INFECTION AND IMMUNITY, Nov. 1979, p. 668-679 Vol. 26, No. 20019-9567/79/11-0668/10$02.00/0

Isolation and Characterization of Toxic Fractions fromBrucella abortus

LOUISA B. TABATABAI,* BILLY L. DEYOE, AND ALFRED E. RITCHIENational Animal Disease Center, Agricultural Research Service, Ames, Iowa 50010

Received for publication 6 August 1979

Two types of toxic fractions, protein-rich and carbohydrate-rich, were isolatedfrom attenuated (strain 19) and virulent (strain 2308) Brucella abortus organisms.Polyacrylamide gel electrophoresis of the protein-rich fraction, in the presenceand absence of sodium dodecyl sulfate, revealed qualitative and quantitativedifferences in the protein bands derived from the attenuated and virulent strains.Sodium dodecyl sulfate-gel electrophoresis indicated that the major differencesbetween these protein fractions were in the molecular weight range from 14,000to 40,000. Immunoelectrophoresis of these fractions from the attenuated andvirulent strains revealed differences in the antigenic spectrum. Polypeptides inthe carbohydrate-rich fraction could be visualized on polyacrylamide gels onlywhen reacted with fluorescamine before electrophoresis. Immune sera did notprecipitate the components of the carbohydrate-rich fraction. Intradermal injec-tion of the protein and carbohydrate-rich fractions resulted in different types ofskin lesions in guinea pigs, i.e., edematous/erythematous and necrotic lesions,respectively. Fractions derived from attenuated and virulent strains of B. abortuswere equally toxic in the guinea pig skin test. The toxic activity of both types offractions was susceptible to pronase and heat treatment.

Numerous reports exist of the isolation ofcellular components from Brucella and theirrelationship to observed toxic effects in labora-tory animals (3, 14, 19, 30). In most instances,cells were extracted with hot aqueous phenol asoriginally described by Westphal et al. (43), orcells were fractionated after total disruption withabrasives (10), with pressure (11), or by sonica-tion (5, 23). Often Brucella fractions were lesstoxic in dermal hypersensitivity tests than wholecell wall or intact killed organisms (17, 30). Evi-dently, Brucella lipopolysaccharide (LPS) withtypical endotoxic reactions is extracted mainlyin the phenol phase, unlike enterobacterial LPS,which partitions into the aqueous phase (17, 24,30, 35).Very few differences in chemical composition

or toxicity have been observed in fractions ob-tained from smooth and rough organisms, i.e.,virulent and attenuated strains of Brucella (8,17, 24, 30). It is conceivable that by using ace-tone- or methanol-killed cells or by using hotaqueous phenol-extracted cells, important anti-genic or potentially toxic components are ex-tracted, denatured, or discarded. Also, thesetoxic or antigenic surface components might es-cape detection among the numerous compo-nents found in extracts (4). Our study was con-ducted in an attempt to demonstrate antigenic

surface components from virulent and atten-uated Brucella abortus by a gentle extractionprocedure and to determine their chemical com-position, antigenic spectrum, and dermal toxic-ity. Preliminary results were presented recently(L. B. Tabatabai, B. L. Deyoe, and S. S. Stone,Fed. Proc. 37:3054, 1978).

MATERIALS AND METHODSGrowth of bacteria and harvesting of cultures.

B. abortus was grown on tryptose agar (Difco Labo-ratories, Detroit, Mich.) in Roux flasks for 72 h at37°C. Frozen stock cultures of strains 2308 (virulent)and 19 (attenuated) were maintained as a source ofinoculum. Frozen material was reconstituted and pas-saged once on tryptose agar before Roux flasks wereseeded. Cells were harvested by gentle washing of theagar surface with phosphate-buffered saline.

