INFECTION AND IMMUNITY, Feb. 1978, p. 421-429

Vol. 19, No. 2

0019-9567/78/0019-0421$02.00/0 Copyright i 1978 American Society for Microbiology

Printed in U.S.A.

Unsustained Multiplication of Treponema pallidum (Nichols Virulent Strain) In Vitro in the Presence of Oxygen PAUL L. SANDOK, HOWARD M. JENKIN,* HERBERT M. MATTHEWS, AND MARK The Hormel Institute, University of Minnesota, Austin, Minnesota 55912

S. ROBERTS

Received for publication 11 October 1977

Treponema pallidum (Nichols virulent strain) was incubated with or without using a modified medium supplemented with reduced glutathione and a variety of nutrients (PRNFjo-B). Two- to fourfold increases in treponemal numbers were observed in cultures without mammalian cells within 96 h of incubation under 5 to 6% oxygen. Treponemal motility and multiplication were maintained more satisfactorily in cultures that were diluted and transferred daily, using an equal volume of fresh medium. Treponemes incubated without oxygen did not significantly increase in number. Virulent microorganisms were detected for at least 96 h in the cell-free system. In the presence of 3 to 4% oxygen, two- to fivefold increases in treponemal numbers were observed in the supernatant fluids of cultures containing human prepuce cells after 48 to 120 h at 35°C. Without oxygen, treponemal numbers rarely approached a threefold increase. Virulent treponemes were detected by the rabbit skin lesion test after at least 120 h in vitro. Regardless of the system of incubation, increases in treponemal numbers could not be sustained for longer than 120 h, and treponemal virulence decreased as a function of time in vitro. oxygen,

Oxygen uptake by Treponema pallidum in vitro was reported by Cox and Barber (6) and Harris et al. (Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, D4, p. 52). Lysko and Cox have demonstrated that T. pallidum has a flavoprotein-cytochrome electron transport system catalyzed by reduced pyridine nucleotides that may function in aerobic respiration (19). Baseman et al. demonstrated that carbohydrate degradation and amino acid utilization by T. pallidum were inhibited under anaerobic conditions and enhanced under aerobic conditions of incubation (3). In light of the growing body of evidence suggesting a critical role for oxygen in the metabolism of T. pallidum, it was necessary to devise systems of cultivation for the treponeme within which the microorganism was not killed in the presence of oxygen. Inactivation or killing of T. pallidum in the presence of oxygen has been retarded in the past by using cell-treponeme co-incubation cultures with or without medium having a low electronegative potential or cultures containing tissue explants (1, 9, 11, 12, 17, 22). However, the microorganism has not been satisfactorily and consistently subcultured in a practical sense under any conditions of incubation described to date (1). Several reasons can account for this failure, including the lack of defined nutritional and physical parameters that affect treponemal survival and lack of good control and monitoring of the P02, pCO2, and

electronegative potential of the medium. The purpose of this investigation was to modify and optimize our systems of treponemal incubation (24) by using low levels of oxygen in the gaseous phase of the cultures in attempts to stimulate treponemal multiplication and maintain virulence. Further screening for treponemal growth factors should be facilitated by using the procedures outlined in this report if oxygen proves to be functional in the growth and metabolism of virulent T. pallidum in vitro. MATERIALS AND METHODS Gases. Three gases, one containing 75% N2 + 20% H2 + 5% C02, one with 95% 02 + 5% C02, and one with 100% N2 were obtained from Matheson Gas Co., Elk Grove, Ill. The 100% N2 and the mixture containing hydrogen were deoxygenated by using a heated copper column (15). Two cannulas, one releasing the oxygen-carbon dioxide mixture and the other releasing the nitrogen-hydrogen-carbon dioxide mixture, were bound together so that both could be inserted simultaneously into culture vessels using a V.P.I. anaerobic culture unit (15). Before use, the flow rate of each gas mixture was adjusted with a gas flow meter (Dwyer Instruments, Inc., Michigan City, Ind.) attached to each cannula. The flow rate of deoxygenated nitrogen, hydrogen, and carbon dioxide was 1,250 ml/min. The flow rate of oxygen and carbon dioxide was between 80 and 90 ml/min to obtain 5 to 6% oxygen or 50 to 60 ml/min to obtain 3 to 4% oxygen in the gas phase of the cultures. Percentage oxygen in the gas phase

421

422

INFECT. IMMIJN.

SANDOK ET AL.

of the tubes was estimated on the basis of the flow rates for each gas mixture and on the assumption that the gases formed a homogeneous mixture within the culture vessels. The 100% N2 was used during preparation of the prereduced medium. Preparation of prereduced medium, PRNF,o-

B. Prereduced new formula medium (PRNF,o) has been described previously (24). The medium used for this investigation was markedly modified from the original formulation for PRNFIo. The modified medium is designated PRNFjo-B (Table 1). After preparation, the medium was dispensed in appropriate vol-

TABLE 1. Preparation of 0.5 liter ofprereduced medium (PRNF1o-B)a Concn of stock solution (g/liter) I

Componentb

Double glass-distilled water Mixture of: NaCl (1) KCl (6) MgSO4 anhydrous (2) KH2PO4 (2) Glucose (2) Phenol red (9) NaHCO3 (8) CaCl2 anhydrous (1) HEPES (7)e Newborn calf serum (11, 13)d

85.00 4.06 1.50 1.85 25.00 0.02 88.00 10.00 477.00 Undiluted

Amt of stocK so-

uton (tml)n

Componentb

260.5

L-Tryptophan (3) Sodium pyruvate (10Ox concentrate) (13) L-Glutamine grade B (4) Mixture of: DL-Alanine (5) L-Asparagine (3) L-Aspartic acid (3) L-Glutamic acid (3) Glycine (4) L-Proline (3) Eagle vitamins (10Ox con-

