STUDIES ON THE BIOLOGG OF STREI?TOCOCC I.ANTIGENIC~LA~TI~N~HI~S BETWEENSTaAINs OF%REPTOC EiEMoLYTICrJs. By A. R. DOCHEZ, M.D., 0. T. A-YY, M.D., AND R. C. LANCEFIELD. ; (From the Zhs#tal of The Rockejd&v I&&& for Medical Research. . (Received for publication, June 1, 1919.) / The importance of the problem of the systematic classification of bacteria for the proper understanding and control of infectious diseases is becoming increasingly evident. Such study is necessary not only in the elucidation of the biological relationship existing between varieties of the same species of bacterium, but is also essential to the working out of epidemiological problems and to the de&l: opment of knowledge useful in the &ort to control infectious piseases by means of specitic therapeutic and prophylactic measures. Bac- teria closely resembling those responsible for the pathological process in many acute .infections have been found to be present and to live, apparently without harm to the host, on the mucous membranes of. a large proportion of normal individuals. The resemblance of the pathogenic to the harmless variety of microorganism is frequently so close that in many instances tests of particular specificity are re- quired to show the existing biological differences. In fact, the problem in etiology .today is to determine not only the bacterial species cak- ing a given disease, but in addition the number of varieties of the same species that qe pathogenic, and whether common and important non-pathogenic varieties exist. The study is one of varying com- plexity, and methods suitable to one species do not give the desired information when applied to another. The purpose of such studies may be broadly defined as an effortto relate fixed and determfna ble characteristics of bacteria to patho- genicity. Though fluctuating variations of bacteria probably occur, it seems not unlikely that in most diseases a sutIiciently constant equi- librium has been attained to justify the usefulness of the effort. .-The 179 180 BIOLOGY OF STREPTOCOCCUS. I methods employed in these studies are numerous. In some species morphological and cultural characters give `important though some- what limited information, and in many the biochemical reactions are of great significance. The most serviceable method, however, for obtaining the particular kind of knowledge desired `is the study of immunological relationships. For some as yet rrnexplained reason the latter specitic reactions are very constant among the pathogenic varieties. In the following study of Streptococcus hamolyticus chief dependence has been placed on the knowledge obtained from the study of these immunity reactions. In the various czhsifhtions of the streptococcus group as a qhoIethat have been proposed, the custom has been to consider the strains that effect hemolysis of red blood cells as constituting a single or unit type (1). The validity of this assumption has been questioned and there has been much study and discussion of the probability of the existence of variations within this group, some evidence of which has been obtained from the study of sugar fermentations (2). Recent studies (3) indicate that certafn broad lines of differentiation may be shown between hemolytic streptococci of human origin, and those of bovine origin whether found in milk or cheese. Hemolytic streptococci of the human type are usually found in association with some pathological process such as puerperal sepsis, septicemia, erysipe-las, bronchopneumonia, or other conditions, and hemolytic activity is generally considered as an indication of pathogenicity. With the development of our knowledge (4), however, these organkns have been found with iucreasing frequency when no pathological lesion has been apparent. Investigators for many years have been interested in these strains, and dkcuasion has centered about the unity or multiplicity of the group (5). The evidence in general favors the belief that hemolytic streptococci pathogenic for human beings comprise a single type.. In the present paper it will be shown that by the use of properly controlled immune reactions dXerential characters between individual strains &n be shown to exist. During the winter of 191748 in the United States, there occurred in numerous localities a great increase in the incidence of a previously rather infrequent type of bronchopneumonial The highest morbidity rate and earliest appearance of this disease were in military can- tonments. In the spring of 1918, however, the type of infection under consideration was commonly observed in the civilian popu- !lation. The disease first appeared as a secondary pneumonia fol- lowing measles, but soon instances of apparently primary infection .of the lungs were observed. Numerous studies of the bacteriology A. R. DOCHEZ, 0. T. AVERY, AND IL C. LANCEFIELD 181 of this condition have demonstrated that the infectious agent re- sponsible for the lung lesion was in almost every instance Strepto- coccus hamolyticus. As a result of the widespread incidence of the disease the latter organism became extensively distributed and was frequently found as a secondary invader in acute lobar pneumonia, and as a common inhabitant of the normal throat. The material used in the present study was obtained from the military establishments in the neighborhood of Fort Sam Houston, Texas. The sources of the individual strains were the throats of patients suffering from acute measles, the sputum of patients with bronchopneumonia both primary and secondary to measles, patho- logical material obtained from cases of bronchopneumonia and acute lobar pneumonia, and the throats of healthy individuals who had been directly or indirectly exposed to infection in a variety of ways. All the strains of hemolytic streptococcus employed in this study possess the typical characteristics of the group. They are hardy organisms and grow readily in meat infusion broth and on blood agar slants. They survive for many months when grown for 18 hours in rabbit blood broth and subsequently placed at refrigerator tem- perature. In meat infusion broth the growth has been of two types -a granular sediment with clear supernatant fluid and a flocculent sediment with turbidity throughout the remainder of the tube. All. the organisms are strongly Gram-positive, grow in chains of varying. length, and are bile-insoluble. Capsule formation has not been ob- served. On plates two types of colonies are seen-a small, round, smooth colony and a moist ameboid colony with a slightly roughened surface. AU the strains are actively hemolytic, showing a wide zone of hemolysis on the surface of rabbit blood agar plates; hemolysis is complete in 2 hours, when a 5 per cent suspension of washed rabbit blood cells suspended in salt solution is mixed with an equal quantity of a 24 hour broth culture. The power of the tierent strains to ferment the usual test substances for streptococcus has been studied. The majority fall in the group of Streptowuxs pyogms according to Holman's classification of streptococcus on a basis of sugar fermen- tations. None of the strains ferments inulin and about 20 per cent of these actively hemolytic strains possess the power of fermenting mannite. The latter