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Brit. Jf. vener. Dis. (1972) 48, 87

Serial ultrathin sectioning demonstrating theintracellularity of T. pallidumAn electron microscopic study

VIVIAN LAUDERDALE AND JEROME N. GOLDMAN

Eye Research Laboratories, The Washington Hospital Center, Washington, D.C.

The possibility that Treponema pallidum could be-come engulfed within the cytoplasm of a host cellwas suggested when early light microscope findingspresented the appearance of intracellularity (Sauvageand Levaditi, 1906; Nyka, 1934; Nicol, Rios-Monte-negro, and Smith, 1969). Several investigators,using ultrathin sectioning and electron microscopytechniques, which eliminate those artefacts of lightmicroscopy which are due to tissue thickness andlow resolution, have also reported finding treponemeswhich appeared to be enclosed within the cytoplasmof cells because of their proximity to mitochondria,Golgi apparatus, and intracellular vacuoles (Abe, 1967;Azar, Pham, and Kurban, 1970; Ovcinnikov, andDelektorskij, 1969b; Goldman, 1969, 1970, 1971).

But, as suggested by Goldman (1969, 1971), thepossibility that a treponeme could be located withinan invagination of the host cell membrane or be-between folds of cytoplasm must be considered inevaluating the possibility that Treponema pallidummight occupy an intracellular habitat. Serial sectioningeliminates the one-dimensional view of the singlesection and allows a more accurate interpretationof the relationship of the treponeme to its apparenthost cell.

This is a report of studies of experimental rabbitorchitis caused by Treponema pallidum. The organismhas been shown to invade a variety of host cell types,some of which are generally considered to be phago-cytic.

Material and methodsApproximately 108 Nichols 1 strain Treponema pallidumwere inoculated into each testis of male New Zealandrabbits and allowed to develop a firm orchitis (6-13 days)

Received for publication July 14, 1971Reprint requests to: Jerome N. Goldman, M.D., 106 Irving Street,N.W. Washington, D.C. 20010, U.S.A.This work was supported by USPHS grant EY-00641 from theNational Institutes of Health, and in part by grants-in-aid from Fightfor Sight, Inc., New York City, and in part by Research to PreventBlindness, New York, N.Y.

The animals were killed with chloroform and 1 x 1mm.3pieces of testis were fixed in one of two fixatives: chromeosmium (Dalton, 1955; Glauert, 1965) or 3 per cent.gluteraldehyde in cacodylate buffer and post-fixed inbuffered 2 per cent. osmium tetroxide (Sabatini, Bensch,and Barrnett, 1963). After initial dehydration in a seriesof ethyl alcohols of increasing concentration, the tissueswhich had been fixed with Dalton's chrome-osmiumfixative were stained en bloc with 20 per cent. uranylacetate. All tissues were finally dehydrated in propyleneoxide and embedded in the epon mixture of Luft (1961).The blocks of tissue were sectioned on an LKB Ultro-tome III and picked up on 2 x 1 mm.2 single slot gridscovered with formvar and carbon films. The sectionswere stained with lead citrate (Reynolds, 1963) for 10minutes or double-stained (Frasca and Parks, 1965) withuranyl acetate (15 min.) and lead citrate (5 min.). AEI 6Band 801 electron microscopes were used for observationand photography.

ResultsCells with seemingly intracellular treponemes wereextremely rare, and the myriads of invading Tre-ponema pallidum were almost exclusively extra-cellular. They were concentrated around fibroblastsand mesenchymal cells (Fig. 1), and were observedaround the interstitial cells of Leydig and smallblood vessels, but were never found within semini-ferous tubules. The pathogens were also seen withinthe lumina of capillaries (Fig. 2) and small venules.The inflammatory response was moderate and

noteworthy because of the disproportionately highnumber of plasma cells in the testicular interstitium.

