ment, dr. colebrook challenged it stating that in his laboratory only

STUDIES ON HEMOLYTIC STREPTOCOCCI

VIII. STREPTOCOCCUS EQUISIMILIS

ALICE C. EVANS

National Institute of Health, Bethesda, MarylandReceived for publication April 10, 1944

If the key in the 5th edition of "Bergey's Manual of Determinative Bacteri-ology" is followed when strains of Streptococcus equisimilis are being identified,they will be found to fall into the pyogenic group of aerobic streptococci whichdo not hydrolyze sodium hippurate. This group includes all strains of strepto-cocci of Lancefield's serologic groups A and C. In an accompanying paperthe specific characters which distinguish S. equisimilis from other species of thegroup are outlined as follows: Precipitation by serum of group C; sensitivityto filtered phage B; fermentation of trehalose, positive; fermentation of sorbitol,negative; fermentation of lactose, irregular.

In this paper the type-strain of S. equisimilis is described; the variations ofcharacter within the species as determined by a study of 53 strains are discussed;immunologic relationships with other species are shown; habitat and patho-genicity are considered, together with a review of the literature.

CULTURES

Our collection of strains of groups A and C was assembled throughout 15 yearsof interest in streptococci. The strains came from many states and from foreigncountries. They were obtained when interest was focused first on one diseaseand then on another. At one time scarlet fever strains were collected; at an-other time strains from septic sore throat; at another time puerperal strains; atanother time strains from suppurative diseases; at other times strains were addedto the collection without reference to any particular source. As the collectiongrew, some pathogenic strains of S. equisimilis from human sources were ac-quired incidentally. Others were received in response to requests for strainsdescribed in the literature as possessing the characters of S. equisimilis. Intable 1, the strains of S. equisimilis which were received from other investigatorsare identified by their previous designations and the name of one or more in-vestigators. The writer gratefully acknowledges indebtedness to all who con-tributed strains for this study. In table 2 all of the 53 strains are listed withtheir source and other information. The fact that only 4 puerperal strains areincluded in this study requires comment because in an earlier paper (1938) it wasreported that fourteen out of thirty-six puerperal strains received in 1932 fromDr. Leonard Colebrook were found to be of group C. After reading that state-ment, Dr. Colebrook challenged it stating that in his laboratory only one of thosestrains had been found to belong to group C. The discrepancy of results doesnot appear to be due to errors in carrying out the precipitative test in our labora-

267

ALICE C. EVANS

tory, for our strains were shown to be of group C in tests repeated after the dis-cussion arose. Further, all of them were agglutinated by serums of the typesbelonging to group C. It appears that in transferring the strains from onelaboratory to another an unexplainable error may have occurred. Hence, onlythe one of Dr. Colebrook's strains which was placed in group C in his laboratory(his strain "Nash," N.I.H. No. 1006) was included in this study.

TABLE 1Identification of strains of S. equisimilis studied by previous investigators

Birkhaug; Williams;Spicer and Bloom;and others

Griffith

Edwards

Amoss and Craven

}Edwards

Stableforth

Fisher

Gibbs; Edwards

Fisher; Edwards

Griffith

NIHNO.

1006

1045

1053

PREVIOUSDESIGNATION

Nash

Cs

Cole

1064 Lucey

113611371138113911401220122312241241

1253125412891296

DejeanJonesK.L. No. 4Daunais1436Horse 27GreenbergSiggins

767

B 102-13B 124-45DAustin

PREVIOUS INVESTIGATORS

Lancefield and Hare

Birkhaug; Williams; andothers

Andrewes and Christie

Plummer

Birkhaug; (AmericanType Culture Coll.)

Frost and EngelbrechtPilotHare

METHODS

The methods employed in this study were described in previous papers (1936,1941, 1943).

BIOCHEMIC CHARACTERS

In table 2 the 53 strains of S. equisimilis are grouped according to variations insensitivity to bacteriophage and fermentation of trehalose, sorbitol, lactose, andsalicin. Thirty-two (60.4 per cent) agreed in. all characters with strain 1180,which was selected to represent the type. The typical strains of S. equisimilisare sensitive to filtered phage B, and ferment trehalose and salicin, but do notferment sorbitol or lactose. Seven varied from the typical strains in their failureto ferment salicin. Seven varied in their ability to ferment lactose. Fourvaried in their resistance to lysis by filtered phage B. Three single strains varied

NO PDIEVIONS PREVIOUS INVESTIGATORS

649 Bs

655

728

729

790875885887927928929946.947

948976987988

Azgazar-dah

4 (TypeBO)

5 (TypeB2)

Dittes126UTB51S 38Var I

IIIIII

II

IIIH 26NielAngel

I

IS

268

STUDIES ON HEMOLYTIC S3TREPTOCOCCI 269

TABLE 2Significant characters and sources of strains of S. equi8imilis

FERMENTATION OF:STRAIN PRECIPITIN SENSITIVITY AGGLUTI-

NO. GOUI? TO PEAGE -NATIVE HOST SPECIES DISEASENO. GRClLOUPX | B FILTRATE Treha- Sorbi- Lac- Salicin TYPE

