Diversity among abortion strains of Chlamydia psittaci demonstrated by inclusion morphology, polypeptide profiles and monoclonal antibodies

veterinary microbiology Veterinary

Microbiology

5 1 (1996)275-289

Diversity among abortion strains of Chlavnydia psittaci demonstrated by inclusion morphology, polypeptide profiles and monoclonal antibodies Evangelia Vretou a3*, Heleni Loutrari a, Laura Mariani a, Ka.lliopi Costelidou a, Petros Eliades a, Giorgia Conidou a, Stavroula Karamanou a, Olga Mangana b, Victoria Siarkou ‘, Orestis Papadopoulos ’ ’ Department ofBiotechnology, Hellenic Pasteur Institute, 115 21 Athens. Greece h Veterinap Institute for Infections and Pcrrasitic Diseases. I.53 10 Athens. Greece ’ L.abora:oT of Microbiology and Infectious Diseases. Veterinap Medicine. Aristotle University. 54 006 Thessulortiki. Greece Received 2 October

1995; accepted 25 March 1996

Abstract Twenty eight C. psittaci abortion strains had been previously classified in to 4 immunologically distinct groups on the basis of cross-protection experiments in a mouse model. To identify the molecular basis of their immunological divergence 4 representative strains were investigated by cellular, molecular and immunological techniques. An identical pattern was obtained by AluI digestion of the amplified major outer membrane protein gene (MOMP) by the polymerase chain reaction (FCR) of the 4 strains. However, inclusion morphology and polypeptide profiles clearly distinguished one strain, named LLG, and its homologous strain POS from the other prototypes by the presence of a unique protein at 26.5 kDa and the absence of a polypeptide at 23 kDa. Six out of 10 monoclonal antibodies (mAbs) raised against abortion strains failed to react with inclusions of the 2 strains. All 6 mAbs reacted with the chlamydial outer membrane complex (COMC). Two of these mAbs, one against the MOMP and one against an antigen at 90 kDa, did not react with immmunohlots of LLG and POS. The data provide direct demonstration of the existence of strain variation in the field and classify strains LLG and POS as a distinct C. psitfuci serotype l-subtype. The antigenic diversity among abortion strains should be taken into consideration when designing a subunit vaccine.

* Corresponding

author. Fax: +30-l-6423498;

0378-l 135,‘96/$15.00 Copyright PII SO378- 1 135(96)00048-X

e-mail: [email protected]

0 1996 Elsevier Science B.V. All rights reserved.

Keywords: C’h/an~u/irr prirfrzci: Mouse: PCR-RFLP: Monoclonal antibodies

Abortion;

Antigenic

variation:

Inclusion

morphology:

SDS-PAGE:

1. Introduction Enzootic abortion in ewes (EAE) is an economically important disease affecting many agricultural countries worldwide. Pregnant ewes and goats abort late in gestation or give birth to weak or stillborn neonates as a result of placental pathology associated with the infection (Storz. 1971). The causative agent of the disease was identified in 1950 as a member of the psittacosis-lymphogranuloma venereum group of organisms (Stamp et al.. 1950). This animal pathogen has recently gained renewed attention since it was demonstrated to cause abortion and severe illness in pregnant women (Herring et al., 1987). Previous serological and cellular studies classified the strains causing ovine and caprine abortions into a distinct subgroup within the species Chlum~din psittaci. named serotype I (Schachter et al., 1974; Spears and Storz, 1979: Perez-Martinez and Storz. 1985; Eb and Orfila. 1983). Although abortion strains were considered similar or very closely related. the fact that a monovalent vaccine provided inefficient protection to vaccinated flocks suggested the possibility of strain variation in the field (Aitken et al.. 1986). However, isolates from the vaccinated flocks investigated by cellular, serological and limited molecular methods could not be distinguished by any of these techniques (reviewed in Herring, 19931. Antigenic variation among abortion isolates, detected by protection experiments in mice inoculated intracerebrally, was reported by Johnson and Clarkson (19861. but the variant strains were not further characterized. A recent study using restriction patterns of the amplified MOMP gene has reinforced the current concept of homogeneity among abortion strains (Denamur et al.. 199 I). In a previous study 28 Greek isolates from placentae or aborted embryos were initially investigated on the basis of their potential to colonize spleens of mice inoculated with the vaccine strain A22. In successive experiments 4 immunologically distinct groups have emerged (Siarkou, 1993). Representative strains of each group protected mice from subsequent challenge with a series of homologous strains. while little or no protection was observed against heterologous strains. According to this scheme, prototype strain FAS. homologous to A22, protected mice from challenge with 13 different strains, strain FAG was homoIogous to 7 strains. strain VPIG protected mice from challenge with 5 strains and strain LLG was homologous to two more strains named ARG and POS (Siarkou, 1992). In the present study we have analyzed the molecular characteristics of the prototype strains that may account for the specific immunological divergence observed in vivo.

