Experimental syphilis: guinea pig model

Br J VenerDis 1983;59:157-68

Experimental syphilis: guinea pig modelCAROL S PIERCE,* KONRAD WICHER,t§ AND SHAHEEN NAKEEB*From the *Division of Clinical Microbiology and Immunology, Erie County Laboratory, Buffalo; theDepartments of tMicrobiology and *Pathology, State University ofNew York, Buffalo; and the §Centerfor Laboratories and Research, New York State Department of Health, Albany, New York, USA

SUMMARY Outbred Hartley male guinea pigs (300-400 g) were infected intradermally withvarious concentrations of Treponema pallidum Nichols strain in the pubic region. The medianlethal dose (ID,) was approximately 10g. The animals produced lesions visible by darkfieldmicroscopy, treponemal antibodies (IgG only), and histopathological changes in the lymphoidorgans. Though less susceptible to T pallidum infection than the rabbit when infected with asufficient number of organisms, the guinea pig may be a useful model in experimental syphilis.

Introduction

The guinea pig has been known to respond toTreponema pallidum infection since Bertarelli'ssuccessful eye infection. Truffi2 was successful ininfecting the scrotum of guinea pigs, and a numberof researchers confirmed Truffi's results.3-7 Kolle andEvers,8 who were the first to use the Nichols strain ofT pallidum, established the time required todisseminate T pallidum in guinea pigs infectedthrough abraded skin, subcutaneously, or intratest-icularly. Tani et a19 contributed to the guinea pigmodel by observing that males were more susceptibleto infection than females and that younger animalswere more susceptible to infection than olderanimals. Dold and Worms'0 were unable to produceclinical symptoms in guinea pigs infected intrader-mally but did find that the lymph nodes and testes ofthese animals were infective. The infectivity (usingrabbit testes) could still be shown after three con-secutive blind passages of the lymphoid organs intohealthy guinea pigs. Other researchers" 12 alsoreported difficulty in producing symptomaticinfection in guinea pigs but did demonstrate the in-fectiveness of various organs, including the brain.After infecting guinea pigs with various strains of Tpallidum in multiple sites Turner and Hollander'3concluded that the incidence of symptomaticinfection varied depending on the strain of Tpallidum and the site of infection.

Wicher et a'4 and Lesinski et al5 infected maleguinea pigs with Tpallidum Nichols strain (obtainedAddress for reprints: Dr K Wicher, Infectious Disease Institute,Center for Laboratories and Research, State of New YorkDepartment of Health, Governor Nelson A Rockefeller EmpireState Plaza, Albany, NY 12201, USA

Accepted for publication 5 December 1982

from the Fournier Institute of Paris, France) intra-venously, intratesticularly, and intradermally. Intra-venous injection did not produce symptomaticinfection nor were the inguinal lymph nodes infectivethree months after Tpallidum injection. Intratestic-ular infection did not produce typical orchitis, asseen in infected rabbits, but the testes and inguinallymph nodes were still infective six months afterinfection. Intradermal infection in the pubic regionproduced darkfield-positive lesions in 67% of theanimals appearing between 13 and 23 days afterinfection. Regardless of whether the animaldeveloped lesions the lymph nodes and testesremained infective six months after infection. Wicherand Jakubowski'6 17 tried without success to man-ipulate the immune mechanism of T pallidum-infected guinea pigs by injecting cortisone (7*5mg/kg body weight) for various lengths of time.Immune responses of Tpallidum-infected guinea

pigs have been studied only sporadically. Lesinski etaP8 examined guinea pigs infected intravenously,intratesticularly, and intradermally with T pallidumNichols strain for antibodies using the complementfixation, flocculation, and T pallidum immobil-isation (TPI) tests. The tests for cardiolipin anti-bodies gave equivocal results. Treponemal antibodiesstarted to appear in several animals two to threemonths after infection, were still present at four tofive months, but in the animals infected intraven-ously and intratesticularly they disappeared beforethe eighth month. In the guinea pigs infected intra-dermally the treponemal antibodies remained beyondthe tenth month after infection.Wicher et al'9 using a Tpallidum strain from the

