Biology of the oral spirochetes: A review

ORAL SURGERY, ORAL MEDICINE and ORAL PATHOLOGY

Quarterly

Review

of the Literature

Associale Editor THOMAS

J. COOK

Corresponding

Editors

AUSTRALIA A. J. Arnott Sydney, Australia

ITALY Alexander G. Nutlay Halifax, Nova Scotia

CENTRAL AMERICA Claudio Funcia Cornell Havana, Cuba

SCANDINAVIA Reidar F. Sognnaes Belmont, Mass.

SOUTH AMERICA Guillermo A. Ries Centeno Buenos Aires, Argentina

SWITZERLAND AND Erik P. Steinmann Hans Miiblemann Raoul H. Boitel Ziiricb, Switzerland

ENGLAND Paul A. Tolkr St. Albans, England

JULY,

1949

Collective BIOLOGY

Review

OF THE ORAL SPIROCHETES:

HEINER

HOFFMAN,

GERMANY

B. S., D.M.D.,

COLUMBUS,

T

A REVIEW OHIO

HE presence in the mouth of spirochetal forms which closely resemble the I’repbnema pallidurn in morphology but which are involved in a different complex of lesions is a curious fact of interest to both dentist and syphilologist. IJp to the present, perhaps the principal motive in working with the more easil? cultivable oral forms has been to utilize the similarities in an effort to develop further information in the cultivation and serologic investigation of T. pallidurn. However, it is evident that a study of the oral spirochetes in their own right> as complex biologic entities with their own peculiar characteristics and importance should eventually bring results of the greatest value for b,oth the syphilologist, and the dentist. In the following review, some of the more pertinent information available on the group of oral spirochetes has been presented from this point of view. The recent literature has been especially considered, but some of the older work has been included also. From

the

Ohio

State

University,

Department 925

of

Bacteriology.

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LITER:\TTJRE

Taxonomy The order Xpirochaetulcs, which includes the oral spirochetes, is a large, ubiquitous group consisting of two families, the Spirochaetacene, which contains three genera, Spirochaetn, Sctprospira, Cristispirn, urld the Treponematacene, which contains the genera Borrelirc, Twpon~m~~, and Leptospirn (Table I) .I

Class : Schizomycetes Nlgeli Order: Spirochaetales I3ucl~rnnn Family I: Spirochnetacene Svellengrebel benus I: Spirochaeta Ehrenberg Genus II: Saprospirn Gross Genus III : Cristispira Gross Fxmilv II: Treponemataceae Rchxutlinn Genus I : Borrelin Rwellengrebel Genus II : Trepotaemn Hchaudinn Genus Tl I : Lrpto~pirn Noguch i

All members of the order multiply by transverse division, and show motility without anteroposterior polarity. No definito nucleus is present, and no coloring matter; thev stain gram negative. The antigens of the spirochetes behave much like those’ of the t,rue bacteria, rather than the protozoa, giving rise to and other protective antibodies. Cultural needs agglutinins, spirocheticides, At one time the order was closely resemble those for the parasit,ic protozoa2 considered verv close to the t,r,vpanosomcs, butt t,he evidence points rather to a close relationship to the true bacteria.” The Spiroch.aetaceae contains no pathogens for man, so only the second family is of immediate interest. Borrelin has open, irregular coils, stains readily with aniline dyes other than Uiemsa, and shows active lashing movements and slow rotation. On the other hand, Treponema shows close, permanent coils of about, 1 micron in pitch, with movements of bending and rotation. This group is strictly anaerobic. Leptospira, which is aerobic, differs from Treponema by having finer coils and by either one or both ends being curved back. It differs from both T,reponenaa and Borrclia by the absence of the axial filament. Both Treponema, and Leptospira stain with difficulty except with Giemsa’s stain and silver impregnati0n.l According to Noguchi,* motilit,y is due in all cases to an axial filament, which is present even if stainin, ~7fails to demonstrate one. He claimed that if the periplast is subjected to dissolution by bile salts and the protoplasm allowed to escape, a slender hyaline elastic filament is left behind which shows spirals. The relaxed organism takes the shape of its axial filament; but when movement occurs the protoplasm contracts and thus stretches the axial filarnent with resulting movement, He explained varieties of movement as due to varying zones of protoplasmic contract,ion, such as unipolar, bipolar, rnedian, or irregularly regional. As will he pointed out later, this entire concept is now being seriously questioned.

Recent Taxonomic Studies Because of the difficulties of culturin g the oral spirochetes, a large number of species has been named in the literature wit,h little more basis than morphology and habitat, and with little regard for possible relationships to previously published descriptions. The appendices of Bcrgey’s manual1 list over forty names of oral spirochetes, many of which refer to the same organism, and all of them so inadequately described that it is impossible to accept them. After making a careful study of the literature, Appleton6 was willing to admit only five spirochetal species resident in the mouth: Borrelia buccde

927

COLLECTIVE REVIEW

(Steinberg), Treponema” naicrodentium (Noguchi), Treponema m,acrodentium (Noguchi) , Treponema mucosum (Noguchi) , and Leptospira dentium (Hoffmann). He believed that perhaps two other species may have to be recognized: Bo,rrelia vincentii (Blanchard) and Borrelia bronchialis (Castellani). These two, however, may be identical. Bergey’s manual recognizes Treponema microden&m, Treponema naucosunl, Borrelicn huccale, and Borrelia cincentii. IIampp” was even more reluctant to recognize clear17 defined species since on the basis of morphology, the only method of identification available, he could only distinguish three by dark field-a smaller oral treponema that corresponds to Treponema naicro~drntium, a larger double-contoured form referred to as I1orrelin buccale, and an intermediate form, a large, single-contoured type which corresponds to descriptions in the literature for Rorrdicl zince?atii (Table II). TABLE ORGANISM

Treponemu microdentium Borreliu buccale aincentii Borrelin Treponema mzccosum Treponema mncmdentium dentkm Leptospira Borrelia

bronchialis

II.