Preparation of cell fractions. Cells were washedtwice with phosphate-buffered saline and centrifugedat 20,000 X g for 20 min at 5°C. Cells were thensuspended at 0.1 g/ml (wet wt/vol) in 60% methanol,stirred gently for 4 h at 5°C to inactivate the cells, andcentrifuged. The aqueous methanol supernatantserved as the source for the carbohydrate-rich fraction.The methanol-extracted (and inactivated) cells werethe source for the protein-rich fraction. The inacti-vated cells were then washed twice with distilled wa-ter, suspended in 1 M NaCl-0.1 M sodium citrate at0.2 g/ml, and stored at 5°C. The methanol inactiva-tion-extraction procedure was repeated until culture

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BRUCELLA ANTIGENS AND TOXIC FRACTIONS 669

tests indicated that there were no viable cells present.Protein and carbohydrate contents of cell washes weremonitored at each step. The cells were then agitatedwith (i) an equal volume of glass beads (0.11 mm) for60 min at 5VC, using a Mickle tissue disintegrator(Mickle, Gomshall Surrey, England), and (ii) 30% byvolume of glass beads (0.11 mm) from 2 s to 4 minunder a stream of liquid carbon dioxide, using theBraun model MSK homogenizing cell (Bronwill Sci-entific, Inc., Rochester, N.Y.). The fractions obtainedas outlined in Fig. la and lb were stored at -40'Cwithout loss of activity.

a WA

6CENTI

SUPERNATANT FILTEREDO.45p FILTER WASH T|

CENTI

SUPERNATANTS FILTERED0.45p FILTER F

1MCENTI

SUPERNATANT DISCARDEDF

1M Na

AGITATE INWITH 0.1mm

ALLOWCENTRIFUGE

PELLETSAMPLED FOR PRECIPITATEELECTRONMICROSCOPY DIAL

b

Electron microscopy. Samples for electron mi-croscopy were obtained from the pellet after centrifu-gation of the Mickle-treated cells. For negative stain-ing, cells were added to a spot-plate well containing 1drop of 4% potassium phosphotungstate adjusted topH 6.8, ca. 20 drops of distilled water, and 1 drop of1% bovine serum albumin (Cohn fraction V). Aftermixing gently, the dispersion was sprayed onto carbon-coated, collodion-filmed grids with a Vaponefrin-typeall-glass nebulizer (Ted Pella Co., Tustin, Calif.). Con-trast, cell distribution, and spreading were optimizedby varying the final concentrations of potassium phos-

ASHED CELLS IN PBS

50%METHANOL.4 HRRIFUGE 20,000 xg 20 MIN

PELLETWICE WITH DISTILLED WATER'RIFUGE 20,000 zg .15 MIN

PELLETRESUSPEND IN COLDNaCI/0.1M Na CITRATE'RIFUGE 20, 000 X g. 15 MIN

PELLETRESUSPEND IN COLDaCI/0.1M Na CITRATE

MICKLE TISSUE DISINTEGRATERGLASS BEADS FOR 60 MIN, 5'CGLASS BEADS TO SETTLESUPERNATANT 17, 000Xg, 30 MIN

SUPERNATANTWITH (NH14)SQ4 AT 70%SATURATIONLYZE VS.5mM NH4 HCO3

SUPERNATANT FILTERED0.45p FLTER

PELLET (SEE FIG. 1)

FLASH EVAPORATE TO DRYNESSDISSOLVE RESIDUE IN 1 TO 4m1 DISTILLED WATERREPEAT TWICE

DIAYLZE AGAINST 2 CHANGES OF 250m1DISTILLED WATER, 50C

FLASH EVAPORATE DIFFUSATE TO DRYNESSDISSOLVE IN 1 TO 4m1 DISTILLED WATER

FIG. 1. (a) Scheme for extracting protein-rich fraction from B. abortus. (b) Scheme for extracting carbohy-drate-rich fraction from B. abortus.

VOL. 26, 1979

WASHED CELLS IN PBS

60%METHANOL,4 HRCENTRIFUGE 20,000 xg 20 MIN

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670 TABATABAI, DEYOE, AND RITCHIE

photungstate, sample, or bovine serum albumin asrequired (31). Grids were examined immediately in aPhilips EM-200 electron microscope operated at 60 kVwith double-condenser illumination and a 30- to 35-t2mcopper disk objective aperture.Chemical determinations. Protein content was

measured by the protein-dye binding method of Brad-ford (6) or by the Folin-phenol method described byLowry et al. (20), using bovine serum albumin (ArmourPharmaceutical Co., Phoenix, Ariz.) as a standard.

