INFECrION AND IMMUNITY, Feb. 1971, p. 200-208Copyright @ 1971 American Society for Microbiology

Vol. 3, No. 2Printed in U.S.A.

Sensitization and Recall of Anti-Brucella Immunityin Guinea Pig Macrophages by Attenuated and

Virulent BrucellaDORIS J. RALSTON AND SANFORD S. ELBERG

Division of Medical Microbiology and Immunology, School of Public Health, University of California,Berkeley, California 94720

Received for publication 17 September 1970

Peritoneal macrophages from guinea pigs vaccinated with strain Rev I of Brucellamelitensis were only moderately activated thereby to limit, in an in vitro system,the intracellular growth of Rev I bacilli. Nevertheless, the appropriate memorycells had been primed, as demonstrated by the observation that reinfection of ani-mals with virulent B. melitensis followed by intraperitoneal inoculation of mineraloil called forth macrophages in immunized guinea pigs which inhibited strongly theintracellular growth of brucellae. These macrophages slowed the growth of brucellaein the absence of immune serum. The intensity of the recall response was relatedto the challenge route and to the virulence of the challenge strain. After equal dosesof attenuated or virulent brucellae, resistance was highest in macrophages recalledby the virulent strain. An important basis for the attenuation of the Rev I strain maylie in its initially low degree of macrophage activation during primary infection, al-though still retaining the capacity to prime stem cells. This property is associatedwith a protein found in fraction I, because 600 jig/ml in Freund's adjuvant primedguinea pigs so that challenge by strain 6015 evoked activated macrophages. Thiswas seen microscopically as a reduced spread of infection in and amongst the macro-phage population. Immune serum further reduced this spread and limited the numberof viable intracellular brucellae.

The Rev I vaccine strain of Brucella melitensishas been highly satisfactory in protecting severalanimal species against challenge with virulentbrucellae (1). During immunization it multipliesslowly in spleen and lymph nodes and disappearsfrom the tissues at about the 12th week. Thisstrain is one of the first organisms involved inchronic infections for which a degree of cellularimmunity was described (9). We have approachedthe study of the vaccine strain with the aim ofunderstanding what events, in terms of changes incellular and serum components, are related toand essential for a prolonged and successful im-munization against brucella infection.Our studies have shown that immunization is

accompanied by changes in both macrophagesand immune serum, each of which plays a role inin vitro inhibition of brucella growth. Cultures ofimmune peritoneal macrophages infected in thepresence of immune serum are more capable ofsurviving, i.e., resisting the necrotoxic action ofvirulent brucella (9), more able to ingest bacillirapidly within streptomycin-protected loci (18),and more capable of reducing the intracellular

growth rate of bacilli surviving initial exposureto the macrophages (19), than are immunemacrophages in normal serum or normal macro-phages in either type of serum.

In rabbit studies, primary doses of living Rev I(109/ml) were not characteristically followed by aperiod of intense or generalized macrophageactivation despite the fact that bacilli spread fromthe subcutaneous site of inoculation to other tis-sues of the reticuloendothelial system (19; inpreparation) and induced delayed hypersensitivitythat lasted for many months. Yet during thistime, the serum accumulated high titers of ma-terials which stimulated macrophages to sequesterbacilli more rapidly into streptomycin-protectedsites (macrophage-stimulating factor, MSF) andwhich (in the absence of streptomycin) reducedthe growth rate of brucella in infected macro-phage cultures (brucella-inhibiting factor, BIF).Between 8 and 16 weeks, such sera were active indilutions greater than 100,000 (19; in preparation).We tentatively regarded the minimal amount of

macrophage activation after infection by an at-tenuated strain as causally related to smaller

200

RECALL OF BRUCELLA IMMUNITY IN MACROPHAGES

amounts of toxic antigenic mass. Nevertheless,such amounts might be sufficient to prime thecells to respond to a virulent challenge infection.To investigate this matter, we turned our attentionto the guinea pig, an animal much more sensitiveto brucella infection than the rabbit and thereforemore likely to exhibit marked macrophage re-sponses. This report is concerned with tests madeon guinea pigs both during primary immunizationwith Rev I vaccine and fraction I, a soluble pro-tein from Rev I (7), as well as after challenge withthe virulent B. melitensis 6015 strain. Challengeby strain 6015 was also compared with challengeby strain Rev I. In addition, we have followedserum responses with respect to their capacity toaugment in vitro macrophage defenses.