589 C + + _ - + 21 Human Scarlet fever790 C + + - - + 21 Human Pneumonia927 C + + - - + 21 Human928 C + + - - + 21 Human (eorai929 C + + - - + Not test- Human [(Semarlhagi

ed976 C + + - - + 21 Human988 C + + _ - + 21 Human Scarlet fever1045 C + + - - + 21-7* Human Erysipelas1053 C + + - - + 7 Human None1064 C + + _ - + 21-20-7 Human Lymphadenoma1136 C + + - _ + 21-20 Human Nasal Discharge1137 C + + - - + 21 Human None1138 C + + - - + 21-7 Human None1139 C + + - - + 21 Human None1140 C + + - - + 21-7 Human None1180 C + + - - + 21-20 Human Abscess1244 C + + - - + 20-21 Human Septicaemia1253 C + + - - + 21 Human None1280 C + + - - + 21-20 Human Septicaemia982 C + + - - + 20-21 Simian None

(monkey)875 C + + - - + 21 Bovine Metritis887 C + + - - + 21-20-7 Bovine Mastitis728 C + + - - + 7-21 Equine None885 C + + - - + 20-21 Equine None895 C + + - - + 20 Equine ?959 C + + - - + Untyped Equine Strangles960 C + + - - + Untyped Equine Strangles1189 Not + + - - + 21-20-7 Equine Strangles

tested1220 C + + - - + Untyped Equine Strangles946 C + + - - + 21 Avian

(chicken) Bronchitis947 C + + - - + 21 Avian

(chicken) Bronchitis948 C + + - - + 21 Avian

(chicken) Bronchitis

649 C + + - - - 21-7 Human Erysipelas655 C + + - - - 7 Human Puerperal fever1006 C + + - - - 21 Human Puerperal fever1224 C + + - - - 21 Human Puerperal fever1241 C + + - - - 21 Human Erysipelas1298 C + + - - - 21 Human ? ?886 C + + - - - 7 Equine Metritis

270 ALICE C. EVANS

TABLE 2-Continued

FERMENTATION OF:STRAIN PRECIPITIN SENSITIVITY AGGLUTI-

NO. GROUP TO PHAGE -NATIVE HOST SPECIES DISEASENO. GROUP B FILTRATE Treha- Sorbi- Lac- Salicin TYPElose tol tose

987 C + + - + + 20 Human Scarlet fever1223 C + + _ + + 21 Human Puerperal fever1296 C + + + + 21 Human None1393 C + + - + + 21-20 Human None729 C + + - + + 21 Equine Metritis878 C + + - + + 21-20 Porcine Abortion1289 C + + - + + 20-21 Canine Distemper

1254 C + _ _ - + 21-20 Bovine ?

1297 C + - + - + 21 Human None

1142 C - + - - + Untyped Human None1163 C - + - - + Untyped Human Empyema1193 C? - + - - + Untyped Human Septicaemia897 C - + - - + 20-21 Equine ?

1337 A and C - + - + - 21 Bovine Mastitis

* When two or more type-numbers are given, the order of the numbers indicates thecomparative strength of the agglutinative reactions, the strongest being in serum of thetype indicated by the first number.

irregularly from the type, one in its failure to ferment trehalose; another in itsfailure to ferment trehalose and its ability to ferment sorbitol. The third strain,of animal origin, resembled S. pyogenes in its resistance to filtered phage B and inits fermentative reactions; nevertheless it was precipitated in serum of group Cas well as in serum of group A, and it agglutinated in serum 790 of type 21 (groupC).

AGGLUTINOGENIC RELATIONSHIPS

Agglutinating serums were prepared with strains representing the three ag-glutinative types of group C established by Griffith. These strains, 655, 987,and 988, received directly from him, were the ones with which he prepared hisserums of types 7, 20, and 21. Agglutinating serum was prepared also withstrain 790 of group C, type 21, isolated from a case of human pneumonia.Among 52 strains tested only six failed to agglutinate in one or more of the four

serums of group C. The greatest number of strains reacted with the serum oftype 21 (See table 2). On the other hand there was no evidence of agglutinogenicrelationship between the strains of S. equisimilis and the lactose-negative strainsof group A when forty-four strains were tested for agglutination in serums oftypes 6, 14, 15, 17, 18, 19, and 23, of group A, with negative results. (Thesetypes include most of the lactose-negative strains of group A.) Furthermore, 38lactose-negative strains of group A were tested for agglutination in antiserum


produced by the human strain 790, of group C, with negative results. Thus theagglutinative reactions indicate a serologic diversity among lactose-negativestrains paralleling that shown by precipitin reactions.

