Veterinary Microbiology, 24 (1990) 21-27 Elsevier Science Publishers B.V., Amsterdam
21
Efficacy against ovine enzootic abortion of an experimental vaccine containing purified elementary bodies of Chlamydia psittaci I.E. Anderson, T.W. Tan, G.E. Jones and A.J. Herring Moredun Research Institute, 408 Gilmerton Road, Edinburgh, EH17 7JH (Great Britain) (Accepted 12 December 1989)
ABSTRACT Anderson, I.E., Tan, T.W., Jones, G.E. and Herring, A.J., 1990. Efficacy against ovine enzootic abortion of an experimental vaccine containing purified elementary bodies of Chlamydia psittaci. Vet. Microbiol., 24:21-27. A vaccine prepared from purified, inactivated elementary bodies of Chlamydia psittaci protected sheep against abortion after subcutaneous challenge with live chlamydiae. Immunoblot analysis of serum samples revealed a consistently dominant antibody response against the chlamydial major outer membrane protein in all vaccinated sheep. Reactions to other chlamydial antigens were also detected but were less pronounced or inconsistent. Serological responses detected by complement fixation were variable and did not correlate with immunity.
INTRODUCTION
Ovine enzootic abortion (OEA), which is caused by Chlamydia psittaci, is the most common form of infectious abortion in sheep in Great Britain and is of major economic importance (Aitken et al., 1986). C. psittaci invades the placental tissue of sheep but does not appear to proliferate until after 90 days of gestation (Buxton et al., 1990), eventually causing a necrotising placentitis leading to abortion in late pregnancy, premature lambing or the birth of weakly lambs (Aitken, 1983). Since 1956, an egg-grown, formalin-inactivated vaccine has been available to aid in the control of the disease in Great Britain (Foggie, 1973 ). However, outbreaks of OEA in vaccinated flocks (Linklater and Dyson, 1979), prompted experiments which suggested the involvement of field isolates differing antigenicaUy from the vaccine strain or having enhanced virulence (Aitken et al., 1986). Those findings led to the incorporation of a second strain of C. psittaci ($26/3) in the sole OEA vaccine currently available corn0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
22
I.E. ANDERSON ET AL.
mercially in Great Britain (Ovine Enzootic Abortion Vaccine, Coopers Animal Health Ltd, Crewe, Cheshire). In this study we sought to determine whether a vaccine containing purified chlamydial elementary bodies (EB) could protect against OEA in a challenge experiment. The specific antibody responses of sheep to this vaccine were analysed by the complement fixation test (CFT) and by immunoblotting, to identify those antigens that might correlate with protection, and which could therefore be useful for further evaluation as potential subunit vaccines. MATERIALS AND METHODS
Preparation of purified EB vaccine A strain of C. psittaci ($26/3) isolated from a severe outbreak of OEA in a vaccinated flock was used throughout (Anderson, 1986a). Chlamydial EB were purified from cell culture harvests by gradient ultracentrifugation as previously described (McClenaghan et al., 1984), but using Urografin 370 (Schering AG, F.R.G. ) instead of Renografin as the density medium. Negative-stain (phosphotungstic acid) electron microscopy was used to check the EB preparation was pure and free from reticulate bodies. Purified EB (800/~g) suspended in Tris-HC1 buffer pH 7.4 (20 mM Tris, 150 mM KC1 ) were inactivated using the method of Zaia and Oxman ( 1977 ). The inactivated EB were pelleted at 53 0 0 0 × g at 4°C for 1 h, resuspended in 4 ml of 0.15 M NaCI then adsorbed with aluminium hydroxide ("Alhydrogel", Miles Laboratories, Slough, Great Britain, diluted 1 : 10 in distilled water) until the solution turned flocculent. This preparation was emulsified with an equal volume of Bayol 82 and Arlacel A mixed in a 9:1 ratio.
Vaccination and challenge of ewes Thirteen Scottish Blackface ewes ( 5-6 years old ) screened and negative for serum antibodies against chlamydia by the CFT were mated after synchronisation of oestrus. Ten and 31 days later, 6 of them were injected subcutaneously with 1 ml of EB vaccine. The remaining 7 ewes were not vaccinated. Serum samples taken at intervals were analysed by the CFT and by immunoblotting. All 13 ewes were challenged at 70 days of gestation (60 days after the first vaccination) by subcutaneous injection of 1 ml of live $26/3 containing 1055 50% chick embryo lethal doses. The 2 groups were kept in separate pens during the experiment.
