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The immune response to dietary antigens and its influence on disease susceptibility in farm animals
Veterinary Immunology and Immunopathology, 17 (1987) 413-423 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
413
THE IMMUNE RESPONSE TO DIETARY ANTIGENS AND ITS INFLUENCE ON DISEASE SUSCEPTIBILITY IN FARMANIMALS
C.R. STOKES, B.G. MILLER, M. BAILEY, A.D. WILSON and F.J. BOURNE University of Bristol, Department of Veterinary Medicine, Langford House, Langford, Bristol, BS18 7DU, U.K.
ABSTRACT Stokes, C.R., Miller, B.G., Bailey, M., Wilson, A.D. and Bourne, F.J., 1987. The immune response to dietary antigens and i t s influence on disease susceptibility in farm animals. Vet. Immunol. Immunopathol., 17: 413-423. Transient hypersensitivity reactions of the intestinal immune system to dietary antigens result in increases in enterocyte turnover and villous atrophy. These changes occur in the intestine of the post weaned piglet and precede the p r o l i f e r a t i o n of E. coli and the development of post-weaning diarrhea. We therefore postula~ed-t'h-~-t a transient cell mediated immune response to dietary antigens may increase susceptibility to disease. The interaction of dietary and microbial antigens upon the gut immune system has been investigated in mice and pigs and i t has been shown that both exert powerful regulatory effects upon each other. INTRODUCTION The immune system of the gastrointestinal tract is presented with a vast array of different antigens ranging from potential pathogens to dietary antigens; which are in themselves harmless.
The nature and magnitude of the
responses they e l i c i t differ markedly, for on the one hand an individual must respond with vigour to potential pathogens, whilst on the other overreaction to harmless dietary antigens would be both wasteful and potentially harmful i f i t resulted in hypersensitivity reactions.
I t is however increasingly clear that
the simultaneous presentation of dietary and microbial antigens can influence the animal's a b i l i t y to respond to either antigen and i t interaction that this paper is in part directed.
is to this
Following the introduction of a new dietary protein antigen a small proportion (<0.002%) of that protein is absorbed intact (Swarbrick, Stokes and S o o t h i l l , 1979) and a serum antibody response may be stimulated (Lippard, Schloss & Johnson, 1936).
Whilst
the systemic antibody
response is
subsequently suppressed (Thomas and Parrott, 1984) a local mucosal antibody response persists a~d this is associated with the development of a specific a b i l i t y to reduce the subsequent absorption of antigen (immune exclusion), (Swarbick etal., 1979). 0165-2427/87/$03.50
The f a l l in serum antibody is associated with the
© 1987 Elsevier Science Publishers B.V.
414
development of oral
tolerance, a specifically acquired capacity to prevent
responses to any antigen that does gain entry.
Delayed hypersensitivity and
i t s subsequent suppression have also been described in mice following feeding of contact sensitizing agents (Asherson et a l . , 1977), erythrocytes (Kagnoff, 1978) and soluble proteins
(Stokes et a l . ,
1983).
It
is therefore well
established that following the introduction of a new dietary antigen, animals pass through a brief phase of s e n s i t i v i t y prior to the development of the protected transient
state of tolerance. period of
We have investigated the influence of t h i s
s e n s i t i v i t y to dietary antigens upon enteric disease
s u s c e p t i b i l i t y in post weaned pigs. MATERIALS AND METHODS Animals Mice:
CBA male mice (LAC grade 3) aged 6-8 weeks were obtained from OLAC
Ltd., Oxford. Pigs:
Each experimental group contained eight mice.
Pregnant g i l t s
S h i f n a l l , Shropshire).
were obtained from a PHCA herd (Brockton Court,
Forty eight hours following birth all piglets received
injections of iron and t h e i r teeth were clipped.
In all cases, except where
indicated otherwise, weaning was performed abruptly at three weeks of age. Diets Mice were fed on commercial diets throughout. Pigs were weaned onto a 22% protein diet which contained a f u l l fat soy as the sole protein source.
The soy (Trusoy - British Soya Products) was a heat
treated f l o u r , free of a n t i - n u t r i t i o n a l factors. Xylose absorption Mice were given a single feeding of 10 mg xylose (0.2 ml) and were bled 30 minutes later.
Piglets were given orally a 10% xylose solution (I ml/kg - I ) and
the xylose concentration in serum measured one hour later (Trinder, 1975).
Antibody assay Serum IgG antibody to soya was measured by the microtitre ELISA method (Voller et a l . ,
1976) using plates coated with an extract of soya flour (10
ug/well) and a sheep anti-serum specific for the heavy chain of pig IgG. Radioimmunoassay for soya Absorbed soya in pig serum was measured by a l i q u i d phase, double antibody radioimmunoassay (Swarbrick et a l . , 1979) using a rabbit antiserum to the soy protein conglycinin.
415 Fed SRBC
0
5
I.V. SRBC
0 +
5 +
14 +
lO¢ ,'T'
]!
E u
'~
J~
~
8(:
I
4(]
~o
i
o
Figure 1.
Increase in mouse skin thickness 24 hours after challenge in animals previously given 30 ul (2 x 103) sheep erythrocytes (SRBC) for O, 5 or 14 days. Three groups of mice also received a sensitizing dose of SRBC following feed. Each point represents the mean of eight animals + ISD.
EXPERIMENTS AND RESULTS A. 1.
Mice fed sheep red blood cells ISRBC) Effect of oral
immunization on the induction of specific delayed type
hypersensitivity Groups of eight CBA male mice were orally immunized by infusing on each of five days 2 x 109 SRBC by stomach tube.
Five days later they were given a
subcutaneous injection of 2 x 108 SRBC into the l e f t hind f o o t pad, and the increase in foot pad thickness was measured after 24 hours. that feeding SRBC for five days sensitized (Figure 1).
