INFECTION AND IMMUNITY, Sept. 1990, p. 3101-31080019-9567/90/093101-08$02.00/0Copyright X) 1990, American Society for Microbiology

Vol. 58, No. 9

Protection of Sheep against Chlamydia psittaci Infection with a

Subcellular Vaccine Containing the Major Outer Membrane ProteinTIN -WEE TAN,t ALAN J. HERRING,* IAN E. ANDERSON, AND GARETH E. JONES

Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JH, United Kingdom

Received 22 February 1990/Accepted 29 June 1990

An outer membrane (OM) preparation from elementary bodies (EBs) of Chlamydia psittaci (ovine abortionstrain) was used to vaccinate pregnant ewes in a single subcutaneous dose and was found to achieve protectionafter subcutaneous challenge with infectious organisms. Inactivated purified EBs used as a single-dose vaccinealso gave protection. The ratio of live to dead lambs was significantly higher in the vaccinated groups (16:1 and15:1, respectively) than in the placebo group (8:9). Polyacrylamide gel electrophoresis and immunoblottingshowed that a 40-kilodalton protein was the main protein constituent of the OM preparation, and this was

positively identified as the major outer membrane protein by protein microsequencing. Electron microscopyrevealed that fine particulate structures on the outermost surface of the EB were also present in the OMpreparation. The findings suggest that the major outer membrane protein is an important immunoprotectivedeterminant in ovine abortion vaccines.

Ovine chlamydial abortion, also known as ovine enzooticabortion (OEA) or enzootic abortion of ewes, is an econom-ically important disease in many countries (1). Infection ofpregnant ewes results in a necrotizing placentitis and conse-quent abortion (40). Vaccines prepared from egg-grownChlamydia psittaci inactivated with Formalin induced immu-nity in ewes against OEA (30) and form the basis of a productthat has been in use for several decades (20).

Recently, the efficacy of this vaccine has been variableand there have been outbreaks in vaccinated flocks (26).Heterologous challenge experiments have suggested thepossibility of strain variation in the field (2, 3). However,attempts to distinguish between OEA isolates have notrevealed any obvious differences (4, 5, 22, 27). Confirmationthat the OEA agent can also cause abortion and severeillness in pregnant women (10, 22) has added impetus toefforts to understand and control the disease. An importantobjective has been the identification of the immunoprotec-tive antigens that can account for the efficacy of OEAvaccines.

Evidence that the major outer membrane protein (MOMP)of C. psittaci may be useful for protection in sheep has beenreported (25, 44; I. E. Anderson, T. W. Tan, G. E. Jones,and A. J. Herring, Vet. Microbiol., in press). However, itwas not possible to obtain pure MOMP in sufficient quanti-ties for further vaccine studies without using strongly dena-turing procedures. In this study, a modified procedure forproducing chlamydial outer membrane complexes (6, 12) hasbeen used to produce a subcellular vaccine highly enrichedfor undenatured MOMP. This preparation, given as a singledose, protected sheep against OEA, as did a single dose of avaccine prepared from purified elementary bodies.

MATERIALS AND METHODS

Chiamydial culture and purification. The ovine abortionisolate of C. psittaci, S26/3, was used in this study (4). Anegg-grown organism was used (4) for the complement fixa-

* Corresponding author.t Present address: Department of Biochemistry, National Univer-

sity of Singapore, Kent Ridge, Republic of Singapore 0511.

tion test. Chlamydial elementary bodies (EBs) for vaccineproduction were purified from infected 5'-iodo-2'-deoxyuri-dine-treated BHK-21 cell monolayers as described previ-ously (5, 27; Anderson et al, in press). Live organisms inplacental samples were detected by culture (4).

Preparation of vaccines and placebo. Purified organismswere divided into two aliquots of 2 mg each. One aliquot wasinactivated and formulated with adjuvant as described byAnderson et al. (in press) to produce the purified EB vaccinecontaining approximately 160 pug of protein per dose. Theother aliquot was subjected to a two-step detergent extrac-tion procedure based on a procedures for preparing chla-mydial outer membrane complexes (6, 12, 14). Elementarybodies were incubated in 100 mM phosphate buffer, pH 7.4,containing 10 mM EDTA and 2% sarcosyl (sodium N-lauroyl-sarcosinate; Sigma Chemical Co., St. Louis, Mo.) for 1 h at37°C with occasional mixing and bath sonication (10-sbursts) to prevent aggregation. The mixture was then cen-trifuged at 100,000 x g for 45 min to pellet the insolublematerial. The pellet was resuspended and further incubatedin the same solution containing 10 mM dithiothreitol, underthe same conditions. This mixture was then centrifuged asbefore. The resultant pellet, the outer membrane (OM)preparation, was suspended in phosphate-buffered saline,pH 7.4, inactivated and formulated into vaccine by themethod used for the first vaccine. Each dose of this vaccinecontained about 20 ,ug of protein.To produce the placebo vaccine, uninfected cell monolay-