characteristic will be shown to have an inter- 182 BIOLOGY OF STREPTOCO-CCUS. `I esting relationship to the immunological classitication of these organisms. . The virulence of this group of hemolytic streptococci is low for the ordinary laboratory animals in comparison with such an or- ganism, for instance, as pneumococcus. Doses of 1 cc. or more of a 24 hour-broth culture administered intraperitoneally are required to hill guinea pigs and rabbits. Furthermore, repeated passages through these animals fail to bring about a considerable accession of `viruIence. The fatal dose for white rats and mice is smaller, usually in the neighborhood of 0.1 cc. of a broth culture. By continuous passage through rats and mice it has been possible to raise the virulence of a certain number of strains to a point where 0.001 cc. of broth culture is lethal for a rat in 24 hours and O.OOO$MjOl cc. for a white mouse. On the other hand, many strains cannot be raised to this high degree of virulence even after the most persevering eiXort. Once the max- imum of virulence is attained, this quality persists without renewed animal passages for an indeCnite period of, time. The sources of the strains of Streptococcus hamdytkus studied and some of their common characteristics are shown in Table I. Although the finer differential classification of single species of bacteria by means of immune reactions is still in the earlier stages of development, enough evidence has been gathered to indicate that the more highly parasitic varieties of the species are more likely to consist of a limited number of unit types than are the less parasitic or the saprophytic members. In other words, unity of type seems to characterize the disease-producing microorganisms, whereas hetero- geneity is more common among the non-pathogens. If this'assump- tion is true it then becomes important to choose for purposes of classiiication the immune reactions which bring out most sharply the kind of differences sought, rather than a reaction which develops the basic relationship existing between all strains of the same species. For this purpose we regard the reactions of agglutination and pro- tection as of superior usefulness to those of precipitation and com- plement fixation. The validity of any final classification arrived at depends, of course, upon the possibility of fitting accurately into such a classitication a large number of strains freshly obtained from their natural environment. A. R. DOCHEZ, 0. T. AVERY, AND R. C. LANCEFIELD 183 TABLE I. soz4rce and co?nmo?l Chamcters of Strains of Stre~t~c0ccu.s hmwlyticus Studi4d. "Es? soorce. clini diagno8ie. Hcmolysia. s29 s 114 s 118 S 124 SIB5 SM.6 Sl49 s 151 Sputum. " FleuraI fluid. sputum. Throat. chest fluid. Blood. PIeuraI fluid. s2 s3 s 14 s 111 Autopsy 0. " yd. " -Chest fluid. S53 564 SC54 S80 s140 s II S 16 s31 S44 SS3 s95 s 125 Sl44 Ttma+ u `1 `4 `L Sputum. " Throat. `6 `I " `, `I `# I` " Types3. following ++++++--- measles. " u ++++++--- `8 `6 ++++++--- " " ++++++--- " " ++++++--- " " ++++++--- u u ++++++--- " U ++++++--- 44 " ++++++--- Bronchopneumonia. " " " ++++++--- ++++++--- ++++++--- ++++++--- MePsla. ++++++--- . 64 ++++++--- `& ++++++--- Germanmeades. ++++++--- " " ++++++--- Lobarpn~llia(TypeI). ++++++--- " u ++++++--- " " ++++++--- " `4 ++++++--- 46 " ++++++--- ,* " ++++++--- " u ++++++--- 4` 6` ++++++--- Rmmonia. ++++++--- " ++++++--- Incipient turn ++++++--- 184 BIOLOGY OF STBJWTOCOCCUS. I . TABLE I-Continued. Type S 23. S39 folIowing s 78 s 101 s 107 Pleural fluid. 4t 1` sputum. Bronchopneumonia measles. `6 " " " " " s 27 S 67 s 120 Autopsy (blood). Blood. Autopsy (blood). Bronchopneumonia. " " S98 S 116 s 117 Tbruat. " `6 MC.&=. 1` 1` s9 S 23 S 56 S6S s 7s s 133 1` 1` Autopsy (lung). sputum. Throat. " Lobar pneumonia. 6` " `I l` S` `4 `6 " " " s104 s 130 S46 s 122 Pneumonia. " folIowing ++++ + + - - ++++++-- " ++++++-- ++++++-- " ++++++-- ++++++-- " ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- +++-t++-- +++-t++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++++-- ++++-i-+-- ++++-i-+-- - . . . . . . . . . . Incipient tuberculosis. 6` " - A. R. DOCJiJZZ, 0. T. AVERY, AND R. C. LANCEFIELD 185 TABLE I-continwd. Sf37 S6 s 35 s55 s 10 s37 S43 S60 S66 s 71 s 86 s 88 S 89 Sloe s109 S 123 S 127 S 128 s4 s 19 s 62 S 72 s 21 s 141 s IX S 265 s26s Pkual fluid. AU~WJY 0. Plealralfhid. sputum. Throat. id " 4` II u " " `4 `6 " Id " 4` Foot. . Blood. Type s 60. Bronchopneumoti folloming measles Bronchopneumonk " `4 Measles. d` " " " " " " " " 4` u u " Germanmeasles. Cibar pneumonia. 6` `4 " " blcipient tuberculosis. 1` 6` " " CelbllitiS. Erysipelss. +++++++-- +++++++-- +++++++-- t++++++-- +++++++-; ++++-f-++-- +++++++-- ++++++-l--- +++++++-- +++++++-- +i-+++++-- t++++++-- t++++++-- t++++++-- t++++++-- t++++++-- t++++++--. t++++++-- t++++++-- t++++++-- +++++++-- t++++++-- t++++++-- t++++++-- t++++++-- +++++++-- +++++++-- 186 BIOLOGY OF STREPTOCOCCUS. I TABLE 1-&?&d. Sl AUWSY Wd. s20 s 110 S 138 " wad). sputum. `8 SSO S84 s 139 Pleural fluid. " `6 Throat. s 11s " s 15 6` S32 Autopsy Ouw$. 559 s93 s97 S 136 S 142 `, (blood). sputum. Pleural fluid. Autopsy Owd. sputum. S49 `1 s 24 S 18 S 26 S42 s 51 s63 S% s 102 SlOt s 1oe s 142 Throat. 4` " " " " " `6 u I` - - Bronchopneumonia foBowing measles. " u u u " *< " u " `I Bronchopneumonia Germanmeasles. following Measles. `1 " 6` " " " ,` " " - TypeS84. Bronchopneumonia following ++++ + + - measles. I` u ++++++- u " +-l--i-+++- " u ++++++- Bronchopneumonia. ++++-b+- `4 ++++++- " ++++++- German measles. ++++++- Lobar pneumonia. ++++++- UnclassZed. . . . . . , . . . . . -- -- -- -- -- -- -- -- -- - ++++++--- +i-++++--- ++++++--- ++++++--- ++++++--- ++++++--- +++++.-i---- ++++ +:-k - - - +++++-l---- +-t+++-i---- +-t++++--- ++ ++--+ ++++*+--- ++++++--- ++++++--- ++++++--- ++++*+--- ++++++--- A. R. DOCEJX, 0. T. AVEBY, AND R. C. LANCEFIELD 187 s 17 s47 S48 S54 S68 S69 S 87 S90 S99 S34 S36 s 121 s129 s 155 s264 S 271 S 272 S 273 S 276 S 277 s286 s288 Throat. 6` " sputum. Throat. Autopsy (lung). sputum. Thmat. sputum. Throat. u u (I 64 B&d. u Pus. 6C u 4` .(abdumen). Pleural fluid. sputum. Gwmanmeasles. " `C u " " " " " Lobar pneumonia. " `L " "` " " Iucipimt tu~osis. " `L II u 6` `6 U u Osteomyelitis. septicemia. Abscess (mea&s). scarlet few+ Pelvic abscess. Peritonitis. Pneumonia. U* + -- +-- +++ +---- t+++++--- t+++++--- t+++++--- t+++++--- t+++++--- t++i++--- t+++++--- t++++++-+ t+++++--- t++++---- t+++*++-- t+++++--- t+++++--- t+++++---. t+++++--- t+++++--- t+++++--- t+++++--- t+++++--- t+++++--- 188 BIOLOGY OF STREPTOCOCCUS. I The Riaction of Agglutiization. Specific agglutination has been found to be one of the most ser- viceable immune reactions for purposes of the biological classif% cation of bacteria. In the typhoid and pneumococcus groups, for instance, it serves to distinguish clearly the difIerent varieties from one another. It is likewise applicable to the classification of many other microorganisms. Efforts to classify the streptococci by means of this reaction apparently have not illuminated materially the relationship of one strain to another, nor have they shown a definite relationship between certain strains and a particular pathological process. A number of