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The effect of parenteral immunisation on antibody production in the pig colon
Veterinary Immunology and Immunopathology, 23 (1989) 171-178 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
171
T h e E f f e c t of P a r e n t e r a l I m m u n i s a t i o n on A n t i b o d y P r o d u c t i o n in the P i g Colon A.S. REES 1, R.J. LYSONS 2, C.R. STOKES 1 and F.J. BOURNE 2
1Department of Veterinary Medicine, University of Bristol, Langford House, Langford, Avon BS18 7DU (Great Britain) 2AFRC Institute for Animal Health, Compton, Berkshire RG16 ONN (Great Britain) (Accepted 24 April 1989)
ABSTRACT Rees, A.S., Lysons, R.J., Stokes, C.R. and Bourne, F.J., 1989. The effect of parenteral immunisation on antibody production in the pig colon. Vet. Immunol. Immunopathol., 23: 171-178. Local and systemic antibody production was studied in pigs to compare responses to live and killed bacterial antigen and purified protein antigen, with and without prior mucosal stimulation. Recovery from challenge with live bacteria and intramuscular injection with killed bacteria gave rise to similar high levels of serum IgG antibody, but the ratio of specific IgA to IgG in the colon was significantly higher after infection than following vaccination with killed bacteria. Vaccination with a protein antigen gave rise to serum and local antibody production. Prior feeding of the antigen had a tolerising effect on the serum antibody response, but production of IgG and IgA antibody by the colon was not suppressed.
INTRODUCTION
Treponema hyodysenteriae, the primary agent in the aetiology of swine dysentery, colonises only the colon. Previous studies (Rees et al., 1989) have shown that prolonged infection leads to serum antibody production and local antibody production by mucosal tissues including the colon and the small intestine; the amount of serum antibody produced increases with the amount of exposure to T. hyodysenteriae. Pigs which had recovered from a number of infections were resistant to further challenge. Parenteral vaccines are a convenient form of vaccination against many diseases, but are not always effective against mucosal infections. Such vaccines against swine dysentery have been described (Fernie, 1983; Parizek et al., 1985), but the protection afforded is less than that following recovery from infection. The purpose of the present study was to compare the local and systemic response to infection with T. hyodysenteriae with that stimulated following parenteral immunisation. 0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
172
A.S. REES ET AL.
The potential of the mucosal immune system to respond to parenteral immunisation can be influenced by prior mucosal stimulation, as has been shown with cholera vaccination of humans (Svennerholm et al., 1977). When infection by an organism such as T. hyodysenteriae is maintained at sub-clinical levels by the use of antibiotics on pig farms, the level of mucosal stimulation prior to parenteral immunisation is unknown and could have implications for any vaccine administered parenterally. In addition, the effectiveness of any vaccine employing whole organisms rather than purified antigens may be influenced by prior mucosal stimulation with cross-reacting antigens on other bacteria such as commensal spirochaetes. For this reason we investigated the effect of parenteral immunisation with a pure protein antigen which would not previously have been encountered by pigs, namely human serum albumin (HSA), with and without prior mucosal stimulation by oral dosing. MATERIALS AND METHODS
Animals Large White pigs from a swine-dysentery free herd were used in this study and were fed an antibiotic-free ration. Five groups of pigs were studied: (A) five pigs challenged three times with live T. hyodysenteriae; (B) five pigs injected intramuscularly with killed T. hyodysenteriae; (C) four pigs injected intramuscularly with HSA; (D) four pigs orally dosed with HSA followed by intramuscular injection; (E) four control pigs injected intramuscularly with saline.
Bacteria T. hyodysenteriae strain P18A was grown in a liquid medium as previously described (Lemcke et al., 1979). For preparation of coating antigen for ELISA assays, the bacteria were grown to stationary phase, then washed in carbonate coating buffer and sonicated in this buffer to disrupt the cells (three 30-s bursts at 1.5 A). Debris was spun down at 30 000 g for 20 min, and the approximate protein concentration of the supernatant was estimated at OD 280 nm to retain consistency between assays, using an extinction coefficient of 10. The yield was typically 4 mg protein per 109 bacterial cells. For infection of pigs, broth cultures of bacteria grown to log phase were administered by stomach tube.
