left unweaned. Group 2 piglets received the same creep diet while with the sow and were weaned on to this diet at three weeks old. The animals of group 3 were treated as those of group 2, except that between 10 days old and weaning they were removed from the sows daily for four hours. During this period they were confined in a small heated pen where they were offered and encouraged to consume additional quantities of the creep food. Piglets of groups 4 and 5 were not offered creep food before weaning. Group 4 piglets were left unweaned, while group 5 animals were weaned on to the creep diet when three weeks old. The piglets of group 6 differed from those of group 5 only in that at eight, nine and 10 days old they were each force-fed with 10 g of the creep food; they were then not given any further access to creep food until they were weaned on to it at three weeks old. 63
64
D. J. Hampson. W. C. Smith
Accommodation after weaning In each trial 23 piglets were weaned simultaneously at three weeks old. They were individually penned in random order in a battery of single tier wire mesh cages with polypropylene slatted flooring. The cages were housed in a controlled-temperature room maintained at 28°C with lighting kept at a low intensity. The creep diet and fresh water were available ad libitum. Before and between trials the room and cages were thoroughly cleaned and disinfected and then rested. The piglets that were left unweaned at three weeks of age remained in the farrowing house, but only one from each pair of sows remained with them.
Bodyweights and meal intakes All piglets were weighed at birth and at 20, 28 and 35 days of age. It proved impossible to measure creep food intakes before weaning because of spillage and wastage, but individual daily meal intakes were recorded after weaning.
Xylose absorption tests All 70 piglets were subjected to xylose absorption tests at 20 and 28 days of age using the procedure described by Miller et al (1984a).
Collection offaeces It was difficult to collect individual faeces samples from pigs up to 21 days old, so pooled faeces from these unweaned piglets were collected from the floor of the farrowing pen. From three weeks fresh individual faeces samples were collected daily from the caged weaned pigs. It remained difficult to obtain individual faeces samples from the unweaned animals, so representative fresh samples were again removed daily from the farrowing-pen floor.
Faecal water content A subjective judgement was made as to whether a piglet had diarrhoea or not on anyone day. Where the samples were thought 'loose', a representative portion was weighed and then dried to constant weight in a hot-air oven; samples with a water content higher than 80 per cent were recorded as diarrhoea (Kenworthy and Allen 1966).
Bacteriology Fresh faeces samples were plated out on to split layer nutrient agar plates containing 5 per cent sheep red blood cells in the upper layer and were incubated
aerobically overnight at 37°C. The approximate percentage of haemolytic coliform colonies among the aerobic flora grown on the plate were recorded. Representative faeces samples were also plated out on to bile lactose agar plates, and representative colonies which were thought to be Escherichia coli had their identity confirmed using commercial API 20E tests. Representative haemolytic E coli colonies from weaned and unweaned pigs were subjected to restriction endonuclease analysis of their DNA using the technique of Marshall et al (1985) in an attempt to identify different BRENDA types. The O-types of these isolates were also determined using E coli 0 antisera (01 to 0166) as described by Bettelheim and Reeve (1982) and their ability to produce heat stable toxin using a suckling mouse assay (Giannella 1976) and heat labile toxin using Y-I adrenal cells (Bettelheim et al 1980) was also assessed.
Virology A commercial enzyme-linked immunosorbent assay (ELISA) system using antisera directed against human rotavirus (Dakopatts rotavirus ELISA kit; Dakopatts) was used to detect common antigens of group A rotaviruses in faeces samples. Representative positive and negative samples were also examined under the electron microscope to exclude the possibility of atypical rotavirus infection (Bridger 1980), and selected positive samples were subjected to electrophoresis using the technique of Herring et al (1982) to determine the electrophoretic mobility of the double stranded viral RNA.
Statistical analysis Data from each trial pertaining to plasma xylose concentrations, piglet bodyweights and food intakes between groups were subjected to one-way analysis of variance. Regression analysis was used to compare food intake over the two weeks after weaning between animals that developed diarrhoea and those that remained healthy. Initially a single regression line (dietary intake on days after weaning) was drawn for all the data and then the significance of the improvement in fit when separate lines were drawn for the two groupings was calculated. Other comparisons between pairs of groupings of pigs were made using Student's t test. Results Mean plasma xylose concentrations one hour after administration, piglet bodyweights at 20 and 28 days old and food intakes between 21 and 28 days old for the six groups ofpigs in the two trials are presented in Table I.
