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Characterization of Brucella abortus Soluble Antigen Employed in Immunoassay

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0095-1137/80/04-0355/08$02.00/0

Characterization

of Brucella

abortus

Soluble

Antigen

Employed in

Immunoassay

DAVID T. BERMAN,'*BAMBI L. WILSON,' EDGARDO MORENO,' ROBERTD.ANGUS,2 AND

LOIS M.JONES'

Department ofVeterinary Science, University of Wisconsin-Madison,Madison, Wisconsin53706,1and

Veterinary ServicesLaboratory, U.S.Department of Agriculture,Ames, Iowa500102

Asoluble antigen extractof Brucella abortus (BASA) hasbeen prepared by

the National Veterinary Services Laboratories and furnished to a number of

workers who are examining antibody-mediated and cell-mediated immune

re-sponses ofcattle infected with B. abortus. Three lots of BASAwere examined.

Therewerequantitative butnotqualitative differencesamonglots bycontentof protein, total carbohydrate, hexose, fatty acid, and 2-keto-3-deoxyoctonic acid.

Thepresenceofsmooth lipopolysaccharidewasdemonstratedby thepresenceof

2-keto-3-deoxyoctonicacid and lipid, by Limulus lysate gelationactivity, and by

formation ofcharacteristiclipopolysaccharideprecipitates in immunoelectropho-resis. A polysaccharide antigen as well as two nonsurface antigens, A2 and C,

werealsoidentified. BASA isasatisfactory antigen forusein theenzyme-linked

immunosorbentassaysince thesmoothlipopolysaccharide componentboundto

polystyrene and functioned in the test. Normal murine spleen cells showed a

mitogenic response toBASA similartothat produced by purified smooth

lipo-polysaccharide.BASA has beenused in otherlaboratoriestostimulate peripheral

blood leukocytes from cattle infected with B. abortus.Because BASA isamixture

ofantigenic components showntohave mitogenic effects in the mouse system,

questionsonthenatureofitsstimulatory effectonbovinecells areraised.

Stabilization of the

prevalence

of bovine

bru-cellosisnationally, and indeedanincrease in its

prevalence in the southernstatesduringthepast

decade

(15, 34),

have stimulated increased

re-searchonthe disease.

Investigations

on

improve-ment of

diagnostic

methods have received

par-ticular

attention,

in part because cattle

immu-nized withthe attenuated strain 19ofBrucella

abortusmay have residualantibody

specific

for

the smooth

lipopolysaccharide

(S-LPS)

com-plex, which cannot be

distinguished

from that

resulting from infection

(34,

40).

Approaches

to

the

problem

haveincludedexamination ofboth

antibody-mediated

(9, 31,

33)

andcell-mediated

immuneresponses (17-19, 21,

27)

of cattleto a

variety of soluble

antigen

extracts.Becausemost

of theresidual

antibody

from strain 19

vaccina-tion is

immunoglobulin

M

(IgM),

various tests havebeen

designed

whichdetect

antibody

ofthe

IgG class (2, 6, 26). These tests include the

automatable

enzyme-linked

immunosorbent

as-say

(ELISA)

(7, 8, 22, 29,

36).

Ina number of

investigations

withboth

cell-mediated immunoassays and

ELISA,

asoluble

antigenextractof B. abortus

(BASA)

produced

by the Reagents

Section,

National

Veterinary

Services

Laboratories,

U.S.

Department

of

Ag-riculture has been used (7, 18, 29,

36).

This

extractis the

supernatant

fluid from autoclaved

cells

ofB. abortus strain1119-3, whose

compo-sition has been

partially

described (18). The

objectives of the work reported herewere (i) the

chemical characterization ofthreedifferentlots

of BASA, (ii) the antigenic analysis of BASA

relative to content of S-LPS and non-S-LPS

antigens, (iii) thedetermination of theextentof

variability ofcomposition among the lots, and

(iv) the examination ofsomeofBASA's

biolog-ical activities.