Vaccination For intramuscular immunisation (in the neck), sodium azide (BDH) was added at 0.1% w/v to the culture broth. The bacteria were then washed extensively in sterile saline and emulsified with Freund's complete adjuvant (Difco), each pig in group B receiving 3 × 109 cells in 1 ml emulsion. Two boosters at 1week intervals were given in Freund's incomplete adjuvant. H u m a n serum albumin (Sigma) was administered in the same way to groups
ANTIBODY PRODUCTION AFTER PARENTERAL IMMUNISATION IN THE PIG
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C and D, each pig receiving I mg HSA per injection. Both groups were injected at the same times using the same vaccine preparation. Pigs in group D had been orally dosed on 10 consecutive days with 200 mg HSA in 5 ml 0.45% saline per day, the last dose being administered 2 weeks before the first intramuscular injection. For the controls (group E), saline in adjuvant was injected following the same protocol as the bacterial/HSA injections. All pigs from groups B, C, D and E were slaughtered 1 week after the last injection, at 6 months of age.
Infection Group A pigs were orally dosed with log phase T. hyodysenteriae broth culture containing on average l0 s colony forming units per ml, allowed to recover from the dysentery which developed, and challenged a second and third time. By the third challenge, the pigs showed little or no sign of clinical disease compared with previously unchallenged controls. The challenge dose was dependent on body weight. The pigs were challenged at 4, 18 and 31 weeks of age when their weights were approximately 5, 50 and 120 kg, respectively. They were dosed accordingly with 20 ml broth culture on four days for the first challenge, 100 ml on two days for the second challenge and 250 ml on two days for the third challenge. The pigs were slaughtered 4-5 weeks after the third challenge for determination of serum and colon antibody levels.
Post-mortem samples The pigs were slaughtered by electrical stunning and exsanguination. Blood was collected and allowed to clot at room temperature, centrifuged, and the serum stored at - 2 0 ° C until assayed by ELISA. A piece of proximal colon approximately 5 cm × 2 cm was taken from each pig, avoiding areas of lymphoid aggregates, rinsed gently under a running tap, and placed in 10 ml R P M I ] 640 (Flow) supplemented with 2% fetal calf serum (FCS; Gibco) on ice.
In vitro antibody production Local antibody production was assayed by a method adapted from Svennerholm and Holmgren (1977). The pieces of colon tissue were rinsed in two further changes of R P M I + 2% FCS. Using a scalpel, the lamina propria was sliced off the underlying muscle and connective tissue, and chopped into pieces approximately 2 X 2 mm. 300 mg of these pieces were placed in each of two 10-ml volumes of tissue culture medium (RPMI 1640 Dutch modification; Flow), supplemented with 10% FCS (Gibco), 2 m M glutamine, 50/tg/ml gentamicin, 1 ]~g/ml fungizone (Flow) and 2 X 10 -~ M 2-mercaptoethanol (BDH). One of these 10 ml volumes was placed at - 2 0 ° C immediately, as a control for preformed antibody; the other was incubated in a tissue culture flask for 18 h at 37 ° C with 5% CO2, then placed at - 2 0 ° C. Both 10-ml cultures were thawed to disrupt the tissue and release antibody. The suspensions were centrifuged (3000 rpm, 10 rain) and the supernatants assayed by ELISA. Results for the
174
A.S. REES ET AL.
colon tissue are expressed as both the control culture values, i.e. the amount of antibody present at the time of slaughter, including locally-produced and serum-transudated antibody, and the values for the incubated cultures, which show in addition the amount of antibody produced overnight.