MATERIALS AND METHODSBacterial strains and immunization procedure. The

vaccine strain B. melitensis Rev I from lyophilizedseed was reconstituted in distilled water and grown onTrypticase soy agar (TSA) for 5 days. Suspensions forinjection were prepared in Z0Bell buffer (20). Maleguinea pigs, Hartley strain, weighing 400 to 500 g,were given primary subcutaneous injections rangingfrom 102 to 108/per animal. For recall of macrophages,secondary challenge materials were prepared in asimilar manner, but suspensions were made in 0.85%saline. Bacilli to be used in in vitro macrophage testswere cultured on Albimi Brucella agar (5 days at 37 C)and treated as previously described (18). B. melitensis6015 was maintained on TSA. Suspensions wereadjusted by turbidimetric standards prepared withthe use of a Klett photoelectric colorimeter as pre-viously described (18).Macrophage harvest. Peritoneal macrophages were

collected on the 5th day after injection of 5 ml ofsterile mineral oil (Klearol; Sonneborn Chemical Co.,Inc., distributed by L. H. Butcher, Hayward, Calif.).When animals were to be given intraperitoneal recallinjections, bacilli were injected into the left side of theperitoneal cavity and the oil was injected into the rightside. Uninfected control animals were given injectionsof 0.85% saline. Macrophages were harvested intochilled Tyrode solution containing 0.01 M phosphatebuffer, pH 7.4. They were routinely washed six times.The last supernatant fluid was negative when testedfor residual agglutinating antibodies at a 1:2 dilutionand in tests for incomplete antibodies. Macrophageswere adjusted for the in vitro test by direct count. Theeosin dye exclusion technique was used to determinemacrophage'viability'(12).

Assay of macrophage resistance by in vitro culture.Washed macrophages were suspended in medium 199plus 15% fetal bovine serum (FBS) (heat-inactivatedfor 15 min at 65 C) to attain 6 X 105/ml. Test inoculaof bacteria were added to attain 6 X 103/ml, giving amacrophage to bacterium ratio of 100:1. When theeffect of immune serum was to be tested, autologousheat-inactivated serum was added to immune cells ata 1 :10 dilution before infection. Portions (1 ml) wereplaced in glass vials. For microscopic examinations,

flying cover slips were added. The course of infectionwas followed by removing replicate samples after 0, 1,and 2 days and plating in triplicate on TSA. Theprocedure for recovering intracellular brucella fromchilled samples has been described (19). The modifiedMacchiavello stain was used to determine the presenceof intracellular organisms (17). From the colonycounts, a resistance index was calculated, based onthe inhibition produced in 48 hr. The resistance index,logi0 (growth in control/growth in test system), equalslog1o[B] increase per milliliter (control) on day 2minus log1o[B] increase per milliliter (test system) onday 2. Samples of fraction I prepared by T. H. Chen(7) were injected into guinea pigs (600 Ag in 0.5 ml ofcomplete Freund's adjuvant) intramuscularly 8 weeksbefore harvesting macrophages. This dose had beenshown to protect 90% of animals against B. melitensis6015 (7).

Serological tests. Passive hemagglutination testswere performed with tanned sheep red blood cellssensitized with fraction I antigen as described (7). Tubeagglutination tests were carried out in 1.0-ml totalvolumes with B. abortus antigen.

Clearance of brucellae. Immediately after harvestingmacrophages, the spleen and the cervical and inguinallymph nodes of the animal were removed and ex-amined for the presence of brucellae (either Rev I or6015 or both) by plating on TSA, TSA plus 2.5,ug ofstreptomycin per ml, and TSA plus 2.5 units of penicil-lin. The antibiotic agars permit differentiation of theRev I and 6015 strain.DHR. Tests for delayed hypersensitivity were per-

formed on the shaven flanks with 8.0,ug per 0.1 ml ofpurified skin test antigen (2). Agglutination titersbefore and after skin testing remained unchanged inevery instance. The calculation of the volume of thelesions was based on the formula H(7rDd)/4, where Hrepresents skin thickness and D and d represent thediameters of the erythema (3). No erythema or in-duration was observed at 3 hr, and delayed hyper-sensitivity reaction (DHR) values were recorded forthe 24-hr observation period. Measurements weremade with a vernier caliper.

