- Email: [email protected]
Humoral immunity in the ewe 3. The influence of adjuvants and immunisation regimes on Immune reactivity in the breeding ewe and her progeny
Veterinary Immunology and Immunopathology, 25 (1990) 167-175 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
167
H u m o r a l I m m u n i t y in the E w e 3. The I n f l u e n c e of A d j u v a n t s and I m m u n i s a t i o n R e g i m e s on I m m u n e R e a c t i v i t y in the B r e e d i n g E w e and her P r o g e n y G.E. R E Y N O L D S and J.F.T. G R I F F I N
Microbiology Department, University of Otago, Dunedin (New Zealand) {Accepted 14 November 1989)
ABSTRACT Reynolds, G.E. and Griffin, J.F.T., 1990. Humoral immunity in the ewe. 3. The influence of adjuvants and immunisation regimes on immune reactivity in the breeding ewe and her progeny. Vet. Immunol. Immunopathol., 25: 167-175. Breeding ewes were immunised with clostridial vaccine using different inoculation schedules. Results, showing differences in the class of antibody produced, were heavily dependent on the vaccination regime used. Immunoglobulin M (IgM) levels were significantly lower in ewes given double doses of vaccine compared to ewes given a single inoculation or no treatment at all ( P < 0.01 }. Neonatal lambs showed significant de novo IgM production with interference in this antibody production in the lambs of ewes vaccinated with the clostridial vaccine {P < 0.05 }. Immunoglobulin G l ( IgG 1 ) levels were significantly increased in all lambs which had mothers vaccinated with the clostridial vaccine prior to or during pregnancy (P < 0.025). The greatest quantity ()f IgG l was transferred to lambs when their mothers were given a double injection with primary" inoculation prior to conception and booster prepartum (P < 0.025). No antigen specific immunogl()bulin G2 (IgG2) was detected in the lambs. Ewes were also immunised with BSA and their isot)-pe specific serum antibody response was compared with their respective lambs. There was no detectable anti-BSA IgM in the lambs of all groups of ewes though specific IgG1 antibodies could be readily detected in the lambs of hyperimmunised ewes. The efficiency of transfer was related directly to the ability of the adjuvant to maximise IgG1 production in the ewe. Although immunised ewes produced high levels of IgG2, this was not transferred passively to the lamb.
INTRODUCTION
Vaccination of the pregnant ewe in the weeks prior to lambing is a common practice used to ensure passive transfer of immunity to the newborn. Antibody to Salmonella typhimurium lipopolysaccharide in colostrum has been shown to correlate with the specific immunoglobulin serum titre in the neonate
0165-2427/90/$03.50
© 1990 Elsevier Science Publishers B.V.
168
G.E. REYNOLDS AND J.F.T. GRIFFIN
(Fleenor and Stott, 1983). Material vaccination of pregnant animals with Escherichia coli or rotavirus induce high antibody titres in colostrum and milk which protect the offspring against enteric disease (Acres et al., 1979; Myers, 1978; Wells et al., 1978). Because IgG1 is the predominant class of antibody passively transferred via colostrum (Butler, 1983), vaccination for enhanced passive transfer of immunity is directed towards stimulating maximal IgG1 production in the dam to produce elevated colostral specific IgG 1 levels (Fleenor and Stott, 1983). Using the analysis of isotypic antibody produced, this study examines the effect of primary immunisation and secondary boosting of pregnant ewes to define conditions for maximal transfer of specific IgG1 antibody from the ewe to the lamb. The use of different immunisation protocols and different adjuvants allowed for the definition of optimal conditions to evoke memory and maximise IgG1 antibody titres. Two distinct antigens, bovine serum albumin (BSA) and clostridial toxoid vaccine were selected to elucidate the impact of pre-existing memory on passive immunity. Clostridial vaccines consist of a complex antigenic mixture which may evoke a variable primary and secondary response due to the level of endemic exposure of sheep flocks to clostridial organisms. Bovine serum albumin is a novel and well characterised primary immunogen to which the ewe should have no pre-existing memory. This allowed for the study of primary immunity and the development of secondary cellular memory and effector function under the influence of pregnancy. MATERIALSAND METHODS
The antigens The protein antigen BSA (Sigma, U.S.A.) was made up to 1000 ttg/ml in saline. The clostridial vaccine (Coopers Animal Health, N.Z.) was supplied at a dose used for proprietory vaccination.
The adjuvants The three adjuvants used in this study were aluminium hydroxide (alum), diethylaminoethyl dextran (DEAE dextran) and Freund's complete adjuvant {FCA), prepared as previously described (Reynolds et al., 1990). Clostridial vaccination A flock of 132 Romney ewes were divided into four groups and each exposed to different vaccination regimes (Table 1 ). Three groups were inoculated with the multivalent clostridial vaccine as a single injection prior to conception or prepartum, or as a double vaccine preconception and boosted prepartum. A further group received no treatment. Three weeks postpartum, blood samples were taken from both ewes and lambs. The serum was collected and stored at
INFLUENCE OF ADJUVANTSAND IMMUNISATIONREGIMESON RESPONSE IN THE EWE
169
TABLE 1 The immunisation regime for pregnant ewes inoculated with Clostridial vaccine preconception a n d / o r prepartum Group
Number of animals
Primary inoculation
Secondary inoculation
1 2 3 4
29 32 46 25
preconception prepartum preconception no t r e a t m e n t
no t r e a t m e n t no t r e a t m e n t prepartum no t r e a t m e n t
TABLE 2 The immunisation regime for pregnant ewes inoculated with BSA in alum for their primary injection and BSA in different adjuvants for their secondary inoculation Groups
Number of animals
Primary inoculation
Secondary inoculation
1
10
0.5ml BSA ~+0.5ml alum
2
10
0.5ml BSA ~+ 0.5ml alum same as above
3 4
10 4
same as above no t r e a t m e n t
0.5ml BSA 1+0.5ml DEAE dextran 0.5ml BSA ~+ 0.5ml FCA no t r e a t m e n t
11000p.g/ml.
