INFECTION AND IMMUNITY, Aug. 2011, p. 3204–3215
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 8
Multigenic Control and Sex Bias in Host Susceptibility to
Spore-Induced Pulmonary Anthrax in Mice?†
Jagjit S. Yadav,1* Suman Pradhan,1Renuka Kapoor,1Hansraj Bangar,1
Benjamin B. Burzynski,2Daniel R. Prows,2and Linda Levin3
Microbial Pathogenesis Laboratory, Department of Environmental Health, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267-00561; Division of Human Genetics, Cincinnati Children’s Hospital Medical Center,
University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-30392; and Division of
Epidemiology and Biostatistics, Department of Environmental Health, University of
Cincinnati College of Medicine, Cincinnati, Ohio 45267-00563
Received 30 December 2010/Returned for modification 25 February 2011/Accepted 14 May 2011
Mechanisms underlying susceptibility to anthrax infection are unknown. Using a phylogenetically diverse
panel of inbred mice and spores of Bacillus anthracis Ames, we investigated host susceptibility to pulmonary
anthrax. Susceptibility profiles for survival time and organ pathogen load differed across strains, indicating
distinct genetic controls. Tissue infection kinetics analysis showed greater systemic dissemination in suscep-
tible DBA/2J (D) mice but a higher terminal bacterial load in resistant BALB/cJ (C) mice. Interestingly, the
most resistant strains, C and C57BL/6J (B), demonstrated a sex bias for susceptibility. For example, BALB/cJ
females had a significantly higher survival time and required 4-fold more spores for 100% mortality compared
to BALB/cJ males. To identify genetic regions associated with differential susceptibility, survival time and
extent of organ infection were assessed using mice derived from two susceptibility models: (i) BXD advanced
recombinant inbred strains and (ii) F2 offspring generated from polar responding C and D strains. Genome-
wide analysis of BXD strain survival identified linkage on chromosomes 5, 6, 9, 11, and 14. Quantitative trait
locus (QTL) analysis of the C?DF2 population revealed a significant QTL (designated Rpai1 for resistance to
pulmonary anthrax infection, locus 1) for survival time on chromosome 17 and also identified a chromosome
11 locus for lung pathogen burden. The striking difference between genome-wide linkage profiles for these two
mouse models of anthrax susceptibility supports our hypothesis that these are multigenic traits. Our data
provide the first evidence for a differential sex response to anthrax resistance and further highlight the
unlikelihood of a single common genetic contribution for this response across strains.
Anthrax is one of the most ancient and lethal human dis-
eases caused by the virulent (toxigenic and encapsulated)
strains of Bacillus anthracis. In humans, anthrax may take three
forms depending upon the route of infection, namely, pulmo-
nary (inhalational), cutaneous, and gastrointestinal, with pul-
monary being the most lethal and difficult to treat. While the
naturally acquired pulmonary anthrax is relatively rare (occur-
ring only ?5% as often as cutaneous anthrax), this form has a
high potential for misuse as a weapon of bioterrorism, consid-
ering that environmental dissemination is the most expected
mode of release of the agent in mass attacks (26). Pulmonary
anthrax often proves fatal, with mortality approaching 100% if
not treated early. Anthrax spores have long been considered a
potential agent of biological warfare (25, 50, 58). Prior to the
11 cases of pulmonary anthrax that occurred via the U.S. mail
delivery system in 2001 (29), fatal cases of pulmonary anthrax
in the United States and other countries have been associated
with occupational and accidental exposures (37, 60). But the
bioterrorism attack in 2001 exposed a new cause for serious
concern for future and more widespread attacks on military
and civilian populations.