RESULTSResponse to primary vaccination. The general

course of infection with living Rev I has beendescribed (15), and again we found clearance ofthe organisms from the tissues to be accomplishedbetween 8 and 12 weeks.The log10 difference between intracellular

growth of Rev I in a culture of normal macro-phages and growth in macrophages from im-munized animals has been used to calculate aresistance index (RI), comparing the relativeabilities of the two types of macrophages to limitmultiplication of the brucella. Macrophages re-moved from guinea pigs over the period 5 days to5 weeks postvaccination and before clearancefrom spleen and lymph nodes showed littlemacrophage stimulation (Table 1). Because of thesmall numbers of animals involved, we have not

VOL. 3, 1971 201

RALSTON AND ELBERG

TABLE 1. Response of guintea pigs during earlyweeks of immunization with Rev I bacilli"

Testno.

Time

afterimmuni-zation(weeks)

Delayedhypersen-sitivityresponse(mm')

1 e 5f NT

2e

3

2

3

5

NT

NT

72v22678 (98)62 (48)5 (15)

Logio increaseof viable Rev Iin macrophages

at 48 hr

ImmunecNormalcontrolb

3.188

3.904

3.8873.9283.8483.8873.9283.848

Relativeinhibitionof Rev Iby IGPMin medium199 + FBS(NGPM!IGPM) d

2.932 0.256

3.700

3.4903.445

3.5293.5773.5553.4203.768

0.204

0.3970.442

0.3560.3100.3320.4670.118

- As measured in vitro by tests of the capacityof macrophages from normal and vaccinatedanimals to limit growth of Rev I in the absence ofantiserum. Abbreviations: IGPM, macrophagesfrom unchallenged immune guinea pigs; NGPM,macrophages from normal animals; FBS, fetalbovine serum; NT, not tested.

b Standard deviation was 3.887 i 0.039 (meanof 5-week data).

c Standard deviation was 3.569 i1 0.041 (meanof 5-week data).

d Resistance index. Calculated as log10 AB permilliliter, in NGPM, minus logo0 AB per milliliter,in IGPM, where AB per milliliter designates theincrease in brucella per milliliter from day 0 today 2. Standard deviation was 0.316 4 0.112(mean of 5-week data).

e Tests conducted on pools of 2 guinea pigs.Other tests reflect data for individual animals,all plated in triplicate from replicate samples.

f Days.Tested at week 3, values in parentheses desig-

nate tests at 5th week postimmunization.

subjected this data to statistical analysis. How-ever, we have considered as significant only thosetests in which the growth of Rev I in macrophagesfrom normal and immune animals differed by a

factor of 3, equivalent to a log value of 0.47. Wenote that, although the recorded differences inTable 1 fall below this value, they were con-sistently in one direction, suggesting that minimaldegrees of macrophage activation had occurred.We point out that a negative value for RI wouldresult from an instance in which growth of Rev Iin the "immune" macrophages was greater thanthat in normal control cells. In our experimentsthus far, this has only occurred in macrophagesfrom nonimmunized animals infected for 5 dayswith virulent 6015 (Table 2).

In instances in which we have tested primaryimmune animals in the period just before majorclearance (8 weeks) or at a time when all animalshad eliminated cultivable Rev I from spleen andlymph nodes, we found that some yielded macro-phages with elevated RI values, ranging from0.508 to 1.142 (Table 2); the guinea pig with thelatter value had not yet eliminated Rev I, a pointwhich is discussed below.Response to challenge in vivo with living virulent

brucelhae. The general procedure for these experi-ments was to reinfect immunized guinea pigsintraperitoneally with strain 6015 and to harvesttheir peritoneal macrophages 5 days afterward.The ability of these macrophages to limit Rev Iinfection was tested in vitro in medium 199 plusFBS. The bacterial growth in guinea pig macro-phages from challenged immune animals (IGPM-6015) was compared with that obtained in macro-phages from unchallenged, immune animals(IGPM), in macrophages from normal animals(NGPM), or in macrophages from nonimmuneanimals infected for 5 days with virulent 6015(NGPM-6015). Each group was infected underidentical conditicins, 6 x 103 bacilli per 6 X 105macrophages, in 1-ml volumes.From the 12th to the 32nd week, 104 cells of

