Localization of Chlamydial Group Antigen in McCoy Cell Monolayers Infected with Chlamydia trachomatis or Chlamydia psittaci

Transcription

1 IFECTIO AD IMMUITY, ov. 1981, p /81/ $02.00/0 Vol. 34, o. 2 Localization of Chlamydial Group Antigen in McCoy Cell Monolayers Infected with Chlamydia trachomatis or Chlamydia psittaci SHIRLEY J. RICHMODIt* AD PEY STIRLIG2 Public Health Laboratory, Kingsdown, Bristol BS2 8EL,' and Department of Bacteriology, University of Bristol, BS8 1TD,2 England Received 15 June 1981/Accepted 9 July 1981 Chlamydial inclusions were demonstrated by indirect immunofluorescence (IF) with antiserum to the chlamydial group antigen when McCoy cell monolayers infected with either Chlamydia trachomatis or Chlamydia psittaci were fixed in formaldehyde or paraformaldehyde, provided the monolayer was not allowed to dry. If these monolayers were then air dried and restained by IF with the same antiserum but with a different fluorescence conjugate, group antigen associated with inclusion-containing McCoy cells but independent of the inclusions was revealed. This antigen was not restricted to infected cells but appeared to radiate out from them, suggesting that group antigen was released from infected cells. Similar host cell-associated antigen could be shown by IF of glutaraldehyde-fixed, air-dried monolayers, but inclusions could not be stained by IF before these preparations were dried, presumably because antibody could not penetrate glutaraldehyde-fixed cells. Electron microscopic immunoperoxidase studies of paraformaldehyde-fixed, wet monolayers located group antigen within inclusions on the outer membrane of chlamydial organisms and on single-membrane vesicles. However, when dried monolayers were labeled with the same immunoperoxidase technique, no intracellular labeling occurred, but dense staining was seen at the surface of infected cells and on adjacent membranous material. These observations are compatible with the postulate that replicating chlamydiae produce outer membrane blebs containing group antigen, which are excreted by the host cell during the chlamydial developmental cycle. Chlamydiae are obligate intracellular procaryotic parasites. They possess a double-unit membrane cell envelope similar to that of gramnegative bacteria (4), and they multiply within eucaryotic cytoplasm in phagosomes known as inclusions. These inclusions can be demonstrated in infected eucaryotic cells fixed in methanol or acetone by immunofluorescence (IF) with antiserum to the group antigen (10). This is a lipopolysaccharide antigen common to all members of the genus (7, 8) and is probably located in the outer membrane (OM) of both infectious chlamydial particles (elementary bodies) and replicating forms (reticulate bodies) (6). Recent IF studies of glutaraldehyde (GTA)- fixed, air-dried McCoy cells revealed group antigen independent of inclusions that is associated with infected host cell cytoplasm and appears to be released from infected cells (9). It has been suggested that OM blebs which contain group antigen are produced during chlamydial replit Present address: Public Health Laboratory, Withington Hospital, Manchester M20 8LR, England. cation and that excretion of such blebs by the host cell would account for this host cell-associated antigen. This hypothesis was strengthened when single-membrane vesicles, compatible in appearance with OM blebs, were seen consistently in chlamydial inclusions examined by transmission electron microscopy (EM) (14). In the present work, further IF studies on chlamydia-infected McCoy cell monolayers were carried out in parallel with immune EM. Within inclusions, group antigen was located both on the outer membrane of chlamydial organisms themselves and on single-membrane vesicles, and it was confirmed that group antigen, revealed only after aldehyde-fixed monolayers were air dried, was released from chlamydiainfected McCoy cells during the chlamydial developmental cycle. MATERIALS AD METHODS Chlamydial strains. Two Chlamydia psittaci strains were studied: an ovine strain, the agent of enzootic abortion (EAE), and an avian strain,