Detection and isolation of chlamydia At abortion or lambing, smears of placental membranes, if available, otherwise vaginal swabs, were stained by the modified Ziehl Neelsen method (Stamp et al., 1950) and examined for EB. The same samples were cultured in baby hamster kidney cells treated with 5-iodo-2'-deoxyuridine (Anderson, 1986b).
AN EXPERIMENTAL VACCINE CONTAINING ELEMENTARY BODIES OF CHLAMYDIA PSITTACI
23
Electrophoresis and immunoblotting Purified EB were solubilised and subjected to sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) according to the m e t h o d of Laemmli et al. ( 1970 ). The immunoblotting procedure of Sharp and Herring (1983) was used with the following modifications. The wash solution and diluent were replaced with phosphate buffered saline pH 7.4 containing 0.5% Tween 20 (PBST). Blocking was achieved by a wash in PBST. The membranes were immunologically probed with serum samples diluted 1 : 100 in PBST.
CFT The CFT used was a microtitre modification of the m e t h o d of Stamp et al. (1952). RESULTS
Efficacy of the experimental vaccine No abortions occurred in the vaccinated ewes and chlamydiae were not detectable in these animals following lambing. In contrast, chlamydiae were isolated from 5 of the 7 unvaccinated sheep, 4 of which aborted. TABLE 1 C o m p l e m e n t fixation test responses (logz titres) and isolation o f Chlamydia in vaccinated and unvaccinated ewes challenged at 70 days gestation (between 8 and 9 weeks after 1st vaccination) with 10 ELD 50 m l - 1 o f C. psittaci strain (strain ( $26 / 3 ) ) Ewe no.
Weeks after initial vaccination
Chlamydia positive
0
2
4
6
10
12
14
18
21
-
V
V
V
C
C
C
P
P
Vaccinated ewes 1 2
1
5
7
7
7
6
5
51
6
-
3 4 5
1 1 l 1
1 3 5 1
1 1 8 1
1 3 7 1
1 1 9 3
1 3 8 6
1 1 6 3
11 71 NS 41
1 4 NS NS
-
6
1
1
7
7
8
8
6
4j
5
NV
NV
NV
NV
C
C
C
P
P
U n v a c c i n a t e d ewes 7 1 NS 8 1 NS 9 1 NS 10 1 NS 11 1 NS 12 1 NS 13 1 NS
NS NS NS NS NS NS NS
1 1 1
3 3 1
3 4 1
1 1
1 3
3 4
1 71 1 51
81 6 61 NS
NS NS NS NS
3
11
NS
1
3
4
1
3
6
5 71
NS NS
NS NS
-
+ + (A) + (A) + (A) + (A)
+ , positive; - , negative; A, aborted; 1Sample taken at or near parturition; NS, not sampled; NV, not vaccinated; V, response to vacination; C, response to challenge; P, response at or near parturition.
24
I.E. ANDERSON ET AL.
The differences between groups in respect of abortions and isolation of the agent were significant ( P < 0.05, Fisher One Tail Exact Test ).
Serological analysis Only CFT responses at titres greater than or equal to 5 log2 ( 1/32) were considered to be significant (Jones and Anderson, 1988 ). The vaccinated ewes gave variable responses by the CFT (Table 1 ). Before challenge, 3 of the 6 were positive; following challenge but before parturition 4 had significant titres, and after parturition 3 ewes remained positive. No significant titres were observed in non-vaccinated ewes before challenge (Table 1 ), but thereafter a significant rise in titre occurred in 4 ewes. After abortion or lambing, 5 of the 7 ewes were positive by the CFT. Of the 60 to 80 chlamydial components resolved by SDS-PAGE and stained with Coomassie brilliant blue R250 or by silver staining (Morrisey, 1981 ), up to 24 are immunoreactive when tested with sera from non-vaccinated sheep convalescent from OEA (unpublished observation). The predominant rela.
lb. E w e No
1 2 3 4
W e e k s Post Vaccination 5
6
02
•
17
~1
12
4 8 10121421
Fig. 1. (a) Immunoblot showing response 8 weeks after initial vaccination with the purified EB vaccine. (b) Immunoblot showing typical time course of response to vaccination in a single vaccinated unchallenged ewe.