Dietary intake and porcine post-weaning diarrhoea
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In the first trial, the ability of the piglets to absorb xylose before weaning (20 days of age) was not influenced by creep feeding treatment but eight days later the four weaned groups (groups 2, 3, 5 and 6) all showed similar reductions in xylose absorption compared to the two unweaned groups (groups I and 4). Xylose absorption for the latter two groups did not differ significantly and, although absorption was somewhat lower at 28 than at 20 days of age, the difference was not significant. There were no significant differences between the groups in bodyweights at 20 days old and no check in bodyweight was seen in the weaned groups one week after weaning. Consumption of meal between 21 and 28 days old differed between groups, with 'abruptly' weaned pigs of group 5 consuming significantly less than pigs of the other three weaned groups. In the second trial plasma xylose concentrations were not significantly different between the groups at 20 days old, but were all similarly reduced in the four weaned groups at 28 days. At this time values for the two unweaned groups differed from each other, although each had not altered significantly from the 20-day value. Group bodyweights were not significantly different at 20 days old but by 28 days weaned pigs of groups 2, 3 and 6 showed significant growth checks compared with unweaned animals. Values for the abruptly weaned pigs (group 5) were not significantly different from any of the other groups. Mean dietary intakes by the weaned groups were similar over this first week after weaning but for groups 2, 3 and 6 meal intakes were less than in the first trial. The number of animals affected with diarrhoea, the number excreting haemolytic E coli as more than 50 per cent of the aerobic faecal flora and the number of pigs with rotavirus in their faeces at some point between 21 and 35 days old are recorded in Table 2, with the mean time of first appearance of these events and their duration. In neither trial did unweaned pigs (groups I and 4) suffer from diarrhoea or excrete rotavirus. A proportion of these unweaned animals did excrete haemolytic coliform organisms but BRENDA-typing and O-typing revealed that these orgaisms were of at least three different non-enterotoxigenic types, which were all different from the single type proliferating in the intestines of weaned littermates at this time. The haemolytic E coli seen in the weaned pigs had O-type 138 and produced heat labile enterotoxin, but not the subtype 'a' heat stable toxin. In the first trial diarrhoea occurred in most weaned pigs but was not seen in those animals that were abruptly weaned (group 5). Pigs from all the weaned groups excreted haemolytic enterotoxigenic E coli and rotavirus at some stage in the two weeks after weaning. In the second trial, diarrhoea was uncommon in the weaned pigs but again most
D. J. Hampson, W. C. Smith
66
TABLE 2: Number of pigs developing dierrhoea and excreting haemolytic E coli and, or, rotavirus in the period between three and five weeks old, and the mean first appearance and duration of these events in the six groups of pigs from both trials Trial 2
Trial 1 1 6
Group Number of animals Weaned or not Creep food or not
+
Piglets developing Number affected First appearance diarrhoea (days after weaning) Duration (days)
0
Piglets excreting haemolytic E coli
Piglets excreting rotavirus
p
E
Number excreting First appearance (50% faecal flora) Duration (days) Number excreting First appearance (days after weaning) Duration (days)
2 6
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3 6
4 6
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1 6
2 6
3 6
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6
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6·8 2·0
4
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6
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4
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4·8 8·2
6·8 5·0
3·0 1·0
8'5 4·2
5·8 6·4
2·8 7·8
4·8 2·0
4-4 3·2
0
5
5
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4
3
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6
6
11·3 1·4
11·3 2·0
4·0 4·0
4·0 3·7
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6·0 2·8
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3·6 4·0
3·3 4·3
Small 'priming' dose of creep feed Extra'creep food
excreted haemolytic enterotoxigenic E coli and rotavirus. Results for those weaned animals from both trials that developed diarrhoea are compared in Table 3 with results for the pigs that remained healthy. There were no significant differences between these groupings in their ability to absorb xylose one week after weaning, nor in their bodyweights at this time. Diarrhoeic and healthy animals began to excrete haemolytic enterotoxigenic E coli at a similar time after weaning but those animals that went on to develop diarrhoea shed these organisms in their faeces for a significantly longer period. Most animals also excreted rotavirus; in the first trial this occurred later than the excretion of haemolytic E coli and the appearance of diarrhoea, but in the second trial it preceded the appearance of the haemolytic E coli, and excretion persisted for longer than in the first trial. 'Atypical' rotaviruses were not detected by electron
microscopy, and the RNA segments of rotaviruses isolated from both trials had the same electrophoretic mobility. A comparison of the mean daily dietary intake during the two weeks after weaning of those animals that developed diarrhoea and those that remained healthy is presented in Fig I. The slopes of the regression lines fitted to daily intake in the diarrhoeic and healthy animals respectively were 51 . 6 g d -I and 31 '6 g d -I (zero intercepts). The overall data fitted these separate lines signi ficantly better (F640 = 125' 5; P
TABLE 3: Comparisons of mean (SO) xylose ebsorptions end excretion of heemolytic E coli and rotaviruses in weaned pigs either developing diarrhoea or not Animals developing diarrhoea Number of animals First appearance of diarrhoea (days after weaning) Duration of diarrhoea (days) Plasma xylose at 28 days (mmot lltre l) Number of pigs shedding haemolytic E coli First shedding of haemolytic E coli at more than 50% aerobic flora (days after weaning) Duration of shedding (days) Number of pigs shedding rotavirus First shedding of rotavirus (days after weaning) Duration of shedding (days) r
NS Not significant
Animals not developing diarrhoea
Significance in Student's t test
18 6·8 (1'0) 2·6 (1-4) 0·60 (0'21) 18
28 0·68 (0'15) 24
NS
6·05(1'76) 5·5 (2'5) 15 8·4 (4·4) 2·5 (1'2)
6·39 (2·04) 2·8 (2·0) 24 5·5 (3'5) 3·4 (1-6)