ELISA The sonicated bacterial preparation, or HSA as appropriate, was diluted to 5 gg/ml in carbonate coating buffer, and 150 gl/well were used to coat ELISA plates (Dynatech) overnight at 4°C. The plates were then washed with P B S + 0 . 5 % Tween 20 (PBS-T20). A high-titre anti-HSA or anti-T, hyodysenteriae serum was used in all assays as a standard at six 10-fold dilutions, and the OD values obtained for test samples were read off these standard curves as antibody units. Culture supernatants were diluted 1 in 5; sera were diluted by three 10-fold dilutions. All dilutions were in PBS-T20, and 100 gl were applied to each well. After 2 h at 37°C, plates were washed in PBS-T20, and 150/~l of mouse monoclonal antibody to pig IgG, IgA or IgM at 10 ~tg/ml PBS-T20 were applied to appropriate wells and incubated for 2 h at 37 ° C. After washing, 150 /~l sheep anti-mouse IgG conjugated with alkaline phosphatase and diluted 1 in 500 in PBS-T20 were applied to each well. The plates were incubated and washed as before, then 150 #l of phosphatase substrate (Sigma) at 1 m g / m l carbonate buffer were added to each well. The plates were read on a Titertek ELISA reader at OD 405 n m when a suitable amount of yellow colour had developed in the positive control {usually 2-3 h at 37 °C ). The OD readings for samples were read off the linear portion of the standard curve and expressed as antibody units. These units were log-transformed for statistical comparisons by t-test. RESULTS
Response to T. hyodysenteriae Pigs challenged three times with live T. hyodysenteriae produced similar levels of serum IgG antibody to those injected with the killed organism in adjuvant (Fig. 1 ). These levels, however, were not reflected in the colon; parenterally immunised pigs had higher levels of colon IgG (P < 0.01 after incubation) while pigs receiving the live challenge had lower initial IgG levels in the colon (P < 0.05) and showed little or no additional in vitro synthesis. One of the four control animals, which had received intramuscular injections of saline in adjuvant alone, produced high levels of specific IgG following in vitro culture of its colon tissue. This was reflected in the culture of small intestine tissue that was assayed totally independently {data not shown). IgA levels in the colon tissue of the pigs which had had the live challenge were higher than in those after parenteral immunisation (P < 0.05 ). Colon IgA
ANTIBODY PRODUCTION AFTER PARENTERAL IMMUNISATIONIN THE PIG
175
colon IgG
serum IgG
colon IgA
4.5
<
4.0
/
3.5
/
3.0
2.5 /
/
2.0
_o 1.5
1.0
~
N
A
B
i
E
A
B
E
A
B
E
Fig. 1. Antibodies to T. hyodysenteriae in pigs challenged three times with the live bacteria (group A), in pigs parenterally immunised with killed T. hyodysenteriae (group B ) and in pigs shami m m u n i s e d with saline (group E).
levels in parenterally immunised pigs were not significantly higher than in the control animals.
Response to HSA Parenteral immunisation with HSA gave rise to high levels of serum IgG antibody (Fig. 2), but those pigs which had been fed HSA prior to immunisation produced significantly (P < 0.05) lower levels of serum antibody than those receiving injections alone. The serum antibody levels were reflected in the colon IgG levels before incubation, the colon from HSA-fed pigs having significantly (P < 0.05) lower levels of IgG antibody than that from pigs not fed HSA before immunisation. After overnight incubation of the tissue pieces the difference between the two groups was not significant. A similar trend was observed for IgA, though differences were not significant at the 5% level, and incubated cultures showed a lower rate of synthesis of IgA than of IgG. Saline-injected pigs did not produce detectable levels of anti-HSA antibody either in the serum or in the colon (data not shown). DISCUSSION
Intramuscular immunisation of pigs with killed T. hyodysenteriae in complete Freund's adjuvant results in an immune response which differs from that
176
A.S. REES ET AL. colon 19A
colon IgG
serum IgG 5
4
•
"J
3
! / //
/' S o
o
/J
/j
/ ,'
~i j
?
2
/
./
/,/ I : / C
D
C
D
C
D
Fig. 2. Antibody to Human Serum Albumin in pigs parenterally immunised with HSA in adjuvant (group C ) and in pigs orally dosed with HSA prior to parenteral immunisation (group D ).
induced by infection of the colon. Parenteral immunisation gives rise to high levels of serum IgG antibody but is a poor stimulator of mucosal immunity (Newby and Stokes, 1984). Parenteral vaccination is used against cholera, a mucosal infection in man, but in the pig such vaccines only give partial protection against swine dysentery (Fernie, 1983; Parizek et al., 1985). Better protection appears to be achieved after recovery from disease induced by exposure to live T. hyodysenteriae, which results in similar serum IgG antibody levels to those produced after intramuscular immunisation but a higher ratio of IgA to IgG antibody in the colon. In the gut, many immune functions can be performed by IgG and by IgA, such as inhibition of adherence of microorganisms and toxins, and antibodydependent cellular cytotoxicity (for review see Newby and Stokes, 1984). It
ANTIBODYPRODUCTIONAFTER PARENTERALIMMUNISATIONIN THE PIG
177