MATERIALS

AND METHODS Preparationofantigenextracts.BASAwas

pre-paredattheNational VeterinaryServices

Laborato-ries, Ames,Iowa, from strain 1119-3cellswhich had

beenpropagatedinafermentorasdescribedbyAlton

etal. (3). Harvested cellsweresuspendedin

double-distilledwater at aratio of1g(packedwetweight)to

4ml ofdouble-distilled water, shaken for2 h,

auto-claved for20minat121°C, cooled,andcentrifugedat

17,000 xgfor20minat40C.Thesupernatant fluid

wasremoved, stored at-25°C withoutpreservative,

anddesignatedBASA.

Aninitial lot (lot 10) of BASA hadbeenprepared

and distributed to several research laboratories. A

portion of lot10and portionsoftwosubsequentlots

(lots 12and 13) were sent toMadisonin the frozen

state. A 40-mlamount ofeach lot was thawed and

355

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lyophilized so that the chemical analyses could be

referredtoBASAon adry weightbasis. Theyieldof

each preparationisgiveninTable1. The dried

prep-arationswerestored inadesiccatoratroom

tempera-ture.

Preparationand compositionofsolubleextractsof

brucellae employed for comparative purposes have

beendescribed in detailpreviously.LPSpreparations

includedcrude (f5)andpurified(f5p)S-LPS,extracted

in the phenol phasefrom B. abortus strain1119-3,and

crude (R3) and purified (R3a) rough LPS (R-LPS),

extracted in theaqueousphasefromB.abortusrough

strain45/20 (25).CrudeS-LPSwasobtained from B.

melitensis strain 16M by hot saline extraction (31),

and LPS-freepolysaccharideB(poly B)wasobtained

by trichloroacetic acid extraction fromroughB. meli-tensis strain B115asdescribedbyDiaz et al.(9).

LPS-free, protein-rich preparations extracted from rough

B. abortus45/20have been described (31). They in-cluded asonicextractandahypertonicNaCl extract

referredtoasbrucellin(24).

TwoLPSpreparationsfrom Escherichiacoli 0128:

B15 were purchased from Difco Laboratories; the

phenol-waterextractwasused in thechemical

analy-sesand in the Limuluslysate gelation activity (LLGA);

the trichloracetic acidextractwasused inthemitogen

assays.

Chemical analyses. Protein content was deter-minedbythe method ofLowry (23)with bovineserum

albuminasstandard. Hexosedeterminationswere

per-formed by the anthrone method (14), and total

car-bohydratewasdeterminedbythetryptophan sulfuric

acid (11) method. The standard forthesugar assays

wasanequimolar mixture of D-glucoseandD-mannose,

which was selected on the basis of gas

chromato-graphic analysesofBrucella LPS (unpublisheddata).

Inpreviously published assaysof LPS, the standard

was an equimolar mixture of D-glucose, D-galactose,

L-rhamnose and D-mannose (25). Amide-linked fatty

acidsplusester-linked fattyacids weremeasuredby

the procedure of Haskins (13) with tripalmitin as

standard,and resultswerecalculatedaspercent

pal-miticacid.

The method describedbyAshwell (5)wasused for

estimationof2-keto-3-deoxyoctonicacid (KDO) with

amodificationtoaccount for thepresenceof interfer-ing 2-deoxy-D-ribose or other 2-deoxy-aldoses (37).

The standardswere2-deoxy-D-ribose (Sigma)and

am-moniumsalt of KDO(Sigma)andwerenotsubjected

to preliminary hydrolysis. Precipitateformation was

commonwiththecomplexextractsandwasprevented

by addition of dimethyl sulfoxide to assay mixtures

and standards after addition of thiobarbituric acid

(20). Absorbance was read atboth 552 and 536 nm.

The appropriate extinctioncoefficientswereusedto

calculate percent KDO.

Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis. Sodiumdodecyl

sulfate-polyacryl-amide gel electrophoresis was performed by the

method of Weber and Osborn (39). The lower gel

consisted of 15% acrylamide, and the stacking gel

consisted of 4%acrylamide.A constant current of 10

mAwasapplieduntil thebromophenolbluetracking

dye had traveled 8 cm. The gels were stained for

proteinwithCoomassie brilliant blue R-250 (Sigma).

