further observations on thepersistence treponema … · experiment 2 human studies (chart 2,...

Brit. J. vener. Dis. (1968), 44, 116.

FURTHER OBSERVATIONS ON THE PERSISTENCE OFTREPONEMA PALLIDUM

AFTER TREATMENT IN RABBITS AND HUMANS*BY

A. R. YOBSt, J. W. CLARK, JR., S. E. MOTHERSHED,J. C. BULLARD, AND C. W. ARTLEY

Venereal Disease Research Laboratory, Venereal Disease Program, National Communicable Disease Center,Public Health Service, U.S. Department of Health, Education, and Welfare,

Atlanta, Georgia 30333

Syphilis in humans was first treated with penicillinmore than 23 years ago when four patients withdarkfield-positive, sero-positive syphilis were con-sidered cured on the basis of lesion regression andserological conversion (Mahoney, Arnold, andHarris, 1943). One of these men became re-infectedand was cured by repeat penicillin treatment(Mahoney, Arnold, and Harris, 1949). The use ofpenicillin with satisfactory clinical results spread,and by 1954 this drug was being used as the soleform of treatment of venereal syphilis and othertreponematoses almost everywhere in the UnitedStates (Willcox, 1954).Although penicillin has won wide acceptance,

there have been occasional reports of patients withearly syphilis who have not been cured by treatmentwith recommended schedules of this antibiotic. Suchreports have generally been poorly documented andhave not excluded the possibility of re-infection.Attempts in vivo to develop a strain of Treponemapallidum resistant to arsenic or penicillin were notsuccessful (Probey, 1948, 1953).

Collart and his co-workers reported the persis-tence of treponemes in lymph nodes and cerebro-spinal fluid of rabbits 1 year after penicillintreatment for late latent syphilis (Collart, Borel, andDurel, 1962a, 1964). These authors reported similarfindings in lymph nodes and cerebrospinal fluidfrom humans treated for late latent syphilis orneurosyphilis with combinations of penicillin,arsenic, and heavy metals (Collart, Borel, andDurel, 1962b; Collart, 1964; Collart and Durel,1964; Collart, 1965). According to their reports,these treponemes were not all morphologicallytypical of T. pallidum but did infect rabbits ontransfer in some cases. Lesions, one of them

* Received for publication July 17, 1967.t Present address: Pesticides Program, National Communicable

Disease Center.

NOTE- Trade names are used for identification only and do notrepresent an endorsement by the Public Health Service or theU.S. Department of Health, Education, and Welfare.

darkfield positive, developed in two of the penicillin-treated rabbits after a course of cortisone. DelCarpio (1959, 1963), who gave penicillin to rabbitswith early latent syphilis and observed them for5 months, also reported data suggesting treponemalpersistence.

Research was undertaken to study treponemespresent after treatment of late latent syphilis in orderto determine their potential clinical significance.Preliminary results of a study in humans havealready been published (Yobs, Rockwell, andClark, 1964a; Yobs, Olansky, Rockwell, and Clark,1965). This paper reports results of further study ofthe human material and of a 4-year study of rabbitstreated with penicillin for late latent syphilis. Thework consisted of four principal experiments, thefirst two being performed at the same time. Themethod and results are presented for each experi-ment.

Material, Methods, and Results

Experiment I Penicillin- Treated Late LatentExperimental Syphilis in Rabbits (Chart 1, opposite)The experimental group included 34 rabbits with

a history of inoculation with Nichols virulent strainTreponema pallidum 14 to 22 months previously,followed by the development of darkfield-positivelesions and appropriate serological response. Allrabbits were infected by inoculation into the testes,except for one in which intracutaneous inoculationwas performed; none had received any antibiotics.No lesions attributable to syphilis were present, andthere had been no evidence of cuniculosis. All werereactive in the Treponema pallidum immobilization(TPI) test at this time. Reactivity in the quantitativeVDRL slide test varied from non-reactive to amaximum reactivity of eight dilutions in one rabbit.VDRL and TPI tests were performed according tothe methods recommended in "Serologic Tests forSyphilis" (USPHS, 1964).

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OBSERVATIONS ON THE PERSISTENCE OF TREPONEMA PALLIDUM 117

PENICILLIN TREATED

CONTROL 4UR ADDED FOR EXP. 3

YLK'KK K K a XKXEs1:i~~~~80E8:ViC 08 8 i1X i @1~~~~~~~~~LBIX6Xi8i-XB-B 1! @-B-1@~~~~~~~~~~~~~~~~~-t 418

NEGATIVE PROOF OF INFECTIVITY TRANSFER

' POSITIVE PROOF OF INFECTIVITY TRANSFER

UNINFECTED RECIPIENT, NODE TRANSFERRED12 MONTHS AFTER TREATMENT OF TEST ANIMALS

INFECTED RECIPIENT, NODE TRANSFERRED

12 MONTHS AFTER TREATMENT OF TEST ANIMALS

HISTOPATHOLOGY OF DONOR'S NODE AFTER CORTISONE

o UNINFECTED BLOOD TRANSFER RECIPIENT

E) UNINFECTED BLOOD TRANSFER RECIPIENT

o) WITH HISTOPATHOLOGY OF NODE AT DEATH

* INFECTED BLOOD TRANSFER RECIPIENT

BBLOOD TRANSFER RECIPIENT WITH TPI WEAKLY REACTIVE ONCE ONLY

- INFECTED BLOOD TRANSFER RECIPIENT WITH* NEGATIVE HISTOPATHOLOGY OF NODE AT DEATH

CHART 1.-Penicillin-treated late latent experimental syphilis in rabbits. Experiment 1.Cortisone effect on penicillin-treated late latent experimental syphilis in rabbits. Experiment 3.

PROOF OF INFECTIVITY To confirm the presence ofactive syphilis, the left popliteal lymph node of eachanimal was extracted and transferred in suspension tonormal rabbits by intratesticular inoculation. Suspen-sions were prepared by mincing nodal tissue in a smallamount of sterile 50 per cent. normal rabbit serum-50per cent. physiological saline (0 * 85 per cent.) solution asdescribed (Yobs and others, 1964a). The development ofdarkfield-positive lesions and of serological response inthe recipients proved that 29 donors (8 53 per cent.) hadbeen actively infected.METHOD The 34 donor rabbits were divided into

two groups. Each of 23 rabbits (nineteen with darkfield-positive tissue recipients) received benzathine penicillinG, four injections of 50,000 Oxford units/kg. bodyweight at 5-day intervals to a total of 200,000 Oxfordunits/kg. body weight. A control group of eleven rabbits(ten with darkfield-positive tissue recipients) was nottreated. Both groups were then followed clinically for12 months, and VDRL and TPI tests were performed at6 and 12 months.One year after penicillin treatment, eighteen treated

and eleven control rabbits survived and were stillTPI-reactive. The right popliteal lymph node wasremoved from each animal. Suspensions prepared fromapproximately two-thirds of each node were inoculatedinto the testes oftwo additional TPI-non-reactive rabbits.These recipients were also followed for 12 months using

a regimen of regular clinical observation, frequentdarkfield examination of testicular aspirates and fluidsfrom any suspicious areas, and serological testing (VDRLslide test at 1i, 3, 6, and 12 months; and TPI at 6 and12 months).The remaining nodal tissue was used for histopatho-

logical study. A total of twelve sections of nodal tissueand twelve smears of inoculating suspension, preparedby a silver-staining method (Krajian, 1939; Dieterle,1927) and a fluorescent-antibody (FA) technique (Yobs,Brown, and Hunter, 1 964b), was examined for aminimum of 8 to 9 hours. In addition, darkfield studyof residual inoculating suspension was carried out byseveral microscopists for a total of 2 hours.