seems logical that organisms which rely on adherence to display their pathogenicity could therefore be eliminated by IgG antibody arising in the gut after parenteral vaccination. IgA, however, has certain unique properties which can make it more suitable than IgG for eliminating pathogenic organisms from mucosal surfaces. Secretory IgA, which predominates in intestinal secretions, does not activate complement, recruit phagocytic cells or produce inflammatory responses. Such reactions are more appropriate for eliminating systemic infections and are mediated by IgG. They are potentially damaging to the host, and are unnecessary in the gut which is constantly in contact with antigenic material, either nutritive, or harmful, or harmless; immune mechanisms must simply be capable of preventing entry of harmful antigens to the intestinal mucosa. IgA antibody reduces absorption of antigen in orally immunised animals (Stokes et al., 1975); the antigen may be trapped by IgA in the mucus layer where it can be degraded by proteolytic enzymes. The ratio of IgA to IgG in the small intestine secretions of the pig is 13:1 while in pig serum IgA is present 2.4 mg/ml and IgG at 24 mg/ml (Bourne, 1973 ). The lamina propria of the small and large intestines contains few IgG plasma cells (Brown and Bourne, 1976 ) and IgG in intestinal tract secretions appears as a result of passive transudation (Newby and Stokes, 1984). We found that the ratio of IgA to IgG in colon secretions was 25:1 but the amount of immunoglobulin released from freeze-thawed colon tissue was 0.7 mg IgA per g and 0.5 mg IgG per g (unpublished observations), supporting the view that only IgA is actively secreted and IgG is serum-derived. The effect of oral immunisation on the subsequent response to parenteral challenge is variable. On the one hand, it has been shown that feeding cholera toxin primes for subsequent parenteral challenge (Svennerholm et al., 1977). Similarly, pigs fed lipopolysaccharide produce a greater serum response to intramuscular immunisation with that antigen (Chidlow and Porter, 1978 ). This contrasts with the suppressive effect of feeding soluble proteins such as ovalbumin fed to mice (Stokes et al., 1983) or dinitrophenylated bovine gammaglobulin fed to pigs (Newby et al., 1979). Tolerance arising from feeding has been shown to suppress systemic immune responses whilst allowing continued mucosal responses (Stokes et al., 1983). Such studies focussed on the small intestine. In the present study we have shown that prior feeding suppresses the serum antibody response, which is reflected in the lower levels of antibody in the non-incubated colon tissue cultures of the orally dosed group. In contrast, colonic tissues incubated for the detection of locally-produced antibody showed no evidence of suppression. This demonstrates that, as has been shown for the small intestine, local antibody production in the colon is unaffected by suppression of the parenteral response that follows antigen feeding. REFERENCES Bourne, F.J., 1973. The immunoglobulin system of the suckling pig. Proc. Nutr. Soc., 32:205-215. Brown, P.J. and Bourne, F.J., 1976. Distribution of immunoglobulin-containing cells in the all-
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mentary tract, spleen, and mesenteric lymph node of the pig demonstrated by peroxidaseconjugated antiserums to porcine immunoglobulins G, A and M. Am. J. Vet. Res., 37: 9-13. Chidlow, J.W. and Porter, P., 1978. The role of oral immunisation in stimulating Escherichia coli antibody of the IgM class in porcine colostrum. Res. Vet. Sci., 24: 254-257. Fernie, D.S., 1983. A review of swine dysentery vaccines. The Pig Vet. Soc. Proc., 10: 63-69. Lemcke, R.M., Bew, J., Burrows, M.R. and Lysons, R.J., 1979. The growth of Treponema hy odysenteriae and other porcine intestinal spirochaetes in a liquid medium. Res. Vet. Sci., 26: 315-319. Newby, T.J. and Stokes, C.R., 1984. The intestinal immune system and oral vaccination. Vet. Immunol. Immunopathol., 6: 67-105. Newby, T.J., Stokes, C.R., Huntley, J., Evans, P. and Bourne, F.J., 1979. The immune response of the pig following oral immunisation with soluble protein. Vet. Immunol. Immunopathol., 1: 37-47. Parizek, R., Stewart, R. and Brown, K., 1985. Protection against swine dysentery with an inactivated Treponema hyodysenteriae bacteria. Vet. Med., 80: 80-86. Rees, A.S., Lysons, R.J., Stokes, C.R. and Bourne, F.J., 1989. Antibody production by the pig colon during infection with Treponema hyodysenteriae. Res. Vet. Sci., 47: 263-269. Stokes, C.R., Soothill, J.F. and Turner, M.W., 1975. Immune exclusion is a function of IgA. Nature (London), 255: 745. Stokes, C.R., Newby T.J. and Bourne, F.J., 1983. The influence of oral immunisation on local and systemic immune responses to heterologous antigens. Clin. Exp. Immunol., 52: 397-406. Svennerholm, A.M. and Holmgren, J., 1977. Immunoglobulin and specific antibody synthesis in vitro by enteral and nonenteral lymphoid tissues after subcutaneous cholera immunisation. Infect. Immun., 15: 360-369. Svennerholm, A.M., Holmgren, J., Hanson, L.A., Lindblad, B.S., Quereshi, F. and Rahimtoola, R.J., 1977. Boosting of secretory IgA antibody responses in man by parenteral cholera vaccination. Scand. J. Immunol., 6: 1345-1349.