CORRELATION OF CHARACTER

Table 2 shows no evidence of correlation between agglutinative typing andgrouping according to fermentative reactions. On the other hand, there ap-pears to be a correlation between resistance to filtered phage B and failure toagglutinate in the type serums, for three of the four resistant strains failed to ag-glutinate in any of the antiserums, whereas only three of the remaining 49 strainsfailed to agglutinate in one or another of them.The limited data offer evidence of correlation between certain variant char-

acters and pathogenicity. There is an indication that the two subgroups char-acterized by failure to ferment salicin, and positive fermentation of lactose,respectively, have a predilection for the genital tract, for four of seven strains ofone group, and three of seven strains of the other, as compared with one of thirtyof the typical strains of S. equisimilis, were isolated from such a source. Theevidence in regard to the lactose-fermenting group (type B2 of Ogura) is strength-ened by the report of Lancefield and Hare that all seven of their strains of groupC isolated from parturient women were capable of fermenting lactose.

DESCRIPTION OF THE TYPE STRAIN

Strain 1180, selected as the type strain, was isolated in 1935 from pus from theleg of a child. It has been deposited in the American Type Culture Collection.Our strain 1244, which is strain 4433 of the third edition of the A.T.C.C. cata-logue, is also typical of S. equisimilis although it was listed as Streptococcuspyogenes.

Strain 1180 is precipitated by serum of Lancefield's group C; it is sensitive tophage B filtrate; it ferments trehalose and salicin, but not lactose, mannitol, orsorbitol. On blood agar plates incubated for 48 hours the colonies are 1.75 mmor less in diameter, discrete, smooth, convex, opaque in transmitted light, andsurrounded by a zone of beta hemolysis. Other characters are as follows: there isslight growth on blood agar containing 10 per cent bile, but no growth with 40per cent bile; human fibrin is lysed rapidly; growth in plain infusion broth"is good.Under the conditions of the test the final pH in glucose broth was 5.2. Strain1180 is agglutinated to full titer in serums of Griffith's types 20 and 21. Smearsfrom broth culture incubated 24 hours show gram-positive diplococci from 0.6to 1.0 ui in diameter, with many in chains of moderate length.

9COMPARISON OF S. EQUISIMILIS WITH OTHER SPECIES

Certain other characters of S. equisimilis which lack the precision required of adifferential test may be useful sometimes for purposes of identification.Appearance of growth of the lactose-negative strains of groups A and C on

blood agar suggests their identification. The strains of group C (S. equi andS. equisimilis) produce a stronger hemolysis, resulting in a zone wider than that

271

ALICE C. EVANS

which surrounds colonies of group A. (See the third paper of this series for adescription of Streptococcus equi.) A difference between the colonies of S. equiand S. equisimilis is generally apparent, the watery or thin matt confluent coloniesof S. equi being quite different from the discrete, opaque colonies of S. equisimilis,which often become dark before the end of the 48 hours' period of incubation.The strains of S. equisimilis are more tolerant of bile than strains of S. equi

or of group A. Among 37 strains of S. equisimilis tested, 28 (75.7 per cent) grewon blood agar containing 10 per cent bile. Twenty-one of the bile-tolerant strainswere later tested on blood agar containing 40 per cent bile, and seven were foundto produce slight growth. On the other hand none of 20 strains of S. equi grew inthe presence of 10 per cent bile. An occasional strain of group A grew on the

TABLE 3Lysis of human fibrin by strains of various species of groups A and C

SER(OLOGIC NUMBER OF DEGREES OF FIBRINOLYSISSEROLOGI STRAINS ________________________GOP TESTED - + ++ +++ ++++

....... ...... C 13 11 2 0 0 0S.equi............ C 8 7 1 0 0 0S. equisimilisC...... 33 9 4 7 3 10S.-b .A............ 18 0 1 11 5 1S. pyogenes.A 19 1 5 8 1 4

a The unnamed species known as "animal pyogenes."b Lactose-negative strains of group A.

TABLE 4Correlation between fibrinolysis and source of strains of S. equisimilis

LYSIS WITHIN 3 HOURS:HOST SPECIES NI1UMBER OFHOSTSPECIES ~STRAINS TESTED

Number Per cent

Man....................... 22 20 90.1Animal....................... 11 0 0

medium containing 10 per cent bile, but with 40 per cent bile only a very fewstrains gave a sparse growth.Although Gunsalus and Sherman reported that under their experimental con-

ditions strains of S. equisimilis fermented glycerol, whereas the strains of group Aand the sorbitol-fermenting strains of group C did not, in this study fermentationof glycerol, and also appearance on chocolate agar, and,reduction of methyleneblue diluted in milk 1: 5000-characters which have been recommended as usefulfor distinguishing the species of groups A and C-were found to be insignificant.They did not distinguish the strains of S. equisimilis from those of other species,nor did they distinguish the subgroups of S. equisimilis from each other. Testsmade on many strains of our collection confirm the statement made by Shermanthat the final pH in glucose broth does not differentiate any of the species withingroups A and C.

