Ultrastructural studies of chlamydial infection in guinea-pig urogenital tract

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1982.

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ULTRASTRUCTURAL STUDIES OF CHLAMYDIAL INFECTION IN GUINEA-PIG UROGENITAL TRACT

B. L. .Vedicnl

SOLOFF,

R. G.

RANK*

and A. L.

BARRON*

Research

Service, L’eterans A4dministration Medical Center, Little Rock, drkanras ?ZL’O6, Department of .4natorny and *Department of Microbiolou and Immunology, lizir,ersi
1 ‘.&P.rl.

INTRODUCTION

Chlamydiae are obligate intracellular prokaryotic parasites that possess a distinctive developmental cycle and are responsible for numerous disease syndromes in animals (Shewen, 1980) and man (Schachter and Caldwell, 1980). Chlumydia truchomatis is principally a human pathogen, and has attained prominence as a major cause of genital tract infections (Taylor-Robinson and Thomas, 1980). A convenient animal model that utilizes guinea-pigs and the guinea-pig inclusion conjunctivitis (GPIC) agent, a Chlumydiu psittuci organism (Mount, Biggazi, and Barron, 1972), has been developed to study chlamydial genital infection. Light and electron microscopical study of biopsy specimens obtained from the cervix of women infected with C. truchomutis showed chlamydial inclusions within columnar cells of the endocervix and within cells that could not be classified with certainty (Swanson, Eschenbach, Alexander, and Holmes, 1975). Studies with the guinea-pig model have shown that the epithrlium of the exocervix and the transitional zone of the guinea-pig cervix contained characteristic inclusions during GPIC-induced genital tract infection (Barron, White, Rank, and Soloff, 1979; Soloff, Barron, White, and Rank, 1977), but that endocervical involvement occurred under special circumstances only; e.g. immunosuppression (White, Rank, Soloff, and Barron, 1979). The present study extends these observations and presents an ultrastructural analysis of infection by GPIC agent of various cells of the guinea-pig urogenital tract, including cervix, fallopian tube, penile urethra, and bladder. MATERIALS

AND

METHODS

Male and female mature (400 to 500 g weight) Hartley-strain guinea-pigs were obtained from Simonsen Laboratories, Gilroy, California. The animals were housed individually in plastic cages with overlying fibreglass filters. This stock has been found to be free of natural GPIC infection; nevertheless, each animal was screened for antibodies to GPIC by indirect immunofluorescence before use (Rank, White and Barron, 1979). Inoculation

GPIC inoculum was prepared from 50 per cent yolk sac homogenate concen002 l-9975/82/040547+

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trated by centrifugation at 192 g and 5 “C for 10 min to remove debris, followed by 30 900 g for 30 min to sediment the elementary bodies. The particles were resuspended in sucrose-potassium glutamate (Rank et al., 1979) and administered in a volume of 0.05 ml. The ELD,, of the inoculum (50 per cent egg lethal dose of the infective agent calculated by the method of Reed and Muench, 1938) ranged from 1.4 x lo4 per ml to 1 *O x log per ml during the course of these experiments. Female guinea-pigs were infected by delivery of the inoculum into the vagina via a syringe fitted with a solder-blunted 23-gauge needle (Barron et al., 1979). Male guinea-pigs, with one exception, were infected by inoculation of the GPIC suspension (O-05 ml volume) into the urethra.

Animals Examined Cervices were obtained from 3 animals. Two of these animals were killed at day 2 of infection (animals CP 20, CP 21), and one was killed at day 4 (CP 19). Fallopian tube tissue was taken from guinea-pigs which had received cyclophosphamide (150 mg per kg body weight) at nine-day intervals in order to effect immunosuppression (White et al., 1979) and which showed gross evidence of salpingitis. The tissue from one animal (number 0077), killed on day 29 of infection, was examined. Five males were examined for the presence of GPIC infection of the urethra. One of these had been killed on day 4 of infection, 2 on day 5 and 2 on day 7. In one animal only (number 422) was the search by electron microscopy successful. This animal had been killed on day 7 of infection. In addition, one male (number 0264) received cyclophosphamide (150 mg per kg body weight) at g-day intervals before being killed on day 20 of infection. It was infected by administering the 0.05 ml inoculum dropwise at the external meatus (Rank, White, Soloff, and Barron, 1981). The urinary bladder from this animal was examined.