The marker proteins(5,ugeach)werephosphorylase

b (molecular weight, 94,000), bovineserum albumin

(molecular weight, 68,000),catalase(molecular weight,

58,000),fumarase(molecular weight, 49,000),aldolase

(molecular weight, 40,000), carbonic anhydrase

(mo-lecularweight, 29,000), hemoglobin (molecular weight,

16,000), andcytochromec(molecular weight, 12,000).

LLGA. Assays were performed as described by

Sullivan and Watson(35)exceptthatthe rack of tubes

wasremovedfrom the 37°Cwaterbath after 1h and heldat4°C for 15minbeforedetermination of

gela-tion. Thetestmixtureconsisted of 0.1ml of Limulus

lysate and 0.1 ml of antigen solution. Activity was

TABLE 1. Chemicalcomposition and LLGA of BASA, LPS from B. abortus, and commercial LPS from E.

coli

Protein Total

car-PreparationPrpao(%roftdry bohydrate Hexose (% FAA + KDO (% of LLGAh Dry wt Protein'

f (%ofdry ofdrywt) FAE" drywt) (ng/ml) (mg/mil) (mg/ml)

Wt) wt)

BASA lot10 20.5 16.6d 14.3d 13.9 0.58 2 11.43 8.9

BASA lot12 16.7 8.9d 6.4d 13.0 0.86 2 10.49 7.2

BASA lot13 16.0 10.6d 7.5d 14.3 0.28 2 8.03 6.0

f5-crudeS-LPS' 24.0 10.7f NDW 19.0 0.67h 1

f5w-pure

S-LPS' 6.3 11.6f ND 26.4 0.86h 0.1

R3-crudeR-LPSe 1.3 13.6f ND 10.0 1.5h 10

R3a-pureR-LPSe 1.5 6.5f ND 27.0 30h 1

LPS fromE.coli 4.4 18.4f ND 43 1.7 0.2

aFAA,Amide-linkedfatty acids; FAE, ester-linked fatty acids. The numbers represent percent, calculated as

palmitic acid,ofdry weight.

bLowest concentrationneeded to form a solid gel.

'Micro-Kjeldahl analyses performed at National Veterinary Services Laboratory, Ames, Iowa.

dStandard is equimolar mixture of D-glucose and D-mannose.

e Dataforthispreparation previously published (25).

fStandardisequimolar mixtureofD-glucose, D-galactose,L-rhamnose, and D-mannose.

g ND,Notdone.

hCorrected fromthose previously published (25).

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B. ABORTUS SOLUBLE ANTIGEN 357

expressed as the lowestantigen concentration in

nan-ograms permilliliter needed to form a solid gel.

Serological analysis. Barbital buffer (pH 8.6) and

0.8% agarose wereemployed in

immunoelectrophore-sis. For double immunodiffusion, gels were prepared

with 1% agarose in borate buffer (pH 8.3) containing 10% sodium chloride to enhance precipitation with

bovine antisera(9).

Sera from B. abortus-infected cattle,hyperimmune

rabbit sera, and monospecific sera were those previ-ously described by Schurig et al. (31).

ELISA. BASA was used in comparison with a

prep-arationof S-LPS from B. abortus strain 1119-3 in the

ELISA described by Lamb et al. (22) with sera from

infectedcows.Separate enzyme conjugates specific for

IgM,IgGI,andIgG2 were used. The S-LPS preparation contained 30% protein by the Lowry method (23) and had been employed by Lamb et al. (22) at a

concen-trationof10ng/ml of carbonate buffer. In the present

study, tests were also done with S-LPS concentrations

of10ng, 100ng, 1,ug, and 10

jug/ml.

Dried BASA lot

10 was reconstituted incarbonate buffer at 10mg/ml

and employed in the ELISA at 1:1,000 dilution, as

reported bySaundersetal. (29), and also at 1:100,000

dilution.

Mitogen assay. Assays of mitogenic activity of

extractsformousespleen cellsweredoneasdescribed

by Moreno and Berman (24) withspleen cells from

the endotoxin-resistant C3H/HeJ (38) and the

con-genic, endotoxin-sensitiveC3H/HeAU mice provided

by R. Auerbach (Zoology Department, University of

Wisconsin).