ResultsNodes from seven of eleven control rabbits

produced darkfield-positive orchitis in recipients.At the end of 12 months, five of these recipients wereTPI-reactive, four were non-reactive, and two haddied. No recipients of nodes from the penicillin-treated group developed darkfield-positive orchitisor other lesions and, after 12 months, the sixteensurvivors were both negative to TPI- and VDRL-tests. No treponemes were demonstrated in thenodes of control or treated rabbits by histo-pathology.



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BRITISHJOURNAL OF VENEREAL DISEASES

Experiment 2 Human Studies (Chart 2, oppo-site)

Summary of Previous WorkAs reported in detail (Yobs and others, 1964a,

1965), a right inguinal lymph node was removedfrom each of 45 TPI-reactive male volunteers withdocumentable medical histories of treatment forsyphilis. Previous treatment, which had consisted ofarsenic, bismuth, and/or penicillin in variouscombinations, was adequate by current standards in44 cases.One man (Patient 20) gave a history of inadequate

treatment. An additional (46th) case (Patient 41) haduntreated darkfield-positive secondary syphilis onthe day before node excision.These nodes were studied intensively by histo-

pathological techniques and by animal inoculation,and nodes from five of the men who had been treatedat some previous time, including the inadequately-treated patient, were shown to contain T. pallidum.Two of these five nodes infected TPI-non-reactivenormal rabbits on transfer. Three nodes, including

CLINICAL DATA

one of those which had infected, were shown tocontain T. pallidum by FA staining, and the fifthnode was positive for T. pallidum by silver staining.(The node from the secondary syphilitic did notproduce darkfield-positive lesions, but one recipientwas TPI-reactive at 6 months.) Possible re-infectioncould not be excluded.These five men were re-treated. Depending on

clinical criteria, treatment consisted of a total of 4 *8or 7 2 million units of benzathine penicillin G,given as intramuscular injections of 2 *4 million unitsat weekly intervals, or 30 g. erythromycin by mouthover 15 days. (The volunteer with secondary syphiliswas treated with 2 4 million units of benzathinepenicillin G and was then dropped from the study.)A left inguinal lymph node was excised from each ofthe five men 3 to 3j months after treatment, and allwere negative for treponemes when studied asbefore; 15 months after the re-treatment, serologicaltesting of the four still available in this group showedno significant difference from initial test results.Clinical data on these five men are summarized inthe upper portion of Table I.

TABLE IFOR PATIENTS FOUND POSITIVE (11 of 45) BY SOME STUDY PROCEDURE

Cerebrospinal FluidYear and Anti- at Surgery

Year and Drug at biotics Serum Testst SerologyPatient Age First Drug at Last since Protein Re- 15 mths after TPINo. (yrs) Diagnosis First Treatment Last Content treatment Re-treatment

Adequate for Syphilis VDRL CeU (mg.Treatment Syphilis Treatment Slide Count per

VDRL dils KRP cent.) VDRL dils KRP

10 41 Early 1932 1960 R 1 R4+ N 0 40 4-8 m.u. R 1 R4+ Rlatent As, Bi Pen Bicillin

12 37 Early 1947 1960 WR 0 R4+ N 0 51 7-2 m.u. WR 0 R4+ Rlatent Pen Pen Bicillin

20 42 Late 1947* Same 1960 Ery- N 0 R1+ QNS 0 27 30 gm. N 0 R2+ Rlatent As, Bi thromycin Erythro-

1961 Terra- mycinmycin

22 34 Con- 1944 1955 R 4 R3+ QNS 0 17 4 8 m.u. N.A.genital As, Bi Pen Bicillin

29 36 Secondary 1951 1959 1961 WR 0 R4+ N 0 41 4-8 m.u. WR 0 R4+ RPen Pen Penicillin Bicillin

16 51 Primary 1931 1941 WR 0 N N 0 32 N.D. N.D. N.D.As As, Bi

21 53 Late 1948 1961 WR 0 R1+ QNS 0 50 N.D. N.D. N.D.latent Pen Pen

*23 29 Con- 1940 1949 WR 0 N QNS 0 16 N.D. N.D. N.D.genital Pen Pen

26 34 Primary 1950 1954 1961 WR 0 N N 0 24 N.D. N.D. N.D.As, Hi Pen Penicillin

38 38 Secondary 1939 Same R 1 R4+ N 0 35 N.D. N.D. N.D.As, Bi

43 43 Secondary 1943 1960 N 0 N N 1 25 N.D. N.D. N.D.As, Bi Pen

* White.t All TPI-reactive.N.D. Not done.

* Inadequate treatment.N.A. Not available.

As, Bi = Arsenic and bismuth.Pen = Penicillin.WR = Weakly reactive.

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OBSERVATIONS ON THE PERSISTENCE OF TREPONEMA PALLIDUM

FIRST HUMAN VOLUNTEERS

t S~~ ~ ~ ~ ~10 15 20 23

(E)~~~~~~(De (D 13(D

25 30 35 4D 45

RETRE1ATED HUMAN VOLUNTEERS

12 26 22 29

CONTROLS MONTH SYPHILIS) ADDED FOR EUP. 3

922

HUMAN VOLUNTEER WITH HISTOPATHOLOGY OF NODE VRECIPIENT INFECTED BY HUMAN NODE

22Itlutltl 212 212 321 E 3 tINOCULATION AND DEAD BEFORE TRANSFER

HUMAN VOLUNTEER WITH SECONDARY SYPHILIS

oRECIPIENT UNINFECTED EY HUMAN NODE 0INFECTED BLOOD TRANSFER RECIPIENT

INOCULATION. 1,2+ MAONTH OBSERVATION (DUNINFECTED BLOOD TRANSFER RECIPIENT

RECIPIENT UNINFECTED RY HUMAN NODEWITH RISTOPATHOLOGY OF NODE AT DEATH

INOCULATION. 6.21 MO.NTH OBSERVATION. BLOOD TRANSFER RECIPIENT WITH TPI WEAKLY

9RECIPIENT UNINFECTED BY HUMAN NODE INOCULATION.ATV NEOL

E 12+ MONTH OBSERVATION WITH HISTOPATHOLOGY OF NODES3 INFECTED BLOOD TRANSFER RECIPIENT WITH

WITH HISTOPATHOLOGY Of NODE AT DEATH

RtECIPIENT INFECTED BY HUMAN NODE INOCULATION

CHART 2.-Human studies. Experiment 2.Cortisone effect on rabbit recipients of human nodes. Experiment 3.

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120BRITISHJOURNAL OF VENEREAL DISEASES

Continued Studies of Material from HumansMETHOD A 6-month observation period was

originally planned for animal recipients of the humanlymph nodes. This was extended to 12 months when twoof those recipients developing darkfield-positive orchitisshowed atypical serological response (non-reactive TPIat 6 months), even though microscopical study of thefour histologically positive human nodes had shownseveral fields with numbers of treponemes. After 12months, the recipients of nodes from Patients 10 and 12were still the only ones obviously infected. In an effortto detect latent infections, popliteal nodes from the 42survivors (representing 32 patients) were then transferredto additional sero-negative rabbits which were alsofollowed for 12 months by the routine described.Recipients of lymph nodes from the re-treated patientswere not included, since the prescribed 12-monthobservation had not been completed.