Centers for Disease Control, Atlanta, GA, andhousing the animals under more controlledconditions, were unable to find cardiolipin anti-

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Carol S Pierce, Konrad Wicher, and Shaheen Nakeeb

bodies in intradermally infected guinea pigs by thecomplement fixation test. Treponemal antibodieswere detectable by the fluorescent treponemalantibody-absorbed (FTA-ABS) test in the firstmonth after infection.To our knowledge the cellular response of infected

guinea pigs to T pallidum antigen has not yet beenexplored. Wicher et alt9 found that peritonealexudate cells from T pallidum-infected guinea pigs(obtained four days after injection of oil), whenexamined by the capillary leucocyte migrationmethod in the presence of Reiter antigen, producedenhanced leucocyte migration, which reachedsignificant values between six and 24 weeks afterinfection. The leucocyte enhancement-migrationfactor, the production of which was confirmed bythe indirect leucocyte-migration test, was not,however, characterised.The present study was undertaken to expand the

knowledge of the rarely used animal model forsyphilis research and to set up immunopathologicalindices which would be useful in future explorationof inbred strains of guinea pigs for experimentalsyphilis. Studies of syphilis in genetically definedguinea pigs may have far reaching consequences.

Materials and methods

ANIMALSAlbino male Hartley guinea pigs (300-400 g, 6-8weeks), an outbred strain, were obtained fromBuckberg Animal Farm, Tompkins Cove, New York.The animals were housed individually at a controlledtemperature of 19-220C and given antibiotic-freefood and water in unlimited amounts. Allexperimental and control guinea pigs were drawnfrom the same source.

BACTERIAL STRAINSTpallidum (Nichols) was obtained in 1970 from theCenters for Disease Control (CDC) and maintainedby intratesticular passage in New Zealand rabbits.Treponema phagedenis biotype Reiter, also obtainedfrom the CDC, was maintained in the spirochetalmedium described by Hanson and Cannefax.20 Theorganisms were grown for several days at roomtemperature (20-240C), centrifuged at 12 000 x g,washed three times with phosphate-buffered saline(PBS), and counted. The organisms were used assuspension for in-vitro lymphocyte transformationtests.

INFECTION OF ANIMALSInocula of Tpallidum (Nichols) were obtained from

the testes of infected New Zealand rabbits at the peakof orchitis (9-12 days). The treponemes in thesupernatant were enumerated by the method ofMorgan and Vryonis21 as adapted by Magnuson etal.22 For all experiments the animals were infectedintradermally in the pubic region.

DETERMINATION OF INFECTIVE DOSEPortions (0 25 ml) of the suspension, containing5 x 104 to 5 x 107 Tpallidum, were used to infect34 guinea pigs. The virulence of the treponemes wasexamined by duplicate intradermal injections into theclipped backs of rabbits. Infected guinea pigs wereexamined daily until the lesions appeared and everytwo days until the lesions disappeared.

HUMORAL IMMUNE RESPONSETo study the kinetics of the antibody response 20guinea pigs were each infected with 5 x 106organisms. Blood for serum was obtained by cardiacpuncture under light ether anaesthesia. Sera obtainedat nine days and at 3, 6, 12, 27, and 52 weeks afterinfection were examined for antibody content. Forcardiolipin antibody the Venereal Disease ResearchLaboratory (VDRL)23 and cardiolipin complement-fixation24 tests were used. For treponemal antibodiesthe slightly modified FTA-ABS test25 was used.Commercial T pallidum-coated slides and sorbent(Palomer Chemicals, Carlsbad, CA) were used in aquantitative test in which test sera were diluted with asorbent. As conjugate, fluorescein-conjugatedantisera to guinea pig IgG and IgM were prepared inrabbits in our laboratory. Only the antiserum to IgMwas rendered class-specific by absorption with in-solubilised, electrophoretically pure guinea pig IgG,a2 protein, and human C3, which cross reacts withguinea pig C3. Globulin fractions of the rabbitantisera were conjugated with fluorescein isothio-cyanate by a standard procedure. A positive testresult was considered to be a reaction from 4+ to 1 +and the end point titration as the last dilution of atest serum exhibiting 1+ reaction (normal guinea pigsera did not demonstrate ± or 1 + reaction).Two groups of five animals each served as controls

