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1987

Investigation of the neutralizing activity for Treponema Pallidum Investigation of the neutralizing activity for Treponema Pallidum

of neonatal rabbit basal serum taken at 2, 3, and 4 weeks of age of neonatal rabbit basal serum taken at 2, 3, and 4 weeks of age

Helen Ceclie Mercier

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INVESTIGATION OF THE NEUTRALIZING ACTIVITY FOR

TREPQNEMA PALLIDUM OF NEONATAL RABBIT BASAL SERUM

TAKEN AT 2, 3, AND 4 WEEKS OF AGE

A Thesis

Presented to the

Faculty of

California State University

San Bernardino

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

in

Biology

by o

Helen Cecile Mercier

August 1987

INVESTIGATION OF THE NEUTRALIZING ACTIVITY FOR

TRRPQNEMA PALLIDUM OF NEONATAL RABBIT BASAL SERUM

TAKEN AT 2, 3, AND 4 WEEKS OF AGE

A Thesis

Presented to the

Faculty of

Callfornla State Un1vers1ty

San Bernardino

Helen Cecile Mercier

August 1987

Approved by:

son. Ph. D.. Biology DateRuth C. Wilson, Ph. D., BioVogyGraduate Program Coord inator

Dar1ene Gamboa, Ph. D.

Alexander Sokoloff, Ph. D.,/W[ology

Charles McCammon, M.D.^ Pathology

ABSTRACT

Evidence of neonatal rabbit resistance to symptomatic

infection with TreDonemapallidum. the etiological agent

of human syphilis, at one week of age, and its decline as

the animal approaches five weeks of age has been demon'"

strated. The present study was designed to examine the

possible influence of a neutralizing factorCs) on neonatal

resistance by determining the neutralizing activity of basal

sera from 18 neonatal rabbits 2, 3, and 4 weeks of age^

Three experimental runs, with two sera each from rabbits 2,

3, and 4 weeks of age were performed. A total of 15 adult

New Zealand white rabbits were inoculated with suspensions ■ .. ■ 3' . ■

containing a final inoculum of 1x10 T. pallidum (Nichols

strain) per site. Positive and negative controls were run in

parallel for a total of 18 inoculation sites per adult rab

bit. Results of the Study demonstrated an absence of detect

able neutralizing activity for X* oal1idum in the sera of 2,

3, and 4 week old rabbits. Serum neutralizing activity may

not necessarily contribute to the resistance demonstrated by

neonatal rabbits. The definitive mechanism(s) of natural

resistance of nepnates to syphlTitic infection has yet to be

defined.

i ii

ACKNOWLEDGEMENtS

I am pleased to have the opportunity to thank the very

special people who have assisted me with this research

project.

Dr. Darlene Gamboa, my major research professor, has

been a motivating force throughout, providing the guidance I

needed when learning new syphilis research techniques, while

at the same time, leaving me "on my own" so that I could de

velop a sense of independence and self-confidence ih the

area of experimental animal research.

Dr. Ruth Wilson, my academic advisor and good friend,

has been a constant source of encouragement, as well as a

totally honest critic. Her delving questions into my re

search methods, results, and conclusions, have provided

invaluable insights into the skills needed to write a paper

that is not only informative, but understandable, as well.

Dr. Wilson took the photographs used in this thesis manu

script, in addition to others used as data vouchers.

A special thank-you to Dr. Alexander Sokoloff for his

patient and very careful reading of the manuscript, as well

as for the academic excellence that he constantly demon

strated as a professor at CSUSB.

Dr. Charles McCammom has been a constant source of

inspiration and praise during the seven plus years that I

iv

have worked with him in the clinical laboratory. As a former

syphilologist and member of my thesis committee, he has

provided valued insights into the acquired form of the human

disease.

Lastly, I would like to thank my mother and father,

Rita and Lucien Mercier, for having instilled in me a sense

of pride in a job well done, and a dedication to persevere

in spite of difficulties. They have been the best and most

important teachers of my life.

TABLE OF CONTENTS

Page

List of Tables vii

Rabbits ^

List of Figures viii

Introduction 1

Materials and Methods 8

Treponema pal1idum 8

Control and Test Sera 9

Micro-neutralization Assay 10

Inoculations 10

Statistical Analysis 11

Results 19

Discussion 31

Literature Cited 39

vi

LIST OF TABLES

Table I. Neutralization activity of neonatal basal sera from 2, 3, and 4 week old

' rabbits''.'i .V'.;. .... ^ : .:2-2

Table 2. Comparison of basal seta neutralizing activity and Treponema P^lU^Vtff lesion development among neonates 2, 3, and 4

.>e'ekS;;;of"age^. ..> l. .... .■.';,: . '>"^ . . '-;:24;

-v ;.

V IX

LIST OF FIGURES

Page

Figs. 1-2. Extractloa of Treponema palIidum from rabbit testes..... 12

Figs.

Fig .