NS <0·001 <0·05 NS
Dietary intake and porcine post-weaning diarrhoea 700
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FIG 1: Mean daily dietary intake in the two weeks after weaning in 18 pigs that developed diarrhoea ( . - - . I and in 28 healthy littermates (e--el
(0'63),6'69 (0'90) and 7'39 (1'06) kg respectively, and for the animals that remained healthy were 5' 95 (0'95),6'11 (I.()() and 6·95 (1'07) kg respectively. At one and two weeks after weaning the animals that developed diarrhoea were on average about O' 5 kg heavier than those that remained healthy, but these differences were not statistically significant. Discussion Six groups of pigs were used in these experiments; two groups were left unweaned as controls, with or without access to creep food, while the other four groups were encouraged to consume varying amounts of creep food before weaning. According to the findings of Miller et al (1984a), group 6 animals which were 'primed' with small amounts of creep food should have had the most severe response following :-veaning, followed by group 2 pigs which were weaned In a conventional fashion, with the least severe reactions occurring in group 5 (abruptly weaned), and, or, in group 3 (receiving extra creep food before weaning), provided that the latter group ate sufficient meal before weaning to induce a state of immunological tolerance to dietary antigens. In the current experiments, however, although all the weaned groups had a significantly depressed capacity to a?sorb xylose one week after weaning, this capacity did not differ significantly between the weaned groups. The testing was done one day earlier than that described by Miller et al (1984a), but was thought to be close enough to detect the obvious effect described by these workers. The failure to detect group differences in the depression of xylose absorption in weaned pigs in the present study is difficult to explain if the theory of Intestinal hypersensitivity to dietary antigens at
67
weaning is valid. One explanation of the differences from the results of Miller et al (l984a) could be that the additional depressed absorption in their 'primed' pigs was caused by diarrhoea, which was mainly restricted to this group. The diarrhoea in turn may have been stimulated by some other factor, such as overconsumption by these animals. Unfortunately no details of dietary intake by the groups were recorded by these workers. This explanation is not complete, however, since in the current study xylose absorption was no more depressed in pigs that developed diarrhoea than in those that stayed healthy. Again, despite the greater incidence of diarrhoea in the first of the current trials and the earlier and more prolonged proliferation of rotavirus in the second trial, a similar depression in the capacity to absorb xylose was seen in all weaned groups in both trials. This may reflect the relative insensitivity of the test as a general indicator of absorptive capacity, as well as its limitation in indicating total xylose absorption, especially where only one plasma sample is taken after administration. Sampling one hour after administration does, however, appear to give a reasonable estimate of peak xylose absorption in weaned piglets (Hampson and Kidder 1986). Overall, a similar incidence of diarrhoea was seen in animals from all groups except those that were abruptly weaned (group 5). Comparing only paired cross-suckled litters, five out of II 'primed' pigs suffered diarrhoea, whereas none of II abruptly weaned littermates were affected; this is in general concordance with the suggestions of Miller et al (I 984a). The 'protective' effect of abrupt weaning seen here was also in agreement with field observations on a large commercial piggery (Miller et al 1984b). In the first trial of the current investigation, however, this protective effect on the development of diarrhoea was associated with a group meal intake in the first week after weaning which was significantly less than those of other groups containing animals which developed diarrhoea; these included 'primed' littermates in group 6. Diarrhoea was uncommon in the second trial and this was associated with generally lower intakes by animals from all groups after weaning than in the first trial. When the individual meal intakes of those animals that developed diarrhoea during the course of the study were compared with those of animals which remained healthy (Fig I), it was apparent that individuals that developed diarrhoea tended to eat significantly more than healthy pigs after weaning. Despite the occurrence of diarrhoea, the larger intakes by the animals that developed diarrhoea resulted in mean bodyweights after weaning that were greater, although not significantly so, than those of the healthy pigs. The view that overconsumption of the weaner diet can predispose pigs to diarrhoea after weaning is not
68
D. J. Hampson, W. C. Smith
new. Restricted access to the weaner diet, or the use of diets low in protein and, or, high in fibre have previously been shown to limit proliferation of haemolytic E coli and to reduce the incidence and effects of diarrhoea after weaning (Palmer and Hulland 1965, Smith and Halls 1968, Bertschinger et al 1979, Leece et al 1983). The pig may normally develop a 'malabsorption syndrome' after weaning (Kenworthy and Allen 1966) and this has been associated with alterations in small intestinal structure and brush-border enzyme activities (Hampson 1986, Hampson and Kidder 1986). In the face of reductions in the ability to digest and absorb nutrients, overconsumption in the immediate period after weaning may thus potentially result in undigested and unabsorbed dietary materials initiating an osmotic diarrhoea. In this study, the protective effect of abrupt weaning appeared to reside in its encouragement of smaller dietary intakes after weaning. Abrupt weaning did not result in any lesser loss of ability to absorb xylose at one week after weaning, and in previous studies (Hampson 1986, Hampson and Kidder 1986) did not alter the normal sequence of structural and enzymatic changes in the small intestine from that seen in conventionally weaned pigs. In the first trial, as in previous studies (Hampson et al 1985), rotaviruses tended to proliferate later after weaning than did haemolytic E coli and also after diarrhoea had resolved. In the second trial rotaviruses proliferated in most animals within a few days of weaning but diarrhoea was uncommon. These results cast doubt on the importance of rotaviruses in the aetiology of the diarrhoea observed after weaning. They do not support the view of Leece et al (1982) that rotavirus infection after weaning precedes and then favours colonisation of the intestines with enteropathogenic E coli. The difference in timing of the proliferation of rotavirus in the two trials was unexplained; it was not simply a litter effect, since four litters were involved in each trial. Contact between weaned caged pigs was limited to their immediate neighbours, so if the earlier and more prolonged proliferation in the second trial was due to degree of challenge with the virus, this would probably have had to be airborne. Virtually all weaned animals shed a single common serotype of haemolytic enterotoxigenic E coli after weaning. There was no significant difference in the onset of this shedding between animals that developed diarrhoea and those that remained healthy. The proliferation after weaning of a single pathogenic serotype in pigs from different litters, transferred three months apart to new, cleaned and rested accommodation, suggested that these organisms were present undetected in low numbers in the intestinal