Preparationof Specimens Tissue preparation was performed as in Barron et al. (1979). All tissue specimens were obtained from guinea pigs anaesthetized with pentobarbital sodium before being killed by an overdose of the same drug. Cervices were divided into exocervical and endocervical/exocervical transitional areas. Samples of fallopian tube tissue were obtained from each of the animals that exhibited gross evidence of salpingitis. In the males, urethral samples were taken from stretched penises. Bladder-wall tissue was removed, promptly dropped into fixative, and diced. Tissues were fixed originally in a formaldehyde-glutaraldehyde mixture (Karnovsky, 1965) or glutaraldehyde buffered with phosphate. Before post-fixation in osmium tetroxide buffered with s-collidine or sodium cacodylate, all tissues were rinsed at least overnight in a buffered 7 per cent sucrose solution. Following post-fixation, a few specimens (Figs 4, 5) were treated with tannic acid (Wagner, 1976) and some (Figs 1, 2, 3) were soaked en bloc with uranyl acetate before dehydration (Karnovsky, 1967). All tissue specimens were dehydrated in an ascending series of methanol, then soaked in propylene oxide and embedded in epoxy resin. Thick (1 to 2 pm) sections were stained with toluidine blue for light microscopy. Thin sections were cut with a diamond knife on a PorterBlum Sorvall model MT-2 ultramicrotome and stained with either a saturated solution of uranyl acetate in ethanol (Gibbons and Grimstone, 1960) followed by lead citrate (Venable and Coggeshall, 1965) or a routine of potassium permanganate, uranyl acetate and lead citrate (Soloff, 1973). Hitachi model HU-11B and Model 300 electron microscopes were used for observation. RESULTS

Cervical Epithelium Several

morphological

types

of cells present

in the epithelium

of the cervix

Fig.

1. Juxtaluminal cervical squamous cell shows large, interconnected phagosomes bordered with tonofibrils and filled with CPIC particles in various stages of their developmental cycle. ‘. 16 000 (Bar= 1 pm). Fig. 2. Cross-sectioned array of profiles (arrows) revealed by tangential section of CPIC agent. : 36 000 (Bar=0.5 pm). Fig. 3. Longitudinally sectioned projections (white arrow) appear to extend from the EB surface through its cell wall. In one case, these projections impinge on the outer membrane of an RB (short arrow). A tangentially sectioned form (double arrow) shows an array of profiles adjacent to an EB on whose surface several of these same profiles appear (arrowheadj. \’ 34 000 (Bar-O.5 pm).

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4. RB scattered among mucous granules (MG) within a cervical mucous cell. The constriction seen in several RB (arrows) provides evidence of binary fission. x 11 000 (Bar= 1 pm). 5. Chlamydial forms within a columnar cell from the transitional zone of cervical epithelium. EB (arrow), RB and IB, some with multiple nucleoids (N), can be identified. Adjacent uninfected columnar cell demonstrates usual distribution and complement of organelles. x 10 000 (Bar= 1 pm).

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were affected by the infectious process. These cells were classified as squamous, mucous, and columnar by light microscopic examination of sections stained with toluidine blue. Squamous cells. Juxtaluminal squamous cells were readily susceptible to infection by GPIC agent. The organisms were present in phagosomes, many of which were quite large and apparently interconnected. Because of the volume displaced by the enlarged inclusion vacuoles, cell organelles localized centrifugally in the remaining cytoplasm. Dense bundles of cytoplasmic filaments rimmed the inclusion vacuoles and were identified as constituents of partial barriers between the adjacent vacuoles. Often these cytoplasmic filaments were condensed into tonofibrils. The vacuoles contained all stages of the chlamydial developmental cycle: elementary bodies (EB), reticulate bodies (RB), and intermediate bodies (IB) (Fig. 1). Approximately oval profiles with average dimensions in cross-section of 19 nm x 26 nm (13 to 25 nm x 16 to 32 nm), arranged in a hexagonal pattern with an average centre-to-centre spacing of 59 nm (50 to 71 nm), were detected on the surface of some tangentially sectioned chlamydial forms (Fig. 2). Other angles of section revealed projections extending apparently from the inner membrane or the intermediate layer of the EB and projecting through the cell wall. These averaged 60 nm centre-to-centre (53 to 67 nm 1 and were approximately 18 nm in diameter (15 to 19 nm). Their lengths varied with the angle of section. In some cases, the projections appeared to impinge on adjacent organisms (Fig. 3). Mucous cells. Light microscopy of thick sections stained with toluidine blue revealed the presence of violaceous granules in many cells adjacent to the lumen. The presence of these granules was the basis for classifying such cells as mucous cells. Darkly staining particles were observed interspersed among the granules, and electron microscopy of thin sections selected from such areas disclosed the particles to be GPIC in the RB stage of their growth cycle. The RB were arranged in groups of 2 to 12 organisms, or sometimes as individual bodies. Fission of RB (Fig. 4) was sometimes evident. Although RB represented the predominant form observed in mucous cells, other stages of the chlamydial growth cycle were observed occasionally in cells with a limited complement of‘ mucous granules. Columnar cells. Tall columnar cells were observed in sections from the transitional zone (Fig. 5). Inclusion vacuoles containing chlamydiae occupied much of the apical cytoplasm of these juxtaluminal cells. The remainder of the apical cytoplasm was occupied by the cellular organelles, e.g. granular endoplasmic reticulum, Golgi apparatus, and mitochondria. The oval nucleus of these cells xi-as located in the basal area of the cell. Fallopian

