Vol. 46, No. 1INFECTION AND IMMUNITY, OCt. 1984, p. 224-2300019-9567/84/100224-07$02.00/0Copyright © 1984, American Society for Microbiology

Ingestion and Intracellular Survival of Brucella abortus in Humanand Bovine Polymorphonuclear Leukocytes

LELA K. RILEYt AND DONALD C. ROBERTSON*Department of Microbiology, University of Kansas, Lawrence, Kansas 66045

Received 10 April 1984/Accepted 2 July 1984

Bovine polymorphonuclear leukocytes (PMNs) were found to be significantly more bactericidal than humanPMNs against a smooth-intermediate strain of Brucella abortus (45/0), whereas there was no difference inbactericidal activity of the two kinds of PMNs against a rough strain of B. abortus (45/20). Electron microscopyof thin sections of PMNs revealed that both strains of B. abortus were readily ingested; however, the extent ofdegranulation was significantly less than in PMNs incubated with an extracellular parasite, Staphylococcusepidermidis. Amounts of myeloperoxidase and lactoferrin released through exocytosis by PMNs incubated withS. epidermidis were 4.7- and 1.2-fold greater, respectively, than those released from PMNs incubated with B.abortus 45/0. When azurophil and specific granules were isolated after incubation of PMNs with either B.abortus 45/0 or S. epidermidis, results showed that the extent of degranulation by both types of granules was

greater in PMNs incubated with S. epidermidis than in those incubated with B. abortus 45/0. Amounts ofdegranulation by azurophil and specific granules were similar in PMNs incubated with either the smooth-intermediate strain 45/0 or the rough strain 45/20. Degranulation was not stimulated when glutaraldehyde-killed strain 45/0 was substituted for viable cells. These data suggest that B. abortus does not stimulate an

effective level of degranulation after ingestion, as observed with extracellular parasites, and that the smoothintermediate strain 45/0 is more resistant to intraleukocytic killing systems than the rough strain 45/20.

The survival of intracellular parasites within phagocyticcells depends on their ability to inhibit or resist intraleuko-cytic killing systems effective against extracellular parasites(8, 28). One mechanism of intracellular survival is mediatedby inhibition of fusion between lysosomes and phagocyticvacuoles (phagosomes) containing microbes. If fusion oc-curs, azurophil and specific granules discharge two groups ofkilling systems into phagosomes: (i) oxygen-dependent re-actions mediated by oxygen metabolites (2, 3, 14), and (ii)oxygen-independent reactions (e.g., cationic proteins, pro-teases, lactoferrin, and phospholipase A2) (32). Lysosomesdo not fuse with phagosomes containing Mycobacteriumtuberculosis due to the presence of acidic sulfatides (12),whereas Mycobacterium microti blocks degranulation byincreasing levels of intracellular cyclic AMP (21, 22). Themechanisms that Toxoplasma gondii (13) and Chlamydiapsittaci (11, 36) use to inhibit lysosome-phagosome fusionare unknown. Alternatively, due to unique surface macro-molecules, parasites can escape detection and not stimulatedegranulation or the respiratory burst that usually followsingestion (7, 15). A third mechanism of intracellular survivalis used by Mycobacterium lepraemurium and Salmonellatyphimurium, which multiply within phagolysosomes despiteextensive degranulation (1, 5).Numerous studies have described the survival of Brucella

sp. within mononuclear cells (4, 10, 23, 25, 31). In an attemptto identify factors that determine intracellular survival ofBrucella sp., we determined the composition of cell walls oftwo isogenic strains of Brucella abortus that'differ in viru-lence and infectivity (18). There were no significant differ-ences between the two strains with respect to total lipids,proteins, and peptidoglycan content; however, the smoothstrain 45/0 contained a phenol-soluble lipopolysaccharide

* Corresponding author.t Present address: Department of Biochemistry, University of

Kentucky Medical Center, Lexington, KY 40536-0084.

not present in the rough strain 45/20, and the phenoliclipopolysaccharide was the only cell wall component toxic tomice (16). Guinea pig polymorphonuclear leukocytes (PMNs)were not brucellacidal to the smooth strain (45/0) with thephenolic lipopolysaccharide, and they killed only 35% of therought strain 45/20 after 2 h of incubation (17). Moreimportant, there was no stimulation of the hexosemonophosphate pathway when guinea pig PMNs were in-cubated with each strain, yet guinea pig granule lysates werebrucellacidal to both strains of B. abortus when supple-mented with hydrogen peroxide (H202) and potassium iodide(KI). Thus, it was not clear whether the increased survival ofB. abortus 45/0 was due to decreased ingestion, inhibition ofdegranulation, or the ability to survive within phago-lysosomes.To characterize mechanisms of intracellular survival by