tracts of all the piglets and that they specifically proliferated after weaning. This proliferation was probably facilitated by withdrawal of milk-derived specific antibody at weaning. On average, diarrhoea started shortly after haemolytic E coli were first detected at a level of more than 50 per cent of the cultured aerobic faecal flora (around the sixth day after weaning). The significance of the timing of this appearance was questionable, however, because these organisms also started to proliferate in healthy animals at this time. The shedding of these organisms by animals that developed diarrhoea was significantly more prolonged than in animals that remained healthy. Interestingly, in diarrhoeic animals shedding continued at this level for an average of 5· 5 days, whereas the diarrhoea resolved after only 2· 6 days. These observations suggest a greater intestinal proliferation of the organism in diarrhoeic than in healthy animals. The greater proliferation may itself have been stimulated by the development of diarrhoea, which could for example have flushed out competing microorganisms. This could explain why excretion continued on average for three days after diarrhoea had resolved, since it would probably take some time for the normal microflora to re-establish itself. Alternatively, the greater dietary intake of the pigs that developed diarrhoea may itself have stimulated a greater proliferation of haemolytic E coli. Overall, in healthy and diarrhoeic animals, a positive linear relationship did appear to exist between dietary intake in the first week after weaning and duration of excretion of haemolytic E coli, the correlation coefficient between them (r = O' 60) being highly significant (t=4'93; P
Dietary intake and porcine post-weaning diarrhoea Acknowledgements This work was supported by the Massey University Veterinary Research Fund and the New Zealand Pork Industry Board. Thanks are due to Dr K. A. Bettelheim, National Health Institute, Porirua, New Zealand for O-typing coliform colonies and for enterotoxin assays, and to Dr R. B. Marshall and Mr P. J. Winter, Massey University for BRENDA typing E coli isolates. Mrs L. Denby provided technical assistance and Mr K. Best was responsible for the care of the animals on experiment. References BERTSCH INGER, H. U., EGGENBERGER, E., JUCKER, H. & PFIRTER, H. P. (1979) Veterinary Microbiology 3, 281-290 BETTELHEIM, K. A. & REEVE, K. G. (1982) New Zealand Medical Journal 95, 215-216 BETTELHEIM, K. A., WILSON, M. W., SHOOTER, R. A. & O'FARRELL, S. M. (1980) Journal of Hygiene 84,411-414 BRIDGER, J. C. (1980) Veterinary Record 107, 532-533 GIANNELLA, R. A. (1976) Infection and Immunity 14, 95-99 HAMPSON, D. J. (1986) Research in Veterinary Science 40,
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HAMPSON, D. J., HINTON, M. & KIDDER, D. E. (1985) Journal of Comparative Pathology 95,353-362 HAMPSON, D. J. & KIDDER, D. E. (1986) Research in Veterinary Science 40, 24- 31 HERRING, A. J., INGLIS, N. F., OJEH, C. K., SNODGRASS, D. R. & MENZIES, J. D. (1982) Journal of Clinical Microbiology 16,473-477 KENWORTHY, R. & ALLEN, W. D. (1%6) Journal of Comparative Pathology 76, 31-44 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 MARSHALL, R. B., WINTER, P. J., ROBINSON, A. J. & BETTELHEIM, K. A. (1985) Journal of Hygiene 94,263-268 MILLER, B. G., NEWBY, T. J., STOKES, C. R. & BOURNE, F. J. (1984a) Research in Veterinary Science 36, 187-193 MILLER, B. G., NEWBY, T. J., STOKES, C. R. & BOURNE, F. J. (1984b) Veterinary Record 114, 296-297 PALMER, N. C. & HULLAND, T. J. (1965) Canadian Veterinary Journal 6, 310-316 SMITH, H. W. & HALLS, S. (1968) Journal of Medical Microbiology 1,45-59
Accepted July 25, 1985
.Influence of creep feeding and dietary intake after weaning on malabsorption and occurrence of diarrhoea in the newly weaned pig D. J. HAMPSON, Department of Veterinary Pathology and Public Health, W. C. SMITH, Department of Animal Science, Massey University, Palmerston North, New Zealand
ships between the pattern of creep feeding, dietary intake after weaning, the proliferation of enteropathogens in the intestine and the occurrence of diarrhoea in the newly weaned pig.
The influence the pattern of creep feeding has on the ability of pigs to absorb xylose after weaning at three weeks old was investigated. All weaned pigs showed a reduction in their ability to absorb xylose one week after weaning, but this was not influenced by the pattern of creep feeding before weaning. Diarrhoea occurred in some animals after weaning, but did not occur in pigs which did not have access to creep food before weaning. This protective effect of withholding creep food was associated with a low dietary intake after weaning with this regimen. Pigs which developed diarrhoea tended to be those which consumed more meal after weaning than their contemporaries. Haemolytic enterotoxigenic Escherichia coli and rotaviruses were present in the faeces of most pigs after weaning, but, in those animals that ate too much and developed diarrhoea, excretion of the E coli continued for approximately twice as long as in animals that remained healthy.
Materials and methods
Animals Seventy crossbred piglets from five White-cross sows served by Hampshire boars, and three similar sows mated to Landrace boars, were obtained from the Pig Research Centre, Massey University. Two batches of 35 piglets, each divided into six groups, were used in separate but otherwise identical trials conducted three months apart. In each trial piglets of groups I, 2 and 3 were selected randomly from the progeny of two sows whose litters were allowed to mix from three days old; piglets of groups 4, 5 and 6 were selected from the litters of a further two similarly managed sows.