2. Material and method

The chlamydial strains used in this study were (i) reference strains purchased from ATCC, (ii) strains isolated and characterized in other European laboratories, and (iii)

Table 1 Characteristilzs

of chlamydial

strains investigated

Strains

Host/site

Disease

Origin

Source/Reference

A22 AB7 F MA/23 11% MB/312 ME/4004 MH/21 Francis. Mn IC-Cal-3 Tl3 FAG VPIG LLG ARG POS FAS

lamb sheep sheep cheep goat sheep goat ferret/lungs human/cervix lamb/faeces goat goat

abortion abortion abortion abortion abortion abortion abortion asymptomatic urethritis asymptomatic abortion abortion abortion abortion abortion abortion

Scotland France England Macedonia. Attica. GR Attica, GR Attica. GR California California England Macedonia. Macedonia. Macedonia. Macedonia, Macedonia, Macedonia.

Stamp et al., 1950 Souriau et al.. I994 Griffiths et al.. 1992 Papadopoulos Mangana Mangana Mangana ATCC VR- 122 ATCC VR-346 Griffiths et al.. 1992 Siarkou. 1992 Siarkou, 1992 Siarkou. 1992 Siarkou. I992 Siarkou. 1992 Sinrkou. 1992

OWdt

goat sheep sheep

GR

GR GR GR GR GR GR

several Greek strains isolated from placentae and aborted sheep or goat embryos. Characteristics of the strains used. their origin and source or reference are summarized in Table 1. All isolates were provided as yolk sac cultures. They were adapted to cell culture in McCoy cells in Eagle minimal essential medium (MEM) supplemented with 2.5% fetal calf serum, 2.57~ horse serum, 2 mM glutamine, 2 mM non-essential amino acids, gentamicin and vancomycin. Briefly, exponentially growing McCoy cell monolayers in 150/225 cm’ flasks were inoculated at a multiplicity of infection of I with 18/27 X I O6 inclusion forming units (IFUs) diluted in 2 ml Hepes-sucrose-cation buffer (HSC; 10 mM Hepes pH 7.3, I mM MgCl,, 0.1 5 mM CaCI,, 90 mM NaCI. 0.2 M sucrose) For I h at 37°C (Vretou et al.. 1989). The cultures were incubated for 24 h in cycloheximide-containing medium (0.001 mg/ml) and for another 48 h in medium without cycloheximide. The cells were harvested with glass-beads in ice-cold HSC. ruptured by a probe sonicator (Vibracell) and centrifuged at 500 X K for 10 min at 4°C. The supernatant was centrifuged at 30,000 X g in a SS34 rotor (Sorvalll at 4°C for 20 min and the pellet was resuspended by sonication in 2 ml HSC and stored at -80°C. Sham-infected cells were also prepared. Chlamydial stocks and cultured cells were routinely checked by standard methods for bacterial and mycoplasma contamination. Crude stocks of elementary bodies (EB) were digested with DNAse (0.1 mg/ml final concentration in 50 mMTris-HCl pH 7.6, IO mM MgCl, ) for 30 min at room temperature and centrifuged through 30 ml 35% v/v Gastrografin (760 mg/ml. Schering) in PBS for I h at 45,000 X g. The pellet was layered on a discontinuous gradient of Gastrografin (5 ml 52’%, 8 ml 44% and 13 ml 40% v/v in PBS) and centrifuged in a SW28 rotor at 50,000 X R at 4°C for 60 min. The layer containing the EB’s observed at the interface between 44% and 52% Gastrografin was collected. washed with 20 ml HSC, pelleted (30.000 X s. 4°C. 30 min), resuspended in HSC and

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frozen in aliquots at - 70°C. Protein concentration was determined by the bicinchoninic acid protein assay (Pierce) with bovine serum albumin (BSA) as a standard. 2.2. h4onoclonal antibodies Five-week old female BALB/c mice were immunized with 60 kg/mouse of partially purified formalin-inactivated EB of strains ME/4004 or MH/2 1. Immunization was repeated twice intraperitoneally at an interval of 7 days with the same amount. Three days after the last injection the spleens were removed and fused with NS-0 cells following two different protocols. Spleen cells from mice immunized with strain ME/4004 were fused in the presence of an electrical field using a Zimmerman apparatus. Cells from mice inoculated with strain MH/21 were fused with 40% polyethyleneglycol 1600. In both cases hybridomas were propagated using mouse macrophages as feeder layers. Supematants were screened for antibody against chlamydiae by an immunofluorescence test on fixed chlamydial inclusions (Vretou et al.. 1992). C. psittaci specific hybridomas were cloned by limiting dilution in OPI Media supplement (Sigma) containing 10% conditioned medium from LPS-stimulated P388 cells (Bazin and Lemleux, 1987). Ascites were prepared in pristane-primed mice. The isotype of mAbs was determined either by double immunodiffusion assay or by a commercial ELISA kit (Pierce). Reactivity of mAbs with chlamydial antigens was determined by immunoperoxidase stain on fixed chlamydial inclusions, by immunoblotting and by ELISA using COMC as the coating antigen. Mab 4/l 1. kindly provided by Dr G. Jones. MRI, Edinburgh. has been recently described in McCafferty et al. (199.5). mAbs against the antigens at 90 kDa (Souriau et al., 1994). were a generous gift of Dr. A. Rodolakis, INRA, Tours. mAbs raised against a chlamydial parakeet strain were provided by Dr. W. Schuy, Behringwerke. Marburg. These mAbs cross-reacted in immunoblotting with the antigens at 90 kDa from abortion strains. 2.3. Immunization

