TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW 1 5

Schereschewsky, J. (1909a) Zuchtung der Spirochaeta Volpino, G. & Fontana, A. (1906) Einige Verunter- pallida (Schaudinn). Dtsch. med. Wschr., 35, 835 suchungen uber kunstliche Kultivierung der Spiro- Schereschewsky, J. (1909b) Weitere Mitteilung uber die chaeta pallida (Schaudinn). Zbl. Bakt., L Abt. Orig.. Zuchtung der Spirochaeta pallida. Dtsch. med. Wschr., 42, 666-9 35, 1260-1 Wassermann, A., Neisser, A. & Bruck, C. (1906) Eine Schuberg, A. & Schlossberger, A. (1930) Zum 25. Jahres- serodiagnostische Reaktion bei Syphilis. Dtsch. med. tag der Entdeckung der Spirochaeta pallida. Klin. Wschr., 32, 745-6 Wschr., 9, 582-6 Wenyon, C. M. (1926) Protozoology, London, Bailliere, Sequeira, P. J. L. (1956) The morphology of Treponema Tindall & Cox, vol. 2 pallidum. Lancet, 2, 749 Willcox, R. R. (1960) The evolutionary cycle of the Stokes, J. H. & Beerman, H. (1934) The fundamental treponematoses. Brit. J. vener. Dis., 36, 78-91 bacteriology, pathology and immunity of syphilis. Wilson, G. S. & Miles, A. A. (1955) The spirochaetes. In: Modern clinical syphilology, 2nd ed., Philadelphia, In: Topley & Wilson's Principles of bacteriology and Saunders, pp. 17-57 immunity, 4th ed., London, Edward Arnold, chapter 38, Swain, R. H. A. (1955) Electron microscopic studies of pp. 1031-56; Syphilis, rabbit syphilis, yaws and pinta... the morphology of pathogenic spirochaetes. J. Path. chapter 81, pp. 2027-4 Bact., 69, 117-28 Zabolotnyj, D. K. (1905) [Spirochaetes in syphilis]. Thiel, P. H. van (1959) Are spirochaetes bacteria or Russk. Vrac, 23 protozoa? Antonie van Leeuwenhoek, 25, 161-7 Zabolotnyj, D. K. (1906) [The pathogenesis of syphilis]. Turner, T. B. & Hollander, D. H. (1957) Biology of the Russk. Vra6, 42 treponematoses, Geneva (World Health Organization: Zabolotnyj, D. K. (1909) [The pathogenesis of syphilis]. Monograph Series, No. 35) Arh. biol. Nauk, 14, 263-82, 389-438 Vanoye, M. (1840-41) Note sur un animalcule trouve Zabolotnyj, D. K. & Maslakovec, N. N. (1907) [Observa- dans le pus syphilitique. Ann. Soc. Sci. nat. (Bruges), tions on the movement and agglutination of Spiro- 2, 39-42 chaeta pallida]. Russk. Vrac, 11

3. MORPHOLOGY: I. METHODS OF EXAMINATION

The morphology of T. pallidum may be examined der (1957) succeeded in staining T. pallidum with no in slide preparations of stained smears, by dark- less than 402 different dyes. field under the light microscope, and by phase- contrast and electron microscopes. With the light Impregnation methods microscope without dark-field and with the electron Contrary to general belief treponemes are easily microscope, the treponemes are examined in the stained (Turner & Hollander, 1957; Wheeler, 1960) dead state: under the dark-field and phase-contrast but they are difficult to see under the light microscope microscopes, live organisms can be studied. Using because of the small amount of protoplasm possessed the light microscope, the unstained organism is by the organism and also because the suspending extremely difficult to see. medium frequently contains too much tissue debris which also takes up the stain (Wheeler, 1960). Early STAINED SMEARS 1 investigators used aniline dyes or coal tar derivatives (e.g., methylene blue, azure eosinates, Victoria blue, Stained smears are examined under the con- cresyl violet, Giemsa and similar mixtures). A ventional light microscope, and two basic techniques mordant (i.e., a protein precipitant such as phenol, of staining are used: (a) the treponeme is im- tannic acid, acetic acid, phosphotungstic acid, etc.) pregnated with a dye, or a metallic ion such as has to be used to make the stain effective. Schaudinn silver, to render the organisms visible against a pale & Hoffman (1904-05) employed a modified Giemsa background; or (b) the background may be stained stain (see Wilson & Miles, 1955), with which T. palli- black, leaving the unstained treponeme pale by dum and other