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Identification by Western blots of virulence specificantigens of Chlamydia psittaci isolated from ewes

K Layachi, A Rodolakis, D Buzoni-Gatel

To cite this version:K Layachi, A Rodolakis, D Buzoni-Gatel. Identification by Western blots of virulence specific antigensof Chlamydia psittaci isolated from ewes. Veterinary Research, BioMed Central, 1993, 24 (1), pp.55-65.�hal-00902106�

Original article

Identification by Western blots of virulence specificantigens of Chlamydia psittaci isolated from ewes

K Layachi A Rodolakis D Buzoni-Gatel

INRA, Pathologie Infectieuse et Immunologie, 37380 Nouzilly, France

(Received 31 March 1992; accepted 16 July 1992)

Summary ― Specific antigens of virulent strains of Chlamydia psittaci isolated from ruminants werecharacterized by western blotting. The immunoblot analyses were performed with chlamydial ele-mentary bodies from abortive (AB7 strain) or intestinal (iB1 strain) chlamydiae using mouse and rab-bit immune sera raised against viable chlamydiae or proteic extracts. Eleven antigens were found tobe AB7-specific, but only 4 (96, 90, 88 and 49-50 kDa) were recognized by both mouse and rabbitsera against viable AB7 strains. These could be candidates for specific diagnosis of caprine andovine abortive chlamydiosis. No iB1-specific antigen recognized by mouse and rabbit anti-sera couldbe identified.

Chlamydia psittaci antigen / virulence / ovine abortion

Résumé ― Identification par immuno-transfert des antigènes de virulence de Chlamydia psit-taci isolée de ruminants. Des antigènes spécifiques des souches virulentes de Chlamydia psittaciont été caractérisés par immuno-transfert en comparant les réponses obtenues avec des corps élé-mentaires purifiés de la souche AB7 (souche abortive) et de la souche i81 (souche intestinale) enprésence de sérums polyclonaux de souris ou de lapins immunisés avec des Chlamydia vivantes oudes extraits protéiques de ces mêmes souches. Onze antigènes spécifiques de la souche AB7 ontpu être identifiés, mais seulement 4 antigènes de masse moléculaire respective 96, 90, 88 et 49-50kDa ont été reconnus également par les sérums polyclonaux de souris et de lapin immunisés avecdes Chlamydia vivantes. Ces antigènes pourraient donc être utilisés pour le diagnostic de la chlamy-diose abortive ovine ou caprine. Aucun antigène spécifique de la souche i81 reconnu par les anti-sérums de lapin et de souris n a pu être identifié par cette technique.

Chlamydia psittaci lantigène / virulence / avortement ovin

* Correspondence and reprints

INTRODUCTION

Chlamydia psittaci, a small Gram-negativeobligate intracellular bacterium, is a majoretiological agent in caprine and ovine abor-tions. The diagnosis of the disease is usual-ly made by the examination of fixed andstained smears from placentae and by ex-amination of sera for chlamydial antibodiesby complement fixation test with genus lipo-polysaccharide antigen. However, clinicallynormal sheep and goats harbor chlamydiaein their intestinal tract and excrete them in

their feces (Omori et al, 1957; Kawakami etal, 1958; Dungworth and Cordy, 1962;Storz, 1963; Wilson and Dungworth, 1963).The epidemiological impact of intestinal

chlamydial strains in abortive chlamydiosisis not known, but the close antigenic rela-tionships between the 2 chlamydiae disturbthe complement fixation test used to diag-nose the abortive infection.

With a murine model we showed thatmost strains isolated from abortion were in-

vasive in mice following subcutaneous inoc-ulation, whereas strains isolated from thefeces of healthy sheep were not (Rodolakis

et al, 1989). This difference in invasive abili-ty is associated with dissimilar protein pat-terns in polyacrylamide gel electrophoresisanalysis between invasive and non-invasivestrains (Buzoni-Gatel et al, 1989).