The etiological agent for anthrax, Bacillus anthracis, is a
Gram-positive bacterial pathogen that belongs to category A in
the CDC list of select agents. The size of its spores (1 to 2 ?m)
makes them optimal for deposition in the alveolar spaces fol-
lowing inhalation. To be virulent, B. anthracis requires two
major components: the capsule and the anthrax toxin. These
components are encoded by the plasmids pXO2 and pXO1,
respectively, in virulent strains such as Ames. Strains such as
Sterne, which lack the capsule (pXO2), are attenuated for
human infection and are therefore used for vaccine applica-
tions. However, these attenuated strains can cause serious in-
fections in mice. While there is increasing information on the
pathogen virulence factors in B. anthracis (3, 7, 12, 34, 71), the
mechanisms of host defense and pathogenesis are poorly un-
derstood for anthrax.
Although anthrax primarily affects herbivores, virtually all
mammals are susceptible to various degrees. Anthrax suscep-
tibility depends on host species (31), strain (69), and the route
of infection (35, 46). Interindividual differences in disease on-
set, survival, and treatment response have been observed in
human cases of pulmonary anthrax. In the 2001 anthrax letter
attacks, only 5 of the 11 individuals infected by the aerosolized
spores succumbed to the disease. The causes underlying these
differences in susceptibility are not known but conceivably in-
volve differences in host factors. It is intriguing that some
* Corresponding author. Mailing address: Microbial Pathogenesis
Laboratory, Department of Environmental Health, University of Cin-
cinnati College of Medicine, Cincinnati, OH 45267-0056. Phone: (513)
558-4806. Fax: (513) 558-4397. E-mail: Jagjit.Yadav@uc.edu.
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 31 May 2011.
animals (e.g., rats) are very sensitive to B. anthracis toxins yet
are difficult to infect by spores, while other animals (e.g.,
guinea pigs) are more resistant to the toxins but can be killed
with relatively few spores (31, 32, 63). Previous investigations
on the genetic susceptibility to B. anthracis have mainly fo-
cused on the role of anthrax lethal toxin (LT) exposure in vitro
(17, 52, 56, 66, 67) and in vivo (36, 40, 45) in laboratory rodent
strains. The toxin-based studies indicated that inbred mice vary
in their sensitivity to anthrax LT and that host genetic factors
underlie the differences in LT susceptibility (4, 36, 40). More
importantly, previous observations have revealed a reciprocal
trend for murine susceptibility to purified anthrax LT-induced
pathology versus spore-induced infection (36, 69). Since spore-
induced anthrax better represents a real world infection sce-
nario for bioterrorism, there is an immediate need to fully
understand the basis of host defense and susceptibility to B.
anthracis spores. Knowledge on host susceptibility to lethal
infection during pulmonary anthrax can be a key initiator in
the development of new countermeasures.
Lethal anthrax infection caused by B. anthracis spores is a
multistep process expected to require spore germination,
pathogen multiplication, and systemic dissemination, culminat-
ing in death (15, 22, 23, 46). Mechanisms of host resistance
could interfere with any of these steps. Hence, there is a need
to understand the genetic loci conferring host resistance/sus-
ceptibility to anthrax infection. In this context, there is also a
continuing need to identify and characterize more appropriate
animal models of resistance or susceptibility for dissecting the
role of these host factors. Initial investigations in this direction
have examined questions of pathogenesis and host resistance
in inbred mice by using spores from avirulent anthrax strains,
such as Sterne or 34F2 (55, 68–70). While inbred mice offer a
valuable tool to understand host susceptibility and response to
anthrax infection, little has been done in mice in experiments
with the fully virulent strains of B. anthracis. Limited initial
studies of mice either used fewer numbers of randomly se-
lected inbred strains (35) or were based on nonpulmonary
routes (69) of infection, and they did not identify host genomic
regions governing the underlying susceptibility. Whether males
and females differ in pulmonary anthrax susceptibility is also
The overall goals of this study were to investigate the role of
host genetic background and sex in susceptibility to pulmonary
anthrax infection by using the Ames strain and to identify
putative genomic loci underlying differential susceptibility by
quantitative trait locus (QTL) analysis. The QTL approach,
based on associating genetic variation with phenotypic varia-
tion, has proven useful for identification of genomic loci in-
volved in many complex diseases, including infectious diseases
(16, 54, 57). To begin to examine the role of host genetics, we
screened a phylogenetically representative panel of 14 inbred
mice strains and F1 progeny from select sensitive-resistant
strain breeder pairs. Initial studies established mouse models
of host susceptibility and also revealed evidence of a parent-
of-origin effect and a differential sex response to pulmonary
anthrax. To identify genetic loci responsible for B. anthracis
susceptibility, two strategies were used. First, genome-wide
single-nucleotide polymorphism (SNP) analysis was performed
on a panel of BXD advanced recombinant inbred (ARI) mice.