ResultsThe node of one animal, inoculated with nodal

material from an original recipient of the node ofPatient 23, was shown to contain treponemes by FAstaining but gave no clinical or serological evidence ofovert infection. All other animal transfer and histopatho-logical studies of material from this patient had beennegative.

Experiment 3 Cortisone Effect on Penicillin-Treated Late Latent Experimental Syphilis in Rabbitsand on Rabbit Recipients of Human Nodes (Charts 1and 2)To alter the immune response so that latent

disease, if present, might become manifest, cortisonewas given to all surviving test animals fromExperiments 1 and 2 and to appropriate controls.The course of cortisone was started 16 to 18 monthsafter inoculation of the first human nodes intorabbits and 13 months after nodes from the re-treated humans had been transferred to rabbits. Inthe penicillin-treated rabbits the time lapse frominfection was 30 to 36 months, and from treatment16 months. A new group of rabbits with untreatedsyphilis of 16 to 18 months' duration served ascontrols for the recipients of human nodes. Eightuntreated rabbits with syphilis of 30 to 36 months'duration were added to the controls for the penicillin-treated animals.

METHOD Sera from all control animals wereexamined by the VDRL and TPI tests before cortisonewas started, and all controls were TPI-reactive. The testanimals had all recently undergone serological testingand were not retested at this time. The 91 rabbits whichreceived cortisone were the fifteen treated with penicillinand their seventeen controls, together with the 34 primaryrecipients of the original human nodes, and the eightrecipients of nodes from the re-treated humans and their

seventeen controls. Aqueous cortisone acetate (Merck,Sharp, and Dohme), at a dose of 5 mg./kg./day, wasinjected into the muscle of the upper part of the back legsfor 28 days or until death. For 14 days, the dose wasgiven as a single injection; thereafter the dose wasdivided into two equal daily injections because anunexpectedly high animal mortality threatened thecompletion of the study.

TEST OF INFECTIVITY Since both popliteal nodeshad been removed from the animals of Experiment 1,infectivity was now determined by blood transfer. As aninfectivity control for the procedure, 1 ml. blood, drawnfrom each animal in the two control and the penicillin-treated groups before cortisone was begun, was inocu-lated into each testis of two fresh, TPI-non-reactive,normal rabbits. Blood transfer of all animals was plannedafter the completion of cortisone treatment, but becauseof the high rabbit mortality during the course of corti-sone, blood was transferred from all survivors after 14and 28 days'treatment. The blood samples were tested byVDRL and TPI procedures. Blood was transferred from43 animals at 14 days and from seven at 28 days. Allblood recipients were followed for 12 months as outlined.When possible; popliteal nodes were removed at deathfrom all cortisone-treated animals of the human studyand axillary and inguinal nodes were taken from animalsin the penicillin-treated group. In addition, poplitealnodes were also excised from all blood transfer recipientsdying in less than 12 months. These nodes were examinedmicroscopically. Nodes from animals completing obser-vation were also studied as time permitted.

ResultsJudged by the development of darkfield-positive

orchitis or the appropriate serological response orboth, infections were produced in the recipients byblood from ten control animals. These were asfollows: four ofthe seventeen animals with untreatedsyphilis of 30 to 36 months' duration (one beforecortisone, one after 14 days' treatment, and twoafter 28 days' treatment); and six of the seventeenwith untreated syphilis of 18 months' duration(three before cortisone, and two each after 14 and28 days' treatment). One donor, No. 873, causedinfection both before and after treatment with28 days' cortisone. Infection from two of the tencontrols (Nos. 488 and 922) was manifested onlyby the development 1 year later of a weakly-reactiveresult to the TPI test in each recipient. Blood frompenicillin-treated rabbits and from rabbits whichhad received human nodes did not cause infection.One recipient of blood from the 30- to 36-month

syphilis controls showed treponemes in its lymphoidtissue by FA staining, while its partner had previ-ously developed overt infection. The node of onerecipient of blood from the penicillin-treated groupwas also positive for treponemes by FA staining.

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Treponemes were not demonstrated in nodes of anyother blood recipients in these two groups. Nodesof two recipients of blood from the 18-monthsyphilis control group contained treponemes on FAstaining, and one of these animals developed obviousinfection. Nodes of two non-paired recipients ofblood from rabbits inoculated with human nodetissue were shown by FA staining to containtreponemes; originally, these two human nodes hadbeen positive for treponemes microscopically. Silverstain of the node ofone blood recipient of the treatedhuman group showed treponemes, while FA studyof this node was negative although highly suspicious.The cortisone-treated animals showed no change

in serological response. After cortisone, FA stainingofinguinal nodes ofone control and oftwo penicillin-treated rabbits from Experiment 1 showed trepo-nemes, but blood from these animals did not infect.The node of one additional penicillin-treated rabbitwhich had died several days before the start of thecortisone study was positive only by silver stain;blood was not transferred. Treponemes weredemonstrated by FA staining in the popliteal nodesof two 18-month control animals, after cortisonetreatment. Blood from one of these control animals(No. 873) infected pre- and post-cortisone recipientsand the node of the latter recipient was also positiveby FA staining. Blood from the other controlanimal did not produce active infection, but itspre-cortisone recipient's node had contained trepo-nemes. The node of one other 18-month controlanimal whose blood caused infection after cortisonetreatment contained atypical spiral forms seen bysilver stain. Six rabbits inoculated with human nodetissue (two representing the same patient) harbouredtreponemes as shown by FA staining of nodes aftercortisone. Two of the six surviving to blood transfergave negative results in each case; the 12-monthnode transfer recipient of one of these had beenpositive by FA staining. Partners of two of the sixhad negative nodes after cortisone and survived forblood transfer studies; a recipient of one of these waspositive by FA staining. Nodes from three recipientsof the human nodes from re-treated patients werealso FA-positive with no positive findings in bloodrecipients; however, the partner of one of thesethree had a blood recipient whose node was positiveby silver stain.

During the course of cortisone treatment, onecontrol donor rabbit (No. 488) developed a lesion onthe dorsal surface of the right ear (identificationnumbers had been tattooed on the anterior surfaceof this ear), which was first noted on the 13th day ofcortisone; this area was darkfield negative on

numerous examinations and regressed during thetreatment without other therapy. A scar persistedto the time of the animal's death 7 days after thecompletion of cortisone. Histopathology of the areaat death showed increased fibrosis typical of scarformation but no indication of a recent syphiliticlesion; no treponemes were demonstrated.

Experiment 4 Node Transfer to Cortisone-TreatedNormal Rabbits

METHOD In an attempt to determine the significanceof the presence of immunochemically demonstratedtreponemes without evident infectivity, a group ofnormal rabbits was given a 7-week course of cortisoneacetate (Merck, Sharp, and Dohme); treatment consistedof two daily injections each of 2 * 5 mg./kg. body weightgiven for 5 days, and then omitted for 2 days; this coursewas repeated for 7 weeks. Nodes from selected animalswere injected into the testes of the normal rabbits after10 days of cortisone, and treatment was continued 5 moreweeks. Recipients were followed clinically for 7 monthswith darkfield examination of testicular aspirates andserological testing (VDRL at 1i, 3, and 7 months andTPI at 7 months.). Nodes were removed for histopatho-logical study from animals dying before the end of theobservation period and from animals completing observa-tion, as time permitted.

A total of forty donors was selected from thefollowing groups:

(1) Normal rabbits.

(2) Rabbits with natural Treponema cuniculi infection.

(3) Rabbits with untreated syphilis of 16 to 17months' duration with a history of darkfield-positivelesions or serological reactivity or both (Blood transferrecipients of Experiment 3).