explanations of this fact may be proposed. In many instances no attention has been paid to the broader groupings of streptococci as determined by hemotoxin production and the fer- mentation of test sugars. Also streptococcus most frequently acts as a secondary invader in the production of disease, and it is prob- ably an unwarranted assumption to suppose that type specificity is closely related to the character of the pathological process. One of the chief obstacles to the successful carrying out of the agglutination reaction has been the tendency of all types of streptococcus to undergo spontaneous granulation, and when used for tests to exhibit the phenomenon of non-specifk agglutination. As a result of this, the reactions must usually be read against a more or less granular back- ground, making it dif3icult, if not impossible, to distinguish between the non-specific and the specific influences. The tendency to spon- taneous clumping is occasioned by several factors, only a few of which are understood. For instance, a homogeneous suspension of a granular streptococcus can easily be prepared by washing the organism several times with distilled water, and then resuspending in the same medium. The suspension will remain homogeneous for an indefkite period of time. If sodium chloride in concentrations above 0.06 per cent is added to the suspension, granulation immediately ensues. Many other salts act in the same manner. Substances antagonistic to this salt action may be added to the medium and function even to the extent of suspending the participation of the salt in the immune reaction, so that specific agglutination may be completely inhibited. Fortunately intermediate combinations can be found in which most A. R. DOCEEZ, 0. T. AVERY, AND R. C. LANCEFIELD 189 strains remain diffuse and in which the salt is stiIl able to fuhilI its part in the immune reaction. The most useful substance of this hind is ordinary meat infusion broth to which 1 per cent peptone has been added.' In addition, if streptococci are exposed to too great acidity the tendency to granulation is increased. In order to avoid this, the reaction of the medium may be so adjusted and such quan- tities of balanced phosphate solutions added that during growth an acidity greater than pH 7.1 is not attained. Certain other unde- termined factors cause granulation, which may be defined as a general unsuitability of the medium for growth, and these can be eliminated only by experimenting with di.Berent preparations. Technique. The immune sera used in the agglutination and protection tests were obtained by the immunization of rabbits, sheep, and dogs. The animals were inoculated intravenously with repeated doses of heat- killed organisms, and in most instances a certain number of doses of living organisms was given. The employment for immunization of freshly isolated unpassed human strains, or the use of the same strains after a series of animal passages, does not alter in any recog- nizable way the specific qualities of the serum. The agglutinin and protective titer of the sera has remained undiminished for many months after the time of bleeding. Great care is taken in the preparation of the organisms, to be used in the agglutination reaction. The broth is made from carefully selected meat, and instead of the usual sodium chloride a sticient quantity of a balanced phosphate mixture is added to give the re- quired salt concentration and to adjust the hydrogen ion concen- tration at a pH of 7.4. When Streptococcus hmnoZyticua is grown for 24 hours in a medium to which no sugar has been added, it does not develop an acidity greater than pH 7.2, which is just above the point at which the tendency to granulation appears. The organisms are removed from the culture medium by centrifugalization and are I The authors are greatly indebted to Dr. CharIes Krumwiede, Jr., of the Research Laboratories of the Department of Health of the City of New York, for many helpful sug&stions in this technique. 190 BIOLOGY OF STREPTOCOCCUS. I washed once or twice in broth with a pH of 7.4 prepared in the manner described above. They are then resuspended in the same medium in approximately the concentration that is attained after 24 hours growth and are ready for use in the reaction. The sera to be employed in the tests are made up to the various concentrations by dilution with the same broth used for suspending the bacteria. The reaction of each specific serum is controlled by a complete series of like dilutions of normal serum of the same animal. Equal quantities of serum dilution and bacterial suspension are added to each tube and the tubes are then placed at a temperature of 55oC. for 1 hour. A temperature of 55oC. must be accurately maintained during the entire period and the tests must be read after the lapse of 1 hour, since if they are TABLE IL Puwcr of Antktre~tocouus Stnm, Ty#e.S 3, to Agglutinak Tm Rcpres~w Straim of the Same Tyjw. 1 Dilution. Strain No. s3 s 14 Sl49 595 S80 S 118 s 111 S140 s 16 S83 Types3 NormaL Types3 Nod. Types3 Normal. Types3 NormaL Types3 Normal. Types3 Normal. Types3 Normal. Types3 Normal. Types3 Normal. TypeS3 Nod. :: 2 . . 2 -A- +* +* - - -+* +* - - +* +* * v: z-+ ++ - - +* +* - - +* +* - - +* +* - - +* +* - - +* +* - - ++ ++ - - I f . . .z 2-- t* +* - - t* +* - - t* +* - - t+ +t - - t* +* - - t* +* - - t* +* - - t* +* - - t* +* - - t+ +t - - - : . : - : . . . : . : . : . : . : . . . / 5 333 t; ; : : ---- t*+*+* + - - - - t*+*+* + - - - - t* +* +* +* - - -_ - t+ ++ ++ +t - - - - t* +* +* +* - - - - t*+*+* + - - - - t* +* +* +* - - - - t* +* +* +* - - - - t* +* +* +* - - - - t++++* + - - - - -g -- ;h - dz - t* - t* - + -. * - + - 4-t - + - * - TABLE III. T& of Cross-Agglutindh ~ivns of Antktreptococcus Serum, Tyfie S 3, with &h.s of Streptocotcus myticw 01 Other Ty#s and with Unclussi&d Slrains. - str$n s3 S 23 s 107 S 67 S65 s 75 S 128 s 55 S60 S 267 s4 S84 Sl SSO s 20 s11.5 s 31 s108 S87 S 288 S42 s 121 = s. hzoTy"L. s3 S 23 S23 S 23 S23 S 23 S60 S60 S60 6560 S60 S84 S84 S84 S84 S84 Undassibed. " I` u " `< SCram. Types3 Nod Types3 Normal. Types.3 NormaL Types3 Normal. Types3 Normal. Types3 NotmaL Types3 Nod. Types3 Normal. Types3 Normal. Types3 Normal. Types3 Normal. Types3 Normal. TYPeS3 NormaL Types3 Normal. Types3 Normal. Types3 Normal. Types3 Nod Types3 Nomd. Types3 NormaL Types3 Normal. Types3 Notmal. Types3 Normal. I I + + - s f; - -* -' + - * 191 + * - + - Dilution. + - - 3 f; - -A - - - - - 192 BIOLOGY OF STRJiZTOCOCC!US. I allowed to continue longer, non-specific granulation occurs. If clump- ing deveIops in the broth controls or in more than the Grst two or three dilutions of normal serum, the reaction should be regarded as unsatisfactory and discarded. By the use of this technique, it has been possible to carry out reliable agglutination tests of various strains of StrGptowccus hamdyticm and to show that constant type rela- TABLE IV. Power of AdistreQtowccus Serum, TyQe S 23, to Agglutinde Ten ReQresentdiva Strati of the Same Type. Strain No. S 23 s 107 S 27 s 39 S 56 S67 S98 s 101 s104 s 130 serum. Types23 Normal. Type S 23 Normal. Type S 23 Normal. Types23 Normal. Types23 Normal. Types 23 Normal. Types23 Normal. Types23 Normal. Types23 Normal. Types23 NormaL - 8 1; - 7-t 7-t + + -. + + -* -* + -* - - -. _ . . : . : . : . - s .z - t-i i-t t-l + + + t-t - t-t 4-S - t* - - -_ -_ - _ -_ _ _ _ _ - _ : - - Dilution. 