The BXD ARI lines were derived from the female C57BL/6J
and male DBA/2J parent strains (48), which represent a pro-
genitor strain pair identified in this study for modeling differ-
ential genetic susceptibility. These homozygous, inbred, and
genetically distinct ARI lines approximate the genetic diversity
in human populations and have been used successfully to map
QTLs associated with various clinical conditions and diseases
(2, 8, 42, 47). Second, we performed QTL analysis on an F2
population generated from the sensitive DBA/2J and resistant
BALB/cJ strains. This work represents the first report of sex
bias and a parent-of-origin effect in a murine model of spore-
induced pulmonary anthrax infection. These initial genome-
wide analyses using two separate mouse models of differential
susceptibility, coupled with spore-induced anthrax infection,
identified multiple genomic loci associated with pulmonary
anthrax outcome. These studies lay the groundwork for fol-
low-on studies to further characterize these susceptibility
QTLs to identify the relevant genes.
MATERIALS AND METHODS
Bacterial strain and spore inoculum preparation. Bacillus anthracis Ames, a
fully virulent (toxigenic and encapsulated) strain, was obtained from the Los
Alamos National Laboratory. The organism was cultivated, handled, and stored
in the biosafety level 3 (BSL-3) Laboratory of the University of Cincinnati
College of Medicine, using the protocol and standard operating procedures
approved by the university’s Institutional Biosafety Committee. Spore inoculum
of B. anthracis Ames was prepared using nutrient sporulation medium (13).
Briefly, an isolated single colony of B. anthracis Ames was streaked onto nutrient
sporulation agar (containing, per liter, 3 g of yeast extract, 3 g of tryptone, 2 g of
Bacto agar, 23 g of Lab Lemco agar, and 1 ml of 1% MnCl2), and the plates were
incubated at 37°C. Spore formation was monitored microscopically. When the
sporulation reached ?99% (approximately the fourth day of incubation),
the bacterial growth was harvested in 0.01 M phosphate-buffered saline (PBS).
The suspension was washed 3 times with PBS by centrifugation (12,000 rpm for
15 min at 4°C) followed by heating at 65°C for 1 h to inactivate the residual
vegetative cells. After heating, the spore suspension was washed and resus-
pended in a suitable suspension medium. An aliquot of the spore suspension was
serially diluted and plated out on Trypticase soy agar (TSA) to check for viable
spore count. The remaining monodispersed spore stock was frozen in 1-ml
aliquots at ?80°C in cryovials. On the eve of each infection experiment, a vial of
the frozen spore stock was thawed, resuspended in PBS, and quantified to verify
the actual viable count in the stock at that point of storage. Before use in mouse
inoculations the next day, the suspension medium in the frozen stock (1 ml) was
replaced with an equal volume of sterile PBS by centrifugation and resuspension.
On the basis of the viable count in the frozen main stock, a working stock of the
spore inoculum was prepared in PBS to obtain the desired number of spores per
ml. The fresh working inoculum (prequantified as above) was directly used to
inoculate mice, as described below. The same batch of inoculum was used for a
given treatment group. To generate a stable and consistent spore inoculum for
extended use, the effects of two suspension media, PBS versus Trypticase soy
broth with 15% glycerol (TSB-G), on the viability of Ames spores during frozen
storage (?80°C) were investigated. Due to the significant decrease in spore
viability in PBS, occurring as early as 24 h after freezing (data not shown), all
inocula were stored frozen (?80°C) in TSB-G broth and resuspended in PBS
right before use.