272


Lysis of human fibrin proved to be a useless character for the identification ofS. equisimili8, because some strains gave a positive and others gave a negativereaction. (See table 3.) There was a correlation, however, between fibrinolysis

TABLE 5Comparative virulence for mice of lactose-negative and lactose-positive strains of

groups A and C

KILLED MICE IN A DILUTION OF 1:100 OR HIGHERSPECIES ~~~NUMBER OFSPECIES STRAINS TESTED

Number Per cent

S. equi....................... 8 7 87.5S. equisimilis................... 17 8 47.1S.-- -........................... 18 17 95.5S. pyogenes b.................... 27 6 22.2

Lactose-negative strains of group A.b Strains belonging to Griffith's types, 4, 5, 8, 26 and 29 were included in the summary of

data on S. pyogenes.

TABLE 6Summary of cross-protective reactions between strains of various agglutinative types of group

A, strains of S. equisimilis and S. equi, and their corresponding antiserums

ANTISERUM PRODUCED BY STRAIN:STRAIN

Group A Group C

No. Group Ty 1072 1475 654 1268 660 778 658 790 940 725TPrup2 Type 23 Type 6 Type 1S Type 18iType 19 Type ? Type 14Type 21 S. cqui S. equi

1072 A 23 +* t1475 6 + + (+) - (+) (+) (+) (4)654 15 + (+) + (+) - 4 + (L)1268 18 + + (+ + + 41 + - -660 19 + (+ + - + + + (+W778 ? + (+) - - - + - +658 14 - (+ -- - + + - -

791 14 - 4- - - 41: + -4

790 C 21 =-1 (+) (+) 4 (+) +) + + -729 21 4I +-4- -1189 21-20-7 :4: + -- - - - -

959 ?+940 S. equi - - - - - - - - +

725 .S equi +

* + indicates good protection; 4 indicates evidence of slight protection; - indicatesno protection; (+) or (=I) indicates reciprocal protection between the strain and the serumof the types concerned.

t Blank space indicates that the test was not made.

and source, as shown in table 4. Strains derived from human sources generallylysed human fibrin within 3 hour6. On the other hand strains derived fromanimal sources failed consistently to lyse human fibrin. The fibrinolytic testwas the only one found to indicate the source of strains of S. equisimilis.

273

ALICE C. EVANS

Data on the comparative virulence for mice of lactose-positive strains of groupA, as compared with lactose-negative strains of group A and strains of S. equiand S. equisimilis, are presented in table 5. The table shows that the virulencefor mice of lactose-negative strains of group A (discussed in an earlier paper) iscomparable with the high virulence for mice of strains of S. equi, whereas that ofstrains of S. equisimilis is on the average notably less, although it is higher thanthe average virulence of strains of S. pyogenes, provided the strains of type 3,which are exceedingly virulent for mice, are omitted from the consideration.

CROSS-PROTECTIVE RELATIONSHIPS BETWEEN STRAINS OF GROUPS A AND C

The status of S. equisimilis between S. equi and the lactose-negative strains ofgroup A, as determined by biochemic and serologic characters, is shown in the ac-companying paper (Evans, 1944). Its intermediary position is well demon-strated also by cross-protective reactions.Aronson reported that the serum of a horse immunized with a scarlatinal strain

of streptococcus protected experimental animals against infection with an equinestrain from a case of strangles, and also against strains from a variety of humanpathologic sources. Then Marxer showed that a monovalent antiserum pro-duced by a strain from strangles protected mice against strains from scarlatinaland puerperal infections. He reported positive results also in the reciprocal ex-periments. He stated that his equine strain was not the same one as that whichAronson used in his experiments.

Further confirmation of cross-protective reactions between strains of groups Aand C is found in the work of Spicer and Bloom. They reported that vaccinationof mice with a certain strain, B3R, originally isolated from a case of erysipelas,gave protection against experimental infection with 12 strains from a variety ofhuman pathologic sources-from cases of scarlet fever, erysipelas, puerperalfever, and general septicaemia. Although the infecting strains were unclassified,it may be assumed safely that, coming from such sources, most of them must havebelonged to group A, and that they must have represented several agglutinativetypes. Likewise the strain used for the production of the vaccine was unclassi-fied by Spicer and Bloom, but it is a well-known strain, later shown by other in-vestigators to belong to group C. Erysipelas strain B3, from which their strainB3R, used for the production of the vaccine, was derived, is one of those isolatedby Birkhaug and distributed widely in laboratories of this and other countries as"Streptococcus erysipelatis." Williams identified it with her agglutinative typeE II, and Griffith placed it in his type 7. It is S. equisimilis strain 649 of ourcollection (see table 2). Boroff and Lackman confirmed the observation ofSpicer and Bloom that mice treated with vaccine prepared with strain B3R wereprotected against experimental infection with streptococci of Group A.Although it is more than 40 years since Aronson's work was published, one of

his strains isolated from a case of scarlet fever is still available for study. Thisstrain, for which our number is 1072, is probably the one with which Aronsonproduced the antiserum which protected animals against heterologous strains.In a study previously reported (1943) his observation that animals may be pro-