3-6. Dilution procedures for the micro-NZ assay

o

7. Anaerobic atmosphere jar, 34 C incubator, and syringe labels

13

14

Figs. 8-9. Rabbit back characteristics 15

Fig . 10. Inoculation pattern for test rabbits 16

Figs. 11-12. Inoculation procedure 17

Fig . 13. Example of measurement of lesion diameter and inspection of lesion development 18

Figs. 14-24. Representative examples of post-inoculation lesion development on rabbit backs . 25

Figs. 14-15. Day 15 26

Figs. 16-17. Day 22 27

Figs. 18-19. Day 32 28

Figs. 20-21. Day 43 29

Figs. 22-24. Day 48 30

V 1 1 1

INTRODUCTION

Scholars and medical historians have debated the

mysterious origin of syphilis for nearly 500 years. Although

this academic dispute over whether syphilis originated in

the New World (Columbian Theory) or had been present in the

Old World from time immemorial (Pre-Columbian Theory)

continues, neither theory is entirely satisfactory. Whatever

its origin, a great pandemic of syphilis occurred in all

parts of Europe between 1493 and 1494, and by 1497 appeared

even in the remote areas of Scotland (PHS 1968). At this

time syphilis was a very acute disease, frequently fatal in

the secondary stage. Physicians throughout Europe recognized

it as a new and previously unknown condition and were

reporting and diagnosing its Symptoms as early as 1500.

Fortunately, the extremely acute, severe form of syphilis

quickly lessened to the more chronic form of today.

The causative organism of syphilis is a member of the

order Spirochaetales of the family Treponemataceae. The many

species of this order are widely distributed in nature with

the overwhelming majority being free-living and saprophytic.

Species have been described from a diversity of ecological

habitats including soil, water, and the alimentary tracts of

insects and amphibians. The spirochetes are generally

defined as actively motile by means of a twisting corkscrew

-1ike rotation.

The family Treponemataceae contains three genera,

Borrelia. Treponema. and Leptospira. Within all three

genera there are species parasitic or pathogenic for man,

other mammals and/or birds. The genus Treponema contains

four principal species of pathogenic organisms. These in

clude T. pallidum. responsifale for human syphilis, T.

pertenue, the etioilogic agent of yaws, T. carateum. respon­

sible for pinta. and T. paraluis subsp. cuniculi. the causa­

tive organism of rabbit syphilis.

Although the individuals of all Treponema species are

morphologically and serologically similar, the pathogenic

forms can be distinguished ahtigenically from the non­

pathogeniC cultivatable strains of Treponema (PHS 1968).

The suggested evolutionary history of the pathogenic species

of the Treponema is a matter of speculation. Most authors

believe that the pathologic species developed from free

-living, non-pathogenic forms, and later adapted to their

human or animal hosts. Although most suggest that all

pathogenic Treponema were derived from a single source,

speculators differ on how far back in the evolutionary scale

thfi differentiation between the species occurred. Many

contend that the Treponema pathogenic for humans are the

same species modified only by various factors in the

environment and the host <PHS 1968).

In 1905 two German scientists, Fritz Shaudinn and Eric

HoffmanV d1seevered Treponema pal1Idum (Splrochaeta

pallIda) in the primary sores of persons infected with

syphilis. Treoonema pallidum is a thin, delicate, spiral

bacterium with 6 to 14 tight-body coils and is motile by

means of endoflagella. It ranges in size from 6 to 15

microns in length, and has a uniform cylindrical thickness

of about 0.25 microns. (Wistreich and Lechtman 1984).

The clinical manifestations of acquired syphilis are

divided into three stages: primary, secondary, and tertiary.

During the primary stage> a chancre develops within two to

six weeks at the site of Treponema contact from which

the treponemes quickly invade the blood stream and lymph

atics, and are distributed throughout the body. Six to eight

weeks after the appearance Of the primary chancre, the sec

ondary stage is characterized by the development of cutan

eous and mucous membrane lesions. Additional symptoms in

clude headache, fever, and generalized lymphadenopathy. A

latent period follows which marks the end of the infectious

period of syphilis. Although serological tests are positive

during this latent phase, clinical symptoms of the disease

are absent. The tertiary stage (one third of the cases go on

to this) may take five to twenty years to appear. During

tertiary syphilis, T. pallidum can invade and damage any

organ of the body, e.g., gummas (lesions that may appear on

any part of the body); and aneurysms of the aorta. Person­

ality disorders and/ or paralysis may also occur due to

invasion of the central nervous system.

Acquired syphilis has been well documented and de­

scrifaed, but additional attention needs to be focused on the

unborn victim, the conceptus. Congenital syphilis begins

when T. pallidum Crosses the placenta and infects the

fetus. Although this Infection was believed to occur only

after the 18th week of gestation, when atrophy of the

Langhan's eel1 layer (cytotrophoblast) of the placenta takes

place (BroWn and Moore 1963, Peterson 1973, Sokol and Aroujo

1973), recent evidence indicates that treponemes can invade

fetal tissue as early as the first trimester (Harter and

Behirschke 1976, Grossman 1977). Stillbirth is likely if

pregnancy occurs during the primary and secondary stages of

syphilis. When pregnancy occurs during the tertiary stage,

infected newborns may exhibit a variety of clinlcal manifes

tations ranging from asymptomatic infection, cold Or flu

-like symptoms, to fatal disease. On the other hand, the

newborn may be totally unaffected (Stokes et al. 1944a,

Crissey and Denenholz 1984).