30.0

13.3 10.4 4.8 50.0

centrate) (11, 13)f Mixture of additional cofactors-vitamins:' NADP (4) Pyridoxal P04 (4) a-Lipoate (4)j CoA (7) B12 (7) Thiamine pyrophosphate chloride (7) Biotin (3) NADH (4) Folinic acid (16) Mixture of:

Amt of Concn of stock stock sosolution lution (g/liter) (ml)

4.0 11.0

4.0 8.0

2.92

4.0

1.78 3.00 2.66 2.94 1.50 2.30

8.0 4.0

Mixture of: 10.00 Choline chloride (4) 10.00 0.4 4.0 Ethanolamine (4) 0.1 10.00 Inositol (3) 4.0 2.10 0.1 L-Serine (4) 2.00 DL-Ornithine (3)e 4.0 0.25 8.0 Eagle minimum essential 0.125 4.0 amino acids (50x con0.25 centrate) (11, 13)f Mixture of: 0.01 0.0005 CoCI2 6H20 (1) 0.70 0.0010 MnCl2 4H20 (1) 0.10 ZnSO4 7H20 (2) 0.0140 4.0 0.0810 Fe(NO3)3 9H20 (2) Reduced glutathione (4)" 12.3 0.5000 (NH4)eMo7024 4H20 (1) NaCl (1) 6.8 (NH4)2SO4 (10) 4.0 9.6 KCl (6) 0.4 Mixture of: MgSO4 7H20 (2) 0.2 Adenine (4) 1.5 0.06 KH2PO4 (2) 25.0 4.0 0.06 Uracil (4) Na2HPO4 (2) 1 Glucose (2) Oleic acid (14) 1.00 0.4 4.0 Palmitic acid (14) h 0.4 4.0 0.14 CaCl2 2H20 (1) FAF-BSA (15) 200.0 Resazurin (1) 8.0 0.0025 Ox serum ultrafiltrate (12) Undiluted Sodium ascorbate (4)' 17.0 5.0 5.0 Galactose (7) 190.8 4.0 I a Ten milliliters of the completed prereduced medium was withdrawn from the vessel and placed into an anaerobic culture tube under 100% N2. The redox potential (-275 ± 25 mV Eva1) and pH (7.3 ± 0.1) of the completed medium were measured by using a saturated KCl calomel electrode and a pH probe as described previously (23). "All stock solutions were sterilized by using a prerinsed 0.45-Lm membrane filter (Millipore Corp.) except as noted below. Components were added to the medium in the sequence shown in the table. Number in parentheses following each component corresponds to the vendors of each reagent: (1) Fisher Scientific Company, Fairlawn, N.J.; (2) Mallinckrodt Chemical Works, St. Louis, Mo.; (3) Nutritional Biochemicals Corp., Cleveland, Ohio; (4) Calbiochem, Los Angeles, Calif.; (5) Eastman Kodak Co., Rochester, N.Y.; (6) Merck & Co., Inc., Rahway, N.J.; (7) Sigma Chemical Co., St. Louis, Mo.; (8) Matheson Coleman and Bell, Norwood, Ohio; (9) J. T. Baker Chemical Co., Phillipsburg, N.J.; (10) Schwarz/Mann, Orangeburg, N.J.; (11) International Scientific Industries, Cary, Ill.; (12) Colorado Serum Co., Denver; (13) Grand Island Biological Co., Grand Island, N.Y.; (14) Nu-Chek-Prep, Inc., Elysian, Minn.; (15) Miles Lab, Inc., Elkhart, Ind.; (16) ICN and K & K Laboratories, Inc., Plainview, N.Y. Stock HEPES (N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid) solution was adjusted to pH 7.3 with NaOH and autoclaved before use. c

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423

TABLE 1-Continued d Newborn calf serum was heat inactivated at 56°C for 30 min. e DL-Ornithine (200 mg) was dissolved in 10 ml of 1 N HCl and then diluted to 100 ml with glass-distilled water before filtration. f See reference 8. " Sterile oleic acid (100 mg) was combined with 3.9 ml of sterile 0.1 N NaOH and rapidly heated to 75°C until dissolved. The warm solution was immediately diluted with 15.7 ml of calcium- and magnesium-free saline (20). The sodium oleate solution (0.8 ml) was complexed to 9.2 ml of 20% fatty acid-free bovine serum albumin (FAF-BSA). The BSA-oleate complex was directly incorporated into the medium. Excess BSA-oleate was stored at 4°C. h Sterile palmitic acid (16 mg) was combined with 1 ml of 0.1 N NaOH and heated rapidly to 750C until dissolved. The resulting solution was immediately complexed to 9 ml of warm (48 to 50°C) 20% FAF-BSA. The BSA-palmitate complex was diluted with 30 ml of 20% FAF-BSA at 37°C, and the final solution was directly incorporated into the medium. Excess BSA-palmitate was stored at 40C. i NADP, Nicotinamide adenine dinucleotide phosphate; CoA, coenzyme A; NADH, reduced nicotinamide adenine dinucleotide. - a-Lipoate (100 mg) was dissolved in 1 ml of 0.2 N NaOH, and the volume was adjusted to 10 ml with glassdistilled water before incorporation into the concentrated solution. k The prereduced solution was made in 50- or 100-ml volumes. Powdered reduced glutathione was dissolved in the salts solution after the solution was degassed, using an autoclave at 215 lb/in2 for 7 min. This mixture was adjusted to pH 6.8 with 10 N NaOH under 100% N2; the vessel was stoppered and autoclaved for 25 min at 15 lb/in2, using an anaerobic tube press (Beilco, Vineland, N.J.). The autoclaved solution was added to the unreduced medium under a constant flow of 100% N2. If necessary, the unreduced medium was adjusted to pH 7.3 with 0.2 N NaOH before addition of the prereduced, anaerobically sterilized salts solution. 'The sodium ascorbate was dissolved in glass-distilled water, filter sterilized, and added to the completed medium immediately.