171
T h e E f f e c t of P a r e n t e r a l I m m u n i s a t i o n on A n t i b o d y P r o d u c t i o n in the P i g Colon A.S. REES 1, R.J. LYSONS 2, C.R. STOKES 1 and F.J. BOURNE 2
1Department of Veterinary Medicine, University of Bristol, Langford House, Langford, Avon BS18 7DU (Great Britain) 2AFRC Institute for Animal Health, Compton, Berkshire RG16 ONN (Great Britain) (Accepted 24 April 1989)
ABSTRACT Rees, A.S., Lysons, R.J., Stokes, C.R. and Bourne, F.J., 1989. The effect of parenteral immunisation on antibody production in the pig colon. Vet. Immunol. Immunopathol., 23: 171-178. Local and systemic antibody production was studied in pigs to compare responses to live and killed bacterial antigen and purified protein antigen, with and without prior mucosal stimulation. Recovery from challenge with live bacteria and intramuscular injection with killed bacteria gave rise to similar high levels of serum IgG antibody, but the ratio of specific IgA to IgG in the colon was significantly higher after infection than following vaccination with killed bacteria. Vaccination with a protein antigen gave rise to serum and local antibody production. Prior feeding of the antigen had a tolerising effect on the serum antibody response, but production of IgG and IgA antibody by the colon was not suppressed.
INTRODUCTION
Treponema hyodysenteriae, the primary agent in the aetiology of swine dysentery, colonises only the colon. Previous studies (Rees et al., 1989) have shown that prolonged infection leads to serum antibody production and local antibody production by mucosal tissues including the colon and the small intestine; the amount of serum antibody produced increases with the amount of exposure to T. hyodysenteriae. Pigs which had recovered from a number of infections were resistant to further challenge. Parenteral vaccines are a convenient form of vaccination against many diseases, but are not always effective against mucosal infections. Such vaccines against swine dysentery have been described (Fernie, 1983; Parizek et al., 1985), but the protection afforded is less than that following recovery from infection. The purpose of the present study was to compare the local and systemic response to infection with T. hyodysenteriae with that stimulated following parenteral immunisation. 0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
172
A.S. REES ET AL.
The potential of the mucosal immune system to respond to parenteral immunisation can be influenced by prior mucosal stimulation, as has been shown with cholera vaccination of humans (Svennerholm et al., 1977). When infection by an organism such as T. hyodysenteriae is maintained at sub-clinical levels by the use of antibiotics on pig farms, the level of mucosal stimulation prior to parenteral immunisation is unknown and could have implications for any vaccine administered parenterally. In addition, the effectiveness of any vaccine employing whole organisms rather than purified antigens may be influenced by prior mucosal stimulation with cross-reacting antigens on other bacteria such as commensal spirochaetes. For this reason we investigated the effect of parenteral immunisation with a pure protein antigen which would not previously have been encountered by pigs, namely human serum albumin (HSA), with and without prior mucosal stimulation by oral dosing. MATERIALS AND METHODS
Animals Large White pigs from a swine-dysentery free herd were used in this study and were fed an antibiotic-free ration. Five groups of pigs were studied: (A) five pigs challenged three times with live T. hyodysenteriae; (B) five pigs injected intramuscularly with killed T. hyodysenteriae; (C) four pigs injected intramuscularly with HSA; (D) four pigs orally dosed with HSA followed by intramuscular injection; (E) four control pigs injected intramuscularly with saline.
Bacteria T. hyodysenteriae strain P18A was grown in a liquid medium as previously described (Lemcke et al., 1979). For preparation of coating antigen for ELISA assays, the bacteria were grown to stationary phase, then washed in carbonate coating buffer and sonicated in this buffer to disrupt the cells (three 30-s bursts at 1.5 A). Debris was spun down at 30 000 g for 20 min, and the approximate protein concentration of the supernatant was estimated at OD 280 nm to retain consistency between assays, using an extinction coefficient of 10. The yield was typically 4 mg protein per 109 bacterial cells. For infection of pigs, broth cultures of bacteria grown to log phase were administered by stomach tube.