Tube Epithelium

The epithelium of the oviducts was composed of both ciliated and nonciliated cells. Infected cells were seen infrequently. Chlamydiae were noted within cells that could be identified as ciliated (Fig. 6), but no infected cell could be identified unequivocally as non-ciliated. The pattern of infection in the

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Fig. 6. Infected fallopian tube cell that contains an inclusion vacuole (P) and exhibits cilia (arrow) at its surface. X 14 000 (Bar= I ym). Fig. 7. Phagosome containing GPIC agent located in cell from the urethra of male guinea pig. An RB shows evidence of binary fission. The remaining forms appear crenated. ~28 000 (Bar=O.S

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oviductal cells differed somewhat from that in cervical cells: a single inclusion vacuole, positioned supranuclearly, was present in each infected cell, and the enclosing membrane of the phagosome usually was not collapsed. This gave the impression that these inclusion vacuoles were more rigid than those observed in cervical cells. lirethral

Epithelium

(Male)

Chlamydiae were detected only sporadically during electron microscopic examination of the juxtaluminal epithelial cells of the penile urethra. The phagosomes were located in the supranuclear area of the infected cells. They were small and did not ramify extensively throughout the cell as had been observed frequently in cervical cells. In contrast to the organisms observed in cervical cells, many of the chlamydiae within urethral cell phagosomes presented with crenated borders (Fig. 7). The architecture of the urethral cells was not disrupted by the infection and cell organelles were unremarkable in appearance.

Fig. 8. A large phagosome that is crowded with chlamydial forms is located adjacent to the nucleus of this superficial bladder cell. MRB (arrowhead) are intermixed with the other chlamydial forms. Occasionally, an MRB is seen attached to an RB (arrow). Y 16 000 (Bar= 1 pm).

554 Bladder Epithelium

B.

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SOLOFF~~~~.

(Male)

Electron microscopy revealed the presence of GPIC in superficial bladder epithelial cells from animals with cystitis. Usually only a single large inclusion vacuole was observed, within which all stages of the chlamydial growth cycle were present. Furthermore, an additional morphological form was observed that resembled the profiles termed miniature reticulate bodies (MRB) by Tanami and Yamada (1973). This form had not been found previously in our studies of genital tract tissues infected with GPIC agent. The MRB were distributed in the interstices between other stages of the chlamydial growth cycle (Fig. 8). Occasionally, MRB appeared to be attached to an RB, but neither the outer nor the inner RB membrane could be identified as ensheathing the MRB. Infected bladder epithelial cells appeared to be intact morphologically and the organelles were normal in appearance and distribution. Polymorphonuclear Leucocytes and Macrophages Although numerous polymorphonuclear leucocytes were observed during electron microscopic observations of the various tissues from infected animals, no profiles recognizable as chlamydiae were observed in these cells. In one instance, examination of a section of cervical tissue revealed a macrophage that contained structures resembling remnants of chlamydiae. DISCUSSION

Four features of our results stand out: hexagonally arrayed profiles on the surface of chlamydial forms located within squamous cells; the presence of the GPIC agent in the RB stage of its life cycle within mucous columnar cells; presence of Chlamydia within oviductal cells; and the detection of chlamydial particles within male urethral and bladder epithelial cells. Both the circular profiles and the projections shown on EB within phagosomes of squamous cells in this investigation resemble those illustrated by Matsumoto, Fujiwara and Higashi (1976) in thin-sectioned samples of meningopneumonitis strain (C. psittaci) EB isolated from L cells. The dimensions and centre-to-centre spacing of the circular profiles conform to those features termed B structures by Matsumoto (1973, 1979). The fine structure of the major features that decorate the plasma membrane and cell walls of Chlamydia have been energetically investigated not only by thin-section techniques, but also by scanning electron microscopy (Gregory, Gardner, Byrne, and Moulder, 1979) high-voltage electron microscopy (Stokes, 1978), freeze-fracture technology (Louis, Nicolas, Eb, Lefebvre, and Orfila, 1980), or both (Matsu1975 ; Matsumoto and Nagatomo, 1976). This appears moto and Higashi, to be the first report concerning observation of these structures in vivo by thin-section methodology. Visualization of the structures may have been facilitated by the additional density and clarity gained through en bloc staining with uranyl acetate. The finding by Matsumbto (1981) that surface projections on RB within isolated inclusions traverse the phagosomal membrane caused