Congenital syphilis is divided into two principal

stages, early and late. This terminology refers to the time

in the child's life when symptoms appear. The effects of

early syphi1is, analogous to acquired secondary syphilis,

appear before the age of two years and include skin and

mucous membrane lesions/ hemolytic anemia, hepatospleno­

megaly, and involvement of the skeletal and central nerv

ous systems (Wistreich and Lechtman 1984). Over 50% of

these infants have 'snuffles', a thick white or blood-tinged

nasal discharge teeming with treponemes (Woody et al. 1963,

Grossman 1977, Crissey and Denenholz 1984).

The clinical disease of late congenital syphilis is

comparable to the tertiary manifestations of the acquired

form. It becomes evident after the age of two years, and in

many cases not until puberty. Pathognomic manifestations of

congenital syphilis include interstitial keratitis, eighth

nerve deafness, and dental deformities (Hutchinson's triad).

Suggestive manifestations include bone destruction (sabre

shins, saddle nose, frontal bossing, perforation of the

palate, Clutton's joints), cutaneous lesions (rhagades,

gummas), and, rarely, neurologic and cardiovascular involve

ment (Grossman 1977).

The discovery of penlci11in by Fleming in 1929 and its

development by Florey and his associates in 1941 (Stokes et

al. 1944b) as a powerful chemotherapeutic agent during World

War II brought about a dramatic reduction in the incidence

of congenital syphilis between the years 1947 and 1957

(Saxoni etal. 1967, Peterson 1973). However, we are begin

ning to see a re-emergence of this form of the disease

(Brown and Moore 1963, Robinson 1969, Tan 1973, Teberg and

Hodgman 1973, ASRMM 1983), perhaps because of a decline in

the use of routine screening procedures and, in part, be

cause of inadequate diagnostic training of medical students

regarding sexually transmitted diseases (Woody et al. 1963,

Saxoni et al. 1967, Peterson 1973, Sokol and Aroujo 1973).

In approximately 60% of human cases, congenital

syphilis is latent (PHS 1968), identified only by reactive

serological tests. This asymptomatic period contributes to

the difficulty in early diagnosis of congenital syphilis.

Perhaps this is a pattern of human resistance similar to

that seen experimentally in fetal and neonatal rabbits. The

works of earlier researchers, Uhlenhuth and Mulzer (1913),

Grigoriew (1929), Bessemans and Van Canneyt (1932), Seiffert

(1934), Kemp and Rosahn (1937), Kemp and Fitzgerald (1938),

and Pautrizel et al. (1957) represent efforts to understand

the course of the experimental congenital and neonatal

disease. The results of the past reports are confusing at

best because differences in experimental design hamper

comparison. However, one point shines through the confusion,

either from careful data interpretation or by each author's

declaration -- fetal and neonatal rabbits demonstrate

resistance to X- oal1idum infection. This was defini

tively demonstrated by Gamboa and Miller (1984) in neonatal

rabbits. In 1985, following high doses of repeated intrave

nous injection of X' oal1idum. Fitzgerald unequivocally

demonstrated the passage of £. pal1idum from infected

does to fetal rabbits. Again, Fitzgerald's results lend

themselves to interpretation as resistance. In contrast,

Festenstein and Bokkenheuser (1961), and Festenstein et al.

(1967), upon inoculation of the newborn animals, demon

strated a runting syndrome indicative of susceptibility.

The studies of Gamboa and Miller (1984) and Gamboa et

al. (1984) provided evidence of neonatal rabbit resistance

to symptomatic infection at one week of age, and monitored

its decline as the animal approached five weeks of age. The

possible influence of a serum neutralizing factor(s) of one

week old neonatal basal serum upon resistance was presented

(Gamboa and Miller 1984). This potential correlation was

based on the presence of neutralizing activity in sera of

one week old neonates and its absence in sera of five week

old animals.

The present study was designed to examine the potential

influence of neutralizing activity on neonatal resistance by

determining the neutralizing activity of basal sera from

neonatal rabbits 2, 3, and 4 weeks of age.

MATERIALS AND METHODS

Rabbits

Adult <> 6-month-old) male New Zealand white (NZ^W)

rabbits with nonreactive Venereal Disease Research

Laboratory (VDRL) serologic tests were used throughout the

Study, the rabbits were maintained at 18 to 20 C and were

given antibiotic-free food and water ad libitum.

