TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW
Kast, C. C. & Kolmer, J. A. (1943) A note on the culti-
vation of Treponema pallidum with the preservation of
virulence. Amer. J. Syph., 27, 309-13
Kolmer, J. A. (1930) Failure of vaccination of rabbits
against syphilis with a note on selective localization of
Spirocheta pallida. Amer. J. Syph., 14, 236-40
Lancet, 1926, 1, 882 (Annotations-Berlin: Cultivation of
Spirochaeta pallida)
Levaditi, C. (1908) Les spirochetes pathogenes. In:
Bericht iiber den XIV. Internationalen Kongressen far
Hygiene und Dermographie, Berlin... 1907, Berlin,
Hirschwald, vol. 2, pp. 160-78
Levaditi, C. (1913) Presence du treponeme dans le sang
des paralytiques generaux. C. R. Acad. Sci. (Paris),
157, 864-6
McLeod, C. P. & Magnuson, H. J. (1951) Development
of treponemal immobilizing antibodies in mice follow-
ing injection of killed Treponema pallidum. J. vener.
Dis. Inform., 32, 274-9
McLeod, C. P. & Magnuson, H. J. (1953) Production of
immobilizing antibodies unaccompanied by active
immunity to Treponema pallidum as shown by injecting
rabbits and mice with the killed organisms. Amer.
J. Syph., 37, 9-22
Magnuson, H. J., Halbert, S. P. & Rosenau, B. J. (1947)
Attempted immunization of rabbits against syphilis
with killed Treponema pallidum and adjuvants. J. vener.
Dis. Inform., 28, 267-71
Manteufel, P. & Herzberg, K. (1929) Zur Syphilis-
Framboesiefrage. Zbl. Haut- u. GeschlKr., 30, 299-301
Mizumoto, R. & Hayashi, 0. (1956) Influences of P32
upon the antibody titer of late rabbit syphilis. Tohoku
J. exp. Med., 64, 21-26
Musumeci, V., Cantone, B., Chiarenza, A. & Auri-
sicchio, A. (1958) Ricerche di terapia sperimentale
della infezione sifilitica; ricerche sperimentali, sull'in-
fluenza della penicillina sul comportamento del
bismuto nell'organismo del coniglio, trattato con
salicilato marcato con isotopo 210 Bi. G. ital. Derm.
Sif., 99, 143-55
Nakajima, T. & Hayashi, 0. (1956) Influences of P32
upon the antibody titer of incurable latent syphilis.
Tohoku J. exp. Med., 64, 17-19
Noguchi, H. (191 la) A method for the pure cultivation
of pathogenic Treponema pallidum (Spirochaeta
pallida). J. exp. Med., 14, 99-108
Noguchi, H. (191lb) A cutaneous reaction in syphilis.
J. exp. Med., 14, 557-68
2. TAXONOMY 1
The name " spirochaete " was first given by
Ehrenberg in 1838 to large, free-living, flexible
1 See Wilson & Miles (1955), Wenyon (1926), Jordan
& Burrows (1945).
9
Noguchi, H. (1912) The luetin reaction. J. Amer. med.
Ass., 59, 1262-3
Noguchi, H. (1913) Paralysie generale et syphilis. Presse
mid., 21, 805-7
Noguchi, H. & Moore, J. W. (1913) A demonstration
of Treponema pallidum in the brain in cases of general
paralysis. J. exp. Med., 17, 232-8
Rosahn, P. D. (1948) Radioactive tracer techniques and
their possible application to studies in syphilis. Amer.
J. Syph., 32, 307-16
Schaudinn, F. & Hoffmann, E. (1904-05) Vorlaufiger
Bericht fiber das Vorkommen von Spirochaeten in
syphilitischen Krankheitsprodukten und bei Papillo-
men. Arb. GesundhAmte (Berl.), 22, 527-34
Schereschewsky, J. (1909) Zuchtung der Spirochaeta
pallida (Schaudinn). Dtsch. med. Wschr., 35, 835
Schobl, 0. (1930) The duration of anti-treponematous
immunity in Philippine monkeys originally conveyed
by immunization with killed yaws vaccine. Philipp.