- 2 0 °C until required. Isotype specific IgM, IgG1 and IgG2 antibody levels to
tetanus toxoid (TT) antigen (Coopers Animal Health, NZ) were determined using ELISA. Using a specific assay system to T T antigen allowed the detection of antibody from the non-specific multivalent anti-clostridial vaccine.
Adjuvancy study A group of 34 pregnant Perindale ewes treated with progesterone implants to synchronise oestrus, were used in this study. At day 80 of gestation, 30 of the animals were inoculated with 500/~g of BSA (Sigma, U.S.A. ) in alum adjuvant to evoke primary sensitisation. At day 56 post primary injection, corresponding to 136 days of gestation, animals were divided into three subgroups for the secondary inoculation (Table 2 ). Each group received 500/~g BSA emulisfied in FCA, DEAE dextran or alum adjuvants. A group of four non-immunised pregnant ewes were included as controls. Ewes were bled at parturition, lambs were bled 3 weeks postpartum and the sera stored at - 2 0 °C until required. The levels of anti-BSA antibody belonging to the different immunoglobulin isotypes IgM, IgG1 and IgG2 were assayed using ELISA.
170
G.E. REYNOI,DS AND J.F.T. GI~IFFIN
The E L I S A assay A four step indirect ELISA system was developed for the detection of classspecific antibodies to BSA and TT. The optimal concentrations of antigen, immune sheep serum, anti-sheep monoclonal antibody and anti-mouse peroxidase labelled conjugate were determined earlier for BSA (Reynolds et al., 1990 ). Tetanus toxoid was used at a concentration of 100 gg/ml for IgM assays and 10 ~g/ml for IgG1 and IgG2 assays.
RESUIJTS Clostridial vaccination All groups of animals immunised with clostridial vaccine, produced significant antibody titres. Comparison of IgM levels (Fig. 1) in the ewes revealed a significantly lower titre in animals given a primary dose preconception and a booster prepartum, compared with all other groups, ( P < 0.01 ). There was no difference between IgM levels in non-treated control ewes and animals given one injection of clostridial vaccine only (preconception or prepartum). Lambs born to non-treated mothers had significantly higher IgM titres than all other groups of vaccinated ewes {P<0.05) though all ewes had higher IgM titres than their lambs (P < 0.025 ). Comparison of IgG1 titres (Fig. 2) showed significantly higher {P < 0.01) anti-TT antibody responses in animals given a double dose of vaccine prior to conception and boosted prepartum, than other groups except the group vaccinated with a single inoculation prepartum. The ewes vaccinated prepartum produced an IgG1 response which was higher than non-vaccinated control ewes and the group of animals vaccinated with a single injection prior to conception (P<0.05). Lambs from mothers vaccinated twice with clostridial vaccine (preconception and prepartum) gave IgG1 titres higher than all other lamb groups (P < 0.025). Lambs born to mothers that were vaccinated prepartum only produced significantly higher IgG1 levels than the group of lambs from mothers immunised prior to conception only and the non-treated control animals ( P < 0.025). The IgG1 titres of lambs born to mothers immunised with a single injection prior to conception were significantly higher than lambs from non-vaccinated control ewes (P < 0.025 ). Ewes given a double vaccination prior to conception and prepartum, had IgG 1 titres equivalent to those found in their lambs, while all other ewes had significantly higher titres of anti-TT IgG 1 than their respective lambs (P < 0.05 ). Non-vaccinated control ewes had lambs that had the lowest relative level of anti-TT IgG1 compared to their mothers. The anti-TT IgG2 titres (Fig. 3) showed that ewes given a double vaccination prior to conception and prepartum produced higher IgG2 levels than all other groups (P < 0.01 ). The animals given a single injection of clostridial vaccine prior to conception or prepartum only, produced equal IgG2 levels. Both groups injected with a single dose only, produced higher IgG2 titres than un-
INFLUENCE OF ADJUVANTSAND |MMUNISATION REGIMES ON RESPONSE IN THE EWE
171
50 4O •-< ¢n ._% ~11
30 20
[] •
Ewe titre Lamb titre
[] •
Ewe titre Lamb titre
[] •
Ewe titre Lamb titre
10 0 1000
800 600 400 200 0 100
Y/~
80
#///
z///
60
//// //// ////
40 3~3 ~t~
1///
~/A ~/z ///¢
20 1~/~
..._ :;};
0 Non-treated PrePre-par~um PreControls Conception Conception & Pre-partum Time of inoculation
Figs, 1, 2 and 3. T h e effect of i m m u n i s a t i o n schedules on IgM, IgG1 and IgG2 antibody transfer frow ewe u) lamb using Clostridial vaccines, preconception a n d / o r prepartum. Antibody levels were measured 3 weeks p o s t p a r t u m in b o t h the ewes a n d lambs.
vaccinated control ewes ( P < 0.005). Little anti-TT IgG2 was produced in lambs (Fig. 3) above background control levels. The group of lambs with mothers given the double vaccination produced IgG2 titres that were higher than animals from all other groups ( P < 0.01 ).
