INFECTION AND IMMUNITY, Nov. 1993, p. 4743-47490019-9567/93/114743-07$02.00/0Copyright © 1993, American Society for Microbiology

Vol. 61, No. 11

Pathology of Congenital Syphilis in RabbitsM. KENT FROBERG,lt* THOMAS J. FITZGERALD,1 THOMAS R. HAMILTON,2

BETTE HAMILTON 2 AND MEHDI ZARABJI2Department ofMedical Microbiology and Immunology, School ofMedicine, University of

Minnesota, Duluth, Minnesota 55812,1 and Department ofPathology, SchoolofMedicine, University ofMissoun, Kansas City, Missouri 641082

Received 6 March 1992/Returned for modification 7 April 1992/Accepted 2 August 1993

We have developed a model for congenital syphilis in the rabbit. This report provides additional informationon newborn tissue pathology in animals that were infected in utero. A total of 35 pregnancies were evaluated,each containing 6 to 12 newborns. In the infected group, the mortality was approximately 50o%; of the livenewborns, half appeared normal and half were hyperreflexic, weak, and runty. Gross pathology in the sicklynewborns was quite prevalent and involved enlarged spleens with isolated spots of necrosis; enlarged livers thatwere overtly congested and hemorrhagic and had numerous granular, white spots; and brains with hemorrhagein the occipital area. Histopathology was apparent in different tissues. Lymphocytes, plasma cells, andvacuolated macrophages were prominent in livers, spleens, brains, and bones. A few actively motiletreponemes were visualized by dark-field microscopy within extracts of spleen and within cerebrospinal fluid.Low numbers of treponemes were also demonstrated in sections of brain and liver by using the Warthin-Starrysilver stain technique. Blood hematocrits were decreased, and extrameduliary hematopoiesis was prominentwithin spleens and livers; this is consistent with anemia. This rabbit model exhibits many ofthe same pathologicfeatures commonly found in human congenital syphilis.

Penicillin was initially used to treat syphilis in the late1940s. This drastically reduced the incidence of both adultand congenital syphilis. After years of decline, adult infec-tion has recently begun to rise. It has now reached a levelthat represents a 41-year high, equal to that of the prepeni-cillin era. Alarmingly, in the past 4 years the incidence ofcongenital syphilis has increased more than threefold. Fur-thermore, the case definition for this disease was just sim-plified, and this will add to the overall incidence (34).

Syphilitic infection of the fetus during pregnancy producesdevastating consequences with three separate clinicalcourses. In one, either the fetus dies in utero or the newborndies shortly after birth. In another, clinical manifestationsare apparent at birth or shortly thereafter. These includemucous membrane lesions, long-bone deformation, dactyli-tis, anemia, enlarged spleens and liver, central nervoussystem defects, and incomplete palate development. Finally,in the third course the child appears healthy at birth withoutovert clinical manifestations. Two to ten years later, syphi-litic infection progresses, producing late manifestations ofinterstitial keratitis and blindness, malformed teeth andbones, eighth-nerve deafness, neurologic defects, hydroar-throsis, and cardiovascular lesions.We have developed an animal model for congenital syph-

ilis that exhibits the clinical courses seen in human infection(8). In a more severe course, the rate of stillbirths ornewborn deaths rises above 50% and birth weights dramat-ically decrease. In a less severe course, newborns appearnormal at birth and grow and develop at expected rateswithout overt clinical manifestations of syphilis. These new-borns, however, have activated immunologic defenses at 2and 5 weeks of age, indicating low-grade infection (9).The pathology of human congenital syphilis has been well

* Corresponding author.t Present address: Department of Laboratory Medicine, Geis-

inger Medical Center, Danville, PA 17822.

documented. To further solidify our rabbit model, it wasnecessary to characterize newborn rabbit pathology in ourmodel and to relate it to that of human infection. Accord-ingly, adult rabbits were infected during pregnancy andthose newborns that either died or were quite sickly wereexamined for gross pathology and histopathology. Survivingexposed newborns were also examined, but as expected, thelesions were of lesser frequency and severity.

