JOURNAL OF BACTERIOLOGY, Sept., 1966 Vol. 92, No. 3Copyright @ 1966 American Society for Microbiology Printed in U.S.A.

Mechanisms of Pathogenesis inListeria monocytogenes Infection

I. Influence of Iron'C. P. SWORD

Department of Microbiology, The University ofKansas, Lawrence, Kansas

Received for publication 15 June 1966

ABSTRACTSWORD, C. P. (The University of Kansas, Lawrence). Mechanisms of patho-

genesis in Listeria monocytogenes infection. I. Influence of iron. J. Bacteriol. 92:536-542. 1966.-The effects of ferric andferrous ironaswell asother cationson Lis-teria infection in mice were studied. Iron compounds caused a reduction in the LD5dose of Listeria, and, when added to a synthetic medium, proved stimulatory for invitro growth of the organism. Bacterial counts on spleen and liver tissue from iron-treated mice showed that iron injections caused more rapid growth of bacteria andresulted in higher numbers of organisms in the tissue. The reticuloendothelial sys-tem did not appear to be impaired by this treatment. Immunized animals were notaffected by iron treatment during challenge. Mice with experimentally inducedhemolytic anemia showed increased susceptibility to listeriosis, whereas thosetreatedwith Desferal, a specific iron-chelating agent, appeared more resistant. Iron provedstimulatory for the avirulent strain, 9037-7, and resulted in an LD50 of 1.3 X 104organisms in iron-treated animals. Growth of L. monocytogenes and mortality fromexperimental infection appeared to be correlated with availability of iron to thebacteria. The results suggest that host iron metabolism may play a part in the onsetand progress of Listeria infections.

Evidence which suggests possible participationof host and microbial iron metabolism in thepathogenesis of microbial diseases has accumu-lated. Conalbumin, a constituent of egg white,was shown by Schade and Caroline (23) to inhibitin vitro growth of many pathogenic and non-pathogenic iron-dependent bacteria by chelatingFe+++. A similar effect was demonstrated for the,B2 iron-binding serum protein, transferrin (22,24). Schade (22) showed that growth of Staphylo-coccus aureus, pigmentation, catalase content, andcertain other metabolic activities were all influ-enced by the availability of iron during growth.Jackson and Burrows (11) found that nonpig-mented mutants of Pasteurella pestis with reducedmouse virulence and inability to proliferate freelyin vivo could be restored to full virulence if sub-lethal amounts of iron were injected with theorganisms. This effect appeared specific for iron.

1 A preliminary report of these data was presentedat the 65th Annual Meeting of the American Societyfor Microbiology, Atlantic City, N.J., 25-29 April1965.

Martin et al. (16) showed that prior or concomi-tant administration of iron salts enhanced thevirulence of strains of Klebsiella pneumoniae andPseudomonas aeruginosa in rats and mice. Injec-tion of small doses of purified iron-free transferrincaused a slight degree of protection against acuteinfections, but could not suppress iron-enhancedinfection with these organisms. It has also beenshown that accumulation of iron in cells of thereticuloendothelial system (RES) occurs subse-quent to hemolytic phenomena, injection of iron,and during some infectious diseases (28).The appearance of increased amounts of hapto-

globin and transferrin in the serum of mice duringthe acute stage of experimental infection withListeria monocytogenes (27) lead us to investigatethe possibility that iron may be involved in thepathogenesis of listeriosis. The present investiga-tion deals with the effect of injected iron com-pounds on mouse virulence of L. monocytogenes,the effect of iron on in vitro growth of L. mono-cytogenes, and the effect of experimental altera-tions in host iron metabolism on in vivo growthand mou$e virulence of Listeria.

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VOL. 92, 1966 INFLUENCE OF IRON IN PATHOGENESIS OF LISTERIOSIS

MATERIALS AND METHODSBacteria. Virulent L. monocytogenes strains A4413,

JHH, and 34S, as well as the avirulent rough strain9037-7, were employed. Strains A4413, JHH, and9037-7 were obtained from the U.S. Army BiologicalLaboratories, Fort Detrick, Md. Strain 34S was aserotype 4b isolate of ovine origin (1). The organismswere maintained on Tryptose Agar (Difco) slantsstored at -20 C. Infecting inocula were prepared bygrowing the organisms in broth containing 2% tryp-tose and 0.5% NaCl for 24 hr. Suitable dilutions wereprepared in the above medium, and the bacteria countof the inoculum was determined by quantitativeplating on Tryptose Agar. Inocula employed intesting for in vitro stimulation by iron were grown inthe synthetic medium described by Welshimer (29)containing salts, amino acids, and vitamins, andwere washed once in deionized distilled water prior touse.