J. Sci., 43, 599-601
Schobl, O., Tanabe, B. & Miyao, 1. (1930) Preventive
immunization against treponematous infections and
experiments which indicate the possibility of anti-
treponematous immunization. Philipp. J. Sci., 42,
219-37
Tani, T., Inoue, R. & Asano, 0. (1951) Studies on the
preventive inoculation against syphilis. Jap. med. J.,
4, 71-86
Turner, T. B. & Hollander, D. H. (1957) Biology of the
treponematoses, Geneva, World Health Organization
(World Health Organization: Monograph Series, No. 35)
Volpino, G. & Fontana, A. (1906) Sulla cultivazione
artificiale della S. pallida (Schaudinn). Riv. Ig. San.
pubbl., 17, 462-6
Waring, G. W., jr, & Fleming, W. L. (1951) Further
attempts to immunize rabbits with killed Treponema
pallidum. Amer. J. Syph., 35, 568-72
Wheeler, A. H. (1960) Preliminary studies on active
immunization in experimental syphilis in rabbits. In:
Eleventh Annual Symposium on Recent Advances in the
Study of Venereal Diseases. Digest of proceedings,
Item 20, Atlanta, Ga., United States Public Health
Service
Willcox, R. R. (1960) The evolutionary cycle of the
treponematoses. Brit. J. vener. Dis., 36, 78-91
World Health Organization, Expert Committee on
Venereal Infections and Treponematoses (1960) Wld
Hlth Org. techn. Rep. Ser. 190
organisms floating in fresh and marine water, e.g.,
Spirochaeta plicatilis. Spirally-shaped organisms
were for a long time grouped under the common
name of Spirillum, Spirochaeta or Vibrio. Later
10 R. R. WILLCOX & T. GUTHE
some were shown to be bacteria, and these are now
grouped under Vibrio and the various species of
Spirillum. The remainder are actively motile not by
flagella, but by means of a screw-like rotation of the
organism. All these spiralled organisms have
commonly been loosely termed " the spirochaetes ".
This term, however, is now strictly confined to
members of the family Spirochaetaceae.
There are four main genera of " spirochaetes " in
the loose usage of the word: Spirochaeta, Cristispira,
Leptospira and Treponema; from the last-named
Borrelia have now been separated. The first two
genera are the Spirochaetaceae; the others form the
family Treponemataceae.
SPIROCHAETA
The various species of this genus are large slender
organisms 200F-500,u long and 0.5,u-0.74,u thick
with 100-250 spirals, and they are not capable of
causing disease. The term Saprospira is used for
smaller free-living forms which may be encountered
in foraminiferous sand (Jordan & Burrows, 1945).
CRISTISPIRA
Members of this group are characterized by a
spirally arranged veil-like membranous appendage
or crista round the body of the cell. The crista may
splay out to resemble the undulating membrane of
a trypanosome-in fact, it was first named Trypano-
soma balbiani (Bradfield & Cater, 1952). Cristispira
are saprophytic in certain molluscs (e.g., Cristispira
balbiani, found in the crystalline style of the oyster,
which is 45u-lOO1 long and l1,-1.5,u thick; its body
is divided into chambers by septa of thickened
cytoplasm).
LEPTOSPIRA
Some members of this group are pathogenic to
man and are the cause of Weil's disease, or infectious
jaundice, and also of certain fevers. They have a
large number of closely-wound spirals, and the ends
are frequently turned round at a sharp angle; at
rest they are characteristically hooked. They are
the thinnest of all spirochaetes, being only 0. l,u-0.2,
thick and 5,u-18,u long. Included in the group are
L. icterohaemorrhagiae, the cause of Weil's disease,
or infectious jaundice; L. canicola, a pathogen of
dogs; and L. biflexa, an easily-cultured, free-living
leptospire said to inhabit ponds, gutters and dripping
taps (Bradfield & Cater, 1952).