Culture mediumwasRPMI-1640(GIBCO

Labora-tories, Grand Island, N.Y.) containing 2 mM

gluta-mine,100U ofpenicillinperml,100yIgofstreptomycin

per ml(GIBCO),5 x 10-5M2-mercaptoethanol, and

25 mM HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid,MicrobiologicalAssociates,

Walk-ersville,Md.).

Antigenextracts weredissolved in culture medium

with the aidof sonication (Sonicator 180, British

In-struments Inc.) at aconcentration of2.5 mg/ml and

filtered through 0.45-ytm-pore size filters (Millipore

Corp.,Bedford, Mass.).Avolume of0.05ml ofantigen

extract diluted in medium was placed in triplicate wells offlat-bottomed microtiter plates (Linbro 1S-MRC-96-TC). Controlwells had medium alone.

Con-canavalin A (Con A) (Sigma) wasusedas a control

mitogen in allexperiments.Spleencells obtained from

two orthreemice,7weeks of age,werepooled,washed

two times with0.1 Mphosphate-bufferedsaline (pH

7.4) at4°C,andsuspendedinculture mediumat2.5 x 106 cellsperml. A volume of0.2ml(5 x 105cells)

wasplaced in each well. Cultures wereincubated at

37°C inanatmosphereof5%CO2in air for42h. Two

microcuries of[3H]thymidine(NewEngland Nuclear,

Boston, Mass.; specific activity, 2.0 Ci/mmol) was

addedtothewells for6hbeforeharvesting.Thecells

wereharvestedonglassfilter paper witha

multiman-ifoldsample harvester, washedwith0.85% saline

so-lution,andfinallydried for 12 hat37°C.

[3H]thymi-dine uptake was measured by liquid scintillation

countinginaTri-Carbliquidscintillation

spectrome-ter(PackardInstrumentCo.,DownersGrove, Ill.)and

recorded incountsperminute.

RESULTS

Chemical analytical data andLLGA, given in

Table1, show the extent ofvariationamong the

three lots of BASA in protein, total carbohy-drate, and hexose content. All three lots con-tainedlipidandKDOandhad the same LLGA. The dataobtained withcrude B. abortus S-LPS (f5) were similar to thatseen with the three lots

of BASA,with the exception of LLGA and

con-tentof proteinandlipid.

The presence of S-LPS in BASA was also

demonstrated by immunodiffusion (Fig. 1). On

thebasis ofdilutiontoextinction ofthe S-LPS

line, as well as LLGA, BASA lot 10 contained

approximatelyhalf thequantityofS-LPSasf5.

Attempts were made to show identity of the

S-LPScomponent withdifferentpreparationsof

smooth Brucella, including crude S-LPS from

B. melitensis; the latter diffuses better than

S-LPS from B. abortus.However,theS-LPS com-ponents did not diffuse farenoughfor thelines

tojoin(Fig.2).Poly B,the second component in

Fig. 1, showedcomplete identitywithpolyB in other S-LPS preparations (Fig. 2), as well as

withpolyBprepared fromaroughstrain of B.

melitensiswhichlacksS-LPS

(Fig.

3).

BASAlot 10 was also examined for the

pres-enceofantigens distinct from the S-LPS

com-I.

...te..

FIG. 1. Immunodiffusion of doubling dilutions of

BASA lot 10(A) and B. abortus crudeS-LPS

fi,

(B)

reactedwithserumfroma cowwith brucellosis

(cen-tertrough). Thehighestconcentrationofeachantigen

was 20 mg/mi in the well to the right. The diffuse

lines closesttotheserumtrougharecharacteristicof

poly B. The sharp lines closer to the antigen wells

are S-LPS. At lower antigen concentrations, the

5-LPS line is intheedge ofthewell.

FIG. 2. Immunodiffusion ofB.melitensiscrude

S-LPS (A),BASA lot 10(B), and B. abortus crude

S-LPS(C) reacted withserumfroma cowwith

brucel-losis.

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358 BERMAN ET AL.