Animals. White female Swiss Webster mice (CD-1strain, pathogen-free, from the Charles River Farms,North Wilmington, Mass.) weighing 18 to 20 g wereused in all experiments. Animals were infected by theintraperitoneal (ip) route in 0.2-ml volumes. Blood forhematocrit determinations was obtained by cardiacpuncture. Immunized mice received approximately 103viable organisms of strain A4413 followed in 6 days by106 viable organisms of the same strain. Experimentswere performed 14 days after the second injection.

Treatment with iron salts and other cations. Thefollowing cations were tested for their effect on viru-lence: Fe+++ (ferric ammonium citrate), Fe++ (FeSO4),Mn++ (MnSO4), Mg++ (MgSO4), Ca++ (CaCl2), Na+(NaCl). Solutions were prepared to contain 400 /Ag/mlfor each cation and were sterilized by autoclaving at121 C for 15 min, except Fe++, which was filter-sterilized. Animals received three injections by the iproute, each injection containing 80 ,g of the respectivecations. The first injection was given 24 hr prior toinfection, the second on the day of infection, and thethird 24 hr after infection. Injection of 1,000 ,g of ironin incomplete Freund's adjuvant by the subcutaneous(sc) route 24 hr prior to infection appeared to producecomparable results. Groups of six mice received eachdilution of Listeria. The animals were observed for 10days after infection, and LD50 values were determinedby the method of Reed and Muench (21). The doses ofcations employed caused no deaths in noninfectedcontrols.

Antimouse erythrocyte serum. Rabbit antimouseerythrocyte serum was prepared by injecting a salinesuspension of washed pooled mouse erythrocytes bythe intravenous, sC, and ip routes at weekly intervals.The titer of hemagglutinins was determined by prepa-ration of twofold serial dilutions of serum in 0.5 ml ofsaline and adding 0.5 ml of a 1% suspension of mouseerythrocytes to each tube. Titrations were incubated atroom temperature for 2 hr, followed by an additionalovernight period at 4 C. The highest dilution of serumresulting in macroscopic agglutination was defined ascontaining 1 hemagglutinating unit (HU). The seraemployed in this study contained 320 HU/ml.

Hemolytic anemia. Hemolytic anemia was producedin mice prior to infection by injection of phenylhydra-

zine and by injection of rabbit antimouse erythrocyteserum as described by Kaye and Hook (13). Hemolyticanemia was produced by ip injection of 2.5 mg ofphenylhydrazine hydrochloride in 0.1 ml of distilledwater 24 hr prior to infection. Hemolytic anemia wasalso produced by ip injection of 0.1 ml of antimouseerythrocyte serum diluted in saline to contain 16 HU/dose 24 hr prior to infection. Control groups receivingsaline and comparable dilutions of normal rabbitserum in saline were included. Groups of 10 animalsper dilution of inoculum were infected, and LD50values were determined by the method of Reed andMuench (21).

Effect ofiron on in vitro growth. Bacteria prepared aspreviously described were inoculated in 0.5-mlvolumes containing approximately 106 organisms into50 ml of synthetic medium (29) supplemented withconcentrations of iron ranging from 0.1 to 100 ,g/ml.Nephelo-Culture flasks (Bellco Glass, Inc., Vineland,N.J.) were employed for these cultures. Cultures wereincubated at 37 C. Optical density readings were re-corded periodically at 620 m, on a Bausch & LombSpectronic-20 colorimeter. The medium contained theamino acid mixture described by Welshimer (29)rather than the alternate acid-hydrolyzed casein. Thebasic formula did not contain iron, except as a possiblecontaminant in the reagents employed. For someexperiments, the medium was absorbed with 8-hy-droxyquinoline as described by Nicholas (19) to re-move iron contamination contributed by the compo-nents of the medium. Slightly lower control valueswere obtained with the absorbed medium.