TREPONEMATA
Treponemata are widely distributed in nature.
Numerous species have been described in water, in
the gut of certain insects (e.g., the ant and the cock-
roach) and in the large gut of the toad. In man they
are found in the mouth, alimentary tract, bronchi,
around the urethral orifice, in certain ulcerating
conditions of the skin, in condylomata, in the blood
of patients with relapsing fever (although these are
now classified as Borrelia), and in many of the
lesions of syphilis. They are considerably thicker
than leptospirae, are less closely wound, and vary
considerably in size. T. termitidis is 20,u-60,u long,
while T. parrum may be only 3,u in length. A
saprophytic water form, T. elusum, has been de-
scribed.
Borrelia
Most treponemata have a low refractive index
and stain poorly with aniline dyes. Those associated
with Vincent's angina and relapsing fever in man
and with avian spirochaetosis have a refractive
index like that of bacteria, and stain more readily.
On these and other less distinctive grounds they are
assigned by many authors to a separate genus,
Borrelia (Wilson & Miles, 1955; Swain, 1955;
Breed et al., 1948; Eagle, 1948).
Those now classified as Borrelia include B. vincentii,
the cause of Vincent's angina, B. anserinum which
affects geese and turkeys (McNeil et al., 1949), and
the relapsing fever borreliae. The latter include
B. recurrentis, agent of cosmopolitan relapsing fever
which is transmitted by lice and is found in Europe,
Africa, Asia and South America, and other species
transmitted by soft ticks (B. duttoni, found in
Africa, B. hispanica, in Europe, B. turicata, B. par-
keri, B. venezuelensis, B. mazzotitii and others in the
Americas). (Some authors still describe these as
treponemes with the generic prefix " T." instead of
" B.".) Some writers include also as Borrelia certain
of the saprophytic genital treponemes.
B. vincentii is 7,u-18p in length and 0.23,u-0.64,u
(average 0.42,u) thick. It has 3-8 shallow spirals of
varied amplitude and, unlike B. duttoni and B. re-
currentis, is stated to have no external amorphous
layer but is enclosed by a clearly-marked cell
membrane (Swain, 1955).
B. duttoni is 9,u-16,u thick and 0.24p-0.60,u wide.
It is tapered at the end and is not of constant thick-
ness. Short forms with up to four spirals of in-
constant amplitude and longer forms of 5-8 spirals
 TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW
are encountered. There is an outer covering of
structureless material. B. recurrentis closely re-
sembles B. duttoni in size and general morphology
(Swain, 1955).
Treponemata of man and animals
Those remaining for classification as treponemata
include T. cobayae, found in the blood of guinea-
pigs; T. microdentium, T. macrodentium and T. muco-
sum (Noguchi, 1912c), which are apparently non-
pathogenic and are found in the mouth of man
(being smaller than the pathogenic B. vincentil also
found in that site 1); a number of saprophytic genital
treponemes found in increased numbers in septic
conditions (T. refringens, T. phagedenis (T. balaniti-
dis), T. pseudopallidum, T. gangrenosa nosocomialis,
T. minutum (T. genitalis), and T. calligyrum);
T. cuniculi, which is responsible for a venereal
disease in rabbits; T. pallidum, the cause of human
syphilis, and T. pertenue and T. carateum, the
organisms responsible for yaws and pinta, respec-
tively.
Of those infecting man, T. microdentium, the small
mouth treponeme, resembles T. pallidum in morpho-
logy and is also cultivable with least difficulty.
According to Rosebury & Foley (1941) it is the only
member of the oral group which can be accepted
without doubt as a distinct species. Of the human
genital treponemata, Schaudinn & Hoffmann (1904-
05) also described T. refringens, at the time of their
original description of T. pallidum, as a broader,
less regular, more refractile organism, but did not
decide whether it was a separate species (see also
Levaditi, 1906). It has a right-handed spiral (Sequeira,
1956). T. phagedenis, with its variety of alternative
names, was described by Noguchi (1912a), who
also (1913) described T. calligyra (calligyrum) and
T. minutum or T. genitalis (see also Noguchi, 1918;
Noguchi & Kaliski, 1918): all may be obtained from
genital sores or condylomata (see Moreau & Giuntini,
1956; Coutts et al., 1952).