FIG. 3. Immunodiffusion ofBASA lot 10 (a) and

threedifferent preparations of LPS-free polyB (b,c,

d) reacted with serumfrom a cow infected withB.

abortus. The ring of precipitate around well A is

attributedtothe S-LPS.

plex, which had been identified by means of

immunoelectrophoresis and immunodiffusion

(31). Theuseof referenceserafromtheprevious

study showedthatantigensA2 andCarepresent

in BASA; there were also additional antigenic

components which could not be identified. As

shown inFig. 4,BASA and the LPS-free extracts havea common component whichdevelops

re-actions ofidentity with infected cow sera and

withmonospecific anti-A2 serum. Theinfected cowserumpool also revealsa secondline with

BASA, but not with the protein antigens

pre-paredfromroughBrucella strains.

Antigen C had been identified as positively

charged on the basis ofits migration in

immu-noelectrophoresis of sonic extracts or protein

extractsofrough brucellae (31). Thepresenceof

antigen CinBASAwasinferred fromformation ofaprecipitate in immunoelectrophoresis in a

position correspondingto that ofantigen C in 45/20 sonicextract(Fig. 5). TheS-LPS linesare

also evidentneartheantigenwellscharged with

S-LPS (f5) and BASA, butnotwith45/20 sonic

extract.The linesproduced by the anodally

mi-gratingantigens in BASAwere diffuse, as

con-trasted withthewell-definedlines producedby theantigensinthe45/20 sonicextract.Joneset al. (16) observed a similar loss ofdefinition of

antigens in protein extracts which had been heatedto100°C.

Sera taken from cattle before, and 4 to 20 weeksafter, conjunctivalinoculation witha

vir-ulent strain of B. abortus were tested in the

ELISA with two concentrations ofBASA and four concentrations ofS-LPS. Ingeneral, with

all three conjugates (anti-IgM,

anti-IgG,

and

anti-IgG2), the levels of binding of antibodyto

BASA ata 1:100,000 dilution and to S-LPSat

10ng/mlwereequivalent. The optical densities

in thesetestswerelow. Higher levels of binding were observed with BASA at 1:1,000 dilution,

and these were equivalent to those obtained with S-LPSat 1or 10

lig/ml.

As both the S-LPS preparation and BASA had high protein content, it is possible that antibodyintheserummight be bindingto

com-ponents inaddition toS-LPS. To examine this

question,serumfromaninfectedcowbefore and

after absorption with livingsmooth B. abortus

cellsorwith sonicextractfromrough B. abortus

45/20 cells was tested. The results in Table 2

show that absorption with living smooth cells removed agglutinins, anti-S-LPS precipitins, and antigen binding capacity in ELISA with either theIgMortheIgG,reagents. Incontrast,

serum absorbed with sonic extract from rough

cells retained its agglutinins, precipitins to S-LPS, and itsbinding capacity in the ELISA with bothIgM and

IgG,

reagents.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the three lots of BASA is illustratedinFig.6. The absence ofsharp protein

:.

FIG. 4. Immunodiffusion ofBASA lot 10 (A), B. melitensisBl 1 proteinantigen (B), and B. abortus 45/20 sonic extract(C) reacted with apooi ofsera

fromcowsinfectedwith B.abortus(D)and monospe-cificanti-A2serum(E).

FIG. 5. Immunoelectrophoresis of BASA lot 10(a),

45/20sonicextract(b), and B. abortus crude S-LPS

F5 (c) reacted with serum from a cow infected with B. abortus.

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B. ABORTUS SOLUBLE ANTIGEN 359

TABLE 2. Serological tests with serum from a B. abortus-infected cow

Precipitation lines in immunodiffu- OD1 in ELISAwith

Serum

absorbed

with: Cardtest tion

titer

sion

with:u

S-LPS BASA Sonicate IgM IgG,

Notabsorbed + ++1:6,400 + + + 0.73 0.93

Live cells B.abortus - -1:100 - - + 0.06 0.06

smooth strain 19

B. abortus rough strain + ++1:6,400 + + - 0.69 0.92

45/20sonicextract

aOpticaldensity at490nm.

-94000 .lL_.;| E -68000

r

Z!11

2

E

58000

-40000

-29000

.-16000 -12000

FIG. 6. Sodiumdodecylsulfate-polyacrylamide

gel

electrophoresis ofmarkerproteinsandBASA in 10%

polyacrylamide

gel.