umes into anaerobic tubes (15 by 145 mm; Bellco, Vineland, N.J.) under 75% N2 + 20% H2 + 5% C02, stoppered, and maintained at 350C until used the same day. Preparation of treponemal inocula. Viable T. pallidum (Nichols virulent strain) were obtained after intratesticular challenge of New Zealand rabbits (24). The testes were minced twice and rinsed each time with 5 to 10 ml of medium, and the eluates were discarded. The eluates from the third and all subsequent mincings, 3 to 4 ml each, were combined and centrifuged at 250 x g for 10 min at 220C, and the supernatant fluids were withdrawn and diluted with an equal volume of prereduced medium. Intact tissue cells in the treponemal suspensions did not exceed 103 cells/ml when examined by dark-field microscopy. The suspension was diluted by using prereduced medium under 75% N2 + 20% H2 + 5% C02 to obtain 3 x 107 to 6 x 107 T. pallidum per ml for use in cell-

free and cell-associated experiments. Cell-free treponeme incubation system. Anaerobic culture tubes containing 4.5 ml of prereduced medium were inoculated with 0.5 ml of the treponemal suspension described above, gassed with 75% N2 + 20% H2 + 5% C02 with or without 95% 02 + 5% C02 to obtain 5 to 6% oxygen, and incubated under static conditions at 350C. Serial passage was accomplished after dilution of the cultures with an equal volume of fresh prereduced medium and transfer of 5 ml of the diluted cultures to new tubes. Treponemal numbers were determined by dark-field microscopy as described earlier (24). Cell-treponeme co-incubation system. Leighton tubes containing removable cover slips were inoculated with 5 x 104 to 6 x 104 human prepuce cells (20th to 50th passage) as previously described (24). The cultures were inoculated with 1 ml of prereduced

medium containing 3 x 106 to 6 x 106 T. pallidum, gassed with 75% N2 + 20% H2 + 5% C02 with or without 95% 02 + 5% C02 to obtain 3 to 4% oxygen, stoppered, and incubated at 350C. Every 24 h, cover slips and supernatant fluids from two to four cultures, each incubated with or without oxygen, were examined by dark-field microscopy (24). All cultures, media, and reagents were checked for microbial contamination

(24).

Virulence test. Samples for virulence testing, 0.8 ml each, were withdrawn from the supematant fluids of cell-free and cell co-incubation cultures and diluted into 0.2 ml of 50% glycerol + 50% medium in 1-dram vials. The samples were stored at -77°C until used for intradermal inoculation (0.1 ml/site) into the shaved backs of 3.5- to 4-kg male Dutch Belt rabbits (13). The cell monolayers with attached treponemes were rinsed with a calcium- and magnesium-free salt solution (20) and removed from the Leighton tubes by using 0.5 ml of a solution containing 0.05% trypsin (Difco, 1:250) and 0.05% ethylenediaminetetraacetate (Difco). The tryptic reaction was inhibited by using 1.5 ml of the prereduced medium. An 0.8-ml amount of the resulting suspension of cells and treponemes was stored and used for virulence testing as described for the supernatant fluids. Cultures without treponemes were treated in the same manner and served as controls against potential nonspecific inflammatory responses due to cells or medium. Sites of inoculation were checked daily for induration for up to 40 days after inoculation. Serous exudates from well-developed lesions were checked for motile T. pallidum by dark-field microscopy. Statistical evaluation of the data. Two to four cultures were prepared for each condition of treponemal incubation within an experiment in order to obtain sufficient numbers of replicate samples for valid sta-

424

INFECT. IMMUJN.

SANDOK ET AL.

tistical analysis by Student's t test (24). All estimates of numbers of T. pallidum were obtained by counting at least 160 treponemes in a total of 20 to 40 fields from each of four to eight wet mounts, using 18-by18-mm cover slips. Two wet mounts were counted from samples withdrawn from each culture in order to decrease variations due to pipetting errors.

RESULTS Cell-free incubation system. Table 2 lists data representative of results obtained in five independent experiments. Increases in treponemal numbers (30 to 130%) were observed in all cultures during the first 24 to 48 h of incubation under 5 to 6% oxygen. When the cultures were diluted and transferred to new tubes, the increases were more pronounced, and a larger proportion of the treponemes remained motile after 96 to 168 h of incubation compared with untransferred cultures. Treponemes incubated under deoxygenated gas retained high motility (95 to 100%), but increases in treponemal numbers usually were not significant (i.e., less than 50% increase). As in the case for untransferred cultures, serial transfer of treponemes incubated under deoxygenated gas did not stimulate treponemal multiplication. In other experiments, treponemes incubated under 9% or more 02 did not survive longer than 48 h, and no increases TABLE 2. Serial passage of T. pallidum (Nichols virulent strain), using a cell-free incubation system with and without oxygena Motile T. pallidum/ml x 10-6 ± 1 SEM' Incubation time Dilution" Transferred

Untransferred cultures

cultures,d

(h)

+ 02e No 02e + 02e 5.3 ± 0.4 5.0 ± 0.5 5.2 ± 0.2 8.4 ± 0.1 8.9 ± 0.1 8.5 ± 1.2 12.1 ± 1.3 6.0 ± 0.6 1:4 16.2 ± 1.4 9.3 ± 0.4 5.1 ± 0.2 1:8 18.8 ± 1.1 1:16 11.8 ± 2.5 12.0 ± 0.7 3.4 ± 0.5 1:16 18.6 ± 5.1 9.2 ± 0.4 1.4 ± 0.3 1.7 ± 0.3 1.9 ± 0.2 1:16 8.5 ± 1.0 a Cultures and treponemal inocula were prepared as described in Materials and Methods, using medium PRNF,o-B (Table 1). The temperature of incubation was 350C. 'Cumulative dilution of transferred cultures from 0-h sus0 24 48 72 96 120 168

