CULTURAL AND ANTIGENIC STUDIES ONSALMONELLA GALLINARUM AND

SALMONELLA PULLORUM'

HENRY G. MAY AND KENNETH GOODNERAgricultural Experiment Station, Rhode leland State College, Kingston,

Rhode Il8and

Received for publication, April 15, 1926

Since it has been shown by Gage (1914) and others that anti-gens prepared from different strains of Salmonella pullorum willgive different results when used in testing for ovarian infection inadult fowls and since the senior author and his associates foundthis to be true to a very marked degree when certain S. pullorumstrains were used, it appeared probable that separate antigenicgroups might be found within this species. This work was, there-fore, undertaken to establish the presence or absence of suchgroups, to help in establishing the position of S. pullorum andS. gallinarum, and to throw more light on the identification ofthese organisms as well as on the agglutination test for the identi-fication of carriers of S. pullorum.

CULTURAL REACTIONS

The 83 cultures chosen for this study are listed in table 1 andinformation is given as to source, type of infections, original des-ignation and previous descriptions.The order of arrangement was established after a study of

experimental data, and, in general, related strains are groupedtogether.The organisms studied are all gram-negative, non-motile rods,

varying considerably in size but being always less then lit inwidth and usually less than 2u in length. Although some data

Contribution No. 335 of the R. I. Agr. Exp. Sta.129

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TABLE 1Strains of S. pullorum and S. gallinarum studied

ISOLATEDNUMBER SOURCE REMARKS OR

RECExVED

S. pullorum

Gage, Mass. Agr. Coll.

Gage, Mass. Agr. Coll.Gage, Mass. Agr. Coll.Gage, Mass. Agr. Coll.Hadley, R. I.Gage, Mass. Agr. Coll.Rettger, Yale Univ.Hadley, R. I.Hadley, R. I.Rettger, Yale Univ.Rettger, Yale Univ.Rettger,Yale Univ.Gage, Mass. Agr. Coll.Gage, Mass. Agr. Coll.Gage, Mass. Agr. Coll.Gage, Mass. Agr. Coll.Hadley, R. I.Hadley, R. I.Hadley, R. I.Hadley, R. I.May, R. I.May, R. I.May, R. I.May, R. I.May, R. I.May, R. I.May, R. I.May, R. I.May, R. I.May, R. I.

May, R. I.May, R. I.Rettger, Yale Univ.Hadley, R. I.Hadley, R. I.Hadley, R. I.Hadley, R. I.Rettger, Yale Univ.

Yolk of chick inoculated withstrain 2

Strain 5Strain 6Strain 25-1Chick lungStrain 25-2. Chick yolkStrain C-'16Chick lungChick lungStrain "Cosgr."Strain B-'15Strain R-'15Strain C-1Strain L-1Strain T-1Strain W-1Adult fowlChick liverChick heartUnabsorbed yolkChick heartChick heartChick heartChick heartChick heartChick heartChick heartChick heartYolk of egg in ovaryLiver of chick inoculated with257

Heart of adult maleChick liverStrain W-'15Liver of adult fowlHeart, same fowl as 102AChick heartAdult fowl (N. Y.)Strain A-'16

12*

13*14*15*17*t182034*t99*123124125126127128129162*167170220223224225226t227t228T229t230257258

27227719102A*102B*117118*122

1915

19151915191519101915191619141914191619161916191619161916191619171915191619201921192119211921192119211921192119241924

19241925191619141914191519151916

I

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S. GALLINARUM AND S. PULLORUM 131

TABLE 1-Continued

ISOLATEDNUMBER SOURCE REBMRKS OR

RECEIVED

S. pullorum-continued

165 Hadley, R. I. Adult fowl 1916168 Hadley, R. I. Liver of adult fowl (Mass.) 1915169 Hadley, R. I. Chick heart 1915224A Tittsler, R. I. Chick inoculated with 224 1923223 May, R. I. Chick heart 1922234 May, R. I. Chick liver 1922244 May, R. I. Chick heart. Same flock as 1923

224245 May, R. I. Heart of adult fowl 1923246 May, R. I. Chick liver. Same flock as 1924

224247 May, R. I. Chick heart 1924271 May, R. I. Heart, adult fowl inoculated 1924

with 102A273 May, R. I. Yolk in ovary, fowl inoculated 1924

with 118275 May, R. I. Liver, adult fowl, no epidemic 1924276 May, R. I. Chick lung 1925