MATERIALS AND METHODS

The Nichols strain of Treponemapalidum was maintainedby injection of 2 x 107 organisms per testis in adult NewZealand White rabbits. Organisms were extracted fromtestes by the procedure outlined by Fitzgerald (8). Toproduce congenital infection, treponemes were adjusted to108/ml and 2.5 ml was injected into the marginal ear vein ofpregnant does at days 3, 10, 17, and 24 of gestation.Pregnancy averages 29 to 31 days. Control does were notinjected. These observations were based on evaluations of25 pregnancies from infected does and 10 pregnancies fromcontrol does. Each pregnancy produced 6 to 12 newborns.At birth, each neonate was weighed and its appearance

was recorded as healthy, sickly (cold, lethargic, or cyanot-ic), or dead. Stillborn neonates or neonates that died within1 to 3 days after birth were examined grossly at necropsy.Also, surviving neonates were sacrificed at 1 to 2 weeks ofage and necropsied, and tissues were sectioned for exami-nation. Tissue samples preserved in 6% formalin were sec-tioned and stained with hematoxylin and eosin (H and E).Some sections were stained with Warthin-Starry silver stainfor identification of spirochetes in host tissues. Brain, liver,and spleen tissues were finely minced and extracted for 15min. Tissue debris was sedimented by low-speed centrifuga-tion. Supernatants were examined microscopically for T.pallidum. Samples of cerebrospinal fluid (CSF) were alsoexamined microscopically.

Treponeme-specific immunoglobulin M (IgM) was quanti-

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TABLE 1. Influence of in utero infection on hematocrits and IgMsynthesis at 2 weeks of age

Hematocrit (%) Treponeme-specific IgMAvg + SEMI Range (cpm + SEMb)

Control 43 ± 1 36-51 458 ± 212Infected in utero 32 + 1 26-39 2,885 ± 322

a For 10 newborn controls and 17 newborns infected in utero.b For five newborns in both groups. Background control levels represent

nonspecific binding.

tated by radioimmunoassay. T. pallidum was adjusted to 5 x107 organisms per ml, and 20 pl was added per well. Afterdrying, wells were fixed in 10% ethanol and dried overnight.To reduce nonspecific binding, 100 ,ul of 5% bovine serumalbumin was added and the mixture was incubated for 30min. Wells were washed three times with phosphate-buff-ered saline (PBS). Sera were isolated from five newborncontrols and five sickly newborns infected in utero. Sincethis was difficult to do on neonates without sacrificing them,they were not included in this assay. Each sample wasdiluted 1/100, and 100 pl was added to triplicate wells. After1 h at 37°C, wells were washed with PBS. Goat anti-rabbitIgM (Cappel Laboratories, West Chester, Pa.) was diluted1/10,000 (optimal concentration), and 100 ,ul was added perwell. After 1 h at 37°C, wells were washed with PBS. Avolume of 100 ,ll of 1"I-protein A (New England Nuclear,Boston, Mass.) containing 100,000 cpm was then added, andthe mixture was incubated for 1 h at 37°C. Wells werewashed with PBS to remove unbound radioactivity. Driedwells were counted with a Beckman gamma counter. Back-ground levels obtained from wells not incubated with thegoat anti-rabbit IgM were subtracted from those obtained fortest wells;

Statistical analysis. Data for Table 1 were analyzed byusing the Student t test. Significance was based on a P valueof less than 0.05.