Bacteria in tissue of nontreated infected and iron-treated infected mice. Groups of mice receiving varyingdoses of Listeria were killed periodically throughoutthe experiment, and the livers and spleens wereaseptically removed. Pooled tissue from five animalswas homogenized by grinding with sterile sand andTryptose Broth (Difco) in a mortar and pestle. Dilu-tions of the homogenates from each infecting dosewere plated on Tryptose Agar, and the colonies werecounted after incubation at 37 C for 48 hr to determinethe number of bacteria per gram of tissue at differentstages of infection.

Circulatory clearance experiments. Groups of fiveiron-treated and five nontreated mice were injected inthe tail vein with approximately 108 viable cells ofListeria strain A4413 contained in 0.1 ml of saline.Blood samples were collected immediately and 40 minafter injection by cutting the tail and collecting 20Mliters of blood in a heparinized micropipette. Thesewere diluted in saline and plated on Tryptose Agar;colony counts were then done to determine relativeclearance of L. monocytogenes from the circulation iniron-treated and nontreated mice.

Peritoneal clearance experiments. Groups of fouriron-treated and four nontreated mice were injectedip with approximately 108 viable cells of Listeria strainA4413 contained in 0.1 ml of saline. Four iron-treated and four nontreated animals were killed im-mediately and the same number 40 min after injection.The peritoneum was washed with 2.5 ml of Hank'sbalanced salt solution. Peritoneal washings were cen-trifuged at 225 X g for 1 min to remove host cells and

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debris. The supernatant fluid which contains Listeriacells not removed by phagocytosis was diluted insaline and plated on Tryptose Agar; colony countswere done to determine relative clearance of L. mono-cytogenes from the peritoneum in iron-treated andnontreated mice.

Iron chelation. Desferal (desferrioxamine B methanesulfonate; CIBA, Summit, N.J.), a specific iron-chelating agent used for treatment of pathological irondeposition, was employed to deplete mice of iron be-fore and during infection. Desferal was dissolved iniron-free saline and given in 0.2-ml doses by the scroute (three injections per day, 5 mg per injection),starting 3 days prior to infection and continuing 5 daysafter infection.

Plasma and tissue iron. Plasma iron was determinedby the method of Schade et al. (25) on pools of hep-arinized plasma from the opthalmic venous plexus of10 to 15 mice. Tissue iron was determined by themethod of Schade et al. (25) on livers and spleens pre-pared by wet washing with HNO3-HCLO4 accordingto the method of Ballantine and Burford (3), and wasneutralized with NaOH prior to assay.

RESULTSEffect of iron compounds on virulence. Table 1

shows a comparison of the LD50 dose of Listeriastrain A4413 for mice under various conditionsof treatment with cations. Treatment with iron,either Fe++ or Fe++, and manganese reduced theLD50 dose. In four repeated experiments with iron-treated and nontreated animals, the mean LD5o(4 SD) was 1.5 (±t0.8) X 102 for Fe-++treated,0.5 (-0.4) x 10 for Fe++-treated, and 2.9 (i2.4)X 103 for nontreated mice. Similar results wereobtained with strains JHH and 34S. Strain 9037-7,reported by Friedman and Kautter (9) to be aviru-lent, did not cause death in nontreated mice, buthad an LD50 of 1.3 X 104 organisms in iron-treated mice.Hemin was also employed in some preliminary

experiments and caused a similar effect; however,the low solubility under physiological conditions

TABLE 1. LD60 values of Listeria monocytogenesA4413 for mice treated with ferric, ferrous, and

other cations

Treatmente LIDo valueb

None (control) ...................... 7.2 X 103Fe++(FeSO4) ......................... 0.6 X 10Fe+++ (Ferric ammonium citrate) ..... 1.8 X 102Mn (MnSO4) ...................... 3.1 X 10Ca++ (CaCl2) ........................ 2.5 X 103Mg++ (MgSO4) ...................... 1.5 X 104Na+ (NaCI) ......................... 2.5 X 104

a Three injection series: 80-/Ag dose of cation in0.2 ml of saline 24 hr prior to infection, on day ofinfection, and 24 hr after infection.

b LD60 values calculated by the method of Reedand Muench (21).

caused accumulation of crystalline hemin andprobable blockade of the RES when injected indoses containing iron concentrations comparableto those employed for other compounds. There-fore, further tests with hemin were not attempted.Table 2 shows the result of iron treatment of

immunized animals during challenge. The resis-tance acquired by immunization with livingorganisms was not overcome as a result of irontreatment.