Brucella sp., we examined the ingestion, degranulation, andkilling steps of phagocytosis by PMNs incubated with thesmooth and rough strains of B. abortus (45/0 and 45/20)described above. The properties of strains 45/0 and 45/20have been summarized previously (18). Briefly, strain 45/20is a rough variant with decreased virulence isolated from the20th passage of the parent strain in guinea pig and has beenused as a killed vaccine. Although the two strains differ ininfectivity, strain 45/20 is pathogenic for humans and ani-mals. This study concentrated on PMNs because the PMN islikely the first phagocyte to challenge the bacteria afterinfection through a cut, the alimentary tract, or via theconjunctival route. The short-lived PMN, if not brucel-lacidal, will transport bacteria to regional lymph nodes andthroughout the reticuloendothelial system (17, 26). The datashow that both smooth and rough strains of B. abortus arereadily ingested by human and bovine PMNs. Intracellularsurvival of B. abortus appears to be due to minimal de-granulation after ingestion and the ability to withstandconditions within phagolysosomes.

224

on April 30, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

PMNs AND B. ABORTUS 225

MATERIALS AND METHODS

Bacterial strains and growth conditions. A smooth inter-mediate strain of B. abortus 45/0 was obtained from M.Meyer, University of California, Davis, and a rough strain,B. abortus 45/20, was provided by B. L. Deyoe, NationalAnimal Disease Center, Ames, Iowa. The origins of bothstrains, maintenance of stock cultures, and growth condi-tions have been described previously (18). Cells for experi-ments were harvested in the mid-log to late-log phase ofgrowth.

Isolation of PMNs. Human PMNs were obtained fromfresh heparinized peripheral blood (5 to 10 U/ml) of healthyvolunteers, and purified as described by Rest and Spitznagelet al. (30, 33). Viability was greater than 99% as determinedby trypan blue exclusion. Purified leukocyte suspensionscontained less than 1% erythrocytes and 90 to 95% neutro-phils. Bovine PMNs were isolated from fresh heparinizedblood obtained from a local slaughterhouse and purified byusing a modification of the technique described by Carson etal. (6). Whole blood was diluted with cold Hanks balancedsalts solution (60:40 [vol/vol]). Diluted blood (22 ml) waslayered in a 50-ml centrifuge tube over 15 ml of Ficoll(Pharmacia Fine Chemicals, Piscataway, N.J.)-Hypaque-M(Winthrop Laboratories, New York, N.Y.) solution pre-pared by mixing 8% Ficoll (84 ml) and 38% Hypaque (35 ml).Gradients were centrifuged at 600 x g for 70 min at 4°C.After centrifugation, lymphocytes and serum were removedby aspiration. PMNs present as a buffy coat layer werecollected and washed two times in 0.85% NaCI to removeplatelets. Contaminating erythrocytes were lysed with dis-tilled water, and then hypertonic saline was added to restoreisotonic conditions. Purified PMN suspensions containedless than 1% erythrocytes and 90 to 95% neutrophils. Vi-ability was greater than 95% as measured by trypan blueexclusion.

Bactericidal activity of purified PMNs. Bactericidal activitywas determined in reaction mixtures containing: PMNs(107), 1.0 ml; bacteria in Hanks balanced salts solution (108),1.0 ml; heat-inactivated horse serum, 0.3 ml; and Hanksbalanced salts solution (pH 7.2), 0.7 ml. Reaction mixtures in25-ml siliconized Erlenmeyer flasks were incubated withgentle shaking (100 rpm) at 37°C. At timed intervals, 0.1-mlsamples were removed and diluted with 9.9 ml of deionizedwater to lyse PMNs. Intact PMNs were not detected bystaining with Wright-Giemsa after distilled water lysis. Di-lutions were made in 0.5% tryptose (Difco Laboratories,Detroit, Mich.)-0.5% sodium chloride, and survival wasdetermined by plating 0.1-ml samples on Trypticase soy agarplates (BBL Microbiology Systems, Cockeysville, Md.).

Electron microscopy studies. Bactericidal mixtures wereprepared as described above and incubated on a shakingwater bath at 37°C. At timed intervals, reaction mixtureswere centrifuged and the pellet was fixed in a solution of 3%glutaraldehyde (Sigma Chemical Co., St. Louis, Mo.). Aftercentrifugation, the cells were washed four to six times with1.5% glutaraldehyde-0.1 M sodium cacadylate (pH 7.4) andpostfixed in 1% osmium tetroxide (OS04) (Ernest F. Fullam,Inc., Schenectady, N.Y.). Samples were dehydrated througha graded series of increasing concentrations of ethanol,carried through propylene oxide, and embedded in Epon.After polymerization, thin sections were cut, stained in leadcitrate and uranyl acetate, and examined with an RCA EMU3-H electron microscope.