AN EXPERIMENTAL VACCINE C O N T A I N I N G ELEMENTARY BODIES OF CHLAMYDIA PS1TTACI
25
sponse 8 weeks after vaccination of all 6 vaccinated ewes was against the major outer membrane protein (MOMP) (Fig. I a). Antibody responses to other antigens were observed but were less pronounced or inconsistently present. The time course of antibody responses in a vaccinated unchallenged sheep as detected by immunoblotting is shown in Fig. 1b. The response shown before challenge (0-8 weeks) was typical of vaccinated challenged sheep. Pre-challenge sera from the unvaccinated sheep were entirely free from chlamydia-specific antibodies detected by immunoblotting. Reaction to challenge became apparent only a few days before or at abortion when faint bands of reaction to several antigens including MOMP were observed. After abortion, a strong antibody response to these bands was detected. No such response occurred in the 2 unvaccinated ewes that did not abort. DISCUSSION
A vaccine containing a purified preparation of chlamydial EB prevented abortion in vaccinated ewes. Immunoblot analysis of the antibody response to vaccination indicated that the predominant response was against MOMP, and that these anti-MOMP antibodies persisted throughout the period of the experiment without diminution. Their likely importance in protection against OEA is supported by their marked and persistent occurrence in the sera and lymph of ewes convalescent from OEA (Huang et al., 1990 ) It is possible, however, that the marked anti-MOMP response was simply a mass effect, since MOMP is the predominant chlamydial protein, and that other protective immunogens, both proteinaceous and non-proteinaceous, were at suboptimal concentrations for the level of sensitivity of the immunoblot procedure used. Antibodies to chlamydial MOMP have been shown to be neutralizing in vitro and protective in vivo. Antibodies raised in rabbits (Caldwell and Perry, 1982) and murine monoclonal antibodies (Peeling et al., 1984) against the MOMP of C. trachomatis strains neutralized their infectivity for HeLa 229 cells. Murine monoclonal antibody against the immunoaccessible MOMP of C. trachomatis serovar B neutralized the infectivity of the organism for monkey eyes and protected mice from toxic death after intravenous injection (Zhang et al., 1987 ). MOMP has recently been implicated in the attachment of C. trachomatis during host cell infection (Su et al., 1988 ). The immunoblot procedure used did not detect antibodies to non-proteinaceous components such as lipopolysaccharide (LPS) (Batteiger et al., 1982 ), but chlamydial LPS is detected by the CFT (McEwen and Foggie, 1954; Dawson et al., 1986). The variability in CFT responses to vaccination seen in these studies was not unexpected, and confirmed the lack of correlation between this test and immunity (Dawson et al., 1986). Why antibodies against chlamydia in the non-vaccinated ewes could not be
26
I.E. A N D E R S O N ET AL.
detected by the CF test until at least 36 days after experimental infection is not clear. Preliminary data from studies on experimental lymph node infections suggest that the initial antibody response at the lymph node is transient and difficult to detect at the systemic level by the CFT or even by the sensitive immunoblotting technique (Huang et al., 1990). It is more likely that inoculation at 70 days of gestation and the delay in proliferation in the placenta until after 90 days (Buxton et al., 1990) may explain this effect. The high efficacy shown by the EB vaccine was encouraging considering that unpurified egg or cell culture-grown vaccines usually give only between 40 and 80% protection depending on the challenge system used (Aitken et al., 1986). The reason for the better performance of this vaccine may have been due to the use of a double vaccination regime. Alternatively, it may have been due to the purity of the material used in its preparation, and thus the greater accessibility of MOMP to the immune system. This would imply that contaminants in unpurified vaccines have an adverse effect on stimulating protection. In either case, MOMP was strongly implicated in this study as an immunogen of importance in OEA. Work currently in progress with a MOMPenriched vaccine preparation should clarify the role of this protein in protection against OEA. ACKNOWLEDGEMENTS
T.W. Tan is in receipt of the Wooldridge Farm Livestock Fellowship from the Animal Health Trust, Great Britain. We thank A. Dawson and P. Dewar for expert technical assistance in the purification and production of the reagents used in immunoblotting, K. Hall and K.W. Quinn for cell culture support, and the department of Clinical Studies for the welfare of the animals.