Treponema pallidum

Treponema pallidum (Nichols strain) were obtained

from infected animals provided by Dr. James N. Miller, UCLA

Treponemal Research and WHO Laboratory, Los Angeles,

California, where they are maintained by intratesticuiar

passage. Normal animals were infected by inoculation of 1.0

ml/testis of a suspension containing a minimum of 2x10

treponemes/ml. At the height of orchitis development,

approximately 9 days, the animals were sacrificed by intra

cardiac injection of a lethal amount of T-61 Euthanasia

Solution (National Laboratories Corporation> Siomerville, NJ)

and the testes were aseptically removed (Fig. 1).

The testes were sliced longitudinally and the trepo­o

nemes were harvested in heat~inactivated (56 C for 30

minutes) normal rabbit seruin (HI-^NRS). The suspension was

centrifuged at 250xg for 7 minutes to remove gross cellular

debris (Fig. 2). Treponemal concentration was calculated

using darkfield microscopy and the suspension was adjusted

to 1x10 T. Dallidum/ml in HI-NRS.

Control and Test Sera

The test sera consisted of eighteen serum samples, each

from a different neonatal rafabit (6 each from 2> 3, and 4

week old neonates). These sera were taken from test rabbits

used by Gamfaoa and Miller (1984) and stored at -76 C. Three

separate experimental runs, with two sera each from rabbits

2, 3, and 4 weeks old, were performed.

ImmUne rabbit serum <IRSj obtained from male NZ-W

rabbits infected with X.. pallidum a minimum of 3 months

prior> and immune to intradermal challenge, was used as the

positive neutralizing control. Normal rabbit serum (NRS)

from VDRL nonreactive male NZ-W rabbits susceptible to

T.pallidum infection was used as the negative neutraliz

ing control. Paralleling test sera, IRS and NRS control sera o

were stored at -76 C until needed, at which time they were

brought to room temperature for further manipulations. <Both

IRS and NRS were kindly supplled by Dr. James Miller.)

Viability controls were suspensions of 1x10 X.

pal1idum/ml in HI-NRS Inoculated at 0 hours and following

the 16 hour incubation period. These were used to gauge the

viability of virulent organisms prior to and following the

incubation period.

Micro-neutralization Assay (micro-NZ)

Micro-neutralization assays were performed by the meth

od of Gamboa and Miller <1984). Briefly, test and control

sera were aliquoted into appropriately labeled sterile and

stoppered test tubes (Fig. 3) and kept on Ice (Fig. 4). Test

and control suspensions were prepared with 90 pi sera

together with lOpl of a 1x10 T. pallidum/ml suspension

XFigs. 5, 6). These were incubated for 16 hours at 34 C in

an anaerobic atmosphere of H and GO (Fig. 7) (BBL Gas Pak : 2 • • 2

Anaerobic Systems, Becton Dickinson Co., Cokeysville, MD).

Test and control suspensions were diluted by the addition of

0.9 ml HI'NRS just prior to injection for a final suspension

of 1x10 T. pallidum/ml. Each site received 0.1 ml suspen­

sion for a total inoculum of 1x10 T. pal1idum/site. Posi

tive (+) neutralization was indicated by the absence of

lesion development, negative (-) neutralization by the

appearance of typical lesions within the appropriate

incubation period established by the (-) Controls. Delayed

lesion development was indicative of partial (+7-)

neutralizatipn, Representative lesions of the mlcro-NZ assay

are pictured in Figures 14-24.

Inoculations

Five VDRL nonreactlye male NZ-W rabbits with "good"

backs (Fig. 8) (as opposed to "bad" backs. Fig. 9) were

obtained in advance of each experimental run (Bio Robotics,

10

Van Nuys, CA) and their backs were shaved just prior to the

time of inoculation. Each rafabit was inoculated in the

designated pattern illustrated in Figure 10. Each of the

test and control suspensions were drawn into sterile 1 ml

leurlok tuberculin syringes (Fig. 11), and administered for

a total of five replicate inoculation sites, one site on

each rabbit back (Fig. 12). All animal backs were clipped

and monitored daily for lesion development. Incubation

periods and lesion diameters (Fig. 13) and durations were

recorded daily. Aspirates of representative lesions were

examined for motile treponemes by darkfield microscopy. VDRL

serology tests were performed on all test animals upon

termination of each experimental run.

Statistical analysis

the incubation periods of neutralization lesions were

analyzed by the Student's t test. The differences in the

results were considered to be significant if p< 0.05,

11

Figs. 1-2. Extraction of Treponema pal 1idum from rabbittestes.--!. Testes infected with a minimum suspension of

72x10 T. pal 1idum/ml/testis removed for maceration.— 2.T. pal 1idum suspension following extraction from testes, centrifugation, and removal of gross cellular debris.

12

Figs. 3-6. Dilution procedures for the micro-NZ assay.6

— 3. Pipeting of I0)il aliquots of a 1x10 Treconena pal 1idum/ml suspension.--4. Serum samples were immedi­ately placed on ice.--5. The addition of 90yl of test or control serum.— 6. Preparation of test and control suspensions for 16 hour incubation period.