Although they were few in number in relation tothe large number of extracellular organisms, it wasnot difficult to find treponemes included in cyto-plasmic folds and invaginations. Some of theseappeared to be intracellular until serial sectioningproved them to be otherwise (Figs 3 to 6).

Figs 7 to 11 are micrographs of serial sections of aof a fibroblast within which a treponeme appears.Figs 7 and 11 are from sections taken beyond each

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88 British Journal of Venereal Diseases

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FIG. 1 A treponeme (arrow) is seen coilingin the extracellular space close to a fibroblast(FB). Fibrils and ribosomes can be seen inthe spirochaete, but the typical spirals areabsent. Chrome-osmium fixative. X 25,000

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end of the organism; these indicate that the spiro-chaete is entirely enclosed by cellular matrix andmay be considered to be intracellular.The intracellularity of T. pallidum was also demon-

strated by serial sectioning in a Leydig cell (Fig. 12),in a monocyte, and within mesenchymal cells (Fig.13).Multiple bacterial invasion of a single-cell type was

FIG. 2 Spirochaetes appear inside the lumenof a capillary. The endothelial cell appearsto have been invaded by a treponeme butserial sectioning was not done so that theintracellularity could be clearly defined.Gluteraldehyde-cacodylate fixative.x 10,000

seen several times. In Fig. 13, two out of four tre-ponemes found in a single mesenchymal cell arevisible.

Serial sections could not be obtained for exami-nation of the capillary endothelial cell of Fig. 2, but atreponeme appears to be located within the cell.The morphology of the extracellular spirochaetes

generally conformed to previous descriptions

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El-ectron microscopy of T. pallidum 89

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FIGs 3 to 6 Serial sections of an a,pparentintracellular treponeme.

In Figs 3 and 4 the spirochaete appears to be located

within a vacuole in the cell cytoplasm. A diffuse edge

of microfibrils (MF) (Fig. 3) is sectioned through to

reveal a typical membranous edge of the fibroblast

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Figs 5 and 6 reveal that the treponeme had beenenclosed within a membranous invagination and inthese figures the single spirochaete appears as two;one intracellular and one extracellular. The arrowindicates the position of the overlapping cellular edge.The presence of axial filaments in Figs 3 and 4 aremore characteristic of extracellular treponemes.x 100,000

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FIGs 7 to 11 This series reveals a spirochaete(arrow) enclosed within a fibroblast. The c toplasmremains uninterrupted by an entering treponemalprocess at the beginning (Fig. 7) and end (Fig. 1 1)

of the series. The host cell cytoplasmic membrane isintact throughout the series, thus fulfilling theclefinition of intracellularity. Chrome-osmiumfixative. x 62,500

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Electron microscopy of T. pallidum 91

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FIG. 12 Leydig cell containing a spirochaete(arrow). This cell was serially sectioned and the

treponeme was found to be intracellular.Chrome-osmium fixative. x 37,500

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FIG. 13 This mesenchymal cell was found upon serialobservation to contain four treponemes. Two of the organismsare visible in the plane of sectioning. (E, extracelleluartreponemes; I, intracellular treponemes.) Gluteraldehyde-cacodylate fixative. X 12,500

FIG. 14 An extracellular treponeme appearsto be burrowing into a mesenchymal cell.Typical spirals are not present, but ribosomesand fibrils (F) are clearly seen. Chrome-osmium fixative. X 100,000

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Electron microscopy of T. pallidumn 93

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FIGS 15 and 16. Two examples from a series

demonstrate the shortened, thickened, electron dense,

folded appearance of the intracellular treponemes (I).