REFERENCES Aitken, I.D., 1983. Enzootic (chlamydial) abortion. In: Diseases of Sheep. Blackwell Scientific Publications, Oxford, pp. 119-123. Aitken, I.D., Anderson, I.E. and Robinson, G.W., 1986. Ovine chlamydial abortion: limitations of inactivated vaccine chlamydial diseases of ruminants. In: I.D. Aitken (Editor), CEC Report, No. EUR 10056 EN, Luxembourg, pp. 55-65. Anderson, I.E., 1986a. Comparison of the virulence in mice of some ovine isolates of Chlamydia psittaci. Vet. Microbiol., 12:212-220. Anderson, I.E., 1986b. Comparison of five ovine isolates of Chlamydia psittaci: an evaluation of three cell culture treatments. Med. Lab. Sci., 43: 241-248. Batteiger, B., Newhall V, W.J. and Jones, R.B., 1982. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods, 55: 297-307.
AN EXPERIMENTAL VACCINE CONTAINING ELEMENTARY BODIES OF CHLAMYDIA PSITTA~7
27
Buxton, D., Barlow, R.M., Finlayson, J., Anderson, I.E. and Mackellar, A., 1990. Observation on the pathology of Chlamydia psittaci infection of pregnant sheep. J. Comp. Pathol. Caldwell, H.D. and Perry, L.J., 1982. Neutralization of Chlamydia trachomatis infectivity with antibodies to the major outer membrane protein. Infect. Immun., 18: 745-754. Dawson, M., Zaghloul, A. and Wilsmore, A.J., 1986. Ovine enzootic abortion: experimental studies of immune responses. Res. Vet. Sci., 40: 59-64. Foggie, A., 1973. Preparation of vaccines against enzootic abortion of ewes. A review of the research work at the Moredun Institute. Vet. Bull., 43: 587-590. Huang, H.S., Tan, T.W., Buxton, D., Anderson, I.E. and Herring, A.J., 1990. Antibody response of the ovine lymph node to experimental infection with an ovine abortion strain of Chlamydia psittaci. Vet. Microbiol., 21: 345-351. Jones, G.E. and Anderson, I.E., 1988. Chlamydia psittaci: is tonsillar tissue the portal of entry in ovine enzootic abortion? Res. Vet. Sci., 44:260-261. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Linklater, K.A. and Dyson, D.A., 1979. Field studies on enzootic abortion of ewes in south-east Scotland. Vet. Rec., 105: 387-389. McClenaghan, M., Herring, A.J. and Aitken, I.D., 1984. Comparison of Chlamydia psittaci isolates by DNA restriction endonuclease analysis. Infect. Immun., 45: 384-389. McEwen, A.D. and Foggie, A., 1954. Enzootic abortion of ewes: Comparative studies of different vaccines. Vet. Rec., 60: 393-397. Morrisey, J.H., 1981. Silver stain for proteins in polyacrylamide gels. A modified procedure will enhance sensitivity. Anal. Biochem., 117:307-310. Peeling, R., Maclean, I.W. and Brunham, R.C., 1984. In vitro neutralization of Chlamydia trachomatis with monoclonal antibody to an epitope on the major outer membrane protein. Infect. Immun., 46: 484-488. Sharp, J.M. and Herring, A.J., 1983. Sheep pulmonary adenomatosis: demonstration of a protein which cross-reacts with the major core proteins of Mason-Pfizer monkey virus and mouse mammary tumour virus. J. Gen. Virol., 64: 2323-2327. Stamp, J.T., McEwen, A.D., Watt, J.A.A. and Nisbet, D.I., 1950. Enzootic abortion in ewes. I. Transmission of the disease. Vet. Rec., 62: 251-254. Stamp, J.T., Watt., J.A.A. and Cockburn, R.B., 1952. Enzootic abortion in ewes. Complement fixation test. J. Comp. Pathol., 62: 93-101. Su, H., Zhang, Y-X., Barrera, O., Watkins, N.G. and Caldwell, H.D., 1988. Differential effect of trypsin on infectivity of Chlamydia trachomatis: loss of infectivity requires cleavage of major outer membrane protein variable domains II and IV. Infect. Immun., 56: 2094-2100. Zaia, J.A. and Oxman, M.N., 1977. Antibody to Varicella-Zoster virus induced membrane antigen: immunofluorescence assay using monodisperse glutaraldehyde-fixed target cells. J. Infect. Dis., 136: 519-530. Zhang, Y-X., Stewart, S., Joseph, T., Taylor, H.R. and Caldwell, H.D., 1987. Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis. J. Immunol., 138:575-581.