(4) Rabbits with late latent syphilis, 22 to 32 monthsafter inoculation: some of these animals had beentreated with curative amounts of spectinomycin (Upjohn)8 to 17 months after infection and their untreatedcontrols. Nodes from the cured animals had been FA-positive 6 months after treatment when nodes weretransferred.

(5) Negative blood recipients of FA-positive controls(Experiment 3).

(6) Negative recipients of blood from FA-negativedonors whose partners had been FA-positive (Experi-ment 3).

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(7) Selected 12-month recipients ofnodes transferredfrom animals which had been inoculated with humannode tissue proved positive for treponemes by someprocedure in the present group of studies (Experiment 2).

ResultsNodes from four of nine controls produced

darkfield-positive disease in recipients; all fourdonors had been darkfield-positive earlier. Recipi-ents of nodes from two control animals of thespectinomycin treatment group also developeddarkfield-positive disease and appropriate serologicalresponse. Nodes from the T. cuniculi-infectedanimals produced darkfield-positive lesions ontransfer, but the serological response of recipientswas minimal to negative. No other recipientsshowed any indication of overt infection. TPI testsperformed at 7 months were reactive only in thoserabbits which had developed darkfield-positivelesions. No attempt was made to examine nodes fromall these animals; however, histopathological exami-nation on animals selected by chance from all sevengroups showed treponemes by FA staining only inGroups 3, 5, and 6. Interestingly, the FA-positivesin Group 6 represented Patients 12 and 20.

Summary of ResultsTreponemes were demonstrated in four of 21

(19-0 per cent.) experimentally-infected rabbitsafter penicillin therapy. Although no overt diseasedeveloped, treponemes were successfully trans-mitted 12 months after penicillin treatment by nodetransfer from three animals (14 -3 per cent.) and 18months after penicillin by blood transfer fromanother (4 -8 per cent.). Nodes from seven of elevenuntreated controls (63 6 per cent.) with disease ofthe same duration produced darkfield-positive,serologically reactive disease in recipients, and bloodfrom four of eleven controls (36 '4 per cent.) withdisease of 30 to 36 months' duration caused overtinfection in recipients either before or after treatmentwith cortisone. Treponemes were present in thenode of one other control in this group at death.

Eleven nodes from 45 men (24 -4 per cent.)treated for syphilis at some time in the past wereshown to harbour treponemes. Five human nodes(11 * 1 per cent.) were shown to be positive bysuccessful animal transfer (2=4 4 per cent.) or bymicroscopic study (4=8 9 per cent.); positivity ofthe other six (13 -3 per cent.) was determined bymicroscopical study of tissues of subsequent animalrecipients. Treponemes were demonstrated micro-scopically, after cortisone treatment, in nodes of sixoriginal animal recipients of material from five

patients. The node of one of these patients had alsobeen positive microscopically (silver stain).Nodes from two rabbits inoculated 12 months

previously with human node tissue contained trans-missible treponemes demonstrable microscopically;one donor rabbit had been positive microscopicallypreviously. Blood from rabbits representing threepatients contained transmissible treponemes; two ofthese patients' nodes had been positive micro-scopically originally while this was the first positivefinding for the third.Nine of the eleven patients were positive only by

microscopical study of human or rabbit nodes; fourof the nine were positive more than once during thestudy.

Inoculation of nodes from two previously-treatedpatients and from the patient with secondarysyphilis infected rabbits; only two of these infectedon subtransfer.Animal transfer of nodes from the five humans

whom we had re-treated did not produce overtinfection and microscopical study of the humannodes was negative. Microscopical study of nodesfrom the original rabbit recipients was positive inthree (60 per cent.), and blood from the partner ofone of these transmitted treponemes after cortisonetherapy. Therefore, the animal study of one of thefive human nodes (20 per cent.) was positive in thetwo different steps.Three controls with syphilis of 18 months' dura-

tion caused overt infection and one caused micro-scopical infection after blood transfer beforetreatment with cortisone; four infected overtly aftercortisone, and one of these four was also micro-scopically positive. Nodes from two of fifteencontrols were positive and one suspicious aftercortisone treatment (20 per cent.). Three controlswith syphilis of 18 months' duration were positivetwice or more in subsequent study.Nodes from normal rabbits, from rabbits with

proven cuniculosis, and from syphilitic rabbitstreated in the late latent stage with spectinomycinproduced no overt infection in rabbits prepared bycortisone treatment; pathological studies of nodesfrom selected recipients were negative for trepo-nemes.

DiscussionThe need for a rapidly effective non-toxic

therapeutic agent for syphilis was apparently met bypenicillin. With the vast majority of patients clinicalresponse was excellent, with rapid regression of the

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lesions, almost immediate clearing of motile trepo-nemes, and serological conversion as determined byreagin tests. Indeed, cure was so rapid and easy thatre-infection became a problem and a new clinicalsyndrome, "ping-pong syphilis", was recognized inwhich sex partners treated individually continuedto re-infect each other until both were treatedsimultaneously (Stewart, Thomsen, and Gardner,1951). The incidence of infectious syphilis declinedwithout an increased incidence of neurosyphilis orother complications of late syphilis (VD Fact Sheet,1965).

Penicillin was purified, and the development ofnew long-acting preparations reduced the numberof injections required (USPHS, 1960). Theantibiotic produced clinical cure of infection, butdid not repair damaged tissues.For almost 20 years there was no clinical evidence

suggesting that T. pallidum was developing resistanceto penicillin, though other micro-organisms de-veloped marked resistance to this and to otherantibiotic agents (Finland, 1955).The development of procedures such as the Reiter

protein complement-fixation (RPCF) (Cannefax andGarson, 1957) and Treponema pallidum immobiliza-tion (TPI) (Nelson and Mayer, 1949) tests, showedthat, although reagin usually returned to non-reactive levels after treatment, the specific antibodyresponse continued, often until death (Rockwell,Yobs, and Moore, 1964).

In reports in the years 1962 to 1965, Collart andco-workers (1962-65), proposed to explain thepersistence of immobilisins by the continuedpresence of treponemes after penicillin treatment.They demonstrated treponemes in silver-stainedsmears of nodes from nine humans who had beentreated repeatedly, and transfer of these nodes torabbits proved that three were still infectious. Twoof twelve rabbits treated in late latency withpenicillin developed granulomatous lesions of theear when they were given a course of cortisone12 months later. Treponemes consistent with T.pallidum were present in silver-stained smears fromthe lesion of one animal only; findings in the otherwere inconclusive. Asymptomatic re-infection orinadequate treatment, due either to insufficientpenicillin or to unrecognized antagonism betweenthe combined therapeutic agents, offered logicalexplanations of their findings in humans. Results ofstudies with individually caged rabbits were moredifficult to explain.Del Carpio (1963) reported that, while nodes from

four rabbits treated with 1 * 5 million units penicillinfor syphilis of2 months' duration were not infectious,

nodes from rabbits similarly treated 8 to 16 monthsafter infection produced TPI conversion in recipi-ents. Recently, Boncinelli, Vaccari, Pincelli, andLancellotti (1966) demonstrated T. pallidum by silverstain in a lymph node from one of eight humans withprimary or secondary syphilis some 6 to 54 daysafter treatment with 15 million units penicillin eitheralone or in combination with bismuth. They alsoreported finding typical T. pallidum in the nodes offour of eight patients with late latent or neurosyphilispreviously treated with arsenic, bismuth, andpenicillin; node transfer resulted in no overtinfections, but one recipient became TPI-reactive.The investigation we report had two facets-one