ers were harvested in a way similar to that used for chla-mydial purification, concentrated by low-speed centrifuga-tion, and formulated into a vaccine as for the other testvaccines. Protein estimation was carried out by using adye-binding assay (8).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) and immunoblotting. The two procedures werecarried out as described before (23, 25; Anderson et al., inpress). Gels were stained with silver by the method ofMorrisey (34).

Protein microsequencing. The OM preparation was re-solved by SDS-PAGE and electroblotted onto a glass fibersupport (Glassybond; Biometra Ltd, Manchester, UnitedKingdom) by a semidry method (18). After the transfer, the

3101

3102 TAN ET AL.

a b

1 2 3 4 5 6 7

0-1..

t.

1 2 3 4

bumwb116937866

Po p

45 _-

36 "~ _0o45

*jm 4m-MOMP-4 *40

29 -

24 Pw

'p

30

20 O

14 _*

FIG. 1. Analyses of the vaccine preparations by SDS-PAGE and by immunoblotting. (a) SDS-PAGE profiles (silver stained) of the vaccinepreparations used: BHK-21 cell preparation used on group A ewes as a placebo vaccine (lane 2); purified EBs used to vaccinate group B ewes(lane 6); and outer membrane preparation used to vaccinate group C ewes (lane 3, 3 ,ug; lane 7, 1 ,ug). Chlamydial components solubilizedfrom the first detergent extraction (sarcosyl) of EBs (lane 5) and the second sequential extraction (sarcosyl-dithiothreitol) (lane 4) are alsoshown. Molecular mass standards are labeled in kilodaltons (lane 1). The stained material at the interface of the stacking and resolving gelsis probably nucleic acid which is not seen in gels stained with Coomassie blue. (b) Distribution of chlamydial antigens during the sequentialextraction procedure as visualized by immunblotting with a serum sample from a postabortion ewe followed by autoradiography. Lane 1,Purified EBs; lane 2, sarcosyl-soluble components from the first detergent extraction of EBs; lane 3, sarcosyl-dithiothreitol-solublecomponents from the second sequential extraction; lane 4, detergent-insoluble outer membrane preparation constituting the subcellular OMvaccine. The position of MOMP is as marked; the molecular mass standards are labeled in kilodaltons.

Glassybond filter was rinsed with distilled water and stainedwith Coomassie blue. The dominant 40-kilodalton (kDa)protein was identified and excised for sequence analysis.Amino acid sequencing was performed on a gas-phasemicrosequencer (model 477A; Applied Biosystems, Inc.,Foster City, Calif.) (courtesy of Linda Fothergill-Gilmore,University of Edinburgh, Edinburgh, United Kingdom).Briefly, Polybrene (2 mg/30 ,ul) was added to the sinteredglass fiber sample disk, which then underwent a 3-h precycleof washes before the protein sample was loaded and sub-jected to 10 automated sequencing cycles.

Electron microscopy. Samples coated on copper grids werestained with either 1% phosphotungstic acid (pH 7.0) or 1%ammonium molybdate (pH 5.3) and examined under a JEOL1200EX transmission electron microscope operating at a

voltage of 80 kV.Serological test. The complement fixation (CF) test was

carried out by the method of Stamp et al. (41) by usingmicrotiter plates.Animal procedures. The widespread occurrence of chla-

mydial infection in sheep flocks necessitated the stringentselection of experimental animals for this study. Ewesobtained from a flock with no known history of OEA were

serologically screened for chlamydia-specific antibodies bythe CF test and by immunoblotting. Following synchroniza-tion of estrus, selected ewes were mated and penned sepa-

rately in three groups. Within a month of mating, ewes fromeach group were vaccinated subcutaneously with 1 ml of theplacebo (group A), purified EB (group B, 160 ,ug of protein),or subcellular OM (group C, 20 ,ug of protein). Serumsamples were taken at regular intervals for analysis by theCF test. After 70 days of gestation, all pregnant ewes were

challenged by subcutaneous injection with 1 ml of live S26/3

organisms (105-5 chick embryo lethal doses). At parturition,placental tissues or vaginal swabs were taken for isolation ofchlamydiae (Anderson et al, in press).

