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JOURNALOF CLINICAL MICROBIOLOGY, Dec. 1990,p. 2647-2652 Vol. 28,No. 12 0095-1137/90/122647-06$02.00/0

Copyright C 1990, American Society for Microbiology

Profiles of

Serological

Reactivity against Cytosoluble Antigens

of Brucella

ovis

in

Experimentally Infected

Rams

J. C. CHIN* AND B.PANG-TURNER

Immunology Section, Elizabeth MacarthurAgriculturalInstitute, CamdenPark, Woodbridge Road,

Menangle, Sydney,

NewSouth Wales

2568,

Australia

Received4December1989/Accepted12July 1990

Serafrom rams infected with and excreting Brucella ovis in the semen (shedders), as well as from animals which had recovered from previousexperimentalchallenge with B. ovis, were analyzed for their serological reactivities against cytosolic antigens of the bacterium. Membrane vesicles, including outer and inner membranecomponents, were precluded from the analyses by subjecting French-pressed bacteria to ultracen-trifugation. The resulting cytosolicsupernatant was fractionated into fourmajorantigenic fractions, fractions A, B, C, and D, by high-pressure liquid chromatography. Temporal enzyme-linked immunosorbent assays with the A antigen revealed that all shedder rams displayed a rise-and-surge response, while rams which recovered fromexperimentalchallenge showed arise-and-fall profile. The B antigen was less discriminatory in detecting adifference between the two ram groups, while C and D antigens were serologically unreactive in the enzyme-linked immunosorbent assay. In contrast to the reactivity patterns shown by native high-pressure liquidchromatography-fractionated cytosolic supernatant antigens, immunoblotting of C and D polypeptides generated by boiling in the presence of sodium dodecyl sulfate and mercaptoethanol wasparticularly useful in distinguishing between sera collected at the mid-surgephase of infected rams from sera obtained at the mid-fall stage ofrecovered animals. It is likely that native or denatured antigens ofdifferentcytosolic fractions may provide useful serological reagents fordifferentiatingbetweeninfected rams and those which have recovered from exposure to B. ovis.

Brucella ovis infectionin rams isa disease ofthe genital

tract (2). Infections are acquired by heterosexual transmis-sion, but recentstudieshaveshownthatitcanalsooccurby

other routes, dependingonthe mating behaviors oframsin thefield(16). Forinstance, intranasal infectionsoccurwhen

rams nose the external genitalia and perineum of carrier

ewes or occur intrarectally when rams mount other rams

(homosexual transmission). Like other Brucella infections

(13), B. ovis is an intracellular pathogen and becomes sequestered from host immune surveillance when it is phago-cytosed. Dependingonthe routeofinfection, thebacterium ultimately localizes in the reproductive tract, where it

em-barks on aproliferative phase. Itisatthis timethatinfected

ramsshed bacteria inthe semen. Attempts todetectB. ovis

in Formalin-fixed, paraffin-embedded genital tissues of in-fected rams by an indirect peroxidase-antiperoxidase

tech-niquehaverevealed afoci of bacteriaininflammatory cells in the epididymis and seminal vesicles, with very little localization in the epithelia(11).

The serological response of rams to B. ovis following intranasal, intrarectal, or preputial challenge has recently

beenexamined by whole-cell enzyme-linked immunosorbent

assay(ELISA) (5).Resultsofthesestudieshave shown that there are threeserological response patterns irrespective of thechallenge route. Exposed butclinically uninfected rams

either showed anull cell ELISA response

(abortive

infec-tion)orelse developed arise-and-fall serology

correspond-ing to apresumptiveexposure-recovery phase of infection. In

contrast,

allclinically infected rams that excreted B. ovis

(shedders)showed arise-and-surge ELISAresponse.

Tem-poral analysis of the serological response can therefore

provide

anindication of the infective status oframs.