None 1:2

pension. ' All estimates were based on counts as described in Materials and Methods. Nonmotile microorganisms comprised less than 10% of the total population for 96 h. These data are typical of five independent experiments. SEM, Standard error of the mean. d Numbers of motile T. paUidum per milliliter were corrected for dilution (cumulative count). Transfer of cultures incubated without oxygen resulted in counts decreasing with dilution, indicating no growth. e + 02, Cultures incubated under 6% 02 with N2, H2, and C02 as described in Materials and Methods. No 02, Cultures incubated under deoxygenated 75% N2 plus 20% H2 plus 5% C02.

were observed. In three experiments using high initial inocula to obtain suspensions containing 2 x 10' T. pallidum per ml, we observed twoto threefold increases in treponemal numbers under 6% 02 after 48 to 72 h of incubation only when the cultures were serially passaged. Cell-treponeme co-incubation system. Data obtained with the cell-treponeme co-incubation system are shown in Table 3. Within 1 to 2.5 h of incubation, 33 to 50% of the treponemal inoculum was removed from the supernatant fluids of the cultures due to treponemal attachment to cells and to the surface of the container. After 24 to 48 h, numbers of treponemes in the supernatant fluids of the cultures equaled or exceeded total numbers of treponemes initially inoculated into the cultures. About 15 to 20% of the treponemes were attached to cells at 24 h and all intervals of time thereafter. Numbers of attached treponemes were not included in the summation of the data presented in Table 3 due to difficulties in obtaining a precise estimate by dark-field microscopy. Increases in treponemal numbers in cultures containing oxygen were usually higher than in cultures without oxygen. Numbers of motile microorganisms often decreased more rapidly after 48 to 72 h of incubation under the oxygenated atmosphere than under the deoxygenated atmosphere. The total numbers of microorganisms in the oxygenated cultures were usually higher than in cultures without oxygen at the end of 96 h in vitro. Failure to observe increases in treponemal numbers occurred with freshly prepared testicular extracts from about one out of every four rabbits. The reason for this is unknown. Attempts to stabilize the reducing environment in the medium in the presence of oxygen were performed by using different concentrations and combinations of glutathione and Lascorbic acid. In earlier experiments before modification of the medium used in these experiments, we found that without glutathione or other reducing agents in the medium, treponemes rapidly lost motility (23). In the presence of 2 and 4 mM glutathione, motility was retained throughout a 5-day observation period. Differences in treponemal responses to changes in the concentrations of glutathione from 2 to 4 mM and of ascorbic acid from 0 to 2.84 mM were not consistently observed. Characteristics of treponemal attachment and motility in cell-treponeme co-incubation cultures. The number of treponemes attached to individual cells varied considerably, from 1 to 80 or more. Attachment usually occurred at one end of the microorganism, although attachment by both ends of the trepo-

VOL. 19, 1978

TREPONEMAL MULTIPLICATION IN VITRO

425

TABLE 3. Increases in numbers of T. pallidum (Nichols virulent strain), using the human prepuce celltreponeme co-incubation systema and different concentrations of glutathione and ascorbic acid with and without oxygen Motile T. pallidum/ml x 10-6 ± 1 SEMW 2 mM glutathione'

4 mM glutathionec

Incubation time

_ 2.84 mM Ascd

(h) +

02

No 2e

0.57 mM Asc

0.57 mM Asc + 02

No02

+

02

No2

0.28 mM Asc, 0 mM Asc, + 02

+ 2

3.4 ± 0.2k 3.5 ± 0.1k 2.5 ± 0.4f 3.2 ± 0.6f 2.3 ± 0.2f 1-2.5 4.4 ± 0.4 3.4 ± 0.2 5.2 ± 0.3 5.2 ± 0.8 5.6 ± 1.0 6.6 ± 0.8 4.2 ± 0.8 24 4.0 ± 0.8 7.6 ± 0.3 9.2 ± 0.1 6.0 ± 0.4 10.6 ± 1.2 8.0 ± 1.6 10.2 ± 0.8 8.8 ± 0.8 48 12.8 ± 2.2 4.5 ± 1.5 3.6 ± 0.2 6.6 ± 0.4 3.8 ± 0.7 5.4 ± 0.3 11.1 ± 0.8 3.1 ± 0.2 72 6.7 ± 0.5 ND NDh 1.8 ± 0.5 0.7 ± 0.1 1.8 ± 0.8 7.9 ± 0.1 96 1.8 ± 0.5 0.8 ± 0.2 aThe treponemal inoculum and cultures containing 5 x 104 prepuce cells per culture were prepared as described in Materials and Methods. 'Estimates of motile T. pallidum per milliliter were based on counts obtained only from the supernatant fluids of the cultures. After 24 h, 15 to 20% of the treponemes remained attached to prepuce cells or to the sides of the container and could not be dislodged to obtain an accurate estimate of total numbers of treponemes per culture and so are not included in the estimates given above. Nonmotile microorganisms ranged from 1 to 10% during 48 to 72 h of incubation and increased to 50 to 95% by 120 to 144 h with or without 02. SEM, Standard error of the mean. c The medium PRNF,-B (Table 1) was modified by adding 10 ml of the reduced salts solution to 90 mil of unreduced medium to obtain 4 mM glutathione. Medium containing 2 mM glutathione contained 5 ml of the reduced salts solution mixed with 95 ml of medium, the same proportions as shown in Table 1. d Asc, L-Ascorbic acid. Concentrated solutions of ascorbic acid were prepared for use in the same manner described for sodium ascorbate in Table 1, footnote 1. One milliliter each of 50-, 10-, or 5-g/liter ascorbic acid was added to 99 ml of PRNF-(r B to obtain 2.84, 0.57, or 0.28 mM ascorbic acid in the final solution. e + 02, Cultures incubated under 3 to 4% oxygen with nitrogen, hydrogen, and carbon dioxide as described in Materials and Methods. No 02, Cultures incubated under deoxygenated 75% N2 plus 20% H2 plus 5% CO2. f Each culture received 4.4 x 106 motile T. pallidun suspended in 1 mi of PRNF,o-B. Within 2.5 h, almost one-half of the treponemes attached to cells and surfaces, leaving 2.3 x 106 to 3.2 x 106 microorganisms in the supernatant fluids. ' Each culture received 5.2 x 106 motile T. pallidum suspended in 1 ml of PRNF,0-B. Within 2.5 h, almost one-third of the treponemes attached to cells and surfaces, leaving 3.4 x 106 to 3.5 x 106 microorganisms in the supernatant fluids. h ND, Not determined.