Statistical test. The Fisher's exact test (one-tailed) wasused to analyze the data for significance in a 2 by 2contingency table (19).

RESULTS

Production and biochemical analyses of the vaccines. Theresults of SDS-PAGE and immunoblotting analyses of thevaccine preparations and the intermediate stages of prepa-ration are shown in Fig. 1. The first detergent extractionsolubilized some chlamydial proteins; most antigens, excepta 40-kDa protein, were removed at this stage. The subse-quent detergent-dithiothreitol extraction removed almost alllow-molecular-mass proteins, leaving a 40-kDa protein in ahighly enriched state (arrow). Some high-molecular-masspolypeptides were still present (Fig. la, lane 7) and particu-larly visible when the well was heavily loaded (lane 3).Densitometric scanning showed that in the final detergent-insoluble preparation, the 40-kDa polypeptide made up inexcess of90% of the protein present. A band migrating at thedye front was also detected in both chlamydial vaccinepreparations by silver staining, and it possessed the mobilityexpected for chlamydial lipopolysaccharide (LPS) (data notshown). Immunoblotting of a gel similar to that in Fig. lawith a postabortion serum sample from an experimentallyinfected convalescent ewe showed that the 40-kDa proteinwas strongly antigenic and comigrated with the dominant40-kDa band from whole organisms (Fig. lb).

Protein microsequencing showed that the 40-kDa proteinpossessed the following amino-terminal sequence:

INFECT. IMMUN.

CHLAMYDIAL MOMP VACCINE 3103

/

lOQnm0 -- +

FIG. 2. Electron micrograph of the purified EB vaccine preparation magnified x 240,000. The arrows indicate obvious particles on the EBsurface. (Bar = 100 nm).

1 2 3 4 5 6 7 8 9 10Leu-Pro-Val-Gly-Asn-Pro-Ala-Glu-Pro-Ser ...

This decapeptide sequence matched the 23rd to 32ndresidue of the amino acid sequence deduced from the S26/3MOMP nucleotide sequence (24), indicating that a 22-residueleader peptide has been cleaved from the precursor S26/3MOMP polypeptide. During the first two sequencing cycles,alternative residues Asp-Gly were also detected. Inspectionof the deduced S26/3 MOMP sequence showed that residues14 and 15 were Asp-Gly. This suggests that an Asp-Glyterminus may have arisen from an alternative processing sitedownstream of Leu-1, but mild acid hydrolysis during thestaining procedure or contamination are alternative explana-tions.

Electron microscopy. Chlamydial EBs observed were typ-ically coccoid and about 200 to 400 nm in diameter (Fig. 2).A granularity of the surface was commonly observed innegatively stained EB preparations. On the edges of the EB,the particulate nature of the surface was clearly visible(arrows).

After a single detergent extraction, the material had theappearance of broken membrane fragments. Following thetwo-step sarcosyl extraction, the preparation had the ap-pearance shown in Fig. 3. The most abundant structureswere fine, tightly packed particles very similar in size tothose seen on the EB surface. Where fragments of mem-brane were visible in an edge-on aspect, knoblike particleswhich appeared to project from a continuous substratumwere seen (open arrow in Fig. 3). Another less abundantstructural feature was a rosette consisting of nine subunits ofabout 3 to 4 nm arranged in a ring about 15 nm in diameterwith a stain-filled central cavity (solid arrow in Fig. 3).

Analyses of the antibody response. Within 3 weeks of thehomologous challenge, most control animals injected withthe placebo vaccine (group A) responded with log2 CFantibody titers ranging from 4 to 6 (Fig. 4). Although threeewes had responded slowly, all control animals eventuallyseroconverted.