274


tected against infection with streptococci from various human pathologic sourcesby a monovalent antiscarlatinal serum were confirmed. The data are sum-marized in table 6, together with further data which show that, as Aronson found,antiscarlatinal serum (1072) may protect mice against infection with an equinestrain from strangles (1189). It gave protection also against strains 790 and 729,of group C. Although the protection given by serum 1072 against strains 790,729 and 1189 of group C was not strong, it appeared to be unquestionable. An-

TABLE 7Protocols of experiments showing protection given to mice by antiserum produced by a strain

of group A, against equine strains of group CSTRAIN (GRLOUP CULTUREC) DILUTION ANTISERUM 1475 (GROUP A) NORMAL SERUM OR NONE*

729 10-8 D3t D6 S Dl D2 D210-4 S S S D2 D4 S10-5 D3 D4 S D2 D2 D210-6 S S S D3 D3 D4

1189 l10- Di S Dl Di10-4 D3 S Dl Di10-6 D7 S D2 D310-6 D2 S Dl S10-7 S S D2 S

* The control mice inoculated with strain 729 received normal serum; those inoculatedwith strain 1189 received no serum.

t D indicates died; the figure followingD indicates the day of death; S indicates survived8 days.

TABLE 8Protocols of tests showing protection given to mice against a strain of S. equisimilis by

antiserums produced by strains of the 6-16-18-19 immunologic group

STRAIN OF S. STRAINS OF GROUP A USED FOR THE STRAINS OF GROUP C USED FOREQUISIHIIS CULTURE PRODUCTION OF ANTISERUM THE PRODUCTION OF ANTISERUM NORMLUSED FOR THE DILUTION SERUM

OF 111CR 1475 654 660 790OF MICE Type 7 Type 15 Type 19 (homologous) 940 S. equi

790 10-4 S S S S D6 S S S Dl Dl Dl Dl10- S S D5 S S S D3 S Dl Dl Dl D510-6 S S D5 S D3 S S S D1 D4 D5 S10-7 S S S S S S S S D2 D4 D5 D7

other antiserum, produced by strain 1475, of group A, type 6, gave good protec-tion against these three strains of group C. The protocols of the tests are shownin tables 7 and 8. (In an earlier paper (1943) it was shown that the two anti-serums, 1072 and 1475, gave protection against strains of many types of group A.),

In the earlier paper there was established an immunogenic group, including"lactose-deficient" strains of types 6, 15, 18 and 19. In this paper it is shownthat the antiserums of that group protected mice against the human strain 790, of

275

276 AUCE C. EVANS

grouxp C (the protocols are shown in table 8), and that protection was given alsoin the reciprocal tests (table 6), excepting that serum 790 gave no protectionagainst infection with strain 1268. Three of the four antiserums of the 6-15-18-19 group did not give protection against infection with the equine strains ofS. equisimilis, 729 and 1189, and this is in accordance with their limited ability toprotect mice against strains of group A not included in the immunogenic group(table 3 of Evans, 1943).Two other strains of group A, 778, of undetermined type, and 658, of type 14,

with their corresponding antiserums, were included in this study. Withoutentering into a discussion of the relationships between these strains and otherstrains of group A, it may be pointed out that the immunologic relationship be-tween strain 790 of group C and strain 778 is the same as that between strain 790and the strains of 6-15-18-19 group, i.e., strain 790 was influenced by antiserum778, and protection was shown also in the reciprocal test. Serum 658 protected

TABLE 9Protocols of tests showing protection of mice against strains of S. equisimilis by antiserums

prepared with strains of S. equi

STRAINS OF S. STRAIN OF S. EQUT USED FOR THE PRODUCTIONEQUISIHILIS CLUEO NIEUUSED FOR TEE CIULTURIOONTSEU NORMAL SERUMINFECTION OF DILUTION

MICE 725 940

959 10-6 D5* D5 S D3 D3 D410-7 S S S S D2 D3 D3 D4

729 10-3 D2 D2 D8 Dl D2 D210-4 D4 D7 S D2 D4 S

10--c D3 S S D2 D2 D2

* See tables 6 and 7 for the significance of the symbols.

mice against infection with strain 790, but in the reciprocal test the results werenegative.Antiserums produced by strains of types 1, 3, 9, and 13, respectively, of group

A gave no protection against strains of group C, and in the reciprocal tests anantiserum produced by strain 790 of group C gave no protection against strainsof the mentioned types of group A. These tests with negative results are not re-corded in the tables.Our first cross-protective tests showing immunologic relationship between

strains of S. equi and S. equisimilis were carried out a number of years ago withantiserum produced by S. equi strain 725, which gave protection to mice infectedwith S. equisimilis strain 959. The unexpected result was confirmed in a secondtest. One of the protocols is shown in table 9. Other work intervened, and itwas several years before this study was resumed. In the meantime strains 725and 959 had become avirulent, and it was necessary to use other strains for theproduction of antiserum and for the production of infection in cross-protective


tests. In these tests the possibility of protecting mice against a strain of S.equisimilis by means of serum produced by a strain of S. equi was again confirmed.The protocol of an experiment in which antiserum produced by S. equi strain 940gave slight protection against infection with S. equisimilis strain 729 is shown intable 9, where delayed deaths, as well as survivals, indicate protection.

