INFECTION AND IMMUNITY, Mar. 1978, p. 755-7620019-9567/78/0019-0755$02.00/0Copyright i 1978 American Society for Microbiology

Vol. 19, No. 3

Printed in U.S.A.

Resistance and Susceptibility of Mice to Bacterial Infection:Genetics of Listeriosis

CHRISTINA CHEERS* AND IAN F. C. McKENZIE2Department ofMicrobiology, University ofMelbourne, Parkville, 3052,' and Department ofMedicine, Austin

Hospital, Heidelberg, 3084,2 AustraliaReceived for publication 21 September 1977

A survey of various strains of mice showed distinct differences in resistance orsusceptibility to Listeria monocytogenes. C57B1, related sublines, NZB, and SJLwere resistant to Listeria, whereas BALB/c, CBA, A, DBA/1, C3H, LP.RIII, 129,andWB were susceptible. The gene(s) responsible for resistance and susceptibilityto Listeria were studied in detail. C57B16/6, BMO.D2, and B1O.A mice were 100times more resistant than were BALB/c, CBA, and A. Resistance of the (C57B1/6x BALB/c)F1 was intermediate between the two parents, suggesting partialpenetration of a dominant gene. Backcross studies in which the (C57B1/6 xBALB/c)F, were crossed with the susceptible BALB/c parent suggested that asingle gene or group of linked genes were the major determinant of resistance,although the possibility that other genes exerted a modifying influence was notexcluded. By using the backcross and various congenic and recombinant mice,linkage of the genes involved to the H-i, H-2, H-3, H-4, H-7, or H-8 loci, to theimmunoglobulin allotype, to the Thy-i gene, to the Hc gene specifying C5, or tocoat color genes (B, c) was excluded. There was no difference in the response ofmales and females. In all studies, the powerful overriding influence of the C57Blgenome was evident.

The availability of inbred strains of mice, in-cluding a wide variety of congenic, recombinant,recombinant inbred, and mutant strains, hasprovided ideal tools with which to study thegenetic basis of the immune response. Muchattention has recently centered on the use ofsuch strains to define the immune responsegenes linked to the major histocompatibility (H-2) complex that control the antibody or delayedhypersensitivity response to a variety of anti-gens, mostly simple polypeptides, but also in-cluding some viral and bacterial antigens (2, 11,23). Genes linked to the immunoglobulin allo-type locus have also been shown to influence theimmune response to certain antigens (12, 19). Byusing other approaches, inbred strains of micehave been developed that are selectively resist-ant or susceptible to several bacterial or viralinfections (6, 24). There are many reports indi-cating that mice and other species (11, 12) varyin their responses to different infectious agentsand that these responses are genetically deter-mined. Such differences are often multigenic.While extensive studies have been made on thegenetics of antiviral resistance, few attemptshave been made to define the precise geneticsand mechanisms of variation to bacteria.

In aiming to examine the genetics of cell-me-diated, rather than humoral, resistance to a bac-

terial infection, we chose Listeria monocyto-genes as an ideal model organism. Intravenouslyinjected Listeria are rapidly phagocytosed bythe macrophages of the liver and spleen. Ac-quired immunity to listeriosis rests on macro-phage activation by stimulated T lymphocytes(10). Antibody is not demonstrable during pri-mary infection (18), nor does it confer immunityon passive transfer (16).We have, therefore, taken advantage of the

known differences in the responsiveness ofinbred strains of mice to L. monocytogenes (15)to study both the genetics and variations inmechanisms of resistance to this organism. Inthis paper, we report on the genetic aspects ofresistance and susceptibility to L. monocyto-genes, and, in subsequent studies (4), use hasbeen made of these defined genetic differencesto study mechanisms at the cellular level.

MATERIALS AND METHODS

Mice. The types of inbred mice used are listed inthe tables. They were maintained in either the labo-ratories at the Austin Hospital or the MicrobiologyDepartment by strict brother-sister mating. The micewere mostly derived from the Jackson Laboratory,Bar Harbor, Me. (from the colonies of M. Cherry, G.Snell, D. Bailey) or from J. Stimpfling, McLaughlinResearch Institute, Great Falls, Mont.; H. McDevitt,Department of Medicine, Stanford University, Stan-

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756 CHEERS AND McKENZIE

ford, Calif.; D. Shreffler, Department of Genetics, Uni-versity of Missouri, St. Louis, Mo.; or from the Walterand Eliza Hall Institute, Melbourne, Australia.