172
G.E. REYNOLDSAND J.F.T. GRIFFIN 50 40
"E
30
[]
<
•
20
Ewe titre Lamb titre
LIJ
tO 0
v 7~., ~
..].
T
1000 8OO
600
[]
//J/
400
•
Ewe titre Lamb titre
[] •
Ewe titre Lamb titre
LU
2O0 0 100 80
6O 40
20
0 Non-treated BSA-alum BSA-DEAE controls
BSA-FCA
dextran Adjuvant inoculated
Figs. 4, 5 and 6. The transfer of anti-BSA IgM, IgG1 and IgG2 antibody from ewe to lamb when ewes were inoculated with BSA in different adjuvants during pregnancy. Antibody levels were measured at parturition for the ewes and 3 weeks postpartum for the lambs.
Adjuvancy study Variable ewe and lamb titres were found following immunisation of the mothers with BSA in alum, DEAE dextran or FCA. The anti-BSA IgM response (Fig. 4) showed that ewes injected with BSA in DEAE dextran produced significantly higher IgM titres compared with other immunised
I N F L U E N C E OF A D J U V A N T S A N D I M M U N I S A T I O N R E G I M E S O N R E S P O N S E IN T H E E W E
173
( P < 0.05) or control (P < 0.01 ) groups. No lamb IgM titres to BSA were evident in any of the groups tested. The IgG1 titres in the ewes (Fig. 5) were significantly higher titres in animals immunised with BSA in DEAE dextran than all other groups ( P < 0.005 ). The ewes immunised with BSA in FCA produced IgG1 antibody titres which were significantly higher than the ewes immunised with BSA in alum and nonimmunised control ewes (P<0.05). Immunisation with BSA in alum gave higher IgG1 levels than non-immunised control animals (P < 0.01 ). Anti-BSA IgG1 titres in lambs from immunised mothers were all higher than the IgG1 levels in lambs from non-immunised control ewes (P<0.05). The group of lambs from mothers that were exposed to BSA in DEAE dextran responded with higher IgG1 titres than all other groups ( P < 0.05). The comparison of titres in ewes given BSA in different adjuvants with their respective lamb IgG1 titres showed detectable but significantly lower titres in the lambs when compared with the ewes (P < 0.025). The anti-BSA IgG2 response in animals given BSA in adjuvant was found to be markedly different from their IgG1 antibody levels (Fig. 6). The ewes immunised with BSA in FCA produced significantly higher titres than all other groups (P < 0.005). The ewes injected with BSA in DEAE dextran produced higher IgG2 levels than the group of ewes immunised with BSA in alum or non-treated controls (P < 0.025). There was no significant titre of IgG2 in any lamb, irrespective of how the ewes were immunised. DISCUSSION These experiments show the amount and the quality of specific antibody produced around parturition in the ewe influence the passive IgG1 antibody titre acquired by the lamb. The vaccination of pregnant ewes with clostridial vaccine using different vaccination regimes produced a high antibody titre in vaccinated animals compared to non-vaccinated controls. The IgM antibody response to T T was particularly strong across all ewe and lamb groups including the non-treated controls. The observation of high titre especially in nonvaccinated ewes reflects the natural exposure to clostridial antigens in New Zealand sheep flocks. Results in ewes show that a double vaccination preconception and prepartum caused a significant reduction in IgM titre compared to all other treatment groups. This would be expected because classical switch from IgM to IgG production would have occurred upon repeated challenge with the clostridial antigen. All lambs showed high IgM antibody levels at 3 weeks of age, especially lambs of non-treated control mothers. These high levels of IgM provide further evidence of the endemic exposure of sheep to clostridial organisms. As antibody of the IgM isotype is not transferred in colostrum (Butler, 1983 ), the IgM titres present in these lambs are likely to be the result of the de novo antibody production. Lambs from vaccinated mothers had lower
174
G.E. REYNOI,DS AND J.F.T. GRIFFIN
IgM levels than lambs born to non-vaccinated ewes inferring that maternal antibody from vaccinated mothers interferes with the lamb's de novo immune response, as documented in other studies (Brar et al., 1978). Using tetanus toxoid vaccine, it was evident that the highest IgG1 antibody titres were in ewes vaccinated prior to conception and boosted prior to lambing. This was also reflected in optimal passive transfer of IgG1 antibody to the lambs. Whereas a single vaccination preconception or prepartum gave a significant ewe titre and passive transfer to lambs, it was at significantly lower levels than found following double vaccination of the ewe. The ewes given a single injection prepartum produced IgG1 antibody titres similar to the ewes inoculated twice (preconception and prepartum) but the amount of antibody transferred to the lambs was significantly higher in the ewes given two doses of vaccine. This reflects qualitative differences in maternal IgG 1 antibody titres in ewes primed pre-conception and boosted prepartum compared with ewes given a primary inoculum prepartum only. Because the untreated control group of ewes produced detectable natural anti-TT antibody it was not possible to consider the vaccine response