How-*Correspondingauthor.

ever, seroconversionatany onetimepoint without

clinically

confirmed lesions or a positive semen culture would, in

itself, be an unacceptable criterion for the assessment of infection status. SinceB. ovis is anintracellular pathogen, it ispossible that infectedramsmaybeexposedto intracel-lularantigens of the bacteriumduring its

proliferative

phase inthereproductivetract. Identificationof seroreactive

anti-gens at this phase of infection might provide useful assay reagentsfor the

asymptomatic

detection ofproductive infec-tions.Toexamine this

possibility,

wecompared theantibody reactivity profiles of experimentally infected rams in the recovery orinfective phase against cytosoluble antigens of

B. ovisfractionatedby high-pressure liquidchromatography (HPLC).

MATERIALS AND METHODS

Growth of bacteria. Lyophilized stock culturesofa

labo-ratory-adapted strain of B. ovis 301 were revived from ampoules by resuspension in brain heart infusion broth

(Oxoid) and subcultured onto sheep blood agar plates (3).

Bacteria were harvested 5dayslater afterincubation at37°C

in a 10% C02-in-air mixture.

Preparation ofcytosolubleantigens. Cytosoluble antigens

were prepared as follows. Saline-washed bacteria were

suspended (1 g [wet

weight])

in 4 volumes of extraction

buffer (25 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7.4], 5 mM phenylmethylsulfonyl fluoride, DNase [5 ,ug

ml-'],

RNase [5 ,ug

ml-'])

andlysed by repeated passage through a French press at an applied

pressure of 845 kgcm-2 (7, 19). The crudehomogenatewas clarified(4)bysuccessive centrifugationsat3,500and20,000

x g for 20 and 30 min, respectively. The supernatant was thencarefully layeredoverasucrosepad

(20% [wt/vol]

in 25 mM HEPES buffer[pH 7.4]) in a 6OTi (Beckman)

polycar-bonate tube. The cytosolic supernatant (Cysup) above the

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sucrose

pad

was recovered after

ultracentrifugation

at

150,000x gfor 90 min andwas

dialyzed

against50mMTris

hydrochloride-0.4

M NaCI

(buffer

A). An Amicon stirred

cell

equipped

with a PM10 membrane was then used to concentrate

Cysup

to aquarter of its

original

volume.

HPLC fractionation. A

protein

PAK 300 sw column (7.5 mm

[inner diameter] by

30cm [length];Waters

Associates,

Milford, Mass.) capable

of

resolving globular proteins

witha molecular mass of 20 to 400 kDa was used to fractionate

Cysup.

Portions of0.1 mlwere

injected

intothecolumn via

anHPLCsystem(model

510)

operating

at aflowrateof1ml

min`

with 0.4 M NaCl in 50 mM Tris

hydrochloride

(pH

8.1)

as the mobile

phase.

Protein

peaks

were detected

by

determining

the

A280-ELISA.

Antigenic

fractions

purified

by

HPLCwerediluted

to 10 Fgofprotein ml1

(determined

by the Lowry

proce-dure

[15])

inbicarbonate

coating

buffer(8). Portionsof 50 ,ul weredispensed into eachwellofa96-wellmicrotiterELISA

plate (Flow Laboratories),

and

antigen coupling

wasallowed to

proceed

for 1 h at

37°C.

Plateswere washed three times

with TSTw (20 mM Tris hydrochloride [pH 7.4], 0.15 M

NaCl,

0.01%

[vol/vol]

Tween 20) and

tapped dry

over an absorbent mat.Antiserum(50 ,ul)at adilutionof 1 in 200 in TSTwwasaddedtoeachwell,and the

plates

wereincubated at

37°C

for 1 h. Plates were washed and dried before the

addition of

enzyme-conjugated

second

antibody

(horserad-ish

peroxidase conjugated

to a monoclonal

antibody

with

specificity against

ruminant

immunoglobulin

G [IgG], IgA,

and

IgM [8]).

Enzymatic activity

was assayed after 1 h at

37°C,

with

freshly prepared o-phenylenediamine

usedasthe

substrate.

Changes

in

optical density

were readat 492 nm.