for the specificity of treponemal antibody produc-tion. One group received supernatant from a prepar-ation of normal rabbit testes (NRT) that had beenprocessed in the same manner as the infected testes.The second group received T pallidum-free super-natant from infected rabbit testes from which thetreponemes had been removed by centrifugation at48 200 x g for one hour. Sera from the controlanimals were obtained every three weeks for threemonths and examined by the FTA-ABS procedurewith conjugated antisera to both IgG and IgM.

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Experimental syphilis: guinea pig model

ABSORPTION OF SERA OF INFECTED GUINEAPIGSSera of four infected guinea pigs with IgG titres bythe FTA-ABS test of 80 or 160 were diluted 1/5 withPBS, and 1-ml aliquots were mixed separately withthrice washed, pelleted T pallidum (1 x 109organisms) or 10 mg of lyophilised NRT protein. Themixtures were left overnight at 6°C on a rotator andcentrifuged at 48 000 x g for 30 minutes. Theabsorbed sera were examined by the FTA-ABS test.Some aliquots of sera were absorbed twice.

IMMUNISATION WITH LIPIDSTwenty-eight control guinea pigs were divided intosix groups of four or five animals each andimmunised with lipid-carrier or carrier preparationsas follows: groups 1 and 2 received the lipid contentof an alcoholic extract of normal guinea pig kidneymixed with porcine serum (group 1) or with PBS(group 2) prepared according to th_ procedure ofSachs et aP6; group 3 received diluted porcine serumalone; and groups 4 and 5 received VDRL antigenthat had been evaporated and resuspended in PBS(group 4) or 0 1/7I methylated bovine serum (group5) to give each animal approximately 60 gAg of cardio-lipin in 0 2 ml of suspension. Bovine serum albuminwas esterified by using absolute methyl alcohol and12 mol/l HCI according to the method of Mandelland Hershey.27 The preparations were givenintravenously in a series of 15 injections, one everyfour days. Group 6 received purified cardiolipinincorporated into Freund's complete adjuvant. Eachguinea pig received three intramuscular injections,each containing approximately 300 Ag of cardiolipin,10 days apart. For each group sera were obtained 10days after the last injection and examined forantibody to cardiolipin in the same manner as serafrom infected animals.

DIRECT PLAQUE ASSAYThe ability of T pallidum-infected guinea pigs torespond to sheep red blood cells (SRBC), a

T-dependent antigen, was determined by the directplaque assay of Jerne et aL2829 Thirty-six animalsinfected with 1 x 107 T pallidum were killed ingroups at two-week intervals for 12 weeks. Sevendays before each killing four infected and twonormal control animals were injectedintraperitoneally with 0'5 ml of washed SRBC(1 x 109). Spleens of the killed animals were

aseptically removed and examined by the plaqueassay in quintuplicate. To distinguish between IgGand IgM plaque-forming cells the slides wereincubated for one hour with 0*2 mmol/l2-mercaptoethanol before treatment withcomplement.