IT has been reported that diarrhoea following weaning in the pig may result from a transient intestinal hypersensitivity to antigenic components of the weaning diet (Miller et al 1984a). These authors demonstrated that piglets given a small amount of creep food as a 'priming' dose before weaning went on to show a significantly greater depression in their ability to absorb xylose eight days after weaning and suffered more diarrhoea than did other weaned pigs. It was suggested that while consumption of small amounts of creep food 'primes' hypersensitivity, consumption of large amounts before weaning causes the animal to become tolerant to the food antigens it meets later. Subsequently, in a commercial piggery, these authors (Miller et al 1984b) demonstrated that withholding creep food before three week weaning had a protective effect against diarrhoea after weaning; they claimed that this 'abrupt' weaning was beneficial because it limited intestinal hypersensitivity responses to the weaner diet. The purpose of the present investigation was to attempt to confirm these potentially important observations and to define more closely the relation-
Treatments and diets Piglets of group I were offered a commercial creep meal (19 per cent protein, no antibiotics) ad libitum from three days old and were left unweaned. Group 2 piglets received the same creep diet while with the sow and were weaned on to this diet at three weeks old. The animals of group 3 were treated as those of group 2, except that between 10 days old and weaning they were removed from the sows daily for four hours. During this period they were confined in a small heated pen where they were offered and encouraged to consume additional quantities of the creep food. Piglets of groups 4 and 5 were not offered creep food before weaning. Group 4 piglets were left unweaned, while group 5 animals were weaned on to the creep diet when three weeks old. The piglets of group 6 differed from those of group 5 only in that at eight, nine and 10 days old they were each force-fed with 10 g of the creep food; they were then not given any further access to creep food until they were weaned on to it at three weeks old. 63
64
D. J. Hampson. W. C. Smith
Accommodation after weaning In each trial 23 piglets were weaned simultaneously at three weeks old. They were individually penned in random order in a battery of single tier wire mesh cages with polypropylene slatted flooring. The cages were housed in a controlled-temperature room maintained at 28°C with lighting kept at a low intensity. The creep diet and fresh water were available ad libitum. Before and between trials the room and cages were thoroughly cleaned and disinfected and then rested. The piglets that were left unweaned at three weeks of age remained in the farrowing house, but only one from each pair of sows remained with them.
Bodyweights and meal intakes All piglets were weighed at birth and at 20, 28 and 35 days of age. It proved impossible to measure creep food intakes before weaning because of spillage and wastage, but individual daily meal intakes were recorded after weaning.
Xylose absorption tests All 70 piglets were subjected to xylose absorption tests at 20 and 28 days of age using the procedure described by Miller et al (1984a).
Collection offaeces It was difficult to collect individual faeces samples from pigs up to 21 days old, so pooled faeces from these unweaned piglets were collected from the floor of the farrowing pen. From three weeks fresh individual faeces samples were collected daily from the caged weaned pigs. It remained difficult to obtain individual faeces samples from the unweaned animals, so representative fresh samples were again removed daily from the farrowing-pen floor.
Faecal water content A subjective judgement was made as to whether a piglet had diarrhoea or not on anyone day. Where the samples were thought 'loose', a representative portion was weighed and then dried to constant weight in a hot-air oven; samples with a water content higher than 80 per cent were recorded as diarrhoea (Kenworthy and Allen 1966).
Bacteriology Fresh faeces samples were plated out on to split layer nutrient agar plates containing 5 per cent sheep red blood cells in the upper layer and were incubated
aerobically overnight at 37°C. The approximate percentage of haemolytic coliform colonies among the aerobic flora grown on the plate were recorded. Representative faeces samples were also plated out on to bile lactose agar plates, and representative colonies which were thought to be Escherichia coli had their identity confirmed using commercial API 20E tests. Representative haemolytic E coli colonies from weaned and unweaned pigs were subjected to restriction endonuclease analysis of their DNA using the technique of Marshall et al (1985) in an attempt to identify different BRENDA types. The O-types of these isolates were also determined using E coli 0 antisera (01 to 0166) as described by Bettelheim and Reeve (1982) and their ability to produce heat stable toxin using a suckling mouse assay (Giannella 1976) and heat labile toxin using Y-I adrenal cells (Bettelheim et al 1980) was also assessed.
Virology A commercial enzyme-linked immunosorbent assay (ELISA) system using antisera directed against human rotavirus (Dakopatts rotavirus ELISA kit; Dakopatts) was used to detect common antigens of group A rotaviruses in faeces samples. Representative positive and negative samples were also examined under the electron microscope to exclude the possibility of atypical rotavirus infection (Bridger 1980), and selected positive samples were subjected to electrophoresis using the technique of Herring et al (1982) to determine the electrophoretic mobility of the double stranded viral RNA.
Statistical analysis Data from each trial pertaining to plasma xylose concentrations, piglet bodyweights and food intakes between groups were subjected to one-way analysis of variance. Regression analysis was used to compare food intake over the two weeks after weaning between animals that developed diarrhoea and those that remained healthy. Initially a single regression line (dietary intake on days after weaning) was drawn for all the data and then the significance of the improvement in fit when separate lines were drawn for the two groupings was calculated. Other comparisons between pairs of groupings of pigs were made using Student's t test. Results Mean plasma xylose concentrations one hour after administration, piglet bodyweights at 20 and 28 days old and food intakes between 21 and 28 days old for the six groups ofpigs in the two trials are presented in Table I.