strain 6015 injected intraperitoneally into Rev I-immunized animals stimulated macrophages ofgreatly increased inhibitory capacity. The RIvalues ranged from 1.122 to 2.733, all uniformlygreater than in macrophages obtained fromprimary immune, nonchallenged animals (IGPM)at comparable periods of immunization and re-flecting reductions of growth ranging from16.6-fold to 540-fold over that obtained innormal macrophages (Table 2). At the 8th weekafter immunization, the response to challengewas variable, some animals yielding macrophageswith no increased brucella-inhibitory capacity,whereas others yielded inhibitory macrophages.The results suggest that there may be a period ofunresponsiveness during the primary immuni-zation, in which the recall of activated macro-phages cannot be effected by secondary challenge,a point which would require further study.

Figure 1 compares the relative degrees ofgrowth inhibition produced by immune macro-phages "recalled" after 12 weeks with those fromnonchallenged and nonimmunized control ani-mals. The response was seen as a steady depres-sion of growth of brucellae beginning on day 1,the most significant depression being producedby the macrophages from immunized animalsthat had been exposed for 5 days to virulent 6015.Table 2 shows that as few as 102 Rev I bacilli

injected subcutaneously in primary immunization

202 INFEC. IMMUN.

RECALL OF BRUCELLA IMMUNITY IN MACROPHAGES

TABLE 2. Response of vaccinatedguinea pigs to challenge wit/h virulent strain 6015, as reflected by the abilityof peritoneal macrophages to limit in vitro inifection with straint Rev I in the abseiice of

immune sernma

Primary immunization

Testno.

Length of Dosetime of

(weeks) Rev I

1 0

2

3

4

5

6

8

8

12

28

32

Resistance to in vitro infection with Rev I by macrophages at 48 hr

Normal, infectedwith 6015 at 5days (NGPM/NGPM-6015) b

Primary immune

Clearance' DHRof Rev I (mm3)

RI(NGPMI/IGPAI)

0 0.063-0.309

105 - 186 1.142

+ <<10 0.004

105 ± i 106& 0.414+ 12 0.508

05 + 328 0.702+ 93 0.306

108 + NT 0.053

102 + NT NT

Immune, challengedwith 6015 at 5 daysc

Clearanceof Rev I

+

+

~12-4+

DHR(mm')

173153

102e

148<10

NT

NT

RI(NGPM/!

IGPM-6015)

0.122'

1.447

2.7331.122

1.6314

1 .833d

a Abbreviations: NGPM, macrophages from normal guinea pigs; NGPM-6015, macrophages fromnonimmune animals infected for 5 days with virulent 6015; IGPM-6015, macrophages from immuneguinea pigs challenged with 6015; DHR, delayed hypersensitivity response; RI, resistance index; NT,not tested.

b RI, calculated as explained in footnote d of Table 1. Macrophages were harvested from nonimmu-nized guinea pigs 5 days after infection by B. melitensis 6015.

c Challenge inoculum was 104 virulent 6015 injected intraperitoneally 5 days before harvest of macro-phages.

d Tests on pools of macrophages from two animals; all others, on individual animals.e Tested at week 6; all others tested at week 3.

were found to be sufficient to prime the guineapig for as long as 32 weeks, the recalled macro-phages depressing the growth by a value of RI =1.833 (equivalent to a 93-fold inhibition as com-pared with normal macrophages).