concerned a group of humans with a history oftreatment for naturally-acquired or congenitalsyphilis, and the other concerned a group of rabbitsexperimentally infected with Nichols virulent strainT. pallidum and treated with penicillin in late latency.While strains of T. pallidum in the human reservoirmay have had some exposure to sub-curative levelsof antibiotics through the treatment of concurrentillnesses in syphilitic patients, the Nichols strain hashad no such exposure. This standard laboratorystrain has been maintained for more than 50 yearsby passage through rabbits never exposed toantibiotics.On the basis of the results of node transfer,

rabbits with late latent syphilis were apparentlycured by penicillin treatment while untreatedrabbits remained infectious. Negative results in nodestudies after 12 months in some of the control groupwere explicable by the small numbers of treponemesthought to be present in late latent syphilis (Besse-mans, Van Haelst, and DeWilde, 1935). Aftercortisone treatment, staining showed nodes fromthree penicillin-treated rabbits and one blood-transfer recipient to be positive for treponemes. Onthe other hand, four recipients of blood from controlrabbits developed darkfield-positive sero-positivesyphilis; one developed (from No. 488) sero-positivesyphilis only; and one was positive only by staining.These recipients represented four different controlanimals; one other control was positive only byhistopathology of its own node.

Results in the human study did not agree with thegenerally accepted concept ofthe response of syphilisto penicillin. Initially, five, eventually eleven, of the45 men with treated syphilis were shown to bepositive by some study procedure; nodes from onlytwo men produced overt clinical infection inrabbits. The medical data of these eleven are sum-marized in Table I. The clinical data of theremaining patients have been published (Yobs andothers, 1964a).

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A group of TPI-reactive prisoners with medicalhistories of at least some reliability were studie4.However, they had last been treated for syphilis atleast one and generally several years previously, andthe possibility of re-infection could not be absolutelyexcluded. The five men initially positive wereinterviewed for possible contacts, re-treated, andre-studied in an attempt to relate the earlier findingseither to re-infection or to inadequate treatment(Yobs and others, 1964a). While study procedureson the post-retreatment nodes were all negative for12 months, after cortisone treatment, the nodes fromrabbit recipients of these human nodes and theirblood transfer recipients, representing three of there-treated patients, were shown to contain trepo-nemes by histopathological techniques.When a person adequately treated for syphilis is

re-infected, he may develop a darkfield-positivelesion, depending on the stage at which the earlierinfection was treated. Generally, a temporaryincrease in reagin antibody is detectable, althoughthe duration and degree of this rise is variable. Whilethe histories of the 45 men did not absolutely ruleout re-infection, their serum test results had beenstable with no sudden or significant fluctuationssince the last treatments; certainly, the results in

TABLE IIRABBIT PASSAGE OF TREPONEMA PALLIDUM

those whom we re-treated showed no change such as

might occur after the treatment of active syphilis(Yobs, Olansky, Rockwell, and Clark, 1965). Despitethis, three of five nodes removed after re-treatmentwere eventually shown to contain treponemes.

It has been difficult to correlate the positivefindings by staining in nodes of humans and rabbitswith the failure to infect. Prolonged darkfieldexamination of human node suspensions showed notypical motile treponemes and only one non-motiletreponemal form. The three human nodes positiveby FA-staining showed several fields with three tofive or more treponemes; silver stain showedfragments of several treponemes in one node. Yet,only two human nodes produced overt infection andone of these had been negative by histopathologicalstudy.The treponemes isolated from Patients 10 and 12

were apparently less virulent for rabbits in earlypassages than the Nichols virulent strain of T.pallidum; darkfield-positive lesions developed moreslowly and were generally more transient. Recipientsdid not show the anticipated serological response;those surviving 6 months were TPI-non-reactive andVDRL-non-reactive or reactive in low titre. Bothisolates were carried through several subsequent

ISOLATES FROM TWO PATIENTS

Transfer I Transfer 2 Transfer 3 Transfer 4

Human-to-Rabbit Rabbit-to-Rabbit Rabbit-to-Rabbit Rabbit-to-Rabbit

l Recipient Recipient Recipient RecipientPatient Material Inocu- -

i

Material Inocu- - Material Inocu- I Material Inocu- -No. Trans- lation Trans- lation Trans- lation Trans- lation

ferred Site DF VDRL TPI ferred Site DF TPI ferred Site DF TPI ferred Site DF TPIat 6 mths 6 mths 6 mths 6 mths

10 Node IT 35 2*5 N Testis IT 17 R No transfer*dils (est'd

2 x 106 IT 18 N.D. Testis IT 9 N.D. No transferTp per (est'dsite) 107 Tp IT 9 N.D. Testis IC 10 R

per site) (est'd1*7 x 106 IC 10 WRTp persite) IC 10 N.D.

Testis IT 10 N.D.(est'd

17X 107 IT 10 N.D.Tp persite)

Node IT 29 Dead* No transfer

12 Node IT 36 N N Testis IT cct Dead No transfer

IT cc N Testis IT co N No transfer

IT _c N No transfer

Node IT 36 Dead No transfer __

DF = Days incubation to darkfield positivity.IT = Intratesticular.

* Tissues frozen to preserve isolate.N.D. = Not done.

t Never found darkfield positive.IC = Intracutaneous, six sites on each animal.

t TPI-reactive at 3 months.

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transfers (Table II). The isolate from Patient 12 wasimmediately lost, but that from Patient 10 has hadeight animal passages, during which it eitheradapted to the new host species with increasedvirulence or the numbers transferred increased andstabilized. Whatever the reason, the development ofdarkfield positivity and of measurable treponemalantibody has been more predictable in these laterpassages of this strain.Brown and Pearce (1920) stated that most

investigators were of the opinion that the virulenceof T. pallidum for rabbits might be increased bypassage or by adaptation, but that the time for whichthis increase might be kept up or the extent to whichit might be carried was unknown. Chesney suggestedthat adaptation to a new host might be an importantfactor in the failure of some animals to develop overtdisease (Chesney and Kemp, 1925).

Determination of the virulence of demonstrabletreponemes has been a primary consideration inthese studies. Since individual host variation isrecognized as an important factor in the successfuldemonstration of virulence, the original study wasexpanded to include intensive efforts to demonstratevirulence by prolonged observation, by subtransferto other animals, and by alteration of host response.Twelve months' observation of human noderecipients and penicillin-treated rabbits followedby subtransfer showed no additional rabbits withsymptomatic infection, but one subtransfer re-cipient's node was shown by FA-staining to containa spiral organism which resembled T. pallidum.