0 s no o 1 ml a I; 1; .. : --A- t+ ++ ++ +i - - - - t+ ++ ++ +t - - - - t++++* + - - - - t*++-i-* + - - - - t*+*+* + - - - - t*+* + + - - - - t+ ++ ++ +* - - - - t+ ++ ++ s't - - - - t+ ++ ++ +-t - - - - t*+++++* - - - 7 q ;' - + r* * * * * -* -* -* + - f N 2 - + + + + + - a 3 . . 1. * * - + - tionships exist, and that the types are sharply differentiated from one another. The results of the application of the method to strains of streptococcus described above are shown in Tables II to IX. In these tables are presented the agglutination reactions of a certain proportion of the total number of strains of Streptococczcs hmdyticzcs tested. An analysis of the results shows that in the collection of TABLE V. Test of Cross-Agghti?tation Reacths of AntistreQtococcus Set%m, TyQe S 23, WUJ Strains of StreQtowccut ho%aolyticus of 0th TyQw and tith S 23 s95 s 149 S80 s146 s 125 s 55 S60 s4 s 19 S6 s 20 s 50 S84 s 115 Sl s 15 S 116 s49 St59 s 155 S 18 S99 S 24 S 23 s3 s3 s3 s3 s3 S60 S60 S60 S60 S60 S&L S84 S84 S84 S84 unclassified. " 1, " " " " " SWUOI. Types23 Normal. Type S 23 $223 Normal. Types23 Normal. Types23 Normal. Types23 Normal. Types23 Normal. Types23 Normal. Type S 23 Normal. Type S 23 Normal. Types23 Normal Types23 Normal Types23 NormaL Types23 NormaL Types23 Normal. Types23 NormaL Type S 23 NormaL Type S 23 NormaL Type S 23 Nomal. Types23 Normal Types= Normal. Type S 23 Normal. Types23 NormaL Types23 Normal. - a 2 - * - - SI 5; - + - 193 - P ; - b* - - - P 3 ; - -A -. - % 2 ; - t - , - 194 BIOL?GY OF STBEpToCOCCUS. ' I organisms studied it has been possible to detect four Merent types of Streptococcus hux&yt&~. These types have been noted as Types S 3, S 23, S 60, and S 84, from the number of the chosen represen- tative. In addition to the type strains,' there remains a residue of unclassified organisms. The summary for the total number of strains studied is given in Table X. TABLEi VI. Power of AntiskGptoGoGcvs Sewn, TyQe S 60, to AggW Ten Reprcsmiatioc 3rais.s of the Same Type. Strain No. %, .c 0 8 a N. % :: 2 v) 'I t; 1 t; 2 ; 1; -- -- .- -- S60 WSm ++ ++ +t +t +* i-* + NormaL - - - - - s269 Types60 +* +* +* t* + + * Normal. - - S 267 Types60 +=k ++ ++ - - - - t++* t* + + NormaL - - - - - - s 55 %Fsm -I-`t ++ +t +* * * * S 128 gtz%o - - - - - ++ ++ +t +t +* : . t* + Nd -.- - - - s 72 Types60 +* +* t* t* + + * NormaL c-t +T- - - - - S43 Typesa t* t* +* : . t* + NormaL - - - - - - s123 TjpeS60 ++ ++ t+ t++* : . 4-k + NormaL - - - Y - - S66 Typesa ++++ t+ t* +A + + NormaL I - - - - - - S 127 ++ ++ I I t+ t+ +* : . t* + - - - - - - - Dihltion. The total number of strains of Streptococcus hmnolyticw studied was 125. Of these, 85, or 68 per cent, are comprised in the types mentioned above, and 40, or 32 per cent, remain unclassified. Work with the unclassified strains is being continued and the indications are that a certain number of other types will be discovered. In fact, two new types have already been encountered, one comprising five TABLE VII. rest of Cross-Agglutination Redions of Antistreptococcus Serum, Type S 60, with Struins of Streptococcus h@mdyticw of Other Types and with Urula.Wi&d Strains. S60 s3 s 111 s146 S 80 s 14 S 23 s 78 S 98 s 107 s 122 S84 s 115 s 15 Sl s 20 SW S% S2M s47 s 10: S48 s17 - S60 s3 s3 s3 s3 s3 S 23 S 23 S 23 S 23 S 23 S84 St34 S84 S84 S84 l&h&d. " " " `6 u 6` s-. Types60 Normal. Types60 NormaL Typesa Nod Types60 NomuL Types60 Nod. Types60 Normal. Types60 Normal. Typesa Normal. Typesa Normal Typesa NormaL Types60 NormaL Typesa NormaL Types60 NormaL Types60 Nod Types60 Normal. Typesa Nod Types60 Normal. Typesa Nomd. Types60 Nomd. Types60 Nod Types60 NormaL Types60 Normal. Typed 60 NormaL - z s - --t -. f * * - + -I- * l-2 * - s t; - --t * * - - - * * + + - s ; - --I - - 3 ; - .* i - - - * + DilUthl. - 5 2 - -A - - - - - * - - * - - $ @!. I : - * - - - - - - - :: x ;' - * - - w a ;; - - - i E9 - - - . - - 195 196 BIOLOGY OF STREPTOCOCCIJS. I strains and another four strains, the immune reactions of which have not as yet been completed. The antistreptococcus sera used in the agglutination reaction were obtained in the main by the immunization of sheep, a species of animal which yields a highly specific agglutinating serum for Slre~`&cocclcs hceneolyticus. Agglutination occurred in all the type sera in dilutions of 1 : 1,000 or higher, with the exception of Type S 84, of which the TABLE VIII. Poww of Antistreptococcus Serum, Type S 84, to AggZutinute Eight Represetztutive Strains of the Same Type. Strain No. S&4 Sl S.50 s 20 s 15 s 115 $139 S 138 S-. Types84 Normal. Types84 Normal. Types84 Normal. Type.584 Normal. Type S 84 Normal. Types&Q Normal. Types84 Normal. Types84 Normal. = I - w s - t* t* t* t+ + t* t* t* - $ i3 2 ; -- +* i-t +* - - t* +* - - t+ +* - - t* +* - - t* +* - - t* +* - - t* +* - - - .- i - - . : - DilUtiOlt. 0 2 E z ; -- + -t - - t* +* - - t* +* - - t* * - - t* + - - t* * - - t* + - - t* + a - - - - - 0 a ;; - * - Et + - - 3 N. c.i - - - - - 2 p1 - - agglutination titer has been consistently lower, usually not above 1: 320. The agglutination titer of all the type sera for each strain of the same type has been approximately equal to the titer for the or- ganism used for purposes of immunization. There has been strikingly little cross-agglutination between serum of one type and strains belonging to another. The same lack of crossing is observed among the unclassiIied strains with the few eXceptions in which certain of these strains have shown some degree of agglutination in the type TABIJZ IX. Test of Cross-Agghthatioion hadon.~ of AktistrefltococclLs Serum, Type S 84, with Strains of Streptococcus holytinrs of Other Types ami with Urulustifid Strains. S84 s3 s 14 S80 5146 s95 S 23 s 107 s 122 s 78 S98 S60 S 128 s 19 s 141 s4 S 108 Sd7 SS4 s34 ~ S 106 ~ Type of S. hanwlyticUs. S84 s3 s3 s3 s3 s3 S 23 S23 S 23 S 23 s 23 S60 S60 S60 560 S60 Undassified. " Serum. Types84 Normal. Types84 Normal. Types84 Normal. Types84 Normal. Types84 Normal. Types84 Normal. Types&Q Normal. Types84 Normal. Types84 Normal. Types84 NormaL TypeS8-4 Normal. Types84 Normal. Types84 Normal. TypeS84 NormaL Types84 Normal. Types84 NormaL Typesa NormaL -S&4 EM Normal. Types&Q Normal. Types84 Normal. - :: .s - I-= * * * * * r; * ;' + 3 T + - 197 - E2 ; - I-; - * z * * * * * * - Q z?l 5; - -t - - * T * t - - si 2 ; - + - - - w a 3 - - - $ p1 - -' i -' - 198 BIOLOGY OF STREPTOCOCCUS. I sera, but not in su&.iently high dilutions to justify their inclusion within the types. These facts show that it is possible by a series of carefully conducted agglutination experiments to determine speciiic type relationships between strains of Streptococcus hmnoZyt;czcS and to show that the different types are immunologically distinct from one another. The clearness of the agglutination reactions presented is somewhat deceptive as to the ease and simplicity of the test. It must be remembered that Skeptococcjcs humolyticus is notoriously TABLE X. Sumnurry of Agglutinutim Reactions. No. of stmfas. s3.................................................. 29 S23.......;......................................... 