Effect of iron compounds on in vitro growth ofbacteria. A stimulatory effect proportional to ironconcentration was noted in synthetic mediumsupplemented with varying levels of iron (Fig. 1).The results of plate counts were consistent withthe turbidimetric results presented. Fe+,++ (ferricammonium citrate) and Fe++ (ferrous sulfate)caused similar growth curves. Insolubility ofFeSO4 in the medium at concentrations above 10Ag/ml of ferrous ion prevented the use of higherFe++ concentrations. Similar results were ob-served with strains A4413, JHH, 34S, and theavirulent strain 9037-7.

Because of its ability to alter the LD5o dose,Mn+ was added to the synthetic medium to de-termine its effect on in vitro growth of Listeria.Concentrations of 0.1, 1.0, and 10 ,ug/ml ofMnHmarkedly inhibited growth in the medium.

Growth of bacteria in iron-treated mice. Figure2 shows relative bacteria counts in liver tissue ofmice receiving 6.0 X 10 cells of Listeria strainA4413. A similar effect was consistently observedin both liver and spleen tissue of mice receivingdifferent levels of Listeria infection ranging from105 to 10 organisms. The organisms appeared togrow more rapidly in both liver and spleen oftreated animals, regardless of the infecting dose.The maximal number of organisms occurring intissue of treated animals receiving comparableinfecting doses was consistently greater than thatin nontreated infected animals.

Figure 3 shows the results of a similar experi-

TABLE 2. Effect of immunization on iron-enhancedinfectioni with Listeria moniocytogenes

A4413

Treatment' LD6o valueb

None (control).2.5 X 103Fe++-treated................... 3.5 X 10Immunized..................... 1.8 X 106Fe++-treated immunized ........ 4.9 X 106

a Three injection series: 80-,Ag dose of cation in0.2 ml of saline 24 hr prior to infection, on day ofinfection, and 24 hr after infection.

b LD50 values calculated by the method of Reedand Muench (21).

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INFLUENCE OF IRON IN PATHOGENESIS OF LISTERIOSIS

140

° 1200

x

:300Elo

0c'J

(0

. 80

0

z

o 60

0

(I)

< 40

20L=16 20 24 23 32 36

HOU RS

FIG. 1. Effect of various concentrations ofFe+++ on

in vitro growth cuirves of Listeria monocytogenesA4413.

ment with the avirulent strain 9037-7. This strainwas not capable of sustained growth in eitherliver or spleen of nontreated mice, but multipliedin both tissues in iron-treated animals. Althoughstrain 9037-7 multiplied and caused deaths in iron-treated mice, it did not reach the same numbersper gram of tissue as the virulent strain.

Circulatory andperitoneal clearance experiments.Bacterial clearance experiments were performedto determine whether the increased in vivo growthand reduction in LD50 dose were the result ofdamage to the RES in iron-treated mice. Nodifferences in either circulatory or peritonealclearance of Listeria cells were noted betweeniron-treated and nontreated animals.

Hemolytic anemia. Hemolytic anemia was in-duced to increase the availability of iron to thebacteria during infection. Within 24 hr afterinjection of 2.5 mg of phenylhydrazide or 16 HUof antimouse erythrocyte serum, the hematocritvalues were reduced. Injection of saline or com-parable dilutions of normal rabbit serum did notappreciably alter the hematocrit values of controlanimals. Varying dilutions of Listeria were in-jected into groups of animals with hemolyticanemia induced by injection of phenylhydrazine

LIVER TISSUEO IRON TREATED (Fe++)A - UNTREATED

LISTERIA INOCULUM:60 ORGANISMS- STRAIN A4413

w

I5-

0

m4 2

0

2

2 3 4 5

TIME (Days post infection)

FIG. 2. Growth of Listeria monocytogenes A4413 inliver of iron-treated and nontreated mice.

9

LIVER TISSUE8 - o - IRON TREATED (Fe*)

a- UNTREATED

LISTERIA INOCULUM:7 r 4 x 103 ORGANISMS- STRAIN 9037-7

< 6-

5

0 4

0

.J3

2 3 4

TIME (Days post infection)

5

FIG. 3. Growth of Listeria monocytogenes 9037-7 inliver of iron-treated and nontreated mice.

or antimouse erythrocyte serum. Mice with ane-mia induced by either method reacted with in-creased susceptibility to infection (Table 3).