It is noted (see table 6) that antiserum produced by S. equi strain 940 reactedonly with an equine strain of S. equisimilis (729). It did not protect mice againstinfection with the human strain 790. Likewise the results were negative in thereverse experiment with serum 790 and strain 940.The close relationship which may exist between strains of S. equi and S.

equisimilis is particularly manifest in S. equisimilis strains 959 and 960, fromcases of strangles. According to their reactions in the differential tests thesestrains could be identified with S. equisimilis. Nevertheless in many respectsthey differed from the typical strains of S. equisimilis and resembled S. equi.They grew sparsely in broth culture, produced watery colonies on blood agar;they were originally highly virulent for mice, and, as shown in table 9, strain 959was influenced by the antibodies in an antiserum produced by S. equi strain 725.Apparently strains 959 and 960 are variants of S. equi which have acquired theability to ferment trehalose. On the other hand the equine strains 1189 and1220, which also were isolated from cases of strangles, resembled closely thehuman strains of S. equisimilis. They resembled the type-strain of S. equisimilis,1180, from a human pathologic source, in every test excepting that for lysis ofhuman fibrin (only strain 1220 was tested), and excepting the failure of one of thestrains (1220) to agglutinate in the type-serums.

HABITAT OF S. EQUISIMILIS

S. equisimilis may be identified in the literature by the description of one ormore distinctive characters. Strains from human pathologic sources belongingto group C, or strains which agglutinated in serum of Griffith's types 7, 20, or 21,or strains which were reported as belonging to the same agglutinative type asstrains identified in later investigations with one of those types, or strains ofanimal origin reported to be capable of fermenting trehalose but not sorbitolmay be regarded as S. equisimilis. (See Evans, 1944, for the distinguishing char-acters of S. equisimilis.) If these criteria are accepted, the observations madeby many investigators on the habitat of S. equisimilis are found to be extensive.

S. EQUISIMILIS IN ANIMALS

Data on the frequency of occurrence of S. equisimilis on the mucous membranesof healthy aniimals are meager. A few strains were available, however, for thisstudy. They include strains from a monkey (1)1 and from horses (2). Seele-mann reported that he isolated S. equisimilis frequently from healthy horses.The following review of the literature and the data in table 2 agree in indicating1 In this review the number in parenthesis refers to the number of cases reported by

the mentioned author.

277

ALICE C. -EVANS

that S. equisimilis may cause diseases of varying manifestations in many speciesof animals. The strains of S. equisimilis from animal pathologic sources in ourcollection were received from various investigators (mentioned below) who hadobtained them from bovine (1), equine (5), porcine (1), canine (1), or avian (3)sources.

In their survey of beta-hemolytic streptococci in equine infections, Bazeleyand Battle found S. equisimilis (trehalose-positive strains of group C) less fre-quently than trehalose-negative strains, but they found it repeatedly in strangles(10) and in various other diseases (44) which included respiratory catarrh, pneu-monia, sinusitis, and wound infections. In the latter disease they found it intwelve out of thirty-one cases. Other investigators have reported finding S.equisimilis in equine diseases as follows: Ogura found it in respiratory catarrh(8); Edwards found it in cervicitis (2); Plummer found it in abscesses (3); Seele-mann found it in infectious catarrh (7), and in nasal secretion in a case of strangles(1).

Infection of other animals with S. equisimilis has been reported as follows:Edwards found it in bovine metritis (1); Gibbs found it in baby chicks during anepidemic of laryngo-tracheitis. He reported that the strains fermented lactose.Edwards obtained representative strains from Gibbs, and reported them to belactose-negative strains of group A. In our laboratory Edwards' finding thatGibbs' strains (Nos. 946, 947, and 948 of table 2) were incapable of fermentationof lactose was confirmed. Contrary to Edwards' results, however, in our teststhe precipitative reactions identified the strains with group C, and agglutinationin serum of type 21 confirmed that identification.

S. EQUISIMILIS IN MAN

Many investigators (Lancefield and Hare; Hare; Davis and Guzdar; Reid andBrowne; Frost and Engelbrecht; Wright; Plummer; Wu; Schwentker and others)have reported the isolation of strains with the characters of S. equisimilis fromthe normal human throat or vagina. Ten strains in our collection were from thethroats of healthy human subjects.