Animals. Adult healthy pathogen-free mice of both sexes (6 to 8 weeks of age)
were purchased from the Jackson Laboratory (Bar Harbor, ME). Fourteen
inbred mouse strains (129S1/SvImJ, A/J, BALB/cJ [abbreviated as C in crosses],
BPL/1J, C3H/HeJ, C57BL/6J [abbreviated as B or B6 in crosses], C57BL/10J,
CAST/EiJ, DBA/1J, DBA/2J [abbreviated as D or D2 in crosses], FVB/NJ,
NOD/ShiLtJ, NON/ShiLtJ, and SPRET/EiJ) were used, which were representa-
tive of the distinct mouse phylogenetic groups recently described (49). The
inbred mice were challenged with 500 spores/mouse of B. anthracis Ames.
B6D2F1/J mice (6 weeks old), derived from crossing resistant strain C57BL/6J
females and sensitive strain DBA/2J males, were purchased from the Jackson
Laboratory (Bar Harbor, ME). Reciprocal F1 crosses from BALB/cJ and
DBA/2J strains (i.e., CD2F1 and D2CF1; female strain listed first) were gener-
ated by in-house breeding at the Laboratory Animal Medicine Services facility of
the University of Cincinnati. Age-matched F1 mice (6 weeks each) and their
VOL. 79, 2011MULTIGENIC CONTROL OF MURINE SUSCEPTIBILITY TO ANTHRAX 3205
respective parents were transferred to the BSL-3 facility and inoculated with a
lethal dose of B. anthracis Ames spores (500 spores/mouse for B6D2F1 progeny
and 2,000 spores/mouse for CD2F1 and D2CF1 progeny).
BXD advanced recombinant inbred (ARI) strains of mice were purchased
from the Jackson Laboratory (Bar Harbor, ME) for genome-wide SNP analysis
to identify QTLs. The BXD ARI strains were originally made by repeated
intercrossing for at least 20 generations of F9 to F14 progeny from the C57BL/6
and DBA/2 parental strains (48). The following 16 genotypically distinct ARI
strains were obtained for genetic studies: BXD44, BXD48, BXD50, BXD55,
BXD62, BXD66, BXD68, BXD69, BXD86, BXD87, BXD89, BXD90, BXD96,
BXD97, BXD98, and BXD100. These 16 BXD ARI strains, along with the
C57BL/6J and DBA/2J progenitor strains, were screened for differences in sus-
ceptibility to infection by B. anthracis Ames spores. Five mice per BXD ARI
strain were challenged with a lethal dose of 500 spores/mouse of B. anthracis
Ames. For traditional QTL analysis, a segregant population of 258 F2 mice (146
females, 112 males) was generated in-house from resistant BALB/cJ and sensi-
tive DBA/2J strains. All F2 mice came from intercrossing CD2F1 mice (BALB/cJ
females crossed with DBA/2J males).
All mice were housed in autoclaved, individually ventilated static microiso-
lator cages (not more than 4 mice per cage) in the specific-pathogen-free
animal facility at the University of Cincinnati. Animals were fed standard
pellet diet and autoclaved water ad libitum. The animals were allowed to
acclimate for 7 days in the BSL-2 and BSL-3 facility prior to their use in
experimental studies. All animal procedures were performed according to the
Institutional Animal Care and Use Committee (IACUC)-approved protocols
at the University of Cincinnati.
Mouse inoculations. All inoculations and housing of mice postinfection were
carried out in the animal BSL-3 containment area at the University of Cincinnati.
For each experiment, mice were age and sex matched, unless noted otherwise.
Mice were anesthetized by intraperitoneal injection of Avertin (0.4 mg/g of body
weight). An incision was made on the ventral region of the neck, and the trachea
was carefully exposed. To deliver the desired spore dose to the lungs, a 50-?l
volume of the appropriate working stock was introduced intratracheally followed
by 50 ?l of air, using a bent needle on a BD ultrafine insulin syringe (Becton
Dickinson, San Jose, CA) and placing the animal on an inclined board (?30°
angle). The incision was then closed using 3M Vetbond tissue adhesive.