~CEPTED

RPECIFS APPI,ETON

OF ORAL

SPIROCHETES BERGEY

IIAMPP +

I

t +

t

?

f

t

t

t t

f

i 8

Proske and Sayers’ described the three organisms accepted by Hampp as follows : 1. Treponema microdentium.-Delicate spiral filament with regular, shallow convolutions. Length 4 to 8 microns, width 0.25 microns and less. In pathologic material and in young cultures the short forms predominate; in older cultures larger forms are found in abundance, often measuring up to 15 microns. The organism tapers sharply toward the extremities, and occasionall,y delicate caudal Blaments ma?: be observed on one 01 Ijoth ends. The latter, however, are not independently motile, but rather seem to be propelled by the organism itself and cannot therefore be looked upon as true flagella. Granular particles have been repeatedly observed. Motility is active and rotating. The body is flexible; division transverse. 2. Borrelia buccale.-A spiral filament of rather large dimensions. Length 5 to 20 microns, width 0.6 to 0.8 microns. When viewed by dark field, it presents doubly contoured outlines. The ends are sharply pointed and devoid of flagellar extensions. The Motility is generally sluggish and undulating. curves are wide and irregular. 3. Borrelia vincentii.-This organism varies in size, the majority being from 6 to 10 (In relation to this variation of size note microns in length and from 0.3 to 0.5 in width. The coils are shallow and irregular, and Culwick ant1 Fairbairn’s work, given below.) motility is quite active. Motion is scrp(xnt,ine rather tllan rotating. Granules have frequently He finds that motility is not I,een observed. Hamppx disagrees with this description. serpentine in character, but consists of rapid flexion, looping, translation, and rotaCon, and that the coils are uniformly ant1 widely spaced.

Investigating another aspect of morphologic differentiation, Culwick and Fairbairn measured the lengt,hs of 778 Borreliu vincentii from a throat swab. On analysis of the data they found that the length distribution curve deviated markedly from normality, but that it was possible to dissect it into’ three svmmetrical curves which did not depart significantly from a gaussian distribution. This was in conformity with results obtained with l’repon,enaa recurrentis and with Trypanosonaa rhodesiense. It was found that Treponema recurrentis and Trypunosoma rhodesiense may possess either a negative or positive charge, thus resulting in six different]\- characterized groups each. Because of the charact,er

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OF

LITERATURE

On of the specimen, the charges on BoweZin zincentii could not be determined. the basis of their findings and drawing upon analogy with l’rypunosoma they postulated the possibilitv of sexual reproduction among rhodesiense, ZTowever, since they seem Treponemn recurrentis, as well as Bovrelicr lq¢ii. to have made their measurements from smears, rather than from living organisms by dark field, one must be reluctant to accept their data, in view of the commonly recognized morpho’logic distortions found in fixed smears of the spirochetes. By careful comparative studies with dark-field apparatus and stained smear techrnqucs of specimens from the lesions of Vincent’s infect,ion and of pure cultures, HamppX has shown that stained smears not only distort or obscure fine structural differences between the various oral spirochetes, but also fail to reveal the presence of the smaller spirochetes in any appreciable numbers.

Recent Morphologic Studies Because of the extreme tenuit,y of the oral spirochetes, approaching in size the limit of vision under the conventional microscope, and because of their low refractive index, dark-field and filtrat,ion techniques were necessarily used in morphologic studies, but very little was learned. However, certain techniques such as special stains and the elect,ron and phase microscopes have been developed in the past few years which have enabled recent new gains in knowledge to be made, and have also reopened certain problems. Mudd, Polevitzky, and Anderson lo have made electron photomicrographs of two strains of oral treponemata which fail to confirm the presence of Noguchi’s ‘ ‘ axial filament. ’ ’ Hyar’s studyll of Spi,rochaeta plicatilis, Ehrenberg’s saprophytic organism found in water, also failed to demonstrate the axial filament by either the phase or electron microscope. She is of the opinion that this structure is simply an artifact created by the twisted cell wall. According to the electron photomicrographs of Mudd, Polevitzky, and Anderson, the inner proto8plasm of the oral treponemata is enclosed in a delicate periplast, which may be continuous between incompletely divided cells and may extend beyond the cell protoplasm as a terminal filament. However, Hampp, Scott, and Wyckoff’2 were unable to observe definite cell membranes in their study of eight strains of spirochetes. h’lagellar-like structures are seen, often in groups of four, distributed along the sides or near the cell ends. Hampp, Scott, and Wyckoff confirmed this observation, and also noted that the terminal filamenm consist of fine intertwined strands, but these did not appear to be a continuation of the cell wall. They also confirmed the presence of internal granules which seemingly represent local accumulations of cytoplasm, as well as small end granules which lack internal strnct~ural detail. The ‘ ‘ granules spirochetogenes ’ ’ described by many previous investigators are evident in the photomicrographs of Mudd, Polevitzky, and Anderson, being 150 to 500 millimicrons in diameter, and attached to the cell wall by a short stalk, or closely adherent. Some granules are seen free. Hampp, Scott, and Wyckoff found the free granules are roughly circular in outline and sharpIy bounded. They appear t,o consist, most,l,v of short sections of spirochetes closely packed t,ogethcr. Another type of free granule repeatedly found by Hampp, Scot,t, and WyckoR consists of tangled masses of spirochetes or spirochetal segments. These dense spheroidal free granules ma,v well be asexual reproductive bodies. Hampp’” has consistently found t,hat agin, (7 cultures of the smaller oral treponemata give rise to spirochetal granules, and hc is of the opinion that the granules represent a constant, and rhythmic process in the life cycle of these organisms. As evidence, he reported that in his pure cultures the granules appear at about three weeks and continue to increase in number until about seven