13

Fig. 7. Anaerobic atmosphere jar, 34®C incubator, and syringe labels.

Figs. 8-9. Rabbit back characteristics.--8. Test rabbitwith a good back, i.e., smooth skin with no hair patterns after shaving. Black dots mark inoculation sites.--9. A rabbit with hair patterns that make lesion interpretation

15

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for

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abbits.

-—

-—­

--

— -—

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—­- - — ­

--

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-

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---

Figs. 11-12. Inoculation procedure. — 11. Sterile, labeledluer lok syringes each containing either incubated test or

4control suspensions of 1x10 Treponema cal Iidum/ml. Only 17 syringes appear because zero hour viability control injections had been administered.--12. Inoculation of one test animal with 0.1 ml L. pallidum suspension at the VC site.

16

17

Fig. 13. Example of measurement of lesion diameter and inspection of lesion development.

18

RESULTS

Micro-neutralization assays performed with neonatal

basal sera from 2, 3/ and 4 week old rabbits failed to

demonstrate neutralizlnq activity for Trepohema pallldum.

Table 1 summarizes the results of this study. Among the

neonate groups 96-100% of Inoculated s1tes developed les1ons

from unheated serum preparations. There were no significant

differences among incubation periods (number of days from

the day of Inoculation to the first day of lesion develop

ment) of lesions using sera from the three neonatal age

gfroups, nor were there any significant differences among

sera from adult NRS control groups and neonatal groups. The

mean incubation period for lesion development among neonate

groups ranged from 16.4 +1.8 to 17.4 ±2.1 days and the mean

incubation period of the NRS was 18.6+4.4 days. However, in

two Instances <3 wk-HI and 4 wk-HI) the examination of data

On Individual sera showed either notably delayed or totally

absent lesion development which is not evident from the sum

marized data on Table 1.

Aliquots of both control and test sera were examined

for a heat-labile component(s) by heat-inactivating <56 C

for 30 minutes) sera prior to use in the micro-neutraliza­

tlon assays. As expected, the Immune rabbit serum controls

neutralized T. pal1Idum (no lesions) when serum was not

heated and resulted in either no lesions (54% of sites) or

19

delayed lesions when heat-inactivated. Interestingly, in one

experimental run, one serum sample from each of the 3 and 4

week old heat-inactivated neonatal age groups demonstrated

partial neutralization by the absence of lesions at 3 of 5

sites and 2 of 5 sites respectively. Lesions that did devel

op from these serum samples were notably delayed.

The neonates whose basal sera were used for these

assays were infected with X- callidum following extrac

tion of their basal serum by Gamboa and Miller (1984). The

resistance among the neonates they inoculated was not uni

form. Some neonates developed atypical dermal lesions at

one or both inoculation sites while others remained free of

lesions. The atypical designation was defined as any lesion

that was small, indurated, nonulcerative and of short dura

tion as compared to adult controls inoculated similarly. As

shown in Table 2, no apparent correlation was demonstrable

between the development or absence of atypical lesions among

neonates and the neutralizing activity of their basal sera

in any of the age groups. Both heat-inactivated and unheated

serum samples from the seven which developed atypical le

sions <+ neonates), failed to neutralize the treponemes at

95% of the inoculated sites. Likewise, sera from 11 that had

not developed lesions <- neonates), failed to demonstrsite

neutralizing activity at 97% of the inoculated sites.

Figures 14-24 follow the progressive lesion development

20

of three representative animals from a total of fifteen used

to test for the neutralizing activity in neonatal rabbit

serum. These figures illustrate the first appearance of ery

thema (day 15 post-inoculation. Figs. 14, 15) and continue

through the healing stages (day 48 post-inoculation. Fig.

24).

Aspirates of representative lesions routinely drawn

just prior to ulceration (Fig. 17), and selected from both

test and control sites, demonstrated actively motile trepo­

nemes by darkfield microscopy. Upon termination of the ex

periments, all test animals had converted to reactive VDRL

aerologies.

21

TflBLE 1- Neutra 1 izing act1Vity of neonatal basal sera from 2, 3, and 4 week . ; a

old rabbits.

Unheated Sera Heat-I nacti vated Sera

No. of No. of

No. Lesions/No. I ncubat ion Lesions/No. Incubation

of of Sites c Period of Sites Period

Rabbit Serum Inocu 1ated Inocu1ated

flge Serum Samp1es Mean + SD Mean X SD

rv)

17.0 ± 2.4 30/30 (100) 16.6 ±2.52 wk basal 6 29/30 (96)

3 w k basal 6 26/26(100) 17.4 + 2. 1 27/30 (90) 16.9 ± 1.6

4 wk basal 6 30/30 (100) 16.4 ± 1.8 28/30 (93) 18.5 ± 2. 1

>6 mo MRS 11/15 (73) 18.6 ±4.4 15/15 (100) 17.6 ± 2.7

0/15 (0) 7/15 (46) 27.6+2.0>6 mo IRS

c

Table 1. Continued.