Vaccination with purified EBs (group B) or the subcellularOM (group C) vaccine induced a primary response in most

VOL. 58, 1990

3104 TAN ET AL.

~~t :';t . *' ; '^ '

-L~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4

-40~ ~ ~ *~

*~~~ ~~ott*M_

b , . ;0 ',,"u-.- .!, j. ,,0.. t;,

I Y

%t4*~'~'&~-A

WOI~~~~~~~~~~~~~~~Iv

FIG. 3. Electron micrograph of the subcellular OM preparation magnified x240,000. The solid arrow indicates a rosette visible as a

nine-membered ring. The open arrow indicates a membrane fragment with protruding knoblike particles. (Bar = 100 nm).

sheep within 42 days. Titers generally declined by the time ofchallenge, whereupon a secondary response was induced inall vaccinated sheep (Fig. 4b and c). One ewe from group Band three from group C did not produce a detectable CF titerbefore challenge, but none of them became infected or

aborted. As previously reported (11, 17, 20, 29), no signifi-cant correlation was found between the presence of prechal-lenge CF titers and immunity.

Figure 5 shows the immunoblots of prechallenge sera fromewes of all three groups 4 weeks postvaccination. Lysates ofwhole EBs were used as antigen. No chlamydia-specificantibodies were detected in the sera of ewes injected withthe placebo. In contrast, ewes vaccinated with the EB andOM vaccines produced chlamydia-specific antibodies, pri-marily directed against MOMP.Lambing and isolation results. Table 1 shows the final

result of the study. All ewes in group C were protectedagainst abortion; however, infectious organisms were iso-lated from one ewe. One of the live lambs from the singleinfected ewe died. In group B, 6 of 7 ewes were protectedand a similar low ratio of lamb mortality was recorded. Incontrast, 7 of the 12 control animals were infected, of which5 subsequently aborted. The lamb mortality was significantlyhigher compared with either of the vaccine groups (P <

0.005).

DISCUSSION

In comparison with attempts to vaccinate against chla-mydial infections in humans and in animal models by usingwhole organisms or specific components as immunogens (16,21, 45-47, 50), vaccination against abortion in ewes causedby C. psittaci has had a long and successful history untilrecent years (2, 20, 26, 29, 30). Vaccines against OEA haveevolved from placental and yolk sac preparations of the1950s (20, 29, 30) to cell-cultured vaccines (48). Recently,purified chlamydial EBs from cell culture have been success-

fully used in a two-dose vaccination-challenge experiment toestablish that the immunoprotective components of thesevaccines resided in the chlamydial EB (Anderson et al., inpress). This study has extended that finding by showing thatEBs are effective when given as a single dose.As the next step in the logical progression of vaccine

development, the subcellular component(s) of the EB re-

quired for protection has been shown to reside in the OM as

described above. SDS-PAGE and immunoblotting analysesshowed that the protective subcellular preparation consistedmainly of a 40-kDa protein directly identified as MOMP byamino acid microsequencing. The sequence was identical tothe first 9 residues of Chlamydia trachomatis L2 MOMP(35), confirming that leader peptide cleavage occurs at thesame site in C. psittaci. A component with the mobility of

*8A -Z$

.i IV* .; ik #

JZ1. 0#'.o.,,4 .

INFECT. IMMUN.

CHLAMYDIAL MOMP VACCINE 3105

8

aL

Ai.-I

Ai

0

6

4

2

e

aL

Ai

U-

a,13

a

6

4

e

8

L.

4*

_L

U-

010

SK

a 39Sa 90

doWs post-vaccination

FIG. 4. Kinetics of the CF antibody response of cin groups A (a, placebo control; n = 12), B (b, purifin = 7) and C (c, subcellular outer membrane vaccinmean log2 CF titers (± standard deviation) of ewes irplotted against number of days postvaccination. Thegous challenge with live C. psittaci is indicated with

chlamydial LPS, a characteristic component of the OM (37),was also present (data not shown).The appearance of the subcellular preparation in the

electron microscope was consistent with that reported for,a OM preparations by Matsumoto (32, 33). Fine particles some/ to 6 nm in diameter appear to be the major structural

component of the outermost surface of the EB. It must beassumed that these particles are aggregates of MOMP. Thisis consistent with the known properties of MOMP, namelyits predominance in the OM (12), surface exposure (12, 14,51), porin function (6), and close relationship with LPS (7).The nine-membered rosettes have also been observed pre-viously in both C. psittaci (31) and C. trachomatis (15).Since these structures are not numerous, they may beformed by the minor protein constituents detected in thepreparation.The nature and significance of the immune mechanisms

elicited by chlamydial infections are not fully understood.XM> The most detailed studies have been made with naturalU/u chlamydial infections of small rodents, and both cell-medi-

ated and humoral immune responses have been shown topossess a role, as reviewed by Williams (49) and by Rank(39). A study of infection with OEA strain C. psittaci in amouse model showed that T-cell-mediated immunity wasmore efficiently transferred than humoral immunity (11).Recently, studies on cellular immune responses have beenreported for ovine abortion strains in sheep (17, 25; H.-S.Huang, M. Phil. thesis, University of Edinburgh, Edinburgh,United Kingdom, 1988), but the immunological mechanismsoperating remain undefined. Whether the anti-MOMP anti-bodies that feature prominently in immunoblots of sera fromvaccinated sheep (44; Anderson et al., in press) are media-tors of immunity or merely indicators of immunity needs to