Vaccination For intramuscular immunisation (in the neck), sodium azide (BDH) was added at 0.1% w/v to the culture broth. The bacteria were then washed extensively in sterile saline and emulsified with Freund's complete adjuvant (Difco), each pig in group B receiving 3 × 109 cells in 1 ml emulsion. Two boosters at 1week intervals were given in Freund's incomplete adjuvant. H u m a n serum albumin (Sigma) was administered in the same way to groups
ANTIBODY PRODUCTION AFTER PARENTERAL IMMUNISATION IN THE PIG
173
C and D, each pig receiving I mg HSA per injection. Both groups were injected at the same times using the same vaccine preparation. Pigs in group D had been orally dosed on 10 consecutive days with 200 mg HSA in 5 ml 0.45% saline per day, the last dose being administered 2 weeks before the first intramuscular injection. For the controls (group E), saline in adjuvant was injected following the same protocol as the bacterial/HSA injections. All pigs from groups B, C, D and E were slaughtered 1 week after the last injection, at 6 months of age.
Infection Group A pigs were orally dosed with log phase T. hyodysenteriae broth culture containing on average l0 s colony forming units per ml, allowed to recover from the dysentery which developed, and challenged a second and third time. By the third challenge, the pigs showed little or no sign of clinical disease compared with previously unchallenged controls. The challenge dose was dependent on body weight. The pigs were challenged at 4, 18 and 31 weeks of age when their weights were approximately 5, 50 and 120 kg, respectively. They were dosed accordingly with 20 ml broth culture on four days for the first challenge, 100 ml on two days for the second challenge and 250 ml on two days for the third challenge. The pigs were slaughtered 4-5 weeks after the third challenge for determination of serum and colon antibody levels.
Post-mortem samples The pigs were slaughtered by electrical stunning and exsanguination. Blood was collected and allowed to clot at room temperature, centrifuged, and the serum stored at - 2 0 ° C until assayed by ELISA. A piece of proximal colon approximately 5 cm × 2 cm was taken from each pig, avoiding areas of lymphoid aggregates, rinsed gently under a running tap, and placed in 10 ml R P M I ] 640 (Flow) supplemented with 2% fetal calf serum (FCS; Gibco) on ice.
In vitro antibody production Local antibody production was assayed by a method adapted from Svennerholm and Holmgren (1977). The pieces of colon tissue were rinsed in two further changes of R P M I + 2% FCS. Using a scalpel, the lamina propria was sliced off the underlying muscle and connective tissue, and chopped into pieces approximately 2 X 2 mm. 300 mg of these pieces were placed in each of two 10-ml volumes of tissue culture medium (RPMI 1640 Dutch modification; Flow), supplemented with 10% FCS (Gibco), 2 m M glutamine, 50/tg/ml gentamicin, 1 ]~g/ml fungizone (Flow) and 2 X 10 -~ M 2-mercaptoethanol (BDH). One of these 10 ml volumes was placed at - 2 0 ° C immediately, as a control for preformed antibody; the other was incubated in a tissue culture flask for 18 h at 37 ° C with 5% CO2, then placed at - 2 0 ° C. Both 10-ml cultures were thawed to disrupt the tissue and release antibody. The suspensions were centrifuged (3000 rpm, 10 rain) and the supernatants assayed by ELISA. Results for the
174
A.S. REES ET AL.
colon tissue are expressed as both the control culture values, i.e. the amount of antibody present at the time of slaughter, including locally-produced and serum-transudated antibody, and the values for the incubated cultures, which show in addition the amount of antibody produced overnight.
ELISA The sonicated bacterial preparation, or HSA as appropriate, was diluted to 5 gg/ml in carbonate coating buffer, and 150 gl/well were used to coat ELISA plates (Dynatech) overnight at 4°C. The plates were then washed with P B S + 0 . 5 % Tween 20 (PBS-T20). A high-titre anti-HSA or anti-T, hyodysenteriae serum was used in all assays as a standard at six 10-fold dilutions, and the OD values obtained for test samples were read off these standard curves as antibody units. Culture supernatants were diluted 1 in 5; sera were diluted by three 10-fold dilutions. All dilutions were in PBS-T20, and 100 gl were applied to each well. After 2 h at 37°C, plates were washed in PBS-T20, and 150/~l of mouse monoclonal antibody to pig IgG, IgA or IgM at 10 ~tg/ml PBS-T20 were applied to appropriate wells and incubated for 2 h at 37 ° C. After washing, 150 /~l sheep anti-mouse IgG conjugated with alkaline phosphatase and diluted 1 in 500 in PBS-T20 were applied to each well. The plates were incubated and washed as before, then 150 #l of phosphatase substrate (Sigma) at 1 m g / m l carbonate buffer were added to each well. The plates were read on a Titertek ELISA reader at OD 405 n m when a suitable amount of yellow colour had developed in the positive control {usually 2-3 h at 37 °C ). The OD readings for samples were read off the linear portion of the standard curve and expressed as antibody units. These units were log-transformed for statistical comparisons by t-test. RESULTS