a Summation of three experiments. See Materia1s and Methods for details of micro-NZ assay-

b 56 C for 30 minutes-

Inoculum from each seru'" injected into five sites for unheated and heat-inactivated sera <exception: unheated test serum from one 3 ueek

old neonate was inoculated i nto only one site due to lost inoculum).

d Number of days from inoculation to first appearance of erythema and induration. Values are mean one standard dev i atIon.

e P > 05 Student's t Test; comparison among nepnata1 sera, neonata 1 sera and MRS, and unheated and heat-inactivated samp1es.

f Normal rabb it serum obta i ned from non-infected ODRL non>-reacti ve adu11

rabbits susceptib1e to symptomatic infection with Treponema pal 1idum.

g Immune rabbit serurn obtained from infected adult rabbits immune to symptomatic reinfection upon challenge with T. pa11idum.

h P < .05 comparison with neonate and IRS control sera.

THBLE 2. Comparison of basal serurn neutralizing activity and Treponema a,

pallidum 1esion development among neonates 2, 3, and 4 weeks of age.

Serurn Incubati on

Pr i mary Neutra1izi ng Period

fl ge No. of Lesion b flcti V ity (Days) c

Groups Rn i m a 1s Development Mean ± SD

17.7+ 1.62 wks 2

16.6 + 2.04

17.3 + 2-43 wks 5

17.6 ± 1.71

ro

16.4 + 1.84 wks

Neonata1 rabbits 2, 3, and 4 weeks of age were i nocu1ated with I X 10 T. pal 1 idurn at each of two sites (Gamboa and Miller 1984). One day prior

to inoculation the same animals were bled for basal serum samples which were stored at -76^C until needed for these micro-NZ assays-

ResuIts of tests performed by Gamboa and Miller (persona1 commun icati on) Neohate Iesions, where developed <+), were atypica1, indicative of resistance.

Results from the present neutra1ization study which tested basal serum samples from 10 neonates.

Figs. 14-24. Representative examples of post-inoculation

lesion development on rabbit backs, demonstrating the

results of micro-NZ assays for Treponema pallidum

neutralizing activity by basal sera from 2, 3, and 4 week

old neonatal rabbits.

25

Figs. 14-15. Day 15. — 14. Rabbit no. 3. Erythema clearly demonstrable at most test sites, except at 3C-HI and 4B-HI. Development is absent at IRS and IRS-HI sites as predicted. — 15. Rabbit no. 4. Erythema development replicates that seen in rabbit no. 3.

26

16”17. Day 22.~“16. Rabbit no. 3. Test sites areconsistently erythematous and indurated, except 3C-HI and4E-HI. Controls NRS, NRS-HI, IRS, IRS-HI, VC and VC

0 16are as predicted.--17. Rabbit no. 4. Lesion developmentclosely replicates rabbit no. 3. Note site VC is near

16

27

ulceration.

3C-HI andFigs. 18-19. Day 32.--18. Rabbit no. 3.4E-HI sites are erythematous, indurated; other test sites and, NRS and NRS-HI are near ulceration; IRS and IRS-HI sites remain lesion free.--19. Rabbit no. 4. Test sites are ulcerated, except 3C-HI and 4E-HI; NRS and NRS-HI are near ulceration; and IRS and IRS-HI remain lesion free.

28

Figs. 20-21. Day 43.--20. Rabbit no. 3. All sites ulcerated, except positive NZ control sites, IRS and IRS-HI.--21. Rabbit no. 4. Most lesions are beginning to heal.

29

30

Figs.

22-24. D

ay 4

8.—

22.

Rabb

it n

o. 3

. Ul

cera

tion

s persist, n

o le

sion

s at p

osit

ive

NZ c

ontrol s

ites

.--23.

Rabb

it n

o. 4

. Le

sion

s ha

ve n

earl

y he

aled

.—24.

Rabb

it n

o. 1.

Exam

ple

of h

eale

d back.

DISCUSSION

Results of this study show an absence of detectable

neutralizing activity in the sera of 2, 3, and 4 week old

rabbits. A correlation between neutralizing activity of

neonatal basal sera and resistance to symptomatic infection

was suggested in a previous study by Gamboa and Miller

(1984). Their sugges.tion was based on 1) the presence of

neutralizing activity in basal sera from one week old

rabbits and their resistance to symptomatic infection fol

lowing ihtradermal inoculation with Irejaoiigjn^ EsXLi^iliiL

and 2) the absence of neutralizing activity in basal sera

and the waning resistance to T. oallidum infection of

five week old neonates. The resistance to symptomatic

infection following intradermal inoculation with T.

palliduih in the same animals from which our sera is de

rived throws suspicion on the influence of a neutralizing

factor(s) on their resistance. Apparently, serum neutraliz

ing activity 1) may contribute nothing to the resistance

demonstrated by neonatal animals, 2) may only partially

contribute, or 3) the sensitivity of the micro-neutraliza

tion assay is insufficient to detect the full in vivo poten

tial of serum as concentrations of its activity begin to

drop in neonates after one week of age.