230C be resolved.Data presented here suggest that the immunoprotective

element(s) of previous OEA vaccines resides within the OM,and the role of each constituent of the OM preparation nowremains to be assessed. Several lines of evidence suggestthat MOMP is a protective immunogen. Polyclonal andmonoclonal antibodies to MOMP neutralize C. trachomatis

'~---~---~----' infectivity in vitro (13, 38) and in vivo (51). Studies of126 186 efferent lymph have indicated that MOMP is recognized

early in the immune response (25). Sheep protected byvaccination with a purified EB preparation expressed a

hallenged sheep strong antibody response directed almost exclusively toied EB vaccine; MOMP (44; Anderson et al., in press). Functionally, MOMPie; n = 11). The is involved in the developmental cycle (6, 36) and may havei each group are a role in infectivity (43). A recent experiment testing SDS-day of homolo- extracted MOMP as a vaccine by oral adminstration showedia solid arrow. only a slight effect against ocular C. trachomatis infection in

monkeys (46). However, the use of SDS-denatured protein

TABLE 1. Effect of vaccinating ewes against challenge with infectious C. psittaci, OEA isolate S26/3

Total no. ChaIIgd No. infected: No. aborted: Lamb ratio,Group Vaccine vacci- Nonpregnant ened uninfected unaborted dead:live

nated ewes' ewes (p <)b (p <)b (p <)b

A Placebo; uninfected 12 0 12 7:5 5:7 9:8BHK cells

B Purified EBs 12 5 7 1:6 (0.080) 1:6 (0.238) 1:10 (0.004)

C Subcellular OM 12 1 11 1:10 (0.019) 0:11 (0.024) 1:16 (0.003)preparation

a Nonpregnant ewes were not challenged.b Probability values quoted were calculated by using Fisher's exact test (one-tailed), testing each vaccine group against the placebo group.

VOL. 58, 1990

2

e

3106 TAN ET AL.

A B C

45

IMOMNA* 0

30

4 + n npfnp np np + np+ np

FIG. 5. Immunoblots of prechallenge sera from ewes of all three groups, 4 weeks postvaccination. (A) Immunoblots of four sets of pooledsera from three placebo-injected animals each (group A); (B) immunoblots of sera from individual animals vaccinated with purified EBs (groupB); (C) immunoblots of sera from individual animals vaccinated with the subcellular OM preparation (group C); (+), immunoblot of serumfrom a postabortion ewe. Lysates of whole EBs from the S26/3 OEA isolate were used throughout as antigen. Blots from nonpregnant eweswhich were subsequently not challenged are indicated at the bottom as np; each + represents a ewe which was subsequently found infectedor aborted. The position of MOMP is indicated; the molecular mass standards are marked in kilodaltons.

may have destroyed epitopes necessary for protection (51).Other protein components present in the subcellular vaccinemay also be important in immunity, but it seems unlikelythat they were present in sufficient amounts to contribute asignificant effect.Chlamydial LPS is generally not thought to be important

as a protective antigen in C. trachomatis infections (45, 51).Serum prepared against heat-killed C. psittaci organisms,which is likely to contain antibodies against the thermostablegenus-specific epitope(s) of LPS, did not confer protection inthe passive transfer experiments of Buzoni-Gatel et al. (11).Complement-fixing antibodies, which do not correlate withprotection against ovine abortion strains of C. psittaci (11,17, 20, 29), are also thought to be directed against genus-specific epitope(s) of LPS. The immunoblotting techniqueused in this study and in previous studies (28; Anderson etal., in press; M. McClenaghan and A. J. Herring, unpub-lished data) did not detect a consistent antibody reactionagainst LPS. Nevertheless, LPS may have an adjuvant rolein the OEA vaccine.