20 s60................................................. 27 . s&l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 uIldassised.......................................... 40 Total typed.......................................... 85 - " stlains studied.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 I Percent. 21.6 7.2 32.0 68.0 variable in its reactions, and that very slight and indeterminable changes in technique frequently obliterate almost completely the specificity of the agglutination reaction. In addition, a considerable number of strains is invincibly granular under all conditions and cannot be used, and occasionally strains are encountered which may occupy intermediate positions, the exact understanding of which needs a technique for the conduction of absorption experiments. The Reactiun of Protection. Study of the power of antistreptococcus serum to protect animals against experimental infection with this organism has given rise to a number of different points of view, both regarding its action against strains from different sources, and against the same strain before and after animal passage, and also concerning the kind and the different A. R. DO-Z, 0. T. AVERY, AND B. C. LANCEFIELJI 199 effect of varying antigens used in the process of immunization. For a full discussion of these matters the reader is referred to the general articles on streptococcus and to the. more important papers dealing with these particular points (6). In this work they will only be con- sidered where they have a particular bearing upon the subject under investigation. Although in the present paper the classification of Streptococcw hremoZyticzcs by means of the agglutination reaction has been presented first, practically we have obtained our primary indi- cations of the degree of antigenic dXerences between strains by means of the reaction of protection. Later each reaction has been used to confirm the information obtained by means of the other. In the successful carrying out of protection experiments two points are of especial importance: first, the production of a serum of high potency; and second, the possibility of raising the virulence of the test strains of streptococcus to such a point that very minute doses of culture are suf.Ecient to kill white mice in a limited period of time. We have been able to produce sera in the manner alluded to above of such potency that 0.5 cc. administered intraperitoneally is sufficient to protect a white mouse against 100,000 lethal doses of a highly virulent streptococcus. In order to produce such a serum many animals must be used, only a few of which may give the desired result. It has been possible to raise the virulence of many strains by continuous passage through white mice and rats to such a point that doses of from 0.000001 to 0.00000001 cc. of broth culture are suflicient to kill the former animals in from 24 to 48 hours. These seemingly difficult conditions must be attained in order that &Ii- ciently long-range protective titers may be carried out to insure the reliability of the information obtained. Protection against one or two lethal doses of a series of strains of streptococcus by a mono- valent serum is subject to so many interpretations that the evidence gained cannot be considered of much value in judging accurately the antigenic relationship of the different strains. The technique observed in the protocols given below has been as follows: The potency of all sera has been titrated for the homologous strain of organism and only the sera which gave a wide range of pro- tection have been used. For infection, virulent streptococcihave been used which have been grown for approximately 18 hours in either 200 BIOLOGY OF STRJ3PTOCOCCUS. I plain broth or ascites broth. In the inoculation of animals the technique advised by Neufeld (5) has been followed with only a minor variation. The test animals have been injected intraperi- toneally with 0.5 cc. of serum 24 hours before the conduction of the experiment. Tentative trials have shown that if the serum is given simultaneously with the infecting dose, no protection results, and that to insure success the serum must be given at least 8 hours before infection. On the following day a series of virulence controls is inoculated intraperitoneally, and the serum z&m& are injected in the same manner with doses of cultures ranging from 0.001 to 0.00000001 cc. of broth culture. Animals surviving for a period of 5 days are considered to be adequately protected. By the use of this method, it has been possible to test the antigenic relationship of a considerable number of virulent strains of Stre$&xucczls hmwlyticus, and the results of these tests are set forth in the following protocols. Protocol 1. In this protocol is shown the titration of the serum of a sheep immunized against Strain S 23. The culture employed for infection was an 18 hour broth culture of No. S 23, which had received eighteen passages through white rats and mice. Each mouse had received 0.5 cc. of immune serum 24 hours previous to infection. vfirulence controls. Protective power of Serum S 23. Dose of culture. Result. Dose of culture. Result. cc. . CC. 0.001 S. 0.00001 D.* i, 24 hrs. .O.oool D.in4days. O.ooooOl S. O.ooooOl I` " 24. " O.oooooOl `< O.oooooOl " 6I 24 u O.OOOOOOl `L *In the tables D. indicates died, S. survived. Protocol 2. In this protocol is shown the protective power of Immune Serum S 3 for two virulent strains of the homologous type, for two strains of each of the heterologous types, and for two unclassified strains. The technique was the same as that in the previous protocol. = I Dose of culture. D.ooooOoo1 cc. ? P Streptocwcus No. 5w of atreptoLnccu9. Type of serum. 0.001 cc. o.ooo1 cc. 0.00001 cc. 0.000001 cc. o.oooooO1 cc. s 3.w* s3 s3 D. 14 hrs. S. S. S. S. No serum. D. 20 hrs. D. 23 hrs. s 149.18 s3 s3 S. S. S. S. No serum. D. 24 hrs. D. 36 hrs. D. 24 Ins. s 39.3 S 23 s3 D. 16 hrs. " 16 " " 7 " L` 19 " No sentm. " 16 " " 16 I` " 21 " S 6t.Z S 23 s3 D. 8 hrs. `L 8 " " 17 " " 1, " D. 19 hrs. No serum. u 23 u " 21 I` S 128.14' S60 s3 D. 24 hrs. D. 24 hrs. D. 48 hrs. 61 24 u No serum. u 24 Lt " 36 `1 " 36 I` s 60.106. S60 s3 D. 18 lirs. " 36 " " 36 I` " 20 " No serum. " 18 " S. 6` 24 u S 1.8' S84 s3 D. 22 hrs. " 22 " D. 22 hrs. " 22 " No serum. " 21 6` " 21 " L` 21 " s 84.17' S84 s3 " 13 1` `1 19 " " 19 " S. No senn+ " 18 " D. 21 hn. S 24.25' UllClaSSilid. s3 D. 14 hrs. D. 16hrs. D. 19 hrs. " 31 " `1 19 L` No serum. `I 21 " " 28 " s 266-e " s3 D. 16 hrs. D. 16 hrs. D. 19 hrs. (` 21 !