Bacteria. L. monocytogenes was originally ob-tained from a fresh human isolate. Listeria was sub-cultured not more than 50 times on horse blood agarbefore the culture was renewed from a fresh lyophi-lized ampoule. No loss of virulence occurred, and the50% lethal dose (LD,5o) values remained constantthroughout the study.

Infection of mice. Twenty-four-hour activelygrowing cultures were washed from their plates with1% horse serum in distilled water. The inoculum wasstandardized turbidometrically by using an EEL col-orimeter and injected intravenously. The dose waschecked retrospectively by using Miles and Misra (17)viable counts.

LD5, determinations. Groups of five mice eachwere injected intravenously with fivefold increasingdoses of L. monocytogenes. They were observed over14 days, after which no more deaths occurred. TheLD.5o was calculated by the method of Reed andMuench (22).Determination of H-2 haplotype in the back-

cross ([C57B1/6 x BALB/cJF1 x BALB/c). In thisbackcross there was segregation of H-2b specificities.The mice were accordingly typed for H-2.33, the pri-vate H-2KY specificity. The antiserum was producedas (B10.2D2 x A)F1 anti-B1O.A (5R) as describedelsewhere (14). The typing was performed on thespleen of the backcross mice, removed as soon aspossible after the death of the susceptible mice, orfrom the resistant mice at the conclusion of the exper-iment. The spleens were frozen (-20'C) in saline andlater tested by an absorption technique. Thus, thespleens were passed through a sieve, washed twice insaline, and then exposed for 30 min at 4VC to 0.1 ml ofthe anti-H-2 serum at a 1/50 dilution. The serum wassubsequently titrated on C57BI/6 spleen cells by usinga cytotoxicity test and rabbit complement (14). Usingthis method, it was possible to distinguish between H-2.33+ = H-2b/H-2c mice and H-2.33- = H-2d/H-2d micein the backcross.Determination of immunoglobulin allotype

in the backcross ([C57BI/6 x BALB/cJFi xBALB/c). The immunoglobulin allotyping was per-formed by Noel Warner, Walter and Eliza Hall Insti-tute, Parkville, Melbourne, Australia, as describedelsewhere (8).

RESULTSLD5o of Listerw in C57B1/6, BALB/c,

CBA, A, B1O.D2, and B1O.A strains and in(C57B1/6 x BALB/c)F1. The LD50 values ofListeria in a variety of mouse strains were de-termined (Table 1). The C57B1/6, BlO.A, andBMO.D2 were highly resistant when comparedwith BALB/c, A/J, and CBA, with a 100-folddifference in their LD50. The resistance of(C57B1/6 x BALB/c)F1 hybrids was intermedi-ate between the two parental strains.Strain distribution of resistance and sus-

ceptibility to Listeria. The resistance or sus-ceptibility to Listeria of a number of different

TABLE 1. LD50 of Listeria for several mouse strainsMouse strain LDr,

C57B1/6 9.0 x 105B10.D2 2.2 x 105B10.A 2.2 x 105BALB/c 3.9 x 103CBA 5.0 x 103A/WySn 8.0 x 103(C57Bl/6 x BALB/c)F1 3.4 x 104

a Groups of five female mice each were injectedwith graded doses of Listeria organisms, and the LD5owas determined over 14 days.

strains was determined by using a single dose ofbacteria that was capable of distinguishingbetween susceptible BALB/c and resistantC57B1/6 mice on the basis of death or sur-vival. This choice was made after cumulativedeaths of C57B1/6, BALB/c, and (C57B1/6 xBALB/c)FI were plotted over a given range ofdoses (Fig. 1). For example, a dose of 3.8 x 105provided good discrimination between resistantC57B1/6 mice on the one hand and intermediateF1 and susceptible BALB/c on the other, butdid not distinguish between the latter two. Adose of 1.5 x 104 discriminated between BALB/con the one and C57B1/6 and F1 on the other, butdid not distinguish between the latter two. Adose of 7.5 X 104, however, provided good dis-crimination among all three situations: resistant,susceptible, and intermediate. This dose was,therefore, chosen as optimal for the strain sur-vey.Of necessity, experiments were performed at