neme to the same cell was not uncommonly observed. One out of 4 to 20 cells had one or more treponemes exhibiting rapid, gliding motility similar to that described for other spirochetes (5). Whether or not the gliding treponemes were actually intracellular could not be

established by dark-field microscopy. Treponemes exhibiting translational movement were often observed to egress from the boundaries of the cell periphery, and one end of the microorganism appeared to firmly attach to the cellular membrane. Thereafter, the treponemes ex-

hibited the rapid undulations, entwinements, and lashing motility typical of the rest of the attached microorganisms. The same microorganisms could yield their point of attachment to resume their translational motility. Translational motility of T. pallidum was observed as early as 1.5 h after inoculation and for at least 48 to 72 h in vitro in the presence or absence of oxygen. Virulence test results. Results obtained by using the rabbit skin lesion test for treponemal virulence are shown in Tables 4 and 5. These data complement the results presented in Tables 2 and 3. Samples prepared at each incubation time from two to four randomly chosen cultures were inoculated intradermally into rabbits.

With the cell-free system of treponemal incubation with oxygen, virulent microorganisms were detected after at least 96 h in transferred and untransferred cultures (Table 4). Virulent microorganisms were detected in the supernatant fluids of the cell-treponeme co-incubation system after at least 120 h with or without oxygen (Table 5). Trypsin-treated celltreponeme suspensions also contained virulent microorganisms for at least 120 h of incubation time with or without oxygen. The mean days of appearance of induration (MDIA) were very similar throughout the observation period regardless of whether the samples used for intradermal inoculation into rabbits were obtained from the supernatant fluids or from the trypsinized cell-treponeme suspensions from the same cultures. In both the cell-free and the cell-treponeme co-incubation systems, the MDIA was roughly related to incubation time, becoming progressively later as incubation time and/or dilution of the sample increased. DISCUSSION Systems of treponemal incubation that limit or eliminate oxygen may be unsatisfactory for further investigation of the physical and nutri-

TABLE 4. Effect of serial transfer on the multiplication, survival, and virulence retention of T. pallidum (Nichols virulent strain) incubated in PRNF1o-B under oxygena without cells Transferred cultures IncubaIncubation time Diluinvitro (h) tionb

0 24 48 72 96 120 144

None 1:2 1:4 1:8 1:16 1:16 1:16

Motile T. pallidum/ml x _+ lSM 106±1 SEMe ~~~10-6

Untransferred cultures

Motile T. pallidum/ml X 1061 ± 1 SEM oieObalidm/

V~netsd Virulence test' Stsps

SitesposExpt A

Expt B

4.2±0.1 13.4 ± 0.6 21.3 ± 0.2 24.0 ± 2.6 23.5 ± 3.0 5.1 ± 1.4 4.2 ± 0.8

4.8±0.2 9.0 ± 0.6 12.6 ± 0.5 16.1 ± 0.5 11.3 ± 1.0 5.2 ± 0.4 0.2 ± 0.1

itive/sites MDIA inoculated 6/6

7±0

6/6

14 ± 2

1/6 0/4

19 >40

Vrlnets

Virulence test

Sitespos-

|

Expt A

Expt B

itive/sites

4.1±0.2 12.4 ± 0.4 22.9 ± 1.0 15.9 ± 0.6 8.2 ± 1.6 0.2 ± 0.1 0.0

5.2±0.2 7.6 ± 0.4 11.4 ± 0.4 9.5 ± 0.3 10.3 ± 0.6 0.7 ± 0.3 0.0

6/6

7±0

6/6

14 ± 2

6/6

20 ± 1 ND

inoculated

NDf

MDIA

a PRNFo-B, Prereduced new formula medium (Table 1). Cultures were incubated at 35'C under a gas mixture obtained by combining 75% N2 + 20% H2 + 5% CO2 mixed with 95%02 +5% C02 to obtain 6% 02 as described in Materials and Methods. bCultures were transferred to new tubes after a 1:2 dilution in fresh medium each 24 h to obtain the final dilution given in

the table. c Data were corrected for dilution. Data were derived from duplicate sets of tubes in experiment A and quadruplicate sets of tubes in experiment B. SEM, Standard error of the mean. d Cumulative data for experiments A and B. For controls against nonspecific inflammation, two sites were inoculated with samples withdrawn at each interval of time from cultures without T. pallidum. No indurations were observed at the control sites. Virulence retention after 24, 72, and 144 h was not determined. eMDIA, Mean number of days for induration appearance after inoculation. f ND, Not done.

