cultivation of virulent treponema pallidum in tissue culture

Vol. 32, No. 2INFECTION AND IMMUNITY, May 1981, p. 908-9150019-9567/81/050908-08$02.00/0

Cultivation of Virulent Treponema pallidum in Tissue CultureA. HOWARD FIELDSTEEL,* DAVID L. COX, AND RANDOLPH A. MOECKLI

Life Sciences Division, SRI International, Menlo Park, California 94025

In a series of seven experiments, the virulent Nichols strain of Treponemapallidum was shown to attach and replicate on the surface of tissue culture cellsof cottontail rabbit epithelium (SflEp) growing in conventional monolayer cul-tures under an atmosphere of 1.5% oxygen. Five days after inoculation of 106 T.pallidum, the number of treponemes had increased to between 8 x 106 and 2.59x 107. The viability of harvested organisms ranged from 86 to 97%. The numberof T. pallidum continued to increase, generally reaching a plateau between days9 and 12 of incubation, with increases ranging up to 100-fold and averaging 49-fold. There appeared to be a ceiling of multiplication of about 2 x 108 irrespectiveof the inoculum, which ranged from 106 to 108 T. pallidum. Concurrent deoxyri-bonucleic acid assays were performed on the cultures containing T. pallidum toobtain further evidence of replication. Significant increases in treponemal deoxy-ribonucleic acid were observed when the inocula ranged from 106 to 107, with thegreatest increases, as might be expected, being in the former group. There wasalso excellent correlation in the amount of deoxyribonucleic acid per treponeme;the averages for the 106, 2.5 x 106, and 107 groups were 3.46 x 10-14, 3.28 x 10-14,and 2.79 x 10-14 g per treponeme, respectively (3.14 ± 0.72 x 10-14 g pertreponeme). In each experiment, organisms were harvested from the groupinoculated with 106 T. pallidum after 7 days of incubation to test for virulence. Inall instances, the organisms were virulent; erythematous, indurated, treponeme-containing lesions were produced from an average of six to seven organisms.Scanning electron microscopy revealed that during the course of replication manymicrocolonies of treponemes formed on the surface of the cells.

As an almost natural sequel to the discoverythat Treponemapallidum is the causative agentof syphilis, attempts were made to cultivate it.Although many investigators have claimed tohave cultivated it in vitro, none of the isolatedstrains have been pathogenic or have beenshown to have a causal relationship to trepone-mal disease in humans (25). Furthermore, in1948, Eagle and Germuth (4) carried out sero-logical studies on the cultivable Nichols, Nogu-chi, Reiter, Kazan, and Kro6 strains of supposedT. pallidum; they found that these strains fellinto three distinct serological groups and thatthey were morphologically and antigenically dif-ferent from T. pallidum. Hence, they questionedthe identification of these treponemes as strainsof T. pallidum. More recently, biochemical anal-ysis was utilized to determine whether therewere genetic relationships between virulent T.pallidum and five avirulent cultivable trepo-nemes isolated from humans (19). Not only didthe deoxyribonucleic acid (DNA) base compo-sition (guanine-plus-cytosine content) of the cul-tivable treponemes differ markedly from that ofT. pallidum, but saturation reassociation assaysrevealed no detectable DNA sequence homol-ogy. Consequently, the nonpathogenic cultivable

treponemes studied were neither variants normutants of T. pallidum and were actually ge-netically distinct organisms.

Originally it had been generally assumed thatT. pallidum was an anaerobic organism becauseit survived poorly under aerobic conditions.Therefore, in attempts to obtain survival or rep-lication (or both) of T. pallidum in vitro, anaer-obic conditions were maintained, and reducingagents were added to media to lower the oxida-tion-reduction potential (1, 20, 24). Although the50% survival time of T. pallidum under theseconditions was as long as 16 days (24), thereapparently was no correlation between the 50%survival time and virulence for rabbits, whichwas lost after 6 days in vitro.

Despite the apparent anaerobic nature of trep-onemes, Kawata (16) demonstrated the presenceof cytochromes in the nonpathogenic cultivableReiter treponeme (Treponema phagedenis bio-type Reiter) which suggested possible utilizationof molecular oxygen under certain conditions.More recently, Cox and Barber (2) showed thatT. pallidum consumed oxygen at the same rateas did a known aerobic spirochete, Leptospira.The oxygen uptake of the former was cyanidesensitive, indicating that T. pallidum contained