` No serum. `I 1, " `, 21 " u 24 u * The integer indicates serial number of the culture, the decimal shows the number of animal passages, and the exponent the number of transplants since the last animal passage,, . I E Q* D. 20 hrs. " 60 " In this protocol is shown the protective power of antistreptococcus setllfh, Type S 23, against two strafns of the homologous type, against two strains of each of the heterologous types, and against two unclassi6ed strains. The technique was the same as that employed in the previous protocols. Streptococcus No. S 23.181 s 107.12' S 3.21' S 80.7' s 55.22' s 60.10' S 84.18' s 50.4' S 276.31' S 24.31' S23 SW s 3 s 3 560 S60 S&L S&M UndaSStied. `4 Type of rerwn. S23 No sernm. S 23 No serum. S 23 No serum. S 23 No serum. S 23 No serum. S 23 No serum; S 23 No serum. S 23 No serum. S23 No serum. S23 No serum. = I 0.001 cc. D.llhrs. S. D. 18 hrs. D. 18 hrs. D.2Ohi-S. D. 20 hrs. D. 12 hrs. D. 18 hrs. D. 16 hrs. D. 16 hrs. o.ooo1ae. S. S. D. 18 hrs. D. 18 hrs. D. 36 hrs. " 21 " " 20 " u 3-j " " 20 `L D. 36 hrs. D. 40 hn. " 20 " " 16 `6 Dose of culture. O.owol cc. S. D. 19 hrs. `< 16 1` u 24 46 " 18 " `I 18 `I " 64 " S. D. 20 hrs. " 22 " `6 24 dd `L 20 " " 18 `6 " 16 `4 `I 18 " `I 18 6` `C 66 6` " 18 6` I` 16 `I 4, 16 `I o.oowo1 cc. S. D. 24 hrs. S. D. 24 hrs. `I 36 " `I 36 4` " 96 " " 36 `8 `I 30 L` " 20 " S. D. 36 hrs. `t 36 " `, 20 `I (6 18 " " 36 " `I #-J `< " 4(-J `4 D. 20 hrs. O.oooooOl cc. D. 2Ohrs.. S. D. 24 hrs. " * " " 36 " S. D. 36 hrs. D.18hrs. _ S. D. 18hm. " 18 I` Protorol 4. In thii protocol is shown the protective power of antistreptococcus serum, Type S 60, against two strains of homologous type, against two strains of each of the heterologous types, and against two unclassified strains. The technique was the same as that employed in the previous protocols. Streptocoam No. s 6O.W s 55.221 S 3.22 s 80.b s 75.8 S 23.196 s g4.12' s 50.9 S 24.23? S 276.31' S60 S60 s3 s 3 S23 S 23 S84 S84 Unclassified. `# lype of saum. S60 No serum. S60 No serum. S60 No serum. S60 No serum. S60 No serum. S60 No serum. sa No serum. S60 NOWNHL S60 No serum. S60 No serom: 0.001 cc. D. 19 hrs. S. D. 18 hrs. D. 18 hrs. D. 18 hrs. D. 18 hrs. D. 18 hrs. D. 18 hrs. D. 17 Ins. D. `9hrs.` o.oim cc. S. s. ' D.18hrs. . D. 18 hn. D. 21 hrs. 66 23 u " 18 U D. 18 hrs. " 18 `6 " 18 #` D. 17hrs. D. 20 hrs. u 23 66 Dose of cultnre. o.oOOo1 cc. S. D. 17 hrs. S. D. 23 hrs. " 38 " " 18 " " 18 " U 18 " " 20 L` " 17 " `I 18 4` U 18 " " 18 U I` 18 6` " 18 `I `C 18 " " 17 `L " 17 u 6;. 24 66 `t 48 " o.ooooO1 cc. S. D. 19 hn. S. D. 16 hrs. " ,jo " " 18 " " 22 " " 18 `< D. 23 hrs. 66 24 t* " 60 I< " 22 u " 20 " " 18 " " 18 " u 24 66 " 65 " " 41 " " 33 `4 O.oaoclool cc. D. 24 hrs. " 18 `L $6 24 46 " 18 " D.6Ohl.s. I` 18 " D. 18 hrs. `4 18 " D.31hrs. Protocol 5. In this protocol is shbwn the protective power `of antistreptococcus serum, Type S 84, against two strains of the honiologous type, against two strains of each of the heterologous types, and against two unclassified str&. The tech- nique was the same as that employed `iti previou's protocols. s I& , Streptocoicus No. S 84.16' .s 20.51 S 3.16' S 14.29 `S 23.161 s 107.7' ' s 128.142 `S io..loa `. I : I . . : s: 152: 5' `S 266.5' Type of streptococcus. . s 84 S 84 s 3 s 3 i3 23 S 23 s&l S60 Unclassified. Type of serum. s 84 No serum. s 84 No serum. s 84 No serum. S84 No serum. s 84 No serum. S84 No,serum. . S84 No s&q. S84 No serum. s 84 No strum. SM No serum. 0.001 cc. S. s. D. 17 hrs. D. 16 hrs. D. 18 hrs. D. 18 hrs. D. 24 hrs. i D. 18 hrs.- D. 20 hrs. D. 15 hn. 0.0001 cc. S. D. 16 hrs. s. * D:30 hrs. D. 18 hrs. S. D. 18 hrs. D. 24 hrs. Dose of culture. 0.00001 cc. S. D. 26 hrs. S. D. 55 hrs. `( 20 " (I 3-J " D. 33 hrs. LL 22 (6 D. 29 hrs. S. D. 23 hrs. tc 3s 4c , D. 16 hrs. tt Jo `L D. 24 hrs. r-l A. R. DOCHXZ, 0. T. AVERY, AND R. C. LANaFIELD 205 Consideration of the above protocols shows that the type rela- tionships manifest from the aggIutination reactions have been sub- stantiated by the evidence obtained from the protection tests. As a matter of fact, each reaction has been used to supplement the other, the first clue as to `the position of an organism sometimes being ob- i tained by protection and sometimes by agglutination. On the I whole, we are inclined to place- greater confidence in the reaction of ! protection than in that of agglutination, and would be slow to draw 1 general conclusions concerning type specificity from agglutination / alone with such a variable organism as streptococcus, unless the \ results of this test could be confirmed by some other specific reaction \ such as protection. The sera prepared, as is seen from the protocols, \ have afforded a high degree of protection to white mice against in- 1 fection with organisms of the homologous type. Little or no pro- tection results when serum of one type is employed against organisms of heterologous types. There are, of course, some exceptions to this general rule. Occasionally strains of Streptococcus hamolyticus are encountered against which all type sera afford a varying degree of protection, and sometimes a serum is obtained from one strain which will protect against an organism of another type, and when the re- action is reciprocally reversed no protection results. `At present our k,nowledge is insufficient to discuss these intermediate reactions in- telligently, and their elucidation must await further development of the technique. In all it has been possible to raise the virulence of 31 strains to a point where protection experiments could be per- formed. Of these, 7 belonged to Type S 3,8 to Type S 23,6 to Type S 60, 7 to Type S 84, and 3 to the unclassified group. In view of the difficulty of raising the virulence of -the. organisms it has been found. advantageous to perform the reaction in two ways: first, to I test a single monovalent serum against a number of strains; and second, to test a number of sera prepared from strains of the same type against a single virulent strain of that type. In Tables XI to XIV is shown a summary of: the total number of protection experi- I ments performed. 4 206 BIOLOGY OF STREPTOCOCCUS. I TABLE XI. Summury of the Pro&&e Pmver of Antistreptococcus Serum, Type S 3, agaifwt Stra&s of the Homologow and Heterologws Tj ts. Type of serum. s3 s3 s3 s3 s3 s3 s3 s3 s3 s3 53 s3 s3 s3 s3 s3 53 s3 s3 s3 53 s3 53 s3 s3 s3 s3 s3 s3 53 Undassiiie.d. " " stmfn of s. hcmolyl- icl`s used for pmduc- tion of serum. S3 (RabbitI). s 3 ( " 1). S3 (Dog 1). s 3 ( " 1). s3 ( " 1). s 3 ( " 1). s 3 ( " 1). s3 ( " 1). S 111 (Rabbit 2) S 118 ( " 3) s 2 ( " 4) Sll ( (a 5) S29 ( " 6) S 16 ( " 7) s3 ( " 1) s3 ( " 1) S3 (Dog 1). s 3 ( `l 1). s3 ( " 1). s 3 ( " 1). s 3 ( " 2). s 3 ( " 1). s 3 ( " 1). S 3 (Rabbit 1) s 3 ( " .l) s 3 ( " 1) s 3 ( " 1). s 3 ( " 1). S3 (Dogl). s 3 ( " 1). S 24 (Rabbit 8). S 24 (Sheep 2). S 276 (Rabbit 9) s3 (Type s31. o.ooooooO s 14 ( " S3). O.OooOOl s3 ( " S3). o.ooooooO s95 ( " S3). 0.00001 s80 ( " S3). O.OoOOol s 149 ( " S3). o.ooOOO1 s 146 ( " S3). o.OOOOO1 s 144( " S3). O.aOOOl 53 ( " S3). O.OOoOOOl s3 ( " S3). O.OOOoOOl s3 ( " S3). O.OoOOOOl s3 ( " S3). 0.0000001 s3 ( " S3). o.alooLmo s3 ( " S3). o.ooooooO s 107 ( " S23). O.OOOOOOl S 23 ( " S23). O.OOCMOl S27 ( " S23). 0.000001 S 67 ( " S23). 0.0000000. S 39 ( " S23). 0.000001 s 75 ( " S23). O.OOOOOl S 56 ( " S23). O.OOOOO1 s 128 ( " S60). o.Ooom s60 ( " S60). O.OOoOol s84 ( " S84). o.ooooooo: Sl (" S&Q). o.OOOoO1 S 24 (unclassitied). O.OOOOOOO: S 276 ( " 1. O.OooOOOl S61 ( " 1. O.OOoOl s 1.52 ( " I. O.OoOOOl S266( " 1. O.OOOOol S 3 (Type S 3). 