Exocytosis studies. Degranulation was measured by thetechnique described by Leffell and Spitznagel (20). Bacte-

ricidal reaction mixtures were prepared as described above.Samples (0.5 ml) were removed at timed intervals andcentrifuged at 500 x g to remove cells. The supernatant wasfiltered (0.22-,um Millipore filters, Millipore Corp., NewBedford, Mass.) and assayed for myeloperoxidase (MPO),P-glucuronidase, lactoferrin, and lactate dehydrogenase.Enzyme assays. MPO was measured by observing the

reduction of o-dianisidine as described by Worthington (34).,-Glucuronidase was assayed by the technique of Fishman(9).

Determination of lactoferrin. Lactoferrin was assayed bysingle radial immunodiffusion (24). Antiserum was preparedby subcutaneous injection of either human or bovine lac-toferrin (50 jig) in complete Freund adjuvant along theshaved back of a rabbit. Animals received booster doses of50 ,ug of lactoferrin in incomplete Freund adjuvant 2 weeksand 3 months after the primary immunization. Blood wascollected 2 weeks after the last booster dose. Serum wasstored at -70°C. Agar plates were prepared by mixing 0.1 Mbarbiturate buffer (pH 8.6), 3.0 ml; anti-lactoferrin, 0.3 ml;and 3% Noble agar, 3.3 ml. Samples (5 ,ul) were applied to 3-mm circular wells. Plates were incubated in a humidifiedchamber for 48 h, and diameters of precipitin rings weremeasured with a micrometer. Lactoferrin concentrationswere calculated by using a plot of area versus amount (innanograms) of lactoferrin. Human lactoferrin was isolatedfrom human colostrum as described by Querinjean et al.(27). Bovine lactoferrin was kindly provided by F. L.Schanbacher, Wooster, Ohio.

Isolation and purification of granule populations. Purifiedpopulations of azurophil and specific granules were isolatedby the method of Rest et al. (29, 30). Neutrophils (108 to 109)were suspended in 3 ml of 25% sucrose and homogenized byusing 25 strokes of a Teflon homogenizer or until PMNswere 90% lysed. The homogenate was centrifuged at 126 xg for 10 min to remove cellular debris. The 126 x gsupernatant was carefully layered over a 30-ml, linear, 30 to53% (wt/wt) sucrose gradient and centrifuged with a Beck-man SW27 rotor at 25,000 rpm for 120 min at 20°C. Fractions(1 ml) were pumped from the bottom of each tube at aconstant rate (3 ml/min). Subcellular fractions were locatedby measuring the absorbance at 450 nm. Purified granuleswere extracted with hexadecyltrimethylammonium bromideto release latent enzymes.

Degranulation studies. After incubation of PMNs witheither S. epidermidis or B. abortus for 2 h, PMNs werehomogenized and the remaining granules were purified byusing sucrose density gradients as described above. Thelevels of MPO, ,-glucuronidase, and lactoferrin after frac-tionation and extraction of the granules were used to quan-titate the amount of degranulation.

Preparation of glutaraldehyde-killed B. abortus. B. abortuscells (1010) were centrifuged and suspended in 2.5 ml of 2.5%glutaraldehyde-0.1 M sodium cacadylate buffer (pH 7.2).After 10 min of incubation at 40C, cells were pelleted bycentrifugation at 6,000 x g, washed twice with Hanksbalanced salts solution, (pH 7.2), and resuspended to aknown concentration.

RESULTS

Bactericidal activity of PMNs. The brucellacidal activity ofhuman and bovine PMNs was assessed by incubating smooth-intermediate and rough strains of B. abortus with intactPMNs in the presence of heat-inactivated horse serum.Previous data indicated that serum was not required for

VOL. 46, 1984

on April 30, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

226 RILEY AND ROBERTSON

20-~~~

O' 30 60 90 1i0 30 60 90 120

Time (min) Time (min)

FIG. 1. Bactericidal activity of bovine (A) and human (B) PMNsagainst B. abortus and S. epidermidis (albus). Symbols: *, B.abortus 45/0; A, B. abortus 45/20; 0, S. epidermidis. Results are

shown as the mean of three experiments, with duplicate data pointsper experiment.

ingestion of B. abortus (17); thus, horse serum was usedinstead of bovine serum to facilitate comparison of thebrucellacidal activities of the two kinds of PMNs, andbecause it was difficult to obtain serum from unvaccinatedcattle. Bovine PMNs were more bactericidal than humanPMNs against the smooth-intermediate strains of B. abortus45/0, whereas there were no differences in killing capacityagainst the rough strain 45/20 (Fig. 1). Bovine, PMNs were

slightly more bactericidal against Staphylococcus epider-midis than were human PMNs.