2 562 RICHMOD AD STIRLIG EAE (kindly supplied as a cell culture harvest by D. Hobson, University of Liverpool, Liverpool, England) had been passaged 35 times in yolk sacs and 10 times in McCoy cell cultures since being isolated from an aborted ewe (13) before it was used in this work. 352 was isolated in this laboratory in emetine-treated McCoy cells (see below) from a specimen of pooled cloacal swabs obtained from domestic ducks. (This specimen was supplied by the Central Veterinary Laboratory, Weybridge, England). 352 was passaged only twice in cell culture before use. Chlamydia trachomatis serotype E (12) and several untyped strains isolated directly from clinical material obtained from patients attending a clinic for the treatment of sexually transmitted disease were also studied. Growth of chlamydiae. Chlamydiae were grown in McCoy cell monolayers prepared on 10-mm-diameter cover slips in flat-bottomed plastic tubes ( , GIBCO Bio-Cult, Paisley, Scotland). C. trachomatis strains were grown in McCoy cells treated with cytochalasin B (12), and C. psittaci strains were grown in monolayers treated with 0.5,ug of emetine per ml of cell maintenance medium for 5 min immediately before inoculation. The infection of all strains was initiated by centrifugation of the monolayers at 3,000 x g for 60 min at 35 C. Inocula were adjusted to yield about 500 inclusions per monolayer for IF, and to infect from 20 to 100% of cells per monolayer for immune EM. Sera. Rabbit serum which contained antibodies to the chlamydial group antigen, made by immunizing a rabbit with yolk sac-propagated C. trachomatis serotype E (9), was used throughout. Pre-immunization serum from the same rabbit was used as the negative serum control. Both sera were used at a dilution of 1: 20, diluted in phosphate-buffered saline (PBS) for IF and in 0.2 M phosphate buffer (ph 7.3) for immune EM Ċonjugates. Fluorescein-conjugated anti-rabbit immunoglobulins produced in sheep (Burroughs Wellcome, Beckenham, England) and rhodamine-conjugated anti-rabbit immunoglobulins produced in swine (Dako Immunoglobulins, Copenhagen, Denmark) were used as fluorescence conjugates, diluted 1:50 and 1:20, respectively, in PBS. Peroxidase-conjugated antirabbit immunoglobulin G produced in goats (.E. Biomedical Laboratories, Windham, Maine), diluted 1:20 in phosphate buffer, was used for immune EM. IF. Between 40 and 68 h after inoculation of chlamydiae, monolayers were rinsed once in PBS, fixed for ca. 20 min at room temperature in either 4% (vol/vol) formaldehyde (F), 3% (wt/vol) paraformaldehyde (paraf), or 2.5% (vol/vol) GTA, and then rinsed thoroughly in PBS. In some experiments, 0.05% (wt/vol) saponin was added to these fixatives. F and GTA were diluted in PBS, and paraf (6%) was made immediately before use by the method of Robertson et al. (11) and diluted to 3% in PBS. Indirect IF tests were then performed either on monolayers which were not allowed to dry (W-IF test) or after monolayers were air dried (D-IF test). The W-IF test was carried out in the culture tubes: serum (0.1 ml) was added to each monolayer, which was then incubated for 30 min at 35 C. Monolayers were thoroughly rinsed with PBS and then reincubated with 0.1 ml of fluorescein or IFECT. IMMU. rhodamine conjugate for 30 min. They were again rinsed well in PBS, and cover slips were removed from the tubes and mounted cell-side downward on 0.8- to 1-mm glass slides in a solution of glycerol and PBS (9 volumes of glycerol to 1 volume of PBS). For the D-IF test, fixed monolayers were removed from the tube, mounted cell side upward on glass slides in DePeX, and left ovemight at room temperature to dry thoroughly. The D-IF test was then carried out by incubation of each monolayer with serum and then with conjugate in a humidified container for 30 min at 35 C. Slides were rinsed thoroughly in PBS between each incubation stage, and monolayers were air dried before the conjugate was added. After the test, a drop of the PBS-glycerol solution was placed on each monolayer, which was then covered with a cover slip (22 by 22 mm) for examination by fluorescence microscopy. These monolayers were compared with similarly infected monolayers fixed in methanol and stained by conventional methods (Giemsa stain and IF) to show chlamydial inclusions. Some F- and paraf-fixed monolayers were stained by IF both before and after the preparations were dried; cover slips were rinsed in PBS after the W-IF test, mounted cell-side upward, and left to dry overnight. They were then restained by the D-IF method with the same serum but with a different fluorescence conjugate from that used for the initial W-IF test. Photomicrographs were taken on a Leitz Ortholux II microscope fitted with Ploem incident fluorescence illumination and a Combiphot photographic attachment. Immune EM. Monolayers infected with either EAE or C. trachomatis serotype E for 40 to 50 h and fixed in 3% paraf with or without 0.05% saponin were labeled with a preembedding immunoperoxidase technique. Immune reactions were carried out without allowing monolayers to dry (W-EM) and after monolayers were air dried (D-EM). For W-EM tests, monolayers were rinsed once in phosphate buffer, fixed in 3% paraf with 0.05% saponin for 5 min at room temperature, fixed for a further 25 min in 3% paraf alone, and finally rinsed well in phosphate buffer. For D-EM tests, monolayers were fixed in 3% paraf for 30 min at room temperature, rinsed thoroughly in phosphate buffer, and allowed to dry in the culture tube overnight. Indirect immunopero:idase reactions were performed on both wet and dry monolayers as described for the W-IF test above, except that preparations were washed in phosphate buffer, not PBS, between each stage. After incubation with peroxidase conjugate, 0.03% (wt/vol) diaminobenzidine tetrahydrochloride (BDH, Poole, England) dissolved in 0.05 M tris(hydroxymethyl)aminomethane buffer (ph 7.6) which contained 0.01% (vol/vol) hydrogen peroxide was added to each monolayer for 5 min at room temperature. Monolayers were then rinsed well again, placed in 2.5% (vol/vol) GTA in 0.1 M cacodylate buffer (ph 7.2), and kept at 4 C until postfixation. They were then rised in cacodylate buffer, postfixed in 1% (wt/vol) osmium tetroxide for 90 min, and dehydrated through a graded ethanol series into propylene oxide. After 1 h in a solution containing equal volumes of propylene oxide and catalyzed Araldite resin, the cover slips were left in catalyzed Araldite for