of mice

Groups of 4 BALB/c mice received two i.p. injections at one week interval of viable IO’ IFU/mouse of LLG, FAG, FAS and VPIG. Antisera were collected 3 days after the second infection and antibody titers were examined by the immunoperoxidase assay and by immunoblotting with the homologous and heterologous strains. 2.4. Immunoperoxidase

assay

McCoy cells grown in 24- or 96-well tissue culture plates and containing about 30-50s infected cells were fixed with methanol. mAbs or serial dilutions of mouse antisera in phosphate buffered saline (PBS. 0.14 M NaCl, 0.026 M KCI, 1.4 mM KH,PO,, 8 mM Na,HPO,) containing 0.05% Tween 20 (PBS-T) was added to each well and incubated for 1 h at 37°C. After four washes with PBS-T, the cells were incubated with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins diluted I:300 in PBS-T (Dakopatts) for 1 h at 37°C. The wells were washed as above.

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rinsed once with PBS and immunocomplexes were visualized with 0.0006% diaminobenzidine (DAB) and 0.03% H,O, in the presence of 0.003% NiCl,. The reaction was stopped with tap H,O. All experiments were run in triplicate. 2.5. Inclusion

morphology

and quantitation

of chlamydial

infectirity

24-well plates of McCoy cells monolayers were infected in duplicate with IO-fold serial dilutions of EB of each of the 4 strains purified from stocks of the same or close cell passage. Inclusions were visualized by the immunoperoxidase assay with a genusspecific anti-LPS mAb and counted at a magnification of X40 with an inverted microscope (20 fields per well). Specific activity was expressed as IFU/ mg purified EB. 2.6. SDS-PAGE Whole lysates of purified EB were analyzed by discontinuous sodium dodecyl sulphate ,polyacrylamide gel electrophoresis (SDS-PAGE) according to currently used protocols (Sambrook et al., 1982) using a 12.5% separating and a 5% stacking gel. Samples containing about 20 Fg of purified EB or sham-infected McCoy cells were solubilized in sample buffer (0.05 M Tris-HCl, pH 6.8, 2.5% SDS, 5.5% 2-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue), boiled for 10 min, sonicated, and loaded after centrifugation at 30,000 X g for 10 min. Protein bands were visualized by staining with Coomassie brilliant blue. The apparent molecular sizes of proteins were estimated according to the following standards (Pharmacia): phosphorylase b (94.000), BSA (67,000), ovalbumin (43,000), carbonic anhydrase (30.000), soybean trypsin inhibitor (20,100), and a-lactalbumin (I 4,000). 2.7. Immunoblotting Following SDS-PAGE, proteins were transferred electrophoretically from gels to nitrocellulose membranes (Hybond, Amersham) using a Biorad apparatus. The membranes were blocked with Tris-buffered saline (TBS, 20 mM Tris-HCl pH 7.4, 0.05 M NaCl) containing 0.05% Tween-20 (TBS-T) and 5% non-fat dry milk for 2 h at room temperature. washed twice with TBS-T and incubated overnight at 4°C with an appropriate dilution of mAb or antisera from mice in TBS-T containing 1% BSA (TBS-T-IBSA). After five washes with TBS-T the membranes were exposed for 1 h at 37°C to rabbit anti-mouse alkaline phosphatase-conjugated immunoglobulins (Dakopatts) diluted 1:lOOO in TBS-T. Strips were washed five times with TBS-T and twice with alkaline phosphatase buffer (100 mM NaCl, 100 mM Tris. 5 mM MgCl,, pH 9.5). Color development was observed on the addition of fresh substrate solution containing 0.33 mg/ml nitro blue tetrazolium and 0.165 mg/ml bromochloroindolyl phosphate (GIBC~, BRL). 2.8. Isolation of chlamydial

outer membrane

complex (COMC)

Sarkasyl treatment of purified EB was performed essentially as described by Caldwell et al. (1981). EB’s (approximately 2 mg/ml, strain ME/40041 were incubated for