Source ofsera. "Infected sera" were collected from six

rams

(In

1 to In 6) that

actively

excreted B. ovis in their

semen. This was confirmed

by

staining

semen smears

by

a modified Ziehl-Nielson

technique

(20) to detect semi-acid-fast

organisms

that

morphologically

resembled B. ovis and

by microbiological

culture of the bacteria. Infected rams

were serologically assessed

by

the

complement

fixationtest and

possessed

titers

ranging

from16to 64.

"Recovery

sera"wereobtainedfromrams20weeksafter

they

were mated to ewes which had been

previously

in-stilled, intravaginally,

with infected semen(16). Serumwas

collected

only

from

clinically

normal or uninfected rams which

displayed

a rise-and-fall whole-cell ELISA response

(n

=

4;

Rv1toRv4). Semen

ejaculated

from theseramswas

culture

negative.

Three of the fourrams hada

complement

fixationtiterof

8,

while the

remaining

ramhada

complement

fixation titer of 16. In

addition, reproductive

tissues from

these rams were also culture

negative

at the time of nec-ropsy.

Source oftemporalserafrom

experimentally

infectedrams.

Temporal

serawerecollected from

eight

rams

(TS

1toTS

8)

inaseparatetrial. These animalswereselected fromalarger

group which was

previously challenged intrapreputially,

intraconjunctivally,

and

intravenously

with B. ovis as de-scribed

by

Chin and Plant(5).

Sodium dodecyl sulfate (SDS)-polyacrylamide gel

electro-phoresis (PAGE).Electrophoresisofproteinswascarriedout

in

mini-polyacrylamide

gels (Hoeffer) underreducing

condi-tionsasdescribed

by

Laemmli(14). Sampleswereheatedat

100°C

for 5 min in the presence ofmercaptoethanolbefore

they

were loadedonto a 10%

(wt/vol)

running gel witha4%

(wt/vol)

stacking gel. Electrophoresis was carried out at a constantvoltageof120 Vuntil thebromophenolblue

track-ing dye

had

migrated

tothe bottom of the gel.

Immunoblots. Proteins were

electrophoretically

trans-Co

B

E

-p mins

FIG. 1. Profile ofconcentratedCysupfollowingHPLC fraction-ationon aPAK 300 swcolumn. The mobile phase buffer was50 mM

Trishydrochloride and 0.4 MNaCl (pH7.4).

ferredfrom the acrylamide gel to nitrocellulose in a Trans-Blot apparatus (Bio-Rad Laboratories, Richmond, Calif.) under conditions essentially similar to those described by Towbinetal. (21), except that isopropanolwasused instead of methanol. Immunoblots were blocked by incubation in high-salt-Tween (25 mM Tris hydrochloride [pH 8.9], 0.15 M NaCI, 0.5% [vol/vol] Tween 20), washed in TSTw, and then reacted with ram antisera diluted in TSTw. Ovine antibodies which bound topolypeptides on the nitrocellulose were located by reaction with alkaline phosphatase-conju-gated monoclonalantibody with specificity against ruminant IgG, IgA, and IgM (8).

Protein estimation. The protein content of fractionated cytosoluble antigenswere determined by a modified Lowry procedure(15).

RESULTS

HPLC fractionation ofCysup. The A280profile of HPLC-fractionated Cysup is presented in Fig. 1. Five distinctive

peaks, peaks A, B, C, D, and E, were individuallypooled andfurtheranalyzed by SDS-PAGE, ELISA, and immuno-blotting.