Response to reinfection with Rev I: differencesbetween virulent and attenuated brucellae in recallof activated macrophages. In the rabbit, we foundthat intravenous reinfection of 4-month immuneanimals with 108 Rev I bacilli did not recall acti-vated peritoneal macrophages (19). In the guineapig, we have injected 104 Rev I intraperitoneallyand 105 subcutaneously in comparison withequivalent numbers of strain 6015. In each in-stance, strain 6015 evoked a more highly activated(brucella-inhibitory) macrophage (Fig. 2). Thatthe response was immunological in nature wassuggested by the observation that macrophagesfrom nonimmune animals challenged with 6015 orRev I failed to inhibit test Rev I organisms (Table3). The data indicate that although Rev I vaccineis capable of priming the guinea pig, even when

injected in quantities as low as 103, it has a verylow capacity to produce generalized macrophageactivation on recall, whether reinfected by eitherthe subcutaneous or intraperitoneal routes. Thisis true despite the fact that 5 days after primaryinfection with Rev I bacilli (104) intraperitoneallywe recovered organisms in peritoneal macro-phages, spleen, and lymph nodes, indicating thatthe organism could spread rapidly and multiply.After "recall" in immune guinea pigs, the or-ganism was cleared and we did not recover it inthe macrophages.The data suggest that the degree of activation is

dependent upon the virulence of the strain, whichin turn may govern the antigenic mass producedin vivo. However, against the idea of mere anti-genic mass is the earlier observation that 108 Rev Igiven to the immune rabbit failed to recall acti-vated macrophages. It is probable that total anti-genic mass may not be the only factor in causinggeneralized macrophage activation, but thatspecific antigens present in greater proportions in

203VOL. 3,9 1971

204 RALSTON AND ELBERG INFEC. IMMUN.

N G PM Relationship to delayed cutaneous hypersensi-7 - p tivity. Preliminary observations indicate that the

intensity of the cutaneous reaction is not a goodGP M indicator of eventual success in clearance of Rev

I by the 8th week, the time clearance had begunin a large proportion of animals; nor does it fore-tell the acquisition of the capacity to respond to

z virulent challenge in terms of the degree of gen-06 eralized macrophage activation. At 8 and 12 weeksz_ / /postimmunization, clearance occurred in someo animals with less than 10 mm3 DHR in the4j 10PM -6015O absence of macrophage activation. On the otherU. / / /hand, one animal failing to clear the vaccine at 8

weeks produced a DHR of 186 mm3 at 3 weeks5 - and activated macrophages at 8 weeks (Table 2).

Xj / / /At 12 weeks, animals responded with activatedw // / macrophages irrespective of the size of the earlier

cutaneous lesions (<10 to 148 mm8). All hadcleared Rev I (and also 6015) at the time of

0 sacrifice.0 Those animals which failed to give positiveJ 4 - cutaneous responses ( <10 mm3) and yet produced

activated macrophages on challenge would ap-pear to have been immunized in the absence of

8 _ SUBCUTANEOUS ROUTE INTRAPERITONEAL ROUTE

32

DAY

FiG. 1. Response of guinea pig macrophages fromprimary immune (12 week) and immune animals chal-lengedfor 5 days with virulent strain 6015, as tested bythe capacity of peritoneal macrophages to limit in-fection with Rev I in medium 199 plus fetal bovineserum. Data show average values and ranige of plate - 6 -

counts for individual animals. 0

each 6015 cell may be involved in producing an Dessential degree of response to infection. SinceRev I has been shown to multiply in the guinea mpig, the exact antigenic concentration necessary -to produce the threshold response is not known.

Differences stemming from the route of chal- &NGPM(605)lenge infection may be an additional factor in the A IG.PM(REVI)degree of generalized activation produced. Re- 4

0NGPM(CONTROL)infection by the subcutaneous route with 105 S 1tGPbl (6015)bacilli yielded macrophages of lesser inhibitorycapacity (Fig. 2) than those reinfected with 104via the intraperitoneal route. These differencesmay have been influenced by the size of the pri- 0 2 2mary immunizing dose (10$ for animals challenged DAY DAYsubcutaneously and 108 for those challenged FIG. 2. Ability of virulent straini Brucella melitensisintraperitoneally). However in other tests we 6015 and attenuated B. melitensis Rev I to "recall"I I " . . >activated macrophages in guinea pigs immunized byfound that 102 Rev I induced protection for over the subcutaneous and intraperitoneal routes. Tests with28 weeks and still yielded highly activated macro- peritoneal macrophages (6 X 105/ml) infected withphages when challenged intraperitoneally with Rev I bacilli (6 X 103/ml) were dlone in medium 199101 virulent brucellae. plus fetal bovine serunm.