COLLECTIVE

REVIEW

929

months, when no vegetative forms can be discerned by dark-field examination. Yet these aged cultures all gave vigorous growth of typical spirochetes when transferred. On the other hand, Akatsu’” stated that the granules observed in old cultures of treponemata are simply a phase of plasmoptysis, associated with the unfavorable surroundings existing prior to degenerat,ion. If positive experimental confirmation of the interpretation made by Hampp and others of the significance of the granules in the life cycle is obtained, a new aspect may be thrown open of the problern of chronicity and t,reatment in the trcponernatoses.‘j Serologic

Typing

As Robinson and Wichelhausen stated,lG many authors agree that morphologic descriptions are not reliable for differentiation since they depend upon environment and are subject to variation. Motility is a characteristic valuable only as a supplement for diagnosis in the hands of experienced workers. Recognizing this, attempts have been made to develop cultural and biochemical characterizations in classification, rather than relying on morphology alone. Since some of the organisms in question may have never been cultured on artificial media and since the various workers have used different culture media and different biologic tests, the limitations of this approach are obvious. In addition, it seems that the oral spirochetes have only a limited power to attack various activity sugars,14 so fermentations are of little value. However, proteolytic Serologic studies with cultivable may be shown and used in differentiation. strains have been largely on the basis of agglutination, although precipitin and complement-fixation tests have been used. Robinson and Wichelhausen did not find the agglutination test satisfactory in their efforts to differentiate serologically ninet,een strains of oral spirochetes and four strains of Treponema pallidurn (Reiter, Kasan, Nichols, and Noguchi) . Cross-reactions were found between distinct morphologically different strains, and it was also found that normal rabbit sera agglutinated spirochetal suspensions. They therefore turned to the precipitin test. Using an extract of spirochetal cells, they found that on the basis of the precipitin reaction sixteen strains of oral spirochetes morphologically and culturally identical could be divided into five groups. None of these groups gave a positive precipitin reaction with one strain which was morphologically and culturally different from all the others tested. This organism was placed separately as group 6. It also did not react with the t,wo strains of larger oral spirochetes (group 7) or with the pallidunr strains, which were placed in groups 7 and 8. The two strains of larger oral spirochetes were found to be serologically identical and morphologically and culturally similar to the Reiter and Kasan strains. The Nichols and Noguchi strains were found serologically related to each other, but different serologically, morphologically, and culturally from the Reiter and Kasan strains; and serologically different from any of the tested strains of oral spirochetes. These findings leave open the question of whether or not the groups established demonstrate species or whether they constitute “sub-types. ” The fact that seven groups were formed from only nineteen strains indicates the possibility of additional serologically distinctive organisms among the cultivable forms.

Other Antigenic Studies BeckI has investigated t,he question of specificity of the complement-fixat,ion reaction of different types of spirochetes (Treponema pallidurn strains Reiter 36, Krcio, Kasan II and Noguchi, and a strain of mouth spirochete), the question

930

QIJARTERLY

RFXIEW

OV LITERATIJRE

of the relation between complement-fixation and agglutination react,ions with spirochetes, and the question of the nature of the spirochetal antigen. His examination of human svphilitic sera, using both the Wassermann reaction and the complement-fixation test with spirochetes, showed a superior sensitivity of the latter and practically equal specificity. Two different antibodies were found in human syphilitic serum, one reacting with the ubiquitous lipoid antigen of the Wassermann reaction, and the other with a specific antigen in the spirochete. The lipoid Wassermann antigen is present in the spirochetes, as well as a fraction, probably not protein, which reacted actively with spirochetal antisera from rabbits in conlplernent-fixation and precipitation t&s, but which failed to react with syphilitic sera. He found that the difference of agglutination titers between normal and s@ilit,ic scra with cultured l’repone?na The reactivity of the mouth spirochetes with the pallidunL is not pronounced. human syphilitic sera was such as to make one suspect that there exists some relationship to the specific complement-fixation reaction given by the palliduna organism. Kolmer’” later confirmed this group ant,igen relationship of the oral spirochetes to various strains of l’reponenm pcdlidunl.