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CELL RESPONSE TO MITOGENS AND ANTIGENSplenic lymphocytes of infected and control animalswere used for the standard assay of in-vitrolymphocyte transformation in the presence ofoptimal concentrations of concanavalin (Con A,12-5 pg/culture; Sigma Chemical Corporation, StLouis, MO), phytohaemagglutinin (PHA, 2 5jug/culture; Wellcome Reagents Ltd, Beckenham,England), or a suspension of 3 x 10 to 3 x 106 Tphagedenis biotype Reiter. The test was done intriplicate samples using 2 5 x I0 cells in 0 2 ml andreagents in 10 Ml volume. The samples were incubated(37°C, CO) for three days for mitogens and fivedays for antigen. Six hours before harvest eachculture was pulsed with 0 4 IACi of [3H]TdR,harvested, washed (MASH II) and the incorporationof [3H]TdR determined in liquid scintillation counter(Mark II). Results were expressed as a difference ofcounts per minute (Acpm) obtained by subtractingthe cpm of non-stimulated cultures from the cpm ofstimulated cultures. The stimulation index, was alsocalculated and the results analysed.

HISTOPATHOLOGYInguinal lymph nodes and spleens from infected(5 x 106 organisms) and control guinea pigs wereobtained in the second week of infection and at two-week intervals for 12 weeks. The organs were placedin 10% buffered formalin immediately afternecropsy. Sections were prepared and stained withhaematoxylin and eosin for histology and by theWarthin-Starry method for detection of treponemes.

Results

INFECTIVE DOSEThe number of demonstrable lesions, their severity,and their time of development varied with theinfective dose. Lesions started to appear as early asday 6 after infection in guinea pigs infected with5 x 107 organisms but not until day 42 in animalsinfected with 5 x I04 organisms (table I). Themedian infective dose (ID50) for these outbred guineapigs was approximately I05 Tpallidum.

TABLE I Susceptibility of guinea pigs to infection by Tpallidum (Nichols)

No of animalsInfective Onset ofdose* Infected With lesions % Infected lesions (days)

5 x 107 8 7 87-5 6-145 x 106 8 6 75 6-155 x 105 8 6 75 17-305 x 104 10 2 20 42

*Virulence of infective doses was determined by intradermalinjections into shaved rabbits.



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FIG 1 (a) Darkfield-positive lesion (developing nine days after infection in the pubic region of a Tpallidum-infected(5 x 106) guinea pig) showing formation of centralised necrosis; (b) healing lesion covered with scales decreasing insize.

FIG 2 Tpallidum in a skin lesion of an infected guinea pig. Characteristic treponemes are evident at theepidermodermaljunction. Warthin-Starry stain4x 1000).

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Each lesion first appeared as a barely discernibleerythematous area 2-3 mm in diameter and pro-gressed to a definite induration, similar to a chancrein man, with centralised necrosis (fig 1). Thediameters of the lesions reached a maximum of 1.5cm in animals infected with 5 x 107 or 5 x 106organisms but only 1-0 cm in those infected with5 x 10' organisms; animals infected with 5 x 104organisms showed only erythema and induration of4 mm after six weeks.Exudates from lesions contained motile

treponemes, as shown by dark field microscopy.When tissue sections from the lesions were stained bythe Warthin-Starry method (fig 2) treponemes couldbe seen in all layers of the skin, but the largestconcentration was at the epidermodermal junction.Histologically the lesions were characterised (figs 3and 4) by degeneration and necrosis of the epidermiswith considerable mononuclear cell infiltration. Thedermis showed pronounced histiocytic, lymphocytic,


and polymorphonuclear infiltration, and theinflammatory reaction extended deep into the fattysubcutaneous layer.

HUMORAL IMMUNE RESPONSENo IgM-antitreponemal antibodies were detected,but IgG antibodies started to appear as early as ninedays after infection (two animals). By 12 weeks afterinfection all 15 surviving animals (five had diedduring cardiac bleeding) showed IgG antibodies bythe FTA-ABS test. Sera of control guinea pigs thathad been injected with NRT or T pallidum-freesupernatant showed no antibody activity whenexamined by the FTA-ABS test periodically for threemonths after injection.