SDS-PAGEprofiles. Thesubunitpolypeptide compositions

ofcytosoluble antigensfractionatedbyHPLCwereanalyzed by SDS-PAGE andarepresented inFig. 2. Coomassie blue

staining revealed 7, 11, 16, and 18 majorbands in fractions A, B,C,andD,respectively. ComponentsinfractionEwere notstained with Coomassie blue. Twobroadlystainedzones with apparent molecular mass ranges of 49 to 50.5 and 54to 57 kDa were prominent in fraction B. The presence ofa 98-kDa bandwasalsocharacteristic of fraction B. Intensely stained bands infraction Cwerelocatedat67, 56, 51, 47, 34, 29.5,26, 24, 20.5, and 18 kDa. Distinctivebandingpatterns forfraction D werecentered around 52, 42.5, 40, 38.5, 35, 31, 29,28, 25, 20, and 18 kDa. A 12-kDa band that migrated close to the front was evident in fraction D but was not presentin theotherfractions. Allfractions alsoappearedto possess bands located at 22.5 and 18 kDa. Overall, the

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B. OVIS CYTOSOLUBLE ANTIGEN PROFILES 2649

S A B C D

--i

--»-W

S»~~-b «ne

ou * *

*~ ~ 2

FIG. 2. SDS-PAGE of HPLC-fractionated cytosolic fractions stained with Coomassie blue. Lane S, Standard molecular mass

markers of 92, 66, 45, 31, 21, and 14 kDa, fromtop tobottom; lanes A, B,C, and D, the corresponding HPLC fractions(see Fig. 1).

subunit banding compositions of fractions A, B, C, and D

were sufficiently different to provide a polypeptide

finger-print characteristic of each fraction.

Antibody profiles ofserafrom infected andrecoveryrams.

Infectedserafrom sixramsthatshed B. oviswerecompared

with sera from four abortively infected rams that were

exposed but that remained clinically uninfected. Although minor differences in theimmunoblots were visible between

infected andrecoveryantisera, thereactivityprofiles of only

a representative ram from each of the two categories is depicted in Fig. 3. In this particular example, the

comple-mentfixation titers oframs In 3 and Rv 4 were 32 and 16,

respectively. Reactivities of both antisera against

unfraction-c

io

o A

o A B C D

D °

o

UA B C D

92r

86

4

5---_

=-45

---...-_

21L -

-a4à

FIG. 3. Immunoblot responseofan abortively (Ram Rv 2) and

productively (Ram In 3) infectedramsagainstcytosolic (or Cysup)

antigen and HPLC-fractionated antigens A, B, C, and D,

respec-tively. (A and B) Immunoblots of Rv 2(A)and In 3 (B). (C andD)

Diagrammatic representations of panels A and B, respectively, including molecularmassstandards (in kilodaltons).

TABLE 1. ELISA reactivities ofserafrom infectedand recovery

ramsagainstHPLC-fractionated cytosolicantigens

Ram Complement ELISA reactivity of fraction:

no. fixation titer A B C D

In 1 16 ++ + -

-In 2 32 +++ ++

In3 32 +++ ++

In 4 64 +++ ++ +

In 5 32 +++ + -

-In 6 32 ++ ++ -

-Rv1 8 + + -

-Rv 2 16 + + -

-Rv 3 8 + + -

-Rv 4 8 + + -

-aELISA reactivitiesaregivenasoptical densities,asfollows:-,<0.2;+,

0.2 to0.6;++, 0.6 to1.2;+++, >1.2.

atedcytosolicantigens accountedfor all detectableactivity against HPLC-fractionated antigens A, B, C, and D. The mostobvious differencebetween recovery and infected sera wastheabilityofthelatter tobind strongly topolypeptides

with apparent molecular massesof 22, 23, 24, 28.5, and 29.5

kDa, while reactivities ofrecoveryserain these zoneswere poor or nonexistent. Both infected and recovery sera

showed strongreactivities againstbands with apparent

mo-lecularmassesof30, 44,48, 54,and 60 kDa.Commonalityin thereactionprofile ofrecoveryandinfected antiseraagainst polypeptidesinthe rangebetween40 and 60 kDaaswell as the 23-kDa band extended across allfour HPLC fractions. Unique reactivities of infected sera occurred primarily against polypeptides with apparent molecular masses of22 kDa infractionC and 22, 28.5, and 42 kDa in fraction D.