The last option is of particular interest. As mentioned

in the results, there appears to be a hint of residual

31

neutralizing activity in the unheated serum of two neonatal

samples <5 of 6 inoculation sites developed lesions

following approximately a 21 day incubation period).

Heat-inactivation of thshepnatal basal serum seems to

slightly enhance this neutralizing activity C5 of 10 lesions

Were absent, the remaihlng were notably delayed). This

heat-inactivation enhancement is a consistent finding, also

seen in previous studies (Gamboa and Miller, personal com

munication). These results are not surprising in light of

the fact that we are examining an age frame in which declin

ing neutralizing activity can be expected. Therefore, though

the numbers are small and only suggestive of residual activ

ity, perhaps they should not be Ignored. Possibly, the con

centrations of the neutralizing factorCs) present in the

serum may have diminished to levels such that the assay

Sensitivity may not be CapaEile of detecting it. Once heated,

however, we may be enhancing that minimal amount of neutral

izing factor(s), and we are able to pick it up in some indi

vidual cases, it is also possible that the reduction of T«

pallidum over the 16 hour in vitro incubation period of

the micro-NZ assay Is masked in the in vivo portion of the

assay. Fitzgerald (1981) Inoculated rabbits with divergent

ranges of T. pallidum. Sites receiving 10 viable organ

isms demonstrated an accelerated incubation period of ap

proximately 15 days when the rabbits had been additionally

32

Injected with 10 viable organisms at other sites^ (The

normal incubation period for 10 organisms averages approx

imately 24 days.). The delayed incufaatioh periods indicative

of partial neutralizing activity may have been masked by

sites on our animals receiving the full viable inoculum of

10 T. nal1idum; therefore, partial neutralization, which

could be expected as the factor declines in concentrations,

is not detected in our assay.

The delayed incubation periods and absence of lesions

at sites receiving HI~NRS is a consistent finding (Bishop

and Miller 1976, Blanco et al. 1984, Gamboa and Miller

1984). Blanco et al. (1984) have demonstrated that the IgG

nature of neutralizing activity in immune rabbit serum, and

its largely abrogated activity upon heating, is most likely

due to the elimination of complement. It has been suggested

that endogenous complement from the extraction of J..

pa11idum frcm rabbit testicles may account for the resid­

ual neutralization seen in these suspensions (Bishop and

Miller 1976). On the other hand, it is possible that some

neutralizing activity in IRS is independent of complement.

It has been demonstrated that HI-MRS enhances phagocytosis

of X* pallidum bv proteose peptone-induced rabbit peri

toneal macrophages (Lukehart and Miller 1978). Therefore it

is feasible that opsonization may account for the residual

neutralization seen in these suspensions.

33

Interest was focused on the fetal disease as early as

19i3j Uhlenhuth and Mulzer (1913) set up studies on the in-^

heritance of syphi1is in rafabits, controlling the experimen

tal conditions to resemble human syphilis whenever possible.

Unfortunately, the ir methods were not sufficiently out!ined

to permit comparisons with later studies. Other research

groups fol1owed, Grigoriew (1929) described a single experi­

mental case of congenital transmission of syphi1is from one

doe to her offspring. In yet another study, Bessemans and

Van CanneytC1932) concluded that, although many suggestions

of congenital infection resulted, they Could not prove con

clusively the existence of congenital syphilis in 34 rabbits

born from parents having ocular syphilitic lesions. Seiffert

(1934) briefly described eight experiments dealing with in

fection With X* sis a result of cohabitation or

cross-placental transmission but failed to describe the

route of infection of his experimental animals (mice and

rabbits). Kemp and Rosahn (1937) did not sufficiently des

cribe their experimental methods, making questionable their

conclusions that a placental barrier prevented the spread of

infection from doe to offspring, or the existence of a trep­

onemicidal factor(s) in the fetus. In addition, and in rath

er forceful terms, Kemp and Fitzgerald (1938) concluded that

syphi1is is not transmitted from an infected doe to her off­

spring. In 1957, Pautrizel et al. concluded that 1) the

34

rabbit fetus possesses a natural immunity to infection by

T. pallidum and 2) maternal antibodies play only a sec

ondary role in the prevention of transmission. Interesting

ly, nearly twenty years elapsed after the results of Kemp

and Fitzgerald (1938) were published before additional work

using the rabbit as a possible model for congenital syphilis

was again presented. Festenstein and Bokkenheuser (1961) and

Festenstein et al. (1967) attempted to tolerize neonatal

rabbits to X. pal1idum and found an increased suscep­

tibilty as defined by the appearance of a runting syndrome.