Cortisone treatment may lower the natural andacquired resistance of laboratory animals to certaininfections (Fischel, 1952); however, cortisone altersthe host's immune system in a complex manner notas yet wholly understood (Turner and Hollander,1950, 1954; Delamater, Sauzino, and Urbino,1952). In experimental syphilis in rabbits, cortisonegreatly enhances the infection when administeredduring the time the disease is developing (Turnerand Hollander, 1950, 1954; Delamater and others,1952; Germath, Ottinger, and Oyama, 1952). Itseffect on the titre of circulating antibodies in earlysyphilis has not been clearly defined, but preliminarystudies indicated that the development of TPIantibody was not affected. Collart and others (1962a)found that disease in penicillin-treated latentsyphilitic rabbits was more manifest after cortisonetreatment.McLeod and Magnuson (1956) gave cortisone to

thirty rabbits with experimental syphilis of 7 to 9months' duration at a dose of 5 mg./kg./day for29 days; eighteen survived the entire course. Onemonth after the injections, one of the cortisone-

treated rabbits had an infectious relapse as shownby the development of a darkfield-positive nodularlesion in scar tissue on the ear. Such infectiousrelapses are extremely rare (Pearce, 1938). Theinfectivity of blood from these animals was en-hanced; blood from 75 per cent. of the cortisone-treated animals was infective compared with only41 per cent. of the controls. The same authors gavecortisone to twelve rabbits with syphilis of 12 to18 months' duration at a dose of 10 mg./kg./day, for17 days; no lesions developed but the VDRL titredecreased more than 2-fold or reverted to negative.Although the cortisone was given in the smallerdose schedule used by McLeod and Magnuson(1956), it was soon obvious that the apparentlyhealthy rabbits in our study, syphilitic control ortest, were extremely sensitive to this regimen.However, our study has revealed no data to indicatesignificant influence by cortisone on the course oflate latent treated experimental syphilis in rabbits oron the receptivity of rabbits to infection.Removal of inguinal or axillary nodes or both

without killing the animal was not practicable in thislaboratory; therefore, since both popliteal nodes hadbeen removed from some animal groups, infectivityafter cortisone therapy was determined by bloodtransfer. This procedure had been used as a measureof infectivity by Frazier, Bensel, and Keuper (1950)who showed that spirochaetaemia persisted inrabbits for as long as 1,287 days after inoculationwith Nichols strain T. pallidum; their data suggestedthat the organisms were not present in the bloodcontinuously (Frazier, Bensel, and Keuper, 1952).In our experiments, blood from four of seventeencontrols (23 -5 per cent.) with syphilis of 30 to 36months' duration and from six of seventeen controls(35 3 per cent.) with syphilis of 18 months' durationcaused infection. Frazier and his co-workers showedthat blood from eleven of 31 rabbits (41 per cent.)with asymptomatic infections for over 24 monthsharboured T. pallidum, and two of these animals hadbeen in latency for over 3 years when their bloodwas found to be infectious. On the basis of dissimilarresponse in paired recipients (only one becominginfected), they concluded that the number ofspirochaetes in the blood at this time was small, andthey suggested that when only one of a pair ofrecipients identically inoculated developed disease,the number of organisms in the inoculum wasinsufficient to overcome the natural resistance of theone recipient. They showed that blood from animalswith negative serological tests could be infectious.The use of histopathological techniques compli-

cated the interpretation of the results of this study.There is little agreement on the morphological

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criteria for identification of T. pallidum in fixedpreparations. Most knowledgeable persons preferto accept only "typical" forms, i.e. those on whicheveryone will agree, although the finding of atypicalforms in darkfield preparations is a recognizedoccurrence. Silver staining is technically difficult toperform and equally difficult to interpret since it isnot a specific stain. Immunochemical techniques,while considerably more specific, incorporatesubjective evaluation of fluorescence intensity. Also,treponemal "group" antigens (Deacon and Hunter,1962) and similar antigens in other groups of micro-organisms which may stain may present problems.The fluorescein-conjugated rabbit antiserum to

T. pallidum, which was used in the direct fluorescentantibody technique for the demonstration oftreponemes, was prepared in our laboratoriesaccording to the procedure described previously(Yobs and others, 1964b). Some pilot work was

performed by staining T. pallidum with conjugateabsorbed with the sorbing agent developed for thefluorescent treponemal antibody-absorption (FTA-ABS) serological test (Hunter, Deacon, and Meyer,1964). This sorbent reduces and frequently elimi-nates the staining of "group" treponemal antigenscommon to many treponemes, including the knownnonpathogenic, cultivable ones (Deacon and

Hunter, 1962). However, since the sorbing agentwas not readily available during this study and sincea change in staining procedure would have addedanother variable, sorbed conjugate was not used inthis study for the FA-staining of treponemes. Allfluorescent stained material was read withoutidentification of source by one of the authors;positives were confirmed by at least two othermicroscopists, also reading without specimen identi-fication. Negative preparations were not routinelyexamined by more than one microscopist.A large number of the positive results in this study

were based on the demonstration of treponemes innodes which produced no detectable disease on

inoculation into normal rabbits. Evaluation of thesensitivity and specificity of the fluorescent stainingprocedure with this conjugate in the demonstrationof T. pallidum was, therefore, undertaken. Materialsstudied included lymph node and testicular tissuesand aspirates and exudate from skin chancres ofrabbits, humans, and the Aotus trivirgatus monkey.Results summarized in Table III show that whileexudate from primary and secondary lesions ofrabbits and humans are positive for treponemes bythis method, lymph nodes were not found positiveuntil more that 6 months after inoculation and were

not consistently positive then. Darkfield-positive

TABLE IIIDEMONSTRATION OF TREPONEMA PALLIDUM BY FA-STAINING COMPARED WITH

INFECTIOUSNESS IN NODE TRANSFER PROCEDURE

Node Transfer Node TransferSpecies Condition Material No. (No. infecting/ No. (No. infecting/

Stained FA+ No. transferred) FA- No. transferred)

Rabbit Normal Node 0 N.D. 7 N.D.

Normal + cortisone Node 0 N.D. 11 N.D.

Lesion exudate 20 N.D. 6 1/1

Primary syphilis Adequately treated* Nodet 0 0/0 18 0/12

Inadequately treated Nodet 0 0/0 5 1/1

14-32 mths Untreated Nodet 5 2/3 13 6/11§

Late syphilis Adequately treated* Node*7 mths after Rx 5 0/5 10 0/108-12 mths after Rx 3 0/3 3 0/313+ mths after Rx 1 0/1 6 0/6

T. cuniculi Lesion exudate 0 0/0 5 2/2Node 0 0/0 17 5/8

Man Secondary syphilis Lesions andexudate 4 2/411 0 0/0

Monkey Normal Node 0 N.D. 2 0/2

Strain from humans Darkfield+node aspirate 0 0/0 2 2/2

N.D. = Not done.* Treatment consisted of varying amounts of spectinomycin.t Node removed 6 months after completion of treatment.* Node removed at times indicated.§ Five infected only one of two recipients.11 Lesion tissue not node suspended and inoculated.

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OBSERVATIONS ON THE PERSISTENCE OF TREPONEMA PALLIDUMmaterial from rabbits with natural cuniculosis didnot stain. Treponemes were also demonstrated inthe small group of infected monkeys studied by thisprocedure. It appears, therefore, that while FA-staining is a specific procedure, it neither reflectsvirulence nor possesses a high degree of sensitivity.The stained material must still contain morpho-logically typical spiral forms to be presumed positivefor treponemes and the procedure does not definitelyidentify the treponeme as T. pallidum.