The results showed
In a further experiment
mice were fed SRBC for five or 14 days and one week later they were given an intravenous injection of I x 105 SRBC. After a further five days they were footpad challenged by footpad injection as described above, and the results compared with non-SRBC fed controls.
Whilst feeding for five days had no
effect on the a b i l i t y to sensitize by the intravenous route, feeding for 14 days completely suppressed the response. These experiments demonstrate that following the introduction of SRBC into the d i e t precedes the development of tolerance. 2.
a phase of s e n s i t i v i t y
The effect of adjuvant feeding upon oral tolerance induction Mice were fed SRBCfor eight days. One group was also fed dextran sulphate
(m.w. 500,000)
at a concentration of 0.1 mg.ml-l.
The effect of feeding
416
Fed SRBC 0 Fed Dx.SO4 too _
E o
8
8
_
+
[
._~
Figure 2.
N
6O
-~
40
Increase in skin thickness 24 hours following foot pad challenge, in mice fed sheep erythrocytes (SRBC) for 0 or 8 days. One group also received dextran sulphate (Dx.S04) in the ~rinking water.
dextran sulphate was to turn a sensitizing dose of SRBC into a t o l e r i z i n g dose (Figure 2). 3.
Gastrointestinal effects Groups of eight male CBA mice were sensitized by feeding 3 x 109 SRBC daily
for two days.
They were then challenged 10 days later on two consecutive days
by feeding SRBC. Their a b i l i t y to absorb xylose was measured one day later and compared with unsensitized mice, tolerized mice ( i . e . those previously fed SRBC for 14 days) and those sensitized with the non-cross reacting antigen horse red blood cells (HRBC). Mice sensitized and challenged with SRBC absorbed s i g n i f i c a n t l y less xylose than did nonsensitized mice, tolerized mice and those sensitized with HRBC. The malabsorption was associated with increases in the crypt cell production rate
(4.2 to
11.7 d i v i s i o n s / h r ) and in the numbers of
intraepithelial
lymphocytes (16 to 23 IEL's/IO0 epithelial c e l l s ) . B.
Pigs fed soy containing diets
1.
Changes following weaning Groups of piglets were weaned abruptly at three weeks of age onto diets
containing soya as the sole protein source.
Separate groups were then skin
tested by intradermal injection into the skin of the ear of an extract of soya and the increase in ear thickness at 24 hours measured.
A transient delayed
hypersensitivity was observed five days after weaning which had disappeared by
417 +4+
PRIMARY
O
SECONDARY
O
0
SRBC HRBC SRBC
SRBC SRBC SRBC SRBC
31111
-z
280
~
•
J
260 -
-~ ¢=.
24o
220
2OO
Figure 3.
Plasmaxylose levels 30 minutes after a single feed of 10 mg xylose. Mice had been previously sensitized by feeding SRBC for 2 days, followed 10 days later by challenge on two consecutive days with sheep erythrocytes (SRBC). Xylose tests were performed the following day and compared with those in unsensitized mice, orally tolerized mice (+++) and those fed horse erythrocytes (HRBC). Each point represents themean of eight mice + ISD.
day 13 (Figure 4). for
Biopsies of skin test sites taken five days after weaning
histological
examination were compatible with
a delayed-type
hyper~eqsitivity reaction. The p i g l e t s '
a b i l i t y to absorb xylose from a single feeding was also
measured in the same group of animals.
The results are shown in Figure 5.
Five days after the introduction of soy at weaning, the piglets' a b i l i t y to absorb xylose was s i g n i f i c a n t l y reduced, but by day 13, t h i s t r a n s i e n t deficiency had been restored to preweaning levels. Histological examination of the intestine of these piglets showed that by two days post weaning there were significant increases in crypt depths and villous atrophy. Whilst subsequently there was a degree of recovery they never returned to preweaning levels. By both l i g h t and scanning electron microscopy,
418
200"
150" E
_= ..=:.
100"
•
o•
50 8
•
";'"'2"'"I
...........................
"~ ...................................
i" .....
-50
Time post-weaning (d)
Figure 4.
Increase in skin thickness 24 hours following intradermal challenge with an extract of soya, in unweaned pigs and in those weaned onto a soya based diet I , 2, 5, 7, or 13 days (d) e a r l i e r .
1.2
o x
E E
O~
Time (d} postweaning
Figure 5.
Plasma xylose levels in pigs 60 minutes after a single feed of xylose. P i g s were examined sequentially at preweaning and on days (d) 2, 5, and 11 postweaning.
419
B -~
lOO
~
80
75%
-7 25%
c
':-
60
e-
~
i
40
2O
0
!
2
5
8
13
days postweaning Each histogram represents the mean of five pigs. Each pig was examined by scanning E.M. at two sites - ?5% and 75~/oalong the small intestine. Ten vitli at each site were examined for adhering bacteria.
Figure 6.
Bacteria adhering to intestinal v i l l i of pigs weaned at 3 weeks of age onto diets containing soya as the sole protein source.
sections of pig intestine taken on days f i v e and seven post weaning had bacteria adhering to the v i l l i . 2.
These were absent by day 13 (Figure 6).
Immunological studies Eleven pigs were weaned abruptly onto a soya containing diet at three weeks
of age. They were then bled daily for up to 20 days post weaning and the serum concentration of soya determined by a l i q u i d phase radioimmunoassay. All blood sampling and feeding was done at the same time of day.
Immunoreactive soya was
not detected in the preweaning serum samples. Following weaning soya protein was detected in the sera of all animals (Figure 7) at concentrations up to 200 ng.ml-l.