Response to T. hyodysenteriae Pigs challenged three times with live T. hyodysenteriae produced similar levels of serum IgG antibody to those injected with the killed organism in adjuvant (Fig. 1 ). These levels, however, were not reflected in the colon; parenterally immunised pigs had higher levels of colon IgG (P < 0.01 after incubation) while pigs receiving the live challenge had lower initial IgG levels in the colon (P < 0.05) and showed little or no additional in vitro synthesis. One of the four control animals, which had received intramuscular injections of saline in adjuvant alone, produced high levels of specific IgG following in vitro culture of its colon tissue. This was reflected in the culture of small intestine tissue that was assayed totally independently {data not shown). IgA levels in the colon tissue of the pigs which had had the live challenge were higher than in those after parenteral immunisation (P < 0.05 ). Colon IgA
ANTIBODY PRODUCTION AFTER PARENTERAL IMMUNISATIONIN THE PIG
175
colon IgG
serum IgG
colon IgA
4.5
<
4.0
/
3.5
/
3.0
2.5 /
/
2.0
_o 1.5
1.0
~
N
A
B
i
E
A
B
E
A
B
E
Fig. 1. Antibodies to T. hyodysenteriae in pigs challenged three times with the live bacteria (group A), in pigs parenterally immunised with killed T. hyodysenteriae (group B ) and in pigs shami m m u n i s e d with saline (group E).
levels in parenterally immunised pigs were not significantly higher than in the control animals.
Response to HSA Parenteral immunisation with HSA gave rise to high levels of serum IgG antibody (Fig. 2), but those pigs which had been fed HSA prior to immunisation produced significantly (P < 0.05) lower levels of serum antibody than those receiving injections alone. The serum antibody levels were reflected in the colon IgG levels before incubation, the colon from HSA-fed pigs having significantly (P < 0.05) lower levels of IgG antibody than that from pigs not fed HSA before immunisation. After overnight incubation of the tissue pieces the difference between the two groups was not significant. A similar trend was observed for IgA, though differences were not significant at the 5% level, and incubated cultures showed a lower rate of synthesis of IgA than of IgG. Saline-injected pigs did not produce detectable levels of anti-HSA antibody either in the serum or in the colon (data not shown). DISCUSSION
Intramuscular immunisation of pigs with killed T. hyodysenteriae in complete Freund's adjuvant results in an immune response which differs from that
176
A.S. REES ET AL. colon 19A
colon IgG
serum IgG 5
4
•
"J
3
! / //
/' S o
o
/J
/j
/ ,'
~i j
?
2
/
./
/,/ I : / C
D
C
D
C
D
Fig. 2. Antibody to Human Serum Albumin in pigs parenterally immunised with HSA in adjuvant (group C ) and in pigs orally dosed with HSA prior to parenteral immunisation (group D ).
induced by infection of the colon. Parenteral immunisation gives rise to high levels of serum IgG antibody but is a poor stimulator of mucosal immunity (Newby and Stokes, 1984). Parenteral vaccination is used against cholera, a mucosal infection in man, but in the pig such vaccines only give partial protection against swine dysentery (Fernie, 1983; Parizek et al., 1985). Better protection appears to be achieved after recovery from disease induced by exposure to live T. hyodysenteriae, which results in similar serum IgG antibody levels to those produced after intramuscular immunisation but a higher ratio of IgA to IgG antibody in the colon. In the gut, many immune functions can be performed by IgG and by IgA, such as inhibition of adherence of microorganisms and toxins, and antibodydependent cellular cytotoxicity (for review see Newby and Stokes, 1984). It
ANTIBODYPRODUCTIONAFTER PARENTERALIMMUNISATIONIN THE PIG