It has been difficult to correlate positive micro-scopic findings in nodes with non-infectiousness ontransfer. These forms probably are not artefacts sincetheir morphology and immunochemical stainingwere typical of T. pallidum. If these were T. pallidumkilled by treatment, they had been dead for sometime and would have been subject to phagocytosis ordegenerative changes. Saprophytic treponemes havenot previously been recognized in nodes or blood insuch numbers; in fact, Public Health Serviceinstructions (USPHS, 1962) suggest examinationof aspirated material from a regional node toestablish a darkfield diagnosis when the demonstra-tion of T. pallidum in lesion exudate is unsuccessful.The possibility must be considered that these

fluorescent treponemes in recipient rabbits may havebeen T. cuniculi. All rabbits in this study werecarefully examined for lesions suggesting thisendemic treponematosis before initial use andregularly thereafter, and each rabbit also underwentVDRL and TPI tests before use and periodicallythereafter. All baseline studies were negative, andthere was no clinical suggestion of cuniculosis inthese animals or others in this colony. In this study,treponemes stained with an intensity typical forT. pallidum; darkfield-positive material from naturalcuniculosis lesions in a separate colony showedsignificantly less fluorescence when stained by thesame procedure. In fact, the staining of T. cuniculiwas so faint as to be termed "negative" in blindreadings when slides of both T. pallidum and T.cuniculi were included. With experimental passageof the T. cuniculi strain, staining became moreintense and virulence increased as shown by morerapid and predictable lesion development (Clark andYobs, unpublished).Accumulated data suggest that the organism

detected in this study by FA-staining was not onlypropagating without causing lesions or detectableserological response but was also transmissible insuspension to new hosts with the same effect.

If it is accepted that altered T. pallidum ofdecreased virulence can persist after the antibiotictreatment of late latent syphilis, questions must be

resolved which concern the mechanism of thealtered virulence, the cause of the persistence, theantigenic significance of these organisms and theirinfluence on the pathogenesis of clinical syphilis. Oneapproach is through considering the mode of actionof antibiotics. Penicillin has been shown to killGram-positive bacteria more readily than Gram-negative ones (Fleming, 1929). It acts on growingcells but not resting ones (Hobby, Meyer, andChaffee, 1942), resulting in the formation of large orfilamentous forms of bacteria (Gardner, 1940;Duguid, 1946). Lederberg (1956), working withbacilli, showed that penicillin selectively inhibitedbacterial cell wall synthesis without causing lethaleffect on protoplasts; the growing protoplastscontinued to enlarge in its presence and reverted tobacilli when the penicillin was removed. Thecontinued enlargement of the protoplast explains therequirement of growth for penicillin action, sincethe growing protoplast emerging through a break inthe wall is then subject to different osmotic pressures.Unless this occurs, the protopla3t remains a stableform within the cell wall, ex en though cell wallsynthesis may have been blocked by penicillin.Antigens present in intact portions of the cell wallcontinue to function.Magnuson, Eagle, and Fleischman (1948) esti-

mated that T. pallidum has a multiplication time forone generation of some 33 hours. If such a slowprocess permitted the protoplast to remain entirelywithin the cell wall, or if the protoplast weresufficiently elastic to adjust to minute osmoticdifferences in its environment, rupture and deathmight not occur. Similarly, if during therapy theextra-cell wall protoplast met an isotonic orhypertonic situation, rupture and death would notoccur. It seems possible that the treponeme may alsobe genetically altered so that its virulence is affected,or that its virulence may be dependent on an intactcell wall.Erythromycin has been shown to inhibit protein

synthesis selectively in Escherichia coli (Brock andBrock, 1959); although the precise mode of actionis unclear, other data suggest that this interferenceresults from interaction with a ribosomal component(Wolfe and Hahn, 1964). Recently spectinomycinhas also been shown to inhibit protein synthesis inE. coli cells and extracts, possibly also by action onribosomes (Davies, Anderson, and Davis, 1965).Another factor in the finding of treponemes after

treatment may be failure of the antibiotic to pene-trate to all parts of the host and to come into contactwith all treponemes, but this seems unli'kely. Whiletissue concentration requirements for cure of

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BRITISHJO0URNAL OF VENEREAL DISEASES

syphilis may vary between drugs, T. pallidum hasbeen shown to be extremely sensitive to minuteamounts of penicillin in vivo and in vitro and some-

what less sensitive to other agents.

Although the effect on bacteria of penicillin andother antibiotics is being studied by numerousinvestigators, little or no work has been reported onthe effects of these agents on the host's cells.Toxicity has been evaluated, but metabolic andgenetic effects are generally unknown. It is possiblethat some obscure enzyme system in the host's cellsmay be altered resulting in the continuation of an

established antibody response or the inhibition ofdevelopment of a detectable response.

Antibodies developed during the course of syphilismay alter T. pallidum, rendering it less virulent butstill transmissible and demonstrable. Eberson (1921)showed that syphilitic serum possesses treponemici-dal properties. Chesney and Kemp (1926) producedsubclinical re-infection upon challenge of immuneanimals. Many investigators as well as the presentauthors (Morgan, 1941; Clark and Yobs, unpub-lished) have observed supposedly normal rabbitswhich did not develop darkfield-positive lesionsand/or reactive serology after inoculation with largenumbers of virulent T. pallidum while other rabbitsinoculated simultaneously with identical inocula diddevelop syphilis. These observations suggest thatantibodies affecting the virulence of the organismmay develop.

Although the human volunteers, the penicillin-treated rabbits, and the control rabbits were allTPI-reactive, nodes from all animals in these groupswere not positive for T. pallidum. Animals in whichtreponemes were demonstrated by FA staining didnot become VDRL-reactive or TPI-reactive unlessthey had also developed darkfield-positive syphilis,and at least one TPI-reactive control, positive byFA-staining reverted to TPI-nonreactive duringthis study. Therefore, the organisms which havebeen demonstrated by FA methods are probably notresponsible for the persistence of immobilisins;certainly, they have not been able to initiate develop-ment of immobilisins to a measurable degree.However, these organisms do remain at least

partially intact antigenically as indicated by theirability to stain in the FA procedure. They also retainsufficient antigenicity to stimulate some antibodyformation, as shown in a small retrospective study ofbaseline and final sera from some of the recipients ofthe eleven positive human nodes and the recipientsof nodes positive for treponemes from penicillin-treated rabbits. Although TPI andVDRL tests wereconsistently nonreactive, the FTA-ABS tests

(Hunter and others, 1964) performed on baselinesera, using fluorescein-labeled anti-rabbit globulin,were nonreactive while those on final sera werereactive. In another study on rabbits inoculatedintracutaneously or intratesticularly with largenumbers of T. pallidum, FTA-ABS reactivity wasdetected 1 week after inoculation (Mothershed,Yobs, and Clark, 1967). The organisms detected bystaining in this study may also stimulate the produc-tion of antibody(s) not detectable with availableserological test procedures and antigens.

If Treponema pallidum or treponemes derived fromT. pallidum continue to live and apparently tomultiply in the host after treatment, it seems logicalto consider that they must have some role in thepathogenesis of clinical syphilis. The persistence ofvirulent T. pallidum would probably be hazardous tothe patient and, if proved, would justify anticipationof a resurgence of late manifest syphilis. As intuberculosis, a state of immunity with delayedhypersensitivity may develop in which a new expo-sure to treponemes triggers renewed clinical activityand late manifestations. The occurrence of such ahypersensitive state was indicated in an earlier study(Magnuson, Thomas, Olansky, Kaplan, De Mello,and Cutler, 1956) in which it was shown that patientswho have once had syphilis, even though cured inthe primary stage, are never the same immuno-logically. Their serological response to re-infectionis typical of those observed in anamnestic rises dueto other antigenic stimuli. Also, if the previousinfection was of sufficiently long duration, itproduced considerable resistance to subsequentchallenge. It is interesting to speculate about theformation and importance of a treponeme-hostcomplex and an auto-immune response to the hostportion of the complex. Of course, these "altered"treponemes may simply invade appropriate tissuesof susceptible hosts where multiplication occursslowly with the development of late lesions depend-ing on exo-effects of the treponemes or metabolicproducts.