S. gallinarum

38 Rettger, Yale Univ. Originally Cornell 1915110* Hadley, R. I. Liver of fowl 1914115* Th. Smith, Harvard Original Bact. sanguinarium 1915

of Moore116* Th. Smith, Harvard Strain F-T II 1915147 Gage, Mass. Agr. Coll. 1917148 Gage, Mass. Agr. Coll. 1917158* Colo. St. Vet. Coll. "B. avisepticus" 1917161 Goldberg, Cornell Isolated by Taylor, in Cali- 1917

fornia163 C. G. Bull, Rockefeller Inst. "B. avisepticus" 1917164 Murray, Iowa Sta. Coll. 1917202 Kral's Laboratory Bact. gallinarum, Klein, Lon- 1919

don206 Pasteur Inst., Paris Seine and Marne 1920207 Pasteur Inst., Paris Aube 1920208 Pasteur Inst., Paris Ain 1920209 Pasteur Inst., Paris Cote Garonne 1920210 Pasteur Inst., Paris Loiret 1920211 Pasteur Inst., Paris Loiret 2 1920

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132 HENRY G. MAY AND KENNETH GOODNER

TABLE 1-Continued

ISOTATEDNUMXBR SOURCE REAURKS OR

RECEIVED

S. gallinarum-continued

212 Pasteur Inst., Paris Doubs 1920213 Pasteur Inst., Paris Aude 1920214 Pasteur Inst., Paris Cote Garonne 1920215 Pasteur Inst., Paris Yonne 1920216 Pasteur Inst., Paris Originally from U. S. Dept. of 1920

Agr.221 Hadley, R. I. Spleen o? fowl 1920235 May, R. I. Fowl liver 1922236 May, R. I. Heart, fowl, same flock 1922237 May, R. I. Fowl spleen 192245* Hadley, R. I. Fowl liver 191047* Pasteur Inst., Paris Bellefontaine Strain 191165* Rettger, Yale Univ. Strain "Simmons, 1910." 191266* Rettger, Yale Univ. Strain "New Haven, 1909,' 191288* Kral's Laboratory Original B. gallinarum of 1914

Klein

* Discussed by Hadley (1918).t Discussed by Mulsow (1919).$ Chicks from same lot.

have been accumulated on the effect of drying, heat and chemicals,these throw no light on the grouping of the different strains andso are omitted from this paper. The reactions in litmus milkshow no striking variation from the usual reactions of culturesof these groups, namely slight acid formation, followed by alkalini-zation and ultimately by saponification.For the purpose of studying the fermentation of carbohy-

drates the organisms were grown in Dunham tubes of meat extractbroth containing 1 per cent of the carbohydrate in question towhich Andrade's indicator had been added. Readings weremade at appropriate intervals as to the presence or absence ofacid or gas up to fourteen days.The results of these tests are summarized in table 2 and certain

peculiarities are discussed in the text. For the sake of brevitythis discussion takes up the carbohydrates in groups accordingto the type of reaction produced.

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In arabinose, xylose, rhamnose, glucose, levulose, mannose,galactose and mannitol most strains of S. gaUinarum and allstrains of S. pullorum regularly produce acid, while the latteras a rule also produce gas. S. gallinarum No. 45 has occasionallyfailed to ferment xylose and 47 and 66 have consistently failedto do so. S. gallinarum also regularly produces acid from maltose,dextrin and dulcitol, while S. pullorum does not attack thesesubstances. Lactose, sucrose, raffinose, inulin, erythritol andsalicin are not fermented by either S. pullorum or S. gallinarum.

In the carbohydrates fermented by both species the productionof acid is usually prompt, being strongly indicated in 24 hours.Xylose alone is an exception to this rule, as acid formation inthis sugar is always weak and may not appear before forty-eighthours.Although there is as a rule a sharp distinction between S.

pullorum and S. gallinarum in the fermentation of maltose,dextrin and dulcitol, there may appear slight positive reactionswith S. pullorum in maltose or dextrin. This is due to the factthat maltose is easily broken down in sterilization and some lotsof dextrin contain sufficient impurities to permit some fermenta-tion. Sterilization in the autoclave at 10 pounds produced lesshydrolysis in maltose than sterilization in the Arnold. Dulcitolalways gave a sharp distinction between the two species.While more than half of the S. pullorum strains regularly

produced gas from all of the carbohydrates fermented, therewere quite a few strains that failed to produce gas from somecarbohydrates and only occasionally produced gas from others.These irregular strains include most of Hadley's (Hadley, Elkinsand Caldwell, 1918) anaerogenic strains, but culture 162 whichwas one of Hadley's typical strains of Bact. pullorum B regularlyproduces gas.The results of these tests do not show any difference between

cultures from diseased adult fowls and from chicks or the ovariesof apparently healthy adult fowls. Most of the more recentlyisolated cultures appear among the irregular strains, while only afew of the older cultures appear there. This indicates that gasproduction from ordinary carbohydrate media tends to increase