Moreau & Aladame (1957), who studied three
oral treponemes (including T. microdentium) and
four genital treponemes (T. refringens, T. phagedenis,
T. calligyra and T. minutum) noted that their
fermentative pattern in culture differed according
to species. It has been suggested from electron
microscope studies of these organisms (Moreau &
Giuntini, 1956) that although T. calligyra and
T. minutum may be considered to be treponemes,
I T.
buccalis, T. dentium and T. spirillum have also been
described-see Wilson & Miles (1955, page 2252).
II
T. refringens and T. phagedenis should be classified
as Borrelia.
Treponemes will resist trypsin digestion for many
days, but 10% bile salts (sodium taurocholate)
cause complete disintegration (von Prowazek, 1907)
and all, with the exception of leptospirae (Noguchi,
1917; Wilson & Miles, 1955) are eventually broken
up by 10% saponin (Jordan & Burrows, 1945).
Relationship of treponemes to protozoa
An argument which has recurred from time to
time is whether treponemes should be classified as
protozoa. McDonagh (1912) so classified spiro-
chaetes and described an apparent life-cycle re-
sembling that of the malaria parasite. At the same
period, on the other hand, Dobell (1912), among
others, pointed out that the one important feature
which distinguished protozoa from bacteria was
motility without flagella. When early workers with
the electron microscope claimed to have found
flagella on T. pallidum even this was in doubt
(Wilson & Miles, 1955). However, it seems that the
apparent " flagella " in T. pallidum arose from
rupture of the treponeme before examination, but,
even knowing this, van Thiell (1959) still inclined
towards protozoa, stating that the presence of
flagella could no longer be used as an argument for
its relationship to bacteria: neither, he considered,
did the movement of T. pallidum fit in with that of
spirochaetes. Bessemans & de Geest (1933), pointing
out the difficulty of culture of T. pallidum in vitro,
also inclined to protozoa. Most workers, however,
like to consider spirochaetes as representing a
phase of life midway between the bacteria and
protozoa (Jordan & Burrows, 1945; van Thiell,
1959).
DISCOVERY OF PATHOGENIC TREPONEMES
At least four different varieties of pathogenic
organisms of the genus Treponema are known:
T. pallidum (syphilis), T. pertenue (yaws), T. carateum
(pinta) and T. cuniculi (venereal treponematosis of
rabbits). They all resemble each other morpholo-
gically.
T. pallidum
Donne, in 1837, is stated (Campbell & Rosahn,
1950) to have been the first to describe a spiralled
micro-organism in the exudate of primary syphilitic
lesions of the genitalia, and he believed he had
found the causative organism of syphilis. His work
 12 R. R. WILLCOX & T. GUTHE
was confirmed by Vanoye (1840-41), who considered
that these organisms could be used in the diagnosis
of syphilis.
According to Stokes & Beerman (1934), Metch-
nikoff stated that Bordet & Gengou in 1903 undoub-
tedly saw the " spirochaete " of syphilis. Klebs is
also stated to have seen it in 1875-77 in syphilitic
material, and also to have transmitted the disease
to the monkey (see Klebs, 1932; Wilson & Miles,
1955). Credit, however, is usually given to Metch-
nikoff and Roux in Paris for having infected mon-
keys and apes experimentally with syphilis in 1903
(Metchnikoff & Roux, 1903, 1904, 1905; see Jordan
& Burrows, 1945). They also showed that calomel
ointment applied to chimpanzees 1-2 hours after
local inoculation would prevent infection.