ELISA reactivities of infected and recoverysera. The rela-tive ELISA reactivities ofsera from six infected and four

recovery rams are summarized in Table 1. The ELISA

responsesagainst HPLC-fractionatedAand Bantigenswere

essentially similar for all sera tested. Generally, serum

samples withhigher complement fixation titers reacted

bet-teragainst antigen Athanthey didagainstantigen B. Both

HPLC-fractionated antigens C and D were poor ELISA substrates againstallantisera.

ELISA profiles against fraction A in experimentally chal-lenged rams. Since the A antigen appeared tobe serologi-cally superiortoother cytosoluble antigens fractionated by HPLC, an attempt was made to test whether this fraction

was able to detect rise-and-surge or rise-and-fall ELISA

responsesfollowing experimental challenge oframs TS 1 to TS 8 with B.ovis.Thetemporalresponsesof individualrams that

exhibited

a rise-and-fallorarise-and-surge ELISAare shown in Fig. 4 and 5, respectively. Although there were

obvious minordifferences betweenindividual animalsin the

timing oftherise-and-surgeorrise-and-fallresponse,thetwo trendswereclearly detectablewiththe A antigen.

Immunoblot profiles of experimentally challenged rams. Because thetimes of thesurge-or-fallresponsesofdifferent rams were different, only sera collected at the midpoint of each surge-or-fall phase wereused in immunoblotting. The

ensuingimmunoblotsagainst antigensC and Daredepicted

in Fig. 6and7, respectively. Withantigen C(Fig. 6), major zonesofreactivitywith surge antiserawerelocatedat63 and 55 kDa (zone P); 41 to42, 38.5 to40.5, 36to37, and 32to 33.5 kDa (zone Q); 25 and 27 kDa(zoneR); and 19 to20.5 and 15 to 18 kDa (zone S). Fall antisera tended to react

discretely and less strongly with polypeptides located in

A

B

..UUM

.

nllem"_

am-M~~

Mals~~~~~~~~M"

.-..._

*_

"__q~~~~~~~ac;

VOL. 28,1990

m

-OE.

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2650 CHIN AND PANG-TURNER

a* b

*9

.ire.""

i

c d

.l

da

0 f

g-

h i

14

;

t',..:

R

.

".. _ 1

0 2 4 6 8 10 12 14 16

Weeks

FIG. 4. ELISA responses of four experimentally challenged rams showing arise-and-fall profile when assayed against

HPLC-fractionated antigen A. TS1 (@), TS2 (A), TS3 (O), and TS4 (A) represent sera collected from rams at weekly intervals over 16

weeks from the time of experimental challenge with B. ovis. OD, Optical density.

zonesQ (63 and 55 kDa) and R (38 and 32 kDa). Three of the

four rams with a surge response (Fig. 7, lanes f, g, and i)

displayed prominent reactivities against D polypeptides with

apparentmolecularmassesof 62,52, and 28 kDa. Prominent

bandsof reactivitywerealso located between 15 and 17, 17

and 19, and 19 and 21 kDa forsurgeantisera. Incontrast,fall antiserawereconsiderably much weaker in their reactivities

andalso reactedmoreerratically against fewer polypeptides

(Fig. 7, lanes athrough d).

-92 -66 p

-45

-31

p

-21

4

FIG. 6. Immunoblot profiles against antigen C with sera col-lectedatthe mid-fall and mid-surge time points oframsfor which results are represented in Fig. 4 and 5, respectively. Lanes: a

through d,serumcollected from the mid-fallpoint oframsTS1to TS 4atweeks 9, 11, 12, and 11 respectively; e,pooled serafrom

prechallengedramsTS 1toTS8;fthroughi,serumcollected from

themid-surge point oframsTS5toTS 8atweeks10, 12, 11, and 10,

respectively. Numbersontherightareapparentmolecularmasses

(in kilodaltons).