If MOMP is the major protective component in OEAvaccines, it is possible, since serovar variation in C. tracho-matis has been attributed to sequence variation in MOMP(14, 42), that similar variation in OEA strains can account forthe recent vaccine breakdowns. We are currently investigat-ing such variability in OEA strains; analysis is complete forfive Scottish isolates, but it has not revealed any sequencevariation (S. Baxter and A. Herring, unpublished results).We are currently investigating further ruminant abortionstrains from continental Europe and Africa.To determine whether MOMP alone is sufficient for pro-

tection against OEA, a recombinant DNA approach is being

pursued. The MOMP gene of an OEA vaccine strain (S26/3)has been cloned, sequenced (24), and expressed in Esche-richia coli (T. W. Tan and A. J. Herring, unpublished data).Expression has also been achieved in a Salmonella typhimu-rium aroA mutant (J. J. Oliver, A. J. Herring, and G. D.Baird, unpublished data), a vector known to be capable ofeliciting both arms of the immune response (9). The recom-binant protein is being tested as a vaccine. It is our hope thatthis and other planned studies of OEA will help to establishthe immune mechanisms involved in both ovine and otherchlamydial infections.

ACKNOWLEDGMENTS

T.W.T. is in receipt of a Wooldridge Farm Livestock Fellowshipfrom the Animal Health Trust, United Kingdom.The authors thank E. Gray and L. Inglis for electron microscopy,

B. J. Easter and A. Inglis for photography, and staff of theDepartment of Clinical Studies for the care of the experimentalanimals.

LITERATURE CITED1. Aitken, I. D. 1983. Enzootic (chlamydial) abortion, p. 119-123.

In W. B. Martin (ed.), Diseases of sheep. Blackwell ScientificPublishers, Ltd., Oxford.

2. Aitken, I. D., I. E. Anderson, and G. W. Robinson. 1986. Ovinechlamydial abortion: limitations of inactivated vaccine, p. 55-65. In I. D. Aitken (ed.), Chiamydial diseases of ruminants.Publication EUR 10056 EN. Commission of the EuropeanCommunities, Luxembourg, Luxembourg.

3. Aitken, I. D., G. W. Robinson, and I. E. Anderson. 1981.Enzootic abortion: experimental infection. Proc. Sheep Vet.Soc. 5:53-60.

4. Anderson, I. E. 1986. Comparison of the virulence in mice of

INFECT. IMMUN.

CHLAMYDIAL MOMP VACCINE 3107

some ovine isolates of Chlamydia psittaci. Vet. Microbiol.12:212-220.

5. Anderson, I. E. 1986. Comparison of five ovine isolates ofChlamydia psittaci: an evaluation of three cell culture treat-ments. Med. Lab. Sci. 43:241-248.

6. Bavoil, P., A. Ohlin, and J. Schachter. 1984. Role of disulfidebonding in outer membrane structure and permeability in Chla-mydia trachomatis. Infect. Immun. 44:479-485.

7. Birkelund, S., A. G. Lundemose, and G. Christiansen. 1988.Chemical cross-linking of Chlamydia trachomatis. Infect. Im-mun. 56:654-659.

8. Bradford, M. M. 1976. A rapid sensitive method for the quan-titation of microgram quantities of protein utilizing the principleof protein-dye binding. Anal. Biochem. 72:248-254.

9. Brown, A., C. E. Hormaeche, R. DeMarco de Hormaeche, M.Winther, G. Dougan, D. J. Maskell, and B. A. D. Stocker. 1987.An attenuated aro-A Salmonella typhimurium vaccine elicitshumoral and cellular immunity to cloned P-galactosidase inmice. J. Infect. Dis. 155:86-92.

10. Buxton, D. 1986. Potential danger to pregnant women of Chla-mydia psittaci from sheep. Vet. Rec. 118:510-511.

11. Buzoni-Gatel, D., A. Rodolakis, and M. Plommet. 1987. T cellmediated and humoral immunity in a mouse Chlamydia psittacisystemic infection. Res. Vet. Sci. 43:59-63.

12. Caldwell, H. D., J. Kromhout, and J. Schachter. 1981. Purifica-tion and partial characterization of the major outer membraneprotein of Chlamydia trachomatis. Infect. Immun. 31:1161-1176.

13. Caldwell, H. D., and L. J. Perry. 1982. Neutralization ofChlamydia trachomatis infectivity with antibodies to the majorouter membrane protein. Infect. Immun. 18:745-754.

14. Caldwell, H. D., and J. Schachter. 1982. Antigenic analysis ofthe major outer membrane protein of Chlamydia spp. Infect.Immun. 35:1024-1031.