TABLE 5. Multiplication, survival, and virulence retention of T. pallidum (Nichols virulent strain) incubated with and without oxygen, using human prepuce cell culturesa Cultures incubated under 02b Cultures incubated without °2 Motile T. pallidum/mil x 10-6 ± 1 SEM in su-

Virulence teste

pernatant fluidsd Incuba. tion time in vitro (h) Expt A Expt B

Superatantfluidsf Sites posi-

Motile T.pallidum/ml x 10-6 ± 1 SEM in supernatant fluids

Virulence test Cel ~~~~~~~~~~~~ ~~Supernatant fluidsCes flid

Cells Sites

Sites posi-

tive/ MDIAh tive/ MDIA

tive/ MDIA

sites

sites

Sites positive/ sites

inoc-

inoc-

posi-

sites

inoc-

inoc-

ulated 4.8 0.3' 6/6 6.9 0.5 8.1 0.4 7/7 12.8 0.4 13.2 1.1 4/4 2.5 0.4 7/8

ulated

Expt A

Expt B

MDIA

ulated ulated 4.2 ± 0.4 9 ± 1 2/3' 10 ± 0 6/6 6.7 ± 0.3 5.9 ± 0.3 4/4 13 ± 1 6/6 14 ± 2 7.7 ± 0.2 6.6 ± 0.3 4/4 20 ± 3 4/4 21 ± 1 0.9 ± 0.2 1/4 21 0/4 >40

1-2.5 3.7 _0.2' 9± 1 2/3j 10 ± 0 3.8 ± 0.3 24 8.7 0.5 6.5 ± 0.4 48 12.5 0.6 15 ± 1 7/7 17 ± 2 11.8 ± 0.1 72 8.3 1.0 10.0 ± 0.3 96 15.5 1.3 20 ± 1 4/4 22 ± 0 8.3 ± 0.3 120 25.2 1.2 20 ± 1 3/8 19 ± 0 6.0 ± 0.5 0.2 0.1 0 NDV 144 ND a Cultures contained prereduced new formula medium (PRNF,o-B, Table 1) and were incubated at 35'C. The cultures were prepared with human prepuce cells and inoculated with a freshly prepared testicular harvest containing T. paUidum as described in Materials and Methods. 'Cultures incubated under a gas mixture obtained by combining 75% N2 + 20% H2 + 5% CO2 with 95% 02+ 5% CO2 to obtain 3% to 4% 02. 'Cultures incubated under 75% N2 + 20% H2 + 5% CO2 without O2. d Data derived from duplicate sets of tubes in experiment A and quadruplicate sets of tubes in experiment B. SEM, Standard error of the mean. 'Cumulative data obtained from the rabbit skin lesion test for experiments A and B, using samples stored at -77'C for 2 to 14 days as described in Materials and Methods. For controls against nonspecific inflammation, two to four sites were inoculated with samples prepared at each interval of time from cultures without T. palidum. No indurations were observed at the control sites. Virulence retention tests at 24, 72, and 144 h of incubation were not done. f Samples withdrawn from the supernatant fluids of the cell-treponeme co-incubation cultures. 'Samples prepared after treatment of the cell-treponeme monolayer with trypsin as described in Materials and Methods. hMDIA, Mean number of days for induration appearance after inoculation. Initial inoculum of T. pallidum at 0 h was 4.2 x 106/ml in experiment A and 5.0 x 106/ml in experiment B. J One of two rabbits with three sites of inoculation died of undetermined causes on the 11th day postinoculation and before the appearance of indurations. 'ND, Not done. 426

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427

tional requirements of T. pallidum in vitro. As creased, and an increasing proportion of the mentioned earlier, there has been much specu- population became nonmotile any time after 48 lation and some evidence that oxygen may be h in vitro. The exact time for the onset of losses beneficial to treponemal metabolism (3, 6, 9, 11, in numbers and motility within the cultures 12, 17-19). Unfortunately, few systems of incu- varied unpredictably between experiments. In the future, it may be possible to improve bation have been devised that allow the microorganism to survive for one doubling (29 to 33 treponemal survival in oxygenated cell cultures h) in the presence of oxygen (28). Systems that by using additional reducing agents. Fitzgerald do allow treponemal survival in excess of 33 h et al. (11) and Fieldsteel (9) maintained the in the presence of oxygen apparently have been microorganism with little loss in virulence for 8 unsatisfactory for sustained treponemal growth and 21 days, respectively, in the presence of (1, 9, 11, 12, 22) with one possible exception oxygen. They used SflEp NBL-11 rabbit epidermal cell monolayers and medium supplemented (17). The data in this report suggest that transient with reduced glutathione and dithiothreitol (9, increases in treponemal numbers resemble un- 11). Ascorbic acid appears to function as a supsustained growth of the microorganism under plementary stabilizing agent in the presence of specific conditions of incubation. The survival suiflfydryl groups in maintaining the redox potime of-the microorganisms has been shown to tential (27). The major conclusion from data in exceed the time required for. one generation of Tables 2 to 5 is that increases in treponemal numbers were consistently observed with oxyT. pallidum in vivo. In the cell-free system of incubation, mainte- gen and various concentrations of reducing nance of the increase in numbers of motile trep- agents in the medium. No definitive conclusions onemes is extended 24 to 48 h when the cultures can be made about the optimum concentrations are serially transferred by using freshly prepared of ascorbic acid and glutathione on treponemal medium. This suggests that adverse environ- growth based on the data to date. mental conditions responsible for treponemal Jones et al. apparently succeeded in cultivatdisintegration occur sooner in untransferred cul- ing T. pallidum at least two times under aerobic tures. Baseman et al. suggested that a dichotomy conditions without using sulfhydryl compounds exists between the functional motility of the (17). The apparent contradiction concerning the microorganism and its protein metabolism (2). necessity for sulflhydryl compounds in most sysIt is possible that treponemal motility retention tems of incubation as opposed to the results of can remain high for some time after protein Jones et al. may be resolved if it can be demonsynthesis stops. This could account for our ob- strated that cells provide appropriate reducing servation that while the treponemes remained conditions for treponemal multiplication in lieu highly motile, after 48 to 72 h no further in- of that provided by the medium. Our attempts to cultivate T. pallidum by using systems of creases in treponemal numbers occurred. With the cell-treponeme co-incubation sys- incubation described by Jones et al. yielded no tem, treponemal numbers in the supematant conclusive results. To our knowledge, the observation of gliding, fluids were usually not significantly higher than those observed in the initial inoculum until after serpentine translational motility by T. pallidum 48 h. Numbers of treponemes in the supematant in cell monolayers has not been documented fluids of the cultures could be affected by de- before. This type of motility has been observed tachment and/or reattachment of the microor- with other spirochetes (5). Translational motilganism to cells during incubation (12, 14). For ity by T. pallidum should be of interest to this reason, and because of the difficulty in ap- individuals specifically investigating mechaproximating numbers of treponemes attached nisms of cell-treponeme interaction and attachper cell, we chose to interpret the data very ment. The observation of translational moveconservatively in Tables 3 and 5. It should be ment by T. pallidum upon cell surfaces may noted that the actual increases in treponemal preclude the possibility that specific binding numbers could have been five- to sixfold higher sites for treponemal attachment are evenly disthan the initial inoculum based on counts ob- tributed upon the cells. There is evidence that the number of binding sites upon cellular surtained using the supernatant fluids alone. Attempts to serially transfer treponemes in faces for T. pallidum may be limited (10, 14). the cell-treponeme co-incubation system are It is possible that treponemes exhibiting transcurrently not possible due to technical difficul- lational motility are within the cell and cannot ties in miniizng losses oftreponemes attaching come into contact with attachment sites on the to cells and to container surfaces. Numbers of surface. However, evidence from studies using motile treponemes in the cell-treponeme co-in- transmission electron microscopy suggest that cubation system in the presence of oxygen de- fewer than 1 treponeme per 100 host cells may