,r - J-..-I,---.

RECALL OF BRUCELLA IMMUNITY IN MACROPHAGES

TABLE 3. Differences between virulent 6015 and attenuated Rev I in capacity to recall activated macrophagesin immune guinea pigs

Index of resistance& for Rev IPrimary Challenge infection in peritoneal macrophagesPrimary ~~~~~~~~~~~~~~afterchallenge by

Macrophage source idspneiganimal

Route animal Virulent 6015 Attenuated

Immune, 28 wk 105 Subcutaneous 105 1.007 0.425Nonimmune 0 Subcutaneous 105 0.318 0.256Immune, 28 wk 108 Intraperitoneal 104 1.631 0.117Nonimmune 0 Intraperitoneal 104 0.063 0.315Nonimmune 0 Intraperitoneal 104 -0.309 NTb

a Calculated from log10 B per milliliter increase on day 2 in macrophages from normal guinea pigsminus log1o B per milliliter increase on day 2 in test guinea pig macrophages.

b Not tested.

delayed hypersensitivity. (However, since theDHR was only tested once, the reaction mighthave been missed.) Thus far we have not definitelydissociated delayed hypersensitivity from thephenomenon of macrophage activation.Response to immunization with fraction I antigen

from Rev I. Guinea pigs were injected with 600-,igamounts of fraction I antigen in Freund's adju-vant. This antigen is a soluble protein fractionobtained from acetone-killed smooth bacilli (7).During the third week after primary injection, theanimals were uniformly hypersensitive to 0.8 ,ug ofskin test antigen, lesions appearing by 24 hrwhich remained as long as 48 hr, in the absence ofany significant 3-hr reaction. At 8 weeks, half ofthe immunized animals were challenged with 104virulent cells (immune-challenge group), and theother half of the animals were retained as immunecontrols (primaryimmunegroup); forcomparison,a nonimmunized group was infected for the firsttime with virulent cells (nonimmune, infectedgroup), whereas a fourth, nonimmunized setserved as normal, uninfected controls (nonim-mune, noninfected group). Macrophages werecollected on the 5th day after challenge.The primary group showed little or no resist-

ance to Rev I, but the macrophages of the im-mune-challenged group were activated and de-pressed the growth of Rev I in vitro (Fig. 3B).Guinea pigs infected for the first time with viru-lent 6015 did not respond with activated macro-phages (Fig. 3A). We concluded that fraction Ihad induced immunologically specific memory inthe appropriate cell systems.Immunization with fraction I also produced a

positive serum-inhibitory titer (BIF), as shown inthe depression of growth curves by primarymacrophages infected in the presence of 10%heated immune serum (Fig. 3A). The effect ofthis immune serum during in vitro infection was

seen microscopically as a slower spread (12.1 %)of bacilli throughout the macrophage population(Table 4). In the presence of immune-challengedmacrophages, immune serum further reduced thespread to 5.9% of the population, and in thesemacrophages (Fig. 3B) the viable count wassignificantly reduced. The reduced spread of in-fection in the presence of immune serum was aresult of both macrophage and serum action,since macrophages alone, in the absence of serum,also reduced the spread of infection albeit to lesserdegrees (49.2 and 28.4% for primary immuneand challenge immune, respectively, as comparedwith 66.8% in nonimmune macrophage cultures).The reduced spread in immune macrophagepopulations probably reflected responses of cellsthat were less susceptible to the macrophage-destroying action of the contained brucellae, amacrophage property which persists during theprimary response for a longer period than thecapacity to inhibit brucella growth. After chal-lenge, the capacity to limit brucella growth wasreinforced and the macrophages also limited thespread of infection.Although not shown in our data, additions of

immune serum to macrophages from animals im-munized with the living bacilli also enhancedmacrophage-inhibitory action. This property,similar to studies in the rabbit (19), appearedwithin the 5th day after immunization and wasdetected in heat-inactivated serum.