21
Efficacy against ovine enzootic abortion of an experimental vaccine containing purified elementary bodies of Chlamydia psittaci I.E. Anderson, T.W. Tan, G.E. Jones and A.J. Herring Moredun Research Institute, 408 Gilmerton Road, Edinburgh, EH17 7JH (Great Britain) (Accepted 12 December 1989)
ABSTRACT Anderson, I.E., Tan, T.W., Jones, G.E. and Herring, A.J., 1990. Efficacy against ovine enzootic abortion of an experimental vaccine containing purified elementary bodies of Chlamydia psittaci. Vet. Microbiol., 24:21-27. A vaccine prepared from purified, inactivated elementary bodies of Chlamydia psittaci protected sheep against abortion after subcutaneous challenge with live chlamydiae. Immunoblot analysis of serum samples revealed a consistently dominant antibody response against the chlamydial major outer membrane protein in all vaccinated sheep. Reactions to other chlamydial antigens were also detected but were less pronounced or inconsistent. Serological responses detected by complement fixation were variable and did not correlate with immunity.
INTRODUCTION
Ovine enzootic abortion (OEA), which is caused by Chlamydia psittaci, is the most common form of infectious abortion in sheep in Great Britain and is of major economic importance (Aitken et al., 1986). C. psittaci invades the placental tissue of sheep but does not appear to proliferate until after 90 days of gestation (Buxton et al., 1990), eventually causing a necrotising placentitis leading to abortion in late pregnancy, premature lambing or the birth of weakly lambs (Aitken, 1983). Since 1956, an egg-grown, formalin-inactivated vaccine has been available to aid in the control of the disease in Great Britain (Foggie, 1973 ). However, outbreaks of OEA in vaccinated flocks (Linklater and Dyson, 1979), prompted experiments which suggested the involvement of field isolates differing antigenicaUy from the vaccine strain or having enhanced virulence (Aitken et al., 1986). Those findings led to the incorporation of a second strain of C. psittaci ($26/3) in the sole OEA vaccine currently available corn0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
22
I.E. ANDERSON ET AL.
mercially in Great Britain (Ovine Enzootic Abortion Vaccine, Coopers Animal Health Ltd, Crewe, Cheshire). In this study we sought to determine whether a vaccine containing purified chlamydial elementary bodies (EB) could protect against OEA in a challenge experiment. The specific antibody responses of sheep to this vaccine were analysed by the complement fixation test (CFT) and by immunoblotting, to identify those antigens that might correlate with protection, and which could therefore be useful for further evaluation as potential subunit vaccines. MATERIALS AND METHODS
Preparation of purified EB vaccine A strain of C. psittaci ($26/3) isolated from a severe outbreak of OEA in a vaccinated flock was used throughout (Anderson, 1986a). Chlamydial EB were purified from cell culture harvests by gradient ultracentrifugation as previously described (McClenaghan et al., 1984), but using Urografin 370 (Schering AG, F.R.G. ) instead of Renografin as the density medium. Negative-stain (phosphotungstic acid) electron microscopy was used to check the EB preparation was pure and free from reticulate bodies. Purified EB (800/~g) suspended in Tris-HC1 buffer pH 7.4 (20 mM Tris, 150 mM KC1 ) were inactivated using the method of Zaia and Oxman ( 1977 ). The inactivated EB were pelleted at 53 0 0 0 × g at 4°C for 1 h, resuspended in 4 ml of 0.15 M NaCI then adsorbed with aluminium hydroxide ("Alhydrogel", Miles Laboratories, Slough, Great Britain, diluted 1 : 10 in distilled water) until the solution turned flocculent. This preparation was emulsified with an equal volume of Bayol 82 and Arlacel A mixed in a 9:1 ratio.
Vaccination and challenge of ewes Thirteen Scottish Blackface ewes ( 5-6 years old ) screened and negative for serum antibodies against chlamydia by the CFT were mated after synchronisation of oestrus. Ten and 31 days later, 6 of them were injected subcutaneously with 1 ml of EB vaccine. The remaining 7 ewes were not vaccinated. Serum samples taken at intervals were analysed by the CFT and by immunoblotting. All 13 ewes were challenged at 70 days of gestation (60 days after the first vaccination) by subcutaneous injection of 1 ml of live $26/3 containing 1055 50% chick embryo lethal doses. The 2 groups were kept in separate pens during the experiment.