The aim of this study was to identifyspecific antigens of invasive chlamydialstrains using an immunoblotting techniqueemploying polyclonal sera in order to pro-pose them for a specific diagnosis.

MATERIALS AND METHODS

Strains

C psittaci AB7 strain was originally obtainedfrom Dr P Faye (Faye et al, 1972). Reisolatedfrom an aborted lamb at the Laboratoire de Pa-

thologie Infectieuse et Immunologie (INRA, Nou-zilly, France), it was considered as the invasivereference strain. Strain iB1 was isolated fromthe feces of a healthy Berrichon ewe from aflock with no previous history of abortion (Buzo-ni-Gatel and Rodolakis, 1983) and was consid-ered as the non-invasive reference strain. Theother chlamydial strains examined are listed in

table I. Stocks of chlamydia (2nd and 3rd egg

passage for AB7 and iBi, respectively) werepropagated in the yolk sacs of developing chickembryos as previously described (Rodolakis,1976), stored at -70 °C and titrated by plaqueassay (Banks at al, 1970). For protein analysisthe strains were propagated in McCoy cells aspreviously described (Rodolakis, 1983) andstored in phosphate glutamine sucrose buffer at- 70 °C. Chlamydiae were purified on renografinas described by Caldwell et al (1981) using 150mM Tris-potassium chloride buffer (pH 7.5). Thepurified chlamydial elementary bodies (EBs)preparation was stored at -70 °C.

Preparation of proteic extract

Purified chlamydial EBs were lysed with 4% so-dium dodecyl sulphate (SDS) and 3 mM EDTAin phosphate-buffered saline as previously de-scribed (Buzoni-Gatel et al, 1989). Proteic con-centration was determined by bicinchoninic acid(BCA) protein assay (Pierce, Rockford) (Smithet al, 1985).

Animals

Six-week-old 20 g, outbred female OF, Swissmice (IFFA Credo; L’Arbresle, France) and fe-male New Zealand White rabbits were used forthe production of polyclonal antibodies.

Preparation of antisera

Viable chlamydiae or proteic extracts were usedto prepare antisera. For production of mouse

antibodies, each mouse (20/group) received 3injections intraperitoneally at 3-week intervals ofeither 105 PFU viable yolk sac propagated chla-mydial strains (AB7 or iBi or 30 pg per mouseproteic extract emulsified in an equal volume ofFreund’s complete adjuvant (Difco Labs, Detroit,MI) at first inoculation and in Freund’s incom-

plete adjuvant for the 2 following inoculations.The mice were bled 7 d after the third inocula-5on, blood was allowed to clot for 2 h at 37 °Cand the serum samples were frozen in aliquotsat -20 °C. Negative sera were obtained from acontrol group of 20 uninoculated mice. For pro-

duction of rabbit antibodies, each rabbit was im-munized 3 times at 3-wk intervals either intrader-

mally with 10! PFU of viable yolk sac propagat-ed chlamydial strains AB7 or iB1, or

subcutaneously with 500 pg of proteic extractemulsified in an equal volume of Freund’s com-plete adjuvant at the first inoculation and inFreund’s incomplete adjuvant for the 2 followinginoculations. Control rabbit sera were takenfrom the same rabbits just before the first immu-nization.

Serum antibody titers were determined byconventional ELISA using AB7 or iB1 proteic ex-tract (250 ng per well) as coating antigens (Fu-kushi et al, 1987).

Separation of chlamydial antigensby SDS-PAGE

Proteins of purified chlamydial EBs were separ-ated by electrophoresis in 8 to 23% gradientacrylamide gel according to the method ofLaemmli (1970). Before electrophoresis the

samples were mixed with an equal volume ofthe solubilizing solution containing 2% SDS, 5%p-mercaptoethanol, 2.5% glycerol and 0.05%bromophenol blue, then boiled for 10 min. Ap-proximately 120 pg of protein was added to thegel. Protein bands were revealed by silver stain-ing (Oakley et al, 1980). Molecular masses wereestimated by using low molecular mass markers(Pharmacia, Uppsala, Sweden): phosphorylase94 kDa, bovine serum albumin 67 kDa, ovalbu-min 43 kDa, carbonic anhydrase 30 kDa, soybean trypsin inhibitor 21 kDa, and lactalbumin14.4 kDa.

