Intracellular Location of Treponema pallidum(Nichols Strain) in the Rabbit Testis
JOHN A. SYKES AND JAMES N. MILLERResearch Department, Soutthern California Caiicer Center, California Hospital Medical Ceenter, and the TreponemalResearch Laboratory, Department of Medical Microbiology and Immunology, U.C.L.A. School of Medicine,
Los Anzgeles, California 90015
Received for publication 24 May 1971
During investigations designed to obtain purified suspensions of virulent Trepo-nema pallidum (Nichols strain), infected rabbit testicular tissue was routinely ex-
amined in the electron microscope. Morphologically typical T. pallidum were foundintracellularly within the cytoplasmic substance of fibroblasts, interstitial and Leydigcells, and of spermatocytes. The importance of these observations to latency andtreatment is discussed.
Early reports dealing with electron micro-scope studies of Treponema pallidum infections ofman and animals failed to show the presence ofthe organisms within tissue cells of the host.More recently, in an electron microscope studyof T. pallidum occurring in a primary humanlesion, Drusin et al. (2) stated, "In each sectionthe bacterial cells are located in the extracellularspace. We have never observed them within thecytoplasm of a host cell." In other recent elec-tron microscope studies, Hasegawa (3) reportedon ultrastructural changes in the cytoplasm ofplasma cells present in the lesions of a patientwith condyloma latum. However, he was unableto demonstrate intracellular T. pallidum in eitherplasma or epidermal cells. Additionally, Azaret al. (1) have reported the engulfment of T.pallidum by human plasma cells which werepresent in a primary chancre. They also demon-strated the presence of the organism withinvacuoles of neutrophils, macrophages, endothe-lial and perivascular connective tissue cells. Inexperimental rabbit syphilis, Jepsen et al. (4)observed that the treponemes were always seenextracellularly and were accompanied by swellingof the amorphous material in the spaces betweenthe interstitial cells. They attributed this altera-tion in morphology to the infectious process.Although the treponemes were seen between thebasal membranes of the tubules, they were neverseen inside either the tubuli contorti nor in thelumen. Recently, Lauderdale and Goldman(personal communication) reported T. pallidwnwithin the cytoplasm of a Leydig cell, a mono-cyte, and within mesenchymal cells from in-fected rabbit testes. During recent studies de-signed to obtain "purified suspensions" of the
Nichols strain of T. pallidun from infected rabbittestes by utilizing density gradient techniques,routine electron microscopy of the starting ma-terial demonstrated T. pallidum within the cyto-plasm of testicular cells. It was felt that the sig-nificance of these findings merited a brief report.
MATERIALS AND METHODS
New Zealand White, adult, male rabbits were inocu-lated in the testes with the virulent Nichols strain ofT. pallidum and kept in air-conditioned quarters,maintained at a temperature of 18 to 20 C. At the timeof development of orchitis (8 to 12 days after inocula-tion), the rabbits were anesthetized and exsan-guinated by cardiac puncture. The testes were removedaseptically, and representative pieces were taken forhistology and electron microscopy. Tissue for elec-tron microscopy was cut on a wax block into smallpieces (1 mm') under drops of 3%C/ glutaraldehydein Millonig's buffer. The tissue was allowed to re-main in the glutaraldehyde for 2 hr at 5 C. Afterthorough washing with the same buffer, the tissuewas postfixed overnight in 1 % osmium tetroxide at5 C, washed with buffer, then dehydrated by usinga graded series of concentrations of ethyl alcoholbefore final dehydration in propylene oxide. The tissuefragments were flat embedded in Epon 812. Sectionsof 50 to 80 nm were cut with an LKB Ultrotome byusing a diamond knife, floated onto distilled water,and collected on Formvar-coated 200-mesh copperscreens. Sections were double-stained, by using uranylnitrate and lead citrate. Before examination in aSiemens Elmiskop IA, grids carrying the stainedsections were lightly coated with carbon. Shadow-castpreparations were made by placing a small dropof the suspension to be investigated on a Formvar-coated copper screen, allowing it to stand for 1 min,and removing the droplet by capillarity to a piece offilter paper. After the screen had air dried, it was
shadowed with Au 60'C: Pd 40" at a shadowingangle of 30°.