as a primary stimulus. However, the low IgG1 titres found in non-vaccinated ewes and the predominance of the IgM isotype in these ewes was compatible with inadequate IgG1 antibody transfer to the lambs. The IgG2 response to the clostridial vaccine correlated with the IgG1 titres in the ewe though this was not reflected in the IgG2 titre in their lambs. A double inoculation regime (preconception and prepartum) was mandatory for efficient anti-TT IgG2 production in the ewe but the material IgG2 antibody was not passively transferred to the lambs. This is consistent with findings in the literature on ruminants which infer that IgG1 is the predominant class of antibody transferred from mother to offspring (Butler, 1983). Some IgG2 was detected in the lambs of ewes given a double inoculum. This may reflect the local production of anti-TT IgG2 antibody in the mammary gland and its subsequent transfer to the lamb (Sheldrake and Husband, 1985). The immunisation of pregnant and non-pregnant ewes with BSA in alum, DEAE dextran or FCA showed the effect adjuvant can have on the quantity and class of antibody produced in the ewe and the subsequent transfer to IgG1 to the lamb. The ewes injected with BSA in DEAE dextran produced the highest IgG1 titres and correlated with higher titres of IgG1 in their lambs. Consistent with the previous experiment, IgG2 was not transferred to the lamb despite significant titres in some immunised ewes. Maximal lamb titres were evident from ewes immunised with BSA in DEAE dextran where IgG1 was the predominant class of immunoglobulin produced. Both the experiments confirm that levels of the IgG1 isotype found at parturition in the ewes correlate directly with antibody transfer to the lamb. The results differ somewhat in that anti-TT antibody (IgG1) titres in ewes given primary and booster clostridial vaccine was equivalent to the titres found in
INFLUENCE OF ADJUVANTS AND IMMUNISATION REGIMES ON RESPONSE IN T H E EWE
175
t h e i r lambs, w h e r e a s in n o i n s t a n c e was t h e a n t i - B S A a n t i b o d y levels in lambs e q u i v a l e n t to t h e ewe titres. T h i s m a y reflect ongoing a n t i b o d y p r o d u c t i o n t h r o u g h o u t late p r e g n a n c y with t h e c u m u l a t i v e c o n c e n t r a t i o n of a n t i b o d y in c o l o s t r u m o v e r t h e weeks p r i o r to p a r t u r i t i o n . In a r e c e n t study, a n t i b o d y titres have b e e n p r o d u c e d in n e w b o r n fawns of a level 10 t i m e s h i g h e r t h a n the t i t r e s f o u n d in d e e r h i n d s s a m p l e d at t h e same t i m e ( H i b m a a n d Griffin, 1989). T h i s identifies t h e u n i q u e facility o f t h e r u m i n a n t to passively t r a n s f e r IgG1 a n d t h e p o t e n t i a l to exploit this p h e n o m e n o n using schedules biased t o w a r d s t h e s t i m u l a t i o n of IgG1 isotype. ACKNOWLEDGEMENTS We wish to t h a n k Mr. M. R o h l o f f a n d t h e A n i m a l P r o d u c t i o n Unit, M i n i s t r y of Agriculture a n d Fisheries, I n v e r m a y for ogranising e x p e r i m e n t a l flocks o f sheep a n d t h e i r c o n t i n u e d access. Our t h a n k s also go to Dr. K. B e h for m o n o clonal r e a g e n t s a n d Coopers A n i m a l H e a l t h (N.Z.) L i m i t e d for the T T reagent. G.R. g r a t e f u l l y a c k n o w l e d g e s scholarship s u p p o r t f r o m S o u t h l a n d Frozen M e a t s L i m i t e d .
REFERENCES Acres, S.D., Isaacson, R.E., Babuik, L.A. and Kapilany, R.A., 1979. Immunisation of calves against enterotoxigenic colibacillosis by vaccinating dams with purified K99 antigen and whole cell bacterins. Infect. Immun., 25: 121-126. Brar, J.S., Johnson, D.W., Muscoplatt, C.C., Shope, R.E. and Meiske, J.C., 1978. Maternal immunity to infectious bovine rhinotracheitis and bovine viral diarrhea viruses. Duration and effect on vaccination in young calves. Am. J. Vet. Res., 39: 241-244. Butler, J.E., 1983. Bovine immunoglobulins: An augmented review. Vet. Immunol. Immunopathol., 4: 43-152. Fleenor, W.A. and Stott, G.H., 1983. Quantification of bovine IgG, IgM and IgA antibodies to Clostridium perfringens fl-toxin by enzyme immunoassay. I. Preparturient immunisation for enhancement of passive transfer of immunity. Vet. Immunol. Immunopathol., 4: 579-591. Hibma, M. and Griffin, J.F.T., 1989. Active and passive immunisation in farmed red deer (Cervus elaphus ). Vet. Immunol. Immunopathol. submitted. Myers, L.L., 1978. Enteric colibacillosis in calves: Immunogenicity and antigenicity of E. coli antigens. Am. J. Vet. Res., 39: 761-765. Reynolds, G.E., Suttie, J. and Griffin, J.F.T., 1990. Humoral immunity in the ewe 1. The influence of adjuvancy and immunogenic substitution on immune reactivity following immunisation with protein antigens. Vet. Immunol. Immunopathol., 25: 1-12. Sheldrake, R.F. and Husband, A.J., 1985. Immune defences at mucosal surfaces in ruminants. J. Dairy Res., 52: 599-613. Wells, P.W., Snodgrass, D.R., Herring, J.A. and McDawson, A., 1978. Antibody titres to lamb rotavirus in colostrum and milk of vaccinated ewes. Vet. Rec., 103: 46-48.