Determination of survival time and organ bacterial load. Survival responses of
inbred strains (male versus female), F1 progeny, BXD ARI lines, and F2 progeny
to pulmonary infection with Ames spores were compared using multiple animals
for each treatment group and a constant spore dose, as indicated for the respec-
tive experiments. Inoculated mice of each treatment group were frequently
monitored for survival to determine the time to death (TTD). The TTD value for
a given mouse strain was calculated by averaging the number of hours required
for death for the individual animals within that group (i.e., mean survival time,
in hours). The BALB/cJ female group was an exception; in this case, the mean
TTD was calculated based on the data from the nonsurviving mice only. The
median survival times for each strain were also calculated. Lungs, livers, and
spleens of the animals who succumbed were immediately harvested and analyzed
for total bacterial load, as indicated by CFU. The tissues were homogenized
(each in 1 ml of TSB-G) and plated on TSA at 37°C for CFU determination.
Dose-response analysis for BALB/cJ mice. Dose-response analysis was per-
formed to select the optimal lethal dose of B. anthracis Ames spores for the
resistant inbred strain, BALB/cJ (females). Various doses (500 to 2,500 spores/
mouse) were used to challenge groups of 10 mice/dose, and the TTD was
recorded for each animal. The survival percentage (number of animals surviving
divided by the total number of animals infected) was plotted against each spore
dose to develop survival curves for the individual doses. The minimum dose that
resulted in 100% mortality in the treatment group was confirmed by probit
analysis and taken as the optimal lethal dose (LD100) for BALB/cJ mice and their
Infection kinetics. A time course experiment was performed to investigate
comparative infection kinetics in the most resistant inbred strain, BALB/cJ, and
the most sensitive inbred strain, DBA/2J. For either strain, female mice were
inoculated with a lethal dose of 2,000 spores/mouse and sampled at increasing
time intervals (?1 h, 12 h, 24 h, 48 h, 72 h, and at death). At each time point,
mice (n ? 3 for each strain) were euthanized using an overdose of Avertin, and
tissues (lung, spleen, and liver) were harvested. Tissue homogenates were sub-
jected to CFU analysis as described above. Methods used to determine organ
infection loads were the same as those described for screening the 14 inbred
strains in the preceding sections.
QTL analysis. QTL analysis, a statistical assessment of the likelihood of
genetic linkage, was performed in two separate cohorts (BXD ARI strains and an
F2 population from BALB/CJ and DBA/2J progenitors) to associate phenotype
with genotype. The BXD ARI strains were exposed to a pulmonary load of 500
anthrax spores/mouse and screened to identify genetic regions linked with the
disparate survival times seen for the C57BL/6J and DBA/2J progenitor inbred
strains. Using the online analysis program WebQTL (http://www.genenetwork
.org) (65) as the mapping engine and the genome-wide SNP source, survival time
and organ CFU data from 16 BXD ARI strains were assessed for putative QTLs.
QTL maps were generated using interval regression, and the log of the odds ratio
(LOD) score was used in linkage analysis to assess significance. Genome-wide
LOD threshold levels for significant and suggestive linkage were determined
using 2,000 permutation tests (9). Peak markers from regression analysis were
fixed one at a time in composite interval mapping (CIM) to identify potential
additional and/or weaker QTLs after variance explained by the first marker was
removed from the analysis (30). Bootstrap analysis, which involved 2,000 itera-
tions of random data set reshuffling (with replacement) and remapping of each
result, was also performed in WebQTL to assess reliability of QTL peaks and to
determine approximate confidence limits.
Genotyping of F2 populations. DNA was isolated from a tail clip of each F2
and progenitor strain mouse by using either the Wizard genomic DNA isolation
kit (Promega, Madison, WI) or the high-throughput DNA extraction service of
the Cincinnati Children’s Hospital DNA Core Facility. DNA was quantitated
using the A260/A280ratio and diluted to ?20 ng/?l for PCR genotyping. Primer
pairs, chosen based on known polymorphisms between the BALB/cJ and DBA/2J
progenitor strains, were purchased from IDT (Coralville, IA). PCR was per-
formed in 15 ?l in 96-well plates (Applied Biosystems, Foster City, CA), as
described previously (51).