S. equisimilis has been reported from cases of puerperal or postabortal infectionby Griffith (1); Lancefield and Hare (1); Congdon (1); Shaw (1); Colebrook andPurdie (2); Gardner (2); Hill and Butler (11); Rosenthal and Stone (1); Plummer(1); Wu (10); Ramsay and Gillespie (12). It has been reported from cases oferysipelas by Birkhaug (31); Williams (1); de Waal (5); from cases of scarletfever by Griffith (1); Bailey (10); Reid and Browne (1); Schwentker, Janney andGordon (38).

It has been reported from various other human diseases. Birkhaug reportedfinding it in cases of mastoiditis (1) and nasal sinusitis (8). Fisher reported culti-vating it from cases of hemorrhagic smallpox (8).. Williams reported finding it insecondary pneumonia (1) and septicaemia (1). Reid and Browne found it inulcerated mouth (1) and septicaemia (1). Gardner found it in tonsillitis (10),hypopyon ulcer (1), otitis (1), and acute rheumatism (1). Pomales-Lebr6n foundit in cases of sore throat (6), rectal ulcer (1), and suppurative infection (1);

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Wright found it in the throat in a case of pyrexia (1); Simmons and Keogh foundit in cases of infection of the urinary tract (1) and suppurative inguinal adenitis(1). Hollander found it in cases of infection of the genito-urinary tract (2).Rantz found it in a case of ulcer (1) and in an abscess (1).

In our collection there are twenty-two strains of S. equisimilis from humanpathologic sources (see table 2). They include three strains from cases of ery-sipelas, one from empyema, three from scarlet fever, one from nasal dischargefollowing scarlet fever, four from puerperal sepsis, four from hemorrhagic small-pox, one from lymphadenoma, one from an abscess, three from septicaemia, andone from pneumonia. Many of these strains were received from the authors men-tioned in the preceding paragraphs (see table 1).

TABLE 10Comparative incidence of streptococci of groups A and C in human infections

GROUP A GROUP CNUMlBERl OFSOURCE STRAINS

Number Per cent Number Per cent

Scarlet fever ........................... 232 229 98.7 3 1.3Tonsillitis and epidemic sore throat.. 52 52 100.0 0 0Rheumatic fever and rheumatic ar-

thritis ................................ 19 19 100.0 0 0Puerperal sepsis ........................ 55 51 92.7 4 7.3Erysipelas .............................. 51 48 94.1 3 5.9Miscellaneous* diseases................. 143 136 95.1 7t 4.9

Total ............................ 552 535 96.9 17 3.1

* Acute suppurative diseases, and various other infections not included in the diseaseslisted above.

t Cases of septicaemia, empyema, lymphadenoma, pneumonia, and abscess.

FREQUENCY OF OCCURRENCE OF S. EQUISIMILIS IN HUMAN INFECTIONS

In table 10 the strains of groups A and C of our collection are tabulated accord-ing to pathologic source. A few strains of S. equisimilis, Nos. 927, 928, 929, 976,and 1136, from human pathologic sources (see tables 1 and 2), were omitted fromthe table because they were received in response to requests for strains of "S.equi." No strain of group C included in table 10 was obtained as a result of re-quest for strains of specified biochemic or serologic characters. Hence the com-parative frequency of occurrence of strains of the two groups in any given humandisease, as shown in the table, should be somewhat indicative of their general oc-currence. The results, which show an average of 3.1 per cent of strains of groupC from human pathologic sources, agree closely with the results of Gardner.Other investigators who have reported that the strains of group C are not com-mon in human diseases are: Lancefield and Hare; Shaw; Bailey; Reid andBrowne; Simmons and Keogh; Plummer; Schwentker, Janney and Gordon.The data in table 10 may be rearranged as in table 11 with infections of the

human throat compared with infections of other parts of the human body. There

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is evidence that diseases caused by infections of the throat with streptococci ofgroup C are rare, whereas diseases caused by infections of other parts of the bodywith streptococci of group C are less rare. In agreement with this observationare the following reports: Wu found a streptococcus of group C in ten out of sixty-eight cases (14.7 per cent) of puerperal infection in China; Hill and Butler foundit in about 9 per cent of 110 cases of streptococcus infection following childbirthand abortion in Australia; De Waal, studying the types of streptococci prevalentin Edinburgh, found six strains of group C among thirty-nine strains from cases oferysipelas, but none among 1,671 strains from other human pathologic sources.Discordant with our limited data on the infrequency of S. equisimilis in infectionsof the throat, however, is the report of Pomales-Lebr6n, who found strains ofgroup C in a considerable percentage of cases of sore throat in Puerto Rico.The three strains from cases of erysipelas included in table 2 were isolated by

Birkhaug, who found 91 per cent of 34 strains from cases of erysipelas to be of thesame agglutinogenic type. He applied the specific name erysipelatis to the group,believing it to be the chief agent of the disease. It appears, however, that he wasmisled in his conclusion concerning the importance of these streptococci as a

TABLE 11Comparative incidence of streptococci of group C in human infections of the throat and of other

parts of the body

GROUP A GROUP CINFECTION NMIBERSOF

Number Per cent Number Per cent

Throat ................. 303 300 99.01 3 0.99Other than throat ....... 249 235 94.4 14 5.6

causal agent of erysipelas because he made his study at a time and place in whichthey happened to prevail. No other investigator has reported finding such ahigh percentage of strains of group C in erysipelas.