Total carbohydrate was determined with thephenol-sulfuric acid method as described by Dubois etal. (9). Presence of 2-keto,3-deoxy sugar acids in thesamples were measured as described by Ashwell (2)except that a 10-min preliminary hydrolysis step with0.25 N H2SO4 at 1000C was included; 2-keto-3-deoxy-octonate (KDO) was used as a standard. Absorbanceat 532 nm (A532) due to 2-deoxy aldoses, althoughminimal, was corrected for as described by Warren(38), using 2-deoxyribose as a standard. Sialic acid,also a 2-keto-3-deoxy sugar acid, was not corrected forseparately, but determined enzymatically (37) on somesamples. Presence of hexosamine was determined bythe phenol-sulfuric acid procedure after the sampleswere deaminated and chromatographed according tothe procedure described by Lee and Montgomery (18).Total nitrogen was determined on 100-ll samples bythe micro-Kjeldahl procedure (21).

Samples for amino acid analysis were hydrolyzed invacuo for 22 h at 1100C with constantly boiling 5.7 NHCl. Samples were analyzed on a Durrum single-col-umn instrument (Durrum Chemical Corp., Sunnyvale,Calif.) at the Department of Biochemistry and Bio-physics, Iowa State University, Ames.

Deoxyribonucleic acid (DNA) and ribonucleic acidwere determined on 1- to 2-mg samples according tothe methods of Webb and Levy (40) and Webb (39).Polyacrylamide gel electrophoresis. Sodium

dodecyl sulfate (SDS)-polyacrylamide gels (7 cm long)contained 7.5% acrylamide and 0.3% bisacrylamide(41). The electrophoresis buffer used was a 1:1 dilutionof the gel buffer (13), which contained 100 g oftris(hydroxymethyl)aminomethane (Tris), 10 g of eth-ylenediaminetetraacetic acid (disodium salt), 3.8 g ofboric acid, and 1 g of SDS per 500 ml and was adjustedto pH 9.3. Electrophoresis was carried out at 7 mA/gel at 15 to 18°C. Samples (not to exceed (100,l) forSDS-gel electrophoresis were prepared as describedby Weber and Osborn (41).

Polyacrylamide gels in the absence of SDS wereprepared as described by Ortec Inc. (27), except thatcylindrical gel tubes (6 mm [inner diameter] by 10 cm)were used. The gels (7 cm long) contained 7.5% acryl-amide and 0.19% bisacrylamide; the stacking gel (0.5cm long) contained 5% acrylamide and 0.13% bisacryl-amide. Electrophoresis was carried out at 2.5 mA/tubeat 15 to 18°C. Samples were prepared either by dialysisagainst Tris-borate tank buffer (27) or by addition ofconcentrated (fourfold) Tris-borate buffer to theproper final concentration. One drop of glycerol and 5,l of 0.3% bromophenyl blue were added before thesamples were applied to the gels. Gels were stained forprotein with Coomassie brilliant blue (41).

Fluorescamine-derivatized fractions were preparedaccording to the method of Rosemblatt et al. (32).

Briefly, 20 ,ul of carbohydrate-rich fraction in distilledwater was mixed with 20,ul of 0.1 M sodium borate ina glass tube (10 by 75 mm). To this solution was added10,lO of fluorescamine (Hoffmann-LaRoche, Inc., Nut-ley, N.J.) at 3 mg/ml in acetone, and the mixture wasallowed to stand at room temperature for 10 min. Acontrol containing distilled water was treated in anidentical manner. One drop of glycerol and 5 ,ul of 0.1%bromophenyl blue tracking dye (the latter only incontrol tubes) was added, and samples were loadedonto 7.5% acrylamide gels. Electrophoresis was per-formed at 5°C for 2 h at 2.5 mA/gel with 0.1 M Tris-0.77 M glycine, pH 8.6, as the electrode buffer (7).Fluorescamine-derivatized bands were visualized un-der long-wave ultraviolet light.