The median level however did not rise above 50 ng.ml-1 and by 20 days
post weaning had f a l l e n
back toward preweaning l e v e l s .
This f a l l
in
circulating soya antigen was associated with the appearance of soya-specific IgG antibody in serum. Twenty days post weaning animals failed to eliminate an intranveous challenge of
1251-1abelled-soya more rapidly than controls,
indicating that the decreased levels of circulating soya were a result of decreased
absorption of antigen (immune exclusion) rather than increased
clearance of absorbed antigen.
In this context isolated gut cells were shown
to be able to synthesize both IgG and IgA antisoya antibody. The other important protective response induced by dietary antigens is the development of systemic immunological tolerance and this was also assessed in
420 serum concentration of soya protein in pigs w e a n e d at three w e e k s of a g e 200
Legend
-
pig 1 "~ pig 2 E] pig 3 [ ] pig 4 150
pig 5
-
0
pig 6 pig 7 (~ pig 8 0
pig 9
0
pig 11
•
median
~oo2
pig 10
0
8 g
(B
50-
0/~
0
Figure 7.
1
2
3
• "-O
~×
4
5
6
~
~
0
0
7
8 9 10 11 12 days post weaning
0
0 x
,~
13
A
14
15
16
17 18
19 20
Serum concentration (ng.ml-1) of soya protein as determined by radioimmunoassay following weaning onto a soya based diet.
the post weaned p i g l e t .
Groups of p i g l e t s were weaned abruptly at three weeks
of age onto e i t h e r diets in which the sole protein was soya or diets which were soya free.
Four weeks l a t e r a l l pigs were Fed the soya free d i e t and a f t e r a
f u r t h e r two weeks they were challenged by i n t r a p e r i t o n e a l i n j e c t i o n of soya with Qui] A adjuvant.
The serum IgG antibody response is shown in Figure 8.
Feeding soya led to high levels of serum IgG antibody being produced.
Upon
parenteral challenge there was no increase in antibody level in the fed group, whilst
in those weaned onto the soya free d i e t there was an increase in IgG
antibody to soya.
We are c u r r e n t l y i n v e s t i g a t i n g the nature of the suppression
in the fed group.
DISCUSSION The introduction of SRBC into the diet of mice induced a brief phase of s e n s i t i v i t y , as indicated by production of positive ~4 h. skin tests, prior to the induction of tolerance.
O r a l challenge during the phase of s e n s i t i v i t y
resulted in gut changes including decreased a b i l i t y to absorb xylose, crypt hyperplasia, villous atrophy and an increase in the number of IEL's.
Failure
to sensitize with the noncross-reacting antigen (HRBC) and the p r o t e c t i o n afforded by prolonged feeding of SRBC ( t o l e r i z a t i o n ) confirmed that the cause of the lesion was immune mediated. Preliminary results from adoptive transfer
421
fed s o y a •
5.0-
ip.soyl
4"5c
c II
4'0"
o v 0
3"5" c el 0
3'0
weeks from injection
Figure 8.
Serum IgG antibody response to soya. Piglets were weaned onto soya based diets (0) or soya free diet (0) for four weeks. All pigs were then placed onto the soya free diet (-2 weeks) and challenged by intraperitoneal (IP) injection of soya (time 0).
studies with mesenteric lymph node cells from sensitizeJ mice (data not shown) indicated that sensitivity could be transferred in this way. The addition of dextran sulphate to the drinking water of mice being fed SRBC enhanced the development of tolerance, and further studies would indicate that both Freund's incomplete adjuvant and E. coli LPS, may have similar potentiating effects. The results support our earlier observations with mice fed contact sensitizing agents (Newby et a l . , 1980) and in the case of LPS may further indicate how gut flora can influence the responses to dietary antigens. The changes in gut morphology, crypt hyperplasia and villous atrophy, and the malabsorption associated with early weaning have been well characterized (Kenworthy and Allen, 1966). We have suggested that these changes occur as a result of a transient hypersensitivity to antigen in the postweaning diet, and evidence has been obtained based on dietary manipulation to support this hypothesis (Miller et a l . ,
1984a & b).
The acceleration of enterocyte
422
development associated with t h i s process is accompanied by i n h i b i t i o n of the capacity of enterocytes to digest and absorb n u t r i e n t s
(Smith et a l . ,
1985).
Such changes in enterocyte maturity might explain the increased s u s c e p t i b i l i t y of post-weaned p i g l e t s to the hypersecretory e f f e c t of enterotoxin (Stevens et a l . , 1972) and the bacterial p r o l i f e r a t i o n that may f o l l o w these changes. In an attempt to characterize the primary lesion we weaned pigs onto diets containing a f u l l
fat soya as the sole protein source.
in delayed type h y p e r s e n s i t i v i t y skin tests decreased
ability
changes.
Feeding large amounts of
to
A transient reactivity
occurred simultaneously w i t h
absorb xylose and the well soya p r i o r
a
established morphological
to
the w i t h d r a w a l
prevented the post weaning malabsorption ( M i l l e r et a l . ,
1985).
of
milk
If a similar
immune mechanism is responsible for the gut changes in the post-weaned pig as that described for mice fed SRBC, the i m p l i c a t i o n of t h i s observation, would be that tolerance had developed. piglets
fed
In f u r t h e r support of t h i s , we have shown that
on soya containing diets
and having high serum levels
of
antibody to soya, f a i l e d to respond to subsequent parenteral challenges.
IgG The
immune response f o l l o w i n g weaning onto soya-containing diets shows a s i m i l a r pattern to that shown in babies weaned onto cows' milk (Lippard et a l . , in
which a n t i g e n
is
subsequent decline, antibody.
first
detectable
in the c i r c u l a t i o n
with the concurrent appearance
1936)
followed by i t s
of serum and s e c r e t o r y
I n t e r e s t i n g l y , as we have shown in mice, the serum antibody produced
f o l l o w i n g feeding does not lead to enhanced immune e l i m i n a t i o n (Swarbrick et a l . , 1979).