1 h at 37°C in PBS containing 2% disodium N-lauryl-sarkosine and 1.5 mM EDTA, pH 8.0 and then centrifuged at 100,000 X g for 1 h. The pellet was resuspended in the same buffer and centrifuged as before. The combined pellets were further treated with DNAse (0.1 mg/ml final concentration in 50 mMTris-HCI pH 7.6. 10 mM MgCl? ) for 2 h at room temperature, centrifuged, and washed several times in PBS. Sarkosyl-soluble and insoluble material were probed in Western blots and in an enzyme-linked immunosorbent assay (ELISA). Polystyrene microtiter plates were coated overnight at 4°C with 100 ~1 of COMC (0.002 mg/ml) in 0.05 M carbonate buffer. pH 9.6. After blocking the wells with 5% non-fat dried milk in PBS-T. they were incubated with mAbs diluted in PBS-T-BSA for 1 h at 37°C. mAbs bound to the COMC were visualized with peroxidase-conjugated rabbit anti-mouse immunoglobulins (Dakopatts) diluted I:1000 in PBS-T (1 h, 37°C) and 3.3’,5.5’Tetramethyl Benzidine (TMB. Pierce) as the chromogen. The reaction was stopped with 1 M H,SO, and absorbance was read at 450 nm. Serial measurements of an unrelated mAb were used to determine the cut-off value at 0.2 AJsO (mean &-3 s). 2.9. PCR PCR amplification of the ollrpl gene was essentially performed as described by Denamur et al. (1991) using the CTU/CTL oligonucleotide primers. Briefly, 10” IFUs of crude EB were lysed in 130 ~1 lysis buffer (SO mM KCl, 20 mM Tris-HCl. 2.5 mM MgCl,. 0.45% Nonidet P40, 0.45% Tween 20) containing 100 kg/ml proteinase K. The mixture was incubated at 56°C for I h and the enzyme was heat-inactivated at 95°C for 10 min. A 12.5 ~1 aliquot of the lysate was subjected to PCR. Amplification products were examined by electrophoresis on 0.88 aparose gel of a 5 ~1 aliquot. They were digested with Alul (Boehrinper Mannheim) according to manufacturer’s instructions using 20 ~1 aliquots of the amplified samples. The digested samples were analyzed on 7% polyacrylamide gels. stained with ethidium bromide and photographed under UV illumination.

3. Results

The morphology of the inclusions of the 4 prototype strains cultured in McCoy cells and stained with an LPS-specific mAb showed considerable variation (Fig. 1A to D). Host cells infected with FAS usually had a single, typically bell-shaped inclusion. Inclusions of FAG and VPIG were polymorphic, and multinucleated syncytia were frequently observed (Fig. ICI. The morphology of inclusions induced by LLG was unique and characteristic for this particular strain. Inclusions were lobed and occupied the whole cytoplasm surrounding the nucleus. Dikaryons were very frequently observed (Fig. 1 D). Of the two LLG-homologous strains, POS and ARG, only POS had a phenotype similar to LLG. Inclusions induced by ARG resembled those of FAS (not shown). On the basis of the inclusion morphology it could be assumed that strain ARG was related to FAS and only POS was homologous to LLG.

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Fig. I. Monolayer cultures of cycloheximide-treated McCoy cells infected with the 4 representative abortive strains: (a) FAS; (b) FAG; (c) VPIG and (d) LLG. Inclusions were stained 72 h post infection with a genus-specific mAb and visualized with the peroxidaae reaction.

Strain LLG differed from the other strains not only by a distinct and characteristic inclusion morphology, but also in its specific infectivity (IFU/ mg purified EB). LLG was repeatedly found to be ten times more infectious than FAS and VPIG, whereas FAG exhibitecl an intermediate specific infectivity (data not shown). 3.2. Ah1 RFLP patterns The phenotypic features of LLG raised the question on the origin of this particular strain and its molecular characterization. The MOMP genes of A22, LLG. VPIG, and several other Greek abortion strains, presented in Table 1, were amplified by PCR and digested with Ah11. This approach has been successfully used for classifying C. psittaci strains since distinct patterns have been observed for abortion, intestinal and avian isolates (Herring et al., 1990, Denamur et al., 19911. All strains examined produced a single DNA fragment of the expected size. Digestion of the amplified MOMP gene with Ah1 ancl analysis of the resulting fragments revealed bands of 198, 171, 159 and 84-80 bp common to all strains examined (Fig. 2). 3.3. Pol,vpeptide profiles The protein profiles on SDS-PAGE of purified EB from the four prototype strains and the LLG-like POS are shown in Fig. 3. Minor heterogeneity was observed in the

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Fig. 2. Ethidium bromide-stained polyacrylamide gels of Ah1 digestion product of chlamydial amplified DNA of selected Greek abortive strains. Lane 2: strain MA/231 184; lane 3: MB/317: lane 4: ME/4004; lane 5: 7’. lane 7: LLG and lane 8: VPIG. Lane I is the molecular size marker V. MH/?l; lane 6: reference strain AL_. Identical fragments are observed at 198. 171, 159 and 83 bp.

profiles of strains FAS, FAG and VPIG. The patterns of LLG and POS were characterized by the presence of a band at 26.5 kDa lacking from FAS, FAG and VPIG and the absence of a polypeptide of 22-23 kDa present in the other strains.

123456789

Fig. 3. Coomassie brilliant blue-stained SDS-PAGE profiles of the polypcptides of purified EBs of C. psittuci abortive isolates: LLG (lane 1 and 9). FAG (lane 2), FAS (lanes 3 and 7). VPIG (lane 4). non-infected McCoy cells (lane 5). POS (lane 6). ARC (lane 8). Molecular weights in kDa are indicated on the left. The position of the two peptides 26.5 and 23 kDa are indicated by arrows on the right.