Immunoelectrophoresis. Protein fractions wereconcentrated with a Minicon B-15 concentrator (Ami-con Corp., Lexington, Mass.) so that the samples con-tained 2 to 5 mg of protein per ml. Electrophoresis of15-pl samples was carried out in 1% agarose in 0.14 Mborate buffer, pH 8.6, at 24 mA/slide at room temper-ature. Slides were developed with hyperimmune bo-vine anti-B. abortus strain 19 serum and immunebovine anti-B. abortus strain 2308 serum. The hyper-immune bovine serum was prepared by multiple injec-tions of a steer with live B. abortus strain 19. Injectionswere initially given intravenously and then subcuta-neously or intramuscularly at biweekly intervals for 16weeks. Organisms given at the third, fourth, and fifthinjections were incorporated into Freund completeadjuvant. Sera collected were concentrated fourfoldby hollow-fiber filtration (Amicon Corp.) through a100,000-dalton filter (B. L. Deyoe, S. S. Stone, andJ. B. Patterson, manuscript in preparation). The im-mune anti-strain 2308 serum was from an experimen-tally infected cow (28).Absorption of sera with whole cells. Absorption

of sera with homologous B. abortus organisms wasperformed with 300 to 400 mg (wet weight) of metha-nol-inactivated cells per ml of serum. Cell suspensionswere incubated for 30 min at 37°C and centrifuged.The supernatant serum was then treated two addi-tional times with a fresh pellet of organisms. The finalabsorbed sera had titers of less than 20 as assayedwith the standard tube test (1). Immunoelectropho-resis was then performed as described above.

Toxicity assays. White female guinea pigs rearedat the National Animal Disease Center, weighing 250to 350 g, were used for the toxicity assays. The lowerlateral surfaces were shaved with an Oster electricclippers equipped with a no. 40 blade. Shaved areaswere then wiped with 2% tincture of iodine. Filter-sterilized, concentrated, protein-rich fractions and car-bohydrate-rich fractions were diluted with sterile sa-line to designated concentrations, and 0.1-ml quanti-ties were injected intradermally with a 26-gauge intra-dermal bevel needle. Guinea pigs were observed at 1,6, 24, and 48 h postinjection for signs of skin lesions.Representative examples of toxic reactions and controlsaline injection sites were photographed and examinedmicroscopically. For histopathological examination,guinea pigs were euthanized, and then a 4-cm2 areaincluding the injection site was excised, fixed in 10%buffered Formalin, embedded in paraffin, sectioned at6 ,um, and stained with hematoxylin and eosin.

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BRUCELLA ANTIGENS AND TOXIC FRACTIONS 671

RESULTS

Characterization of toxic protein-richfractions. After 60 min of shaking in the hyper-tonic salt solution in a Mickle apparatus, themethanol-inactivated Brucella cells were unbro-ken (Fig. 2a,b). Most of the cells were flattened,indicating some plasmolysis as expected. Whenthe Braun apparatus was used at the least con-trollable time, 2 s, most of the cells were dis-rupted with massive protoplasmic extrusion(Fig. 3), precluding its use for extracting thepredominantly surface-localized cell compo-nents.The ammonium sulfate-precipitated protein-

rich fractions yielded approximately 1 mg ofprotein per mg (dry weight) of bacteria. Becauseof this low yield, these fractions were kept insolution rather than lyophilized. Chemical anal-ysis of the protein-rich fraction is reported inTable 1. Carbohydrate and 2-keto-3-deoxy sugar(expressed as KDO) content varied from batchto batch. No marked difference was noted in thetotal carbohydrate content in the protein-richfractions obtained from the two different strains.Total carbohydrate content (expressed as glu-cose) ranged from 0.05 to 0.4 mg per mg ofprotein. The 2-keto-3-deoxy sugar acid content(KDO) of protein-rich fractions from differentbatches of cells ranged from <1 to 10 ,ug per mgof protein. However, no KDO was detected inthe protein-rich fraction used for the toxicitystudies, although carbohydrate was present. Theresults expressed as KDO, however, could in-clude contributions made by sialic acid or neu-raminic acid (a 2-keto-3-deoxy sugar acid) be-cause incubation of some of the samples withneuraminidase released thiobarbituric acid-pos-itive substances.The high A20/20 value of the protein-rich