0.000001 s 14 ( " S3). o.OOoOOOO: s 14 ( " s 3). O.oOOOOl ProOcp~&of . s.0.0001 cc. "0.001 " " 0.001 `< " 0.01 " " 0.001 " " 0.01 `1 " 0.01 `1 " 0.001 " " 0.001 1` "0.0001 " " 0.001 (4 " 0.001 (` " 0.0001 " " o.Om (` D.O.OOOOOOl " " O.OoOOOOl `d " O.OOml (a " O.OOOOOOl u " O.OOoOOl " " 0.00001 " `( o.OOoOO1 `( " 0.000001 " "O.OoOoOl " " o.OoOOOO01 `< " O.OOoOOl " " 0 OoOooOOl " s. 0:OaOOOO1 " " 0.00001 " D.0.OOOOO1 " " O.OOOOOl " `I O.OOOOOl I` s. 0.00001 " " o.oooOO1 " ' A. R. DOCBEZ, 0. T. AVERY, AND R. C. LANCEFIELD 207 TABLE XII. Summary of the Protective Power of Antistreptococcus Serum, Type S 23, ugainst Strains of the Homologous ad Heterologous Types. Type of serum. S 23 S 23 S 23 S 23 S 23 S 23 S 23 S23 S 23 S 23 S23 S 23 S23 S.23 UIl&SS%ed. " " " " " `I `I " " ,` S 23 (Sheep 1). S 23 ( " 1). S 23 ( " 1). S 23 ( " 1). S 23 ( " 1). s 23 ( " I). S 23 ( " 1). S 23 ( " 1). S 23 ( " 1). S 23 ( " 1). S 23 ( " 1). S 23 ( " 1). S23 ( " 1). S 23 ( " 1). S 24 ( " 2). S24(" 2). S 24 ( " 2). S 24 ( " 2). S 24 ( " 2). S 276 (Rabbit 9) S276 ( " 9) S276( "` 9) S276 ( " 9) S 276 ( " 9) S276 ( " 9) S23 (TyyeS23). O.&l s 107 ( " S23). O.OOOOOOl S27 ( " S23). O.OOOOOl s 75 ( (` S23). 0.0000001 S 65 ( " S23). O.OOOOOOl s3 ( " S3). O.coOOOol S80 ( " S3). o.OOoOm s5.5 ( " s.60). O.OOOOOl s60 ( `( $60). O.OOOOOl S 128 ( " S60). O.OOOOol sso ( " S84). O.OOOOOOl s84 ( `L S84). O.ooOOOl S24 (unclassi6ed). O.OOOOOl S276( " >. O.OmOl S27 (Type.523). O.&O001 556 ( " S23). O.OOOOOl s 107 ( " S23). O.OOOOOl s39 ( (` S23). O.OOOOOOO S 23 ( " S23). 0.0000000 S27 ( " S23). O.OOOWl S56 ( " S23). 0.00001 s 107 ( " S23). O.OOOOOO1 s39 ( `( S23). O.OOOOOl S 23 ( " S23). 0.0000000 S67 ( " S23). O.OOOOOl Pmtective power of 0.5 cc. of serum. s. 0.001 cc. " 0.001 " " 0.0001 " " 0.001 " " 0.001 " D.O.OOOOOl " s. O.o5oooOl L1 D. O.OOOOO1 " s. o.cmOO1 " D. O.OOOOOl " (6 O.OOOOOOl u " O.oLMOOOl " " 0.00001 " . " O.OOOOOl " " O.OoOOOl " " O.OOOOOl " " O.ooooOl " `( O.ooooOl ;' 1( o.OOOOm `( " O.OOOOOl u " O.OOoOol " " o.OOOOOO01 `( " O.OOOoOl " " O.ooooooOl " "0.OoOOO1 " 208 BIOLOGY OF STREPTOCOCCUS. I TABLE XIII. Sumnuy of the Protective Power of Antistreptococczcs Serum, Type S 60, against Strains of the Homologous and %eterologous Ty Type of serum. Strain of S. hamolyt- icur used for produc- Strain of S. ham++ tion of serum. and typeo~edfdf;,mfecttlon S60 S 128 (Rabdit 10). S 128 (Type S 60). S60 S 128 ( " 10). S60 ( " S60). S60 S128 ( " 10). s 55 ( " S60). S60 S128( " 10). s4 ( " S60). S&O S 128 ( " 10). S72 ( " S60). S60 S 128 ( " 10). S 267 ( " S 60). S60 S 128 ( " 10). s3 ( " S3). S60 S 128 ( I` 10). S80 ( " S3). S60 S 128 ( " 10). S75 ( " S23). S60 s 128 ( " 10). S65 ( " S23). S60 s 128 ( `( 10). S 23 ( " S 23). S60 S 128 ( `I 10). s84 ( " S&Q). S60 S 128 ( " lo).' s 50 ( " S84). S60 S 128 ( " 10). S 24 (unclassified). 560 s 128 ( u 10). S276( " >. Unclassified. S24 ( " 8). S 128 (Type S 60). " S276 ( " 9). S 128 ( " S 60). Millild e:hsl dose 01 S. hamolyt- icus used fol infection . cc. O.ooOOl O.OOoOOl O.oOooOl O.OOOOol 0.00001 O.oOOOOl O.OOOOOO O.OOOOOO 0300001 O.OooOOl O.OOOOOO O.OOoOOl O.OOOOOO O.OOOOOO O.ooooOl O.OoOOOl O.OOOOOl Is. Protective power of 0.5 cc. of serum. s. 0.01 cc. " 0.0001 " " 0.001 " " 0.001 " " 0.001 . " " 0.001 " D.O.OCOOOOl " " O.OOOOOOl " "0.OOoO1 " s. 0.00001 " D.O.OOOOOOl " " O.OOOOOl " . " O.OoOOOOl (` " O.OoOoOl " " o.ocxloo1 " s. 0.001 " " 0.0001 " A. R.DOCHEZ, 0. T. AVERY, AND R. C.LANCJSFIELD 209 TABLE XIV. Sum?lmry oj the Protective Power of Adstreptococcus Se&m, Type S 84, against Strains of the Homologous and %eterologous Types. strafn of s. /lmOlyc Type of serum. icur used for produc tion of s-. S84 S&l S84 S84 S84 S84 S84 S84 S84 S84 S84 S84 S84 S84 S84 S&l. S84 S84 I-584 S84 S&l S84 S84 S84 S84 S84 s84 S 84 (Sheep 3). s 84 ( " 3). s 84 ( " 3). s 84 ( " 3). s 84 ( " 3). S84 ( " 3). s 84 ( " 3). S 1 (Rabbit 11) s 1 ( " 11) s 1 ( " 11) S 84 (Sheep 3). s 84 ( " 3). s 8q ( " 3). s 84 ( " 3). s 84 ( u 3). 9 84 ( " 3). s 84 ( " 3). s 84 ( " 3). S 1 (Rabbit 11) s 1 ( `f 11) S 84 (Sheep 3). s 84 ( " 3). s 84 ( " 3). s 84 ( " 3). s 84 ( " 3). s 84 ( " 3). s 84 ( " 3). Unclassified. S 24 (Rabbit 8). `1 S 24 (Sheep 2). `1 S 276 (Rabbit 9). . `1 S 276 ( " 9). `1 S276 ( " 9). Minimal Strain of S. hmwlyficus lethal dose o md type used for infection of mice. S. kmoryt- icus used fa infection. cc. S84 (TypeS84). O.OOOOO1 Sl (" S&Q). O.OOoOOl s 20 ( " S&4), O.oouoOl s50 ( " S84). O.OOOOOl s 139 ( " S84). O.OOOOOl s 110 ( `( S84). o.OOOOOO1 s 15 ( " SE%). O.OoOOOOl Sl (`1 S&p). o.ooooooO s84 ( (L S84). o.ofxmoo s20 ( " S84). O.OOoOol s3 ( " S3). s 14 ( " S3). S 23 ( " S 23). S 107 ( " S 23). S39 ( " S 23). S67 ( " S23). S27 ( " S23). S56 ( " S23). S 107 ( " S 23). O.OOOOOl o.ooooooO: o.OOoOOO1 o.oOOOoO1 O.OOoOOl 0.000001 o.OOoOO1 0.00001 O.OOOOoOl S 128 ( " S60). O.OoOOOl S 128 ( " S60). O.OOoOOl s60 ( " S60). O.oOOOOl S 276 (unMed). O.OOOOO1 S 277 ( " ). O.OOOOOl S 152 ( " I. O.OOOOOl S266( " 1. O.OoOoOl S24 ( " 1. oxMOO S84 (Types&Q). 0.00001 Sl ($` S&). O.OOOoOl s84 ( " S84). o.ooooooO1 s20 ( " S84). o.OOOOO1 s.50 ( " S&Q). O.OOOOOl Protective power of 0.5 cc. of serum. s. 0.001 `1 O.-JO05 : " 0.001 " " 0.0005 " "0.001 " " 0.001 " " 0.001 " " O.OOoOl " " 0.0001 " " 0.001 " D.O.OOOOOl " s. O.OOOOOl " D.O.OOOOOOl " s. O.OooOOOl " " O.cQoool " D.0.000001 " " O.OoOOol " " O.OoOOOl " s. O.OOOoOOOl " D.O.OOOOOl " " O.OOOOOl " "0.OOOoO1 " " O.OOOOOl `( s. O.OOOool " D.O.OOOOOl " "0.OOOoO1 " " O.oooOol " " o.OOOOo1 " "0.OOOoo1 " " o.oOOOOO01 (` " O.OOooOl " " O.OOoOOl " 210 BIOLOGY OF STREPTOcoC~ I DISCUSSION. The complete biological classification of any pathogenic micro- organism presents a very complex problem. The first phase of the undertaking concerns itself with the development of reliable methods for the determination of antigenic differences between members of the species and the application of these methods to the discovery of the immunological relationships between a limited number of strains purposefully selected. In this way the degree of similarity and diversity of type is shown and also the probable number of types, and the proportion of classifiable to unclassitiable strains. The next step of necessity is the testing of the adequacy and universality of the in- formation so gained by applying the tentative classification to a large number of strains of the organism obtained under what may be described as normal conditions of pathogenicity. That some sort of equilibrium has been established in nature among microorganisms that have produced disease over long periods of time is not unlikely. Indeed, evidence obtained from the study of pneumococci supports this view (7), although departure from the norm may occur under special conditions (8). After the relationships of the pathogens of the species to one another have been discovered, it then becomes im- portant for purposes of epidemiological study to compare by the same methods the pathogenic with the saprophytic varieties. This task requires years for its completion and many di&ulties and seemingly unexplainable phenomena are encountered. In the beginning, the broader lines of differentiation must be drawn, and divergent results discarded for the time being, since, if the original conception is correct, most of the discrepancies disappear with the advance of. knowledge. In this paper are presented the facts so far obtained in the present study of Stre#ococcus hcemoZyt~us in accordance with the plan out- lined above. The strains were collected in a limited community during the course of what may be considered an epidemic of broncho- pneumonia secondary to measles. Individuals, however, from all parts of the United States were passing rapidly through this com- munity which was a center for primary training of the aviation serv- ice, so that a wider range of territory is represented than the im- A.R. Docmz, 0.