pathogenic treponemes stain rose-red. comparison (see Bessemans et al., 1936; Yamamoto, Noguchi (1918) found that treponemes fixed with 1929a, b). Matsumoto (1930) and Turner & Hollan- Fontana's fixative and Fontana's mordant could be stained by carbol fuchsin. This was further con- I See Campbell & Rosahn (1950). firmed by DeLamater, Wiggall & Haanes (1950), 16 R. R. WILLCOX & T. GUTHE who used a modification of Fontana's technique for Beamer & Firminger, 1955). This was used by photographic study, and to examine the so-called Levaditi & Manouelian (1906) in Metchnikoff's life-cycle of the Nichols pathogenic rabbit-adapted laboratory soon after the discovery of T. pallidum strain of T. pallidum (DeLamater, Wiggall & Haanes, in the histological examination of monkeys infected 1950b). The slides were first placed in Ruge's solu- with syphilis. tion (glacial acetic acid and formalin), then in In general the dye stains are more suited to Fontana's mordant, and subsequently stained with smears, and silver impregnation methods to tissue gentian violet. Takahashi (1922) used a potassium section. Stroesco & Vaisman (1936)-in Levaditi's permanganate stain. laboratory-developed a silver impregnation method Rose & Morton (1952) found Fontana and Giemsa applicable to frozen sections (see Stokes & Beerman, stains useful, especially for virulent organisms. 1948). Tannic-acid-mordanted fuchsin, as in Leifson's flagella stain (Leifson, 1930), was found convenient. Background stains and other staining methods These authors described another modification useful Of the background relief stains, Indian ink, congo for cultured treponemes. A drop of centrifuged red and nigrosin have been used for T. pallidum culture was placed on a cover-slip and immediately (Campbell & Rosahn, 1950). The dark-field, using exposed to osmium tetroxide vapour. Then, after an optical method, is on the same principle. Lenn- prolonged immersion in acetone, the organisms were hoff (1948) described an in vitro method of staining. *' mordanted " in formalin, stained in basic fuchsin It was noted that, if a colloidal mercury preparation and examined in the wet state. was injected into the chancre of a rabbit, and Victoria blue and a carbol gentian violet stain immediately followed by an intravenous injection of have also been reported as satisfactory (Golds- sodium thiosulfate, treponemes stained black would worthy & Ward, 1942; Jordan & Burrows, 1945) be found in the chancre within two minutes. and Rizk et al. (1951) used carbol gentian violet (and also silver impregnation and other methods) to study T. pallidum from cases of bejel; Wright's IMMUNOFLUORESCENCE stain was preferred. Crystal violet and basic fuchsin More recently, staining by immunofluorescence were used by Levine (1952) for a variety of trepo- has been applied to T. pallidum. A new specific nemes including T. pallidum, while Vago (1947a, b,c) technique has been evolved by Deacon et al. (1964) used a method employing mercurochrome. which requires but 30 seconds for staining. The Coutts, Silva-Inzunza & Valladares Prieto (1952) reagent consists of human or rabbit syphilis anti- observed that T. macrodentium and T. micro- serum from which non-specific antibody has been dentium absorbed a dilute carbol fuchsin stain removed by Reiter treponeme sonicate. Yobs et al. rapidly but T. pallidum did so less easily. If T. palli- (1964) have proposed a technique for staining dum and the dental treponemes were stained with T. pallidum in tissues. carbol fuchsin and observed under the dark-field microscope, T. pallidum appeared with an argentine brilliancy whereas the dental treponemes showed THE DARK-FIELD with only a pinkish brilliancy. The use of stained The dark-field, which uses the light microscope smears under the dark-field was earlier described with transmitted rather than direct light, has definite by Hoffman (1921) (see DeLamater, Haanes & advantages in that it permits the examination of Wiggall, 1950). A