Cultivation

and Physiology

There have been a number of reports of succwsful cultivation and isolation of the mouth spirochetes. ,411 agree that at least two conditions must be met: anaerobiosis and the presence of enrichening substances in the media; commonly used are ascitic fluid, serum, or blood.‘” Of the recent methods, the most successful seem t,o rely upon Proske and Sayers” modification of Noguchi’s method, using Huntoon ‘R ” ho’rmone ’ ’ agar medium, which contains egg albumin and ascitic fluid with a veal heart infusion agar base. HamppZO has utilized elements from the methods of both Proske and Sayers and Rosebury and FoleyZ1 to develop a technique to facilitate both primary isolation and mass cultivation. In addition, he has been able to apply successfully the lyophile process of preservation to the strains he has isolated and culturcd.22 At present, it seems that the problem of isolating and culturing a number of the oral spirochetes has been solved to the point where it is possible to handle the organisms for routine investigation. Further advance may perhaps be possible through the precise study of nutritional requirements. Up to the present no attempts have been made to determine the exact needs of any of the oral spirochetes, but there have been two recent studicsZ3, 24 on the requirements of a closely allied strain which may perhaps serve as well for the oral forms. Whiteley and E’razier 23found that growth of t,he Reiter strain of Treponema pallidunz. could be maintained for nine subcultures in a medium consisting of a mixture of amino acids, sodium thioglycolate, glucose, vitamins, dipotassium The vitamins, ascorbic acid, niacin, choline phosphate, and serum albumin. chloride, thiamin, riboflavin, pyridoxinc, and calcium pantothenate, were used, but it seemed that the amino acid source rather than the vitamins is the limiting factor. Eagle and Steinman in a later stud) AY found that a mixture of acetic acid, compounds, and crystalline arginine, any of a number of sulfur-containing serum albumin, supplemented with minute amounts of yeast extract, an enzymatic casein digest, and glucose, permits the multiplication of the Reiter treponema. Little and SubbaRow’j have shown that serum albumin is the essential material in blood, plasma, serum, and ascitic fluid for cultivation of avirulent However, since Whiteley and Frazier found that a Treponema pallidurn. medium containing albumin but with no other source of amino acids did not

COLLECTIVE

REVIEW

931

support growth and since an acid hydrolysate of albumin in a medium containing amino acids did not support growth, they suggested that perhaps the chief function of the albumin is protective, detoxifying fatty acids, the mechanism DubosZ6 had described for Mycobacterium tuberculosis. Akatsu14 found that Treponema micro’dentium, as well as a number of other spirochetes, is indifferent to the presence of sugars, with the exception that Treponenaa nzicrodentium did not grow as vigorously as normally and degenerated earlier when amygdalin, glycogen, b&rcose, or lactose were present. The studies of Okabe*; showed that potassium chloride and potassium nitrate have physiologic effects of the same order as sodium chloride. A 0.5 per cent concentration of sodium chloride was found to be most favorable for growth. The typical appearance of the oral spirochetes in culture media is an opalescent, whitish, homogeneous haze which on aging becomes a pale granular yellow.‘” (:rowth is evident within two days following inoculation into Huntoon’s medium, and the maximum is reached in five days. Okabe*’ found that with Treponemn dentium growth in a serum-horse testicular infusion broth was accompanied by the formation of indole and hydrogen sulfide. The hydrogen-ion concentration suitable for growth, according to Okabe, varies between pH 6.5 and 7.7, but pH 7.0 seemed the most favorable concentration. Wichelhauscn and Wicllelhausen,l!’ on the ot,her hand, found that in liquid media there is little difference in growth response in the range pH 6.4 to 6.6 and pH 7.3 to 7.5, but in solid media the alkaline range is more satisfactory. With continued growth Okabe found a shift in the hydrogen-ion concentration toward the acid side so that after eight days a pH of 6.5 was evident. ScheiF previously had found evidence that a similar shift observed in cultures of Trepo?lema ~~ullidu~~~ (Flopstock) and three other strains of spirochetes is due to lactic acid formation in the metabolism of sugar. He showed that these strains also evolve carbon dioxide. Scheff found the organisms to be anaerobic, but Hampp was of the opinion that the oral spirochetes are not as fastidious in regard to a reduced oxygen tension during cultivation as was formerly believed.”

Resistance The entire order is unable to withstand desiccation. The resistance of the oral spirochetes to heat is slight. Bronfenbrenne?” demonstrated that Treponema microdentium or Treponema mucosum, suspended in physiologic salt solution, dies in seven minutes when subjected to 45’ C. Okabe found that in his serum-horse infusion medium death results at 50” C. after thirty minutes. Disintegration occurs in oxgall or 2 per cent saponin solution. Leadinghams observed that a 2 per cent sodium citrate solution causes instant cessation of mot,ility of the spirochetes of Vincent’s angina, with apparently eventual dissolution. Testing the effects of phenol, saponin, hydrochloric acid, and other chemical aoents Bronfenbrenne?’ found that Treponemu microdentium and Treponema r&co& showed in general a sensitivity twenty times at least and sometimes one hundred times greater than Escherichia coli. Eagle and Musselman”’ have shown spirocheticidal action by penicillin against a strain of a mouth spirochete, and penicillin has proved to be an effective adjunct in the treatment of oral fusospirochetosis.32 Ecology The establishment of the typical flora of the adult human mouth is dependent upon the appearance of the first and second dentitions. In the toothless baby and toothless adult there are few harboring places for microorganisms,