Immunoblot analysis

Western blots were performed according to themethod of Towbin et al (1979). After transfer,the nitrocellulose membrane (NC) was stainedwith Ponceau S red to ensure that the chlamydi-al proteins were transferred successfully. Non-specific reactions on NC were blocked for 1 h at37 °C with Tris-buffered saline (TBS), 1%skimmed milk and 0.01% Tween 20. The NCsheets were then washed with TBS 0.01%Tween 20 (3 times for 10 min each). Thesewere incubated for 2 h at 37 °C with tested sera

diluted in TBS and 0.03% skimmed milk and0.01% Tween 20, washed with TBS and 0.01%Tween 20 (3 times for 10 min each) then incu-bated with biotinylated sheep antimouse or don-key anti-rabbit F(ab’)2 fragments (Amersham lntplc, Amersham, UK) for 2 h at 37 °C. Followinga further wash, the antigen-antibody complexeswere vizualised by the biotin-streptavidin-peroxidase system (Amersham lnt plc Amers-ham, UK).

RESULTS

Serum antibody titers

Immunization of mice or rabbits with viable

chlamydiae induced a higher level of anti-bodies against proteic extracts of AB7

than against those of iBl, even after im-munization with viable iB1 (table II),whereas immunization of mice or rabbitswith proteic extracts induced a similar levelof antibodies against the 2 strains.

The serum dilutions used for immuno-

blotting are indicated in table II. 1.

Immunoblotting with antibodiesagainst viable chlamydiae (fig 1)

Mouse antibodies against viable AB7

(MvAB7) recognized 20 bands of AB7 (fig1A) with apparent molecular masses (MMs)of 27 to 115 kDa. Fourteen bands (28-29,33, 35, 37, 40, 49-50, 62, 68, 74, 78, 88, 96and 110 kDa) appeared as specific for AB7strain, since they reacted only in the homol-ogous system antigens AB7 with MvAB7.

Mouse antibodies against viable iB1

(MviB1) cross-reacted with 7 of AB7 strainbands (fig 1 A), 4 of which were also recog-nized by MvAB7. AB7 or iB1 major outermembrane protein (MOMP) did not reactwith MviBl. MviB1 recognized 9 bands oniB1 protein extracts (fig 1 B), the MMs ofwhich were 30 to 100 kDa; 2 of them (43and 66 kDa) seemed to be iB1-specificantigens since they reacted only in the

homologous system antigens iB1 withMviBl. Only one iB1 band (30 kDa) react-ed weakly with MvAB7.

Rabbit antibodies against viable AB7

strain (RvAB7) recognized 24 of AB7 strain

bands (fig 1A) strain and reacted weaklywith 8 bands of iB1 strain (fig 1B). AsMvAB7 they recognized considerably few-er proteins on iB1 strain than on AB7

strain, but unlike MvAB7 they revealed

about as many bands on iB1 strain as rab-bit antibodies against viable iB1 strain

(RviB1) did on iB1 or AB7 strains. Fourbands of iB1 strain (30, 35, 62 and 66 kDa)reacted specifically with RviB1 (they were

not revealed by rabbit antisera in the het-erologous system) but all of them exceptthe 66 kDa band were recognized bymouse antiserum MvAB7 on AB7 strain.

Eleven antigens of AB7 strain reactedspecifically with RvAB7; 6 of them (37, 49-50, 54, 78, 88, 90 and 96 kDa) were alsofound to be specific with MvAB7, and 5(54, 72, 76, 80 and 82 kDa) reacted onlywith RvAB7.