Sera from nine animals of the infected group werechosen for titration of treponemal antibody (tableII). There was a slow but steady rise in titre up to oneyear after infection. These titres were, however,much lower than those found in sera of infected

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to T pallidum. in nine

Weeks after infectionGuineapig No 2t 3 6 12 27 52

12 <5 <5 20 40 80 8014 10 40 80 80 80 16015 5 20 20 20 80 8018 <5 5 40 40 80 8019 <5 <5 10 40 20 4021 <5 10 20 20 40 8024 <5 <5 <5 20 80 8027 <5 <5 10 20 40 8028 <5 5 10 40 80 80

Mean <5 8.9 23-2 356 644 84-4

*Reciprocal of the serum dilution giving at least a I + positivereaction in the FTA-ABS test.tBlood sample taken on the ninth day after infection.

rabbits or man. Sera of four infected guinea pigswere absorbed with Tpallidum or NRT. Tpallidumlowered the titres from 160 to 80 or from 80 to 20;NRT had no effect. The decrease of titre afterabsorption with T pallidum may be indicative ofspecificity of the antibodies for Tpallidum.No antibodies to cardiolipin were detected in sera

of infected guinea pigs examined by the VDRL orcomplement fixation tests. Guinea pigs immunisedrepeatedly with various lipid preparations also failedto produce antibodies to cardiolipin detectable bythese tests. Antibodies with specificity for the carrierserum protein (porcine) were shown by double-diffusion gel precipitation in sera of all guinea pigsreceiving the protein.

PRODUCTION OF IgM ANTIBODIES TO SRBCBoth infected and control animals showed widevariations in response to SRBC, but the infectedanimals responded as well as the controls. Reductionof the number of plaques after treatment with2-mercaptoethanol suggested that most of theantibodies causing the complement-mediated plaqueswere IgM.

CELLULAR RESPONSESpleen lymphocytes of both infected and controlanimals responded well to mitogens. Infected group:Con A, Acpm 44 482-66 541; PHA, 41 044-57 145;controls: Con A, Acpm 15 167-107 310; PHA,9397-86 580. No proliferative response of spleniclymphocytes to various concentrations of Tphagedenis biotype Reiter was observed in eithergroup of animals.

HISTOLOGYInfection of guinea pigs with Tpallidum resulted inactivation of the germinal centres in the cortex of the

lymph nodes. Hyperplastic and hypertrophic changesin these centres gradually increased and reached amaximum at about six weeks after infection (fig 5).At eight weeks follicular activity started to decrease,but it was still apparent 18 months after infection.Most of the early changes were in the follicles, whichcontain predominantly B cells; with time the T-cellareas of the cortex and medulla became moreprominent.

Splenic changes were not as conspicuous as thosein lymph nodes (fig 6). Ten weeks after infection thenumber of cells in the periarteriolar lymphatic sheathappeared to have increased. At the time extra-medullary haematopoiesis became evident in the redpulp of the spleen.

Discussion

The guinea pig has been a neglected model inexperimental syphilis. The rabbit, whosesusceptibility and humoral immune response aresimilar to those in early syphilis in man, overshadowsthe guinea pig as a model. The lack of concordantresults on the guinea pig's susceptibility to Tpallidum infection and on its immunopathologicalmechanism also makes the model unattractive. Theguinea pig offers some advantages over the rabbit,however, especially the availability of inbred andgenetically defined strains, which may becontributory to the exploration of immunity in a Tpallidum-infected host.The susceptibility of the Hartley strain of guinea

pigs is far less than that of the rabbit, but our resultsindicate that the percentage of symptomaticinfections may be dose-related especially in view ofrecent experiments (K Wicher and V Wicher,unpublished observations) in which 1000o ofsymptomatic infection was obtained with 108/ml of Tpallidum. The animals' susceptibility may, however,depend on their age and sex.9 Young animals aremore susceptible than older ones, and males seemmore susceptible than females. Adler et aP' observedthat baby guinea pigs were susceptible to infectionwith Leptospira interrogans serovar pomona butrapidly became resistant as they matured.