more recent stain is an ink-blue live motile organisms (see Clarkson, 1956). For the dye originally found in " Parker 51 " blue-black recognition of T. pallidum fixed specimens have ink (Gomes, 1953; Turner & Hollander, 1957; definite limitations. Not only may there be agonal Goldman & Sawyer, 1959). Yamamoto (1929a, b) or later distortion of the organisms but also the found that two blue stains would stain T. pallidum study of motility-an essential point of recognition and T. pertenue but not T. cuniculi. -is not possible. Coles (1909) described the use of Methods other than the use of aniline dyes were dark-ground illumination, which was also used by developed because of some lack of consistency in Antoni (1921) and by Hoffman (1921a) (see Camp- the results obtained with stained smears. One was bell & Rosahn, 1950). Hoffmann (1921b) employed the method of silver impregnation improved by the dark-field for the examination of stained and Fontana (1912) (see Campbell & Rosahn, 1950; unstained smears as did Coutts, Silva-Inzunza & TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW 17

Valladares-Prieto (1952) and Wheeler (1960). Coutts, ELECTRON MICROSCOPE Silva-Inzunza & Morales-Silva (1952a, b), although the study of motility was sacrificed, found the The limits of resolving power of the light micro- examination of unstained smears useful by this scope have been extended over 100 times by the method as it could be done at leisure. electron microscope: enormous magnification may Sequeira (1956) made careful dark-field observa- be obtained. With an instrument working with an tions of T. pallidum, T. pertenue and T. refringens. electron optical magnification of 200 000 times, a Rizk et al. (1951) studied T. pallidum from cases of tenfold increase to 2 000 000 diameters may be bejel under the dark-field and made camera lucida achieved by photographic enlargement, although drawings. Methods of counting treponemes under such figures are artificial as it is the resolving power the dark-field have been described by Bessemans rather than magnification which is all-important. & de Moor (1940), Morgan & Vryonis (1938), Moreover, the full potentialities of this instrument Magnuson et al. (1948); see Turner & Hollander have probably not yet been reached. The technique (1957). of examination, however, requires complete immobi- lization of the organism and as a re3ult of desiccation a distortion with lengthening and flattening of the PHASE-CONTRAST MICROSCOPE coils may occur. No motility studies can, therefore, The phase-contrast microscope of Zernicke is be undertaken. (This partial disintegration of the favoured by many investigators for, like the dark- organism has led to some confusion regarding ground, it permits the study of motile organisms. " flagella "; see section 4). The material may, how- It is limited by the powers of resolution of the light ever, be successfully re-examined over a period of microscope but, depending on differences in re- days after implantation in a collodion film (Wile et fractive index, it allows the internal structure of al., 1942). organisms to be better examined than does the Rose & Morton (1952) prepared specimens for dark-field microscope. It is considered, therefore, the electron microscope by placing a drop con- to have advantages even over the electron micro- taining cultured treponemes, concentrated by cen- scope (DeLamater, Newcomer, Haanes & Wiggall, trifugation if necessary, on a collodion membrane 1950), although the magnification obtained is, of mounted on a fine mesh screen. This was then course, much lower. It is useful in interpreting exposed to osmium tetroxide before examination, the findings of the electron microscope, and De- which was facilitated by shadow-casting with Lamater (1952) has described it as "a magnificent chromium or uranium, thus enabling fine surface tool ". structures to be seen more clearly. To observe The phase-contrast microscope was used to study