Electron microscopy studies. The low bactericidal activityof PMNs against B. abortus 45/0 could be due to limitedingestion, decreased degranulation after ingestion, or in-creased resistance to intracellular killing. Thin sections ofhuman PMNs incubated with either B. abortus 45/0 or S.epidermidis in the presence of heat-inactivated horse serumwere prepared for electron microscopy. Samples taken at 30min postinfection showed that both S. epidermidis and B.abortus 45/0 were ingested (Fig. 2A and B). Electron-denseregions at the periphery of phagosomes containing S. epi-dermidis indicated that degranulation had occurred. In con-trast, minimal amounts of fusion between lysosomes andphagosomes were noted in PMNs that had ingested B.abortus 45/0. No attempt was made to determine the numberof bacteria per PMN; however, greater than 90% of thePMNs contained two to five bacteria at 30 min. After 120min of incubation, extensive degranulation was observedonly in human PMNs incubated with S. epidermidis. Only afew granules remained intact, and the integrity of the cyto-plasm was significantly diminished (Fig. 3A). As noted at 30min, PMNs that had ingested B. abortus 45/0 showed onlylimited degranulation (Fig. 3B). Similar results were ob-

tained with bovine PMNs incubated with either S. epider-midis or B. abortus (data not shown).

Degranulation studies. Electron micrographs providedqualitative evidence that phagolysosome formation was lessin PMNs infected with B. abortus than in PMNs that hadingested S. epidermidis; thus, the extent of degranulation byPMNs after ingestion of B. abortus or S. epidermidis wasdetermined by (i) measuring the amounts of granule enzymesreleased into supernatants by exocytosis, and (ii) isolatingintact granules from infected PMNs. When granules fusewith incompletely closed phagosomes, the amounts of gran-ule proteins released into the medium can be correlated withthe extent of ingestion and degranulation (20, 35). Super-natants of reaction mixtures containing human PMNs, bac-teria, and serum were assayed for MPO and lactoferrin,which were used as markers of azurophil and specificgranules, respectively (Fig. 4) (19, 33, 35). The amount ofMPO released at 30 min by PMNs infected with B. abortuswas ca. fivefold less than that released by PMNs incubatedwith S. epidermidis, whereas the amount of lactoferrinreleased by B. abortus-infected PMNs at 30 min was ca; 50%that released by PMNs incubated with S. epidermidis. Thedifferences in amounts of lactoferrin released by exocytosiswere not as pronounced at 90 min, with only 20% morelactoferrin released by PMNs incubated with S. epidermidiscompared with PMNs incubated with B. abortus. Lactatedehydrogenase, a cytoplasmic enzyme, was not releasedfrom PMNs infected with either B. abortus or S. epidermidis(data not shown); therefore, release of granule proteins wasnot due to leukocyte lysis.Although exocytosis studies indicated that degranulation

was less in PMNs infected with B. abortus 45/0 comparedwith PMNs incubated with S. epidermidis, interpretation ofthese results was limited by at least two factors: (i) therelative amounts of degranulation by heavy and light azuro-philic granules (30) could not be determined, and (ii) quan-titation was not entirely satisfactory because only ca. 10% ofthe total granule contents was released by exocytosis (datanot shown). These limitations were avoided by isolation ofintact granules after incubation of PMNs in the presence andabsence of bacteria, and by fractionation of the mixedgranule population by differential centrifugation on linearsucrose gradients. Three discrete bands were visible withinthe gradient. The upper band contained specific granules,and the lower two bands were identified as light and heavypopulations of azurophil granules (30). The results of theseexperiments, expressed as the percentage of each markerprotein present in control PMNs not incubated with bacte-ria, are summarized in Table 1. If degranulation resultedfrom ingestion of bacteria, amounts of MPO and lactoferrinshould be lower relative to controls not incubated withbacteria. The results showed that degranulation by heavyand light azurophilic granules was less in PMNs that hadingested B. abortus than in PMNs that had ingested S.epidermidis. Lactoferrin levels in PMNs after incubationwith S. epidermidis reflected extensive degranulation byspecific granules and were significantly less compared withPMNs incubated with B. abortus 45/0.Because of the differences in bactericidal activity of

human and bovine PMNs against the two B. abortus strains,45/0 and 45/20, which differ in virulence and surface prop-erties, it was of interest to compare the amounts of de-granulation in PMNs infected with each strain. The design ofthese experiments was similar to that described above,except that granules were not fractionated on sucrose gra-dients and bovine PMNs were used for these experiments

INFECT. IMMUN.

on April 30, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

PMNs AND B. ABORTUS 227

t. $. Vw ,

..