Dietary intake and porcine post-weaning diarrhoea
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In the first trial, the ability of the piglets to absorb xylose before weaning (20 days of age) was not influenced by creep feeding treatment but eight days later the four weaned groups (groups 2, 3, 5 and 6) all showed similar reductions in xylose absorption compared to the two unweaned groups (groups I and 4). Xylose absorption for the latter two groups did not differ significantly and, although absorption was somewhat lower at 28 than at 20 days of age, the difference was not significant. There were no significant differences between the groups in bodyweights at 20 days old and no check in bodyweight was seen in the weaned groups one week after weaning. Consumption of meal between 21 and 28 days old differed between groups, with 'abruptly' weaned pigs of group 5 consuming significantly less than pigs of the other three weaned groups. In the second trial plasma xylose concentrations were not significantly different between the groups at 20 days old, but were all similarly reduced in the four weaned groups at 28 days. At this time values for the two unweaned groups differed from each other, although each had not altered significantly from the 20-day value. Group bodyweights were not significantly different at 20 days old but by 28 days weaned pigs of groups 2, 3 and 6 showed significant growth checks compared with unweaned animals. Values for the abruptly weaned pigs (group 5) were not significantly different from any of the other groups. Mean dietary intakes by the weaned groups were similar over this first week after weaning but for groups 2, 3 and 6 meal intakes were less than in the first trial. The number of animals affected with diarrhoea, the number excreting haemolytic E coli as more than 50 per cent of the aerobic faecal flora and the number of pigs with rotavirus in their faeces at some point between 21 and 35 days old are recorded in Table 2, with the mean time of first appearance of these events and their duration. In neither trial did unweaned pigs (groups I and 4) suffer from diarrhoea or excrete rotavirus. A proportion of these unweaned animals did excrete haemolytic coliform organisms but BRENDA-typing and O-typing revealed that these orgaisms were of at least three different non-enterotoxigenic types, which were all different from the single type proliferating in the intestines of weaned littermates at this time. The haemolytic E coli seen in the weaned pigs had O-type 138 and produced heat labile enterotoxin, but not the subtype 'a' heat stable toxin. In the first trial diarrhoea occurred in most weaned pigs but was not seen in those animals that were abruptly weaned (group 5). Pigs from all the weaned groups excreted haemolytic enterotoxigenic E coli and rotavirus at some stage in the two weeks after weaning. In the second trial, diarrhoea was uncommon in the weaned pigs but again most
D. J. Hampson, W. C. Smith
66
TABLE 2: Number of pigs developing dierrhoea and excreting haemolytic E coli and, or, rotavirus in the period between three and five weeks old, and the mean first appearance and duration of these events in the six groups of pigs from both trials Trial 2
Trial 1 1 6
Group Number of animals Weaned or not Creep food or not
+
Piglets developing Number affected First appearance diarrhoea (days after weaning) Duration (days)
0
Piglets excreting haemolytic E coli
Piglets excreting rotavirus
p
E
Number excreting First appearance (50% faecal flora) Duration (days) Number excreting First appearance (days after weaning) Duration (days)
2 6
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4 6
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1 6
2 6
3 6
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0
3
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6
6·3 3·0
6·8 2·0
4
6
6
2
6
5
4
4
5
0·8 3·2
4·8 8·2
6·8 5·0
3·0 1·0
8'5 4·2
5·8 6·4
2·8 7·8
4·8 2·0
4-4 3·2
0
5
5
0
4
3
0
6
6
11·3 1·4
11·3 2·0
4·0 4·0
4·0 3·7
10-4 2·6
7-7 1·3
4 6
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6
7·8 2·2
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5 6
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5 5
6 6
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2 8·0 2·0
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6
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6·0 2·8
5
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3·6 4·0
3·3 4·3
Small 'priming' dose of creep feed Extra'creep food
excreted haemolytic enterotoxigenic E coli and rotavirus. Results for those weaned animals from both trials that developed diarrhoea are compared in Table 3 with results for the pigs that remained healthy. There were no significant differences between these groupings in their ability to absorb xylose one week after weaning, nor in their bodyweights at this time. Diarrhoeic and healthy animals began to excrete haemolytic enterotoxigenic E coli at a similar time after weaning but those animals that went on to develop diarrhoea shed these organisms in their faeces for a significantly longer period. Most animals also excreted rotavirus; in the first trial this occurred later than the excretion of haemolytic E coli and the appearance of diarrhoea, but in the second trial it preceded the appearance of the haemolytic E coli, and excretion persisted for longer than in the first trial. 'Atypical' rotaviruses were not detected by electron
microscopy, and the RNA segments of rotaviruses isolated from both trials had the same electrophoretic mobility. A comparison of the mean daily dietary intake during the two weeks after weaning of those animals that developed diarrhoea and those that remained healthy is presented in Fig I. The slopes of the regression lines fitted to daily intake in the diarrhoeic and healthy animals respectively were 51 . 6 g d -I and 31 '6 g d -I (zero intercepts). The overall data fitted these separate lines signi ficantly better (F640 = 125' 5; P