fraction is believed to be due to nucleic acidcontamination. However, no DNA (determinedas deoxyribose after hydrolysis of samples withtrichloroacetic acid [40]) could be detected inthe samples. The A260 value might also havebeen due to ribose-containing nucleotides orother ribose-containing compounds absorbing at260 nm, since ribose was detected (Table 1) inthe protein-rich fraction by the method ofWebb(39).Gel electrophoresis. Conventional poly-

acrylamide gel electrophoresis and SDS-gel elec-trophoresis of the protein fractions obtainedfrom the two strains of Brucella revealed qual-itative and quantitative differences in the pro-tein bands (Fig. 4): strain 19 contained six majorprotein bands and strain 2308 contained fourbands. The major differences of the proteinbands between the attenuated and virulent

strains were found in a molecular weight rangefrom 14,000 to 40,000.Immunoelectrophoresis of protein frac-

tions. Immunoelectrophoresis of the proteinfractions from strains 19 and 2308 showed almostidentical patterns (Fig. 5) when hyperimmunebovine anti-strain 19 serum was used to developthe plate. When immune anti-strain 2308 serumwas used, fewer precipitin lines were revealed,but the pattern differed considerably (Fig. 5). Inthe homologous reaction with anti-strain 2308serum, an additional arc was visible.The absorption experiment was designed to

determine whether the components in the pro-tein-rich fraction were derived solely from thesurface of Brucella cells. Absorbed sera resultedin removing only one of four arcs, the mostcathodal migrating arc (Fig. 6a,b, upper panel,arc 4), from strain 19 protein-rich fraction (thefifth arc did not show up in this experiment);two of three arcs were removed (the most cath-odal and anodal migrating arcs, Fig. 6a,b, lowerpanel, arcs 1 and 3). The arcs near the antigenwells were not removed from either protein-richfraction by the absorption experiment.Toxicity assay of protein-rich fractions.

Protein-rich fractions at a concentration of 40,ug of protein per intradermal injection causederythematous and edematous skin reactionswith a diameter of 14 to 17 mm, which wasmaximal after 6 h (Fig. 7); with 4 ,Ig of proteinper injection, this type of reaction was not con-sistently observed. Histopathological examina-tion of the skin lesions showed polymorphonu-clear cell infiltration into adipose and musclelayers, but no vascular damage was observed(Fig. 7). When protein-rich fractions weretreated with pronase or heated at 100°C accord-ing to Leong et al. (19), the dermal reaction wasreduced to less than 5 mm at the 40-,Lg level 6 hpostinoculation.Characterization of toxic carbohydrate-

rich fractions. The aqueous methanol extractof Brucella cells, after concentration, dialysis,and further concentration, was deeply yellowcolored. Chemical analysis (Table 2) showedthat it was predominately carbohydrate. Proteincontent of this fraction, measured by the Folin-phenol method (20), varied from batch to batch,ranging from 0.5 to 2.0 mg per mg of carbohy-drate. Comparision of total nitrogen with proteincontent indicated that protein or peptides didnot account for all of the nitrogen present in thesamples. Qualitative tests for hexosamines werepositive. Amino acid analysis of the carbohy-drate fraction revealed peaks with identical re-tention times as standard amino acids. The chro-matogram (not shown) indicated that most ofthe common amino acids were present, with two

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672 TABATABAI, DEYOE, AND RITCHIE

4-_

it

i02 PM

.1i

,-.s

0.2 pm0FIG. 2. (a) Untreated Brucella cell in phosphotungstate-negative stain. Typical condition is indicated by

retention of surface convolutions and minor protoplasmic retraction. (b) Brucella cell after 60-min agitationin the Mickle apparatus. Surface convolutions are still present without marked protoplasmic retraction.Fractured cells were not noted.

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BRUCELLA ANTIGENS AND TOXIC FRACTIONS 673

0 I. .11

FIG. 3. Brucella cells after minimum (2 s) agitation in the Braun apparatus. Ten to 20%o of cells werefractured, releasing excessive protoplasmic mass.