single organisms the slide was covered with human the action of penicillin on bacteria by Levaditi et al. skin oil on which was placed a drop of culture and (1950), and specifically for the study of T. pallidum a cover-slip was then pressed upon it. Very small by Poetschke & Kaiser (1951) and of T. pertenue " islands "' of medium resulted which were mapped by Schaeffer et al. (1951). Numerous studies have to serve as a guide to the " geography " of the been made by DeLamater and his colleagues, specimen. particularly with a view to investigating the so-called Untreated treponemata are too dense to reveal life-cycle of the Nichols pathogenic rabbit strain of details of internal structure in an electron micro- T. pailidum (DeLamater, Wiggall & Haanes, 1950a; scope operating at 70 kV. Chemical dissection by DeLamater, Haanes, Wiggall & Pillsbury, 1951); mild partial trypsin digestion can be used to reveal of the Nichols non-pathogenic culture strain in cul- this more clearly (Bradfield & Cater, 1952). ture (DeLamater, Haanes, Wiggall & Pillsbury, 1951; Structural differentiation within bacterial proto- DeLamater, Haanes & Wiggall, 1951a), and in the plasm was studied under the electron microscope embryonated hen's egg (DeLamater, Haanes & by Mudd et al. (1942), and the oral treponemes, Wiggall, 1951b); of the Kazan cultured strain T. macrodentium and T. microdentium, were similarly (DeLamater, Haanes, Wiggall & Pillsbury, 1951; examined by Mudd et al. (1943) and by Hampp DeLamater, Haanes & Wiggall, 1951c); and of et al. (1948), who also included B. vincentii. Other the Reiter cultured strain (DeLamater, 1952); the Borrelia, including B. duttoni and B. recurrentis, Russian cultured strains have been examined by Wave been examined by Babudieri & Bocciarelli Vjaseleva (1957). (1943, 1948), Bradfield & Cater (1952) and Swain 18 R. R. WILLCOX & T. GUTHE

(1955). Leptospirae have been studied by Morton Askura, Z. (1952) Jap. J. Bact., 7, 335-7 & Anderson (1942a), Babudieri (1948, 1949, 1958), Babudieri, B. (1948) Ricerche di microscopia elettronica. Bradfield & Cater (1952), Czekalowski & Eaves III. Studio del genere Leptospira. R. C. Ist. sup. (1955), and Swain (1955), and cristispirae by Sanita, 11, 1046-66 Babudieri, B. (1949) The morphology of the genus Bradfield & Cater (1952). The genital treponemes Leptospira as shown by the electron microscope. T. minutum, T. calligyrum, T. refringens and T. pha- J. Hyg. (Lond.), 47, 390-2 gedenis were compared by Moreau & Giuntini (1956). Babudieri, B. (1952) Ricerche di microscopia elettronica. Virulent T. pallidum from the human and the IV. Studio morfologica del genere Treponema. R.C. rabbit (Nichols pathogenic strain) have been studied Ist. sup. Sanitai, 15, 711-22 under the electron microscope by numerous observers Babudieri, B. (1958) Feinstruktur der Leptospiren und (Wile et al., 1942; Magerstedt, 1943; Mudd et al., anderer SpirochWten. Zbl. Bakt., I. Abt. Orig., 173, 1943; Wile & Kearney, 1943; Mudd & Anderson, 386-406 1944; Levaditi, 1946; Molbert, 1956; Prokoptchouk Babudieri, B. & Bocciarelli, D. (1943) Ricerche di microscopia elettronica. 1I. Studio morfologico del et al., 1951; Bradfield & Cater, 1952; Schmerold genere Spironema. R. C. Ist. sup. Sanita, 6, 305-14 & Duebner, 1954; Swain, 1955; Ovcinnikov & Babudieri, B. & Bocciarelli, D. (1948) Electron micro- Vitjurin, 1961; Ovcinnikov & Delektorskij, 1966). scope studies on relapsing fever spirochaetes. J. 11vg. Spirochaetal lysis caused by penicillin was studied (Lond.), 46, 438-9 under the electron microscope by Kawata et al. Beamer, P. R. & Firminger, H. I. (1955) Improved (1960). methods for demonstrating acid-fast and spirochetal Numerous studies have been made of the non- organisms in histologic sections. Lab. Invest., 4, 9-17 virulent cultured strains, including the Nichols non- Bessemans, A. & de Moor, A. (1940) Ultranumeration pathogenic strains, by Morton & Anderson (1942b), homogene et seuil infectieux de