3 VOL. 34, h. They were then drained and placed on a glass slide, cell-side uppermost. A gelatin capsule filled with fresh catalyzed Araldite was quickly inverted over the cells, and the slides and capsules were left at 70 C for 2 days. They were then removed and immersed completely in tap water for 4 to 7 days. The capsules and cells were then freed from the cover slips and glass slides by applying gentle lateral pressure against the capsules. The capsules were air dried for several hours and then trimmed and sectioned on an LKB III Ultratome with a diamond knife. Sections were mounted on Formvar-coated grids and examined unstained in a Philips 201 electron microscope. Some sections were subsequently double stained with uranyl acetate and lead citrate and reexamined. Some immunoperoxidase-labeled preparations were also examined by light microscopy and compared with similarily infected monolayers fixed and stained by conventional methods. RESULTS IF findings. Similar results were obtained with all chlamydial strains studied. When infected monolayers fixed in F or paraf were processed by the W-IF method, inclusions were stained. The quality of this stain was generally improved by the addition of 0.05% saponin to the fixative, since this both increased the density and evenness of the inclusion stain and reduced the amount of nonspecific extracellular fluorescence (Fig. la). o inclusions could be demonstrated in GTA-fixed monolayers by the W-IF technique, whether or not saponin was added to the fixative. When monolayers fixed in F, paraf, or GTA (with or without saponin) were dried before IF staining, no inclusions were visible, but plaques of fluorescent material could be seen distributed throughout the monolayer (Fig. lb). Each plaque was associated with several McCoy cells, and the number of plaques was comparable to the number of inclusions seen in similarily infected monolayers that were fixed and stained by conventional methods. o plaques were seen either in uninfected monolayers that were fixed, dried, and stained in the same way or in infected monolayers that were stained with the negative control serum. When infected monolayers fixed in F or paraf (with or without saponin) were stained twice by IF, before and after the monolayer was dried, both inclusions and plaques were seen differentially stained by the two fluorescence conjugates (Fig. lc through f ). The plaques of group antigen were associated with inclusion-containing McCoy cells; this antigen appeared to radiate out over the confluent monolayer from infected cells, which suggested that group antigen was being released from infected cells and was ad- LOCALIZATIO OF CHLAMYDIAL GROUP ATIGE 563 hering to adjacent noninfected cells. Immune EM. Inclusions of EAE examined by EM after incubation for 40 to 50 h were densely packed with chlamydial particles, predominantly reticulate bodies, whereas serotype E, incubated for similar periods, in general produced inclusions which appeared emptier, and the proportion of elementary bodies to reticulate bodies was higher in serotype E inclusions than in EAE inclusions. After the immunoperoxidase reaction, similar labeling patterns were seen with both EAE and serotype E. When the immune reaction was carried out without letting the monolayer dry (W-EM test), good ultrastructural preservation and specific labeling within infected McCoy cells were achieved. This labeling occurred only within inclusions (Fig. 2a and d): on the chlamydial particles themselves (both elementary and reticulate bodies) it was restricted to the OM, but in addition, single-membrane vesicles were also clearly labeled (Fig. 2b, c, and e). At least one in three of the inclusions detected by conventional light microscopy in similarly infected monolayers was labeled by this immunoperoxidase technique, and no labeling occurred when negative control serum was used (Fig. 2f). After the D-EM test, the fine structure of the McCoy cells was sufficiently well preserved, despite drying, for the main intracellular components, including inclusions and chlamydial organisms within them, to be recognizable. o labeling after the immune reaction was seen either within inclusions or in host cell cytoplasm, but dense labeling occurred both at the surface of the infected cells and on adjacent membranous material (Fig. 3a, b, d, and e). This labeling was not seen in uninfected monolayers treated in the same way or in infected monolayers when either negative control serum or peroxidase conjugate alone was used (Fig. 3c and f). DISCUSSIO Methanol and acetone are fixatives commonly used for IF of cultured cells since they allow good intracellular penetration of antibody, but membranes are disrupted and the ultrastructure of the cells is destroyed by these reagents, so they are unsuitable fixatives for EM. The group antigen associated with the surface of infected host cells detected by IF and immune EM in this work is likely to be removed by these fixatives, which would explain why it is not seen in acetone- or methanol-fixed preparations stained by IF. Fixation which both preserves ultrastructure and allows antibody penetration is difficult to achieve, but it has been reported in recent stud-