908

CULTIVATION OF VIRULENT T. PALLIDUM IN VITRO 909

a functioning cytochrome-oxidase system andtherefore should be capable of aerobic respira-tion. In a study of the mechanisms involved interminal electron transport by T. pallidum,Lysko and Cox (18) confirmed these observa-tions, leaving little doubt as to the capabilitiesof virulent T. pallidum for aerobic respiration.Therefore, in view of the failure of all attemptsto cultivate T. pallidum under anaerobic con-ditions, it appeared more than likely that oxygenis a requirement for in vitro replication.Between 1913 and 1948, sporadic attempts

were made to cultivate T. pallidum in tissueculture of mammalian tissues, with uniformlynegative results (25). Recently, there has beenrenewed interest in the cultivation of T. palli-dum in mammalian cell cultures. Several inves-tigators, using both aerobic and anaerobic cul-ture systems (5, 6, 8-10, 13, 21, 22), have reportedthe attachment of T. pallidum to tissue culturecells, with periods of survival ranging from 24 hto 23 days. Survival appeared to be best underconditions of low oxygen tension (3%) and whenreducing agents were added to the medium.However, there was little or no evidence thatthe treponemes had replicated.

In 1976, Jones et al. (15) claimed to havedemonstrated replication and subculture ofpathogenic T. pallidum in cultures of baby ham-ster kidney, but Foster et al. (11) failed to cor-roborate these results. Sandok and his col-leagues (21) demonstrated what they called "un-sustained multiplication" of T. pallidum in cul-tures under reduced oxygen tension, both in thepresence and absence of mammalian cells. Vir-ulence ofthe treponemes decreased as a functionof time in vitro. In earlier experiments (6) inwhich gradient cultures of cottontail rabbit epi-thelium (SflEp) were infected with T. pallidum,we and co-workers were able to observe, in all ofthe 25 experiments, apparent increases in num-bers of attached treponemes. These increasesranged from three- to fivefold; however, sinceonly about 5% of the inoculated treponemesattached to the cells, we did not observe anoverall increase above that of the original inoc-ulum. In contrast to the findings of others, wehave never observed in vitro loss of virulence ofT. pallidum. We now report unequivocal multi-plication of virulent T. pallidum in a conven-tional tissue culture system, using SflEp cells assubstrate, under conditions of reduced oxygentension (1.5%).

MATERUILS AND METHODSRabbits. New Zealand white male rabbits (6 to 8

months old) weighing 3 to 4 kg and free of treponemalinfection as determined by nonreactivity to the Ve-

nereal Disease Research Laboratory test were usedfor testicular passage and as a source of treponemesfor inoculation into tissue culture.New Zealand white female rabbits (10 to 12 weeks

old) were used for determining virulence of T. palli-dum after passage to tissue culture. The shaved backsof these animals were inoculated intracutaneouslywith 0.1 ml of various concentrations of T. pallidum(5).

All animals were housed individually at a tempera-ture of 16 to 18°C.

T. pallidurm The virulent Nichols strain was usedthroughout this work, and was maintained and pas-saged as described previously (5).

Fetal bovine serum. We have determined thatcommercial lots of fetal bovine serum, although ca-pable of supporting growth of mammalian cells, varygreatly in their ability to support survival of T. palli-dum (7). Therefore, all lots had to be compared witha lot known to support survival for at least 2 weeks.Acceptable lots were divided into aliquots sufficientfor use in one experiment. The aliquots were thenquick-frozen in a dry ice-alcohol bath and stored at-20°C until thawed at 37°C for use.

Testis extract and tissue culture inocula. Rab-bits were inoculated intratesticularly with 5 x 107 T.pallidum. On days 4 and 9 after inoculation, the rab-bits were inoculated intramuscularly with 4 mg oftriamcinoline acetonide (Sigma Chemical Co.) per mg.The testes were removed aseptically 12 days (±1 day)postinfection of the time of peak orchitis. They werefreed of extraneous tissue and minced finely with irisscissors. An individual testis was extracted with 5 mlof modified basal reduced medium, (see below forpreparation) in a 50-ml Fernbach flask. Air was dis-placed from the flask with 5% CO2 in N2. The flaskwas sealed with a silicone-rubber stopper and placedin a shaking machine (118 oscillations per min) for 30min at 33°C. The extract was then centrifuged at 500x g for 5 min to remove gross particles, and thenumber of treponemes in the supernatant wascounted. This supernatant was then diluted with freshinfected testis extract, which was the supernatant ofthe infected extract centrifuged at 12,000 x g for 10min. (This treatment does not necessarily remove allof the treponemes in the supernatant, but we havedetermined that the residue does not add significantlyto the total number.) Dilution with infected testisextract was such that 0.34 ml of the treponeme-con-taining extract contained the desired number of trep-onemes to be inoculated into each flask. Addition ofthis extract to 10 ml of modified basal reduced me-dium, the amount used in tissue culture bottles, re-sulted in an infected testis extract: modified basalreduced medium ratio of 1:30, which was previouslydetermined to be essential to the survival of T. palli-dum in tissue culture (5).Trypsin-Versene MEM. This solution consists of

Ca2+- and Mg2+-free minimum essential medium(MEM) containing 10 mg of dithiothreitol per 100 ml,20 mg of crystalline trypsin per 100 ml, and 20 mg ofVersene per 100 ml. With this solution it was possibleto not only disrupt the cell monolayer, but also toseparate the treponemes from the SflEp cells. The

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910 FIELDSTEEL ET AL

latter were not removed from the suspension sincethey did not interfere with the counting of the trepo-nemes.