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HENRY G. MAY AND KENNETH GOODNER

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HENRY G. MAY AND KENNETH GOODNER

with the length of time a culture has been kept on artificial media.This is, however, not a universal rule, as some strains that haveformerly produced gas regularly, now fail to produce gas fromsome carbohydrates. Since every laboratory obtains differentresults in regard to gas formation by S. pullorum and since differ-ent workers in the same laboratory or the same workers at differ-ent times do not always get the same results it is probable thatslight variations in the media produce relatively large differencesin gas formation. The authors are taking up a special study ofthis question and hope. to be able to make a report in the nearfuture.

ANTIGENIC STUDY

For the inoculation of rabbits, cultures were selected thatrepresented all of the various peculiarities that were known atthe time. From S. pullorum the following cultures were se-lected: An early and a later strain from Dr. Rettger (Nos. 20 and122); an early and a later strain from Dr. Gage (Nos. 12 and 128);two strains which Hadley had determined to be S. pullorum B(Nos. 118 and 162); a typical and an irregular strain isolatedvery recently in this laboratory (Nos. 224 and 234 respectively).The selections to represent S. gallinarum were Klein's original(No. 88), Moore's original Bact. sanguinarium (No. 115); astrain from the Pasteur Institute (No. 206), and a recent isolationby this laboratory (No. 235). For the sake of comparison -astrain of the human typhoid bacillus was also used.These organisms were grown on meat infusion agar slants for

twenty-four hours, the growth washed from the slants with theaid of sterile saline and the suspensions heated in a water bath forthirty minutes at 600C. The suspension was standardized bythe Gates (1920) method at a density between 1 and 2 cm. andwas then diluted so that the final density was approximately acomputed 8 cm.Young, healthy male rabbits were given five intravenous in-

oculations of killed bacterial suspension at intervals of two tothree days. The amounts used in these injections were: 0.25 cc.,0.5 cc., 1.0 cc., 1.5 cc., and 2.0 cc., respectively. Test bleeding

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was made on the fifth day after the last inoculation. In everyinstance a favorable titer was obtained and the final bleedingwas on the seventh to ninth day after the last injection.

In preparing the bacterial suspensions for the long seriesoftitrations, the organisms were grown in large plates on meat in-fusion agar. The growth was washed off with 0.8 per cent sodiumchloride solution containing 0.5 per cent phenol, and the suspen-sion was diluted to an appropriate density with the same reagent.This density corresponded to approximately 3 cm. by the Gatesmethod.The dilutions of the antiserum were such that each succeeding

tube had twice the dilution of the one just preceding, e. g.,1-40, 1-80, 1-160, etc. Antigen and antiserum were then mixedin equal parts, ginvng a final antigen dilution of 6 cm. and a finalserum dilution of 1-80, 1-160, 1-320, etc. Each antiserum wastitrated against each antigen and controls were uniformly run.The tubes were incubated at 370C. for eighteen hours and thiswas followed by a few hours in the ice box. Tubes were read outfor agglutination with a lens. More than twenty-five hundredtitrations were made in this series. All antigens were titratedagainst the same serum at the same time, in fact the serum wasdiluted in flasks and pipetted out to the agglutination tubes sothat all antigens would be mixed with serum of exactly the samestrength.The results have been summarized in table 3. Only the de-

nominator of the fraction representing the limit of agglutinationbetween a given antigen and serum has been given. The regularS. pullorum strains are listed first followed by those giving someirregular fermentations. The division of the strains of S. gal-linarum is a natural one based on the agglutinative propertiesof the various antigens. Certain strains which were agglutinatedto the same degree by the same antisera are placed above, fol-lowed by the less regularly agglutinated strains.A study of table 3 seems to show that there are no conspicuous

antigenic differences within the groups studied. At the sametime there are some variations which are hard to explain.

In the titration of pullorum antigens against pullorum antisera

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HENRY G. MAY AND KENNETH GOODNER

the uniformity in the degree of agglutination is very striking.In any one serum no antigen agglutinates more than eight timesas well as any other antigen. Furthermore, the weakly aggluti-nating antigens give a rather uniformly low titer with all seratested, while the more readily agglutinating antigens give arelatively high titer with all sera. No indication of grouping istherefore apparent.

In the titration of pullorum antigens against gallinarum anti-sera the degree of agglutination was uniformly somewhat lowerand variations were somewhat greater. There has appearednothing unusual, however, except for occasional serum-antigencombinations that failed to give agglutination even in the lowestdilution of the serum (1-80). These sporadic cases can in no waybe correlated with antigenic groups and must for the presentremain unexplained.