Manipulation of the guinea pig immune system byinjecting cortisone will not provide results similar tothose observed in T pallidum-infected rabbits.Guinea pigs infected intradermally with T pallidumbut given cortisone (7 - 5 mg/kg body weight) a weekbefore infection and throughout the course ofinfection responded with a prolonged incubationtime, a shorter duration, and less pronounced lesionsthan animals not receiving cortisone.'6The reasons for the difference in susceptibility to T

pallidum infection between the guinea pig and the

TABLE II IgG antibody titres*infected guinea pigs

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164 Carol S Pierce, Konrad Wicher, and Shaheen Nakeeb

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FIG 5 Histopathology of the lymph nodes of an infected guinea pig; (a) normal and (b) six weeks after infection(x 40).



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rabbit is unknown. Several factors may play a role.Klein et al,3' using various inbred strains of mice,produced symptomatic lesions regularly only in somestrains, indicating dependence on host susceptibility.The genetically controlled resistance could berestored in irradiated mice by transfer of syngeneicspleen cells, suggesting that a radiosensitivelymphoid cell is involved in the outcome of theinfection.

Differences in the susceptibility of guinea pigstrains to infection with Mycobacterium tuberculosiswere shown by Wright and Lewis.32 They examinedthe genetic factors and concluded that there might besome inherent resistance in guinea pigs to infectionwith this organism.The lower susceptibility of guinea pigs to T

pallidum infection may also be due to the animals'extremely vigorous reaction to rabbit proteins (KWicher, unpublished observations). The Tpallidumused for infection is extracted from orchitic rabbittissue and is covered with a variety of rabbit serumproteins, making it a good target for the guinea pigimmune system. Most of the organisms may beeliminated before they can coat themselves withguinea pig proteins. Thus a guinea pig-adapted strainof Tpallidum may work in guinea pigs as well as theNichols strain does in rabbits.Thus far very little is known about the humoral

response of Tpallidum infected guinea pigs. Positivecomplement fixation results have been observed'8 inthe sera of a few animals infected intravenously,intratesticularly, and intradermally; the positivereactions appeared during the fifth month ofinfection and disappeared after one to three months.Whether this reaction was due to cardiolipinantibodies was not explored, and the results,especially in view of more recent'9 and the presentstudies, must be considered unexplained. In theearlier work14l17 the animals were infected with Tpallidum Nichols from the Fournier Institute. Thatstrain seems to be more virulent than the Nicholsstrain from the CDC, as it also produced much moresevere lesions in rabbits.33 Furthermore, the animalsused in the earlier experiments were raised and keptin a moist colder environment and were fed grass andhay.

In more recent studies'9 conducted with animalsobtained from one source, infected with TpallidumNichols from the CDC, and kept in more controlledconditions, no cardiolipin antibodies were detectedby the complement fixation or VDRL tests for anobservation period of six months after infection with2 x 107 T pallidum. We have now confirmed thelack of cardiolipin antibodies: sera of animalsinfected for a whole year did not show positivecomplement fixation or VDRL iesults.


In human and rabbit syphilis the origin of thecardiolipin that elicits an anticardiolipin response isunknown, but cardiolipin may be either host-derivedfrom tissue destruction-34 3 or present in the Tpallidum itself.36 The reason for the lack ofcardiolipin antibodies in guinea pigs is unknown.Possibly not enough tissue destruction takes placeduring the infection. The guinea pig is not, however,a good responder to lipids, as shown earlier37 andconfirmed by our experiments.