Tissue culture procedures. Because of the manypast failures to successfully cultivate T. pallidum,precise details of the method are presented. The tissueculture medium utilized for growing T. pallidum con-sisted of a modified Eagle MEM (3) plus fetal bovineserum to a final concentration of 20%, and a 1:30dilution of fresh infected testis extract from rabbits.The modified basal reduced medium (the prepara-

tion of which was alluded to above) was made bycombining the following components (in milliliters) indistilled water to a final volume of 100 ml: lOx Earlebalanced salt solution, without phenol red andNaHCO3, 10; 5Ox MEM amino acids, 2; 10Ox MEMvitamins, 1; 200 mM L-glutamine, 1; 10Ox MEM non-essential amino acids, 1; and sodium heparin solution,containing 100 U/ml, 1. The solution was gassedbriefly with CO2, then 3.38 ml of NaHCO3 (7.5%), 3.13ml of 1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 0.63 ml of resazurin (20 mg/100ml), 10 mg of sodium pyruvate, 10 mg of dithiothreitol,and 150 mg of glucose were added. The solution wasfilter-sterilized, and 25 ml of heat-inactivated fetalbovine serum was added. The flask of modified basalreduced medium was then alternately evacuated andgassed with 5% C02-95% N2 mixture three times andstored overnight before use.Two days before infection with T. pallidum, 4-

ounce (ca. 120-ml) prescription bottles were seededwith 5 x 105 cells of SflEp in MEM plus 10% fetalbovine serum and incubated at 33°C. The seed cul-tures of SflEp were maintained as described previ-ously (5). Before inoculation of the treponemes, thegrowth medium was removed from the flasks andreplaced with 10 ml ofmodified basal reduced medium.The cultures were gassed with 5% C02-95% N2 for 1min at 3 liters/min and allowed to equilibrate for 4 to5 h before inoculation. Each flask was then inoculatedwith 0.34 ml of testis extract containing the desirednumber of treponemes. The cultures were gassed for1 min with 1.5% 02-5% C02-93.5% N2 at 3 liters/min.We had previously determined, in gradient culturesincubated under air, that the concentration of dis-solved oxygen was approximately 1.5% in the area inthe gradient that contained the greatest number ofand most active treponemes. Increasing the oxygenconcentration to 3.5% resulted in both loss of viabilityand smaller increases in numbers of T. pallidum.Incubation was carried out at 33°C. Cultures weresacrificed at various intervals up to a maximum of 12days, and treponemal counts were made as follows.The medium was removed and saved. The monolayerwas rinsed with 2 ml of trypsin-Versene MEM whichwas removed and combined with 1.25 ml of fetal bovineserum. Additional trypsin-Versene MEM (3 ml) wasadded to the flask, which was gassed with 5% C02-95%N2 for 30 s, stoppered, and placed on a shaker for 20min to remove the monolayer from the glass. The fluidcontaining suspended cells and treponemes was pipet-ted vigorously over the surface of the bottle to removeany adhering cells. This suspension was added to theprevious rinse-serum mixture. The modified basal re-duced medium that had originally been removed fromthe monolayer was then added. Treponeme counts

were made on triplicate samples from each flask asfollows. A 10-pl sample was placed under a 22-mm2cover slip, and random fields were counted at a mag-nification of x800 until at least 100 treponemes werecounted or 50 fields were covered. The percentage ofactively motile treponemes was calculated. Countswere made on two flasks per group on each day thatcounting was done.