differentes varietes de Treponema pallidum. Rev. belge Sci. med., 12, 254-61 Mudd et al. (1943), Mudd & Anderson (1944), Bessemans, A., Janssens, P., Van Thielen, E. & Hampp et al. (1948), Morton & Oskay (1950), and De Wilde, H. (1936) Affinites tinctoriales et argentiques Morton et al. (1951); the cultured Noguchi strain du treponeme pale. Bull. Soc. franc. Derm. Svph., by Mudd et al. (1943), Mudd & Anderson (1944), 43, 1073-84 Jakob (1947), Hampp et al. (1948); the cultured Bradfield, J. R. G. & Cater, D. B. (1952) Electron- Kro6 strain by Mudd et al. (1943), Mudd & microscopic evidence on the structure of spirochaetes. Anderson (1944); and the cultured Reiter strain by Nature (Lond.), 169, 944-6 Mudd et al. (1943), Mudd & Anderson (1944), Campbell, R. E. & Rosahn, P. D. (1950) The morphology Schmerold & Duebner (1954), Askura (1952), and staining characteristics of Treponema pallidum. Review of the literature and description of a new Takeya & Mori (1953), Greifelt (1955), Greifelt & technique for staining the organism in tissues. Yale Molbert (1955), Kawata (1956), M6lbert (1956), J. Biol. Med., 22, 52743 Babudieri (1952, 1958), D'Alessandro & Zaffiro Clarkson, K. A. (1956) Technic of darkfield examination. (1961), Wallace & Harris (1967) and Ryter & Pillot Med. Techn. Bull., 7, 199-202 (1963). Coles, A. C. (1909) Spirochaeta pallida: Methods of The treponemata of yaws and pinta were examined examination and detection, especially by means of under the electron microscope by Angulo et al. the dark-ground illumination. Brit. med. J., 1, (1951), by Watson et al. (1951) and by Molbert 1117-20 and shadow-cast Coutts, W. E., Silva-lnzunza, E. & Morales-Silva, G (1956), using unshadowed spe- (1952a) Unstained slides for the diagnosis of certair cimens; and T. microdentium was examined under treponematoses. Publ. Hlth Rep. (Wash.), 67, 442 the electron microscope by Listgarten et al. (1963). Coutts, W. E., Silva-Inzunza, E. & Morales-Silva, G, (1952b) Dark-ground illumination of unstained smears REFERENCES and tissue sections for the diagnosis of Treponema pallidum. Brit. J. vener. Dis., 28, 97-9 Angulo, J. J., Watson, J. H. L., Wedderbum, C. C., Coutts, W. E., Silva-Inzunza, E. & Valladares-Prieto, J. Leon y Blanco, F. & Varela, G. (1951) Electron- (1952) Comparative study of certain treponemata micrography of treponemes from cases of yaws, pinta found in human genitalia, specially referring to and the so-called Cuban form of pinta. Amer. J. trop. T. pallidum, T. macrodentium and T. microdentium. 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D'Alessandro, C. & Zaffiro, P. (1961) Sull'esistenza di un Greifelt, A. (1955) Das Treponema pallidum im Elektro- involucro simil-capsulare nel T. pallidum. G. Microbiol., nenmikroskop. Minerva derm. (Torino), 30, 330-2 9, 151-8 Greifelt, A. & Molbert, E. (1955) Elektronenmikrosko- Deacon, W. E., Kellogg, D. S. Wende, R. D. & Moore, pische Untersuchungen zur Morphologie des Trepo- K. B. (1964) Fluorescent antibody darkfield. In: nema pallidum. Hautarzt, 6, 17-20 Digest of proceedings. Fourteenth Annual Symposium Hampp, E. G., Scott, D. B. & Wyckoff, R. W. G. (1948) on Recent Advances in the Study of Venereal Diseases, Morphological characteristics of certain cultured Houston, Texas: 1964, Atlanta, Ga., Public Health strains of oral spirochetes and Treponema pallidum Service as revealed by the electron microscope. J. Bact., DeLamater, E. D. (1952) A study of the life-cycle of 56, 755-69 spirochetes and other micro-organisms by means of Hoffmann, E. (1921a) Ober die Verwendung des Dunkel- phase contrast and routine microscopy. Trans. N. Y. feldes zur Auffindung der Gelbfieber-Gelbsucht- Acad. Sci., 14, 199-201 Syphilis, und anderer Spirochaten in fixierten und DeLamater, E. D., Haanes, M. & Wiggall, R. H. (1950) gefarbten Ausstrich- und Schnittpraparaten. Dtsch med. Studies on the life cycle of spirochetes. II. The Wschr., 47, 65 development of a new stain. Amer. J. Syph., 34, 515-8 Hoffmann, E. (1921b) Die Bedeutung der Leuchtbild- DeLamater, E. D., Haanes, M. & Wiggall, R. H. (1951a) methode zur Darstellung von Mikroorganismen. Studies on the life cycle of spirochetes. V. The life Derm. Z., 33, 1-10 cycle of the Nichols non-pathogenic Treponema Jakob, A. (1947) Neuere Untersuchungsergebnisse in der pallidum in culture. Amer. J. Syph., 35, 164-79 Spirochaetenforschung mit dem Elektronenmikroskop. DeLamater, E. D., Haanes, M. & Wiggall, R. H. (195lb) Ein Beitrag zur Morphologie der Spirochaeta pallida. 