The final confirmation of T. pallidum as the
causative agent of syphilis-indeed, its discovery-
is attributed to Schaudinn & Hoffmann (1904-5,
1905a, b) (see also Hoffmann, 1905, 1906; D'Ago-
stino, 1957) who observed the organism in chancres
of syphilitic patients. Other early workers include
Mansurov (1885), who described a vegetable parasite
of syphilis, and Zabotolnyj (1909), who apparently
saw T. pallidum two years before Schaudinn &
Hoffmann but attached no importance to his dis-
covery. After the discovery by Schaudinn &
Hoffmann, numerous confirmatory papers appeared
within a very short time all over the world (including
Russia-e.g., Zabolotnyj, 1905, 1906, 1909; Ivanov,
1905). See also Minouflet (1906).
The discovery of T. pallidum added enormous
impetus to experimental research. The following
year (1906) claims were advanced that the organism
had been cultured (Volpino & Fontana, 1906;
Schereschewsky, 1909a, b), although the cultures
obtained were not shown to be virulent. In the
same year Bertarelli (1906) showed that syphilis
could be passed from the eye of one rabbit to that
of another and Parodi (1907) first transmitted the
disease to the testicle of a rabbit. Virulent cultures
were, however, claimed by Noguchi (1911 a)-but
this also proved a disappointment as the virulence
soon disappeared.
Around this time, too, the first diagnostic serum
test for syphilis was evolved by Wassermann
(Wassermann et al., 1906) and the dark-field method
of diagnosis was soon introduced (Coles, 1909).
Zabolotnyj & Maslakovec (1907) described the
cessation of movement and the agglutination of
T. pallidum under the effect of serum from a syphi-
litic patient. Ehrlich & Hata (1910) discovered
salvarsan, or 606, to provide what was hoped to be
an effective and rapid treatment for the disease
(and also for yaws). From the cultured treponeme,
Noguchi (191 lb) prepared luetin, which was tested
in many hospitals and which would produce a
positive skin reaction in most cases of late syphilis
(less often in early cases). Moreover, it was con-
sidered to be specific for the disease (Noguchi, 1912b).
Thus, during less than a decade before the First
World War, most of the essential problems of
syphilis appeared to have been solved-at least, it
must have appeared so to syphilologists at the time.
T. pertenue, T. carateum and T. cuniculi
T. pertenue, the causal organism of yaws, was also
discovered in 1905, by Castellani. It is morpholo-
gically indistinguishable from T. pallidum (Wilson
& Miles, 1955). T. carateum, the causative organism
of pinta, although suggested by Menk (1927) and
by Gonzalez-Herrejon (1927) on the basis of a high
incidence of positive Wassermann reactions in
pinta patients (see Turner & Hollander, 1957) was
first demonstrated by Saenz et al. (1938) (see Fox,
1939; Jordan & Burrows, 1945). Leon y Blanco
(1940), who called it T. herrejoni, showed this
treponeme also to be morphologically indistin-
guishable from T. pallidum. For further history see
Schuberg & Schlossberger (1930).
Although T. pertenue was soon shown to be
transferable to rabbits (Nichols, 1910, was already
working on a strain obtained from a coloured soldier
returning from the Philippines), T. carateum, on
the other hand, is not easily passed to animals and
only one such claim has been made, that of Leon
y Blanco & Oteiza (1945) (see Holcomb, 1942).
T. cuniculi, the cause of a venereal treponematosis
in rabbits first described by Ross (1912), was identi-
fied by Bayon (1913) and is also morphologically
similar to T. pallidum. It causes a complaint known
as " rabbit syphilis ", or sometimes as pallidoidosis.
Affected rabbits develop superficial small, scaly,
eroded, sometimes crusted, lesions on the genitalia
and adjacent perineal region: sometimes the nostrils
and eyelids may be affected (see section 17).