E. Vretou et al./ Veterinq

3.4. Characterization

of monoclonal

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51 (1996) 275-289

antibodies

10 mAbs raised against abortion strains were used to study the antigenic relationship of the 4 prototype strains. The characteristics of the 10 mAbs are summarized in Table 2. Emmunoperoxidase staining of inclusions of a series of strains listed in Table 1 suggested that the majority of the mAbs was serotype l-specific, whereas 2 mAbs, 179 and 193, also recognized the meningopneumonitis strain Mn (identical to Cal-lo) classified ;as an avian strain (Herring et al., 1989). Immunoblot analysis using reference strains AEI7 and A22 and several Greek abortive strains identified the antigens of 6 mAbs (Table 2). MAbs 181 and 191 recognized a triplet at 90 kDa plus a band at 85 kDa whereas mAb 192 reacted strongly with the doublet within the triplet at 90 kDa and only slightly with the minor band at 85 kDa. A Western-blot of EB from strain FAS with the 3 mAbs is shown in Fig. 4c. The molecular basis of the pleiomorphic antigenic reaction of the mAbs is still unknown, neither is the nature of the corresponding antigens. They may represent post-translational modifications of products of one or more genes (triplet at 90 kDa) and their proteolytic fragments (85 kDa). They will, therefore, be referred to as the ‘antigens at 90 kDa’. Their apparent molecular sizes are only approximate because of the difficulty of an exact calculation in this region. MAb 188 was specific for the MOMP; mAb 193 recognized a yet unknown antigen at 50 kDa, and finally mAb 179 reacted with a band at 28 kDa. The 28 kDa antigen comigrated with a C. trachomatis-polypeptide recognized by mAb 28.2, raised against the Mip-like protein (Lundemose et al., 1992, not shown). The antigens of mAbs 195, 200, 201 and 204 could not be identified in Western blots under denaturing conditions. Their association with the COMC was tested in an ELISA as described under Material and Methods. Electrophoretic analysis of the COMC prepared in the absence of reducing agents, showed a protein band at about 12 kDa, presumably the small cysteine-rich protein env A (Everett and Hatch, 1995), the MOMP. the large cysteine-rich protein at 60 kDa, the

Table 2 Characteristics

of monoclonal

antibodies

mAb

Isotype

Specificity

179 181 188 191 192 193 195 200 201 204

IgC2a IgG2b IgG2b IgG3 IgG2a IgGl IgG2b IgG2a IgG2b IgG3

Serotype Serotype Serotype Serotype Serotype Serotype Serotype Serotype Serotype Serotype

a

Antigen b

Reactivity

I, Mn ’ I 1

28 kDa 85-93 kDa MOMP 85-93 kDa 90 kDa 50 kDa _d

0.050 0.540 1.00 0.500 0.220 0.110 1.175

1 1 I. Mn

I

1.9

I

1

-

I

a Antigen recognized in immunoperoxidase assay. b Antigen recognized in Western-Blot negative reaction. ’ Reactivity in ELISA using sarcosyl-insoluble pellet as the antigen (AJS,,). d Negative reaction. ’ Positive reaction with the meningopneumonitis strain.

I .45 1.4

with COMC ’

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E. Vwtou et cd. / Veterina~

a 94

b

1234

-mm’

Microhiolo,q.v 51

a-192

1

(IY961275-2(19

234

191-

-

--

II-188

94 67

1931

-

43

-

-

30

4/m-

Fig. 4. Comparison of immunoblots of purified EB “s from the 1 prototype strains with mAbs. a: mAbs 192 and 188 with (1) LLG. (2) FAG, (3) FAS and (4) VPIG. b: mAbs 191. 193 and 4/l 1 with (1) VPIG. (2) FAS. (3) FAG. and (4) LLG Molecular weights in kDa are indicated on the left in (a) and on the right side in (b). The position of the antigens reacting with the respective mAbs is shown by arrows. c: immunoblot of the polypeptides of purified chlamydial EB from strain FAS rtm on 10% SDS-PAGE and probed with mAbs 192, 191 and 18 I (left to right). Only the region at 90 kDa is shown.

antigens at 90 kDa and some minor bands at higher molecular weight. With the exception of mAbs 179 and 193, all mAbs reacted in ELISA with the COMC (Table 2). 3.5. Reactirity

with monoc~lorml nntibodies.

Table 3 summarizes the results of immunoperoxidase staining of methanol-fixed inclusions of the four prototype strains FAS, FAG, VPIG and LLG and their homologous strains POS (LLG-like) and ARG (FAS-like) with the 10 mAbs. The reactivity

Table 3 Reactivity

of mAbs with strains FAS, ARG. FAG, VPIG. LLG and POS in imunoperoxidase

asaay

mAb

FAS

ARC

FAG

VPIG

LLG

POS

179 181 188 191 192 193 195 200 201 204

+ + + + + + + + + +

+ + + + + + + + + +

+ + + + + + + + + +

+ + + + + + + + + +

+ + -

+ + _

+ _

+ -

+ _ _ -

+ -

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pattern of FAS, FAG, VPIG and ARG was very similar. mAbs 188, 192, 195, 200, 201 and 204 failed to react with inclusions of strains LLG and POS. The anti-MOMP mAb 4/l 1 and several mAbs against the antigens at 90 kDa (Souriau et al., 1994) did not differentiate the strains. The same was true for a series of mAbs raised against a chlamydial parakeet strain which we had found to cross-react with the ovine antigens at 90 kDa. The negative reaction of LLG with mAbs 192 and 188 was also observed in immunoblotting. Both antibodies did not react with LLG while a strong reaction was seen with the bands of the other 3 prototype strains (Fig. 4a). Additionally. mAb 191 gave a distinct pattern with LLG, different from that observed with the other strains (Fig. 4b). MAbs 193 and the anti-MOMP mAb 4/l 1 reacted similarly with the four strains. POS showed the same reactivity pattern with the mAbs as LLG. 3.6. Analysis of the antibody response in mice To investigate further the antigenic properties of the 4 prototype strains, antisera against each strain were elicited in mice and their reactivities to homologous and heterologous strains were compared by immunoperoxidase and immunoblotting. Within three day:; after the second injection (10th day of immunization) the animals responded