932

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LITERATGRE

so the bacterial flora is quite scanty. In addition, the teeth favor certain organisms b,y establishing anaerobiosis. However, such factors as saliva, diet, and climate, as well as the interrelationships of the various species present, undoubtedly are important in determining t,he character of the flora, but it is only comparatively recently that serious studies have begun to be made of these problems, and the literature on the ecology of the oral flora is still limited.“” In Appleton ‘s opinion,:‘4 a healthy mouth does not contain oral spirochetes and fusiform bacilli in its residual flora, but the average mouth does. Several studies have shown that the incidence of these organisms is fairly high. Okabe”’ found oral spirochetes present about the teeth in all of a group of 100, consisting of healthy individuals and a number with upper respiratory or oral disease. Stafness has found that 250 men and women with oral conditions varying from healthy to diseased all showed oral spirochetes when specimens were examined by both dark field and stained smear; 57 per cent showed both spirochetes and fusiform bacilli. Brooke3G found spirochetes and fusiform bacilli in 84 per cent of the mouths of medical students and nurses considered normal. Cuthbert and Williams”’ have shown that among naval patients chosen at random and showing no signs of oral disease, both spirochetes and fusiforms were found in small numbers in smears obtained from 63 per cent of the cases. Among 161 men and women in the age group 20 to 30 years, Hotchkiss38 found an incidence of 60 per cent. PiloF9 found that in smears from the mouths of medical students, 50 per cent showed fusospirochetal organisms, whereas all the ward patients of a hospital showed them upon entrance. Breazeale and Greene,4” in examining a group of 719 school children and young adult,s, showed that 3.5 per cent gave posit,ive smears for spirochetes and fusiform bacilli. The lowest incidence was in school children of superior social and financial status; the highest was in a group of Mexican children from homes of low social and economic status. Among young ad&s the incidence was 27 per cent for enlisted men of the Arizona National Guard, 25 per cent for college women, and 36 per cent for college men. Pilot and BramF found spirochetes in 25 per cent of removed tonsils, but Okabe*; claimed to have found them in all tonsils he examined. It was found by Reckord and Baker 42 that 16 per cent of the smears from the teeth of 25 normal individuals showed the two organisms, but smears from diseased teeth showed an incidence of 90 per cent. Rosenthal and GootzeiVs have found that of 212 completely edentulous healthy mouths examined, only two (0.9 per cent) Of 68 partially edentulous showed spirochetes and fusiform bacilli together. jaws examined, 51 (75 per cent) showed fusospirochetal organisms on the gingivae about the remaining teeth. Blaek,4” in a careful study using both stained smears and the dark field, reported that among 33 children spirochetes were found in 36 per cent of those under 3 years of age, and in 77 per cent of those over 3. None were found in the first year of life. Kostecka45 found no spirochetes or fusiform bacilli in the mouths of 50 edcntulous patients and could find none in young infants until the dentition began to appear. It is difficult accounting for the wide variations of incidence reported (Table III), but it is certain that these reports, as well as the many others not cited here, were based upon variations in methods of examination and interpretation which make it impossible to consider them comparable to each other. The failure to use the dark field in most of these investigations constitutes a serious fault in method. Perhaps the only conclusions that may be drawn are that in the very young and in the edentulous, spirochetes are not found, but in those with teeth the incidence is very high.

COLLECTIVE

TABLE III.

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REVIEW

INCIDENCE OF ORAL SPIROCHETES AS REPORTEI) BY HOME INVESTIGATORS

AUTHOR

Stafne Okabe Reckord and Baker Brooke Black Rosenthal and Gootzeit Cuthbert and Williams Hotchkiss Pilot Black Breazeale and Greene Reckord and Baker Rosenthal and Gootzeit Kostecka Kortecka Black

NUMBER EXAMINED

250 100

51 161 719 25 212 50

CHARACTER

OF SMEAR

SOITRCE

Adults, varied oral health Healthy and ill persons Diseased teeth Medical students and nurses Children over 3 years old Partially edentulous cases Naval patients, normal mouths Young men and women Medical st,udents Children under 3 years old School children and young adults Normal individuals ’ teeth Edentulous persons Edentulous persons Infant,s before dentition Infants in first year of life

PERCENT INCIDENCE

100 100 90 81 77 75 63 Ei 36 35 16 0.9 ii 0

The curious association of spirochetes with fusiform bacilli still remains unexplained. Tunnicliff ‘s claim4” that the two forms are simply different stages in the life cycle of one organism has not been accepted. The latest evidence refuting her thesis consists of the demonstration that by choosing cultures of fusiform organisms where atypical forms are abundant and by manipulation of the various factors involved in making smears and staining, artifacts may be produced which resemble the spirals Tunnicliff has observed “developing” from fusiform bacilli.47 It is of interest to note that experimental associations of the oral spirochetes with a number of microorganisms, including the fusiform bacillusJ have been observed to favor spirochetal growth. S&guin 48 has found it impossible to gsow Spirochaeta acuta, a small spirochete, unless Bacillus fusiformis is present in the medium as an associate. Kast and KolmeP9 also observed an enhancement of growth of spirochetes by contaminants. Using a serum agar medium, these workers found that without fresh brain tissue no growth of Treponema microdentium occurred, but when some of the medium tubes became contaminated with Xtaphylococcus aureus, growth took place, the staphylococcus serving as a substitute for tissue. Cocci have also been observed to favor the growth of fusiform bacilli, which may be a factor involved in the fusospiroehetal symbiosis.5o Skguin and Vinsent? believe that in the case of weakly proteolyt,ic strains of oral spirochetes, an association is generally formed with an organism which is strongly proteoIytic. At,tempts to determine the relative numbers of t,hc various species of oral spirochetes found in different conditions are confronted with formidable difficulties. Relative numbers vary with the conditions of the mouth and the localities from which specimens are taken. Cultural methods of investigation are inadequate since the conditions of artificial .cultivation have practically no similarity to those in the mouth. All that is possible is to record general impressions. NoguchP found Treponemu microdentium most frequently at the gingival margin and in the cavities of teeth ; the larger forms were found more frequent12 in the mucus about the tonsils and pharynx, and in great numbers in ulcerative stomatitis. He also stated5” that Treponema mucosum is the only treponema constantly associated with pyorrhea alveolaris. Stafne”” found that the larger forms predominated in unclean mouths in which there were lesions and that Treponema micdentium predominated in the clean healthy mouth. Rorrelia vincentii was found predominant in a greater number of cases than the other