TABLE 1. Composition ofprotein-rich fractionsprepared from B. abortusDetermination Value

Carbohydratea/protein (mg/mg) 0.05-0.40KDO/protein (Iug/mg) <1-10A260/A280 1.3-1.4Ribose +2-DeoxyriboseaExpressed as glucose.

or possibly four additional ninhydrin-positivepeaks presumably representing hexosamines.The carbohydrate-rich fractions contained 2-keto-3-deoxy sugar (expressed as KDO), but thisvaried in different batches from 1 to 10 jig permg of carbohydrate. Absorption at 532 nm dueto 2-deoxyribose (which interferes in the test forKDO) was minimal, but was corrected as de-scribed under Materials and Methods. The KDOcontent of the carbohydrate-rich fractions usedin the toxicity study was 0.05 ug/0.1 ml (Fig.7c,d).The carbohydrate-rich fractions had an unu-

sually high A260/280 value of 3.0 to 4.0, with anapparently high Allm value of 175 to 230 basedon carbohydrate content and an A"~mvalue of100 to 380 based on protein content. The highA260 value of the carbohydrate-rich fraction is inthe range reported for nucleic acids. However,DNA was not detected in the samples. Again, aswas the case for the protein-rich fractions, theA260 value may have been due to ribose-contain-ing nucleotides or other ribose-containing com-pounds which absorb at 260 nm, because ribosewas detected in the samples. The carbohydrate-rich fraction was never obtained in solid form,but was always kept in solution because of itsallergenic properties.Gel electrophoresis of carbohydrate-rich

fractions. Since it was determined that proteinor peptides were present in these fractions, elec-trophoresis in 7.5% gels was performed underdenaturing and nondenaturing conditions at pH9.0 and in 7.5% gels containing 4 M urea at pH4.5. No bands could be visualized with Coomas-sie brilliant blue as the protein stain. Lack ofreaction with Coomassie brilliant blue in the

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41,i.

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674 TABATABAI, DEYOE, AND RITCHIE

WITH SDS NO SDS

--_- -51,000_ -_ 46,000

. -30,000

*_ 5l -17,000

-14,000

A B A B

FIG. 4. Polyacrylamide gel electrophoresis in thepresence and absence of SDS. (A) Protein-rich frac-tion from strain 19; (B) protein-rich fraction fromstrain 2308.

protein-dye binding method of Bradford (6) andthe negative results obtained with polyacryl-amide gels could have been due to the carbo-hydrate content of this fraction (6). However,amino acids and hexosamines were present inthis fraction, and these should react with flu-orescamine. Polyacrylamide gel electrophoresisof this fraction after reaction with fluorescamine(32) resulted in two strong and one weak flu-orescent band when gels were viewed underlong-wave ultraviolet light (not shown).Immunoelectrophoresis ofcarbohydrate-

rich fractions. Results of immunoelectropho-resis of the carbohydrate-rich fractions followedby development of the plate with hyper-immune anti-strain 19 and immune anti-strain2308 sera were negative. Negative results wereobtained also with the double-immunodiffusiontest. Lack of reaction with the immune sera maymean that the carbohydrate-rich fractions arenot immunogenic or that the components in thisfraction are of such size that a visible precipitateis not formed.

Toxicity assays with carbohydrate-richfractions. Intradermal injection of 40 ,ug of car-bohydrate-rich fraction (based on carbohydratecontent) containing 0.05 mg of KDO induced areaction within 5 min, causing a lesion with anecrotic center. Injection of 4 ,ug, however, onlyproduced a slight reddening of the skin at thepoint of injection which disappeared after 24 h(Fig. 7).

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Histopathological examination (Fig. 7) of thetissues revealed coagulative necrosis of the epi-thelium and necrosis of the walls of the arteriolesin dermis. Polymorphonuclear cell infiltration ofthe dermis was also observed, which extendedfrom the primary lesion site, but the collagenfibers were intact. The tissue also was hyperemicwith edema.

Pronase treatment or heat treatment, carriedout as described above, destroyed the ability ofthese fractions to cause a necrotic dermal lesion,although an erythematous reaction was still pro-duced. These results suggest that the activeprinciple could be a protein or peptide or aglycopeptide.