SEVERITY OF HUMAN INFECTIONS WITH STRAINS OF GROUP C

Insufficient data are available for an adequate comparison of the severity ofhuman disease in cases of infection with streptococci of group C with that in casesof infection with streptococci of group A. There is a belief, however, thatstreptococci of group C generally cause mild infections. That they may be as-sociated with severe disease sometimes is shown in the following reports: Rosen-thal and Stone reported a fatal case of puerperal infection with vegetative endo-carditis in which a streptococcus of group C was shown to be the causal organism.They believed it to be the first fatal human case of infection with a strain of groupC on record. There are others, however. Stolpe cited a case of fatal infectionof a man contracted from a horse sick with strangles. The eight cases of hemor-rhagic smallpox from which Fisher obtained blood cultures of S. equisimilis wereall fatal, and Hill and Butler reported a severe puerperal infection with a strepto-coccus of group C.

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THE TRANSMISSION OF EQUINE DISEASE TO MAN

There are in the literature a few references to the transmission of streptococcalinfection from horses suffering with strangles to man. Stolpe reported a case ofmiliary abscesses around the finger nails in a man who had performed an autopsyon a horse which had died of strangles. Stolpe also mentioned three cases, one ofwhich was fatal, all having been reported by Bloch. (The reference was notgiven.) The infected persons, having symptoms of septicaemia, had been in con-tact with a horse sick with strangles. Leipold reported a case of streptococcalinfection of a man who had handled a horse sick with strangles. The pus from anabscess ran over his hand, and a few hours later he noticed itching and reddeningof the skin in the area which had been covered by the pus. Within a few days theinfection had spread to the skin of other parts of the body. The disease wasdescribed as corresponding to streptodermia bullosa. A pure culture of strepto-coccus which resembled culturally the streptococcus of strangles developed inmedia inoculated with fluid from the vesicles. Jopke reported the cases of twoveterinarians who developed abscesses on the hands after opening abscesses onhorses sick with strangles.

Since S. equisimilis may infect man and also may cause strangles in horses (seetable 2), and since S. equi, the organism which is the more common causal agentof strangles in horses, does, not infect man in so far as we know, and no strepto-coccus other than S. equi and S. equisimilis is known to cause strangles, it appearsthat S. equisimilis must have been the species concerned in the human cases con-tracted from horses sick with strangles.

SUMMARY

A study of 53 strains of Streptococcus equisimilis was made to determine thetypical and variant characters of the species.The type-strain, 1180, which was isolated from a human pathologic source,

gave a positive precipitative reaction in serum of group C; it was sensitive tofiltered phage B; it fermented trehalose and salicin, but not sorbitol or lactose.Thirty-one other strains agreed with the type strain in all these characters.There were differences in agglutinative properties among the 32 strains, but mostof them agglutinated in one or more of the serums of Griffith's types 7, 20, and 21.The strains of one subgroup differed from the typical strains in their failure to

ferment salicin; those of another subgroup differed from the typical strains intheir ability to ferment lactose; those of still another subgroup differed from thetypical strains in their resistance to filtered phage B.The intermediary status of S. equisimilis between Streptococcus equi and

lactose-negative strains of group A was demonstrated in cross-protective testswhich showed that the strains of S. equisimilis are immunologically related toboth of these groups. The data show no evidence of immunologic relationshipbetween S. equi and lactose-negative strains of group A.The data presented here, together with a review of the literature, show that

S. equisimilismay occuron the normal mucous membrane of man and animals, andthat it may cause infection in man and in many species of animals including birds.

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In our collection, 3.1 per cent of the strains from human pathologic sources werefound to belong to the species S. equisimilis.

Evidence is presented which indicates that in man the strains of S. equisimilishave a tendency to infect other parts of the body more frequently than the throat.

Evidence is presented which indicates that the two variant groups which arecharacterized by failure to ferment salicin and ability to ferment lactose, re-spectively, possess a greater tendency to infect the genital tract than do thetypical strains of S. equisimilis.

ADDENDUM

After this manuscript was completed, the paper by P. Livoni Hansen onthe same subject came to the writer's attention. [On the differentiation be-tween animal and human forms of the trehalose-positive, sorbitol-negativegroup C streptococci ("Human C" streptococci). Skand. Vet. Tidskr., 33,257-280, 1943. In English.] Hansen gives references to a number of authorsnot included in the following list. He concludes that strains may be identi-fied as of human or animal origin by means of the fibrinolytic test. A tabu-lation of the lactose-fermenting abilities of the strains studied by manyauthors leads to the conclusion that the percentage of human strains is greaterthan the percentage of animal strains possessing that property.

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ment, dr. colebrook challenged it stating that in his laboratory only

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