1234

94 67

43

30

20.1

Fig. 5. lmmunoblot of the polypeptides of purified chlamydial EB’s from strains LLG (I), FAG (2). FAS (3) and VPIG (3) after SDS-PAGE with pooled mouse hyperimmune antisera against native EBs of strain FAS. Molecular weight in kDa are indicated on the left.

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with titers ranging from l/4000-l/l6000 tested in the immunoperoxidase assay. Significant differences in the antibody titers against the homologous and heterologous strains were not observed (not shown). Antigens 28 kDa, 50 kDa and at 90 kDa (with the exception of LLG) were among the most immunogenic proteins.The response against the MOMP was rather weak in all strains. The antigenic pattern of the 4 prototype strains with pooled sera from mice infected with strain FAS is shown in Fig. 5. Anti-FAS antibodies did not react with the antigens at 90 kDa in LLG as strongly as with the homologous and the other two heterologous strains.

4. Discussion We have used cellular, molecular and immunochemical methods to differentiate 4 chlamydial abortion strains from Greece that had been previously classified as prototypes of 4 immunologically distinct groups. The classification had been done on the basis of protection-challenge experiments in a mouse model (Siarkou, 1992). In the present study inclusion morphology, polypeptide profiles and antigenic characterization by mAbs and mice antisera clearly distinguished strain LLG and its homologous POS from the 3 other prototype strains, FAS, FAG and VPIG. Dikaryons. and a central nucleus, were characteristic for the inclusions of LLG and POS, suggesting a specific interference by these microorganisms in the cellular events of the host. Infection with LLG/POS resulted in enhanced fusion of cell membranes and/or disruption of cell division. Enhanced fusion of cell membranes might have further important implications for the generation of chlamydial diversity, e.g. by recombination of different strains, co-existent within the same inclusion. Recombination has been recently demonstrated between 2 different C. trachomatis serotypes, LGV Ll and L3 (Hayes et al., 1994). Very little is known about the formation of the chlamydial inclusion membrane, and identification and characterization of its components has been undertaken only recently (Rockey et al.. 1995). Comparative studies on different phenotypes might possibly contribute to further insights into this particularly important and unique chlamydial feature. The function of the distinct polypeptide at 26.5 kDa (Fig. 3) unique to strains LLG and POS and of the 23 kDa peptide absent in these strains, is unknown at present. The same is true concerning the involvement of these 2 proteins in the protective immune response. Peptides at this range of molecular mass have been previously characterized as histone-like proteins, products of late chlamydial genes (Hackstadt et al., I991 ). Further experiments are needed to identify the nature of the two proteins. Besides their role in chlamydial biology, they could prove useful epidemiogical tools for the identification of LLG-like types of abortion strains. Six out of 10 mAbs from our panel distinguished strains LLG and POS by a negative reaction and confirmed the relationship between these strains observed in vivo. They could, therefore, prove to be useful epidemiological tools. All 6 mAbs reacted in ELISA with the sarcosyl-insoluble pellet, the COMC. The MOMP and a band at 90 kDa were identified as the antigens of 2 of these mAbs. MOMP as a locus of chlamydial diversity was not unexpected. The molecular basis of the diversity of the 15 C. trachomatis