different times, and the dose varied between 6X 104 and 12 x 104 organisms per animal (ratherthan the ideal 7.5 X 104 dose). However, C57B1/6and BALB/c controls were included as stan-dards in every experiment, and these alwaysbehaved consistently. Table 2 classifies thestrains into susceptible or resistant, based on themean time of death: susceptible, 50% or fewermice surviving day 5; resistant, more than 50%surviving day 14. Although in a number of casesboth male and female mice were tested, theresults shown are for female mice, except in thecases of SJL and A/J, in which only males wereavailable. This comparison was possible sincethe sex of the mice did not influence their resist-ance to Listeria (see below). Resistant mice wereC57BI/6, C57Bl/10, and related sublines andcongenic lines (Table 2), but also included NZBand SJL mice. Susceptible strains were BALB/c,A, CBA, DBA/1, C3H, LP.RIII, WB, and 129.None fell into an intermediate category.Resistance and susceptibility to Liuteria

of backcross mice. The aim of this study was,first, to estimate the number of genes determin-ing resistance and susceptibility in C57B1/6 and

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0ES2.

ES

c1ut

a.

2 4 6 8 10 12 14Time since infection (days)

FIG. 1. Cumulative mortality of C57BI/6 ( - - ), BALB/c ( ), and (C57BI/6 x BALB/c) F1(--- ) mice infected with graded doses of L. monocytogenes. Groups of five mice each were injectedintravenously with the doses of Listeria shown, and deaths were recorded daily over a period of 2 weeks. Nodeaths occurred thereafter.

BALB/c mice and, second, to investigate theirlinkage relationships with such readily deter-mined genes as coat color, H-2 haplotype, andjinmunoglobulin allotype. The dose aimed forwas 104 Listeria, chosen to discriminate betweenthe highly susceptible BALB/c and the inter-mediate resistance of (C57B1/6 x BALB/c)F1mice (Fig. 1). The results of a representativeexperiment (Table 3, experiment a) show that ofthe mice backcrossed onto the susceptibleBALB/c strain, i.e., ([C57B1/6 x BALB/c]Fj xBALB/c)NI mice, 41/75 (55%) were resistant toa dose of Listeria, which killed all BALB/c miceand no F1 mice. Overall, in several experimentsdone at different times, 68/131 of ([C57BI/6 xBALB/c]Fl X BALB/c)NI mice were resistant,very close to the theoretical 50% for one gene.The similarity of time of death of BALB/c andsusceptible backcross mice (Fig. 2) lends furthercredence to the strong suggestion of a single

major gene or group of linked genes determiningresistance. In contrast to these results, the back-cross onto the resistant C57B1/6 strain, i.e.,([C57B1/6 X BALB/c]F1 x C57B1/6)N1 mice,showed 43/45 (95%) surviving a dose of Listeria,which discriminated between BALB/c and(C57B1/6 x BALB/c)F, (Table 3, experiment b).Linkage tests for H-2 haplotype, Ig allo-

type, and coat color (c and B) in the([C57B1/6 x BALB/c) X BALB/c) back-cross (Table 4). In the backcross ontoBALB/c, H-2' was segregating, to produce H-21i/H-2b and H-2d/H-2d mice. In pooled experi-ments, with doses of Listeria ranging from 7 x103 to 3 x 104, 126 mice were satisfactorily typedfor H-2. There was no effect of H-2 on suscepti-bility or resistance to Listeria (Q = 0.03, P >0.05). Thirty-eight mice were allotyped accord-ing to whether they were Ig-la/Ig-lb or Ig-la/Ig-la, and, again, there was no linkage of suscepti-

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TABLE 2. Strain survey for resistance andsusceptibility to Listeriaa

% Mice surviving Mediantime to

5 Days 14 Days (deayh

ResistantC57Bl/6J 100 100C57Bl/lOScSn 100 100B1O.D2/Sn 100 60B1O.A/SnSg 100 60(B1O.A x A)FM 100 100NZB/WEHI 100 80B6.C-H-2d By 100 88SJL/WEHIC 100 100B6.PL (74NS)/Cy 100 90