DISCUSSION

Host immune responses are expected to be directed mainly against surface antigens of invading pathogenic bac-teriaduring the primaryencounterbetweenhost and

patho-gen (9). Since all B. ovis strains isolatedto dateare of the

rough phenotype, rough lipopolysaccharide and associated

outer membraneproteins (OMPs) may be considered tobe the major antigenic determinants available for antibody binding. Afzaletal. (1) have demonstrated weak reactivities againstgroup1(91 kDa) andgroup2(54 and 36 kDa) OMPs

(10) andstrongantibody activities againstgroup3OMPs(19,

21, and 25kDa). These OMPswereclassified by the

nomen-ELISAO.D.492nm

2.0_

1.5

1.09

0.5

F

---O--o

,,,O/--e-4-..;1~,e

-a' c rd

-92

-66

45--31

.0L

0 2 4 6 8 10 12 14 16

Weeks

FIG. 5. ELISA responses of four experimentally challenged ramsshowingarise-and-surge profile when assayed against HPLC-fractionated antigen A. TS5 (@), TS6 (A), TS7 (O), and TS8 (A)

represent sera collected from rams at weekly intervals over 16 weeks fromthe time of experimental challenge with B. ovis. OD, Optical density.

FIG. 7. Immunoblot profiles against antigen D with sera

col-lectedat themid-fall andmid-surge timepointsoframsfor which

results are represented in Fig. 4 and 5, respectively. Lanes: a

throughd, serumcollected from the mid-fallpointoframsTS 1 to

TS 4atweeks9, 11, 12, and 11, respectively;e, pooledserafrom

prechallenged ramsTS 1toTS8;fthrough i, serumcollected from themid-surge pointoframsTS 5toTS 8atweeks10, 12, 11,and10, respectively. Numbersontherightareapparent molecularmasses

(in kilodaltons).

=- - - -îim -m

- -

--a - --

-- m

--n- m

a~~~m

- .

-~ ~ =

- - - a

-~~-

--i- -=

»-- -~-m

m a -

--~~~m- =-

--- m mu

-I

J.CLIN. MICROBIOL.

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B. OVIS CYTOSOLUBLE ANTIGEN PROFILES 2651

clature of Santos et al. (18). Asimilarreactivity profile fora

singleB. ovis-infected ramhas beenreported byRiezu-Boj etal.(17). Morerecently, Gamazoetal.(12) havedescribed the presence of outer membrane blebs in B. ovis cultures harvestedfrom agarplates. Althoughwe attempted to pre-clude OMPinvolvement inserological analyses by

subject-ing

cytosoluble extracts ofB. ovis toultracentrifugation, it was still notpossibletoexclude the leakage ofsome mem-brane antigens into the cytosol. Nevertheless, Cysup was

resolved by HPLC intofive fractions (A, B, C, D, and E).

Fraction E was notanalyzedfurther because of insufficient material andasmall nativeapparentmolecularmass. SDS-PAGE comparison of fractions A, B, C, and D revealed

distinctive differences in theirpolypeptide profiles (Fig. 1). HPLC-fractionated antigens A, B, C, and D represent

essentially undenatured antigens and shouldassume anative

configuration. However, boiling of these antigens in the presence of SDS and mercaptoethanol wouldresult in fur-ther denaturation to their subunit polypeptides. Sera of

infected rams that shedB. ovis appearedto react

preferen-tially

with fractions A and B in

ELISAs,

while fractions C

and D were

comparatively

inferior ELISA reagents (Table 1). The possibility that the low ELISA activities of fractions

C and Dmay be due to poorbindingtomicrotiterplatescan be

dispelled

since their reactivitiesasnativeantigensindot blot assays on nitrocellulose

proved

to be

equally

disap-pointing (J.

C.

Chin, unpublished data).

An attempt was

madetovalidatethe

A-antigen

fractionas anELISA reagent

for

monitoring

the

temporal

response of rams that were

experimentally challenged

with B. ovis. The results inFig.4

and 5 confirmed that fraction A was able to

distinguish

between rams that had a

rise-and-surge

or rise-and-fall response.

Antigen

A therefore

performs

as well as the whole-cell ELISA (5) in its

ability

to detect infected or shedderrams from

exposed

butuninfected animals.