Biochemical procedures. In each experiment, tosubstantiate observed increases in T. pallidum, assayswere carried out to determine whether DNA alsoincreased. The treponemal DNA was recovered withthe following procedures. Duplicate cultures, both in-fected and uninfected, were harvested by the methodsdescribed above, and 1 ml was removed from thetreponeme suspension for enumeration. The SflEpcells were pelleted out of the remaining sample bycentrifugation at 500 x g for 15 min, and the top 13 mlof the samples was removed. Since 2.25 ml of thatsample was left in the centrifuge tube, the SflEp cellpellet remained undisturbed. Samples of supernatantwere checked microscopically, and no SflEp cells weredetected. We found less than 5% difference betweencounts made initially and those made after removal ofSflEp cells. The treponemal fractions of duplicatebottles were pooled and centrifuged at 18,000 x g for20 min. The supernatant was removed, and the tre-poneme pellet was quick-frozen immediately in a dryice-alcohol bath and stored at -20°C. Uninfected cul-tures were treated in an identical fashion. DNA assayswere performed simultaneously after all the samplesin the same experiment had been collected.The DNA from the thawed pellet was extracted by

subjecting the treponemes to enzymatic digestion withlysozyme (0.5 mg/ml). The treponemes were thentotally disrupted and homogenized by a brief sonictreatment at 4°C. The DNA was quantitated by thespectrofluorometric technique of Lebarca and Paigen(17). This method involves the reaction of DNA withthe fluorescent dye bisbenzimide. The increase in flu-orescence produced by a test sample was comparedwith that produced by a standard solution of DNA.The net treponemal DNA per flask was determinedby subtracting the quantity of DNA in the controlflasks from the quantity of DNA in the infected cul-tures. Since the pellet represented only 80% of thetotal number of treponemes obtained from the twobottles, the results were multiplied by 1.25 to deter-mine the actual quantity of treponemal DNA presentin the bottles.Scanning electron microscopy. SflEp cells were

cultivated and inoculated with 107 T. pallidum asdescribed above except that cover slips were placed inthe culture vessels. Both infected and uninfected coverslips were removed after 5, 7, 9, and 12 days of incu-bation. They were fixed in 2% paraformaldehyde-2.5%glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at4°C for 45 min. They were then thoroughly washedwith the phosphate buffer and dehydrated in a gradedseries of ethanol solutions (50, 70, 95, 100, 100, and100%; 5 min each). The ethanol was replaced by theintermediate fluid, amyl acetate, through a gradedseries of ethanol-amyl acetate solutions (33, 50, 67,100, 100, 100, and 100%, 5 min each). The amyl acetatewas then replaced by the transition fluid, C02, in thebomb of a critical-point dryer (Technics). After criti-

INFECT. IMMUN.


cal-point drying at 43°C and 1,300 psi, the cover slipswere mounted on aluminum microscope slides withconductive colloidal silver, and coated with a 20-nmlayer of gold-palladium alloy (60:40, wt/wt) by triodesputtering (Polaron). Material thus prepared was ex-

amined in a Cambridge Mark Ila scanning electronmicroscope at an accelerating voltage of 10,000 eV,and a 300 tilt. Micrographs were made with Polaroidtype 55 positive-negative black and white film.

RESULTS

Replication of virulent T. pallidum. A se-ries of seven experiments was carried out inidentical fashion by the procedure outlinedabove. The results are summarized in Table 1.Significant increases in numbers occurred in ev-

ery experiment when the inoculum was 106;these increases ranged up to a maximum of 100-fold, with an average increase of 49.3-fold after9 days of incubation. With increasing inocula,there were decreases in the fold increase until,with the 108 inoculum, little or no increase in thenumber of treponemes was observed. It is moststriking that in all of the groups, irrespective ofthe inoculum, a ceiling of multiplication seemedto be imposed. Despite the 100-fold differencesbetween the 106 and 10' inocula, the intergroupdifferences in the ceiling were remarkably small.The maximum ceilings were 1.0 x 108, 1.25 x108, 1.35 x 108, and 2.37 x 108, respectively, forthe 106, 2.5 x 106, 107, and 108 inocula.DNA assays on cultivated T. pallidumL

The average quantities of treponemal DNA re-

covered from the cultures of the seven experi-ments are also presented in Table 1. In the firstthree experiments, the treponemal DNA contentof cultures inoculated with 106 and 2.5 x 106treponemes and incubated 1 day was slightlyless than or equal to that of the negative con-

trols. Because the treponemal DNA was insuf-ficient to quantitate, these determinations were

omitted from subsequent experiments. The netamount of DNA harvested from the infectedcultures increased concurrently with the in-crease in numbers of treponemes in all inoculumgroups. When the inoculum was 106, treponemalDNA increased from below the detectable limitof 0.05 ,ug per flask to 1.62 ,ug per flask on day 9,representing a minimum of a fold increase of 33

10.The treponemes in the cultures inoculated

with 106 to 107 organisms appeared to enter a

logarithmic phase of growth after 1 to 2 days ofincubation. The average DNA content of T.pallidum during that period was 3.14 x 10-14 g

per treponeme. The treponemes in cultures in-oculated with 10' organisms replicated very lit-tle, if at all, and the organisms appeared to be ina near stationary phase of growth. The averageDNA content per treponeme was 1.88 x 10-14 g,significantly lower than that found in the ac-tively replicating treponemes.Virulence of tissue culture-passaged T.