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(1950) in the rabbit's testis as seen by phase contrast micro- Etude morphologique des bacteries au moyen du scopy. J. exp. Med., 92, 239-46 microscope a contraste de phase. Action de la DeLamater, E. D., Wiggall, R. H. & Haanes, M. (1950b) penicilline. Bull. Acad. nat. Mid. (Paris), 134, Studies on the life cycle of spirochetes. IV. The life 369-77 cycle of the Nichols pathogenic Treponema pallidum Levine, B. S. (1952) Staining T. pallidum and other in the rabbit's testis as visualized by means of stained treponemata. Publ. Hlth Rep. (Wash.), 67, 253-7 smears. J. exp. Med., 92, 247-52 Listgarten, M. A., Loesche, W. J. & Socransky, S. S. Fontana, A. (1912) Metodo per colorare intensamente.e (1963) Morphology of Treponema microdentium as rapidamente il Treponema pallidum ed altri spirocheti. revealed by electron microscopy of ultrathin sections. Pathologica, 4, 582 J. Bact., 85, 932-9 Goldman, L. & Sawyer, F. (1959) A simple portable- Magerstedt, C. (1943) Ein Beitrag zur Morphologie der dark-field microscrope. Arch. Derm., 79, 589-90 Syphilisspirochaete. Arch. Derm. Syph. (Berl.), 185, Goldsworthy, N. E. & Ward, H. K. (1942) A simple 272-80 method of staining T. pallidum. J. Path. Bact., 54, 382-4 Magnuson, H. J., Eagle, H. & Fleischman, R. (1948) Gomes, L. S. (1953) An easy and rapid staining method The minimal infectious inoculum of Spirochaeta for Treponemata.. In: VI Congresso internazionale di pallida (Nichols strain) and a consideration of its Microbiologia, Roma... 1953.-Riassunti delle communi- rate of multiplication in vivo. Amer. J. Syph., 32, cazioni, Rome, vol. 2, sessions 8-16, p. 420 1-18 20 R. R. WILLCOX & T. GUTHE

Matsumoto, S. (1930) Experimental syphilis and fram- Rizk, E., Garabedian, G., Chaglassian, H. & Pipkin, A. boesia with special reference to the comparative (1951) Studies on the treponemes of bejel. I. History, pathology and immunity. Monographiae Actorum morphological characteristics and staining properties. Dermatologicorum, Series B, Syphilidologica, No. 3, Amer. J. Syph., 35, 201-6 Kyoto, Imperial University Rose, N. R. & Morton, H. E. (1952) The morpho- Molbert, E. (1956) Vergleichende elektronenmikro- logic variation of treponema. Amer. J. Syph., 36, skopische Untersuchungen zur Morphologie von 17-37 Treponema pallidum, Treponema pertenue und Reiter Ryter, A. & Pillot, J. (1963) Etude au microscope electro- Spirochaten. Z. Hyg. InfektKr., 142, 510-5 nique de la structure externe et inteme du treponeme Moreau, M. & Giuntini, J. (1956) Etude au microscope Reiter. Ann. Inst. Pasteur, 104, 496-501 6lectronique de quatre especes de treponemes ana6ro- Schaeffer, K., Loughlin, E. & Joseph, A. (1951) The bies d'origine genitale. Ann. Inst. Pasteur, 90, 728-37 effect of orally administered aureomycin on T. pertenue Morgan, H. J. & Vryonis, G. P. (1938) A method for in man. Amer. J. trop. Med., 31, 24-5 the quantitation of inocula in experimental syphilis. Schaudinn, F. & Hoffmann, P. (1904-5) Vorlaufiger Amer. J. Syph., 22, 462-9 Bericht uber das Vorkommen von Spirochaten in Morton, H. E. & Anderson, T. F. (1942a) Observations syphilitischen Krankheitsprodukten und bei Papillo- on the morphology of Leptospira and the Nichols men. Arb. GesundhAmte (Berl.), 22, 527-34 strain of Treponema pallidum with the aid of the Schmerold, W. & Duebner, B. (1954) Elektronenmikro- RCA electron microscope. J. Bact., 43, 64-5 