15. Chang, J.-J., K. Leonard, T. Arad, T. Pitt, Y.-X. Zhang, andL.-H. Zhang. 1982. Structural studies of the outer envelope ofChlamydia trachomatis by electron microscopy. J. Mol. Biol.161:579-590.

16. Cui, Z.-D., L. J. Lascolea, Jr., J. Fisher, and P. L. Ogra. 1989.Immunoprophylaxis of Chlamydia trachomatis lymphogranu-loma venereum pneumonitis in mice by oral immunization.Infect. Immun. 57:739-744.

17. Dawson, M., A. Zaghloul, and A. J. Wilsmore. 1986. Ovineenzootic abortion: experimental studies of immune responses.Res. Vet. Sci. 40:59-64.

18. Eckerskorn, C., W. Mewes, H. Goretzki, and F. Lottspeich.1988. A new siliconized-glass fibre as support for protein-chemical analysis of electroblotted proteins. Eur. J. Biochem.176:509-519.

19. Finney, D. J. 1948. The Fisher-Yates test of significance in 2 x2 contingency tables. Biometrika 35:145-156.

20. Foggie, A. 1973. Preparation of vaccines against enzootic abor-tion of ewes. A review of the research work at the MoredunInstitute. Vet. Bull. 43:587-590.

21. Grayston, J. T., S.-P. Wang, L.-J. Yeh, and C.-C. Kuo. 1985.Importance of reinfection in the pathogenesis of trachoma. Rev.Infect. Dis. 7:717-730.

22. Herring, A. J., I. E. Anderson, M. McClenaghan, N. F. Inglis, H.Williams, B. A. Matheson, C. P. West, M. Rodger, and R. P.Brettle. 1987. Restriction endonuclease analysis of DNA fromtwo isolates of Chlamydia psittaci obtained from human abor-tions. Br. Med. J. 295:1239.

23. Herring, A. J., and J. M. Sharp. 1984. Protein blotting: the basismethod and its role in viral diagnosis, p. 115-124. In M. S.McNulty and J. B. McFerran (ed.), Recent advances in viraldiagnosis. Martinus Nijhoff, Publishers BV, The Hague.

24. Herring, A. J., T. W. Tan, S. Baxter, N. F. Inglis, and S.Dunbar. 1989. Sequence analysis of the major outer membraneprotein gene of an ovine abortion strain of Chlamydia psittaci.FEMS Microbiol. Lett. 65:153-158.

25. Huang, H. S., T. W. Tan, D. Buxton, I. E. Anderson, and A. J.Herring. 1990. Antibody response of the ovine lymph node toexperimental infection with an ovine abortion strain of Chla-

mydia psittaci. Vet. Microbiol. 21:345-351.26. Linklater, K. A., and D. A. Dyson. 1979. Field studies on

enzootic abortion of ewes in south-east Scotland. Vet. Rec.105:387-389.

27. McClenaghan, M., A. J. Herring, and I. D. Aitken. 1984.Comparison of Chlamydia psittaci isolates by DNA restrictionendonuclease analysis. Infect. Immun. 45:384-389.

28. McClenaghan, M., A. J. Herring, I. D. Aitken, and J. R.Honeycombe. 1986. Some comparative biochemical studies onChlamydia psittaci strains of ovine and avian origin, p. 139-147.In I. D. Aitken (ed.), Chlamydial diseases of ruminants. Publi-cation EUR 10056 EN. Commission of the European Commu-nities, Luxembourg, Luxembourg.

29. McEwen, A. D., and A. Foggie. 1956. Enzootic abortion of ewes.Prolonged immunity following the injection of adjuvant vaccine.Vet. Rec. 68:686-690.

30. McEwen, A. D., J. T. Stamp, and A. I. Littlejohn. 1951.Enzootic abortion of ewes. II. Immunisation and infectionexperiments. Vet. Rec. 63:197-201.

31. Matsumoto, A. 1973. Fine structures of cell envelopes of Chla-mydia organisms as revealed by freeze-etching and negativestaining techniques. J. Bacteriol. 116:1355-1363.

32. Matsumoto, A. 1979. Recent progress of electron microscopyand its development in future: from a study of the obligateintracellular parasites, Chlamydia organisms. J. Electron Mi-crosc. 28(Suppl.):S57-S64.

33. Matsumoto, A. 1982. Morphology of Chlamydia psittaci elemen-tary bodies as revealed by electron microscopy. Kawasaki Med.J. 8:149-157.