~. AVERY, m R. c. LANCEFIELD 211 mediate co~tmity itself. All the strains were investigated as to their cultural reactions, bile solubility, capacity to hemolyze red blood cells and to ferment the different test sugars, and as to hydrogen ion concentration limiting their growth, and thus identified as accurately as possible as Streptococcus hmolyticus of the human type. A technique was then developed for studying the immunological reactions of agglutination and protection. By the reaction of `agglu- tination four distinct immunological types and a certain number of unclassifiable strains have been discovered among the 125 strains studied. Individuals of the same type are closely related to one another immunologically, and the different types can be sharply distinguished one from the other. In addition to the four types, study of the reactions of which has been completed, there are in ad- dition two other types, investigation of which is as yet incomplete. The technique of the agglutination reaction demands great care, both as regards the handling of the organisms and the preparation of the medium for their growth. In the medium used by us, a large percentage of strains has grown sufficiently diffusely to permit the preparation of stable suspensions. To what extent continuous growth in this medium has promoted the tendency to diffuseness, and whether the same percentage of freshly isolated strains will grow diffusely, we are as yet unable to say. We have found that by the immunization of sheep a highly specific agglutinating serum is obtained, but that the serum produced from rabbits is not so specific and may show a wider range of crossing, especially in one of the types of streptococcus de- scribed. Variations in the specificity of di.lXerent animal sera have been observed by students of the immunological reactions of menin- gococcui. In order fully to understand this phenomenon, it would be necessary to compare the specifkity of immune sera produced from different species of animals by means of the method of absorption. It is not as yet possible to undertake tbis hind of an investigation of Streptococcers hmwlytkus. The observation has been made, however, that rabbit sera showing non-specifk cross-agglutination reactions in general fail to manifest corresponding cross-protection reactions. Whenever it has been possible to raise the animal virulence of strains of Strept0coccu.s hamolyticus, the evidence obtained from the agglutination tests has been confirmed by that- gained from the pro; 212 BIOLOGY OF STREPTOCOCCUS. I tection reaction. In all instances in which this has been done, one reaction has corroborated the Endings of the other. The performance of reliable protection tests has been made possible by the production of sticiently high titer antistreptococcus sera, and by the possibility of raising the animal virulence of a certain number of strains to a high degree. The types of Strepticoccus hamdyticus have been noted asTypesS3,S23,S60, and S 84, from the serial numbers of the rep- resentative strains. This nomenclature is not put forth as a final one, since we realize that probably many other human types exist, to say nothing of the bovine and cheese varieties, and that the pro- portional distribution of the different varieties pathogenic for man may be very different from that represented by this work. Strep- tococcus is the largest of all pathogenic groups of bacteria and many years will be required to bring out the information necessary to the perfecting of an adequate classification. It is of considerable interest that all the members of Type S 60 . ferment marmite, and that none of the members of the other groups so far encountered ferments this sugar. A few unclassi&ble strains, however, have been found to be mannite fermenters. This work has cleared up a number of points about Streptococcus hmdyticus which have been in dispute for many years. In the first place, Streptococcus hmwlyticus of human origin is not a unit type as was previously supposed, but probably consists of a number of types, at least four of which have been definitely identified. Previous in- vestigators have stated that freshly isolated human strains change their antigenic properties on animal passage, and that the latter pro- cedure for the development of animal virulence gives a common antigenic character to all strains. We have found no evidence to support this contention; in fact immune sera produced with human strains that have never been passed through animals afford a high degree of protection against strains that have received many animal passages. In addition, the antigenic differences between strains of Streptococcus ~molyticzcs which have been passed through animals are as distinct as those between strains which have not been so passed. The types of Streptococcus hmwlyticus studied have been obtained almost exclusively from the respiratory tract and from a limited source of supply, and there is some reason to believe that those which pro- A.R. DOCEEZ, 0. T. AVERY, AND R. C. LANCJDIELD 213 duce cellulitis, erysipelas, and septicemia may be of somewhat dif- ferent character. It is, therefore, readily seen that only a beginning has been made in the classification of Streptococcus hmwlyticus, and that before the classification is complete and the relative dominance of the different pathogenic varieties determined, much work must be done. SUMMARY. 1. Immunological ditrerences have been shown to exist between strains of Streptococcus hamdyticus of the human type. 2. Four biological types have been identified by means of the re- actions of agglutination and protection. 3. At least two other types have been encountered and the indi- cations are that more exist. BIBLIOGRAPHY. 1. Schottmfihr, H., M&u%. nud. IV&., 1903, I, 849. 2. Adrews, F. W., and Horder, T. J., Lancct, 1906, ii, 708. Holman, W. L., J. &fed. Research, 1916, xxxiv, 377. Blake, F. G., ibid., 1917, xxmri, 99. 3. Smith, T., and Brown, J. H., J. Med. Research, 1914-15, xxxi, 455. Jones, F. S., J. Exp. Med., 1918, xxviii, 253. Ayers, S. H., J. Ba~terioL, 1916, i, 84. Avery, 0. T., and Cullen, G. E., J. Exp. Med., 1919, xxix, 215. 4. Smillie, W. G., J. Ivzject. Dia., 1917, xx, 45. Cole, R., and Ma&&m, W. G., J. Am. Med. Assn., 1918, lxx, 1146. Levy, R. L., and Alexander, H. L., ibid., 1918, lxx, 1827. Opie, E. L., Freeman, A. W., Blake, F. G., Small, J. C., and Rivers, T. M., im., 1919, lxxii, 556. 5. Marmorek, A., Anti. Inst. Pasteur, 1895, ix, 593. Aronson, H., Deutsch. med. Woch., 1903, xxix, 439. Neufeld, F., 2. Eyg. Y. Injectionskrankh., 1,?03, , .xliv, 161. Kinsella, R. A., and Swift, H. F., J. Exp. Med., 1918, xxvfii, 169. Howell, K., J. Ifect. Dis., 1918, xxii, 230. 6. Gay,.F. P., J. Lub. und C&a. Med., 1918, iii, 721. 7. Do&z, A. R., and Avery, 0. T., J. Exp. Med., 1915, xxi, 114. 8. Lister, F; S., Publiations South Ajrican Inst. Med. Resewch, No. 2, 1913; No. 8, 1916; No. 10, 1917.