RESULTSMorbidity and mortality. Control groups of newborns from

does not infected during pregnancy had a mortality rate of

A

21% (11 of 53). Live newborns were healthy and grewnormally. Forty of these were sacrificed and autopsied; noovert internal pathology was evident. In contrast, the mor-tality rate in newborns from does infected during pregnancyincreased to 53% (35 of 66). Approximately half of the livenewborns were weak, unthrifty, slow to gain weight, andhyperreflexic and had ruffled coats, while 15% of infectednewborns were stillborn. These unhealthy-appearing new-borns either died within 1 to 3 days or were sacrificed thesecond week. Some exhibited serosal hemorrhages espe-cially prominent in the peritoneum, frank blood in theperitoneal cavity, and frank cyanosis. The other half ofcongenitally exposed newborns appeared healthy and grewnormally over 12 weeks of observation. No gross pathologywas apparent in these newborns when autopsied at 2, 4, or 12weeks of age. This sharp difference between overtly sick andhealthy newborns occurred within litters (17).

Liver. The most dramatic tissue pathology occurred inlivers and was seen in approximately 75% of stillborn andunhealthy-appearing congenitally infected newborns. Theselesions were almost always grossly discernible on the liversurface. The larger (1 to 3 mm) was slightly raised with a firmgritty texture and was yellowish-white. It extended downinto the parenchyma of the liver. Figure 1 shows liver tissuesfrom a control (panel A) and an infected newborn (panel B).There were some smaller areas (1 mm), which were lessnumerous, consisting of focal necrosis and surface erosion.

Infected sections were prepared and stained withH and E.Granulomas or granuloma-like areas were apparent. Tissuedamage involved discrete and necrotizing areas with dystro-phic calcification (Fig. 2A). These areas contained mono-cytic and multinuclear giant-cell infiltrates, basophilic cal-cium spicules, and abundant macrophages with ingestedcalcium spicules. Persistent extramedullary hematopoiesiswas prominent in adjacent fields (arrows). Other areas ofinflammation contained lymphocytes, plasma cells, epithe-lioid macrophages, and giant cells (Fig. 2B). Cords wereproliferating and in disarray, and there was evidence offibrosis, biliary hyperplasia, and perivascular cuffing withdisarray of endothelial cells. Warthin-Starry (silver) stainsrevealed infrequent spiral-shaped treponemes interspersedthroughout the liver sections.

B

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FIG. 1. Gross liver pathology in newborn rabbits. (A) Control rabbit; (B) infected rabbit with numerous light-colored lesions.

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K;aAv'._FIG. 2. H and E stains of liver sections from infected newborns. (A) Necrosis with dystrophic calcification and persistent extramedullar

hematopoiesis (arrow); (B) inflamed areas containing lymphocytes, plasma cells, macrophages, and giant cells.

Spleen. Splenomegaly was prominent in the 2-week-oldnewborns. Rare surface lesions with focal necrosis weredetected. These were small (1 mm) and eroded. H and Estains revealed numerous areas of extramedullary hemato-poiesis (Fig. 3) and some areas of probable T-cell hyperpla-sia. Perivascular infiltration was infrequently observed; itwas composed of lymphocytes, plasma cells, macrophages,and occasional giant cells. Fibroblasts were smaller withshadowy degenerated elements. Ten freshly harvestedspleens were extracted for 10 min; a few actively motiletreponemes were observed by dark-field microscopy in twospleens.

Brain. The calvaria was retracted to expose the brain.Figure 4 shows the control and infected brains. Gross signsof infection included petechial and ecchymotic hemorrhageson the dura (arrow). H and E stains revealed microscopicfocal necrosis on the cerebrum containing lymphocytes andvacuolated macrophages. Figure 5A shows meninges that

are thickened and edematous. This section also shows ameningeal vessel adjacent to the cerebrum. Deeper in thebrain tissue, Virchow-Robins spaces exhibited microscopichemorrhages. Figure 5B shows three such spaces; one isintact, while the other two have numerous external erythro-cytes (microencephalomalacia). Brain sections stained bythe Warthin-Starry technique revealed very low numbers ofwidely scattered treponemes only in damaged areas. Inaddition, CSF samples were taken from 10 infected new-borns and were examined by dark-field microscopy. Onesample of CSF contained an actively motile treponeme.Mesenchymal tissues. Gross pathology of the bone was

rarely encountered. Some suspicious areas of the rib weresectioned and stained with H and E. Microscopic focalnecrosis was apparent, and lymphocytes and plasma cellswere interspersed within the muscle and connective tissueadjacent to the bone (Fig. 6A). Large numbers of plasmacells and monocytes were adjacent to small blood vessels