Effect of Desferal treatment on infection. Des-feral was used to deplete iron before and duringinfection. Desferal treatment appeared to have a

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TABLE 3. LD60 values of Listeria monocytogenesA4413 for mice with hemolytic anemia

Group Hema- LD5o value"tocrit

Control (not treated) ...... 44.6 3.1 X 104Phenylhydrazine (2.5 mg).. 28.8 3.9 X 10Saline.................... 47.6 8.5 X 104Antimouse erythrocyteserum (16 HU).......... 38.9 0.46 X 10

Normal rabbit serumb...... 46.3 1.2 X 104

LDro values calculated by the method of Reedand Muench (21).

b Dilution comparable to that used for anti-mouse erythrocyte serum.

protective effect, as shown by an increase in LD50dose (Table 4). Iron treatment of mice receivingDesferal reversed this effect and reduced the LD50dose to comparable levels with iron-treated micenot receiving Desferal.

Plasma and tissue iron during infection and irontreatment. Plasma iron decreased during pro-gressive infection (Table 5). Table 6 shows thelevels of iron in the liver and spleen. Although themean hepatic iron values increased during infec-tion, the wide range and large standard errorsmade it difficult to establish with certainty the ex-tent of any changes. Splenic iron remainedessentially the same during infection.

Iron treatment of mice increased the meanhepatic and splenic iron values, but did not affectplasma iron. A wide range of tissue iron values,comparable to those during infection, was ob-served.

DIscussioN

Treatment of mice with Fe++- or Fe+ con-sistently reduced the LD50 values for L. monocyto-genes. This effect was produced by three ip injec-tions in saline rather than a single injection of thesalts in oil, as had been done with P. pestis byJackson and Burrows (11). It was feared that useof oil or other such material might induce an in-flammatory exudate. Oral administration of ironcompounds was attempted in preliminary experi-ments, but the normal mechanisms controllingiron absorption by the intestinal mucosa did notmake this method feasible. Dietary iron is usuallyabsorbed only if the body has a specific need for it;otherwise, it is excreted (5, 7). Massive doses ofiron by the oral route would cause some increasedabsorption, but would be difficult to control.Other cations such as Ca++, Mg+H-, and Nat didnot affect the LD60 dose, or raised it slightly. Mncaused a reduction in LD50 dose much like that of

TABLE 4. Effect of iron chelation with Desferal onthe LD50 of Listeria monocytogenes

A4413

Treatment LD5o valuea

None (control).............. 3.7 X 104Feb.................... 0.8 X 10FeHb and Desferal.......... 1.3 X 102Desferal.................... 1.1 X 106

a LDro values calculated by the method of Reedand Muench (21).

b Three injection series: 80-pg dose of Fe+ in0.2 ml of saline 24 hr prior to infection, on day ofinfection, and 24 hr after infection.

TABLE 5. Plasma iron levels during infection withListeria monocytogenes A4413

Hr after infection Plasma ironb

0..........................

24..........................48..........................72..........................

237 ±t 6 (3)163 ±t 9 (5)162 ±t 4 (5)130 ±t 4 (3)

Control (not infected) ...... 233 ± 8 (5)a The infecting dose consisted of 105 organisms.b Mean + SE; parentheses indicate number of

determinations each performed on pooled plasmafrom 10 to 15 mice.

TABLE 6. Iron levels in mice during infection withListeria monocytogenes A4413

Liver SpleenHr afterinfectio& No. of Ironb (pg of No. of Ironb (,.g of

animals Fe/g) animals Fe/g)

0 13 85 ± 8.3 11 101 ± 6.6(45-130) (74-145)

24 11 80 it 10.0 12 98 ±i 7.2(30-120) (66-137)

48 12 101± 7.8 13 99 ±+ 8.0(50-135) (58-145)

72 12 108 ±W 7.8 12 91 ±4 9.3(82-162) (60-158)

a Infecting dose consisted of 105 organisms.b Mean ± SE; the range of values is given in

parentheses.

iron, but was inhibitory to growth of Listeria invitro. The concentration of Mn++ in tissue is nor-mally not of the same magnitude as iron. Thehuman body contains 160 ,ug of Mn § per kg oflean tissue (6), whereas it contains 50 to 60 mg ofiron per kg (8). Manganese has been shown to betransported in the body by a j3 globulin, possibly

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transferrin (6), and, therefore,, may have beeninterfering with iron metabolism in a mannerfavorable to the bacteria, perhaps by replacingiron in transferrin.