Thus the f a l l
in c i r c u l a t i n g antigen would appear to be associated
with a mucosal (immune exclusion) e f f e c t rather than asystemic event. The i n t r o d u c t i o n of new d i e t a r y antigens for example, at weaning, protnotes a series
of
immunological events that
tolerance
and immune exclusion.
protected
state
may r e s u l t
However, p r i o r
in the development of oral to the
induction
animals pass through a phase of h y p e r s e n s i t i v i t y .
of
this
In the
present paper we have described the a n t i g e n - s p e c i f i c e f f e c t of t h i s s e n s i t i v i t y upon
gut
morphology,
and
the
resultant
s u s c e p t i b i l i t y to bacterial p r o l i f e r a t i o n . development bacterial
of
LPS.
the
response to
malabsorption
and
increased
I n t e r e s t i n g l y , in t h i s context the
dietary
antigen may also be modified by
In addition to the a n t i g e n - s p e c i f i c e f f e c t s
we have e a r l i e r
described antigen non-specific effects associated with d i e t a r y change (Newby et a l . 1980; Stokes et a l . ,
1983).
The l a t t e r
results in an altered capacity to
respond to non-related antigens presented at that time.
For example, oral
tolerance induction to a human s p e c i f i c albumin, fed two days a f t e r commencing oral
ummunization w i t h
ovalbumin,
is
prevented.
The precise mechanisms
underlying the events that occur during the t r a n s i e n t period of s e n s i t i v i t y that
follows the i n t r o d u c t i o n of a new d i e t a r y antigen are unclear, but the
immunopathological and disease s i g n i f i c a n c e are however, c l e a r l y apparent.
423
ACKNOWLEDGEMENTS The financial support of the Agriculture and Food Research Council of Great Britain is gratefully acknowledged. REFERENCES Asherson, G.L., Zembala, M., Perera, M.A.C.C., Mayhew, B. and Thomas, W.R., 1977. Production of immunity and unresponsiveness in the mouse by feeding contact sensitizing agents and the role of suppressor cells in the Peyer's patches, mesenteric lymph nodes and other lymphoid tissues. Cell Immunol., 33: 145-155. Kagnoff, M.E., 1978. Effects of antigen feeding on intestinal and systemic immune responses. 1. Priming of precursor cytotoxic T-cells by antigen feeding. J. Immunol., 120: 395-399. Kenworthy, R. and Allen, W.D., 1966. Influence of diet and bacteria on small i n t e s t i n a l morphology, with special reference to early weaning on Escherichia c o l i . Studies with germ free and gnotobiotic pigs. J. Comp. Pathol., 76:-29T-296. Lippard, V.W., Schloss, O,M. and Johnson, P.A., 1936. Immune reactions induced in infants by intestinal absorption of incompletely digested cows milk protein. Am. J. Dis. Child., 51: 562-574. M i l l e r , B.G., Newby, T.J., Stokes, C.R. and Bourne, F.J., 1984. Influence of diet on post weaning malabsorption and diarrhoea in the pig. Res. Vet. Sci., 36: 187-193. M i l l e r , B.G., Newby, T.J., Stokes, C.R., Hampson, D.J., Brown, P.J. and Bourne, F.J., 1984. The importance of dietary antigen in the cause of postweaning diarrhoea. Am. J. Vet. Res., 45: 1730-1733. M i l l e r , B.G., Phillips, A., Newby, T.J., Stokes, C.R. and Bourne, F.J., 1985. In: S.A. Just, H. Jorgensen and J.A. Fernandez (Editors), Proc. 3rd Int. Seminar on Digestive Physiology in the Pig. N a t . Inst. Animal Science, Denmark, p. 65. Newby, T.J., Stokes, C.R. and Bourne, F.J., 1980. Effects of feeding bacterial lipopolysaccharide and dextran sulphate on the development of oral tolerance to contact sensitizing agents. Immunology, 41: 617-621. Newby, T.J., Stokes, C.R. and Bourne, F.J., 1980. Altered po}yvinylpyrrolidone clearance and immune responsiveness caused by small dietary changes. Clin. Ex. Immunol., 39: 349-354. Smith, M.W., M i l l e r , B.G., James, P.S. and Bourne, F.J., 1985. Effect of weaning on the structure and function of piglet small intestine. In: A. Just, H. Jorgensen and J.A. Fernandez (Editors), Proc. 3rd Int. Seminar on Digestive Physiology in the Pig. Nat. Inst. Animal Science, Denmark, p. 75. Stevens, J.8., Gyles, C.L. and Barnum, D.A., 1972. Production of diarrhoea in pigs in respones to Escherichia coli enterotoxin. Am. J. Vet. Res., 33: 2511-2526. Stokes, C.R., Newby, T.J. and Bourne, F.J., 1983. The influence of oral immunization on local and systemic immune responses to heterologous antigens. Clin. Exp. Immunol., 52: 399-406. Swarbrick, E.T., Stokes, C.R. and Soothill, JoF., 1979. The absorption of antigens after oral immunization and the simultaneous induction of specific systemic tolerance. Gut, 20: 121-125. Thomas, H.C. and Parrott, D.V., 1974. The induction of tolerance to a soluble protein antigen by oral administration. Immunology, 27: 631-639. Trinder, P., 1975. Micromethod for the determination of plasma Xylose. Analyst., 100: 12-15. V o l l e r , A., Bidwell, D.E. and B a r t l e t t , A., 1976. Enzyme immunoassays in diagnostic medicine. Theory and Practice Bull., WHO, 53: 55-68.