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OF

LITERATURE

forms. Okab? reported that both the small and large forms were l’ound in almost equal numbers about t,he gingival margins of the teeth, but that the large forms predominated. The opposite relat,ionship was found for the pharyngeal tonsil where all those examined showed spirochetes also. In the mouth and throat, when the pharyngeal tonsil was infected, the! large forms were found in 95 per cent of the cases, and the small forms in only 67 per rent. HamppG found by dark field that mat,erial from lesions of Vincent’s infection showed a large percentage of t,he represent,ativc spirochetes; for example, Treponenaa naicrodentium constituted about 22 per cent of the total spirochetes and Bor,relia b~~c(,nle may far outnumber the Vincent’s spirochetes in such material. From these reports it would seem that as a rule, in obviously pathologic conditions, the larger forms replace t,he smaller forms to a certain degree, becoming predominant. However, this does not seem invariably true, for SCguin and VinzenP have reported that they found the small forms predominant in putrid and gangrenous lesions of the respiratorv tract, as well as in experimental lesions in guinea pigs and rabbits inoculated &th organisms from the mouth. Just what relationship the human oral spirochetes hear to the commensal and pathogenic forms commonly found in other parts of the body has been a subject for considerable controversy but for few experimental investigations. The tendency is to regard at least some of the spirochetes found in the external genitalia, gut, and lungs as very closely related, if not indeed identical with the oral spirochetes. Noguchis4 found a slight immunologic relationship between two strains of Treponema minutuna from the genitalia and Treponema microdentiuna. Vinzent and %guin”” isolated Tyepone,nm nvicrodentium from the pus of gangrenous appendicitis but later were unable to isolate it from fecal cultures. Therefore, t,hey stated, the organism may have its source in t,he oral carit,- and is not normally present in the intestine. Hindle”” stated it, is possible that bronchial spirochetosis is an example of normally sal)rophytic mouth spirochetes becoming pathogenic by mutation and adaptation. This may also be the case for various ulcerous conditions of the alimentary tract, but no clear-cut evidenec exists. Spirochetes have been noted in the oral cavities 01’ a number of animal species. KellyZ7 found that the tonsillar mucous membrane of the rhesus monkey (Maraca nautatta) would sometimes show spirochetes, generally of the Three guinea large variety resembling Borrelia OzLccnle or Romelin ztineentii. pi$s with normal gums, examined by Lichtenberg, Werner, and Lueck,“P showed neither spirochetes nor fusiform bacilli, but, after being traumatized an exudate formed which showed many of the organisms. Phillips and Berry”” found the typical association of fusiform bacilli and spirochrtes in a nccrot,ic lesion of t,he lower lip of a dog. Smears from normal dogs in the same kcnnt~l showed the same organisms, but in relatively small nnmbcrs, and cight,een mont,hs later the same picture was found. The relat,ionship of these spirochetes to those in the human oral cavity is not known.

Pathogenicity Clinical Observations.--Of the naturally occurring oral infections, Vincent’s stomatitis is one of the most commonly seen. The lesions may involve the gingivae, tongue, oral mucosa, and lips. The most common sites are the lower and upper incisor and the third molar regions. The involved gingivae become swollen and ulcers are present wit,11 a necrotic membrane which reveals a raw, bleeding surface when removed. Predisposing factors may be calculus, faulty dental restorations, third molar operculum, poor diet and poor oral hygiene, the presence of mercury or obher heavy metals, or a blood dpscrasia.

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Rosebury”” has given an excellent summary of the etiology of Vincent’s infection which is still largely up-to-date. The histologic picture, as indicated by infected tonsils,61 shows an occasional cocc~~s, fusiform bacillus, and spirochete in the necrotic epithelium; the layer between the necrotic and living tissue shows a few leucocytes, some fibrin, and large numbers of fusiform bacilli radiating out from them. Both organisms are seen invading the living tissue, but the spirochetes are ahead of t,he fusiform bacilli. On the whole Vincent’s infection is a rather mild disease which makes a sharp contrast to noma, a widely distributed but rare malignant form of fusospirochetosis most commonly found among children of poor general physical condition, and usually following an infectious disease such as measles or scarlet fever. The most frequent site of noma is t,he mouth but various other parts of the body are susceptible. This disease is a process of slowly spreading gangrene involving mucous membrane or mucocut,aneous orifices. The final outcome 01 death is due to septicemia, with pulmonary gangrene as a terminal complication, ljrobably due to aspiration of necrotic material from the mouth. AgnewG2 gives a splendid, thorough description of noma in all its phases. For an excellent historical review see the paper by Weaver and Tunnicliff.“” Experimental Findings.-There is no evidence that the oral spirochetes alone can produce disease in man. Efforts by Okabe” to demonstrate toxin production consistently resulted in negative findings, and the inoculation of pure cultures of strains of oral spirochetes has not been found to produce any characteristic results of infection.“* Nevertheless, there are several lines of evidence which indicate that the spirochetes, in combination with fusiform bacilli? as well as vibrios and cocci, may be the etiologic agents in the lesions in which they are characteristically found. According to Appleton,6” these are : 1. The organisms are found in the lesions in enormous number, the more severe the lesion, the greater the number. %. The evidence for infectiousness and contagiousnessindicates a parasitic origin. 3. Healing of the lesions is accompanied by disappearance of the organisms. 4. Histologic pidure of the lesions. 5. Results of animal inoculation.