TABLE 1. Growth of T. pallidum in tissue cultures of SflEpaDay DNA/trep-

ob- Avg. no. of treponemes Avg. fold increase M ( a of DNA/ oneme xInoculum ser- x 107/flask (range) (range) Motflity ( (range) flask (mean + (maser- SD) (mean±va- SD)tion

106 5 1.59 (0.80-2.59) 15.9 (8.0-25.9) 90.5 (86.5-97.2) 0.64 ± 0.25 3.76 ± 0.707b 3.27 (1.44-6.92) 32.7 (14.4-69.2) 87.8 (78.2-93.2) 0.99 ± 0.38 3.25 ± 0.979 4.93 (2.29-9.69) 49.3 (22.9-96.9) 72.2 (38.1-89.1) 1.62 ± 0.54 3.43 ± 0.7412 4.91 (1.52-10.00) 49.1 (15.2-100.0) 30.6 (7.8-59.9) NDC ND

2.5 X 106 5 2.57 (1.81-3.99) 10.3 (7.2-16.0) 90.5 (86.3-94.3) 1.06 ± 0.31 3.91 ± 0.667 4.97 (3.09-8.30) 19.9 (12.4-33.2) 89.2 (85.8-95.1) 1.60 ± 0.33 3.31 ± 0.509 6.88 (4.00-10.0) 27.5 (16.0-40.0) 73.7 (59.9-91.8) 1.99 ± 0.53 2.96 ± 0.4912 6.06 (2.82-12.50) 24.3 (11.3-50.0) 28.4 (4.8-55.2) ND ND

107 1 1.00 ND 0.33 ± 0.04 3.30 ± 0.345 4.95 (3.87-6.00) 5.0 (3.9-6.0) 88.2 (90.1-86.3) 1.51 ± 0.33 3.10 ± 0.807 9.03 (8.10-11.30) 9.0 (8.1-11.3) 89.6 (85.1-93.8) 2.16 ± 0.24 2.41 ± 0.329 11.76 (7.75-15.30) 11.8 (7.8-15.3) 71.8 (54.2-90.8 2.56 ± 0.85 2.32 ± 0.1812 10.04 (6.57-13.50) 10.0 (6.6-13.5) 16.0 (5.3-35.6) ND ND

l0o 1 10.0 ND 2.02 ± 0.23 2.02 ± 0.235 16.04 (13.80-21.00) 1.6 (1.4-2.1) 82.4 (75.2-87.5) 2.89 ± 0.75 1.66 ± 0.307 17.48 (12.10-23.7) 1.7 (1.2-2.4) 76.6 (68.2-89.4) 2.93 ± 0.26 1.76 ± 0.469 16.80 (14.50-18.60) 1.7 (1.5-1.9) 66.9 (60.4-72.9) 3.07 ± 0.55 1.83 ± 0.2012 13.41 (8.02-20.0) 1.3 (0.8-2.0) 13.7 (2.6-36.2) ND ND

a Combined data from seven separate experiments. SD, Standard deviation.bIn each experiment, organisms taken at this time produced treponeme-containing lesions in rabbits. An

average of 6.59 treponemes (range, 1.52 to 18.5) was required to produce a lesion.'ND, Not done.

VOL. 32, 1981


pallidunm In six of the seven experiments sum-marized in Table 1, treponemes harvested onday 7 of incubation were inoculated intracuta-neously into the shaved backs of rabbits, in serial10-fold dilutions. This was done not only toconfirm virulence, but also to determine theminimum number of tissue culture-grown T.pallidum required to produce typical trepo-neme-containing erythematous and induratedlesions (Table 2). In every instance the trepo-nemes were virulent, with an average of 6.59(range, 1.52 to 18.5) organisms being required toproduce a lesion.Scanning electron microscopy observa-

tion of T. pallidum in tissue culture. In anattempt to better understand attachment andreplication of T. pallidum in cultures of SflEpcells, we took a series of scanning electron mi-croscopy photomicrographs at various times.Figures 1 and 2 are typical of what we observed

INFECT. IMMUN.

during the course of incubation. After attach-ment, colonies of treponemes apparently formedon the surface of the cells. After 5 days of incu-

TABLE 2. Virulence of T. pallidum after onepassage in SflEp cellsa

Minimum no. of T.Expt. no. pallidum producing

EIb appearance

1 1.85 x 10' 262 3.02 x 100 303 1.52 x 100 224 6.69 X 100 155 1.75 x 100 356 8.06 x 100 26

a Treponemes were harvested after 7 days in cul-ture, and rabbits were inoculated intracutaneouslywith serial 10-fold dilutions.

b EI, Erythematous, indurated lesions containing T.pallidum.