., ; t.

Al 'Y' (R)'

A.ti i .,, Jffl -:.

s s t3 j; itCr

_

%¢' < _m;3sB.Jr:M,

a <hfa

FIG. 2. Electron micrographs of thin sections of human PMNs after 30 min of incubation with S. epidermidis (A) and B. abortus 45/0 (B).Ingested bacteria are noted by SA (S. epidermidis) and B (B. abortus). Azurophilic and specific granules are noted by A and S, respectively.The electron-dense region (D) at the periphery of the phagosome is an indication of degranulation. Magnifications: (A) x 14,000; (B) x 10,500.

due to their higher brucellacidal activity compared withhuman PMNs. The amounts of MPO and lactoferrin weresimilar in PMNs that had ingested either strain of B. abortus(Table 2). Lactoferrin levels in bovine PMNs incubated withB. abortus 45/0 or 45/20 were ca. threefold higher than inPMNs incubated with S. epidermidis, whereas MPO levelswere ca. twofold greater.

Inhibition of degranulation by some intracellular parasitesdepends on viable cells (11). To determine whether viable B.abortus cells were required to prevent fusion of granuleswith phagosomes, glutaraldehyde-killed B. abortus 45/0 cellswere incubated with human PMNs. After 2 h of incubation,infected PMNs were homogenized and the homogenate wasfractionated on sucrose density gradients. Levels of MPO,

3-glucuronidase, and lactoferrin in isolated granule fractionswere identical to those in PMNs incubated with viable B.abortus cells (data not shown).

DISCUSSIONThe results presented in this report show that bovine

PMNs were more bactericidal than human PMNs against asmooth strain of B. abortus, 45/0. Brucellacidal activities ofhuman and bovine PMNs against a rough strain of B. abortus(45/20) were comparable and significantly higher than thoseobserved against strain 45/0, but less than observed with anextracellular parasite, S. epidermidis. Since the increasedsurvival of B. abortus 45/0 could be due to decreased

TABLE 1. Enzyme levels in granules obtained by differential centrifugation of leukocyte homogenates after incubation with and withoutbacteriaa

MPO in band:Lactoferrin in band III (least dense)

Incubation I (most dense) II%~~onto Amtpaa(U) % of Comparative % of ComparativecultureAmt (U) control Comparative Amt (U) control C b Amt (,ag) control %b

Control 200.2 100 269.5 100 132.0 100S. epidermidis 111.1 55.5 69.1 130.0 48.3 48.2 1.6 1.2 5.4B. abortus 180.5 90.2 78.5 186.2 69.1 75.8 91.2 69.1 56.4

a The granules from approximately 108 PMNs were applied to each gradient.b Mean of three to five experiments with PMNs from different human donors.

0

k"V

VOL. 46, 1984

on April 30, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

228 RILEY AND ROBERTSON

FIG. 3. Electron micrograph of thin sections of human PMNs after 120 min of incubation with S. epidermidis (A) and B. abortus 45/0 (B).Ingested bacteria are noted as in Fig. 2. Magnifications: (A) x9,750; (B) x12,880.

ingestion, decreased degranulation after ingestion, or in-creased resistance to intracellular killing, electron micros-copy of thin sections of human and bovine PMNs was usedto characterize the ingestion phase of phagocytosis. Signifi-cant ingestion of strain 45/0 was observed at 30 min post-infection and did not increase significantly in preparationsexamined at 120 min. Similar experiments with S. epider-midis were performed to assess the relative amounts ofingestion and degranulation by PMNs incubated with anextracellular parasite. After 30 min of incubation with S.epidermidis, electron-dense regions at the periphery ofphagosomes containing bacteria indicated that degranulationhad occurred. Thin sections of PMNs that had ingested

TABLE 2. Degranulation by bovine PMNs incubated withsmooth and rough strains of B. abortus

% of control levela of:Organism and strain MPO Lactoferrin

B. abortus 45/0 67.5 89.0(smooth-intermediate)

B. abortus 45/20 68.8 95.5(rough)

S.epidermidis 36.2 30.0a Control levels are those in PMNs incubated without bacteria.

either B. abortus or S. epidermidis had a similar appearanceat 30 min, yet at 120 min, extensive degranulation wasobserved only in human PMNs incubated with S. epider-midis. There was little evidence of degranulation at 120 minin PMNs that had ingested B. abortus 45/0. These data

3000^ A 40B

z 30-~~~~~

200 zo i'*: 4 9

a.