Efforts to produce fusospirochetosis experimentally have met with a certain degree of success, but much remains to be done. Proske and SayersG4 confirmed I). T. Smith’s findings”” by showing that a typical fusospirochetal abscess may be developed by the injection into guinea pigs of a uniform mixture of pure Treponema mucosum, anaerobic anhemocultures of Treponemu microdentium, lytic streptococcus, Vibrio virida,ns, and Varney type I (subtype 2) and type II of the fusiform bacillus, which had been isolated from the upper respiratory tract of man. They found Treponema microdentium and Treponema mucosum may be substituted for each other. On repeated passage of the symbiotic combination through guinea pigs, it was noted that the individual species, originally nonpathogenic, became capable of setting up local reactions when inoculated separately. These workers suggested further investigation to determine whether or not there are other organisms of the upper respiratory tract which may replace one or the other of the members of the symbiotic group they found. Foley and Rosebury 67 have found that suspensions of gingival scrapings from patients with various clinical forms of oral fusospirochetosis, as well as material from lung ab,scess cases, all gave approximately similar results when injected in to the groins of guinea pigs, thus indicating that fusospirochetal exudates from different human sources, irrespective of the clinical differences in the source diseases, are all approximately equally pathogenic for guinea pigs.

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Discussion As U. T. Smith et aLo emphasized, and as the material here presented indicates, the essential role of the oral spirochetes in Vincent’s infection is as a Since no attempt participant in a complex association of several microorganisms. has been made yet to analyze the mechanisms involved in the association, the following provisional scheme of relationships is formulated as a guide in the experimental investigation of the problem : favor Cocci -----+

Fusiform

favor Bacilli t-----Oral

Vibrios

. Oral Spirochetes On the basis of available evidence, the unknown factors involved may possibly include the establishment or enhancement of anaerobiosis by the cocci and their elaboration of hyaluronidase,69 which may account for the tissue invasion of the normally noninvasive oral spirochetes. Black44 has stated that the streptococci and staphylococci may indeed be the primary cause of Vincent’s stomatitis since these are the only organisms involved which have an independent pathogenicity. The release of simpler nutritional elements by the symbionts, as postulated by X6guin and VinzenP in regard to proteins, may also be a factor favoring the growth of the oral spirochetes. A large numb,er of additional possibilities may be advanced, not only in regard to the symbionts themselves,70 but also in regard to the participation of the host in the association and the mechanisms of host resistance-questions almost completely ignored in this review. A recent review by Stavitsky71 presents much relevant material on this subject, although it has particular reference to leptospirosis, relapsing fevers, and syphilis.

Conclusion It is obvious that the present state of knowledge of the oral spirochetes is far from complete. However, encouraging progress has been made in the past few years, and it seems reasonable to believe that the renewed interest now apparent will eventually result in significant advances in the understanding of the fusospirochetoses and treponematoses.

References Bergey’s Manual of Determinative Bacteriology, ed. 6, Baltimore, 1. Breed, R. S., et al.: 1948, Williams & Wilkins Company, p. 1051. 2. Wilson, G. S., and Miles, A. A.: Topley and Wilson’s Principles of Bacteriology and Immunity, ed. 3, Baltimore, 1946, Williams & Wilkins Company, p. 907. Spirochaetes: A Review of Recent Work With Some Original 3. Bosanquet, W. C.: 1911, W. B. Saunders Company, p. 11. Observations, Philadelphia and London, 4. Noguchi, H.: “The Spirochetes, ” in Jordan, E. O., and Falk, I. S. (Ed.): The Newer Knowledge of Bacteriology and Immunology, Chicago, 1928, The Univensity of Chicago Press, p. 462. Bacterial Infection, With Special Reference to Dental Practice, 5. Appleton, J. L. T.: ed. 3, Philadelphia, 1944, Lea & Febiger, p. 332. 6. Hampp, E. G.: Vincent’s Infection, a Wartime Disease, Am. J. Pub. Health 35: 441, 1945. Pulmonary Infection in Pneumoconiosis. I. A 7. Proske, H. O., and Sayers, R. R.: Bacteriologic and Experimental Study, Pub. Health Rep. 49: 839, 1934.