34. Morrisey, J. H. 1981. Silver stain for proteins in polyacrylamidegels. A modified procedure will enhance sensitivity. Anal.Biochem. 117:307-310.

35. Nano, F. E., P. A. Barstad, L. W. Mayer, J. E. Coligan, andH. D. Caldwell. 1985. Partial amino acid sequence and molecu-lar cloning of the encoding gene for the major outer membraneprotein of Chlamydia trachomatis. Infect. Immun. 48:372-377.

36. Newhall, W. J., V., and R. B. Jones. 1983. Disulfide-linkedoligomers of the major outer membrane of chlamydiae. J.Bacteriol. 154:998-1001.

37. Nurminen, M., M. Leinonen, P. Saikku, and P. H. Makela. 1983.The genus-specific antigen of Chlamydia: resemblance to thelipopolysaccharide of enteric bacteria. Science 220:1279-1291.

38. Peeling, R., I. W. MacLean, and R. C. Brunham. 1984. In vitroneutralization of Chlamydia trachomatis with monoclonal anti-body to an epitope on the major outer membrane protein. Infect.Immun. 46:484-488.

39. Rank, R. G. 1988. Role of the immune response, p. 217-234. InA. L. Barron (ed.), Microbiology of chlamydia. CRC Press,Inc., Boca Raton Fla.

40. Stamp, J. T., A. D. McEwen, J. A. A. Watt, and D. I. Nisbet.1950. Enzootic abortion in ewes. I. Transmission of the disease.Vet. Rec. 62:251-254.

41. Stamp, J. T., J. A. A. Watt, and R. B. Cockburn. 1952. Enzooticabortion in ewes. Complement fixation test. J. Comp. Pathol.62:93-101.

42. Stevens, S. S., R. Sanchez-Pescador, E. A. Wagar, C. Inouye,and M. S. Urdea. 1987. Diversity of Chlamydia trachomatismajor outer membrane protein genes. J. Bacteriol. 160:3879-3885.

43. Su, H., Y.-X. Zhang, 0. Barrera, N. G. Watkins, and H. D.Caldwell. 1988. Differential effect of trypsin on infectivity ofChlamydia trachomatis: loss of infectivity requires cleavage ofmajor outer membrane protein variable domains II and IV.Infect. Immun. 56:2094-2100.

44. Tan, T. W., A. J. Herring, M. McClenaghan, H.-S. Huang, I. E.Anderson, N. F. Inglis, G. E. Jones, and D. Buxton. 1988.Immunoblotting analysis of the humoral response in sheepinfected with the ovine abortion strain of Chlamydia psittaci.Proc. Eur. Soc. Chlamydia Res. 1:136.

45. Taylor, H. R., and R. A. Prendergast. 1987. Attempted oralimmunization with chlamydial lipopolysaccharide subunit vac-

cine. Invest. Ophthalmol. Vis. Sci. 28:1722-1726.46. Taylor, H. R., J. Whittum-Hudson, J. Schachter, H. D. Caldwell,

VOL. 58, 1990

INFECT. IMMUN.

and R. A. Prendergast. 1988. Oral immunization with chlamydialmajor outer membrane protein (MOMP). Invest. Ophthalmol.Vis. Sci. 29:1847-1853.

47. Taylor, H. R., E. Young, A. B. MacDonald, and R. A. Prender-gast. 1987. Oral immunization against chlamydial eye infection.Invest. Ophthalmol. Vis. Sci. 28:249-258.

48. Waldhalm, D. G., W. J. DeLong, and R. F. Hall. 1982. Efficacyof a bacterin prepared from Chlamydia psittaci grown in cellculture for experimental immunization of ewes. Vet. Microbiol.7:493-498.

49. Williams, D. M. 1988. Stimulation of immune response, p.209-216. In A. L. Barron (ed.), Microbiology of chlamydia.CRC Press, Inc., Boca Raton, Fla.

50. Wills, J. M., T. J. Gruffydd-Jones, S. J. Richmond, R. M.Gaskell, and F. J. Bourne. 1987. Effect of vaccination on felineChlamydia psittaci infection. Infect. Immun. 55:2653-2657.

51. Zhang, Y.-X., S. Stewart, T. Joseph, H. R. Taylor, and H. D.Caldwell. 1987. Protective monoclonal antibodies recognizeepitopes located on the major outer membrane protein ofChlamydia trachomatis. J. Immunol. 138:575-581.

3108 TAN ET AL.