RESULTS
In the course of studies designed to obtain cleansuspensions of T. pallidunm (virulent Nicholsstrain) from infected rabbit testes, the electronmicroscope was used to evaluate the startingmaterial and the various steps of the purificationprocedure. The palladium-gold-shadowed or-ganism in Fig. 1 and la and the cross andlongitudinal sections seen in Fig. 2 were repre-sentative of the general morphology of theorganisms used for inoculation of the rabbits. Thesame general morphology was evident for theT. pallidumn seen within cells, whether in vacuoles(Fig. 3) where a cross section of one organismcan be seen lying within a cytoplasmic vacuole in afibroblast of the interstitium, or within the cyto-plasm of other cells, as shown in succeedingelectron micrographs. The organisms had anaverage diameter of 171 nm, with the axial fila-ments or tubules having a diameter of 21.7 nmwith a wall thickness of 6.7 nm. The averageoverall length of intact organisms not showingdegenerative changes (such as detachment ofaxial filaments from the cell wall) was 9 ,um.The cytoplasmic membrane had an averageoverall thickness of 21.0 nm. The same generalmorphology of the organisms was also evidentwhen they were found in extracellular areas(Fig. 4). Here the organisms can be seen lyingamongst cell debris close to some collagenfibrils which appear to form a part of the laminapropria. In the higher magnification seen inFig. 4a, three axial filaments or tubules havebeen cut in a glancing diagonal direction alongtheir long axis. These tubules have the samegeneral dimensions as those more clearly shownin Fig. 2. Spiralling of the axial filaments is welldemonstrated in Fig. 5, which shows parts ofa treponeme within a superficial vacuole, pos-sibly pinocytotic, in a fibroblast lying near thebasement membrane. Outside the cell, collagenfibrils cut in various planes were evident in adiffuse stroma.The location of T. pallidum within the cyto-
plasmic substance of what appears to be adegenerating secondary spermatocyte can be seenin Fig. 6. In this cell the nucleus appears lobu-lated and may possibly be in an incompletestage of division. Some nuclear pores cut acrosstheir diameter are clearly visible. The many darkmasses seen within the cytoplasm are similar tothose seen in other secondary spermatocytes.The condensation of chromatin at the nuclearmargin indicates that the cell was fairly mature.
Some cytoplasmic vacuoles containing neutralfat can be seen, and there has been some degen-eration of mitochondrial cristae, possibly due tothe invasion of the cytoplasm by the treponemesseen lying close to the nucleus. The typicaltreponemal morphology is more clearly seen atthe higher magnification in Fig. 6a. In a similarfashion, treponemes were frequently found withinthe cytoplasm of interstitial cells, such as thatshown in Fig. 7. This cell, despite the rectangularshape of the nucleus usually associated with tubulecells, was in an interstitial area. The cell isjudged to be mature by the degree of condensa-tion of chromatin along the nuclear margin.The nuclear sap is well preserved, showing dif-fuse and condensed chromatin; no nucleolus isvisible in this plane of sectioning. The cytoplasmshows well preserved ribosomes and rough endo-plasmic reticulum. Two vacuoles in the cyto-plasm are filled with material having stainingcharacteristics similar to neutral fat and serumproteins. The darker staining material betweenthe vacuoles is frequently seen in Leydig inter-stitial cells prior to the formation of crystals.Lying along one margin of the nucleus, parts oftwo treponemes are clearly visible. The charac-teristic morphology of the organisms is moreclearly demonstrated at a higher magnification inFig. 7a. Figure 8 shows a Leydig interstitial cellcontaining several dark staining bodies within thecytoplasm; in the large dark staining body,aciculate crystals characteristically found inLeydig cells were seen at higher magnifications.Only a small portion of the nucleus is included,but it shows well preserved diffuse chromatinand well marked condensed chromatin aroundthe nuclear membrane. Three nuclear pores canbe seen cut in cross section. The cytoplasm showsgood preservation of rough and smooth endo-plasmic reticulum; cristae are seen within manyof the mitochondria. In the area of the plasmamembrane there is evidence of active pinocytosis,and the cell is surrounded by the characteristicdiffusely staining material associated with theinterstitium. Within the cytoplasm, parts of atypical treponeme are visible and shown at highermagnification in Fig. 8a. Treponemes were alsofound within fibroblasts of the lamina propria,as shown in Fig. 9, where parts of several tre-ponemes are clearly visible lying adjacent to thenucleus of a fibroblast. Throughout this study,sections have been studied in sequence to makesure that the observed organisms were trulywithin the cytoplasm of the studied cells and notwithin invaginations of the plasma membraneor vacuoles.