Preparationsandconcentrations

wereBASA lot13, 0.3 mg, (A);lot13,0.6 mg(B);lot

10, 0.3

mig,

(C); lot10, 0.6 mg,(D);lot

12,

0.3 mg;(E);

andlot

12,0.6

mg(F).

bands in the

gels

is

attributable,

atleastin

part,

to the

relatively high

content of S-LPS and to thepresenceofsalt in these

preparations.

There

were

quantitative

differences relatedtothe

dif-ferences in

protein

content among the three

preparations,

but

qualitative

differences were

not

apparent.

The

mitogenic

response of

spleen

cells of the

endotoxin-resistant

C3H/HeJ

micetoBASA lot

13 and to other

mitogens

is shown in

Fig.

7.

Similar results were obtained with

spleen

cells

of the

congenic,

endotoxin-sensitive

C3H/HeAU

mice

(data

not

shown).

The

maximnum

stimula-tion levelwasreachedataconcentrationof 200

,tLg

of BASA per

ml;

higher

concentrations of

antigen

did notinhibitor

significantly

increase the

uptake

of

[3

H]thymidine.

The

dose-response

profile

induced

by

BASA was similar to those

produced

with

f5

and

f5p,

however,

the counts perminuteobtainedwerelower than with these endotoxin

preparations

from B.abortusand E.

i

A

RASA

x

V 40

z LU it

I0 x In

I- 20

o1 0.3 10 20 10 20 0.1 510203050

CONCENTRATION OF

MITOGEN

(mg/ml)X10

FIG. 7. [3H]thymidineincorporation byC3H/HeJ

mousespleen cell cultures containing concanavalin

A,trichloroacetic acid-extracted E. coli LPS, B.

abor-tusLPS(fe),orBASA.

coli. The standard error wasbelow 10%o of the

totalcountsforeachpreparation.

DISCUSSION

ThepresenceofS-LPS in BASAwas

demon-stratedby itscontentof KDO andlipid, LLGA,

andbyformation ofcharacteristic LPS

precipi-tatesin

immunoelectrophoresis

and

immunodif-fusion. PolyB (9) wasalso seen in

immunodif-fusion.InadditiontoS-LPSand

poly

B,BASA

was shown to contain the nonsurface

antigens

A2and C which hadbeen identified

previously

inprotein-rich Brucellaextractsbutnotincrude S-LPSpreparations(31).Thesefour

antigens

in BASA are among the

principal

ones to which

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infected cowsproduce antibody (9, 31, 33). It appears that the S-LPS component in BASA is the antigenic component which func-tions in the ELISA test. Lamb etal. (22) have shown thatcrude S-LPScontaining30%protein

and purified S-LPScontaining 6%protein gave

identical results in ELISA with a variety of serum samples. Theabsorption experiment de-scribed in the present study shows that the antibody measured in ELISA is directed against surface antigenswhich are most probably S-LPS rather than against protein components. Lamb et al.(22) proposed that protein present inf5did not bind to the polystyrene, and Stemshorn (Ph.D. thesis, University of Guelph, Guelph,

Ontario, Canada, 1979) was unable to

demon-strate binding ofprotein A2 to polystyrene for usein an ELISA. Thus, ELISAs either with S-LPS preparations or with BASA apparently measureantibody of the samespecificity as that detected in theagglutination and

complement-fixationtests. BASA has theadvantage of being

easier to prepare thanS-LPS.

Spellman and Reed (32) reportedthat crude B. abortus S-LPS was mitogenic for murine spleen cells, and Moreno and Berman (24) con-firmed this with purified phenol-water-ex-tracted LPS, either from smooth or rough B. abortus. The data in that report support the position thatmitogenic stimulationby

purified

Brucella LPS of

spleen

cells of

C3H/HeJ

mice

isattributabletotheunique compositionof

Bru-cellalipid A (25) andnot tothepresence ofan endotoxin protein. In addition, they (24) ob-served that two LPS-free protein preparations extracted from rough B. abortus by different methods (31) showed different biological activ-ity. The sonic extractwasmitogenicfor murine spleen cells;the brucellin was not.These obser-vations are extendedby the present demonstra-tion of a mitogenic effect by BASA on murine spleen cells, with a dose-response curve similar

tothatproducedwithBrucella S-LPS.