Iron has been shown to exert a stimulatoryeffect on in vitro growth of Listeria. The site ofincorporation or utilization within the bacterialcell was not determined. Keeler and Gray (14)showed incorporation of Fe59 into cell wall frac-tions contaminated with cell membrane and intothe protoplasmic fraction of L. monocytogenes. Itis probable that iron is being incorporated intothe cytochromes usually associated with the bac-terial cell membrane and into catalase. In experi-ments to be reported in a later paper, a majorportion of Fe69 from the medium was incorpo-rated into the cells during the initial stages ofgrowth and reappeared in the medium during thelogarithmic phase of growth (Jackson and Sword,unpublished data).

L. monocytogenes grew more rapidly and to agreater extent in iron-treated than in nontreatedmice. This is suggestive evidence that death inthese animals was through mechanisms associatedwith the increased bacterial population ratherthan production of factors having pronouncedtoxic activity. If iron induced or was needed forincorporation into a toxic factor, it would be ex-pected that deaths would have occurred muchearlier and with considerably lower bacterialcounts than in the nontreated groups. This hy-pothesis is consistent with the increased virulenceof the supposedly avirulent strain, 9037-7, in iron-treated mice, and stimulation of growth in iron-supplemented media (Fig. 2).

L. monocytogenes is a facultative intracellularparasite, and immunity to listeriosis appears tobe due, totally or in great part, to cellular ratherthan humoral factors (1, 15). Iron-treated micehad a level of resistance to challenge comparableto nontreated immunized animals. Phagocyticcells are known to remove iron from plasma andto serve as storage depots for iron in the form offerritin (28). Listeria cells have been observed tobe surrounded by ferritin within phagosomes ofmouse spleen macrophages (2). If we assume thatiron exerts a stimulatory effect during intracellulargrowth of Listeria, and that "immune" phago-cytes destroy the organism rapidly, as shown byMackaness (15), then the organisms would per-haps not have time to utilize iron stores of the"immune" cell before they are destroyed.

Phenylhydrazine and antimouse erythrocyteserum increased susceptibility to listeriosis. Theresults are similar to those reported by Kaye andHook (13) with Salmonella typhimurium. How-ever, these authors suggest that phagocytosis ofaltered erythrocytes by reticuloendothelial cells

impairs their capacity to kill ingested bacteria. Ithad been shown previously by others that injectionof particulate material may cause stimulation ofphagocytic activity by the RES and that pretreat-ment of rats with albumin-globulin aggregates,produced by heating, causes a stimulation of theRES capable of protecting animals against experi-mental infection with S. typhimurium (4). Hemo-lytic anemia caused by phenylhydrazine intoxica-tion is known to cause a rise in serum iron levelswithin 3 to 5 hr after injection (8). It is also es-tablished that inflammation and infection maycause fixation and accumulation of iron in thecells of the RES (8, 28). Thus, increased availa-bility of iron rather than decreased bacterialdestruction resulting from damage to the RESmay be responsible for the increased susceptibilityin animals with hemolytic anemia.

Desferal has been shown to remove iron fromtransferrin and to deplete iron stores in tissue (18).This compound appeared to make mice more re-sistant to Listeria infection, perhaps by limitingthe growth of the organisms and by allowing thehost defenses time to overcome the bacteria.The decrease observed in plasma iron during

progressive infection may be a reflection of ironfixation in tissue. The mean values obtained forhepatic iron are suggestive of hepatic iron ac-cumulation during infection. The wide range ofvalues for tissue iron was confirmed in other ex-periments and is consistent with iron values re-ported by others in rabbit liver (26).

L. monocytogenes produces a soluble hemolysinwith properties similar to streptolysin 0 (10, 12,20). The hemolysin is reported to be nontoxic forintact animals, lytic for leukocytes, and to pro-duce opacity in egg-yolk medium, lecithovitellin,or serum. Destruction, in vivo, of erythrocytes orleukocytes could increase the amount of ironavailable to the bacteria, and thereby enhancethe infection. The effect of purified hemolysin onlevels of serum iron, hemoglobin, transferrin,and haptoglobin has not yet been investigated.