TABLE 3: Comparisons of mean (SO) xylose ebsorptions end excretion of heemolytic E coli and rotaviruses in weaned pigs either developing diarrhoea or not Animals developing diarrhoea Number of animals First appearance of diarrhoea (days after weaning) Duration of diarrhoea (days) Plasma xylose at 28 days (mmot lltre l) Number of pigs shedding haemolytic E coli First shedding of haemolytic E coli at more than 50% aerobic flora (days after weaning) Duration of shedding (days) Number of pigs shedding rotavirus First shedding of rotavirus (days after weaning) Duration of shedding (days) r
NS Not significant
Animals not developing diarrhoea
Significance in Student's t test
18 6·8 (1'0) 2·6 (1-4) 0·60 (0'21) 18
28 0·68 (0'15) 24
NS
6·05(1'76) 5·5 (2'5) 15 8·4 (4·4) 2·5 (1'2)
6·39 (2·04) 2·8 (2·0) 24 5·5 (3'5) 3·4 (1-6)
NS <0·001 <0·05 NS
Dietary intake and porcine post-weaning diarrhoea 700
§ Q)
500
"'B"
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~
B
.s Q
300
100 2
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6 8 10 Days after weaning
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FIG 1: Mean daily dietary intake in the two weeks after weaning in 18 pigs that developed diarrhoea ( . - - . I and in 28 healthy littermates (e--el
(0'63),6'69 (0'90) and 7'39 (1'06) kg respectively, and for the animals that remained healthy were 5' 95 (0'95),6'11 (I.()() and 6·95 (1'07) kg respectively. At one and two weeks after weaning the animals that developed diarrhoea were on average about O' 5 kg heavier than those that remained healthy, but these differences were not statistically significant. Discussion Six groups of pigs were used in these experiments; two groups were left unweaned as controls, with or without access to creep food, while the other four groups were encouraged to consume varying amounts of creep food before weaning. According to the findings of Miller et al (1984a), group 6 animals which were 'primed' with small amounts of creep food should have had the most severe response following :-veaning, followed by group 2 pigs which were weaned In a conventional fashion, with the least severe reactions occurring in group 5 (abruptly weaned), and, or, in group 3 (receiving extra creep food before weaning), provided that the latter group ate sufficient meal before weaning to induce a state of immunological tolerance to dietary antigens. In the current experiments, however, although all the weaned groups had a significantly depressed capacity to a?sorb xylose one week after weaning, this capacity did not differ significantly between the weaned groups. The testing was done one day earlier than that described by Miller et al (1984a), but was thought to be close enough to detect the obvious effect described by these workers. The failure to detect group differences in the depression of xylose absorption in weaned pigs in the present study is difficult to explain if the theory of Intestinal hypersensitivity to dietary antigens at
67
weaning is valid. One explanation of the differences from the results of Miller et al (l984a) could be that the additional depressed absorption in their 'primed' pigs was caused by diarrhoea, which was mainly restricted to this group. The diarrhoea in turn may have been stimulated by some other factor, such as overconsumption by these animals. Unfortunately no details of dietary intake by the groups were recorded by these workers. This explanation is not complete, however, since in the current study xylose absorption was no more depressed in pigs that developed diarrhoea than in those that stayed healthy. Again, despite the greater incidence of diarrhoea in the first of the current trials and the earlier and more prolonged proliferation of rotavirus in the second trial, a similar depression in the capacity to absorb xylose was seen in all weaned groups in both trials. This may reflect the relative insensitivity of the test as a general indicator of absorptive capacity, as well as its limitation in indicating total xylose absorption, especially where only one plasma sample is taken after administration. Sampling one hour after administration does, however, appear to give a reasonable estimate of peak xylose absorption in weaned piglets (Hampson and Kidder 1986). Overall, a similar incidence of diarrhoea was seen in animals from all groups except those that were abruptly weaned (group 5). Comparing only paired cross-suckled litters, five out of II 'primed' pigs suffered diarrhoea, whereas none of II abruptly weaned littermates were affected; this is in general concordance with the suggestions of Miller et al (I 984a). The 'protective' effect of abrupt weaning seen here was also in agreement with field observations on a large commercial piggery (Miller et al 1984b). In the first trial of the current investigation, however, this protective effect on the development of diarrhoea was associated with a group meal intake in the first week after weaning which was significantly less than those of other groups containing animals which developed diarrhoea; these included 'primed' littermates in group 6. Diarrhoea was uncommon in the second trial and this was associated with generally lower intakes by animals from all groups after weaning than in the first trial. When the individual meal intakes of those animals that developed diarrhoea during the course of the study were compared with those of animals which remained healthy (Fig I), it was apparent that individuals that developed diarrhoea tended to eat significantly more than healthy pigs after weaning. Despite the occurrence of diarrhoea, the larger intakes by the animals that developed diarrhoea resulted in mean bodyweights after weaning that were greater, although not significantly so, than those of the healthy pigs. The view that overconsumption of the weaner diet can predispose pigs to diarrhoea after weaning is not
68
D. J. Hampson, W. C. Smith