In the titration of gallinarum antigens against pullorum anti-sera the degree of variation is still more pronounced. The divi-sion, however, is again into weakly agglutinating antigens andmore strongly agglutinating antigens. For the sake of comparisonthe antigens showing the lowest degree of agglutination withpullorum sera have been listed at the lower end of the table (cul--tures 45, 47, 65, 66 and 88).The degree of agglutination between gallinarium antigens and

gallinarum antisera is very nearly the same as in case of gallina-rum antigens and pullorum antisera. The-same group of cul-tures is again more or less set off from the rest as showing thelowest degree of agglutination.

In the Eberthella typhosa antiserum both pullorum and gal-linarum antigens agglutinated only to a moderate degree, thepullorum antigens agglutinating somewhat better than the gal-linarum antigens. In fact, some of the gallinarum antigens failedto agglutinate in this serum even in the highest concentrationused. Among these antigens is the entire group at the end ofthe list. The typhosa antigen failed in all cases to agglutinatein the pullorum and gallinarum antisera.

Outside of the five gallinarum strains mentioned, there isnowhere any indication of antigenic groups. These five strains

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may possibly be considered as forming an antigen-ic igroup feebly'set off from the rest. They fail to agglutinate well with pullorumand gallinarum antisera-and;do-agglutinate as a rule fairly readilyw -iththe antiserum of one of their own number, stran 88. Thedivision, however, is not at all dlearly m-aked as strain 65 does notagglutinate strongly with antiserum from strain S88 and othercultures such as 110, 116, 147, 161 and 163 show only slightlystronger agglutination in other sera than do the members ofthis group.

GENERAL DISCUSSION

One of the more important questions in connectio with thesetwo organisms is their position in the system of -classification.The typical aerogenic S. pullorum has long been accepted as amember of the genus -Salmonek-la but the anaerogenic form israther a stumbling block for those who use gas produetiona as .theessential criterion. Depending upo the elassier, S. >galliiirumis likely to be found -either in the :genus EbertheHla or the genusSalmonella.One thing has definitely been shown, and that is that S. gal-

linarum bears an extremely close relationship with S. pullorum.Any classification that seeks to throw the two organims intowidely separated groups is only defeating the purpose of thesystem.

It must be accepted too, that there is a certain antigenicsimilarity between these two organims and Eberthella typhosa,but it has been shown in this paper that this relationship is notas remarkable as certain workers have been led to believe.Although the members of the old colon-typhoid group are be-

coming more and more distinct and defined, there still exists agreat deal of confusion and all of the systems of separating thislarge group into sub-groups or distinctgenera mustifor the presentstill resort to some very artificial barriers. One dof the moreobvious lines of division has been the presence or absence of gasformation in fermentable sugars. This has recently been usedvery much as the presence or absence -of motility was formerly-used. It appears, however, that in the case of -the two -species

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considered the distinction on the basis of the presence or absenceof gas formation is entirely too artificial, separating into twogenera organisms that otherwise scarcely show specific distinc-tions. To avoid this unnatural division it seems necessary toregard the two species as one unit and place them in the genusto which they collectively show the greatest relationship. Thisbeyond a doubt is the genus Salmonella in which Ligni6res(1925) originally placed his organisms of fowl typhoid and inwhich both organisms would fall according to the grouping byKrumwiede and Kohn (1917) and by Park, Williams and Krum-wiede (1924). The grouping of these authors ignores gas pro-duction and excludes from the genus Salmonella organisms whichdo not ferment rhamnose and which do ferment lactose, sucroseor salicin.With this in view the following definitions are advanced for

these organisms.Salmonella pullorum (Rettger) (1909).Gram-negative, non-spore-forming, non-motile rods. Growth

on solid media resembling Esch. coli, but much less vigorous.Acid is produced in media containing arabinose, xylose, rhaamnose,glucose, levulose, galactose, mannose and mannitol. Gas mayor may not be produced. Does not ferment lactose, sucrose,raffinose, maltose, dextrin, dulcitol, inulin, erythritol or salicin.Does not produce indol or liquefy gelatin. Usually produceshydrogen sulphide. Indistinguishable antigenically from Sal-monella gallinarum (Klein). Regarded as the causative agent ofbacillary white diarrhea of young chicks but found in adult birdsin localized infections or as producing a disease that can not bedistinguished from fowl typhoid.