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FIG. 3. H and E stains of spleen sections showing numerous areas of extramedullary hematopoiesis.

with possible vasculitis (Fig. 6B). Total-body X rays takenvarious times postbirth were unremarkable, and no bonedeformities were apparent up to 6 weeks of age.Other tissues. Kidneys in a very few newborns were

grossly hemorrhagic. No gross pathology or histopathologywas observed in lung sections. The pancreas appearednormal except for grossly thickened membranes adjacent tothe spleen. These areas contained vascularized connective

tissue with swollen endothelial cells. Minimal fibrosis wasapparent within the pancreas. Acini and ductules wereintact.

Blood. Hematocrits were significantly decreased in theinfected newborns aged 2 weeks (Table 1). In addition, IgMspecifically reactive with T. pallidum was elevated fourfold.Other observations. Each infected newborn was closely

examined prior to sacrifice at 1 to 2 weeks of age, or in some

FIG. 4. Gross pathology of brain tissues in newborns. Shown are a control and an infected newborn. The arrow indicates a hemorrhagicarea on the dura.

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FIG. 5. Histopathology in brain sections as revealed by H and E stains. (A) This inflamed area of the cerebrum exhibits focal necrosiscontaining lymphocytes and vacuolated macrophages. Meningeal areas were also thickened and edematous. (B) Deeper in the brain tissue,Virchow-Robins spaces contained released erythrocytes; of the three spaces shown, one appears normal while the other two exhibithemorrhage.

cases up to 12 weeks of age. We did not detect any of thefollowing: overt oozing mucous membrane lesions, skinlesions, eye infections, blindness, or deformed limbs.

DISCUSSION

There are a number of important parallels between ourmodel and human congenital syphilis. Variation in pathologywas the norm. Some newborns were obviously sick, whereasothers from the same litter appeared healthy and grewnormally. This reflects human studies of nonidentical twinsborn to syphilitic mothers, in which one twin was infectedand the other was not (6). Rabbits within individual littersalso varied in the extent of tissue damage. Some exhibitedliver damage; others exhibited liver, spleen, and brain dam-age; still others exhibited liver and brain damage.Human congenital syphilis is known for its wide clinical

diversity and like its adult counterpart can be described asthe great imitator (3-5, 10, 14, 18, 20, 22-25, 29-31, 33).Death of newborns, when it occurs, has been attributed tosepsis, hepatitis, and hemorrhage (11, 20). In most cases thecause of death is listed only as complications of infection (11,16, 19). In our model, clinical manifestations were evident inmany tissues. In those animals that died shortly after birth,we were unable to determine the cause of death. The grosspathology and histopathology in livers, spleens, brains, the

pancreas, and mesenchymal tissues were almost identical tothose found in human infections. Hepatomegaly (10, 11, 13,16, 19, 20, 23, 28-30, 32) and hemorrhage were the mostcommon gross findings. Splenomegaly (10, 11, 13, 16, 19, 20,23, 28-30, 32) was encountered less frequently. As in humaninfection, lymphocytes, plasma cells, and macrophages werefrequently evident within infected tissues. Cuffing of bloodvessels and endothelial damage were observed. Persistentextramedullary hematopoiesis was a consistent finding inmost of the livers (10, 11, 20, 28-30) and many of the spleens(20, 29, 30). Coupled with decreased hematocrits, anemia, acommon manifestation of human congenital syphilis (10, 13,20, 23, 29, 30), is suggested.