167
H u m o r a l I m m u n i t y in the E w e 3. The I n f l u e n c e of A d j u v a n t s and I m m u n i s a t i o n R e g i m e s on I m m u n e R e a c t i v i t y in the B r e e d i n g E w e and her P r o g e n y G.E. R E Y N O L D S and J.F.T. G R I F F I N
Microbiology Department, University of Otago, Dunedin (New Zealand) {Accepted 14 November 1989)
ABSTRACT Reynolds, G.E. and Griffin, J.F.T., 1990. Humoral immunity in the ewe. 3. The influence of adjuvants and immunisation regimes on immune reactivity in the breeding ewe and her progeny. Vet. Immunol. Immunopathol., 25: 167-175. Breeding ewes were immunised with clostridial vaccine using different inoculation schedules. Results, showing differences in the class of antibody produced, were heavily dependent on the vaccination regime used. Immunoglobulin M (IgM) levels were significantly lower in ewes given double doses of vaccine compared to ewes given a single inoculation or no treatment at all ( P < 0.01 }. Neonatal lambs showed significant de novo IgM production with interference in this antibody production in the lambs of ewes vaccinated with the clostridial vaccine {P < 0.05 }. Immunoglobulin G l ( IgG 1 ) levels were significantly increased in all lambs which had mothers vaccinated with the clostridial vaccine prior to or during pregnancy (P < 0.025). The greatest quantity ()f IgG l was transferred to lambs when their mothers were given a double injection with primary" inoculation prior to conception and booster prepartum (P < 0.025). No antigen specific immunogl()bulin G2 (IgG2) was detected in the lambs. Ewes were also immunised with BSA and their isot)-pe specific serum antibody response was compared with their respective lambs. There was no detectable anti-BSA IgM in the lambs of all groups of ewes though specific IgG1 antibodies could be readily detected in the lambs of hyperimmunised ewes. The efficiency of transfer was related directly to the ability of the adjuvant to maximise IgG1 production in the ewe. Although immunised ewes produced high levels of IgG2, this was not transferred passively to the lamb.
INTRODUCTION
Vaccination of the pregnant ewe in the weeks prior to lambing is a common practice used to ensure passive transfer of immunity to the newborn. Antibody to Salmonella typhimurium lipopolysaccharide in colostrum has been shown to correlate with the specific immunoglobulin serum titre in the neonate
0165-2427/90/$03.50
© 1990 Elsevier Science Publishers B.V.
168
G.E. REYNOLDS AND J.F.T. GRIFFIN
(Fleenor and Stott, 1983). Material vaccination of pregnant animals with Escherichia coli or rotavirus induce high antibody titres in colostrum and milk which protect the offspring against enteric disease (Acres et al., 1979; Myers, 1978; Wells et al., 1978). Because IgG1 is the predominant class of antibody passively transferred via colostrum (Butler, 1983), vaccination for enhanced passive transfer of immunity is directed towards stimulating maximal IgG1 production in the dam to produce elevated colostral specific IgG 1 levels (Fleenor and Stott, 1983). Using the analysis of isotypic antibody produced, this study examines the effect of primary immunisation and secondary boosting of pregnant ewes to define conditions for maximal transfer of specific IgG1 antibody from the ewe to the lamb. The use of different immunisation protocols and different adjuvants allowed for the definition of optimal conditions to evoke memory and maximise IgG1 antibody titres. Two distinct antigens, bovine serum albumin (BSA) and clostridial toxoid vaccine were selected to elucidate the impact of pre-existing memory on passive immunity. Clostridial vaccines consist of a complex antigenic mixture which may evoke a variable primary and secondary response due to the level of endemic exposure of sheep flocks to clostridial organisms. Bovine serum albumin is a novel and well characterised primary immunogen to which the ewe should have no pre-existing memory. This allowed for the study of primary immunity and the development of secondary cellular memory and effector function under the influence of pregnancy. MATERIALSAND METHODS
The antigens The protein antigen BSA (Sigma, U.S.A.) was made up to 1000 ttg/ml in saline. The clostridial vaccine (Coopers Animal Health, N.Z.) was supplied at a dose used for proprietory vaccination.
The adjuvants The three adjuvants used in this study were aluminium hydroxide (alum), diethylaminoethyl dextran (DEAE dextran) and Freund's complete adjuvant {FCA), prepared as previously described (Reynolds et al., 1990). Clostridial vaccination A flock of 132 Romney ewes were divided into four groups and each exposed to different vaccination regimes (Table 1 ). Three groups were inoculated with the multivalent clostridial vaccine as a single injection prior to conception or prepartum, or as a double vaccine preconception and boosted prepartum. A further group received no treatment. Three weeks postpartum, blood samples were taken from both ewes and lambs. The serum was collected and stored at
INFLUENCE OF ADJUVANTSAND IMMUNISATIONREGIMESON RESPONSE IN THE EWE
169
TABLE 1 The immunisation regime for pregnant ewes inoculated with Clostridial vaccine preconception a n d / o r prepartum Group
Number of animals
Primary inoculation
Secondary inoculation
1 2 3 4
29 32 46 25
preconception prepartum preconception no t r e a t m e n t
no t r e a t m e n t no t r e a t m e n t prepartum no t r e a t m e n t
TABLE 2 The immunisation regime for pregnant ewes inoculated with BSA in alum for their primary injection and BSA in different adjuvants for their secondary inoculation Groups
Number of animals
Primary inoculation
Secondary inoculation
1
10
0.5ml BSA ~+0.5ml alum
2
10
0.5ml BSA ~+ 0.5ml alum same as above
3 4
10 4
same as above no t r e a t m e n t
0.5ml BSA 1+0.5ml DEAE dextran 0.5ml BSA ~+ 0.5ml FCA no t r e a t m e n t
11000p.g/ml.