15 30 t0 901b Sb Sbolime (min) rime (min)

FIG. 4. Exocytosis of MPO and lactoferrin by human PMNsincubated with either S. epidermidis or B. abortus. Symbols: 0, S.epidermidis; A, B. abortus 45/0. Results are representative of threeexperiments, using PMNs from different human donors, with du-plicate data points per experiment.

INFECT. IMMUN.

Al

ALr- Aft

on April 30, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

PMNs AND B. ABORTUS 229

suggested that the smooth strain of B. abortus was readilyingested, but that degranulation was inhibited or not stimu-lated after ingestion.The relative extent of degranulation by PMNs that had

ingested either B. abortus 45/0 or S. epidermidis was deter-mined by (i) measuring amounts of granule enzymes releasedby exocytosis and (ii) isolating intact granules by differentialcentrifugation of phagocyte homogenates after incubationwith bacteria. During ingestion, lysosomes fuse with in-completely formed phagosomes and release some of theircontents into the surrounding medium (20, 35). The amountsof granule proteins released by exocytosis correlate withamounts of ingestion and degranulation. With MPO andlactoferrin as markers of azurophil and specific granules,respectively, the results indicated that the release of MPOby exocytosis was four- to fivefold greater by PMNs incu-bated with S. epidermidis than by PMNs incubated with B.abortus 45/0. However, the release of lactoferrin by PMNsincubated with S. epidermidis was only ca. 20% greater thanthat by PMNs incubated with B. abortus 45/0. These dataindicated that more extensive degranulation of azurophilgranules had occurred in PMNs that had ingested S. epi-dermidis, compared with those incubated with B. abortus45/0. However, the difference between the amounts oflactoferrin released by PMNs incubated with B. abortus andby specific granules of PMNs incubated with S. epidermidiswas less than expected, based on results of examination ofthin sections by electron microscopy (35).To ascertain the fate of heavy and light azurophil granules

after ingestion of brucella, and to obtain a better indicationof the extent of granule fusion with phagosomes containingbacteria, we measured degranulation by isolating intactgranules from infected PMNs. After incubation with bac-teria, PMNs were homogenized, followed by fractionation ofgranules on sucrose density gradients and assays for specificmarker proteins. In all experiments, degranulation byazurophil granules and specific granules was less in PMNsincubated with B. abortus 45/0 than in PMNs incubated withS. epidermidis. The lactoferrin content of granules isolatedfrom PMNs after incubation with S. epidermidis was signifi-cantly less than that from PMNs incubated with B. abortus45/0. The amounts of MPO measured in isolated granulefractions of PMNs after incubation with S. epidermidis weregreater than expected based on their appearance and num-bers detected by electron microscopy. These differences arelikely due to the qualitative nature of the electron micros-copy method, since all experiments were performed underconditions in which there was extensive bactericidal activityagainst S. epidermidis.

Since there was limited degranulation by human andbovine PMNs that had ingested the smooth strain of B.abortus, 45/0, it was of interest to determine whether de-granulation might be more extensive in PMNs incubatedwith the rough strain, 45/20. These experiments were per-formed with bovine PMNs due to their higher brucellacidalactivity compared with human PMNs. Results of theseexperiments showed that residual MPO and lactoferrin lev-els were similar after incubation with the two strains of B.abortus. The data suggest that the higher level of intra-cellular survival of B. abortus 45/0 compared with strain45/20 is due not to decreased degranulation, but to the abilityof the bacteria to resist killing reactions within phago-lysosomes. In contrast to human PMNs, degranulation bybovine azurophil granules was more extensive than that bysecondary granules, but considerably less than in PMNsincubated with S. epidermidis. The increased degranulation

by azurophil granules in bovine PMNs may explain theirhigher brucellacidal activity compared with human PMNs.Although it has not been proven directly, several lines of

evidence suggest that Brucella sp. organisms do not stimu-late extensive degranulation after ingestion. (i) There was norespiratory burst when either strain of B. abortus wasincubated with guinea pig PMNs (17); (ii) preincubation ofguinea pig PMNs with each strain of B. abortus did notinhibit the subsequent oxidation of [1-'4C]glucose whenchallenged with heat-killed, S. typhimurium cells (17); (iii)minimal brucellacidal activity was exhibited by human PMNseven though extensive ingestion was observed by electronmicroscopy, and degranulation was not stimulated by glu-taraldehyde-killed B. abortus 45/0 cells. The unique surfaceproperties of Brucella sp. apparently enable the parasite toescape detection. The cell walls of B. abortus 45/0 and 45/20used in this study-strains that differ in virulence andinfectivity-have been extensively characterized with re-spect to proteins, phospholipids, peptidoglycan content, andlipopolysaccharides (16, 18). The smooth strain 45/0 con-tained a phenol-soluble lipopolysaccharide not present in therough strain 45/20; this was the only qualitative differencenoted in the comparison study. More important, the phe-nolic lipopolysaccharide was the only cell wall componenttoxic to mice.The higher level of survival of B. abortus 45/0, compared