413
THE IMMUNE RESPONSE TO DIETARY ANTIGENS AND ITS INFLUENCE ON DISEASE SUSCEPTIBILITY IN FARMANIMALS
C.R. STOKES, B.G. MILLER, M. BAILEY, A.D. WILSON and F.J. BOURNE University of Bristol, Department of Veterinary Medicine, Langford House, Langford, Bristol, BS18 7DU, U.K.
ABSTRACT Stokes, C.R., Miller, B.G., Bailey, M., Wilson, A.D. and Bourne, F.J., 1987. The immune response to dietary antigens and i t s influence on disease susceptibility in farm animals. Vet. Immunol. Immunopathol., 17: 413-423. Transient hypersensitivity reactions of the intestinal immune system to dietary antigens result in increases in enterocyte turnover and villous atrophy. These changes occur in the intestine of the post weaned piglet and precede the p r o l i f e r a t i o n of E. coli and the development of post-weaning diarrhea. We therefore postula~ed-t'h-~-t a transient cell mediated immune response to dietary antigens may increase susceptibility to disease. The interaction of dietary and microbial antigens upon the gut immune system has been investigated in mice and pigs and i t has been shown that both exert powerful regulatory effects upon each other. INTRODUCTION The immune system of the gastrointestinal tract is presented with a vast array of different antigens ranging from potential pathogens to dietary antigens; which are in themselves harmless.
The nature and magnitude of the
responses they e l i c i t differ markedly, for on the one hand an individual must respond with vigour to potential pathogens, whilst on the other overreaction to harmless dietary antigens would be both wasteful and potentially harmful i f i t resulted in hypersensitivity reactions.
I t is however increasingly clear that
the simultaneous presentation of dietary and microbial antigens can influence the animal's a b i l i t y to respond to either antigen and i t interaction that this paper is in part directed.
is to this
Following the introduction of a new dietary protein antigen a small proportion (<0.002%) of that protein is absorbed intact (Swarbrick, Stokes and S o o t h i l l , 1979) and a serum antibody response may be stimulated (Lippard, Schloss & Johnson, 1936).
Whilst
the systemic antibody
response is
subsequently suppressed (Thomas and Parrott, 1984) a local mucosal antibody response persists a~d this is associated with the development of a specific a b i l i t y to reduce the subsequent absorption of antigen (immune exclusion), (Swarbick etal., 1979). 0165-2427/87/$03.50
The f a l l in serum antibody is associated with the
© 1987 Elsevier Science Publishers B.V.
414
development of oral
tolerance, a specifically acquired capacity to prevent
responses to any antigen that does gain entry.
Delayed hypersensitivity and
i t s subsequent suppression have also been described in mice following feeding of contact sensitizing agents (Asherson et a l . , 1977), erythrocytes (Kagnoff, 1978) and soluble proteins
(Stokes et a l . ,
1983).
It
is therefore well
established that following the introduction of a new dietary antigen, animals pass through a brief phase of s e n s i t i v i t y prior to the development of the protected transient
state of tolerance. period of
We have investigated the influence of t h i s
s e n s i t i v i t y to dietary antigens upon enteric disease
s u s c e p t i b i l i t y in post weaned pigs. MATERIALS AND METHODS Animals Mice:
CBA male mice (LAC grade 3) aged 6-8 weeks were obtained from OLAC
Ltd., Oxford. Pigs:
Each experimental group contained eight mice.
Pregnant g i l t s
S h i f n a l l , Shropshire).
were obtained from a PHCA herd (Brockton Court,
Forty eight hours following birth all piglets received
injections of iron and t h e i r teeth were clipped.
In all cases, except where
indicated otherwise, weaning was performed abruptly at three weeks of age. Diets Mice were fed on commercial diets throughout. Pigs were weaned onto a 22% protein diet which contained a f u l l fat soy as the sole protein source.
The soy (Trusoy - British Soya Products) was a heat
treated f l o u r , free of a n t i - n u t r i t i o n a l factors. Xylose absorption Mice were given a single feeding of 10 mg xylose (0.2 ml) and were bled 30 minutes later.
Piglets were given orally a 10% xylose solution (I ml/kg - I ) and
the xylose concentration in serum measured one hour later (Trinder, 1975).
Antibody assay Serum IgG antibody to soya was measured by the microtitre ELISA method (Voller et a l . ,
1976) using plates coated with an extract of soya flour (10
ug/well) and a sheep anti-serum specific for the heavy chain of pig IgG. Radioimmunoassay for soya Absorbed soya in pig serum was measured by a l i q u i d phase, double antibody radioimmunoassay (Swarbrick et a l . , 1979) using a rabbit antiserum to the soy protein conglycinin.
415 Fed SRBC
0
5
I.V. SRBC
0 +
5 +
14 +
lO¢ ,'T'
]!
E u
'~
J~
~
8(:
I
4(]
~o
i
o
Figure 1.
Increase in mouse skin thickness 24 hours after challenge in animals previously given 30 ul (2 x 103) sheep erythrocytes (SRBC) for O, 5 or 14 days. Three groups of mice also received a sensitizing dose of SRBC following feed. Each point represents the mean of eight animals + ISD.
EXPERIMENTS AND RESULTS A. 1.
Mice fed sheep red blood cells ISRBC) Effect of oral
immunization on the induction of specific delayed type
hypersensitivity Groups of eight CBA male mice were orally immunized by infusing on each of five days 2 x 109 SRBC by stomach tube.
Five days later they were given a
subcutaneous injection of 2 x 108 SRBC into the l e f t hind f o o t pad, and the increase in foot pad thickness was measured after 24 hours. that feeding SRBC for five days sensitized (Figure 1).