Immunoblotting with antibodiesagainst proteic extracts

Immunization of mice with proteic extractfrom AB7 strain (MpeAB7) significantly in-creased the number of recognized bandsfrom iB1 strain, since MpeAB7 reactedwith 10 bands of iB1 strain and 22 of AB7strain (fig 2). The one band of iB1 strain

(30 kDa) recognized by MvAB7 reactedvery weakly with MpeAB7 or MpeiBl. Fivebands of IB1 strain reacted with MpeiB1and did not react with MpeAB7, but noneof them were specific for iB1 strain.

MpeAB7 revealed 2 (60 and 64 kDa) outof 3 proteins of AB7 strain which reactedwith MviB1 but not with MvAB7.

We found only 1 specific antigen on iB1strain, a 66-kDa band which reacted onlywith MviB1 and 11 specific antigens ofAB7 strain, but only one of them (32 kDa)was not previously recognized by MvAB7or RvAB7.

Rabbit antibodies against proteic ex-

tract of AB7 (Rpe AB7) reacted with 30proteins of AB7 strain (fig 3), 16 of whichwere recognized by RvAB7. RpeAB7 didnot react with 78 to 94 kDa bands asdid MpeAB7, but recognized bands ofMMs < 30 kDa. RpeiB1 reacted with 17

proteins of iB1 strain, 4 of which were spe-cific to iB1 strain but were not recognizedby the other sera, MviBl, RviB1 1 or

MpeiB1.

Fifteen AB7-specific bands were foundby rabbit antibodies but only 11 appearedto be truly specific in all systems: MvAB7,RvAB7, MpeAB7, RpeAB7 (table 111); 4 ofthem (MM 22 to 32 kDa) only reacted withsera against proteic extracts. Only 4 (96,90, 88 and 49-50 kDa) were recognizedby both mouse and rabbit sera against vi-able AB7 strains and could be antigens forspecific diagnosis of caprine and ovine

chlamydiosis. The one protein (66 kDa)found iB1-specific by MviB1 reacted with

RpeAB7.In order to determine whether the spe-

cific antigens revealed on AB7 could beused for diagnosis of abortive chlamydiosiswe looked for their evidence in other inva-sive abortive strains (AB13, S26/3, A22),invasive intestinal strain Mo 907, or non-invasive intestinal strains (iB2, iB5). Thesestrains, used as antigens in immunoblot-

ting (figure 4), confirmed the absence ofthe specific antigens on non-invasive

strains and their presence on invasive

strains including the invasive intestinalstrains Mo 907.

DISCUSSION

AB7 specific antigens were found by im-munoblotting with mouse and rabbit sera.As we wanted antigens for diagnosis of

chlamydial abortions in ewes and goats,only antigens which reacted with both seraagainst viable AB7 prepared in mice andrabbits was kept, since the others could bespecific to the host species and could benot recognized by ovine or caprine sera.Ovine or caprine sera were not used herebecause of the antibodies against intesti-nal chlamydia that almost all sheep andgoats have. The one AB7-specific antigenwhich reacted with all the anti AB7 serawas the 49-50 protein, so this could be thepotential antigen for diagnosis of abortive

chlamydiosis. The other specific antigensrecognized by both MvAB7 and RvAB7 butnot by Mpe AB7 or Rpe AB7 were 88, 90and 96 kDa which were previously identi-fied as &dquo;virulence&dquo; proteins by SDS-PAGE(Buzoni-Gatel et al, 1989) whereas the 76-80 doublet specific of non-invasive strainsin SDS-PAGE was not antigenic. No iB1-

specific antigen fitting the previous criteri-on could be found.