- 2 0 °C until required. Isotype specific IgM, IgG1 and IgG2 antibody levels to
tetanus toxoid (TT) antigen (Coopers Animal Health, NZ) were determined using ELISA. Using a specific assay system to T T antigen allowed the detection of antibody from the non-specific multivalent anti-clostridial vaccine.
Adjuvancy study A group of 34 pregnant Perindale ewes treated with progesterone implants to synchronise oestrus, were used in this study. At day 80 of gestation, 30 of the animals were inoculated with 500/~g of BSA (Sigma, U.S.A. ) in alum adjuvant to evoke primary sensitisation. At day 56 post primary injection, corresponding to 136 days of gestation, animals were divided into three subgroups for the secondary inoculation (Table 2 ). Each group received 500/~g BSA emulisfied in FCA, DEAE dextran or alum adjuvants. A group of four non-immunised pregnant ewes were included as controls. Ewes were bled at parturition, lambs were bled 3 weeks postpartum and the sera stored at - 2 0 °C until required. The levels of anti-BSA antibody belonging to the different immunoglobulin isotypes IgM, IgG1 and IgG2 were assayed using ELISA.
170
G.E. REYNOI,DS AND J.F.T. GI~IFFIN
The E L I S A assay A four step indirect ELISA system was developed for the detection of classspecific antibodies to BSA and TT. The optimal concentrations of antigen, immune sheep serum, anti-sheep monoclonal antibody and anti-mouse peroxidase labelled conjugate were determined earlier for BSA (Reynolds et al., 1990 ). Tetanus toxoid was used at a concentration of 100 gg/ml for IgM assays and 10 ~g/ml for IgG1 and IgG2 assays.
RESUIJTS Clostridial vaccination All groups of animals immunised with clostridial vaccine, produced significant antibody titres. Comparison of IgM levels (Fig. 1) in the ewes revealed a significantly lower titre in animals given a primary dose preconception and a booster prepartum, compared with all other groups, ( P < 0.01 ). There was no difference between IgM levels in non-treated control ewes and animals given one injection of clostridial vaccine only (preconception or prepartum). Lambs born to non-treated mothers had significantly higher IgM titres than all other groups of vaccinated ewes {P<0.05) though all ewes had higher IgM titres than their lambs (P < 0.025 ). Comparison of IgG1 titres (Fig. 2) showed significantly higher {P < 0.01) anti-TT antibody responses in animals given a double dose of vaccine prior to conception and boosted prepartum, than other groups except the group vaccinated with a single inoculation prepartum. The ewes vaccinated prepartum produced an IgG1 response which was higher than non-vaccinated control ewes and the group of animals vaccinated with a single injection prior to conception (P<0.05). Lambs from mothers vaccinated twice with clostridial vaccine (preconception and prepartum) gave IgG1 titres higher than all other lamb groups (P < 0.025). Lambs born to mothers that were vaccinated prepartum only produced significantly higher IgG1 levels than the group of lambs from mothers immunised prior to conception only and the non-treated control animals ( P < 0.025). The IgG1 titres of lambs born to mothers immunised with a single injection prior to conception were significantly higher than lambs from non-vaccinated control ewes (P < 0.025 ). Ewes given a double vaccination prior to conception and prepartum, had IgG 1 titres equivalent to those found in their lambs, while all other ewes had significantly higher titres of anti-TT IgG 1 than their respective lambs (P < 0.05 ). Non-vaccinated control ewes had lambs that had the lowest relative level of anti-TT IgG1 compared to their mothers. The anti-TT IgG2 titres (Fig. 3) showed that ewes given a double vaccination prior to conception and prepartum produced higher IgG2 levels than all other groups (P < 0.01 ). The animals given a single injection of clostridial vaccine prior to conception or prepartum only, produced equal IgG2 levels. Both groups injected with a single dose only, produced higher IgG2 titres than un-
INFLUENCE OF ADJUVANTSAND |MMUNISATION REGIMES ON RESPONSE IN THE EWE
171
50 4O •-< ¢n ._% ~11
30 20
[] •
Ewe titre Lamb titre
[] •
Ewe titre Lamb titre
[] •
Ewe titre Lamb titre
10 0 1000
800 600 400 200 0 100
Y/~
80
#///
z///
60
//// //// ////
40 3~3 ~t~
1///
~/A ~/z ///¢
20 1~/~
..._ :;};
0 Non-treated PrePre-par~um PreControls Conception Conception & Pre-partum Time of inoculation
Figs, 1, 2 and 3. T h e effect of i m m u n i s a t i o n schedules on IgM, IgG1 and IgG2 antibody transfer frow ewe u) lamb using Clostridial vaccines, preconception a n d / o r prepartum. Antibody levels were measured 3 weeks p o s t p a r t u m in b o t h the ewes a n d lambs.
vaccinated control ewes ( P < 0.005). Little anti-TT IgG2 was produced in lambs (Fig. 3) above background control levels. The group of lambs with mothers given the double vaccination produced IgG2 titres that were higher than animals from all other groups ( P < 0.01 ).