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seems logical that organisms which rely on adherence to display their pathogenicity could therefore be eliminated by IgG antibody arising in the gut after parenteral vaccination. IgA, however, has certain unique properties which can make it more suitable than IgG for eliminating pathogenic organisms from mucosal surfaces. Secretory IgA, which predominates in intestinal secretions, does not activate complement, recruit phagocytic cells or produce inflammatory responses. Such reactions are more appropriate for eliminating systemic infections and are mediated by IgG. They are potentially damaging to the host, and are unnecessary in the gut which is constantly in contact with antigenic material, either nutritive, or harmful, or harmless; immune mechanisms must simply be capable of preventing entry of harmful antigens to the intestinal mucosa. IgA antibody reduces absorption of antigen in orally immunised animals (Stokes et al., 1975); the antigen may be trapped by IgA in the mucus layer where it can be degraded by proteolytic enzymes. The ratio of IgA to IgG in the small intestine secretions of the pig is 13:1 while in pig serum IgA is present 2.4 mg/ml and IgG at 24 mg/ml (Bourne, 1973 ). The lamina propria of the small and large intestines contains few IgG plasma cells (Brown and Bourne, 1976 ) and IgG in intestinal tract secretions appears as a result of passive transudation (Newby and Stokes, 1984). We found that the ratio of IgA to IgG in colon secretions was 25:1 but the amount of immunoglobulin released from freeze-thawed colon tissue was 0.7 mg IgA per g and 0.5 mg IgG per g (unpublished observations), supporting the view that only IgA is actively secreted and IgG is serum-derived. The effect of oral immunisation on the subsequent response to parenteral challenge is variable. On the one hand, it has been shown that feeding cholera toxin primes for subsequent parenteral challenge (Svennerholm et al., 1977). Similarly, pigs fed lipopolysaccharide produce a greater serum response to intramuscular immunisation with that antigen (Chidlow and Porter, 1978 ). This contrasts with the suppressive effect of feeding soluble proteins such as ovalbumin fed to mice (Stokes et al., 1983) or dinitrophenylated bovine gammaglobulin fed to pigs (Newby et al., 1979). Tolerance arising from feeding has been shown to suppress systemic immune responses whilst allowing continued mucosal responses (Stokes et al., 1983). Such studies focussed on the small intestine. In the present study we have shown that prior feeding suppresses the serum antibody response, which is reflected in the lower levels of antibody in the non-incubated colon tissue cultures of the orally dosed group. In contrast, colonic tissues incubated for the detection of locally-produced antibody showed no evidence of suppression. This demonstrates that, as has been shown for the small intestine, local antibody production in the colon is unaffected by suppression of the parenteral response that follows antigen feeding. REFERENCES Bourne, F.J., 1973. The immunoglobulin system of the suckling pig. Proc. Nutr. Soc., 32:205-215. Brown, P.J. and Bourne, F.J., 1976. Distribution of immunoglobulin-containing cells in the all-
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mentary tract, spleen, and mesenteric lymph node of the pig demonstrated by peroxidaseconjugated antiserums to porcine immunoglobulins G, A and M. Am. J. Vet. Res., 37: 9-13. Chidlow, J.W. and Porter, P., 1978. The role of oral immunisation in stimulating Escherichia coli antibody of the IgM class in porcine colostrum. Res. Vet. Sci., 24: 254-257. Fernie, D.S., 1983. A review of swine dysentery vaccines. The Pig Vet. Soc. Proc., 10: 63-69. Lemcke, R.M., Bew, J., Burrows, M.R. and Lysons, R.J., 1979. The growth of Treponema hy odysenteriae and other porcine intestinal spirochaetes in a liquid medium. Res. Vet. Sci., 26: 315-319. Newby, T.J. and Stokes, C.R., 1984. The intestinal immune system and oral vaccination. Vet. Immunol. Immunopathol., 6: 67-105. Newby, T.J., Stokes, C.R., Huntley, J., Evans, P. and Bourne, F.J., 1979. The immune response of the pig following oral immunisation with soluble protein. Vet. Immunol. Immunopathol., 1: 37-47. Parizek, R., Stewart, R. and Brown, K., 1985. Protection against swine dysentery with an inactivated Treponema hyodysenteriae bacteria. Vet. Med., 80: 80-86. Rees, A.S., Lysons, R.J., Stokes, C.R. and Bourne, F.J., 1989. Antibody production by the pig colon during infection with Treponema hyodysenteriae. Res. Vet. Sci., 47: 263-269. Stokes, C.R., Soothill, J.F. and Turner, M.W., 1975. Immune exclusion is a function of IgA. Nature (London), 255: 745. Stokes, C.R., Newby T.J. and Bourne, F.J., 1983. The influence of oral immunisation on local and systemic immune responses to heterologous antigens. Clin. Exp. Immunol., 52: 397-406. Svennerholm, A.M. and Holmgren, J., 1977. Immunoglobulin and specific antibody synthesis in vitro by enteral and nonenteral lymphoid tissues after subcutaneous cholera immunisation. Infect. Immun., 15: 360-369. Svennerholm, A.M., Holmgren, J., Hanson, L.A., Lindblad, B.S., Quereshi, F. and Rahimtoola, R.J., 1977. Boosting of secretory IgA antibody responses in man by parenteral cholera vaccination. Scand. J. Immunol., 6: 1345-1349.