In earlier experiments'8 treponemal antibodieswere detected by the TPI test in guinea pigs infectedintravenously, intratesticularly, or intradermally.The antibodies started to appear around the thirdmonth of infection, but in the animals infectedintravenously and intratesticularly they disapppearedaround the eighth month. Such a pattern of antibodyresponse is characteristic when heat-killed Tpallidumare injected into rabbits or mice. The TPI antibodiesin intradermally infected guinea pigs persistedthroughout the studies, which lasted for over 12months. In other experiments'9 (not designed forkinetics of antibody response) treponemal antibodies(FTA-ABS) were present from the fourth week (serawere not available earlier) until the termination of theexperiments at 24 weeks. In present experiments onthe kinetics of antibody response, we have estab-lished that the treponemal antibodies (FTA-ABS)started to appear as early as nine days after infectionand that the titres slowly but steadily increasedthroughout the 12 months of observation (table II).The activation of the germinal centres in the cortex ofthe lymph nodes and the persistent changes observedfor over 18 months support the findings of prolongedantibody production. The titres, however, remainedrather low in comparison to the rabbit or man. Inmore recent experiments using an inbred guinea pigstrain infected with a higher dose of Tpallidum, thetitres in some animals exceeded 1 000 (K Wicher andV Wicher, unpublished data), indicating that thetreponemal antibody titres may be dose-dependent,genetically determined, or both.The slow but continuous increase in antibody titres

in the present experiments suggests the possibility ofa constant source of treponemal antigen. This issupported by the work of earlier investigators,"I whodemonstrated infective treponemes in infected guineapig organs for almost one year.The production of IgG treponemal antibody,

detectable by the FTA-ABS test seemingly withoutprevious production of IgM antibody, did not followthe normal immunological response. Since relativelyfew IgG antibodies were produced, as evidenced bythe low FTA-ABS titres, possibly a very little IgMwas also produced, but the FTA-ABS technique wasnot sensitive enough to detect it. Also, the initial time



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of sampling (nine days) may have been too late todetect IgM, even though some animals did not yethave visible lesions and most had no detectable IgGantibodies at that time. Watanabe et aP8 dem-onstrated the presence of a genetically controlledsubclass of T cells which selectively suppresses the Bcells responsible for the production of IgEantibodies. In the guinea pig a similar situation maypossibly occur which selectively suppresses theproduction of IgM antibodies during T palliduminfection. Such a suppression would have to beantigen-specific, since the infected guinea pig is ableto produce IgM antibody to another T-dependentantigen, SRBC; infected and control animals alikeproduced antibodies to SRBC. Baughn and Musher39showed that the number of IgM plaque-formingantibodies to SRBC in T pallidum-infected rabbitswas significantly increased over controls for up toseven weeks after infection. Such enhancement of theIgM response to SRBC was not detected in infectedguinea pigs.The in-vitro cellular response was not well

explored. Because guinea pig peripheral blood is notthe best source of active lymphocytes, we usedsplenic lymphocytes. In the past Reiter antigen hasbeen used successfully to examine the cellularresponse in infected rabbits.40 41 The lymphocytes ofinfected guinea pigs responded to mitogens similarlyto cells from non-infected control animals but didnot respond to Reiter antigen. In recent experimentsusing nylon-wool-purified peritoneal T lymphocytesfrom Tpallidum-infected inbred guinea pigs the lackof reaction to Reiter antigen was confirmed; the Tlymphocytes did react to T pallidum antigen (KWicher and V Wicher, unpublished observations).Evidently the immune response in T pallidum-infected guinea pigs is more specific than in therabbit or man, which do respond to Reiter antigen.The changes in the T-cell areas of the cortex andmedulla in the outbred guinea pigs support theassumption that lymphocytes are activated by theinfective agent.

Thus, the guinea pig is less susceptible to Tpallidum infection than the rabbit and, wheninfected with a sufficient number of T pallidug,responds with darkfield-positive lesions, productionof treponemal antibody, and histopathologicalchanges in the lymphoid organs. The immuneresponse in the guinea pig may be more specific thanin the rabbit or man, since no cardiolipin or cross-reactive antibodies or cellular response to Reiterantigen was detected.

We thank Carolyn Kalinka for her assistance.

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Experimental syphilis: guinea pig model