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serotypes is based on the variance of short sequences within surface-exposed segments of the MOMP, named VS (1 to 41, which are flanked by constant segments, CS 1 to 5 (Stephens et al., 1987). MAbs capable of serotype distinction recognize sequential epitopes within these variable domains (Baehr et al., 1988; Vretou et al., 1992). It is not unlikely, that divergence within the VS’s provides the basis for the antigenic variation of C. psittaci serotypes as well. This, however, is the first report of an anti-MOMP mAb that distinguishes various C. psittaci abortion strains. Although the negative reaction of the anti-MOMP mAb 188 with LLG in Western Blots suggested variation in the sequence of LLG-MOMP. genotyping by AU-RFLP of the amplified MOMP gene did not detect any difference between LLG and the other abortive strains (Fig. 2). This is not surprising if the A/u1 restriction sites may reside outside the epitope of this mAb. Indeed. epitope mapping using synthetic peptides in our laboratory has located the epitope of mAb 188 in VSI . very close to the restriction site of the enzyme (manuscript in preparation). Failure of the AluI genotyping to detect the variant strains of C. psittuci appears, however. as a limitation of this otherwise very useful typing method. The antigen family at 90 kDa has been described as a highly immunoreactive component of the COMC (Cevenini et al., 1991) and of major diagnostic importance (Souriau let al.. 1994). McCafferty et al. (1995) described very recently a sarcosyl-insoluble doublet at 66 kDa that changed its mobility to 98 kDa after heating, and was recognized by post-abortion sheep serum. Our results using several mAbs confirm the presence of this molecule in the sarcosyl-insoluble pellet and underline its complexity. Originally reported as a single band of 89 kDa by Cevenini et al. (19911, this antigen was subsl?quently characterized as a triplet (Souriau et al., 1994). We have observed antigenic heterogeneity between the bands of the 90 kDa triplet. What is far more importanl, is the apparent diversity of this phenotype among different strains. McClenaghan et al. (1991). had indicated quantitative differences between abortion strains in this region. Fig. 4a and b and also Fig. 5 provide evidence for quantitative and qualitative differences in the immunological reactivity of this antigen(s) in strain LLG. A more detailed analysis, e.g., by 2-D electrophoresis and probing with several mAbs should reveal the extent of variation. Their role in the immunological diversity detected by the protection experiments in vivo should be the subject of further studies. In conclusion, our data provide direct demonstration of the existence of variant types within C. psittaci abortion strains. Cellular, immunological and serological techniques have confirmed the classification of LLG and POS as an immunologically and biologically diainct subgroup. We therefore propose that these two strains constitute a subtype of serotype 1 strains. The strain variation in the field observed in this country may represem a general phenomenon that has been underestimated. For this purpose, appropriate tools, such as differentiating mAbs, now available, can be effectively used.

A&now ledgements We thank J.H. Pearce for a sample of mAb 28.2, A. Rodolakis for mAbs against the antigens at 90 kDa and strain AB7. G. Jones for mAb 4/l 1, M.J.Clarkson for strains F

and T13 and C. Sayada for performing the PCR-RFLP assay. A. Staikoglou has subcloned hybridomas, E. Psarrou is acknowledged for technical expertise and S.K. Bose and A.J. Herring for helpful suggestions and comments. L.M is a recipient of a fellowship of the National Foundation of Stipends. This work was supported by the Mediterranean Integral Programmes and by the EC Cost shared research contract AIR3-CT93-0957.

References Aitken, I.D.. Anderson. I.E. and Robinson, G.W.. 1986. Ovine chlamydial abortion: limitations of inactivated vaccine. In: ed. Aitken. Chlamydial disease of ruminants. Publication EUR 10056 EN. Commission of the European Communities. Luxembourg. pp. 55-65. Baehr, W.. Zhang, Y.-X.. Joseph. T.. Su, H.. Nano. F.F.. Everett, K.D.E. and Caldwell. H.D.. 1988. Mapping antigenic domains expressed by Chlangh trcrchonmis major outer membrane protein genes. Proc. Natl. Acad. Sci. USA. 85: 3000-4004. Bazin, R. and Lemleux, R.. 1987. Role of the macrophuge-derived hybridoma growth factor in the in vitro and in viva proliferation of newly formed B cell hybridomas. J. Immunol., 139: 780-787. Caldwell, H.D., Kromhout, J and Schachter. J.. lY81. Purification and partial characterisation of the major outer membrane protein of C. tmchomntis. Infect. Immun., 3 I : I 161 -I 176. Cevenini. R., Donati. M.. Brocchi, E.. De Simone. F and La Placa. M.. 1991. Partial characteriaation of an 89-kDa highly immunoreactve protein from C. psittclci A/ 22 causing ovine abortion FEMS Microbial. Lett.. 81: 11 l-l 16. Denamur. E.. Sayada, C.. Souriau. A.. Orfila. J.. Rodolakia. A. and Elion. J.. 199 I. Restriction pattern of the major outer membrane protein gene provides evidence for a homogenous invasive group among ruminant isolates of Chlarn~dicr pritruci. J. Gen. Microbial., 137: 25352530. Eb. F. and Ortila, J., 1983. Serotyping of Chhrn~dicz psitfai by the microimmunolluorescence test: isolates of ovine origin. Infect. Immun., 37: 1289-1291. Everett, K.D.E. and Hatch, T.P.. 1995. Architecture of the cell envelope of Clrlnnr~dia pittck 6BC. J. Bacterial.. 177: 877-882. Griffiths. P.C.. Philips. H.L.. Dawson. M. and Clarkson. M.J.. 1992. Antigenic and morphological differentiation of placental and intestinal isolates of Clhrnydia psittaci of ovine origin. Vet. Microbial.. 30: 165-177. Hackstadt. T.. Baehr, W. and Yuan, Y., 1991. Chkopdin trclchomcrti.r developmentally regulated protein is homologous to eucaryotic histone HI. Proc. Nat]. Acad. Sci. USA. 88: 3937-3941. Hayes. L.J., Yrarsley, P., Treharne, J.D.. Ballard. R.A., Fehler. G.H. and Ward. M.E.,1993. Evidence for naturally occuring recombination in the gene encoding the major outer membrane protein of lymphogranuloma venereum isolates of Clhmyliu truchomcztis. Infect. Immun., 62: 5659-5663. Herring. A.J., Anderson, I.E., McClenaghan. M.. Inglis, N.F.. Williams. H., Matheson. B.A.. West, C.P.. Rodger, M. and Brettle. R.P.. 1987. Restriction endonuclease analysis of DNA from two isolates of Chlumydicr ysitttrci obtained from human abortions. Br. Med. J.. 395: 1233. Herring, A.J.. 1993. Typing Clhm,vdirr psittoci- a review of methods and recent findings. Br. Vet. J.. 139: 155-375. Herring, A.J.. Tan, T.W.. Baxter. S.. Inglis, N.F. and Dunbar, S., 1989. Sequence analysis of the major outer membrane protein gene of an ovine abortion strain of Cltlamydio prittrrci. FEMS Microbial. Len.. 65: 153-158. Herring, A.J.. Tan. T.W. and Baxter, S.. 1990. Chlamydial abortion in sheep: molecular approaches to vaccination. pathogen detection and strain typing. In: ed. W.R. Bowie. H.D. Caldwell. R.P. Jones. P-A. Mardh. G.L. Ridgway, J. Schachter, W.E. Stamm and M.E. Ward. Chlamydial Infections, Proc. of the 7th Int. Symp. on Human Chlamydial Infetions. Cambridge University Press, pp. 378-382. Johnson. F.W.A.. and Clarkson. M.J., 1986. Ovine abortion isolates: antigenic variations detected by mouse