SusceptibleBALB/cJ 0 0 3CBA/H 0 0 3DBA/lJ 0 0 3C3H.OH/Sf 0 0 3C3H/HeJ 0 0 4C3H.OL/Sf 25 13 4A/JC 0 0 4LP.RIII 40 40 5WB/Re 50 13 5129/J 33 17 5

a Doses of Listeria varied between 6 x 104 to 12 x104 in different experiments. Between 5 and 10 micewere used per group. C57B1/6 and BALB/c controlswere included in each experiment.

I Time taken for 50% of mice to die was noted forsusceptible mice. However, by definition, less than50% of resistant mice died.

c Only male SJL and A/J mice were available. Allothers shown were female.

100A

C

20 it{

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bility or resistance to Ig lb allotype (X2 = 0.004,P > 0.05). In this particular cross, the coat colorgenes for albinism (c) and black (B) could beexamined (25). There was no linkage to albinismin 132 animals examined (X2 = 1.20, P > 0.05).The animals that demonstrated coat color (72 innumber) were either agouti (A) or brown, whichrepresented the segregation and interaction ofthe genes for agouti (A), black (B), and brown(b). There was no linkage of the nonagouti colorwith susceptibility of resistance to Listeria (XI= 0.87, P > 0.05).Resistance and susceptibility ofH-2 con-

genic and recombinant strains. The back-cross data (Table 4) effectively excluded a roleof the H-2 complex in this particular cross. How-ever, in view of the great importance of the H-2complex in many different immune responses,

TABLE 3. Resistance and susceptibility of backcrossmice to Listeriaa

Backcross mice No. died No. survived

Expt aBALB/c 24 0(C57B1/6 x BALB/c)Fi 0 25F, x BALB/c 41 (55%)b 34 (45%)

Expt bBALB/c 1 19(C57B1/6 x BALB/c)F, 18 2F, x C57B1/6 43 (95%) 2 (5%)a Mice were given 7.0 x 103 (experiment a) or 1.3 x 104

Listeria (experiment b) intravenously and observed over 14days.

b X2 Compared with expected 50% = 2.34, not significant.

E

2 4 6 8 10 12 14 STime since infection (days)

FIG. 2. Mice surviving (S) or dying at various times after infection with 7.0 x 10; L. monocytogenes. BALBIc(A), (C57BI/6 x BALB/c)F, (O), and ([C57BI/6 x BALBIc] x BALB/c) backcross (*).

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we examined several different H-2 haplotypesfor resistance or susceptibility to Listeria (Table5). We were able to examine these effects onthree different genetic backgrounds: C57B1/10,A, and C3H. These strains were congenic at theH-2 complex, and a number were also intra-H-2recombinants. Several different haplotypes wereexamined: a, b, d, h, i, k, and s. All the C57BI/10congenic mice were resistant when comparedwith susceptible BALB/c (H-2d), although allexcept B1O.S carried the H-2 region of suscep-tible mice. Thus, on the C57B1/10 background,H-2 genes play little or no role in the resistanceto Listeria. Conversely, when the backgroundwas either A or C3H, the mice were all suscep-

tible. Particularly noteworthy is A.SW, whichhas the H-28 haplotype of the resistant SJL

strain. These results clearly show that loci otherthan H-2 have the strongest influence on theresistance and susceptibility we are studying.Resistance and susceptibility of congenic

strains differing at various non-H-2 loci. Tofurther examine the C57B1/10 genome, variouscongenic strains were examined (Table 6). Thesestrains differ from C57Bl/lOScSn at various non-H-2 histocompatibility loci: H-i, H-3, H-4, H-7,and H-8. They were obtained from crosseswhereby non-H-2 genes from BALB/c, 129, andDBA/2 were placed on a C57BI/10 background.It should be noted that these three strains wereclassed as susceptible to Listeria (Table 2). Allnon-H-2 congenic strains were similar toC57BI/10 in their being resistant, indicating thatthe susceptibility genes were not associated with