DISCUSSIONAn interesting feature of Brucella infection is

the ability of Brucella to exist intracellularly(22). The involvement of microbial cell surfacecomponents in the initiation and maintenance ofintracellular parasites has been well established(33). Brucellar surface components (8, 10, 12, 25,26, 30, 36) and soluble components from whole-cell sonic extracts (23) have been extensivelystudied. These components, whether isolatedfrom attenuated or virulent and rough or smoothforms of Brucella, were usually very similar inchemical composition and less innocuous (in thecase of endotoxin or heat-stable or -labile proteintoxins) than components isolated from othergram-negative pathogens. However, Smith andFitzGeorge (34) reported that the virulence ofBrucella was found to be roughly proportionalto its ability to grow intracellularly. Obviously,the isolation of a component that would be pres-ent on the surface of virulent but not attenuatedorganisms would increase our understanding ofthe pathogenicity of Brucella and might be use-ful in developing a differential diagnostic tech-

ANT

AN'

ANT;

*Ang-----"'.,.--------.-w.-.-o -

FIG. 5. Immunoelectrophoresis of protein-richfractions from strains 19 and 2308. (a) Attenuated;(v) virulent; (anti-a) anti-attenuated strain 19 serum;(anti-u) anti-virulent strain 2308 serum. The anode ison the right.

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BRUCELLA ANTIGENS AND TOXIC FRACTIONS 675

nniu-srnrin 19

10

anti -strain 19 absorbed)

anti -strain 2308 (absorbed)

0

anti-strain 2308

r - -:- _--_ ~~~~~~~~~~~~~~~1

anti-strain 19

19

ant i -strain 19 (absorbed)

_ _,_ ,,_ ,, anti-strain 2308_Lpbsorbed

2 (j 2308

3 1

anti-strain2308FIG. 6. Immunoelectrophoresis ofprotein-rich fraction from strains 19 and 2308 before and after absorption

of antisera with homologous cells. (a) Actual photograph; (b) schematic representation.

nique. In this communication, we have shownthat by using a very gentle extraction procedure,a pronase-susceptible, heat-labile, toxic, protein-rich fraction could be isolated. The protein-rich

fractions isolated from attenuated and virulentorganisms differed quantitatively and qualitita-tively in their polypeptide composition in themolecular weight range between 14,000 and

a

0k

b

+

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676 TABATABAI, DEYOE, AND RITCHIE

4pg

6J4pgH fi~ SalineA_..........................9 n. 6hrs

.."A

;.24'hr!s

'..40NObb-,S'F1

FIG. 7. Gross and histological appearance of dermal lesion caused by protein-rich (a and b) and carbo-hydrate-rich (c and d) fractions. Protein-rich fraction injected contained 40 pg ofprotein, 8 jig ofcarbohydrate,and no detectable 2-keto-3-deoxy sugar. Carbohydrate-rich fraction injected contained 40 ig of carbohydrate,11 pg ofprotein, and 0.05 pg of2-keto-3-deoxy sugar. Magnification of b and d, x40.

40,000. A soluble Brucella allergen that is pre-pared from a rough variant of virulent strain2308 by extraction with 2.5% sodium chloride(16) and does not cause skin lesions in normalguinea pigs contains few protein bands in this

region (L. B. Tabatabai, unpublished data).Based on this observation, it is tempting tospeculate that a protein(s) or glycoprotein(s) ofsubunit molecular weight between 14,000 and40,000 might be responsible for the observed

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BRUCELLA ANTIGENS AND TOXIC FRACTIONS 677

TABLE 2. Composition of carbohydrate-richfractions prepared from B. abortus

Determination Value

N (Kjeldahl)/carbohydrate' (mg/mg) 0.2-2.5Protein/carbohydrate (mg/mg) 0.5-1.6KDO/carbohydrate (jIg/mg) 1-10Hexosamines +Ribose +2-DeoxyriboseA26o/A28o 3.0-4.0A 1' 175-230Allcm ~~~~~~~100-380a Expressed as glucose.Absorbance measured at 260 nm for a 1% solution

based on carbohydrate content.cAbsorbance measured at 260 nm for a 1% solution

based on protein content.