Pk:t*'v;aus6|43 <~~~~i F ., :-

r ,; ''

F ; .^?, ......

a :s,>

^. i ..,e F... R. t f

*-t_ v._F ..

FIG. 1. Scanning electron micrographs of T. pallidum. (A) SflEp cell with attached treponemes after 5days of incubation. Bar, 5 jim. (B) Same cell as in (A), showing large numbers of treponemes forming amicrocolony (arrow in A). Bar, 1 jim. (C) and (D) Colonies of T. pallidum after 7 days of incubation. Bar, I,um.

ouplipw,-l .I ''I.. I

M11-F.:., ..:,. ",:. I

9- .I

I

.I.


k'IG. 2. Scanning electron micrographs of T. pallidum. (A) Low-power magnification view showing manycolonies of T. pallidum after 9 days of incubation. Bar, 20 /im. (B) High-power magnification view of onecolony (arrow) seen in (A). Bar, 1 ji. (C) Low-power magnification view after 12 days of incubation, showingone large colony (arrow), several smaller ones, and many individual treponemes. Bar, 5 jim. Cell sheet isbeginning to break up. (D) High-power magnification view of large colony seen in (C), containing largenumbers of treponemes. Bar, 1 /im.bation, several of these colonies as well as indi-vidual treponemes were seen under low magni-fication (Fig. 1A). Figure 1B is a high-powermagnification view of one of the colonies seen inFig. 1A. Figures 1C and D are a high-powermagnification view of individual colonies, show-ing groups of intertwined treponemes after 7days of incubation. The number of colonies in-creased with time of incubation; Fig. 2A (lowmagnification view) shows many colonies after9 days of incubation, and one of them is seenunder high magnification in Fig. 2B. These col-onies, as well as many individual treponemesthat apparently washed off during the fixationprocedure, were also observed under dark-fieldillumination. At day 12 of incubation (Fig. 2C),the cell sheet started to deteriorate, the coloniesapparently started to break up, and many moreindividual treponemes were seen. Figure 2D is a

high-power magnification view of one of thelarge colonies seen in Fig. 2C.

DISCUSSIONWe have presented evidence for what we be-

lieve is the first successful in vitro cultivation ofT. pallidum. We have shown that under theconditions described herein, virulent T. palli-dum attaches to and multiplies on the surface ofSflEp cells. In a series of seven experiments, wedemonstrated that unequivocal replication oc-curred in each one. The extent of treponememultiplication was dependent on the initial in-oculum of T. pallidum; it was the greatest whenthat inoculum was 106, and it was the least withan initial inoculum of 108. Interestingly, the dif-ference between these two inocula, 100-fold, wasthe maximum multiplication observed with the106 inoculum, although the average was 49-fold.

VOL. 32, 1981


With all of the inocula, the maximum ceiling ofmultiplication was approximately 108 to 2 x 108;therefore, the fold-increases of replicating T.pallidum decreased with increasing inoculumsize. Thus, when the initial inoculum was 2.5 x106 T. pallidum, there was an average increaseof 27.5-fold (range, 16- to 40-fold) by day 9 afterinoculation. When the inoculum was 107, theaverage increase was 11.8-fold (range, 7.8 to 15.3)on day 9 after inoculation. With the 108 inocu-lum, the greatest increase was 2.4-fold, indicat-ing little or no replication.

It is important to know the reason for theapparent ceiling of multiplication, since it isrelevant to further optimization of the culturesystem. It has been theorized by Hayes et al.(13) that there are only limited sites of attach-ment on each cell, which would certainly limitreplication. If this were true, we should havebeen able to increase the yield of treponemes byincreasing the number of cells in the culture.However, when we inoculated 2.5 x 106 trepo-nemes into SflEp cultures that were either 20or 80% confluent, the rates of growth were al-most identical and the maximum increases in T.pallidum were 24.0- and 20.2-fold, respectively(unpublished data), indicating that replicationdid not appear to be dependent on specific at-tachment sites on each cell. In fact, it appearedto be unrelated to cellular attachment per se.The generation time of T. pallidum in rabbits