A

comparison

of the subunit reactivities of

antigens

A and Brevealedlittle differenceintheir

reactivity profiles against

infectedorshedderrams and those that had recovered from

experimental

exposure to B. ovis. In contrast, sera from infectedrams reactedagainstmany subunit determinants in

fractions C andDwhichwere not seenbyserafromexposed

butuninfectedrams

(Fig.

3). However, it is

important

that

immunoblot

profiles

of individual rams

against

different

antigenic preparations

maywellencompassantibody

binding

to several

polypeptides

with identical apparent molecular masses.

Therefore, reactivity against,

for

example,

the

42-and 18-kDa bands located in the same apparent molecular

masszoneinfractions C andDneednotrepresentidentical determinants.

LikeotherBrucella

infections,

B. ovisis

rapidly

phagocy-tosed and has evolved

adaptive strategies

whichresist

intra-cellular

degradation by macrophages.

Intracellular

pathoge-nicity probably provides

a mechanism forthe bacterium to evade

immunological

surveillance. When the bacterium

reaches the

reproductive

tract, itembarks upona prolifera-tive

phase.

Once thisoccurs,

cytosolic antigens

areprobably

released andprocessed

by circulating

macrophages.

Conse-quently,

subunit determinants may provide preferred reac-tive

epitopes symptomatic

ofthe surgephase ofthetemporal

response. Preferential ELISA reactivities

against

native

antigens

A and B and the presence of distinctive bands

against

C- and D-subunit

antigens

in immunoblots tend to support this

hypothesis.

If this is indeed the case, then subunit

antigens

present in fractions C and D may be of greater value than membrane

antigens

in the serological detection of infected rams. Further work is in progress to

ascertain whethera subpopulation of antibodies present in

the surge phase may represent primary reactivities (IgM) against newintracellularantigens of B. ovisorwhether it is

ananamnesticresponse.

ACKNOWLEDGMENTS

Thetechnical assistance of Joanne Loughlin is gratefully acknowl-edged. We alsothankM. Carrigan for interest and involvementin

the program.

Thisstudywasfundedby Australian Wool Corporation as project

DAN43Pontherecommendation of the Wool ResearchTrustFund.

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2. Biberstein,E.L.,B.McGowan,H.Olander, and P. C. Kennedy. 1964. Epididymitis in rams, studies on pathogenesis. Cornell

Vet.54:21-41.

3. Chin, J. C. 1983.Comparisonofdifferentantigenic preparations for the detection of ovineserumantibodiesagainst Brucellaovis

by ELISA. Aust. Vet.J. 60:261-264.

4. Chin, J. C., and Y. Dai. 1990. Selective extraction ofouter

membrane proteins from membrane complexes of Pseudomo-nas maltophilia by chloroform-methanol. Vet. Microbiol. 22: 69-78.

5. Chin, J. C.,andJ.W. Plant.1989.Temporal ELISA response of

rams to Brucella ovisfollowingexperimental infectionor

vac-cination. Res.Vet. Sci.46:73-78.

6. Chin, J. C., J. Plant, and P. D. Claxton. 1983. Evaluation of surface components of Brucellaovis asantigensfor the detec-tion ofprecipitin antibodies inserumfromartificially exposed

rams. Aust. Vet. J.60:264-267.

7. Chin, J. C.,andC.Scully. 1986. Identification of immunoreac-tiveantigens of Brucellaovisby ELISA profiling. Res.Vet.Sci. 41:1-6.

8. Chin, J. C., and J. E. Watts. 1988. Biological properties of phospholipase Cpurifiedfromafleecerot isolate of

Pseudomo-nasaeruginosa. J.Gen. Microbiol. 134:2567-2575.

9. Costerton, J. W.,R. T.Irvin,and K.J. Cheng. 1981. The role of bacterial surfacestructuresinpathogenesis. Curr.Rev. Micro-biol.8:303-383.

10. Ficht,A.F.,S. W.Bearden,B.A.Sowa,and L.G. Adams.1988.

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