with the rough strain 45/20, suggests that the smooth strain ismore resistant to intraleukocytic killing systems. The brucel-lacidal activity of human and bovine granule extracts isgreater against the rough strain 45/20 and is mediated byoxygen-dependent reactions (30a). Thus, intracellular sur-vival of B. abortus appears to be due to more than onemechanism that may be related to cell surfaces of theparasite. It remains to be determined whether the phenol-soluble lipopolysaccharide plays a role in intracellular sur-vival.

ACKNOWLEDGMENTSThis research was supported by the University of Kansas General

Research Fund.We thank R. F. Rest, Hahneman University, Philadelphia, Pa.,

for reviewing the manuscript.

LITERATURE CITED1. Armstrong, J. A., and P. D'Arcy Hart. 1971. Response of

cultured macrophages to Mycobacterium tuberculosis with ob-servations of the fusion of lysosomes with phagosomes. J. Exp.Med. 134:713-740.

2. Babior, B. M. 1978. Oxygen-dependent killing by phagocytes.N. Engl. J. Med. 298:659-668.

3. Badwey, J. A., and M. L. Karnovsky. 1980. Active oxygenspecies and the functions of phagocytic leukocytes. Annu. Rev.Biochem. 49:695-726.

4. Braun, W., A. Pomales-Lebron, and W. R. Stinebring. 1958.Interactions between mononuclear phagocytes and Brucellaabortus strains of different virulence. Proc. Soc. Exp. Biol.Med. 97:393-397.

5. Carrol, M. E. W., P. S. Jackett, V. R. Aber, and D. B. Lowrie.1979. Phagolysosome formation, cyclic adenosine 3':5'-monophosphate and the fate of Salmonella typhimurium withinmouse peritoneal macrophages. J. Gen. Microbiol. 110:421-429.

6. Carson, C. A., D. M. Sells, and M. Ristic. 1975. A method forseparation of bovine blood leukocytes for in vitro studies. Am.J. Vet. Res. 36:1091-1094.

7. DeChatelet, L. R. 1978. Initiation of the respiratory burst inhuman polymorphonuclear neutrophils: a critical review. RESJ. Reticuloendothel. Soc. 24:73-91.

8. Densen, P., and G. L. Mandell. 1980. Phagocyte strategy vs.

VOL. 46, 1984

on April 30, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

230 RILEY AND ROBERTSON

microbial tactics. Rev. Infect. Dis. 2:817-838.9. Fishman, W. H. 1963. 3-Glucuronidase, p. 869-874. In H. U.

Bergmeyer (ed.), Methods of enzymatic analysis. AcademicPress, Inc., New York.

10. Freeman, B. A., and L. Vana. 1958. Host-parasite relationshipsin brucellosis. I. Infection of normal guinea pig macrophages intissue culture. J. Infect. Dis. 102:258-267.

11. Friis, R. R. 1972. Interaction of L cells and Chlamydia psittaci:entry of the parasite and host responses to its development. J.Bacteriol. 110:706-721.

12. Goren, M. N., P. D'Arcy Hart, M. R. Young, and J. R.Armstrong. 1976. Prevention of phagosome-lysosome fusion incultured macrophages by sulfatides of Mycobacterium tuber-culosis. Proc. Natl. Acad. Sci. U.S.A. 73:2510-2514.

13. Jones, T. C., S. Yeh, and J. G. Hirsh. 1972. The interactionbetween Toxoplasma gondii and mammalian cells. II. Theabsence of lysosomal fusion with phagocytic vacuoles contain-ing liver parasites. J. Exp. Med. 136:1173-1194.

14. Klebanoff, S. J. 1975. Antimicrobial mechanisms in neutrophilicpolymorphonuclear leukocytes. Semin. Hematol. 12:117-142.

15. Kossack, R. E., R. L. Guerrant, P. Densen, J. Schadelin, and G.L. Mandell. 1981. Diminished neutrophil oxidative metabolismafter phagocytosis of virulent Salmonella typhi. Infect. Immun.31:674-678.

16. Kreutzer, D. L., C. S. Buller, and D. C. Robertson. 1979.Chemical characterization and biological properties of lipo-polysaccharides isolated from smooth and rough strains ofBrucella abortus. Infect. Immun. 23:811-818.