has been postulated to be 30 to 33 h (23). Hence,the theoretical maximum increase in T. palli-dum during the period of observation in tissueculture could be about 100-fold in the culturescontaining the 106 inoculum. This maximum wasachieved in only one experiment, and althoughthere was great variation in the maximum in-creases in T. pallidum from this inoculum, theaverage increase was approximately 49-fold.Therefore, increases from the 106 inoculum werenot limited by attachment sites, but by the gen-eration time. During the period in which T.pallidum was replicating, the SflEp cell mono-layer progressed from approximately 25% to100% confluency; yet there was little differencein the number of attached organisms with aninoculum of 108. Five days after inoculation, thenumber of treponemes attached to the cells av-eraged 1.6 x 108. By day 9, the number wasalmost identical, 1.7 x 108. On day 12 of incu-bation, it had declined to 1.3 x 108. Perhaps ofgreatest significance is that in all of the inoculumgroups, the viability of the treponemes haddropped off dramatically by day 12 of incuba-tion. It seems likely that the ceiling of multipli-cation is due to a combination of factors, prob-ably primarily to the exhaustion in the mediumof some essential component(s) or to the accu-mulation of toxic products. Since the SflEp

INFECT. IMMUN.

monolayer simultaneously deteriorated at day12 of incubation, it was not possible to ascertainwhether the treponemes, the cells, or both, wereaffected by these factors.

It seems likely that the testis extract is in-volved in the demise of treponemes, since thiswas one factor in the medium that could not beadequately controlled, varying from one experi-ment to another. We regard the testis extract asone of the three most important factors in thesuccessful cultivation of T. pallidum. Addition-ally, we have preliminary evidence that testisextract prepared from normal rabbits may beequally effective.A second, equally important factor in the cul-

tivation of T. pallidum is careful selection of thefetal bovine serum used in both the extractionand cultivation media. Commercial lots of fetalbovine serum vary greatly in their ability tosupport survival of T. pallidum and may be sotoxic as to kill off most of the treponemes aftera few days in culture (7). The third importantfactor in the successful cultivation of T. palli-dum is the amount of oxygen present in thesystem. We have determined, by actual meas-urement, that an atmosphere of 1.5% oxygen isoptimal for replication of T. pallidum.As further evidence that T. pallidum was

replicating in the SflEp cultures, we carried outconcurrent DNA assays on the cultivated trep-onemes. We observed highly significant in-creases in treponemal DNA when the inocularanged from 106 to 107, with the greatest in-creases, as might be expected, being in the for-mer group. There was also excellent correlationin the amount of DNA per treponeme; the av-erages for the 106, 2.5 x 106, and 107 groups were3.46 x 10-'4, 3.28 x 10-'4, and 2.79 x 10-14 g pertreponeme, respectively (3.14 ± 0.72 x 10-'4 gper treponeme). However, the average DNA pertreponeme for the 108 inoculum group (1.88 +0.41 x 10-'4 g) was considerably lower than thatfor the other groups. A phenomenon similar tothis has been observed in other bacteria. Gilliesand Alper (12) reported an increased DNA con-tent per organism during the logarithmic phaseof growth for two strains of Escherichia coli,which contained 1.4 to 1.8 times as much DNAper organisms during logarithmic phase ofgrowth as during the stationary phase. Hodsonand Beck (14) have also reported that vegetativecells of Bacillus megaterium and Bacillus cer-eus contained 2.3 and 3.3 times as much DNA,respectively, as did their spores. In our experi-ments, the average DNA content per treponemefor the cultures inoculated with 106 to 107 orga-nisms was 1.67 times that of treponemes fromcultures inoculated with 108 organisms. This dif-ference is attributed to the different stages ofgrowth the treponemes were in and falls within


the range of increased DNA content reportedfor other bacteria in the logarithmic phase ofgrowth.

In all of the experiments reported here involv-ing single passage of T. pallidum in SflEp cells,the organisms remained highly virulent for rab-bits. The numbers required to produce trepo-neme-containing lesions were consistent withthose reported for virulent T. pallidum passagedin vivo (23).As demonstrated by scanning electron micros-

copy on T. pallidum during the course of repli-cation in SflEp cells, microcolonies of trepo-nemes are formed on the surface of the cells.These colonies, as well as many individual trep-onemes scattered across the cell surface, werealso observed by dark-field illumination. Veryfew individual treponemes were seen by scan-ning electron microscopy before day 12 of incu-bation. This may have been due to their beingwashed off during the processing of the coverslips. However, on day 12 of incubation, manyindividual treponemes were seen in addition tothe colonies. This also coincided with the break-ing up of the cell sheet and loss of motility ofthe treponemes.