TABLE 4. Segregation ofresistance and susceptibility to Listeria with H-2, 1g, and coat color genes in([C57BI/6 x BALB/cIFl x BALB/c)NI backcrossa

Locus Genotype No. of mice No. died No. survived X2 Probability

H-2 H-2 d/H.2d 30 41H dI/H2b 126 25 30 0.03 >0.05

Ig Igla/Igqla 10 11 0.004 >0.05Ig~laIjg~lb 387 10 004 00

Coat colorsAlbinism cc (albino) 132 25 35 1.20 >0.05

Cc (colored) 1321.03>.0

Black bb (nonbrown) 72 22 15 0.87 >0.05Bb (brown) 16 19

a Mice were given between 7 X 103 and 3 x 104 Listeria intravenously and observed over 14 days.b See text for details. This cross involved the segregation of several coat genes for albinism ( c), black ( b), and

brown (B), the latter two occurring as alleles at the same locus (b). These mice were albino (cc) or colored (Cc),as the parental strains were albino (BALB/c) and black (C57Bl/6). In the nonalbino mice, the segregation ofthe brown color was observed.

TABLE 5. Resistance ofH-2 congenic and recombinant strains to ListeriaaH-2 haplotypeb % Mice surviving

Strain BackgroundK I S G D 5 days 14 days

BALB/c BALB/c d d d d d 0 0C57Bl/10 B10 b b b b b 100 100B1O.A B10 k k d d d 100 100B1O.D2 B10 d d d d d 100 100BlO.A(2R) B10 k k/a d ? b 100 100BlO.A(4R) B10 k k/s b b b 100 88BlO.A(5R) B10 b b/d d d d 100 100B1O.BR B10 k k k k k 100 86B1O.S B10 s s s s s 100 100A A k k d d d 0 0A.AL A k k k k d 20 0A.SW A s s s s s 40 10C3H/HeJ C3H k k k k k 0 0C3H.OH/Sf C3H d d d d k 0 0C3H.OL/Sf C3H d d k k k 25 13

a Challenge dose of Listeria was 6 x 104. There were 5 to 10 mice per group.b The regions and subregions of the H-2 haplotype are shown, wherein K, I, S, G, and D refer to regions

within the H-2 complex that are defined by marker loci with known characteristics.

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760 CHEERS AND McKENZIE

the H-1, H-3, H-4, H-7, or H-8 genes of thevarious strains used.

Effect of lack of C5 on resistance andsusceptibility. To study the effect of the Hcgene, we examined the B1O.D2 old and newstrains (Table 7) that carry the Hc and Hc1genes, respectively, Hc being the gene that codesfor C5, a complement component (7). Althoughthe B1O.D2 0/SN mice lack C5, their resistanceto Listeria was as high as was that of B1O.D2and C57B1/6.

Effect of the Thy-i locus on resistanceand susceptibility. Two different congenicpairs of mice were tested. A.AL-Thy-i5 andA.AL-Thy-lb differ at the Thy-I locus as do thesecond pair, C57B1/6 and B6.PL(74NS). In nei-ther case did the Thy-i locus appear to influencethe results, as the Thy-i congenic pair with Astrain background were both susceptible (Table7), and the pair with B6 background were bothresistant.TABLE 6. Resistance to Listeria of congenic strainsdiffering from C57BI/10 at histocompatibility loci

other than H-2aLoci that dif- % Mice surviving

Strain fer fromC57B1/10b 5 days 14 days

BALB/c 0 0C57Bl/10 100 100B10.129 (5M) H-i 100 100BlO.C-H-3c H-3 100 100B1O.C(28NX) H-3 100 100B1O.LP8 H-3 100 100B1O.129 (21M) H-4 100 86B10.C(47N) H-7 100 100B1O.D2(57N) H-8 100 100a Challenge dose of Listeria was 6 x 104. There

were 6 to 10 mice per group.b H-i to H-8 refer to histocompatibility loci with

effects weaker than those of H-2.