skin lesions. The possibility has been consideredthat the inflammatory response observed withthe protein-rich fractions could be due to smallamounts of lipid A-containing LPS contamina-tion. The protein-rich fraction injected (Fig. 7)contained 40 jig of protein, 8 jig of carbohydrate,and no detectable 2-keto-3-deoxy sugar. Thisinjection resulted in inflammatory reactionsvarying from 14 to 17 mm in diameter. Theresults of Jones (14) indicated that 100-, 10-, and1-jig quantities of LPS caused erythematous re-actions of 16, 11, and 5.7 mm, respectively, innormal guinea pigs. Our results suggest thateven though carbohydrate is present in the pro-tein-rich fractions, this carbohydrate may not beof the toxic LPS type, but rather could be of thenontoxic type of polysaccharide referred to byJones (14). Also, heat and pronase treatmentdecreased the dermal response of the protein-rich fractions, suggesting that a protein or gly-coprotein is responsible for the observed reac-tion.

Absorption experiments were performed todetermine whether components in the protein-rich fraction were indeed derived from the bac-terial surface. The results of the absorbed anti-serum experiment suggest that one or more sur-face components were present in protein-richfractions. In strain 19, the surface componentrepresented only a minor part (one out of fouror five components) as judged from the immu-noelectrophoretic pattern. But in the strain 2308protein-rich fraction, two of three componentswere removed, as would be expected if theyrepresented surface components. Interestingly,one of the strain 2308 surface components hadan electrophoretic mobility similar to that of thesingle surface component found in strain 19. Itis doubtful that the protein-rich fraction con-tained LPS, since the precipitin bands near the

wells (Fig. 6) should have been removed by theabsorption experiments if they consisted of LPS.The results suggest that these precipitin bandsprobably are not due to LPS, but rather couldbe due to subsurface polysaccharides. Prelimi-nary experiments using sialidase (type VIII fromClostridium perfringens; Sigma Chemical Co.,St. Louis, Mo.) have shown that sialic acid(which is also a 2-keto-3-deoxy sugar acid) isliberated from the samples after incubation withthe enzyme. These results suggest that the 2-keto-3-deoxy sugar measured and expressed asKDO may be all or partially due to sialic acid.Since KDO is associated with LPS and sialicacid is associated with glycoproteins, furtherwork on the differential analysis of these twoconstituents would be of interest.

It was not altogether surprising to find thatthe pronase-susceptible, heat-labile, carbohy-drate-rich fractions isolated from the attenuatedand virulent strains were similar in their overallchemical composition and dermal toxicity (ne-crotic lesions). It is possible that because we didnot use the appropriate test system the virulencefactor remains elusive, or that both virulent andattenuated strains contain the same toxic factorbut in different quantities. The skin reactionobserved with the water-soluble, carbohydrate-rich fraction is most likely not due to lipid Acontamination for the following reasons: (i) the2-keto-3-deoxy sugar content is very low (at themost, 0.04 to 0.4 jig per 40-,ug injection); (ii) thisconclusion is indicated by solubility properties(soluble in water, 60% methanol, and 70%ethanol and insoluble in nonpolar solvents); and(iii) skin reaction caused by this fraction is re-duced after heat and pronase treatment (seecompilation of endotoxic reactions by lipid A[42]). The interesting feature of the carbohy-drate-rich fraction is the apparent preferentialdestruction of epithelial cells. The observed der-mal response has similarities to the ulcerativeendometritis that occurs in the pregnant bovineuterus infected with B. abortus (29).

In summary, we have shown that by gentleextraction methods, a water-soluble toxic pro-tein fraction is extracted from attenuated andvirulent B. abortus cells which differ in poly-peptide composition. Separation and isolation ofthe surface protein antigens in these protein-richfractions may lead to the development of a dif-ferential diagnostic technique for detecting in-fected animals. In addition, a carbohydrate-richfraction which contains a necrotizing principlewas isolated. Purfication and structural studies(in progress) of the necrotizing principle mayincrease our understanding of the mechanism bywhich Brucella organisms maintain intracellular

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growth and eventually form necrotic lesions ininfected animals.

ACKNOWLEDGMENTS

L.B.T. was the recipient of a National Research Councilpostdoctoral associateship.

The excellent technical assistance of Kathryn Meredithand Linda Schmidt is gratefully acknowledged.

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