172
G.E. REYNOLDSAND J.F.T. GRIFFIN 50 40
"E
30
[]
<
•
20
Ewe titre Lamb titre
LIJ
tO 0
v 7~., ~
..].
T
1000 8OO
600
[]
//J/
400
•
Ewe titre Lamb titre
[] •
Ewe titre Lamb titre
LU
2O0 0 100 80
6O 40
20
0 Non-treated BSA-alum BSA-DEAE controls
BSA-FCA
dextran Adjuvant inoculated
Figs. 4, 5 and 6. The transfer of anti-BSA IgM, IgG1 and IgG2 antibody from ewe to lamb when ewes were inoculated with BSA in different adjuvants during pregnancy. Antibody levels were measured at parturition for the ewes and 3 weeks postpartum for the lambs.
Adjuvancy study Variable ewe and lamb titres were found following immunisation of the mothers with BSA in alum, DEAE dextran or FCA. The anti-BSA IgM response (Fig. 4) showed that ewes injected with BSA in DEAE dextran produced significantly higher IgM titres compared with other immunised
I N F L U E N C E OF A D J U V A N T S A N D I M M U N I S A T I O N R E G I M E S O N R E S P O N S E IN T H E E W E
173
( P < 0.05) or control (P < 0.01 ) groups. No lamb IgM titres to BSA were evident in any of the groups tested. The IgG1 titres in the ewes (Fig. 5) were significantly higher titres in animals immunised with BSA in DEAE dextran than all other groups ( P < 0.005 ). The ewes immunised with BSA in FCA produced IgG1 antibody titres which were significantly higher than the ewes immunised with BSA in alum and nonimmunised control ewes (P<0.05). Immunisation with BSA in alum gave higher IgG1 levels than non-immunised control animals (P < 0.01 ). Anti-BSA IgG1 titres in lambs from immunised mothers were all higher than the IgG1 levels in lambs from non-immunised control ewes (P<0.05). The group of lambs from mothers that were exposed to BSA in DEAE dextran responded with higher IgG1 titres than all other groups ( P < 0.05). The comparison of titres in ewes given BSA in different adjuvants with their respective lamb IgG1 titres showed detectable but significantly lower titres in the lambs when compared with the ewes (P < 0.025). The anti-BSA IgG2 response in animals given BSA in adjuvant was found to be markedly different from their IgG1 antibody levels (Fig. 6). The ewes immunised with BSA in FCA produced significantly higher titres than all other groups (P < 0.005). The ewes injected with BSA in DEAE dextran produced higher IgG2 levels than the group of ewes immunised with BSA in alum or non-treated controls (P < 0.025). There was no significant titre of IgG2 in any lamb, irrespective of how the ewes were immunised. DISCUSSION These experiments show the amount and the quality of specific antibody produced around parturition in the ewe influence the passive IgG1 antibody titre acquired by the lamb. The vaccination of pregnant ewes with clostridial vaccine using different vaccination regimes produced a high antibody titre in vaccinated animals compared to non-vaccinated controls. The IgM antibody response to T T was particularly strong across all ewe and lamb groups including the non-treated controls. The observation of high titre especially in nonvaccinated ewes reflects the natural exposure to clostridial antigens in New Zealand sheep flocks. Results in ewes show that a double vaccination preconception and prepartum caused a significant reduction in IgM titre compared to all other treatment groups. This would be expected because classical switch from IgM to IgG production would have occurred upon repeated challenge with the clostridial antigen. All lambs showed high IgM antibody levels at 3 weeks of age, especially lambs of non-treated control mothers. These high levels of IgM provide further evidence of the endemic exposure of sheep to clostridial organisms. As antibody of the IgM isotype is not transferred in colostrum (Butler, 1983 ), the IgM titres present in these lambs are likely to be the result of the de novo antibody production. Lambs from vaccinated mothers had lower
174
G.E. REYNOI,DS AND J.F.T. GRIFFIN
IgM levels than lambs born to non-vaccinated ewes inferring that maternal antibody from vaccinated mothers interferes with the lamb's de novo immune response, as documented in other studies (Brar et al., 1978). Using tetanus toxoid vaccine, it was evident that the highest IgG1 antibody titres were in ewes vaccinated prior to conception and boosted prior to lambing. This was also reflected in optimal passive transfer of IgG1 antibody to the lambs. Whereas a single vaccination preconception or prepartum gave a significant ewe titre and passive transfer to lambs, it was at significantly lower levels than found following double vaccination of the ewe. The ewes given a single injection prepartum produced IgG1 antibody titres similar to the ewes inoculated twice (preconception and prepartum) but the amount of antibody transferred to the lambs was significantly higher in the ewes given two doses of vaccine. This reflects qualitative differences in maternal IgG 1 antibody titres in ewes primed pre-conception and boosted prepartum compared with ewes given a primary inoculum prepartum only. Because the untreated control group of ewes produced detectable natural anti-TT antibody it was not possible to consider the vaccine response