DISCUSSIONAlthough a number of nonspecific agents, such

as FBS, glycogen, and bacterial endotoxins (5, 10,16), induce the appearance of bactericidal macro-phages in animals, stronger immunologicalchanges accompany infections with intracellularpathogens by producing highly activated macro-

205VOL. 3, 1971

RALSTON AND ELBERG

B. IMMUNEA. PRIMARY MACROPHAGES AFTER

IMMUNE MACROPHAGES RECALL WITH 6015

C. NORMAL MACROPHAGESAFTER INFECTION

WITH 6015

0

NGPS

z

0

0 6

IGSAUTOL. GOL.

4

l l2 2 2

DAY DAY DAY

FIG. 3. Antibacterial activity of peritonteal macrophages fronm guinea pigs immunized with fraction I antigenand challenged with virulent 6015. Tests ofperitoneal macrophages inifected with Rev I were done in medium 199plus fetal bovine serum, conitaining 10% normal guinea pig serum or serum from unchallenged immune guinea pigs.

phages. It is with this aspect of the cellular re-sponse that we have been concerned in thesestudied of the Rev I brucella vaccine.

It is increasingly evident that the developmentof highly activated macrophages and the capacityto resist infection by intracellular bacterial patho-gens are correlated with the onset of delayedhypersensitivity (8), although the exact conditionsprerequisite to the recall of macrophages withheightened antibacterial activity have not beendefined. The stage of the infection before chal-lenge, the size of the primary and secondary infec-tion dose, and the type of immunizing antigen andintensity of antigenic stimulus have been proposedas determinants of activation (8).As with BCG infection in the mouse (6), the

rabbit responds to primary immunization withRev I by the production of minimally activatedmacrophages. In contrast to studies with BCG (4),we found that Rev I immune serum rapidly ac-cumulated high titers of heat-stable antibodies (inaddition to heat-labile factors) which acted inconcert with macrophages to lessen the effects ofbrucella action. Inhibitory action of the serum

was specific for brucella since neither Listeriamonocytogenes nor Salmonella enteritidis wasinhibited (unpublished data). Our results suggestthat the cells concerned in the ultimate disposalof brucellae in the living animal can be aidedsignificantly by factors in the immune serum.We have not yet obtained a combination ofmacrophages and serum from the rabbit whichcould completely sterilize intracellular brucellain an in vitro system. We have only observed in-hibition, indicating that elimination of brucellaein vivo is effected by fixed macrophages of liverand spleen.

Since the guinea pig is more susceptible tobrucella infection and yet is capable of developinga solid immunity, it may show increased primaryimmune cellular responses and produce activatedperitoneal macrophages on recall of the immunitysimilar to that shown during infection of micewith strain 19 or B. abortus (6).We examined animals injected with Rev I at

the beginning (5 and 14 day), middle (3 and 5weeks), and end (8 and 12 weeks) of the periodwhen the attenuated bacilli move from the site of

206 INFEC. IMMUN.

RECALL OF BRUCELLA IMMUNITY IN MACROPHAGES

TABLE 4. Slow spread of Rev I bacilli in macro-phages from immunized guinea pigs after recall

by virulent Brucella melitensis 6015

Macrophage source

Nonimmunecontrol

Nonimmuneinfected

Fraction Iprimary im-mune)d

Fraction I (im-mune chal-lenged) e

Serum sourcea

NGPS

NGPSNGPS (6015)NGPSIGPS

NGPSIGPS (6015)

Per cent ofmacrophagesinfectedb at

0 day 2 days

NTc 66.8

1.8 76.82.4 38.64.4 49.23.1 12.1

18.0 28.43.2 5.9

-Abbreviations: NGPS, normal guinea pigserum; NGPS (6015), serum from nonimmuneanimals infected with 6015; IGPS, serum fromunchallenged immune animals; IGPS (6015), serumfrom immune animals challenged with 6015.

b Data are based on counts of 500 to 1,000 stainedmacrophages for each condition.

c Not tested.d Macrophages and IGFS were collected 8 weeks

after primary immunization.eAnimals were challenged with 104 strain 6015 in-

traperitoneally.

inoculation to nodes and organs and are finallyeliminated and have found that, in general,macrophage activation was minimal. One notableexception involved a guinea pig which was stillinfected (8 weeks) at a time when other membersof the group were cleared, suggesting that thevaccine acted as a more virulent organism for thatanimal. Yet at a time when splenic infection wassubstantial and delayed hypersensitivity had ap-peared, the 14th day, we noted that macrophageactivation was absent. At present, our data indi-cate that the vaccine can prime the guinea pig inthe absence of intense activation.