Detection and isolation of chlamydia At abortion or lambing, smears of placental membranes, if available, otherwise vaginal swabs, were stained by the modified Ziehl Neelsen method (Stamp et al., 1950) and examined for EB. The same samples were cultured in baby hamster kidney cells treated with 5-iodo-2'-deoxyuridine (Anderson, 1986b).
AN EXPERIMENTAL VACCINE CONTAINING ELEMENTARY BODIES OF CHLAMYDIA PSITTACI
23
Electrophoresis and immunoblotting Purified EB were solubilised and subjected to sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) according to the m e t h o d of Laemmli et al. ( 1970 ). The immunoblotting procedure of Sharp and Herring (1983) was used with the following modifications. The wash solution and diluent were replaced with phosphate buffered saline pH 7.4 containing 0.5% Tween 20 (PBST). Blocking was achieved by a wash in PBST. The membranes were immunologically probed with serum samples diluted 1 : 100 in PBST.
CFT The CFT used was a microtitre modification of the m e t h o d of Stamp et al. (1952). RESULTS
Efficacy of the experimental vaccine No abortions occurred in the vaccinated ewes and chlamydiae were not detectable in these animals following lambing. In contrast, chlamydiae were isolated from 5 of the 7 unvaccinated sheep, 4 of which aborted. TABLE 1 C o m p l e m e n t fixation test responses (logz titres) and isolation o f Chlamydia in vaccinated and unvaccinated ewes challenged at 70 days gestation (between 8 and 9 weeks after 1st vaccination) with 10 ELD 50 m l - 1 o f C. psittaci strain (strain ( $26 / 3 ) ) Ewe no.
Weeks after initial vaccination
Chlamydia positive
0
2
4
6
10
12
14
18
21
-
V
V
V
C
C
C
P
P
Vaccinated ewes 1 2
1
5
7
7
7
6
5
51
6
-
3 4 5
1 1 l 1
1 3 5 1
1 1 8 1
1 3 7 1
1 1 9 3
1 3 8 6
1 1 6 3
11 71 NS 41
1 4 NS NS
-
6
1
1
7
7
8
8
6
4j
5
NV
NV
NV
NV
C
C
C
P
P
U n v a c c i n a t e d ewes 7 1 NS 8 1 NS 9 1 NS 10 1 NS 11 1 NS 12 1 NS 13 1 NS
NS NS NS NS NS NS NS
1 1 1
3 3 1
3 4 1
1 1
1 3
3 4
1 71 1 51
81 6 61 NS
NS NS NS NS
3
11
NS
1
3
4
1
3
6
5 71
NS NS
NS NS
-
+ + (A) + (A) + (A) + (A)
+ , positive; - , negative; A, aborted; 1Sample taken at or near parturition; NS, not sampled; NV, not vaccinated; V, response to vacination; C, response to challenge; P, response at or near parturition.
24
I.E. ANDERSON ET AL.
The differences between groups in respect of abortions and isolation of the agent were significant ( P < 0.05, Fisher One Tail Exact Test ).
Serological analysis Only CFT responses at titres greater than or equal to 5 log2 ( 1/32) were considered to be significant (Jones and Anderson, 1988 ). The vaccinated ewes gave variable responses by the CFT (Table 1 ). Before challenge, 3 of the 6 were positive; following challenge but before parturition 4 had significant titres, and after parturition 3 ewes remained positive. No significant titres were observed in non-vaccinated ewes before challenge (Table 1 ), but thereafter a significant rise in titre occurred in 4 ewes. After abortion or lambing, 5 of the 7 ewes were positive by the CFT. Of the 60 to 80 chlamydial components resolved by SDS-PAGE and stained with Coomassie brilliant blue R250 or by silver staining (Morrisey, 1981 ), up to 24 are immunoreactive when tested with sera from non-vaccinated sheep convalescent from OEA (unpublished observation). The predominant rela.
lb. E w e No
1 2 3 4
W e e k s Post Vaccination 5
6
02
•
17
~1
12
4 8 10121421
Fig. 1. (a) Immunoblot showing response 8 weeks after initial vaccination with the purified EB vaccine. (b) Immunoblot showing typical time course of response to vaccination in a single vaccinated unchallenged ewe.