as a primary stimulus. However, the low IgG1 titres found in non-vaccinated ewes and the predominance of the IgM isotype in these ewes was compatible with inadequate IgG1 antibody transfer to the lambs. The IgG2 response to the clostridial vaccine correlated with the IgG1 titres in the ewe though this was not reflected in the IgG2 titre in their lambs. A double inoculation regime (preconception and prepartum) was mandatory for efficient anti-TT IgG2 production in the ewe but the material IgG2 antibody was not passively transferred to the lambs. This is consistent with findings in the literature on ruminants which infer that IgG1 is the predominant class of antibody transferred from mother to offspring (Butler, 1983). Some IgG2 was detected in the lambs of ewes given a double inoculum. This may reflect the local production of anti-TT IgG2 antibody in the mammary gland and its subsequent transfer to the lamb (Sheldrake and Husband, 1985). The immunisation of pregnant and non-pregnant ewes with BSA in alum, DEAE dextran or FCA showed the effect adjuvant can have on the quantity and class of antibody produced in the ewe and the subsequent transfer to IgG1 to the lamb. The ewes injected with BSA in DEAE dextran produced the highest IgG1 titres and correlated with higher titres of IgG1 in their lambs. Consistent with the previous experiment, IgG2 was not transferred to the lamb despite significant titres in some immunised ewes. Maximal lamb titres were evident from ewes immunised with BSA in DEAE dextran where IgG1 was the predominant class of immunoglobulin produced. Both the experiments confirm that levels of the IgG1 isotype found at parturition in the ewes correlate directly with antibody transfer to the lamb. The results differ somewhat in that anti-TT antibody (IgG1) titres in ewes given primary and booster clostridial vaccine was equivalent to the titres found in
INFLUENCE OF ADJUVANTS AND IMMUNISATION REGIMES ON RESPONSE IN T H E EWE
175
t h e i r lambs, w h e r e a s in n o i n s t a n c e was t h e a n t i - B S A a n t i b o d y levels in lambs e q u i v a l e n t to t h e ewe titres. T h i s m a y reflect ongoing a n t i b o d y p r o d u c t i o n t h r o u g h o u t late p r e g n a n c y with t h e c u m u l a t i v e c o n c e n t r a t i o n of a n t i b o d y in c o l o s t r u m o v e r t h e weeks p r i o r to p a r t u r i t i o n . In a r e c e n t study, a n t i b o d y titres have b e e n p r o d u c e d in n e w b o r n fawns of a level 10 t i m e s h i g h e r t h a n the t i t r e s f o u n d in d e e r h i n d s s a m p l e d at t h e same t i m e ( H i b m a a n d Griffin, 1989). T h i s identifies t h e u n i q u e facility o f t h e r u m i n a n t to passively t r a n s f e r IgG1 a n d t h e p o t e n t i a l to exploit this p h e n o m e n o n using schedules biased t o w a r d s t h e s t i m u l a t i o n of IgG1 isotype. ACKNOWLEDGEMENTS We wish to t h a n k Mr. M. R o h l o f f a n d t h e A n i m a l P r o d u c t i o n Unit, M i n i s t r y of Agriculture a n d Fisheries, I n v e r m a y for ogranising e x p e r i m e n t a l flocks o f sheep a n d t h e i r c o n t i n u e d access. Our t h a n k s also go to Dr. K. B e h for m o n o clonal r e a g e n t s a n d Coopers A n i m a l H e a l t h (N.Z.) L i m i t e d for the T T reagent. G.R. g r a t e f u l l y a c k n o w l e d g e s scholarship s u p p o r t f r o m S o u t h l a n d Frozen M e a t s L i m i t e d .
REFERENCES Acres, S.D., Isaacson, R.E., Babuik, L.A. and Kapilany, R.A., 1979. Immunisation of calves against enterotoxigenic colibacillosis by vaccinating dams with purified K99 antigen and whole cell bacterins. Infect. Immun., 25: 121-126. Brar, J.S., Johnson, D.W., Muscoplatt, C.C., Shope, R.E. and Meiske, J.C., 1978. Maternal immunity to infectious bovine rhinotracheitis and bovine viral diarrhea viruses. Duration and effect on vaccination in young calves. Am. J. Vet. Res., 39: 241-244. Butler, J.E., 1983. Bovine immunoglobulins: An augmented review. Vet. Immunol. Immunopathol., 4: 43-152. Fleenor, W.A. and Stott, G.H., 1983. Quantification of bovine IgG, IgM and IgA antibodies to Clostridium perfringens fl-toxin by enzyme immunoassay. I. Preparturient immunisation for enhancement of passive transfer of immunity. Vet. Immunol. Immunopathol., 4: 579-591. Hibma, M. and Griffin, J.F.T., 1989. Active and passive immunisation in farmed red deer (Cervus elaphus ). Vet. Immunol. Immunopathol. submitted. Myers, L.L., 1978. Enteric colibacillosis in calves: Immunogenicity and antigenicity of E. coli antigens. Am. J. Vet. Res., 39: 761-765. Reynolds, G.E., Suttie, J. and Griffin, J.F.T., 1990. Humoral immunity in the ewe 1. The influence of adjuvancy and immunogenic substitution on immune reactivity following immunisation with protein antigens. Vet. Immunol. Immunopathol., 25: 1-12. Sheldrake, R.F. and Husband, A.J., 1985. Immune defences at mucosal surfaces in ruminants. J. Dairy Res., 52: 599-613. Wells, P.W., Snodgrass, D.R., Herring, J.A. and McDawson, A., 1978. Antibody titres to lamb rotavirus in colostrum and milk of vaccinated ewes. Vet. Rec., 103: 46-48.