An extracellular organism exhibiting typical fibrils

(F), and an inner membrane (IM) and outer cell

wall (OW), appears in the same micrograph

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(Fig. 16). Gluteraldehyde-cacodylate fixative.x 75,000

FIG. 17 Encystment (C) appears to have begun inthe extracellular treponemes. Chrome-osmiumfixative. X 37,500

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FIG. 18 Extracellular treponemes were found which FIG. 19 Intracellular encystment (arrow) appearsexhibited typical spiral coils such as that (arrow) to have surrounded one highly convoluted or severalappearing close to the processes of a mesenchymal spirochaetes. This cell was not serially sectionedcell. Gluteraldehyde-cacodylate fixative. X 12,500 in order to validate the intracellularity. Chrome-

osmium fixative. X 22,500

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Electron microscopy of T. pallidum 95

regardless of the fixation technique utilized. Theyranged in size from 0 10 to 0-15p in diameter and 5,u inlength. The characteristic spirals were rarely seenand were especially sparse in organisms which wereclose to host cells (Figs 1 and 14). A few extra-cellular treponemes were observed in the inter-stitium with regular spirals (Fig. 18). These spiralspossessed a wavelength of 1 1,u and amplitude of0 3O0 4uL.

Intracellular treponemes differed from extra-cellular ones in that they were shorter and thicker(1-1-7[i long and 035-0{5p in diameter). They alsolacked the spiral coils and were frequently foldedupon themselves (Figs 7, 11, 15, and 16). Theseintracellular T. pallidum organisms were generallymore electron dense with less distinct ribosomes andnuclear areas. Inner and outer fibril bundles werenever seen associated with the treponemes withincells. In contrast, extracellular treponemes in thesame sections showed characteristic morphology(Figs 1, 3-6, 14, 16, 17).Apparent treponeme encystment was seen, but

serial sections were not available to demonstrate thisphenomenon (Figs 17 and 19).Two fixatives were used and compared. Dalton's

chrome-osmium fixative appeared to preserve tre-ponemal structure well and allowed definition ofribosomes, fine filamentous nuclear areas, and doublecell membranes. But some electron dense cyto-plasmic components of the host cells appeared to belost with this type of fixative. The gluteraldehyde-cacodylate buffer fixative did not appear to preservea fine, sharp, double unit membrane in all instances,giving the treponeme and host cell a general electrondense appearance. Cytoplasmic details of the hostcell, such as fine fibrils and filaments, which wereapparent with this fixative appeared to have beenlost in the chrome-osmium fixed tissues.

DiscussionThe serial section approach has confirmed that T.pallidum does become intracellular. The morphologyof the organisms in the intracellular location differsfrom that of those outside the cell. The intracellularorganisms are smaller and more electron dense andlack fibrils. These changes were found in severaldifferent infected rabbits and with the use of twodifferent fixation techniques. The extracellular tre-ponemes in these experiments, which exhibitedmorphology in general agreement with the descrip-tions of other investigators (Ovcinnikov and Delek-torskij, 1968, 1969a; Jepsen, Hougen, and Birch-Andersen, 1968) were found on the same sectionsas the atypical intracellular organisms describedherein.

It may be that the intracellular organisms do nothave pathogenic potential and that this conditionis terminal and short-lived. But treponemes in dif-ferent stages of lysis were seen only in monocytesand polymorphonuclear leucocytes-cells known fortheir phagocytic function. The treponemes withinfibroblasts always had uniform morphology.

This report of intracellular treponemes shouldstimulate consideration of the possibility that T.pallidum may be 'stored' intracellularly, with re-tention of its antigenicity, viability, or even its patho-genicity, in some host cells. These suggestions havebeen made by Abe (1967) and by Ovcinnikov andDelektorskij (1969b) after they reported finding T.pallidum in plasma cells. Other workers (Azar andothers, 1970) have also reported the finding of tre-ponemes in plasma cells; although plasma cells wereabundant in the inflammatory process of our experi-ment, we saw no organisms within them. The specu-lation of Goldman (1969, 1971) that an intracellularhabitat may provide another protective device forthe treponemal invader against the action of drugs orthe immunological reactions of the host is raisedonce more.