The results showed
In a further experiment
mice were fed SRBC for five or 14 days and one week later they were given an intravenous injection of I x 105 SRBC. After a further five days they were footpad challenged by footpad injection as described above, and the results compared with non-SRBC fed controls.
Whilst feeding for five days had no
effect on the a b i l i t y to sensitize by the intravenous route, feeding for 14 days completely suppressed the response. These experiments demonstrate that following the introduction of SRBC into the d i e t precedes the development of tolerance. 2.
a phase of s e n s i t i v i t y
The effect of adjuvant feeding upon oral tolerance induction Mice were fed SRBCfor eight days. One group was also fed dextran sulphate
(m.w. 500,000)
at a concentration of 0.1 mg.ml-l.
The effect of feeding
416
Fed SRBC 0 Fed Dx.SO4 too _
E o
8
8
_
+
[
._~
Figure 2.
N
6O
-~
40
Increase in skin thickness 24 hours following foot pad challenge, in mice fed sheep erythrocytes (SRBC) for 0 or 8 days. One group also received dextran sulphate (Dx.S04) in the ~rinking water.
dextran sulphate was to turn a sensitizing dose of SRBC into a t o l e r i z i n g dose (Figure 2). 3.
Gastrointestinal effects Groups of eight male CBA mice were sensitized by feeding 3 x 109 SRBC daily
for two days.
They were then challenged 10 days later on two consecutive days
by feeding SRBC. Their a b i l i t y to absorb xylose was measured one day later and compared with unsensitized mice, tolerized mice ( i . e . those previously fed SRBC for 14 days) and those sensitized with the non-cross reacting antigen horse red blood cells (HRBC). Mice sensitized and challenged with SRBC absorbed s i g n i f i c a n t l y less xylose than did nonsensitized mice, tolerized mice and those sensitized with HRBC. The malabsorption was associated with increases in the crypt cell production rate
(4.2 to
11.7 d i v i s i o n s / h r ) and in the numbers of
intraepithelial
lymphocytes (16 to 23 IEL's/IO0 epithelial c e l l s ) . B.
Pigs fed soy containing diets
1.
Changes following weaning Groups of piglets were weaned abruptly at three weeks of age onto diets
containing soya as the sole protein source.
Separate groups were then skin
tested by intradermal injection into the skin of the ear of an extract of soya and the increase in ear thickness at 24 hours measured.
A transient delayed
hypersensitivity was observed five days after weaning which had disappeared by
417 +4+
PRIMARY
O
SECONDARY
O
0
SRBC HRBC SRBC
SRBC SRBC SRBC SRBC
31111
-z
280
~
•
J
260 -
-~ ¢=.
24o
220
2OO
Figure 3.
Plasmaxylose levels 30 minutes after a single feed of 10 mg xylose. Mice had been previously sensitized by feeding SRBC for 2 days, followed 10 days later by challenge on two consecutive days with sheep erythrocytes (SRBC). Xylose tests were performed the following day and compared with those in unsensitized mice, orally tolerized mice (+++) and those fed horse erythrocytes (HRBC). Each point represents themean of eight mice + ISD.
day 13 (Figure 4). for
Biopsies of skin test sites taken five days after weaning
histological
examination were compatible with
a delayed-type
hyper~eqsitivity reaction. The p i g l e t s '
a b i l i t y to absorb xylose from a single feeding was also
measured in the same group of animals.
The results are shown in Figure 5.
Five days after the introduction of soy at weaning, the piglets' a b i l i t y to absorb xylose was s i g n i f i c a n t l y reduced, but by day 13, t h i s t r a n s i e n t deficiency had been restored to preweaning levels. Histological examination of the intestine of these piglets showed that by two days post weaning there were significant increases in crypt depths and villous atrophy. Whilst subsequently there was a degree of recovery they never returned to preweaning levels. By both l i g h t and scanning electron microscopy,
418
200"
150" E
_= ..=:.
100"
•
o•
50 8
•
";'"'2"'"I
...........................
"~ ...................................
i" .....
-50
Time post-weaning (d)
Figure 4.
Increase in skin thickness 24 hours following intradermal challenge with an extract of soya, in unweaned pigs and in those weaned onto a soya based diet I , 2, 5, 7, or 13 days (d) e a r l i e r .
1.2
o x
E E
O~
Time (d} postweaning
Figure 5.
Plasma xylose levels in pigs 60 minutes after a single feed of xylose. P i g s were examined sequentially at preweaning and on days (d) 2, 5, and 11 postweaning.
419
B -~
lOO
~
80
75%
-7 25%
c
':-
60
e-
~
i
40
2O
0
!
2
5
8
13
days postweaning Each histogram represents the mean of five pigs. Each pig was examined by scanning E.M. at two sites - ?5% and 75~/oalong the small intestine. Ten vitli at each site were examined for adhering bacteria.
Figure 6.
Bacteria adhering to intestinal v i l l i of pigs weaned at 3 weeks of age onto diets containing soya as the sole protein source.
sections of pig intestine taken on days f i v e and seven post weaning had bacteria adhering to the v i l l i . 2.
These were absent by day 13 (Figure 6).
Immunological studies Eleven pigs were weaned abruptly onto a soya containing diet at three weeks
of age. They were then bled daily for up to 20 days post weaning and the serum concentration of soya determined by a l i q u i d phase radioimmunoassay. All blood sampling and feeding was done at the same time of day.
Immunoreactive soya was
not detected in the preweaning serum samples. Following weaning soya protein was detected in the sera of all animals (Figure 7) at concentrations up to 200 ng.ml-l.
The median level however did not rise above 50 ng.ml-1 and by 20 days
post weaning had f a l l e n
back toward preweaning l e v e l s .