AN EXPERIMENTAL VACCINE C O N T A I N I N G ELEMENTARY BODIES OF CHLAMYDIA PS1TTACI
25
sponse 8 weeks after vaccination of all 6 vaccinated ewes was against the major outer membrane protein (MOMP) (Fig. I a). Antibody responses to other antigens were observed but were less pronounced or inconsistently present. The time course of antibody responses in a vaccinated unchallenged sheep as detected by immunoblotting is shown in Fig. 1b. The response shown before challenge (0-8 weeks) was typical of vaccinated challenged sheep. Pre-challenge sera from the unvaccinated sheep were entirely free from chlamydia-specific antibodies detected by immunoblotting. Reaction to challenge became apparent only a few days before or at abortion when faint bands of reaction to several antigens including MOMP were observed. After abortion, a strong antibody response to these bands was detected. No such response occurred in the 2 unvaccinated ewes that did not abort. DISCUSSION
A vaccine containing a purified preparation of chlamydial EB prevented abortion in vaccinated ewes. Immunoblot analysis of the antibody response to vaccination indicated that the predominant response was against MOMP, and that these anti-MOMP antibodies persisted throughout the period of the experiment without diminution. Their likely importance in protection against OEA is supported by their marked and persistent occurrence in the sera and lymph of ewes convalescent from OEA (Huang et al., 1990 ) It is possible, however, that the marked anti-MOMP response was simply a mass effect, since MOMP is the predominant chlamydial protein, and that other protective immunogens, both proteinaceous and non-proteinaceous, were at suboptimal concentrations for the level of sensitivity of the immunoblot procedure used. Antibodies to chlamydial MOMP have been shown to be neutralizing in vitro and protective in vivo. Antibodies raised in rabbits (Caldwell and Perry, 1982) and murine monoclonal antibodies (Peeling et al., 1984) against the MOMP of C. trachomatis strains neutralized their infectivity for HeLa 229 cells. Murine monoclonal antibody against the immunoaccessible MOMP of C. trachomatis serovar B neutralized the infectivity of the organism for monkey eyes and protected mice from toxic death after intravenous injection (Zhang et al., 1987 ). MOMP has recently been implicated in the attachment of C. trachomatis during host cell infection (Su et al., 1988 ). The immunoblot procedure used did not detect antibodies to non-proteinaceous components such as lipopolysaccharide (LPS) (Batteiger et al., 1982 ), but chlamydial LPS is detected by the CFT (McEwen and Foggie, 1954; Dawson et al., 1986). The variability in CFT responses to vaccination seen in these studies was not unexpected, and confirmed the lack of correlation between this test and immunity (Dawson et al., 1986). Why antibodies against chlamydia in the non-vaccinated ewes could not be
26
I.E. A N D E R S O N ET AL.
detected by the CF test until at least 36 days after experimental infection is not clear. Preliminary data from studies on experimental lymph node infections suggest that the initial antibody response at the lymph node is transient and difficult to detect at the systemic level by the CFT or even by the sensitive immunoblotting technique (Huang et al., 1990). It is more likely that inoculation at 70 days of gestation and the delay in proliferation in the placenta until after 90 days (Buxton et al., 1990) may explain this effect. The high efficacy shown by the EB vaccine was encouraging considering that unpurified egg or cell culture-grown vaccines usually give only between 40 and 80% protection depending on the challenge system used (Aitken et al., 1986). The reason for the better performance of this vaccine may have been due to the use of a double vaccination regime. Alternatively, it may have been due to the purity of the material used in its preparation, and thus the greater accessibility of MOMP to the immune system. This would imply that contaminants in unpurified vaccines have an adverse effect on stimulating protection. In either case, MOMP was strongly implicated in this study as an immunogen of importance in OEA. Work currently in progress with a MOMPenriched vaccine preparation should clarify the role of this protein in protection against OEA. ACKNOWLEDGEMENTS
T.W. Tan is in receipt of the Wooldridge Farm Livestock Fellowship from the Animal Health Trust, Great Britain. We thank A. Dawson and P. Dewar for expert technical assistance in the purification and production of the reagents used in immunoblotting, K. Hall and K.W. Quinn for cell culture support, and the department of Clinical Studies for the welfare of the animals.
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