17. Kreutzer, D. L., L. A. Dreyfus, and D. C. Robertson. 1979.Interaction of polymorphonuclear leukocytes with smooth andrough strains of Brucella abortus. Infect. Immun. 23:737-742.

18. Kreutzer, D. L., and D. C. Robertson. 1979. Surface macro-molecules and virulence in intracellular parasitism: comparisonof cell envelope components of smooth and rough strains ofBrucella abortus. Infect. Immun. 23:819-828.

19. Leffell, M. S., and J. K. Spitznagel. 1972. Association oflactoferrin with lysozyme in granules of human polymorphonu-clear leukocytes. Infect. Immun. 6:761-765.

20. Leffell, M. S., and J. K. Spitznagel. 1974. Intracellular andextracellular degranulation of human polymorphonuclearazurophil and specific granules induced by immune complexes.Infect. Immun. 10:1241-1249.

21. Lowrie, D. B., V. R. Aber, and P. S. Jackett. 1979. Phagosome-lysosome fusion and cyclic adenosine 3':5'-monophosphate inmacrophages infected with Mycobacterium microti, Mycobac-terium bovis BCG or Mycobacterium lepraemurium. J. Gen.Microbiol. 110:431-441.

22. Lowrie, D. B., P. S. Jackett, and N. A. Ratcliffe. 1975. Mycobac-terium microti may protect itself from intracellular destructionby releasing cyclic AMP into phagosomes. Nature (London)

254:600-602.23. Macrae, R. M., and H. Smith. 1964. The chemical basis of the

virulence of Brucella abortus. VI. Studies on immunity andintracellular growth. Br. J. Exp. Pathol. 45:595-603.

24. Mancini, G., A. 0. Carbonara, and J. F. Heremans. 1965.Immunological quantitation of antigens by single radial im-munodiffusion. Immunochemistry 2:235-254.

25. McGhee, J. R., and B. A. Freeman. 1970. Osmotically sensitiveBrucella in infected normal and immune macrophages. Infect.Immun. 1:146-150.

26. Morris, J. A. 1977. The interaction of Brucella abortus 544 andneutrophil polymorphonuclear leukocytes. Ann. Sclavo19:143-150.

27. Querinjean, P., P. L. Masson, and J. F. Heremans. 1971.Molecular weight, single-chain structure and amino acids ofhuman lactoferrin. Eur. J. Biochem. 20:420425.

28. Quie, P. G. 1983. Perturbation of the normal mechanisms ofintraleukocytic killing of bacteria. J. Infect. Dis. 148:189-193.

29. Rest, R. F., M. H. Cooney, and J. K. Spitznagel. 1977. Suscep-tibility of lipopolysaccharide mutants to the bactericidal actionof human neutrophil lysosomal fractions. Infect. Immun.16:145-151.

30. Rest, R. F., and J. K. Spitznagel. 1977. Subcellular distributionof superoxide dismutases in human neutrophils. Influence ofmyeloperoxidase on the measurement of superoxide dismutaseactivity. Biochem. J. 166:145-153.

30a.Riley, L. R., and D. C. Robertson. 1984. Brucellacidal activityof human and bovine polymorphonuclear leukocyte granuleextracts against smooth and rough strains of Brucella abortus.Infect. Immun. 46:231-236.

31. Smith, H., and R. B. Fitzgeorge. 1964. The chemical basis of thevirulence of Brucella abortus. V. The basis of intracellularsurvival and growth in bovine phagocytes. Br. J. Exp. Pathol.45:174-186.

32. Spitznagel, J. K. 1975. Mechanisms of killing by poly-morphonuclear leukocytes, p. 209-214. In D. Schlessinger (ed.),Microbiology-1975. American Society for Microbiology, Wash-ington, D.C.

33. Spitznagel, J. K., F. G. Dalldorf, M. S. Leffell, J. D. Folds, I. R.H. Welsh, M. H. Cooney, and L. E. Martin. 1974. Character ofazurophil and specific granules from human polymorphonuclearleukocytes. Lab. Invest. 30:774-785.

34. Worthington Biochemical Corp. 1972. Worthington enzymemanual, p. 4345. Worthington Biochemicals Corp., Freehold,N.J.

35. Wright, D. G., and S. E. Malawista. 1972. The mobilization andextracellular release of granular enzymes from human leuko-cytes during phagocytosis. J. Cell Biol. 53:788-797.

36. Wyrick, P. B., and E. A. Brownridge. 1978. Growth of Chla-mydia psittaci in macrophages. Infect. Immun. 19:1054-1060.

INFECT. IMMUN.

on April 30, 2019 by guest

http://iai.asm.org/

Dow

nloaded from