This f a l l
in
circulating soya antigen was associated with the appearance of soya-specific IgG antibody in serum. Twenty days post weaning animals failed to eliminate an intranveous challenge of
1251-1abelled-soya more rapidly than controls,
indicating that the decreased levels of circulating soya were a result of decreased
absorption of antigen (immune exclusion) rather than increased
clearance of absorbed antigen.
In this context isolated gut cells were shown
to be able to synthesize both IgG and IgA antisoya antibody. The other important protective response induced by dietary antigens is the development of systemic immunological tolerance and this was also assessed in
420 serum concentration of soya protein in pigs w e a n e d at three w e e k s of a g e 200
Legend
-
pig 1 "~ pig 2 E] pig 3 [ ] pig 4 150
pig 5
-
0
pig 6 pig 7 (~ pig 8 0
pig 9
0
pig 11
•
median
~oo2
pig 10
0
8 g
(B
50-
0/~
0
Figure 7.
1
2
3
• "-O
~×
4
5
6
~
~
0
0
7
8 9 10 11 12 days post weaning
0
0 x
,~
13
A
14
15
16
17 18
19 20
Serum concentration (ng.ml-1) of soya protein as determined by radioimmunoassay following weaning onto a soya based diet.
the post weaned p i g l e t .
Groups of p i g l e t s were weaned abruptly at three weeks
of age onto e i t h e r diets in which the sole protein was soya or diets which were soya free.
Four weeks l a t e r a l l pigs were Fed the soya free d i e t and a f t e r a
f u r t h e r two weeks they were challenged by i n t r a p e r i t o n e a l i n j e c t i o n of soya with Qui] A adjuvant.
The serum IgG antibody response is shown in Figure 8.
Feeding soya led to high levels of serum IgG antibody being produced.
Upon
parenteral challenge there was no increase in antibody level in the fed group, whilst
in those weaned onto the soya free d i e t there was an increase in IgG
antibody to soya.
We are c u r r e n t l y i n v e s t i g a t i n g the nature of the suppression
in the fed group.
DISCUSSION The introduction of SRBC into the diet of mice induced a brief phase of s e n s i t i v i t y , as indicated by production of positive ~4 h. skin tests, prior to the induction of tolerance.
O r a l challenge during the phase of s e n s i t i v i t y
resulted in gut changes including decreased a b i l i t y to absorb xylose, crypt hyperplasia, villous atrophy and an increase in the number of IEL's.
Failure
to sensitize with the noncross-reacting antigen (HRBC) and the p r o t e c t i o n afforded by prolonged feeding of SRBC ( t o l e r i z a t i o n ) confirmed that the cause of the lesion was immune mediated. Preliminary results from adoptive transfer
421
fed s o y a •
5.0-
ip.soyl
4"5c
c II
4'0"
o v 0
3"5" c el 0
3'0
weeks from injection
Figure 8.
Serum IgG antibody response to soya. Piglets were weaned onto soya based diets (0) or soya free diet (0) for four weeks. All pigs were then placed onto the soya free diet (-2 weeks) and challenged by intraperitoneal (IP) injection of soya (time 0).
studies with mesenteric lymph node cells from sensitizeJ mice (data not shown) indicated that sensitivity could be transferred in this way. The addition of dextran sulphate to the drinking water of mice being fed SRBC enhanced the development of tolerance, and further studies would indicate that both Freund's incomplete adjuvant and E. coli LPS, may have similar potentiating effects. The results support our earlier observations with mice fed contact sensitizing agents (Newby et a l . , 1980) and in the case of LPS may further indicate how gut flora can influence the responses to dietary antigens. The changes in gut morphology, crypt hyperplasia and villous atrophy, and the malabsorption associated with early weaning have been well characterized (Kenworthy and Allen, 1966). We have suggested that these changes occur as a result of a transient hypersensitivity to antigen in the postweaning diet, and evidence has been obtained based on dietary manipulation to support this hypothesis (Miller et a l . ,
1984a & b).
The acceleration of enterocyte
422
development associated with t h i s process is accompanied by i n h i b i t i o n of the capacity of enterocytes to digest and absorb n u t r i e n t s
(Smith et a l . ,
1985).
Such changes in enterocyte maturity might explain the increased s u s c e p t i b i l i t y of post-weaned p i g l e t s to the hypersecretory e f f e c t of enterotoxin (Stevens et a l . , 1972) and the bacterial p r o l i f e r a t i o n that may f o l l o w these changes. In an attempt to characterize the primary lesion we weaned pigs onto diets containing a f u l l
fat soya as the sole protein source.
in delayed type h y p e r s e n s i t i v i t y skin tests decreased
ability
changes.
Feeding large amounts of
to
A transient reactivity
occurred simultaneously w i t h
absorb xylose and the well soya p r i o r
a
established morphological
to
the w i t h d r a w a l
prevented the post weaning malabsorption ( M i l l e r et a l . ,
1985).
of
milk
If a similar
immune mechanism is responsible for the gut changes in the post-weaned pig as that described for mice fed SRBC, the i m p l i c a t i o n of t h i s observation, would be that tolerance had developed. piglets
fed
In f u r t h e r support of t h i s , we have shown that
on soya containing diets
and having high serum levels
of
antibody to soya, f a i l e d to respond to subsequent parenteral challenges.
IgG The
immune response f o l l o w i n g weaning onto soya-containing diets shows a s i m i l a r pattern to that shown in babies weaned onto cows' milk (Lippard et a l . , in
which a n t i g e n
is
subsequent decline, antibody.
first
detectable
in the c i r c u l a t i o n
with the concurrent appearance
1936)
followed by i t s
of serum and s e c r e t o r y
I n t e r e s t i n g l y , as we have shown in mice, the serum antibody produced
f o l l o w i n g feeding does not lead to enhanced immune e l i m i n a t i o n (Swarbrick et a l . , 1979).