4 564 RICHMOD AD STIRLIG IFECT. IMMU. FIG. 1. Chlamydia-infected McCoy cell monolayers stained by indirect IF with antiserum to the chlamydial group antigen (x550). (a) Inclusions of C. psittaci (EAE strain). The monolayer was fixed in paraf and saponin and stained with rhodamine conjugate. (b) C. trachomatis (clinical isolate). The monolayer was fixed in F and air dried and then stained with rhodamine conjugate. (c and d) C. trachomatis, serotype E. The monolayer was fixed in F and stained with fluorescein conjugate; it was then air dried and restained with rhodamine conjugate. (c) Fluorescein-stained inclusion. (d) The same field as (c) showing rhodamine-stained plaque. (e and /) C. psittaci, 352 strain. The monolayer was fixed in paraf and stained with rhodamine conjugate; it was then air dried and restained with fluorescein conjugate. (e) Rhodamine-stained inclusion. (D) The same field as (e) showing fluorescein-stainedplaque surrounding rhodamine-stained inclusion. (In the optical system used, rhodamine-stained antigen was visible with fluorescein filters, but fluorescein-stained antigen was not visible with rhodamine filters)

5 VOL. 34, 1981 ies of virus-infected monolayers with various combinations of GTA, paraf, and saponin (2, 3). In this work, inclusions could not be demonstrated by IF in chlamydia-infected monolayers fixed in GTA, and preliminary immnunoperoxidase studies (not reported here) with the paraf- GTA-saponin combination used by Bohn (1) failed to demonstrate any labeling within inclusions. This may be because the inclusion membrane, which is derived from the host cell but is probably considerably modified by the parasite during the course of the chlamydial developmental cycle, is a more difficult barrier for antibodies to penetrate than is the host cell plasma membrane. However, a high proportion of inclusions in the monolayer were stained by the W- IF technique when monolayers were fixed in paraf or F alone, particularly when saponin, which in combination with aldehydes increases antibody penetration without gross disruption of membranes (1), was added to the fixative. ParaF with 0.05% saponin was therefore used to fix monolayers before immune EM was performed; this fixation technique achieved specific inclusion labeling of a large proportion of inclusions, and definitive proof that group antigen occurs on the OM of chlamydial particles was obtained (Fig. 2). This corroborates the earlier observations of Dhir and Boatman, who used a silvermethenamine stain and EM to locate group antigen at the periphery of purified organisms (6). Group antigen was also located on the singlemembrane vesicles (Fig. 2c and e), which confirms that these vesicles are chlamydial in origin rather than derived from the host cell, and strengthens the view that they are OM blebs produced by replicating chlamydiae in the same way that free-living gram-negative bacteria release blebs during growth (5). The rabbit serum used in this work was obtained by immunizing a rabbit with C. trachomatis serotype E (9); it therefore probably contained antibodies to species- and type-specific antigens in addition to group antibodies. However, this serum gave similar results with C. psittaci and C. trachomatis strains both in this LOCALIZATIO OF CHLAMYDIAL GROUP ATIGE 565 study and in earlier work (9), which suggests that the predominant antibodies were directed against the group antigen. As other chlamydial antigens are characterized and antisera are raised against them, use of this immune EM technique should both enable these antigens to be located in situ within inclusions and indicate at what time during the chlamydial developmental cycle these antigens are present. Drying of aldehyde-fixed monolayers before IF or immune EM dramatically affected the staining of group antigen. o intrainclusion labeling occurred, despite the fact that the morphology of the inclusion remained intact after drying (Fig. 3); this suggests that drying prevents antibody penetration into the host cell. In addition, group antigen which was present but not accessible to antibody in wet monolayers was revealed when monolayers were dried. This antigen occurred both at the surface and in the neighborhood of infected cells, and it appeared to adhere to adjacent uninfected cells, so that, provided