Fitzgerald et al. (8) and Hayes et al. (13) havealso studied T. pallidum attachment to mam-malian cells by scanning electron microscopy;Fitzgerald et al. observed the treponemes onlyafter a 3-h incubation period with cultured cells,and Hayes et al. made observations up to 22 hafter coincubation of cells and treponemes. Bothgroups observed randomly attached treponemes,but in no instance did they observe colony for-mation. We assume that the colonies probablyform as the result of replication and thereforewould not be observed under other conditions.The successful in vitro cultivation of T. pal-

lidum now paves the way for biochemical, phys-iological, and immunological studies on this or-ganism that were not possible previously.

ACKNOWLEDGMENTSThis work was supported by Public Health Service Re-

search grant R01-AI-115113 from the National Institute ofAllergy and Infectious Diseases.We thank Jan C. Terry and Charles Hart for their excellent

technical assistance. We are especially grateful to J. A. Levy,University of California, San Francisco, in whose laboratorythese results were confirmed.

LITERATURE CITED1. Clark, J. W., Jr. 1962. Loss of virulence in vitro of motile

Treponema pallidum. Br. J. Vener. Dis. 38:78-81.2. Cox, C. D., and M. K. Barber. 1974. Oxygen uptake by

Treponema pallidum. Infect. Immun. 10:123-127.3. Eagle, H. 1955. Nutrition needs of mammalian cells in

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tionship between five cultured strains of supposed T.paUidum (Noguchi, Kroo, Nichols, Reiter, and Kazan)and two strains of mouth treponemata. J. Immunol. 60:223-239.

5. Fieldsteel, A. H., F. A. Becker, and J. G. Stout. 1977.

Prolonged survival of virulent Treponema pallidum(Nichols strain) in cell-free and tissue culture systems.Infect. Immun. 18:173-182.

6. Fieldsteel, A. H., J. G. Stout, and F. A. Becker. 1979.Comparative behavior of virulent strains of Treponemapallidum and Treponema pertenue in gradient culturesof various mammalian cells. Infect. Immun. 24:337-345.

7. Fieldsteel, A. H., J. G. Stout, and F. A. Becker. 1980.Role of serum in survival of Treponema pallidum intissue culture. In Vitro 17:28-32.

8. Fitzgerald, T. J., P. Cleveland, R. C. Johnson, J. N.Miller, and J. A. Sykes. 1977. Scanning electron mi-croscopy of Treponema pallidum (Nichols strain) at-tached to cultured mammalian cells. J. Bacteriol. 130:1333-1344.

9. Fitzgerald, T. J., R. C. Johnson, J. A. Sykes, and J.N. Miller. 1977. Interaction of Treponema pallidum(Nichols strain) with cultured mammalian cells: effectsof oxygen, reducing agents, serum supplements, anddifferent cell types. Infect. Inunun. 15:444-452.

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11. Foster, J. W., D. S. Kellogg, J. W. Clark, and A.Balows. 1977. The in vitro cultivation of Treponemapallidum. Corroborative studies. Br. J. Vener. Dis. 53:338-339.

12. Glues, N. E., and T. Alper. 1960. The nucleic acidcontent ofEscherichia coli strains B and B/R. Biochim.Biophys. Acta 43:182-187.

13. Hayes, N. S., K. E. Muse, A. M. Collier, and J. B.Baseman. 1977. Parasitism by virulent Treponemapallidum of host cell surfaces. Infect. Immun. 17:174-186.

14. Hodson, P. H., and J. V. Beck. 1950. Origin of deoxyri-bonucleic acid of the bacterial endospore. J. Bacteriol.79:661-665.

15. Jones, R. H., M. A. Finn, J. J. Thomas, and C. Folger.1976. Growth and subculture of pathogenic T. pallidum(Nichols strain) in BHK-21 cultured tissue cells. Br. J.Vener. Dis. 52:18-23.

16. Kawata, T. 1967. Presence of cytochromes in the Reitertreponeme. J. Gen. Appl. Microbiol. 13:405-406.

17. Labarca, C., and K. Paigen. 1980. A simple, rapid, andsensitive DNA assay procedure. Anal. Biochem. 102:344-352.

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19. Miao, R., and A. H. Fieldsteel. 1978. Genetics of Tre-ponema: Relationship between Treponema pallidumand five cultivable treponemes. J. Bacteriol. 133:1-1-107.

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22. Sandok, P. L., S. T. Knight, and H. M. Jenkin. 1976.Examination of various cell culture techniques for coin-cubation of virulent Treponema pallidum (Nichols IStrain) under anaerobic conditions. J. Clin. Microbiol.4:360-371.

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25. Wilcox, R. R., and T. Guthe. 1966. Treponema palli-dum. A bibliographical review of the morphology, cul-ture and survival of T. pallidum and associated orga-nisms. Bull. W.H.O. (Suppl.) 35:5-139.

VOL. 32, 1981

cultivation of virulent treponema pallidum in tissue culture

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