new. Restricted access to the weaner diet, or the use of diets low in protein and, or, high in fibre have previously been shown to limit proliferation of haemolytic E coli and to reduce the incidence and effects of diarrhoea after weaning (Palmer and Hulland 1965, Smith and Halls 1968, Bertschinger et al 1979, Leece et al 1983). The pig may normally develop a 'malabsorption syndrome' after weaning (Kenworthy and Allen 1966) and this has been associated with alterations in small intestinal structure and brush-border enzyme activities (Hampson 1986, Hampson and Kidder 1986). In the face of reductions in the ability to digest and absorb nutrients, overconsumption in the immediate period after weaning may thus potentially result in undigested and unabsorbed dietary materials initiating an osmotic diarrhoea. In this study, the protective effect of abrupt weaning appeared to reside in its encouragement of smaller dietary intakes after weaning. Abrupt weaning did not result in any lesser loss of ability to absorb xylose at one week after weaning, and in previous studies (Hampson 1986, Hampson and Kidder 1986) did not alter the normal sequence of structural and enzymatic changes in the small intestine from that seen in conventionally weaned pigs. In the first trial, as in previous studies (Hampson et al 1985), rotaviruses tended to proliferate later after weaning than did haemolytic E coli and also after diarrhoea had resolved. In the second trial rotaviruses proliferated in most animals within a few days of weaning but diarrhoea was uncommon. These results cast doubt on the importance of rotaviruses in the aetiology of the diarrhoea observed after weaning. They do not support the view of Leece et al (1982) that rotavirus infection after weaning precedes and then favours colonisation of the intestines with enteropathogenic E coli. The difference in timing of the proliferation of rotavirus in the two trials was unexplained; it was not simply a litter effect, since four litters were involved in each trial. Contact between weaned caged pigs was limited to their immediate neighbours, so if the earlier and more prolonged proliferation in the second trial was due to degree of challenge with the virus, this would probably have had to be airborne. Virtually all weaned animals shed a single common serotype of haemolytic enterotoxigenic E coli after weaning. There was no significant difference in the onset of this shedding between animals that developed diarrhoea and those that remained healthy. The proliferation after weaning of a single pathogenic serotype in pigs from different litters, transferred three months apart to new, cleaned and rested accommodation, suggested that these organisms were present undetected in low numbers in the intestinal
tracts of all the piglets and that they specifically proliferated after weaning. This proliferation was probably facilitated by withdrawal of milk-derived specific antibody at weaning. On average, diarrhoea started shortly after haemolytic E coli were first detected at a level of more than 50 per cent of the cultured aerobic faecal flora (around the sixth day after weaning). The significance of the timing of this appearance was questionable, however, because these organisms also started to proliferate in healthy animals at this time. The shedding of these organisms by animals that developed diarrhoea was significantly more prolonged than in animals that remained healthy. Interestingly, in diarrhoeic animals shedding continued at this level for an average of 5· 5 days, whereas the diarrhoea resolved after only 2· 6 days. These observations suggest a greater intestinal proliferation of the organism in diarrhoeic than in healthy animals. The greater proliferation may itself have been stimulated by the development of diarrhoea, which could for example have flushed out competing microorganisms. This could explain why excretion continued on average for three days after diarrhoea had resolved, since it would probably take some time for the normal microflora to re-establish itself. Alternatively, the greater dietary intake of the pigs that developed diarrhoea may itself have stimulated a greater proliferation of haemolytic E coli. Overall, in healthy and diarrhoeic animals, a positive linear relationship did appear to exist between dietary intake in the first week after weaning and duration of excretion of haemolytic E coli, the correlation coefficient between them (r = O' 60) being highly significant (t=4'93; P
Dietary intake and porcine post-weaning diarrhoea Acknowledgements This work was supported by the Massey University Veterinary Research Fund and the New Zealand Pork Industry Board. Thanks are due to Dr K. A. Bettelheim, National Health Institute, Porirua, New Zealand for O-typing coliform colonies and for enterotoxin assays, and to Dr R. B. Marshall and Mr P. J. Winter, Massey University for BRENDA typing E coli isolates. Mrs L. Denby provided technical assistance and Mr K. Best was responsible for the care of the animals on experiment. References BERTSCH INGER, H. U., EGGENBERGER, E., JUCKER, H. & PFIRTER, H. P. (1979) Veterinary Microbiology 3, 281-290 BETTELHEIM, K. A. & REEVE, K. G. (1982) New Zealand Medical Journal 95, 215-216 BETTELHEIM, K. A., WILSON, M. W., SHOOTER, R. A. & O'FARRELL, S. M. (1980) Journal of Hygiene 84,411-414 BRIDGER, J. C. (1980) Veterinary Record 107, 532-533 GIANNELLA, R. A. (1976) Infection and Immunity 14, 95-99 HAMPSON, D. J. (1986) Research in Veterinary Science 40,
69
32-40
HAMPSON, D. J., HINTON, M. & KIDDER, D. E. (1985) Journal of Comparative Pathology 95,353-362 HAMPSON, D. J. & KIDDER, D. E. (1986) Research in Veterinary Science 40, 24- 31 HERRING, A. J., INGLIS, N. F., OJEH, C. K., SNODGRASS, D. R. & MENZIES, J. D. (1982) Journal of Clinical Microbiology 16,473-477 KENWORTHY, R. & ALLEN, W. D. (1%6) Journal of Comparative Pathology 76, 31-44 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 MARSHALL, R. B., WINTER, P. J., ROBINSON, A. J. & BETTELHEIM, K. A. (1985) Journal of Hygiene 94,263-268 MILLER, B. G., NEWBY, T. J., STOKES, C. R. & BOURNE, F. J. (1984a) Research in Veterinary Science 36, 187-193 MILLER, B. G., NEWBY, T. J., STOKES, C. R. & BOURNE, F. J. (1984b) Veterinary Record 114, 296-297 PALMER, N. C. & HULLAND, T. J. (1965) Canadian Veterinary Journal 6, 310-316 SMITH, H. W. & HALLS, S. (1968) Journal of Medical Microbiology 1,45-59
Accepted July 25, 1985