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take up an intracellular residence in vitro when translational motility is most prominent at 24 h of incubation (unpublished data). In contrast, 1 in every 4 to 20 cells has one or more treponemes exhibiting typical translational movement. This strongly suggests that the treponemes are epicellular rather than intracellular. It has been established by earlier investigators and reconfirmed in our laboratory that an increasing MDIA suggests a progressive loss in numbers of virulent microorganisms (13, 17, 24, 27, 28). The results of the tests shown in Tables 4 and 5 suggest that numbers of virulent microorganisms decrease regardless of the presence or absence of increases in the number of treponemes during incubation. The influence of storage on the samples used for the virulence tests was not directly tested. Virulent microorganisms could have become increasingly cryolabile as incubation time increased. Apparent losses in numbers of virulent microorganisms could not be predicted on the basis of morphological alterations or discernible changes in motility characteristics observed by dark-field microscopy. It is possible that phenotypic changes occur in vitro which effectively dilute virulence factors associated with treponemal surface components in vivo (16, 26, 29). Observation of decreasing virulence in vitro coincident with increases in treponemal numbers cdn also suggest preferential stimulation of growth for an avirulent subpopulation(s) of treponemes normally associated with the virulent microorganism in vivo. This could account for earlier reports on the successful cultivation of T. pallidum in vitro without maintaining its virulence (28). At least two subpopulations of treponemes in infected rabbit testes have been fractionated by Baseman et al. by velocity sedimentation (4). We have not yet determined whether two populations of treponemes exist in our culture system. The lack of significant differences in the MDIAs of the diluted cell-treponeme suspensions compared with the undiluted supernatant fluids from the same cultures could be due to the lack of sensitivity of the virulence test (24). It is also possible that the diminishing subpopulation of virulent microorganisms in the cultures preferentially seeks cellular attachment during incubation. It has been suggested in other reports that only living, virulent treponemes attach to cells (11, 12, 14). It remains possible that improvements in the nutritional and environmental conditions of the culture systems described here may improve treponemal virulence retention as well as growth in future experiments. Variations in unknown

INFECT. IMMUN.

nutritional factors that the treponemes acquired in vivo could account for differences in the rates of increase, peak numbers of microorganisms, and rates of decline in numbers of treponemes between experiments. Variations in nutritional factors from one testicular harvest to another will be difficult to document. There is evidence that the physical environment in which the microorganism proliferates in vivo contains dissolved oxygen. The PO2 iS 11.6 mm of Hg in the uninfected rabbit testes (7). A dynamic equilibrium between oxidation and reduction is maintained in vivo by the biochemical replenishment of oxidized compounds with their reduced counterparts. The electronegative potential of tissue in man is maintained at -40 mV Eh at pH 7.2 in the presence of dissolved oxygen (25). In static systems of incubation, it is not possible to maintain an environment that has both a low redox potential and dissolved oxygen without observing an increase in the redox potential and a decrease in the PO2 with time. The technology exists to develop a new system of treponemal incubation using a chemostat/fermentor wherein the dynamic interplay of oxidation and reduction may be controlled and monitored in the presence of dissolved oxygen in order to obtain optimal conditions for treponemal growth (21). In conclusion, it appears that serial cultivation of T. pallidum in vitro will be accomplished in the future. For the present time, technical problems for incubation of the microorganism in the presence of oxygen must be resolved and additional information must be obtained elucidating the biochemical and physical requirements of T. pallidum in vitro. ACKNOWLEDGMENTS We wish to thank Karan Crilly, Beth Kolb, Kimberly Orr, and other supporting staff at The Hormel Institute for their technical and secretarial assistance. We gratefully acknowledge the constructive comments of Stephen Graves during the preparation of this manuscript. This investigation was supported by Public Health Service contract NOI AI 42537 from the National Institute of Allergy and Infectious Diseases and by The Hormel Foundation. H.M.M. was the recipient of a Public Health Service research fellowship award 1 F32 AI 05354-01 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Baseman, J. B. 1977. Summary of the workshop on the biology of Treponemapallidum: cultivation and vaccine development. J. Infect. Dis. 136:308-311. 2. Baseman, J. B., and N. S. Hayes. 1974. Protein synthesis by Treponema pallidum extracted from infected rabbit tissue. Infect. Immun. 10:1350-1355. 3. Baseman, J. B., J. C. Nichols, and N. S. Hayes. 1976. Virulent Treponema pallidum: aerobe or anaerobe. Infect. Immun. 13:704-711. 4. Baseman, J. B., J. C. Nichols, J. W. Rumpp, and N. S. Hayes. 1974. Purification of Treponema pallidum