E. Vretou et al. / Veterinay

Microbiology

51 (19%)

275-289

289

infection. In: ed. I.D. Aitken. Agriculture. Chlamydial diseases of ruminants. Rep. EUR 10056 EN, Commission of the European Communities, Luxembourg. pp. 129- 132. Lundemose. AC.. Rouch. D.A., Birkelund. S.. Christiansen. G. and Pearce. J.H.. 1992. Chlamyfia trachornatis Mip-like protein. Mol. Microbial., 6: 2539-2548. McCafferty, ‘VLC., Herring, A.J., Andersen, A.A. and Jones. G.E., 1995. Electrophoretic analysis of the major outer membrane protein of C’hlarn~dia psiffaci reveals multimers which are recognised by protective monoclonal antibodies. Infect. Immun., 63: 2387-2389. McClenaghan. M.. Inglis. N.F. and Herring. A.J.,1991, Comparison of isolates of Chlatnydia psittaci of ovine, avian. and feline origin by analysis of polypeptide profiles from purified elementary bodies. Vet. Microbial.. 26: 269-278. Perez-Martinez. J.A. and Storz. J.. 1985. Antigenic diversity of Chhzdia psittaci of mammalian origin determin,:d by microimmunofluorescence. Infect. Immun., 50: 905-910. Rockey. D.D.. Heinzen. R.A. and Hackstadt, T.. 1995. Cloning and characterisation of a Clzlam~dia psittaci gene coding for a protein localized in the inclusion membrane of infected cells. Mol. Microbial., 15: 617-626. Sambrook. _I.. Fritsch, E.F. and Maniatia. T., 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor. NY. Schachter. .I.. Banks, J.. Sugg. N., Sung, M., Storz. J. and Meyer, K.F., 1974. Serotyping of chlamydia.1. Isolates 3f ovine origin, Infect. Immun., 9: 92-94. Siarkou. V.. 1992. Ph.D. thesis. Aristotle University. School of Veterinary Medicine, Thessaloniki. Greece. Souriau. A.. Salinas. J., DeSa. C.. Layashi, K. and Rodolakis, A., 1994. Identification of subspecies and serotype l-specific epitopes on the 80. to 90.kilodalton protein region of Chlaqdiu psittuci that may be useful for diagnosis of chlamydial induced abortion. Am. J. Vet. Res.. 55: 510-514. Stephens, R.S.. Sanchez-Pescador. R.. Wagar, E.A., Inouye. C. and Urdea. M.S., 1987. Diversity of Chhmylic~ truchomatis major outer membrane protein genes. .I. Bacterial.. 169: 3879-3885. Spears. P. and Storz, J.. 1979. Biotyping of Chhmydiu psittuci based on inclusion morphology and response to diethylaminoethyl-dextran and cycloheximide. Infect. Immun., 24: 224-232. Stamp. J.T.. McEwen. A.D., Watt, J.A.A., Nisbet, D.J.. 1950. Enzootic abortions in ewes. I. Transmission of the disease. Vet. Rec., 52: 251-254. Storz. J.. 1971. Chlamydia and Chlamydia-induced diseases. Charles C. Thomas. Springfield, IL. USA. Vretou. E.. Goswami. P.C. and Bose. S.K.. 1989. Adherence of multiple serovars of Cidamydin truchnatis is mediated by thermolabile protein(s) to a common receptor on HeLa and McCoy cell&. J. Gen. Microbial.. 135: 3229-3237. Vretou. E.. Mentis, A.. Pbarrou, E., Taoumaris, L.. Conidou, G. and Spiliopoulou, D., 1992. Unusual prevalence of the rare serovar Da of Chlnm?_dia trachomatis in Greece detected by monoclonal antibodies. Sexually Transmitted Diseases. 19: 78-83.