Cyst-like forms, as described by Ovcinnikov andDelektorskij (1968, 1969a), were seen in our pre-parations. No serial sectioning was performed onthe specimens in Figs 17 and 19 in order to assessthe possibility of intracellular encystment, the numberof spirochaetes within a single cyst, and whether thecyst wall fully enveloped the treponemes. Theappearance of T. pallidum in small vessels has beenpreviously described in older lesions but not earlyin the development of the infection as in theseexperiments, and this may indicate why the earlytreatment of a syphilitic infection is of clinicalimportance.

SummarySyphilitic rabbit orchitis was studied electron micro-scopically, using serial sectioning of cells whichappeared to contain treponemes in order to provetrue intracellularity. The organism was shown toinvade a variety of types of host cell and it wasobserved that morphological changes occurred in thespirochaetes upon cell invasion.

We wish to thank Miss Julie Yin and Mr. Don Coble fortheir technical assistance; Dr. William Dobbins, of theVeterans Administration Hospital Department of Gastro-enterology, for the use of an electron microscope, photo-graphic equipment, and technical assistance; the CatholicUniversity Department of Microbiology and the AEICompany for the use of an electron microscope; and Mr.Robert Saunders, of Walter Reed Army Institute of Re-search, Department of Serology, for tissue specimens. This

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project was performed in the George Hyman MemorialResearch Building at the Washington Hospital Center.

ReferencesABE, S. (1967) Bull. pharm. Res. Inst. (Osaka), 68, 1AZAR, H. A., PHAM, T.D., and KuRBAN, A. K. (1970)

Arch. Path., 90, 143DALTON, A. J. (1955) Anat. Rec., 121, 281FRASCA, J. M., and PARKS, V. R. (1965) J. Cell Biol., 25,

157GLAUERT, A. M. (1965) 'Dalton's chrome-osmium fix-

ative', in 'Techniques for Electron Microscopy',2nd ed., ed. D. H. Kay, p. 173. Blackwell, Oxford

GOLDMAN, J. N. (1969) Discussion in 'Colloquium onOcular Treponemes'. Association for Research inOphthalmology Meeting, April 19-23, 1969. Sarasota,Florida.

- (1970) Trans. Amer. Acad. Ophthal. Otolaryng., 74,509

- (1971) 'A Review of Therapeutic Studies in LateSyphilis'. Presented at the Neuro-OphthalmologyCourse, January 4-8, 1971. Miami Beach, Florida.

JEPSEN, 0. B., HOUGEN, K. H., and BIRcH-ANDERSEN, A.(1968) Acta path. microbiol. scand., 74, 241

LuFT, J. H. (1961) J. biophys. biochem. Cytol., 9, 409

NICOL, W. G., RIos-MONTENEGRO, E. N., and SMITH,J. L. (1969) Amer. J7. Ophthal., 68, 467

NYKA, W. (1934) Ann. Inst. Pasteur, 53, 243OVCINNIKOV, N. M., and DELEKTORSKIJ, V. V. (1968)

Brit. J. vener. Dis., 44, 1- (1969a) Ibid., 45, 87

(1969b) Vestn. Derm. Vener., 43, no. 3, p. 14REYNOLDS, E. S. (1963) J. Cell Biol., 17, 208SABATINI, D. D., BENSCH, K., and BARRNETT, R. J. (1963)

Ibid., 17, 19SAUVAGE and LEVADITI (1906) C.R. Soc. Obstdt. Gynec.

Pidiat. de Paris, 8, 15.

Coupes ultra-fines en serie d6montrant lapr6sence intra-cellulaire de T. pallidum: eRtude aumicroscope 6lectronique

SOMMAIRE

L'orchite syphilitique du lapin fut etudiee au microscopeelectronique par la technique de coupes en series decellules paraissant contenir de treponemes afin de prouverleur presence intra-cellulaire. L'organisme se montraenvahir une variete de types de cellules de l'h6te et il futobserve que des changements morphologiques survenaientchez les spirochetes lors de l'invasion cellulaire. copyright.

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