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from infected rabbit tissue: resolution into two treponemal populations. Infect. Immun. 10:1062-1067. Canale-Parola, E. 1977. Physiology and evolution of spirochetes. Bacteriol. Rev. 41:181-204. Cox, C. D., and M. K. Barber. 1974. Oxygen uptake by Treponema pallidum. Infect. Immun. 10:123-127. Cross, B. A., and I. A. Silver. 1962. Neurovascular control of oxygen tension in the testis and epididymis. J. Reprod. Fertil. 3:377-395. Eagle, H. 1959. Amino acid metabolism in mammalian cell cultures. Science 130:432-437. Fieldsteel, H. A., 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. Fitzgerald, T. J., P. Cleveland, R. C. Johnson, J. N. Miller, and J. A. Sykes. 1977. Scanning electron microscopy of Treponema pallidum (Nichols strain) attached to cultured mammalian cells. J. Bacteriol. 130:1333-1344. Fitzgerald, T. J., R. C. Johnson, J. A. Sykes, and J. N. Miller. 1977. Interaction of Treponema pallidum (Nichols strain) with cultured mamnualian cells: effects of oxygen, reducing agents, serum supplements, and different cell types. Infect. Immun. 15:444452. Fitzgerald, T. J., J. N. Miller, and J. A. Sykes. 1975. Treponemapallidum (Nichols strain) in tissue cultures: cellular attachment, entry, and survival. Infect. Immun.

11:1133-1140. 13. Graves, S. R., P. L. Sandok, H. M. Jenkin, and R. C. Johnson. 1975. Retention of motility and virulence of Treponema pallidum (Nichols strain) in vitro. Infect. Immun. 12:1116-1120. 14. Hayes, N. S., K. E. Muse, A. M. Collier, and J. B. Baseman. 1977. Parasitism by virulent Treponema pallidum of host cell surfaces. Infect. Immun. 17:174-186. 15. Holdeman, L. V., and W. E. C. Moore (ed.). 1972. Anaerobe laboratory nianual. Virginia Polytechnic Institute Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va. 16. Johnson, R. C., D. M. Ritzi, and B. P. Livermore. 1973. Outer envelope of virulent Treponema pallidum. Infect. Immun. 8:291-295. 17. Jones, R. H., M. A. Finn, J. J. Thomas, and C. Folger.

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1976. Growth and subculture of pathogenic T. pallidum (Nichols strain) in BHK-21 cultured tissue cells. Br. J. Vener. Dis. 52:18-23. 18. Kiraly, K., and L Horvath. 1976. Survival of T. paUidum under microaerobic conditions in cell and tissue cultures. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 235:500-505. 19. Lysko, P. G., and C. D. Cox. 1977. Terminal electron transport in Treponema pallidum. Infect. Immun. 16:885-8.

20. Merchant, D. J., R. H. Kahn, and W. H. Murphy, Jr. 1964. Handbook of cell and organ culture, p. 174. Burgess Publishing Co., Minneapolis, Minn. 21. Onderdunk, A. B., J. Johnson, J. W. Mayhew, and S. L. Gorbach. 1976. Effect of dissolved oxygen and Eh on Bacteroides fragilis during continuous culture. Appl. Environ. Microbiol. 31:168-172. 22. Perry, W. L M. 1948. The cultivation of Treponema pallidum in tissue culture. J. Pathol. Bacteriol. 60:339-342. 23. Sandok, P. L, H. M. Jenkin, S. R. Graves, and S. T. Knight. 1976. Retention of motility of Treponema pallidum (Nichols virulent strain) in an anaerobic cell culture system and in a cell-free system. J. Clin. Microbiol. 3:72-74. 24. Sandok, P. L, S. T. Knight, and H. M. Jenkin. 1976. Examination of various cell culture techniques for coincubation of virulent Treponema pallidum (Nichols I strain) under anaerobic conditions. J. Clin. Microbiol. 4:360-371. 25. Shapiro, H. M. 1972. Redox balance in the body: an approach to quantitation. J. Surg. Res. 3:138-152. 26. Smith, H. 1977. Microbial surfaces in relation to pathogenicity. Bacteriol. Rev. 41:475-500. 27. Weber, M. M. 1960. Factors influencing the in vitro survival of Treponema pailidum. Am. J. Hyg. 71:401417. 28. Wilcox, R. R., and T. Guthe. 1966. Treponema pallidum. A bibliographical review of the morphology, culture and survival of T. pallidum and associated organisms. Bull. W.H.O. 35(Suppl.):1-169. 29. Zeigler, J. A., A. M. Jones, R. H. Jones, and K. M. Kubica. 1976. Demonstration of extracellular material at the surface of pathogenic T. pallidum cells. Br. J. Vener. Dis. 52:1-8.