TABLE 7. Resistance to Listeria in mice congenic atthe H, (complement C5) locus and Thy-i locia

Back- % Mice survivingStrain Allele Bkoundground 5 days 14 days

BALB/c Hc' BALB/c 0 0BlO.D2.o/Sn Hc° B10 100 100B10.D2n/Sn Hc' B10 100 100A.AL (0) Thy-la A 0 0A.AL (N) Thy-lb A 20 0B6.PL(74NS) Thy- ia B6 100 90C57Bl/6 Thy-lb B6 100 80

a Challenge dose of Listeria was 6 x 104. Therewere 5 to 15 mice per group. Hc is a locus that codesfor C5 (complement component C5). There are twoalleles Hc' and HcO, indicating the presence or absenceof C5. Thy-i (0) codes for antigen present on T cells(thymus-derived lymphocytes).

Effect of sex on resistance and suscepti-bility. In different experiments with several dif-ferent strains of mice of each sex, there was nosignificant effect on the resistance (Table 8)between males and females. In addition, therewas no difference in susceptibility of male andfemale mice in the backcross -nuce studied (datanot shown).

DISCUSSIONC57Bl/6 and A/J mice were previously re-

ported (15) to vary in their susceptibility to L.monocytogenes, and our studies have confirmedand extended this observation (Table 1).C57BI/6 and B10.A, and B10.D2 mice were 100-fold more resistant than BALB/c, CBA, or A/Jto L. monocytogenes. The differences are almostcertainly due to genetic differences betweenthese strains, and we have, therefore, performedextensive studies to determine the number ofgenes involved and their linkage relationshipswith other genes as the first step in analyzingthe mechanisms responsible for the resistanceand susceptibility effects.To define the number of genes involved, the

resistance and susceptibility to Listeria of(C57Bl/6 x BALB/c)F, mice and their back-cross onto the susceptible BALB/c parent wereinvestigated. The resistance of the F1 mice wasintermediate between that of the two parents,indicating either a gene dose effect or interactionof resistance and susceptibility genes from thetwo parents. The backcross mice ([C57BI/6 xBALC/ciFi X BALB/c) were approximately50% susceptible, like the homozygous BALB/cparent, and 50% relatively resistant, like theheterozygous F1 parent, suggesting that only onegene or a group of linked genes was involved.This interpretation is reinforced by the similar-ity in time of death of BALB/c and susceptiblebackcross mice (Fig. 2) (1). The converse back-

TABLE 8. Resistance of male and female mice to L.monocytogenesa

% Mice survivingStrain Sex

5 days 14 days

BALB/c M 0 0F 0 0

CBA M 10 10F 0 0

C57Bl/6 M 100 72F 100 90

C57BI/6-H-2 d M 100 57F 100 87

B10.129 (21M) M 100 87F 100 85

a Challenge dose of Listeria was 5 x 104. There were7 to 11 mice per group.

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cross onto the resistant C57B1/6 parent pro-duced mice that were virtually 100% relativelyresistant, like the F1 hybrid. Further informationon the behavior of resistant and susceptiblebackcross individuals is being sought by usingthe quantitative parameters described in theaccompanying paper (4). Attempts are also un-der way to produce a strain of resistant micecongenic with BALB/c, which, if successful, willprovide stronger evidence of a single gene.

It was important to determine the role of theH-2 complex in the resistance and susceptibilityeffects noted, particularly as this complex andits associated I region affects so many immuneresponses of a multiple and diverse nature. Therole of H-2 was examined in two ways: (i) byserotyping mice of the ([C57B1/6 x BALB/c]Fjx BALB/c) backcross, wherein the H-2b haplo-type of the resistant C57B1/6 was segregatingand (ii) by examining H-2 congenic and recom-binant strains. In neither case was there evi-dence of any influence of the H-2 complex (Ta-bles 1, 4, 5). The H-2" haplotype was not dispro-portionately represented among backcross miceresistant to Listeria. Furthermore, the congenicmice that had various H-2 haplotypes in aC57B1/6 background were uniformly resistant toListeria (Table 5). However, it is possible thatsome type of gene interaction occurs wherebystrong resistance effects (in the C57B1/6 back-ground) mask or override other effects. Prelim-inary attempts to examine the influence of H-2on susceptible C3H and A backgrounds have sofar failed to reveal such an influence. In thisrespect, an interesting comparison is betweenB1O.S and A.SW (Table 5), both of which areH-?, but differ in their resistance.The immune response effects of the H-2 com-