In their studies on in vitro stimulation of bovine peripheral blood leukocytes (PBL) by Brucella antigens, Kaneene et al. (17) found

thatthe PBLfrom cowsinfected with B. abortus

demonstrated greater thymidine uptake when cultured with BASA than when cultured with LPS-freebrucellinprovided by us. Greater stim-ulation was observedwith BASAeven when the two antigen preparations were used at equal protein concentrations. Although they recog-nizedthepresence of endotoxin in BASA on the basis ofLLGA, they discounted itssignificance andsuggested that". . .theprotein fractionwas primarily responsible for stimulating the

sensi-tized lymphocytes to undergo blastogenesis"

(18). The basis for this conclusion isunclear, and their data can be interpreted equally well if it is postulated thatLPS contributes to the stimula-tion.Significantly, brucellin produces typical de-layed-type hypersensitivity reactions in Bru-cella-infected cows, goats, and guinea pigs (3) and is not mitogenic for normal mouse spleen cells. As shown in the present studies,BASA is mitogenic for normal murine cells at concentra-tions equal to those used by Kaneene et al. in their bovine PBL stimulation assay. At those concentrations, there is at least 100

jig

ofLPS perml.

Several hypotheses may be advanced to ex-plain the nature of the bovine PBL response to BASA observedbyKaneene et al.(17, 18). Some of the proliferation seen in PBL from infected

cattle cultured with LPS-containing Brucella

antigens such as BASA may represent expansion ofclones of BrucellaLPS-committedcells. This is supported by the observation of Kaneene et al. (18) that maximumstimulation in their lym-phocyte transformation system occurred in cul-turesincubated for6daysand by the fact that LPS-free brucellinproduced less stimulation of PBL (17).Alternatively, the increased response of PBLmay be onemanifestationof nonspecific increase insensitivity of Brucella-infected ani-mals to the toxiceffect of enterobacterial as well as Brucella LPSs reported by several investi-gators(1, 10).Consistentwith this hypothesis is the observation ofSchuriget al. (30) that PBL

from Campylobacter-infected cows, but not

those fromnormal animals responded to E. coli

LPS.Also,it isknown that in vitro

transforma-tion responses oflymphocytesfrom humans and other animals during severe infection or after vaccination (4, 12, 28) are largely governed by nonspecific factors. Because of this, studies of lymphocyte responses to antigens and bacterial products should include several controls in ad-dition to cells from normaland infected subjects. Determination of the responses of cells from Brucella-infected cattleto

purified

LPS from E.

coli orSalmonella, inadditionto

purified

Bru-cella LPS,and the responsiveness to BASA of PBL from cattle infected withorganisms other thanBrucella would beappropriatetestsof this hypothesis.

Thepossibilityofusing thelymphocyte

stim-ulation response as a means ofdetecting early Brucella infectionis worthpursuing (19),butit has not yet been proved rigorously that the responseisspecificforBrucella.

Our demonstration that BASA containsa mix-tureofcomponentswhich aremitogenicfor nor-mal mousespleencellsemphasizesthenecessity

ofclarifying the nature of the responses of

bo-J.

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ABORTUS

vine PBLtoBASA. Withoutexamination of the

responsetodefined antigens, conclusionsonthe

mechanisms of the in vitro stimulation tests,

with suchacomplexmixture of antigens, should

beconsideredastentative.

ACKNOWLEDGMENTS

We thank Mary Hayesand Louise A. Boettcherfor their

experttechnicalassistance.

Thisinvestigation wassupportedby the Collegeof

Agri-cultural and Life Sciences, University ofWisconsin; by

Coop-erative Agreements with the United StatesDepartment of

Agriculture, Animal and Plant Health InspectionService, and

theScience andEducational Administration;and byagrant

from theConsejo Nacional de Investigaciones Cientificasy

Tecnologicas (CONICIT) de Costa Rica.

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