If the heightened macrophage response is re-lated more to the toxic components responsiblefor inflammation or to their increase duringgrowth of the bacteria, an explanation would beat hand for the rapid onset of response to L.monocytogenes in the mouse, a strain originallyselected for its virulence by mouse passage (6), asopposed to the lesser and slower effects of B.abortus 19 and the more modest action of theBCG strain of Mycobacterium tuberculosis, bothattenuated vaccine strains.

Conditions for recall of activated macrophagesin the mouse-BCG system involve both the levelof delayed hypersensitivity and the infecting and

recall doses of organisms (6). Mice reinfectedintravenously with 5 X 107 BCG during the peak(23rd day) of hypersensitivity invoked a morerapid recall of macrophages with bactericidalaction for S. typhimurium than those injected at56 days when the sensitivity was waning (6). Thiscontrasts with the time relationships observedwith some of our animals in that reinfection at anearly time (8 weeks), when sensitivity was pro-nounced, failed to invoke activated macrophages,whereas months afterwards, when sensitivity haddropped to lower levels, reinfection with equiva-lent numbers of virulent brucellae produced acti-vated macrophages. Also, macrophages of height-ened capacity could be recalled at 12 weeks in ananimal which had produced a very low, almostundetected cutaneous hypersensitivity reaction(3 weeks after the primary infection). From thesestudies, it is not possible to draw conclusions thatdelayed hypersensitivity is a necessary step in thepriming of the macrophage system.

It has been claimed that a large dose of bac-teria must be injected to stimulate generalizedactivation (as contrasted with smaller numbers tocreate local tissue responses; reference 6). Tothis can be added the present observation thatthe relative ability to stimulate recall dependson the virulence of the reinfecting organism; wefound strain 6015 regularly to produce more ef-fective activation than equal numbers of at-tenuated Rev I.

In the guinea pig, as in the rabbit, infectionwith strain Rev I and, to a lesser extent, immuni-zation with fraction I caused the formation ofserum components which mediated enhancedmacrophage inhibition (BIF), even in macro-phages which themselves were inhibitory forRev I. The serum components appeared within 5days of infection and were contained within serumfractions rich in 19 and 7S globulins. In addition,we found activity associated with a lower-mo-lecular-weight material in a fourth protein frac-tion after Sephadex G200 separation (in prepa-ration).

It is evident that infection invokes a series ofantibodies possessing various potentials to in-fluence the metabolism of brucella and probablyto affect the responses of macrophages oncecombined with antigen. In the immunity whichresults from infection with attenuated Rev I,these special classes of antibodies may appearsequentially, augmenting final clearance andinfluencing the early responses of the animalduring challenge with virulent organisms. Recentstudies of murine pertussis have suggested thatconvalescent serum contributes enhanced abil-ities to immune macrophages (11). These ap-peared at the time infected mice underwent

VOL. 3, 1971 207

RALSTON AND ELBERG

clearance, somewhat similar to those reported formice 14 days after infection with S. typhimurium(13).Presumably these antipertussis and antisal-

monella antibodies have different biologicalfunctions than the heat-stable antibrucella(BIF) antibodies described in our studies. Theyare released (in the pertussis system) mainlyduring final clearance, whereas the brucella-immune serum contains significant amounts asearly as 5 days postimmunization and theselevels remain high long after clearance. We havenot yet been able to assign an essential role tothe brucella antibodies in the in vivo protectioninduced by the vaccine, although it is quite clearthat in vitro they augment the inhibitory activityof recalled immune macrophages as well asmacrophages from normal animals. From this,one can only speculate that they function toraise macrophages to their highest defensecapacities, which may ultimately provide us witha clearer notion of how the vaccine confersimmunity.

ACKNOWLEDGMENTS

We acknowledge with gratitude the technical assistance ofJoan Okimoto and J. B. Cunningham.

This investigation was supported by grant A100022 from theNational Institute of Allergy and Infectious Diseases and by a

grant from the Veterinary Public Health office of the WorldHealth Organization.

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208 INFEC. IMMUN.