Except for the identification of specificantigens, the most notable features of thisstudy were the weak antigenic reactivity ofproteic extract from iB1 strain and non-invasive strains, and the small number ofcommon antigens shared among strains

AB7 and iB1 compared to the great simi-larity of the protein patterns of the 2 strainswhen analyzed by SDS-PAGE (Buzoni-Gatel et al, 1989). The difference ob-served between the antibody responses toinoculation with viable strains AB7 or iB1

may reflect: 1), the difference in virulenceof the strains. We have previously shownthat iB1, as all non-invasive strains, is

quickly eliminated in mice contrary to inva-sive strains such as AB7 (Rodolakis et al,

1989); 2), the difference of presentation ofthe proteins following antigen processingby an antigen-presenting cell; 3), a differ-ence in sensitivity of the proteins to dena-turation by the process used to extract pro-teins and analyze them by SDS-PAGE.

In order to try to answer these differenthypotheses we studied the immunogenicityof proteic extracts of strains AB7 or iB1 in

mice and rabbits. This study indicated thatsome iB1 antigens were denatured by ex-

traction or electropheresis while those ofAB7 were not, as for example the 94 kDaprotein which did not react on iB1 whatev-er the serum, but was revealed on AB7 byMviBl and RviBi. However the responsesof mice and rabbits to immunization with

proteic extracts showed that the denatura-tion of iB1 proteins could not by itself beresponsible for those results, since the an-tibody titers of the anti-proteic extract serawere almost the same whatever the chla-

mydial strain and the host species. Where-as, with viable chlamydiae the anti-AB7 re-sponses were 8 to 10 times higher thanthose against iS1 as the differences in mul-tiplication of the 2 strains reflect. These re-sponses clearly indicated that the antigenprocessing by antigen-presenting cells wasnot the same according to the host speciesand/or chlamydial strain. The case of the90 kDa AB7-specific protein illustrated the

problem of processing: the 90 kDa proteinwas not revealed by MpeAB7, but was ob-viously immunogenic since it was revealed

by RpeAB7. When we compared the ho-mologous response after immunizationwith viable chlamydiae or proteic extractsof AB7 there was > 50% homology in

mouse or rabbit, whereas with iB1 therewas < 33%.

Our results were in accordance with

those previously published on immunoblot-ting of chlamydial proteic extracts fromovine (Mondesire et al, 1989; Baghian etal, 1990; Cevenini et al, 1991) or bovine(Fukushi and Hirai, 1988) abortive strains:the 58-60 kDa antigens common to AB7and iB1 could correspond to the 57-62doublet described by Mondesire et al

(1989) after Sarkosyl extraction, and the89 kDa bands described by Cevenini et al(1991) was likely to be the same as the90 kDa bands we had previously de-scribed (Buzoni-Gatel et al, 1989). Therewas no previously published antigeniccharacterization of intestinal strains of

C psittaci by immunoblotting; neverthe-

less, we noticed that the immunoblots ob-served with iB1 were more similar to thoseobtained with strains of serotype 2

(Mondesire et al, 1989; Baghian et !al,1990), than to those of abortive strains ofC psittaci. In C trachomatis, MOMP wasreadily extracted from intact EBs by SDS(Caldwell et al, 1981 but in C psittaci (Fu-kushi and Hirai, 1988; Mondesire et al,1989; Baghian et al, 1990) or C pneumoni-ae (Campbell et al, 1990), Sarkosyl-insoluble fraction was the best way to ob-tain MOMP. For this reason we only ob-tained a weak or no antibody responseagainst MOMP, but we were not lookingfor these antibodies, since MOMP, whichharbors genus, species and type epitopes,was not an ideal candidate for a specificdiagnosis antigen.

Thus this study showed that it must be

possible to find antigens which are specific

to virulent chlamydiae; but the very smallnumber of bands revealed on iBl, whatev-er the polyclonal serum (less than half thenumber on AB7), indicates that the 2 kindsof strains are more different than SDS-PAGE leads one to suppose: some pro-teins which seem identical when analyzedby SDS-PAGE must be different. Resultsfrom further research on characterization

using more discriminating methods suchas 2-dimensional gel electrophoresis will

clarify the situation, since in 1-dimensionalgel electrophoresis more than one proteincan give the same band.

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