VOL. 4, 1971 INTRACELLULAR LOCATION OF T. PALLIDUM 309
L' __. \v _w * ' -_ o
J n__zL' - <rf f / *$ ./ l
_S - - R ".?* } . . ........ w }
'"''' "' E_W4g;1ggER a'''
s i Sqg ;i;t- r !!t£i_ e t ;e. $ i . +;< A e Z; , ! . t v . ._, z AAs< .:" ,..: yiw ,,:; ' fz%, *we < _ .,,,,,,,.x, ,,,,j,, ,S,t,f, ;,-,,. *,;f, t ;oj, :,,-'d
SP.
.,,.
.ijF?::_
vk
:'.'3e -__ fi | _ I§.Ls| a |__y7 E
1_ I l_C *..-.#_ l | .,:}j_ 11 l' .>.| l l - s ---l B U -;+t z-e
; ., ,,;.e,._ .Fg >1 FA_' v
0.1 pmR . 2
FIG. 1. A typical T. pallidum as usedfor rabbit inoculation, from one of the stages ofpurificationi. Tlhe organismswere fixed in 1.5% glutaraldehyde before shadowing with Au 60%gO: Pd 40%7O at a 30 awigle. X 24,000. (Ja) Thearea within the small rectangle in Fig. 1 at higher magnification to demonstrate the superficial locationi of the axialfilaments. Bar on each illustration represents 1 ,um. X 56,000.
FIG. 2. A composite ofa longitudinal and a cross section of T. pallidumfrom the same negative to show thle axialfilaments and the general ultrastructure of the organism. X 160,000.
FIG. 3. A cross section ofa T. pallidum in a vacuole withinl a fibroblast of the interstitium. The external situationof the axial filaments is well demonstrated. X 80,000.
FIG. 4. A group of extracellular treponiemes lying amongst cell debris close to a bundle of collagen fibrils in thelamina propria. Bar represents 1 ,Lm. X 10,000. (4a) A higher magnification of the area enclosed by the rectanglein Fig. 4 demonstratin2g the characteristic morphology of T. pallidum. Axial filaments cut diagoncally can be seen.X 50,000.
VOL. 4, 1971 INTRACELLULAR LOCATION OF T. PALLIDUM 311
< :,. f \
a a a s Aw¢~~~~4. 4 tW1N1X lll 5M1 v 5t WSb 5'
A4_4
.<'5.is ft . + . s t
FIG. 5. A fibroblast lying niear the basement membrane conztaining a typical treponeme iii what appears to be apinocytotic vacuole. The spirallintg nature of the axial filaments is apparent. X 50,000.
FIG. 6. Part ofa degeneratinig secondary spermatocyte containing a small group of treponiemes within the cyto-plasm. Nuclear pores (ni) cut in cross section cant be seen. Bar represents I um. X 10,000. (6a) The area of therectantgle in Fig. 6 at higher magnification2 to show the ultrastructure of the treponemes. X 40,000.
FIG. 7. A cell of the interstitiuim showinzg parts oftwo treponemes lying within the cytoplasm close to the nucleuts.Bar represenits 1 Aim. X 8,400. (7a) The treponemes in Fig. 7 at a higher magnification. The spirallinig nature ofthe axial filaments is well demonstrated. X 40,000.
FIG. 8. A Leydig interstitial cell with a dark mass composed of aciculate crystals, lying in the ceniter to the leftof the nutcleuis. Parts of a coiled treponeme are seen lying within the cytoplasm. X 8,000. (8a) Tlle area of therectangle in Fig. 8 at a higher magiificationi to show the characteristic morphology of the treponiemes. Bar on eachillustratioln represents 1 ,um. X 29,000.