skopische Untersuchungen an Reiter-Spirochaetales Morton, H. E. & Anderson, T. F. (1942b) I. Some und Nichols-Treponemen. Hautarzt, 5, 511-3 morphologic features of the Nichols strain of T. palli- Sequeira, P. J. L. (1956) The morphology of Treponema dum as revealed by the electron microscope. Amer. pallidum. Lancet, 2, 749 J. Syph., 26, 565-73 Stokes, J. H. & Beerman, H. (1948) Some problems in Morton, H. E. & Oskay, J. (1950) Electron microscope the biology ofthe syphilitic infection. Amer. J. med. Sci., studies of treponemes. II. The effect of penicillin on the 215, 461-9 Nichols strain of T. pallidum. Amer. J. Syph., 34, 34-9 Stroesco, G. & Vaisman, A. (1936) Nouvelles contribu- Morton, H. E., Rake, G. & Rose, N. R. (1951) Electron tions a l'etude de l'infection syphilitique cliniquement microscope studies of treponemes. III. Flagella. apparente des souris. C.R. Soc. Biol. (Paris), 122, Amer. J. Syph., 35, 503-16 399-402 Swain, R. H. A. (1955) Electron microscopic studies of Mudd, S. & Anderson, T. F. (1944) Pathogenic bacteria, the morphology of pathogenic spirochaetes. J. Path. rickettsias and viruses as shown by the electron Bact., 69, 117-28 microscope. Their relationships to immunity and Takahashi, T. (1922) Eine neue Nachweismethode von chemotherapy. J. Amer. med. Ass., 126, 561-71 Spirochdte pallida, die Thee-Permangan-Methode. Mudd, S., Polevitsky, K. & Anderson, T. F. (1942) Jap. Z. Derm. Urol., 22, 1556 Bacterial morphology as shown by the electron Takeya, K. & Mori, R. (1953) Jap. J. med. Progr., microscope. IV. Structural differentiation within the 40, 607-11 bacterial protoplasm. Arch. Path., 34, 199-207 Turner, T. B. & Hollander, D. H. (1957) Biology of the Mudd, S., Polevitsky, K. & Anderson, T. F. (1943) treponematoses, Geneva (World Health Organization: Bacterial morphology as shown by the electron Monograph Series, No. 35) microscope. V. T. pallidum, T. macrodentium and Vago, S. (1947a) Das Verhalten der Mikroorganismen T. microdentium. J. Bact., 46, 15-24 und Formelemente der Mund- und Rachenhohle bei Noguchi, H. (1918) The spirochetal flora of the normal der Spirochatenfarbung nach dem Mercurochrom- male genitalia. J. exp. Med., 27, 667-78 Pyoctanin-Verfahren. Wien. med. Wschr., 97, 62-4 Ovcinnikov, N. M. & Delektorskij, V. V. (1966) Morpho- Vago, S. (1947b) Die Rolle der Serumaffinitiit des Mer- logy of Treponema pallidum. Bull. Wid Hlth Org., 35, curochroms in der Spirochaetendarstellung. Wien. 223-9 med. Wschr., 97, 420-30 Ov6innikov, N. M. & Vtjurin, B. V. (1961) [Some Vago, S. (1947c) Methode de coloration pour l'examen special features of the structure of Treponema pallidum microscopique de spirochetes. Schweiz. med. Wschr., as observed under the electron microscope]. Vestn. No. 17, p. 479 Vener. Derm., No. 4, pp. 48-52 Vjaseleva, S. M. (1957) The action of various penicillin Poetschke, G. & Kaiser, B. (1951) Der Nachweis von preparations on Treponema pallidum. Part II. The T. pallidum mit dem Phasenkontrastmikroskop. change in morphology of Treponema pallidum in cul- Hautarzt, 2, 408-10 ture and attempts to adapt it to penicillin. J. Microbiol. Prokoptchouk, A. J., Prokoptchouk, A. & Bondarovitch, Epidem. Immunobiol., 28, 573-80 (Trans.) A. G. (1951) [Some agents of skin and venereal disease Wallace, A. L. & Harris, A. (1967) The Reiter treponeme. under the electron microscope]. Vestn. Vener. Derm., A review of the literature. Bull. Wid Hlth Org., Suppi. No. 3, pp. 20-3 (in press) TREPONEMA PALLIDUM: A BIBLIOGRAPHICAL REVIEW 21