monolayers stained by IF were confluent, plaques of fluorescent material, rather than individual fluorescing cells, were seen. These plaques were easy to detect at low magnification, and preliminary unpublished observations suggest that IF staining of F-fixed, dried monolayers provides a practical method for detecting chlamydial infection in monolayers inoculated with clinical material. This method is as sensitive as the conventional methods currently used to recognize chlamydial inclusions in diagnostic microbiological laboratories. It is possible that the antigen detected at the surface of infected cells was an artifact caused by the shift of intracellular antigens when aldehyde-fixed monolayers were air dried. We feel that this is unlikely for the following reasons. The antigen would have to move both across the cytoplasm of infected cells and extracellularly in the vicinity of infected cells to produce the plaques of antigen that were consistently seen (Fig. 1); very considerable disruption of both inclusions and host cell cytoplasm would be necessary for this to occur. EM of dried monolayers showed that, on the contrary, ultrastructure was very well retained; inclusion morphology was good, and both nuclear membranes and intracytoplasmic organelles such as mitochondria and lysosomes were well preserved (Fig. 3). Moreover, chlamydial antigen localized within inclusions by IF in wet monolayers was not dispersed after these monolayers were dried, but additional antigen was revealed after the dried preparations were restained by IF (Fig. 1). Exposure of antigen on dehydration, rather than shift of intrainclusion antigen, is therefore the more likely explanation for the extracellular antigen detected by both IF and EM in dried preparations in this work. The presence of group antigen at the surface of infected cells can be explained by postulating that infected cells excrete OM blebs during the chlamydial developmental cycle. However, if this occurs, group antigen should also be found in infected host cell cytoplasm in transit from inclusion to the exterior of the host cell. o such antigen was seen, which may be because intracytoplasmic antigen is not accessible to anti-

6 566 RICHMOD AD STIRLIG IFECT. IMMU. e,.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~- ~~~~~~4# v9 7-.X. U a Downloaded from on September 3, 2018 by guest FIG. 2. McCoy cells infected with C. psittaci (EAE strain) or C. trachomatis (serotype E) and labeled by the immunoperoxidase technique without letting the monolayer dry (W-EM test). (a) Cell infected with EAE showing labeled inclusion (IC). Part ofunlabeled inclusion (arrow) isjust visible in adjacent cell. (b) Portion of EAE inclusion showing labeling at periphery of reticulate bodies (RB). (c) Portion of labeled and stained EAE inclusion showing labeling restricted to OMs of reticulate bodies and to single-membrane vesicle (arrow). (d) Cell infected with serotype E showing labeled inclusion. (e) Portion of serotype E inclusion showing labeling of OM of elementary body (EB) and single-membrane vesicles (arrows). (D) Serotype E- infected cell tested with negative control serum, showing unlabeled EBs and RBs. Single-membrane vesicles are present but barely visible (arrows)., McCoy cell nucleus. Bars represent 0.5 /Im. (o preparations shown in Fig. 2 and 3, apart from that illustrated in Fig. 2c, were stained with uranyl acetate and lead citrate.)

7 VOL. 34, 1981 LOCALIZATIO OF CHLAMYDIAL GROUP ATIGE tt *Wr ~. 4t'IXlC SIz 3f - #3, 9'~~~~~~~~~~l a MAMA.S r En%.Ml A 1- i,!. -,.A4.y Downloaded from /. 4- RB on September 3, 2018 by guest FIG. 2-con't. bodies in either wet or dried monolayers; alternatively, antigen may be present in the cytoplasm only in relatively small amounts, which the preembedding immunoperoxidase technique used in this work was not sensitive enough to detect. More sensitive, postembedding immune EM techniques may in the future locate such antigen. It is not yet understood why group antigen at the surface and in the vicinity of infected cells is only accessible to antibody after aldehyde-fixed monolayers are air dried. Modification of antigen may occur either at the phagosome membrane or at the cytoplasmic membrane of the host cell, which might account for this difference between intra- and extrainclusion antigen.