In our earlier report (8) we sacrificed 4-week-old fetuses ornewborns 1 week postbirth. Whole fetal tissues or newbornspleens were removed and extracted in medium. The ex-tracts were then injected dermally into adult rabbits. Lesionsdeveloped, indicating the presence of virulent treponemeswithin fetal or newborn tissues. Importantly, this demon-strated that treponemes were in fact able to cross thematernal-fetal barriers during pregnancy. The findings in thispaper confirm this earlier observation. Spleens from infectedneonates were extracted and examined by dark-field micros-copy. Low numbers of actively motile spirochetes werepresent. Ten specimens of CSF were examined only forspirochetes; one sample contained an actively motile trepo-

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*~~ ~ ~ ~ ~ ~~.. 1 S %;ts7' 7:.iX

FIG. 6. H and E stains of bone tissue from infected newborns. (A) Perivascular plasma cells and lymphocytes within intercostal muscleadjacent to the bone; (B) higher magnification containing plasma cells and monocytes adjacent to a blood vessel (cuffing).

neme. Although this was only one sample, it indicates that T.pallidum can cross the blood-brain barrier of the fetus.Alternatively, treponemes, like the human immunodefi-ciency virus, could enter the brain ingested in macrophages(1). Spiral-shaped treponemes were also observed by War-thin-Starry staining of brain tissues. Organisms were foundonly in those areas associated with necrosis, lymphocytes,and vacuolated macrophages. This observation has ramifi-cations as a potential model for neurosyphilis. Recently,Marra et al. (15) published the first report of tertiary-likebrain manifestations in rabbits. T. pallidum was injecteddirectly into the spinal fluid of adult rabbits, and neurosyph-ilitic manifestations of uveitis developed 9 weeks later.

Spiral organisms were also found in newborn liver sec-tions stained by the Warthin-Starry technique. The observa-tions of treponemes in CSF and spleen, brain, and livertissues involved very small numbers of organisms. Thisparallels human congenital syphilis. With the exception ofmucous membrane lesions, it is frequently very difficult tovisualize T. pallidum within infected tissues, and whenvisualization is successful, only low numbers are detected(11, 12, 20, 21, 28).The occurrence of lymphocytes, plasma cells, and macro-

phages within different tissues points to immune stimulation1 to 2 weeks postbirth. In agreement, serum IgM reactivewith T. pallidum antigens was also elevated, indicatingnewborn antibody synthesis. In related research we hadreported that congenitally infected newborns exhibited in-creased T-cell proliferation, increased macrophage Ia, andincreased rapid plasma reagent serum antibody (9). Thus, inutero exposure to T. pallidum produces activation of differ-ent immune functions.

In summary, we have now firmly established our rabbitmodel for congenital syphilis. It duplicates many of thoseclinical manifestations found in human congenital infection.Future studies will focus on the immunologic consequencesof congenital exposure to treponemes. We are especially

interested in determining those mechanisms responsible fortissue damage. T. pallidum lacks potent toxins (7), and it hasbeen proposed that syphilitic damage in newborns is afunction of immunologic reactivity (26, 27). Some recentevidence (2) implicates circulating immune complexes andrheumatoid factor, both of which could contribute to patho-logic damage. Our model and observations of human con-genital syphilis support this concept. There are fewtreponemes but many immune cells in areas of damagedtissues. We have recently demonstrated that challenge in-fection of newborns at age 2 or 5 weeks produced moreintense lesions of longer duration in those neonates infectedin utero (9). This further supports the concept of immune-mediated tissue damage.

ACKNOWLEDGMENTSThis research was supported by a grant from the National

Institute of Allergy and Infectious Diseases (AI 18619).We gratefully acknowledge the expert technical assistance of

Mathew Oswald. We also thank Eileen Nelson, Department ofPathology, University of North Dakota, for doing the Warthin-Starry silver stains on fixed tissues.

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