Watson, J. H. L., Angulo, J. J., Le6n y Blanco, F., Wilson, G. S. & Miles, A. A. (1955) The spirochaetes. Varela, G. & Wedderburn, C. C. (1951) Electron In: Topley & Wilson's Principles of bacteriology and microscopic observations offlagellation in some species immunity, 4th ed., London, Edward Arnold, chap- of the genus Treponema Schaudinn. J. Bact., 61, 455-61 ter 38, pp. 1031-56 Wheeler, A. H. (1960) Darkfield microscopy as an aid in Yamamoto, T. (1929a) Studien iiber Spirochatenfarbung. visualization of Treponema pallidum. In: Eleventh I. Untersuchung der pallidafarbenden Farbstoff. Annual Symposium on Recent Advances in the Study Acta derm. (Kyoto), 13, 591 (Cited by Matsumoto, of Venereal Diseases. Digest of Proceedings, Item 1930) No. 24, Atlanta, Ga., United States Public Health Service Yamamoto, T. (1929b) Studien uber Spirochatenfarbung. Wile, U. J. & Kearney, E. B. (1943) The morphology of III. Farberischen Unterschiede zwischen Sp. pallida, T. pallidum in the electron microscope. Demonstration Sp. pallidula, und Sp. cuniculi. Acta derm. (Kyoto), of flagella. J. Amer. med. Ass., 122, 167-8 14, 145 (Cited by Matsumoto, 1930) Wile, U. J., Picard, R. G. & Keamey, E. B. (1942) The Yobs, A. R., Brown, L. & Hunter, E. F. (1964) Fluores- morphology of S. pallida in the electron microscope. cent antibody technique in early syphilis. Arch. Path., J. Amer. med. Ass., 119, 880-1 77, 220-5

4. MORPHOLOGY: II. GENERAL CHARACTERISTICS (A)

For general reviews on the study of treponemes, T. pallidum from both human and rabbit sources had readers are referred to (among others) Noguchi thicker and thinner forms. He described three (1917, 1928); Zuelzer (1928); Hindle (1931); types-the thicker, the normal and the thinner. Ingraham (1932); Eagle (1948); Campbell & Rosahn Whether these are constant forms or fluctuations (1950); Rose & Morton (1952); Turner & Hollander around a mean is not known (Wilson & Miles, 1955). (1957). In culture he grew some extremely long organisms (Mason, 1959; Eagle & Germuth, 1948). SIZE AND SHAPE The adaptability and change of morphology of The morphology and movements of T. pallidum T. pallidum to its environmental conditions in were first described by Schaudinn & Hoffmann culture have been noted by many authors (e.g., (1904-05). Bessemans, 1938; Kast & Kolmer, 1940); treponemes T. pallidum is a thin, delicate spiral organism with thicker and flatter curves being associated with tapering ends. It has a length of 6,u-15,t with culture and the thinner, more mobile forms (Jordan & Burrows, 1945; Swain, 1955) and is of with the struggle for existence in tissue (Stokes & uniform cylindrical thickness. There is great Beerman, 1948). Cultured organisms show marked variability in length (Manouelian, 1940) and the morphological change after prolonged exposure to greatest variations are found at the time of the most penicillin. Vjaseleva & Danilova (1952), using rapid multiplication (Rose & Morton, 1952). micrometer measurements of the Kazan strain, The organisms are held back by gradocol mem- found notably smaller forms under the influence oranes having a pore size of 0.4,u. Their narrowest of penicillin. They described very small forms diameter is therefore about 0.2,u (Hindle & Elford, (length 0.9,u-3.6,u; width 0.36p-0.6,t) with not more 1933; Tilden, 1937; Wilson & Miles, 1955) although than three spirals, compared with 5-9 spirals and other figures such as 0.25,t-0.3,u (Jordan & Burrows, lengths of 7.2,t-18, (width 0.75,u-0.9p) under normal 1945), 0.25tt-0.3ju (Noguchi, 1918) and 0.09,u-0.18,u conditions. The pleomorphism of the organism is (Swain, 1955) are given-the last being based on therefore to a great extent determined by environ- electron-photomicrograph measurements. ment. Although a great variation from long to There is thus great variability in the size of short may be noted in the cultured treponeme, the " normal " treponemes, quite apart from aberrant time of the greatest relative variations coincides forms as yet to be described (Nyka, 1934a, b), not with that of most rapid multiplication (Rose & only in length (Manouelian, 1940; Welferz, 1936) Morton, 1952). but also in thickness-especially in successive T. pallidum has a varying number of regular pri- transplants of the cultured organisms (Eagle & mary spirals which are sharp and angular. The Germuth, 1948). Noguchi (1912) noted that width of the spiral, which is rigid and regular, is