Thus the f a l l
in c i r c u l a t i n g antigen would appear to be associated
with a mucosal (immune exclusion) e f f e c t rather than asystemic event. The i n t r o d u c t i o n of new d i e t a r y antigens for example, at weaning, protnotes a series
of
immunological events that
tolerance
and immune exclusion.
protected
state
may r e s u l t
However, p r i o r
in the development of oral to the
induction
animals pass through a phase of h y p e r s e n s i t i v i t y .
of
this
In the
present paper we have described the a n t i g e n - s p e c i f i c e f f e c t of t h i s s e n s i t i v i t y upon
gut
morphology,
and
the
resultant
s u s c e p t i b i l i t y to bacterial p r o l i f e r a t i o n . development bacterial
of
LPS.
the
response to
malabsorption
and
increased
I n t e r e s t i n g l y , in t h i s context the
dietary
antigen may also be modified by
In addition to the a n t i g e n - s p e c i f i c e f f e c t s
we have e a r l i e r
described antigen non-specific effects associated with d i e t a r y change (Newby et a l . 1980; Stokes et a l . ,
1983).
The l a t t e r
results in an altered capacity to
respond to non-related antigens presented at that time.
For example, oral
tolerance induction to a human s p e c i f i c albumin, fed two days a f t e r commencing oral
ummunization w i t h
ovalbumin,
is
prevented.
The precise mechanisms
underlying the events that occur during the t r a n s i e n t period of s e n s i t i v i t y that
follows the i n t r o d u c t i o n of a new d i e t a r y antigen are unclear, but the
immunopathological and disease s i g n i f i c a n c e are however, c l e a r l y apparent.
423
ACKNOWLEDGEMENTS The financial support of the Agriculture and Food Research Council of Great Britain is gratefully acknowledged. REFERENCES Asherson, G.L., Zembala, M., Perera, M.A.C.C., Mayhew, B. and Thomas, W.R., 1977. Production of immunity and unresponsiveness in the mouse by feeding contact sensitizing agents and the role of suppressor cells in the Peyer's patches, mesenteric lymph nodes and other lymphoid tissues. Cell Immunol., 33: 145-155. Kagnoff, M.E., 1978. Effects of antigen feeding on intestinal and systemic immune responses. 1. Priming of precursor cytotoxic T-cells by antigen feeding. J. Immunol., 120: 395-399. Kenworthy, R. and Allen, W.D., 1966. Influence of diet and bacteria on small i n t e s t i n a l morphology, with special reference to early weaning on Escherichia c o l i . Studies with germ free and gnotobiotic pigs. J. Comp. Pathol., 76:-29T-296. Lippard, V.W., Schloss, O,M. and Johnson, P.A., 1936. Immune reactions induced in infants by intestinal absorption of incompletely digested cows milk protein. Am. J. Dis. Child., 51: 562-574. M i l l e r , B.G., Newby, T.J., Stokes, C.R. and Bourne, F.J., 1984. Influence of diet on post weaning malabsorption and diarrhoea in the pig. Res. Vet. Sci., 36: 187-193. M i l l e r , B.G., Newby, T.J., Stokes, C.R., Hampson, D.J., Brown, P.J. and Bourne, F.J., 1984. The importance of dietary antigen in the cause of postweaning diarrhoea. Am. J. Vet. Res., 45: 1730-1733. M i l l e r , B.G., Phillips, A., Newby, T.J., Stokes, C.R. and Bourne, F.J., 1985. In: S.A. Just, H. Jorgensen and J.A. Fernandez (Editors), Proc. 3rd Int. Seminar on Digestive Physiology in the Pig. N a t . Inst. Animal Science, Denmark, p. 65. Newby, T.J., Stokes, C.R. and Bourne, F.J., 1980. Effects of feeding bacterial lipopolysaccharide and dextran sulphate on the development of oral tolerance to contact sensitizing agents. Immunology, 41: 617-621. Newby, T.J., Stokes, C.R. and Bourne, F.J., 1980. Altered po}yvinylpyrrolidone clearance and immune responsiveness caused by small dietary changes. Clin. Ex. Immunol., 39: 349-354. Smith, M.W., M i l l e r , B.G., James, P.S. and Bourne, F.J., 1985. Effect of weaning on the structure and function of piglet small intestine. In: A. Just, H. Jorgensen and J.A. Fernandez (Editors), Proc. 3rd Int. Seminar on Digestive Physiology in the Pig. Nat. Inst. Animal Science, Denmark, p. 75. Stevens, J.8., Gyles, C.L. and Barnum, D.A., 1972. Production of diarrhoea in pigs in respones to Escherichia coli enterotoxin. Am. J. Vet. Res., 33: 2511-2526. Stokes, C.R., Newby, T.J. and Bourne, F.J., 1983. The influence of oral immunization on local and systemic immune responses to heterologous antigens. Clin. Exp. Immunol., 52: 399-406. Swarbrick, E.T., Stokes, C.R. and Soothill, JoF., 1979. The absorption of antigens after oral immunization and the simultaneous induction of specific systemic tolerance. Gut, 20: 121-125. Thomas, H.C. and Parrott, D.V., 1974. The induction of tolerance to a soluble protein antigen by oral administration. Immunology, 27: 631-639. Trinder, P., 1975. Micromethod for the determination of plasma Xylose. Analyst., 100: 12-15. V o l l e r , A., Bidwell, D.E. and B a r t l e t t , A., 1976. Enzyme immunoassays in diagnostic medicine. Theory and Practice Bull., WHO, 53: 55-68.