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Comparative Study of Darkfield and Stained Smear techniques fol 8. Hampp, E. G.: Identification of Oral Spirochetes on the Basis of Morphologic Characteristics, J. Am. Dent. A. 32: 318, 1945. Polymordhism in Treponema Recurrentis and 9. Culwick, A. T., and Fairbairn, H.: Spiroehaeta Vincenti, Ann. Trop. Med. 41: 1, 1947. 10. Mudd, S., Polevitzky, K., and Anderson, T. F.: Bacterial Morphology as Shown by V. Treponema Pallidum, T. Macrodentium and T. the Electron Microscope. Microdentium, J. Bact. 46: 15, 1943. 11 . Dvar M T . Isolation and Cytological Study of a Free-Living Spirochete, J. Bact. ” h4:

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Characteristics of 12. Hampp, E. b., Scott, D. B., and Wyckoff, R. W. G.: Morphologic Certain Cultured Strains of Oral Spirochetes and Treponema Pallidum as Revealed by the Electron Microscope, J. Bact. 56: 755, 1948. 13. Hampp, E. G.: Morphologic Alteration of Smaller Oral Trepnnemes During Aging of Cultures: Effect of Age on Viability of Spirochetal Cultures, J. Am. Dent. A. 33: 201, 19i6. 14. Akatsu, S.: The Influence of Carbohydrates on the Cultivation of Spirochetes, J. Exper. Med. 25: 375, 1917. Quelques don&es rBcentes sur le spirochCtog&ne en 15. Simon, C:,. and Mollinedo, R.: ,syphlllpraphie, Bull. med., no. 18, 1943, p. 273, Abst. in Bull. Inst. Pasteur 44: la& 1926: L. B., and Wichelhausen, R. H.: 16. Robinson, The Problem of Identification of Oral Spirochetes and Description of a Precipitin Test for Their Serological Differentiation, Bull. Johns Hopkins Hasp. 79: 436, 1946. The Role of the Spirochaete in the Wassermann Reaction, J. Hyg. 39: 298, 17. Beck, A.: 1939. J A: h plrochetal Antigens in Serum Diagnosis of Syphilis, Am. J. M. 18. Kolmer Tehhnil 9’ 3,’ ,943 h. %., and Wichelhausen, R. H.: Cultivation and Isolation of Mouth 19. Wichelhauseil, Spirochetes, J. D. Re,s. 21: 543, 1942. 20. Hampp, E. G.: A Method for Routine Isolation and Cultivation of the Smaller Oral Treponemeq J. Am. Dent. A. 30: 1066, 1943. and Pure Cultivation of the Smaller Mouth T., and Foley, G.: Isolation 21. Rosebury, Spirochetes by an Improved Method, Proc. Sot. Exper. Biol. & Med. 47: 368, 1941. Preservation of Borrelia Vincenti and Cultured Strains of Treponema 22. Hampp, E. G.: Pallidum by the Lyophil Process, J. Am. Dent. A. 34: 317, 1947. H. R., and Frazier, C. N.: A Study of the Nutritional 23. Whiteley, Requirements of the Reiter Strain of Treponema Pallidum, Am. J. Syph., Gonor. & Ven Dis. 32: 43, 1948. H. G.: The Nutritional Requirements of Treponemata. I. 24. Eagle, H., and Steinman, Arginine, Acetic Acid, Sulfur-Containing Compounds, and Serum Albumin as Essential Growth-Promoting Factors for the Reiter Treponeme, J. Bact. 56: 163, 1948. P. A., and SubbaRow, Y.: Use of Refined Serum Albumin as a Nutrient for 25. Little, Treponema Pallidum, J. Immunol. 50: 213, 1945. The Effect of Lipids and Serum Albumin on Bacterial Growth. J. Exper. 26. Dubos, B. J.: Med. 85: 9, 1947. Zentralbl. f. Bakt. (Abt. 1) 136: 485, 1936. 27. Okabe, S.: Studien iiber MundspirochLten, Untersuchungen iiber den Stoffwechsel der SpirochLten in vitro, Zentralbl. 28. Scheff, G.: f. Bakter. (Abt. I) 134: 35, 1935. of Various Spirochetes in J., and Noguchi, G.: On the Resistance 29. Bronfenbrenner, Cultures to the Action of Chemical and Physical Agents, J. Pharmacol. & Exper. Therap. 4: 333, 1912-1913. Spirocheticide, J. Lab. & Clin. Med. 21: 922, R. S.: Sodium Citrate-a 30. Leadingham, 1936. The Spirochetal Action of Penicillin in Vitro and 31. Eagle, H., and Musselman, A. D.: Its Temperature Coefficient, J. Exper. Med. 80: 493, 1944. 32. Goldman, H. M., and Guralnick, W. C.: Some Uses of Penicillin in Dentistry. A Collective Review, Oral Surg., Oral Med., and Oral Path. 1: 116, 1948. 33. Wilson, G. S., and Miles, A. A.: op. cit., p. 1984. 34. Appleton, J. L. T.: op. cit., p. 415. and Fusiformis Dentium in the Mouth, Dental Cosmos 35. Stafne, E. C.: Spirochaetaceae 70: 493, 1928. Regional Study of Bacteria in Clinically Normal Mouths, J. D. Res. 36. Brooke, J. W.: 17: 57, 1938. Gingivo-Stomatitis, With Special Reference to 37. Cuthbert, J., and Williams, F. R. P.: Vincent’s Gingivitis, J. Roy. Nav. M. Serv. 29: 115, 1943, Abst. in Bull. War. Med. (Great Britain) 4: 358, 1944.

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57. 58. 59. 60. 61. 62. 63. 64.

2 67. 68. 69. 70. 71.

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