If the demonstrated treponemes are not acceptedas altered T. pallidum, then they must be explainedin some other way. Several possibilities havealready been considered. It is interesting to specu-late about others. The FA-positive treponemespresent in recipients without overt infection maynot be T. pallidum at all but nonvirulent treponemeswith the same or a related antigenic mosaic. Onemay hypothesize, although there is no supportingevidence at present, that T. pallidum is symbioticallydependent on these for nutritional or other supportto maintain existence or virulence. If conditionswere not favourable to the biochemical functions

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necessary for symbiosis, virulence might be affected;and one or both treponemes might conceivably belost, even though the original inoculum hadcontained both. Or it may be postulated that theseare T. pallidum rendered avirulent by alteration ofmolecular biology through either mutation or as yetunidentified chemical action.One may also speculate that T. pallidum has a life

cycle in only one stage of which the recognizedtreponemal morphology is found and in only onestage ofwhich the organism is sensitive to antibiotics.Many scientists, notably Delamater, Newcomer,Haanes, and Wiggall, 1950; Delamater, Haanes,Wiggall, and Pillsbury, 1951, and Levaditi, Schoen,and Sanchis-Bayarri, 1927; Levaditi and Li YuanPo, 1930, have proposed the existence of a life cyclein which stages differ in morphological characteris-tics, but no one has proved that T. pallidum hassuch a cycle.This research suggests that in syphilis, as in acute

rheumatic fever, it may be possible to demonstratethe causative organism at various intervals aftertreatment when there is no clinical disease. Stoller-man, Siegel, and Johnson (1965) have reportedexamining 1,000 patients with throat cultures posi-tive for Group A betahaemolytic streptococci butwithout rheumatic fever; demonstrating in effecta carrier state. The significance of such a state hasnot been determined in either disease. The virulenceof these organisms is unknown as is the requirementfor the development of clinical disease where theyare found. The magnitude of the immune responseand the duration of convalescent carriage of strepto-cocci seem to be related to the attack rate of rheu-matic fever. Both are related to the virulence of thestreptococcal strain. Interestingly, type-specific anti-bodies persist for as long as 32 years, and thispersistence is not correlated with the severity of thestreptococcal infection or the serological type of theinfecting strain (Lancefield, 1959).

In the past, evaluation of a chemotherapeuticagent's efficacy in curing syphilis has been based onthe clinical response to treatment and the infectivityof tissues transferred at some interval after treat-ment, as shown by the development of darkfield-positive lesions and serological response in recipients.Because of technical difficulty and low sensitivity,failure of histological demonstration of T. pallidumhas not been used as a criterion. Findings in thisstudy do not conflict with these criteria; instead,they add a new dimension to the study of thepathogenesis and immunity of syphilis.As often transpires, this study has posed more

questions than it has answered. We have shown, asdid the teams led by Collart, Del Carpio, and

Boncinelli, that a treponeme is present afterantibiotic treatment in syphilitic humans and inindividually caged, experimentally infected rabbitswhere re-infection is not a problem. However, incontrast to these investigators, we have not demon-strated disease-producing virulence, although wehave shown infectivity in that we have recoveredthese organisms after blood and tissue transfer. Thetreponemes found in this study were present afterantibiotic treatment with penicillin, erythromycin,or spectinomycin. While several possible explana-tions have been proposed, the results are notconclusive. The questions raised might be answeredin two ways:

(1) A similar investigation of nodes from a groupof humans with negative results to TPI testsand no history or indication of previous orcurrent syphilitic infection;

(2) A determination of the virulence oftreponemespresent after treatment in recipients withpermanently impaired immune mechanisms.

Both should include in vitro culture for trepo-nemes, but large-scale studies are not necessary tosecure the required data.

SummaryNodes from eleven of 45 TPI-reactive male

volunteers treated in the past for syphilis wereshown to harbour treponemes. Nodes from three offive of these subjects were shown to harbourtreponemes after re-treatment.Four of 23 TPI-reactive rabbits with syphilis of

14 to 22 months' duration at the time of penicillintreatment were later shown to harbour treponemes.Data presented suggest that these are probably

T. pallidum of decreased virulence and alteredantigenicity. Alternative conclusions and implica-tions are discussed.

The authors wish to thank the Testing and TPIUnits, Standardization and Consultation Services,Venereal Disease Research Laboratory, Venereal DiseaseProgram, National Communicable Disease Center,Atlanta, Georgia, for performing serological tests on allhuman sera and cerebrospinal fluids and for performingthe TPI testing on rabbits in this study.

REFERENCESBessemans, A., Van Haelst, J., and DeWilde, H. (1935).

Amer. J. Syph., 19, 161.Boncinelli, U., Vaccari, R., Pincelli, L., and Lancellotti.

M. (1966). G. ital. Derm., 107, 1.Brock, T. D., and Brock, M. L. (1959). Biochem.

biophys. Acta, 33, 274.Brown, W. H., and Pearce, L. (1920). J. exp. Med.,

31, 475.Cannefax, G. R., and Garson, W. (1957). Publ. Hlth

Rep. (Wash.), 72, 335.

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Chesney, A. M., and Kemp, J. E. (1925). J. exp. Med.,42, 33.

-- - (1926). Ibid., 44, 589.Clark, J. W., Jr., and Yobs, A. R. Unpublished data.-- - In preparation.Collart, P. (1964). "Persistence of Treponema pallidum

in Late Syphilis in Rabbits and Humans, notwith-standing Treatment", in "Proceedings WorldForum on Syphilis and Other Treponematoses,Washington D.C., Sept. 4-8, 1962", p. 285.Public Health Service Publication No. 997. U.S.Communicable Disease Center. Venereal DiseaseBranch, Atlanta, Georgia.

Collart, P. (1965). Proph. sanit. morale, 37, 247.-, Borel, L., and Durel, P. (1962a). Ann. Inst.

Pasteur, 102, 596.-,-, - (1962b). Ibid., 102, 693.-,-,- (1964). Brit. J. vener. Dis., 40, 81.and Durel, P. (1964). Ann. Derm. Syph., 91, 485.

Davies, J., Anderson, P., and Davis, B. D. (1965).Science, 149, 1096.

Deacon, W. E., and Hunter, E. F. (1962). Proc. Soc.exp. Biol. (N.Y.), 110, 352.

DeLamater, E. D., Haanes, M., Wiggall, R. H., andPillsbury, D. M. (1951). J. invest. Derm., 16, 231.

-, Newcomer, V. D., Haanes, M., and Wiggall, R. H.(1950). Amer. J. Syph., 34, 122.

-, Saurino, V. R., and Urbach, F. (1952). Ibid., 36,127.

Del Carpio, C. (1959). Riv. Ist. sieroter. ital., 34, 432.- (1963). Ibid., 38, 166.Dieterle, R. R. (1927). Arch. Neurol. Psychiat. (Chicago),

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Des observations additionnelles au sujet de lapersistance des Treponema pallida apres

traitement chez 1'homme et le lapinR1SUME

Les ganglions lymphatiques de onze volontairesobtenus d'un nombre total de 45 hommes donnant unereaction positive au test TPI qui avaient ete traites dansle passe pour la syphilis ont montre qu'ils hebergeaientdes treponemes apres un second traitement.

Quatre des 23 lapins reagissants au test TPI etatteints de syphilis d'une duree de 14 a 22 mois aumoment oii le traitement a la penicilline avait etcommence avaient montre plus tard qu'ils hebergeaientdes treponemes.Les donnees presentees suggerent que ces treponemes

etaient des T. pallidum de virulence diminuee dontl'antigenicite etait changee. Plusieurs conclusions etleurs implications sont discutees.

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