plex are generally confined to antigens present-ing a very limited number of "foreign" determi-nants to the host (11), so it may not be surprisingthat we have so far failed to demonstrate anyinfluence on the response to such a complexantigen as a whole bacterial cell. However, thereare instances of the resistance to viruses beinglinked to H-2 (13), and adoptive transfer of im-munity to Listeria is restricted to interactionsbetween T lymphocytes and macrophages thatare H-2 compatible (26).Immunoglobulin allotype was also examined

in the ([C57B1/6 x BALB/c]Fi x BALB/c)backcross, and, again, no linkage with resistancewas found (Table 4). This and the high resist-ance of BlO.D2.0/Sn mice lacking C5 (Table 7)are compatible with the known mechanisms ofresistance to Listeria, which do not involve an-tibody (16, 18) or, presumably, complement.Lack of C5 would effect both the classical andbypass pathways of complement fixation, al-

though at a point after C3 fixation.Studies with congenic mice failed to demon-

strate any linkage to the minor histocompatibil-ity loci H-1, H-3, H-4, H-7, and H-8 (Table 6).The absence of linkage to H-3 probably excludesa linkage to the Ir-2 locus (5), which controlsproduction of antibodies to mouse Ea-1 eryth-rocyte antigen. Furthermore, the Thy-l locusgoverning the allelic form of antigen on T cellsdid not affect resistance (Table 7). Backcrossstudies revealed no linkage to immunoglobulinallotype or the coat color genes B or c (Table 6).In addition, sex of the mice had no effect onresistance (Table 8). These linkage studies thusinvolved a number of different chromosomes,namely chromosome 2 (H-3 and Hc)1, chromo-some 4 (B), chromosome 7 (c, H-i, and H-4),chromosome 9 (Thy-i), and the as yet uniden-tified chromosomes for H- 7, H-8, and Ig, as wellas the sex chromosomes.The strain distribution pattern was of interest,

with some strains such as NZB and SJL showingresistance. These strains are known to have de-fects in immune responsiveness, NZB developingautoimmunity and SJL developing spontaneoustumors. These defects apparently do not affectresistance to Listeria.The contrast between our studies of resistance

to Listeria and those of Salmonella typhimu-rium (15, 20, 21) is of particular interest. UnlikeListeria, recovery from S. typhimurium mayinvolve both cellular and humoral immunity (3,9). The studies of Plant and Glynn (20, 21)showed that resistance of mice to subcutane-ously injected S. typhimurium was also con-trolled by a single, non-H-2-linked gene. Strainsresistant to Salmonella showed strong delayed-type hypersensitivity responses to a variety ofantigens, including a Salmonella extract (20). Inour accompanying studies (4), it was the time ofonset of delayed-type hypersensitivity to Liste-ria, as well as of other correlates of cell-mediatedimmunity, that differed in resistant and suscep-tible strains. Once immunity was fully devel-oped, no differences were detected. Most impor-tantly, the fact that the strain distribution ofresistance and susceptibility is totally differentfor Listeria (Table 2) and Salmonella (21; un-published data) emphasizes the fact that theeffects we are observing are not related to ageneral inability of the reticuloendothelial sys-tem to handle bacteria like Listeria and Sal-monella, which, after phagocytosis, are able tosurvive within macrophages and induce cell-me-diated immunity. At least some degree of speci-ficity is indicated.The results of this study indicate that a single

gene or group of linked genes may dictate thespecific resistance or susceptibility effects de-

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762 CHEERS AND McKENZIE

scribed herein. The dominance ofresistance sug-

gests that lack of a particular component leadsto susceptibility, but, like the early tumor trans-plantation immunologists, we cannot yet statewhether the basic defect is qualitative or quan-

titative. The mechanisms are clearly less com-

plex than previously considered and should lendthemselves to analysis. In the next paper (4), wepresent our early studies in this area.

ACKNOWLEDGMENTS

This work was supported by a grant obtained from theNational Health and Medical Research Council, Australia.We are grateful to N. Warner of the Walter and Eliza Hall

Institute for performing the immunoglobulin allotyping andsupplying certain strains of mice. J. F. A. P. Miller providedextremely helpful discussions.

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