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him to suggest that the projections function in information exchange with the host cell. Our present observation of projections from EB impinging on adjacent RB and possibly EB is consistent with the explanation that such structures may be involved in interchlamydial communication as well. The observation of GPIC organisms exclusively in the RB stage of‘ their developmental cycle within functional cervical mucous cells, as judged by their complement of granules, parallels similar observations made by Doughri, Altera, Storz, and Eugster (1973) and Doughri, Young, and Storz (1974) in the goblet cells of bovine calf ileum. That chlamydial development proceeded in mucous cells relatively depleted of secretion was noted by these workers also, and was attributed to the decreasing competition for essential molecules consequent on gradual disruption of host cell metabolism brought about by the parasite (Todd, Doughri, and Storz, 1976). In the present study, examination of fallopian tube tissue revealed clearly the presence of GPIC agent in ciliated oviductal cells, but left unresolved their presence in non-ciliated oviductal cells. However, during a fine structure study involving organ culture of bovine oviduct inoculated with the bovine abortion strain of C. psittaci, frequent infection of epithelial cells that did not bear cilia on their surface were observed as well as occasional infection of ciliated cells (Hutchinson, Taylor-Robinson, and Dourmashkin, 1979). That salpingitis could be induced in vivo in normal guinea pig oviduct was demonstrated by Sweet, Banks, Sung, Donegan, and Schachter (1980). The inclusion vacuole within the oviductal cells in the present investigation appeared more rigid than those observed within other epithelia from this study. The significance of this observation is inapparent at present; however, evidence has been presented that inclusion morphology is a function of the host cell and its environment (Spears and Storz, 1979a). crinary tract infection of male guinea-pigs can be effected by the intra-urethral inoculation of GPIC agent (Mount, Bigazzi, and Barron, 1973: Ozanne and Pearce, 1980), but histopathological exploitation of this system has been minimal. The ultrastructural studies reported here show chlamydial inclusions within epithelial cells of the penile urethra. These inclusions were relatively small in contrast to those observed in cervical cells, and the GPIC agent within the phagosomes appeared crenated. Profiles resembling miniature reticulate bodies, another divergence from the standard developmental forms o!‘Chlamydia, were found in urinary bladder epithelial cells. Both Tanami and Yamada (1973) and Spears and Storz (197913) concluded that MRB were caused by treatment of their cultures with penicillin and cycloheximide, respectively. The cause of the MRB found in our investigation is not readily apparent, but it may be significant that the bladder tissue used in thir, stud\ was obtained from an animal that had received cyclophosphimide. Regardless of the epithelium investigated, infection was observed to be confined to the superficial cells. This is in marked contrast to the invasiveness described by Doughri, Altera, and Storz (1973) upon infection of neonatal bovine gut with C. psittaci agent LW-613. These workers found chlamydial particles not only within cells comprising the gut epithelium, but also within cells beneath the basal lamina, i.e. macrophages, plasma cells, fibroblasts and

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neutrophil leucocytes. The results presented in this report emphasize the marked differences in the infectious process that exist between various strains of C. fisittaci. While these studies on GPIC agent in the genital tract of guinea-pig may not be translatable directly to humans infected with C. trachomatis, the evidence obtained to date demonstrates that the GPIC strain of C. psittuci is capable of replicating in a variety of cell types. Perhaps the accepted specificity of the oculogenital strain of C. truchomatis for columnar cells deserves re-examination (Schachter and Caldwell, 1980). SUMMARY

GPIC agent (C. @sit&) inoculated into the vagina of guinea-pigs produced cervical and oviductal infection. The cells infected were all juxtaluminal in position and included ciliated oviductal cells and squamous cervical cells, many of which were extensively tunnelled with interconnecting phagosomes that contained all stages of the GPIC developmental cycle. Some EB within these phagosomes revealed modifications of their surfaces that were surmised to be involved in chlamydial interaction. Columnar cells, both mucous and nonmucous, constituted the other infected cervical cells. Chlumydia in the mucous cells were noted to be predominantly in the RB stage. Inoculation of GPIC agent into the urethra of male guinea-pigs resulted in infections that were detected in juxtaluminal cells of bladder and urethral epithelium. The Chlumydia observed in both the male urethra and bladder epithelial cells diverged somewhat from the usual forms observed. The organisms in the urethra appeared crenated. In the bladder epithelial cells, miniature reticulate bodies were noted, intermixed with profiles of the chlamydial developmental cycle. ACKNOWLEDGMENTS

We thank Danna Rose11 and Lisa Carter for their excellent technical assistance. This investigation was supported by the Medical Research Service of the Veterans Administration Medical Center, Little Rock, Arkansas and in part by Public Health Service Grant no. AI 13069 from the National Institute of Allergy and Infectious Diseases. REFERENCES

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26, 728-735. [Received for publication,

August 7th, 198 l]