EVOLUTION OF PATHOGENIC TREPONEMES
The evolution of the pathogenic treponemes can
only be a matter of speculation. It is considered by
some (e.g., Cockburn, 1961) that they arose aeons
ago from free-living water forms which have since
become adapted to their human or animal hosts,
 TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW
having been subjected to all the influences concerned
in natural selection. Although it is suggested that
the four treponemes may have come from a single
source (Hoffmann-Bonn, 1953) speculators may
differ as to how far back in the evolutionary scale
the separation between them occurred. Hudson
(1946) considered the treponemes pathogenic to
humans (T. pallidum, T. pertenue and T. carateum)
all to be T. pallidum, the disease picture in man being
modified by various external environmental factors
and by factors within the host. This author (1963)
believed that a common organism became adapted to
man in Africa in palaeolithic times. Hackett (1963)
suggests that the human treponematoses probably
arose long ago from an animal infection. Mutations
subsequently occurred-T. carateum probably being
the earliest-resulting in the diseases now known as
yaws, pinta and venereal syphilis. That the distri-
bution of the disease syndromes of yaws, pinta and
endemic and non-venereal syphilis conforms to some
considerable extent to the environmental background
is well known (Guthe & Luger, 1957; Guthe
& Willcox, 1954). Willcox (1960) has indicated the
evolutionary cycle which may occur in the disease
syndromes caused by human treponematoses result-
ing from pressures in the extrinsic environment,
and in the intrinsic environment of the host.
STRAINS OF T. PALLIDUM
Those strains of T. pallidum which have been
maintained in animals have remained virulent to
both animals and man. These include: the Nichols
(Nichols-Hough or Nichols pathogenic), Truffi,
Gand, Gent, Ami, Fan, Tho, Est, Rei and Pour
strains (see section 15). Strains maintained in
animals for a period of years may undergo modifi-
cation (Turner & Hollander, 1957).
Although virulent treponemes have not been
established in culture outside the body, there are a
number of strains-usually designated T. pallidum
which have been maintained in artificial media for
many years. These include the Reiter, Noguchi,
Nicholsnon-pathogenic, Kazan, Kroo and Vasarhelyi.
Details of these are given below (see section 19).
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3. MORPHOLOGY: I. METHODS OF EXAMINATION
The morphology of T. pallidum may be examined
in slide preparations of stained smears, by dark-
field under the light microscope, and by phase-
contrast and electron microscopes. With the light
microscope without dark-field and with the electron
microscope, the treponemes are examined in the
dead state: under the dark-field and phase-contrast
microscopes, live organisms can be studied. Using
the light microscope, the unstained organism is
extremely difficult to see.
STAINED SMEARS 1
Stained smears are examined under the con-
ventional light microscope, and two basic techniques
of staining are used: (a) the treponeme is im-
pregnated with a dye, or a metallic ion such as
silver, to render the organisms visible against a pale
background; or (b) the background may be stained
black, leaving the unstained treponeme pale by
comparison (see Bessemans et al., 1936; Yamamoto,
1929a, b). Matsumoto (1930) and Turner & Hollan-
I
See Campbell & Rosahn (1950).
15
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pp. 1031-56; Syphilis, rabbit syphilis, yaws and pinta...
chapter 81, pp. 2027-4
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Russk. Vrac, 23
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Russk. Vra6, 42
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chaeta pallida]. Russk. Vrac, 11
der (1957) succeeded in staining T. pallidum with no
less than 402 different dyes.
Impregnation methods
Contrary to general belief treponemes are easily
stained (Turner & Hollander, 1957; Wheeler, 1960)
but they are difficult to see under the light microscope
because of the small amount of protoplasm possessed
by the organism and also because the suspending
medium frequently contains too much tissue debris
which also takes up the stain (Wheeler, 1960). Early
investigators used aniline dyes or coal tar derivatives
(e.g., methylene blue, azure eosinates, Victoria blue,
cresyl violet, Giemsa and similar mixtures). A
mordant (i.e., a protein precipitant such as phenol,
tannic acid, acetic acid, phosphotungstic acid, etc.)
has to be used to make the stain effective. Schaudinn
& Hoffman (1904-05) employed a modified Giemsa
stain (see Wilson & Miles, 1955), with which T. palli-
dum and other pathogenic treponemes stain rose-red.
Noguchi (1918) found that treponemes fixed with
Fontana's fixative and Fontana's mordant could be
stained by carbol fuchsin. This was further con-
firmed by DeLamater, Wiggall & Haanes (1950),