In 1985 Fitzgerald listed four factors that select

against congenital syphilis in rabbits: 1) fetal damage

requires large numbers of T♦ pal1idum that accumulate in

a short period of time (Magnuson et al. 1948, Fitzgerald et

al. 1982j 2) female steroids, which are elevated during

pregnancy (Tietz 1982), diminish rabbit lesion progression

(Frazier et al. 1935); 3) rabbit pregnancy results in multi

ple births, further diluting the numbers of organisms per

fetus (Fitzgerald 1985); and 4) possibly, the heat-stable

treponemicidal factor found in the serum of 4 to 6 day old

rabbits (Gamboa and Miller 1984) begins killing X.

pallidum before birth. Taking these factors into account

Fitzgerald (1985) was successful in demonstrating the

passage of X* pal1idum from infected does to fetal

rabbits, but only after multiple intravenous injections of

35

X. pal1idum over a period of four weeks for a total of

4x10 treponemes.

The large numbers of organisms necessary to demonstrate

overt symptoms of transmission may be a reflection on the

priesenee of additional resistance factors. As discussed by •

Gamboa and MiHer (1984), resistance of 5 to 8 day old neo

natal rabbits to dermal lesion development after intradermal

inoculation with X. pal1idum may be influenced by a num

ber of factors. Group housing (nesting) could create unfav

orable temperatures for the survival of X.. pallidum.

Experimental syphilis in rabbits requires that the animals

be kept in cool quarters <18-21 C) to allow for proper

lesion development following intradermal challenge with X*

pal1idum. In addition, inoculation sites must be kept

clipped. Therefore, higher temperatures due to huddling

of neonates in a nest may contribute to their resistance,

even though this influencing factor was Shown not to be

totally responsible for the absence of lesion deveiopment.

Nursing was also considered as a potential influencing

factor (Brambe11 1970a, b, Wilson and Miles 1975). Colostrum

and milk of several mammals are known to contain factors

which may influence resistance (Reiter and Oram 1967,

Goldman and Smith 1973, Head and Beer 1979). Although there

has been no evidence to substantiate a role for similar fac

tors in rabbits, and several investigators have concluded

36

that the systemic protective factors transmitted in utero to

the rafabit fetus are not supplemented fay nursing after birth

CBramfaell et al. 1951, Kraehenfauhl and Campiche 1969,

Bramfaell 1970c), conclusive evidence for or against this

theory remains lacking.

The association of a "natural antibody with innate re­

sistance has been suggested as anpther factor^ p res

ponsible for neonatal serum neutralizing activity. Several

facts, however, have negated this as a possible explanation.

Natural antibody has classically been associated with the

IgM class of immunogiobulins CSolomon 1971) and,in the

rabbit, IgM is transmitted in utero CHemmings and Jones

1962). Therefore, were natural antibody a participant in

resistance, does' sera would also demonstrate neutralizing

activity and this has not been the case (Gamboa and Miller

1984). "

Gamboa and Miller (1984) also proposed that the absence

of a nutritional factor(s) necessary for optimum survival

and multiplication of the treponemes may influence resist

ance. The inability thus far to cultivate T. callidum in

pure culture makes the direct investigation of nutritional

requirements difficult.

While several hypotheses have been advanced to explain

the natural resistance of heonates to.syphil itic infection,

the definitive mechanism<s) has yet to be identified.

37

Studies on the isolation and identification of the neutral

izing factor(s) of basal sera from dnfe week old rabbits are

ongoing CGamboa, personal communicatioh), but in light of

this study/ may still be only half the story.

Congenital and neonatal human syphilis is not a ghost

of the past, but remains very much a disease of the present.

A total of 159 cases of early congenital syphilis have been

reported in the United States during 1982, an increase of 44

cases in four years (ASRMM 1983). These numbers "without

doubt underestimate the true magnitude of the problem,

because of misdiagnosis, and the occurrence of undocumented

cases manifested by spontaneous abortion or stillbirth"

(Hansfield and Lukehart 1984). Pregnant women and their

health care providers need to be aware of the significance

of the diagnosis of syphilis during pregnancy so that the

truly innocent victims may be spared readily preventable

suffering. :

38

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Bessemans, A. and Van Canneyt, 1932. Heredo-syphi1is Chez les lapereaux issus de parents attaints de manifestations oculaires specifiques. Soc. Beige de Biol. 110; 116-119.

Bishop, N.H. and Miller, J.N. 1976. Humoral immunity in experimental syphilis. II. The relationship of neutralizing factors in immune serum to acquired resistance. J. Immunol. 117: 197-207.

Blanco, D.R., Miller, J.N., and Hanff, P.A. 1984. Humoral immunity in experimental syphilis: the demonstration of IgG as a treponemicidal factor in immune rabbit serum, J. Immunol. 133: 2693-2697.

Brambell, F.W.R. 1970a. Transmission of immunity in the pig and horse. A. Neuberger and E.L. Tatum (eds.). In: Transmission of Passive Immunity from Mother to Young, Frontiers of Biology, vol. 18. North-Holland Publ. Co., Amsterdam, pp. 166-200. Cited in Gamboa and Miller 1984

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43