Salmonella gallinarum (Klein) (1889).Gram-negative, non-spore-forming, non-motile rods. Growth

on solid media resembles that of Esch. coli, is less vigorous, but ismore vigorous than that of S. pullorum. Never produces gas inany carbohydrate media. Acid is produced in media contain-ing arabinose, rhamnose, glucose, levulose, galactose, mannose,maltose, dextrin, dulcitol and mannitol. Does not fermentlactose, sucrose, raffinose, inulin, erytliritol, or salicin. Does

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S. GALLINARUM AND S. PULLORUM

not produce indol or liquefy gelatin. Usually produces hydrogensulphide. Most strains are indistinguishable antigenically fromS. pullorum. Regarded as the causative agent of fowl typhoid butpossibly also associated with diarrhea-like disturbances in chicks.For routine identification purposes the following scheme is in

use in this laboratory.Gram-negative, non-spore-bearing rods from avian sources

are considered. These are inoculated into media containingglucose, lactose, sucrose, and dulcitol respectively. If the glu-cose is fermented with or without gas, but none of the others arefermented the organism is S. pullorum. If both glucose anddulcitol are fermented but not lactose or sucrose the organism isS. gallinarum. The use of the glucose media is necessary toplace the organism in the group. The lactose serves to eliminatecoli-like organisms, the sucrose eliminates cholera-like bacteria,while the dulcitol serves to distinguish between S. pullorum andS. gallinarum. Maltose or dextrin might be substituted fordulcitol but the latter has been found much easier to prepare andto give very reliable results. To reduce cost dulcitol is used in0.5 per cent solution in place of 1 per cent.The results of this investigation do not entirely clear up the

difference in results obtained with various antigens in the routinetest for pullorum reactors. The fact that some antigens willagglutinate in a given serum as much as 8 times as well as othersexplains some of the results, such as the fact that some antigenswill usually detect more reactors than others; but the fact that theweaker agglutinating antigens will in certain flocks detect morereactors than the more readily agglutinated strains still remainsunexplained.

SUMMARY

A study of the carbohydrate fermentation of 52 strains of S.pullorum and 31 strains of S. gallinarum shows that the membersof each group agree very closely in the production of acid fromthese substances. S. pullorum usually produces gas from fer-mentable carbohydrates while S. gallinarum never produces gas.The titration of antigens from these strains against antisera

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146 HENRY G. MAY AND KENNETH GOODNER

from 8 representative strains of S. pulloum and 5 representativestrains of S. gallitarum as well as one strain of Eberthella typhosareveals no definite antigenic differences between the two groupsof avian. pathogens nor does it reveal any sub-groups with theexception of a slight differentiation within S. gallinarum.Both pullorum and gallinarum are regarded as belonging to the

genus Salmonella.A method for the rapid identification of the two species consists

in passing suspected avian pathogens (gram-negative rods) throughglucose, dulcitol, lactose and sucrose.

REFERENCES2

GAGE, G. E. 1914. On the diagnosis of infection with Bacterium pullorum inthe domestic fowl. Mass. Agric. Exper. Sta. Bul. 148.

GATES, F. L. 1920 A method of standardizing bacterial suspensions. Jour.Exper. Med., 31: 105-114.

HADLEY, P., ELKINS, M. W., AND CALDWELL, D. W. 1918 The colon-typhoidintermediates as causative agents of disease in birds: I. The para-typhoid bacteria. R. I. Agric. Exper. Sta. Bul. 174, 216 p.

KLBIN-, E. 1889 Ueber eine epidemische Krankheit der Huhner, verursachtdurch einen Bacillus-Bacillus gallinarum. Centbl. Bakt., 5: 689-693.

KRUMWIEDE, CHAS. AND KOHN, L. 1917 The differentiation of the membersof the paratyphoid enteritidis group from B. typhosus with specialreference to anaerogenic strains and observations on the fermentativecharacteristics of the avian groups. Jour. Med. Res., 36: 509-518.

LIGNItRES, J. 1925 Sur la classification du microbe de la typhose aviaire.Bul. Acad. M6d. Paris, 9S: 500-505.

MULSOW, F. W. 1919 The differentiation and distribution of the paratyphoid-enteritis group. VI. Aviian paratyphoid bacilli: A comparative studyof B. pullorum and B. sanguinarium. Jour. Infect. Dis., 25: 135-162,

PARK, W. H., WILLIAMs, A. W. AND KRUMWIEDE, CHARLES. 1924 Pathogenicmicroorganisms. Lea and Febiger, N. Y., p. 405.

RETTGER, L. F. 1909 Further studies on fatal septicemia in young chickens,or "white diarrhea." Jour. Med. Res., 21: 115-123.

' The recent literature of this subject is reviewed in Bulletin 204 of the R. I.Agr. Exp. Sta.

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