FIG. 9. A fibroblast from the lamina propria showing several treponemes within the cytoplasm. X 8,400. (9a)The area of the rectangle in Fig. 9 at a higher magnification demonstrates the characteristic morphology of theT. pallidum. Bar on each illustration represents I ,um. X 37,800.
DISCUSSIONThe ultrastructure of virulent T. pallidun
(Nichols strain), described in this study as beingwithin cells of the rabbit testis, was in agreementwith that previously described by others (4, 5).The findings of Azar et al. (1), who first describedthe intracellular location of T. pallidum withinplasma cells of a human chancre, and the recentfindings of Lauderdale and Goldman (personalcommunication), who reported the organismswithin the cytoplasm of a Leydig cell, mono-cytes, and mesenchymal cells of infected rabbittestes, have been extended. This report demon-strates that T. pallidum is present not only withinthe cytoplasm of Leydig cells, but also withinfibroblasts, interstitial cells, and in spermatocytes.It is possible that application of the electronmicroscopy techniques used in this investiga-tion, if applied to tissues from other regions ofinfected animals and man, might demonstrate anintracellular location for T. pallidum in cells ofother tissues. Studies along these lines are inprogress.The persistence of T. palliduim in rabbit testicu-
lar tissue, at a time when they are demonstrableby infectivity tests but not by dark-field micro-scopic observation, could be explained on thebasis of an intracellular location. Further, theappearance and intracellular persistence of or-ganisms in the spleen, bone marrow, and lymphnodes of rabbits, if demonstrable, might accountfor the phenomenon of latency which, afterdevelopment, persists for the remainder of thelife of the untreated animal despite the presenceof measurable circulating antibody. Additionally,a cellular immune mechanism for acquired re-sistance in experimental syphilis can be reasonablypostulated, based upon the ability of T. palliduinto reside within cells.The significance of an intracellular location for
T. pallidum in relationship to treatment has beensuccinctly stated in the World Health Organiza-tion Technical Report Series no. 455 by an inter-national group of treponematoses experts (7).
"In lesions, treponemes are commonly foundin the intracellular spaces. It has recently beensuggested-but not proved, that pathogenictreponemes may occupy an intracellular habitatwithin the host. If this is so, the cell wall andcytoplasmic components of the host cell mayprovide additional defences against treatment,as has been found in such diseases as tubercu-losis and brucellosis."
It is felt that our findings support this hy-pothesis.
ACKNOWLEDGMENTS
We appreciate the excellent technical assistance provided byN. Parry, Frank P. Fazzan, and Steven P. Lerner.
This work was supported by the Albert Soiland Cancer Founda-tion, by Public Health Service grant CC-001 15 from the NationalCenter for Disease Control. and by the World Health Organiza-tion.
LITERATURE CITED
1. Azar, H. A., T. D. Phamn, and A. K. Kurban. 1970. Anelectron microscopic study of a syphilitic chancre. Arch.Pathol. 90:143-150.
2. Drusin, L. M., G. C. Rouller, and G. B. Chapman. 1969.Electron microscopy of Treponemna pallidum occurring in ahuman primary lesion. J. Bacteriol. 97:951-955.
3. Hasegawa, T. 1969. Electron microscopic obseivations cn thelesions of condyloma latum. Brit. J. Dermatol. 81:367-374.
4. Jepsen, 0. B., K. H. Hougen, and A. Birch-Andersen. 1968.Electron microscopy of Trepone,na pallidurn Nichols. ActaPathol. Microbiol. Scand. 74:241-258.
5. Ovcinnikov, C. C., and V. V. Delektorskij. 1969. Further studiesof the morphology of Treponzemna palliduni under the electronmicroscope. Brit. J. Vener. Dis. 45:87-116.
6. Willcox, R. R., and T. Guthe. 1966. Treponierna palliduin. Abibliographical review of the morphology, culture, andsurvival of T. pallidun and associated organisms. Bull.W.H.O. (Suppl.) 35:1-169.
7. World Health Organization Technical Report Series No. 455.1970. Teponemiiatoses research, p. 1-91. World HealthOrganization, Geneva, Switzerland.