Endocrine disorders can have a pronouncedeffect on iron metabolism and erythropoiesis (8).Direct and indirect stimulation of the adrenalcortex is reported to cause elevated serum ironvalues. Cortisone was reported by Miller andHedberg (17) to cause a reduction in LD50 of L.monocytogenes from 104 or 105 to less than 50bacteria. Although cortisone may also cause othereffects which would lower resistance of the animal,its effect on iron metabolism is consistent withinvolvement of iron in the pathogenesis of lis-teriosis. If availability of iron in host tissue isinvolved in pathogenesis of naturally occurringlisteriosis, then stress and other factors whichaffect endocrine balance may influence suscep-

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tibility to listeriosis through induction of changesin iron metabolism.

ACKNOWLEDGMENTSThis investigation was supported by Public Health

Service grant AI-04343 from the National Institute ofAllergy and Infectious Diseases, and by The Universityof Kansas General Research Fund.The excellent technical assistance of Kristine

Aldrich and Sondra Frye is gratefully acknowledged.The author is also indebted to W. F. Westlin, Jr., ofCIBA Pharmaceutical Co., Summit, N.J., for theDesferal (desferrioxamine B methane sulfonate) usedin this study.

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Cellular resistance in listeriosis. J. Infect.Diseases 114:258-264.

2. ARMSTRONG, B. A., AND C. P. SWORD. 1966.Electron microscopy of Listeria mon1ocytogenies-infected mouse spleen. J. Bacteriol. 91:1346-1355.

3. BALLENTINE, R., AND D. D. BURFORD. 1957.Determination of metals (Na, K, Mg, Ca, Mn,Fe, Co, Cu, Zn), p. 1002-1035. In S. P. Colo-wick and N. 0. Kaplan [ed.], Methods inenzymology, vol. 3. Academic Press, Inc., NewYork.

4. BIozzI, G., B. N. HALPERN, B. BENACERRAF, AND

C. STIFFEL. 1957. Phagocytic activity of thereticulo-endothelial system in experimental in-fections, p. 204-225. In Physiopathology of thereticulo-endothelial system. Blackwell Scien-tific Publications, Oxford.

5. CONRAD, M. E., AND W. H. CROSBY. 1963. In-testinal mucosal mechanisms controlling ironabsorption. Blood 22:406-415.

6. COTZIAS, G. C. 1961. Manganese versus mag-nesium: Why are they so similar in vitro and so

different in vivo. Federation Proc. 20(Suppl.10, Part II): 98-103.

7. CROSBY, W. H. 1963. The control of iron balanceby the intestinal mucosa. Blood 22:441-449.

8. DEMULDER, R. 1958. Iron. Metabolism, biochem-istry, and clinical pathological physiology-Review of recent literature. A.M.A. Arch.Internal Med. 102:254-301.

9. FRIEDMAN, M. A., AND D. A. KAUT-rER. 1962.Effect of nutrition on the respiratory virulenceof Listeria monocytogenes. J. Bacteriol. 83:456-462.

10. GIRARD, K. F., A. J. SBARRA, AND W. A. BARDA-WIL. 1963. Serology of Listeria molnocytogenes.I. Characteristics of the soluble hemolysin. J.Bacteriol. 85:349-355.

11. JACKSON, S., AND T. W. BURROWS. 1956. Thevirulence-enhancing effect of iron on non-pigmented mutants of virulent strains ofPasteurella pestis. Brit. J. Exptl. Pathol. 37:577-583.

12. JENKINS, E. M., A. N. NJOKU-OBI, AND E. W.ADAMS. 1963. Purification of the soluble hemol-ysins of Listeria monocytogenes. J. Bacteriol.88:418-424.

13. KAYE, D., AND E. W. HOOK. 1963. The influence ofhemolysis or blood loss on susceptibility toinfection. J. Immunol. 91:65-75.

14. KEELER, R. F., AND M. L. GRAY. 1960. Antigenicand related biochemical properties of Listeriamoniocytogeines. J. Bacteriol. 80:683-692.

15. MACKANESS, G. B. 1962. Cellular resistance to in-fection. J. Exptl. Med. 116:381-406.

16. MARTIN, C. M., J. H. JANDL, AND M. FINLAND.1963. Enhancement of acute bacterial infectionsin rats and mice by iron and their inhibition byhuman transferrin. J. Infect. Diseases 112:158-163.

17. MILLER, J. K., AND M. HEDBERG. 1965. Effects ofCortisone on susceptibility of mice to Listeriamoniocytogenies. Am. J. Clin. Pathol. 43:248-250.

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