8 568 RICHMOD AD STIRLIG IFECT. IMMU. -.^ IC 9qip 14 'lot L. w 9.. 6,-: II 9.L IC * IC 4 4. I LJ d p- LJ f FIG. 3. McCoy cells infected with C. psittaci (EAE strain) or C. trachomatis (serotype E) and labeled by the immunoperoxidase technique after monolayers were air dried (D-EM test). (a and b) EAE-infected cells showing unlabeled chlamydial particles within inclusions (IC) and labeling at cell surface and on adjacent extracellular membranous material (c) Cell containing EAE inclusion treated with negative control serum; no labeling has occurred. (d and e) Serotype E-infected cells showing unlabeled chlamydial particles within inclusions and labeling at surface ofmccoy cell and on adjacent extracellular material (t) Cell containing serotype E inclusion treated with peroxidase conjugate only; no labeling has occurred., McCoy cell nucleus. Bars represent 1 tmn. Similar observations were made with all chlamydiae studied in this work, irrespective of the source of the isolate or the extent to which it. I It t, 0 A %.;i.. W" 0 a had been passaged in the laboratory. So it is likely that the behavior of these strains is representative of the genus as a whole and occurs

9 VOL. 34, 1981 LOCALIZATIO OF CHLAMYDIAL GROUP ATIGE 569 IC * :-^t IC., A z_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~, A 0 IC 4, s-i L >.1*....a... Jb IC V., L-J r', LI I... a c FIG. 3-con't. in vivo as well as in vitro. Group antigen released from infected cells during intracellular chlamydial replication in naturally infected hosts may therefore contribute both to the pathogenesis of chlamydial infections and to the immune response of the host. It is also interesting to speculate whether similar excretion of the by-products of parasite replication occurs in eucaryotic cells infected with other procaryotic and protozoal intracellular parasites. ACKOWLEDGMETS We are grateful to Ian Paul, Bristol Public Health Laboratory, for expert technical assistance, and to Leighton Greenham, University of Bristol, for the use of the Leitz Ortholux microscope.

10 570 RICHMOD AD STIRLIG LITERATURE CITED 1. Bohn, W A fixation method for improved antibody penetration in electron microscopical immunoperoxidase studies. J. Histochem. Cytochem. 26: Bohn, W Electron microscopic immunoperoxidase studies on the accumulation of virus antigen in cells infected with Shope fibroma virus. J. Gen. Virol. 46: Chasey, D Investigation of immunoperoxidase-labelled rotavirus in tissue culture by light and electron microscopy. J. Gen. Virol. 50: Costerton, J. W., L. Poffenroth, J. C. Wilt, and. Kordova Ultrastructural studies of Chlamydia psittaci 6BC in situ in yolk sac explants and L cells: a comparison with gram-negative bacteria. Can. J. Microbiol. 21: Devoe, L. W., and J. E. Gilchrist Release of endotoxin in the form of cell wall blebs during in vitro growth of eisseria meningitidis. J. Exp. Med. 138: Dhir, S. P., and E. S. Boatman Location of polysaccharide on Chlamydia psittaci by silver-methenamine staining and electron microscopy. J. Bacteriol. 111: Dhir, S. P., S. Hakomori, G. E. Kenny, and J. T. IFECT. IMMU. Grayston Immunological studies on chlamydial group antigen (presence of a 2-keto-3-deoxycarbohydrate as immunodominant group). J. Immunol. 109: Dhir, S. P., G. E. Kenny, and J. T. Grayston Characterization of the group antigen of Chiamydia trachomatis. Infect. Immun. 4: Richmond, S. J Chlamydial group antigen in McCoy cells infected with Chlamydia trachomatis and Chlamydia psittaci. FEMS Microbiol. Lett. 8: Richmond, S. J., and E. 0. Caul Fluorescent antibody studies in chlamydial infections. J. Clin. Microbiol. 1: Robertson, J. D., T. S. Bodenheimer, and D. E. Stage The ultrastructure of Mauthner cell synapses and nodes in goldfish brains. J. Cell Biol. 19: Sompolinsky, D., and S. J. Richmond The growth of Chiamydia trachomatis in McCoy cells treated with cytochalasin B. Appl. Microbiol. 28: Stamp, J. T., A. D. McEwen, J. A. A. Watt, and D. J. isbet Enzootic.abortion in ewes. I. Transmission of the disease. Vet. Rec. 62: Stirling, P., and S. J. Richmond Production of outer membrane blebs during chlamydial replication. FEMS Microbiol. Lett. 9: Downloaded from on September 3, 2018 by guest