QTL mapping and linkage analyses. All phenotype data (i.e., survival times or
organ CFU) and genotype data were analyzed for main effect QTLs by using the
Scan One function of the freely available R/QTL computer package (5). A total
of 71 polymorphic markers were genotyped for all F2 mice. Threshold values for
significant and suggestive linkages were established using 10,000 permutations of
the data set. To identify potential gene-gene interactions, including additivity and
epistasis, genome-wide scans for all marker pairs were carried out on the total F2
population using the Scan Two function in R/QTL. Empirical threshold signifi-
cance values for pairwise interactions were determined using 100 permutations
of the data set.
Statistical analyses. Weighted least-squares analysis (WLS) was performed to
analyze the TTD data of 14 inbred strains of 167/169 male and female mice
(approximately 99% mortality). Preliminary investigations showed that the dis-
tribution of TTDs approximated normality, based on the Shapiro Wilk test.
Within-strain variances were found to be heterogeneous, based on Levene’s test.
The WLS methodology, combined with a normality assumption, has the opti-
mum power to detect differences between means of strains and susceptibility
groups, compared to nonparametric methods. A censored distribution of TTD
was not analyzed, since the 1% censoring rate (2/169) was believed to be suffi-
ciently low to have a negligible effect on results of hypothesis testing. TTD strain
means were sorted in increasing order of magnitude, and two groups (low [most
susceptible] and high [resistant]) were identified. The remaining strains com-
prised a third group whose means were between the endpoints of the two groups.
Our initial regression model was a multiway analysis of variance (ANOVA) of
TTD that included susceptibility group and strain within susceptibility group as
categorical independent variables. Means and standard errors of TTDs by strain
were calculated. The linearity of the 14 ordered strain means was tested by linear
orthogonal polynomial coefficient analysis within the ANOVA model. In addi-
tion, t tests were performed to test differences between all pairs of strain means.
Following a significant difference between at least one pair of means, as deter-
mined by a global chi-square test evaluating the equality of all strain means (P ?
0.001), Fisher’s protection level was used to adjust for multiple comparisons of
differences between strain means. The same methodology (WLS) was used to
analyze organ CFU values, after a logetransformation was applied to approxi-
mate normality. Geometric means were calculated to estimate the medians of
each strain and of sexes within each strain. Sex-specific analyses of TTDs and loge
CFU determinations were also performed using WLS, with ANOVA models to
compare strain means by sex. Comparisons of between-strain to within-strain
variability were obtained by calculating the ratios of the variance between strains
to the residual variance, or the average variance within strains. These were
calculated for all TTD data and for each sex.
Probit regression was performed to estimate the LD50and the minimum
effective dose approximating 100% mortality for 5 groups of female BALB/cJ
mice (10 mice/group) receiving various spore doses (500, 750, 1,000, 2,000, or
2,500 spores/mouse). Equality of the distribution of TTD values among doses
was tested by the Wilcoxon log rank statistic in a survival curve analysis stratified
by dose. In addition, mean values of TTDs for 16 strains of ARI-BXD mice were
analyzed by WLS, assuming an ANOVA model. The linearity of ordered TTD
3206 YADAV ET AL.INFECT. IMMUN.
means was tested by linear orthogonal polynomial coefficients. The ratio of
between-strain to within-strain variability was calculated from ANOVA by di-
viding the model mean square by the residual mean square.
Interstrain and/or sex differences within strains for survival time were also
evaluated for reciprocal CD2F1 and D2CF1 progeny and B6D2F1 progeny, and
P values were adjusted for multiple comparisons. Means and standard errors for
each outcome were used in the bar graphs to facilitate interpretation of results.
Statistical analyses were performed using SAS for Windows, version 9.2 (SAS
Institute, Cary, NC).
Male and female mice for 14 inbred mouse strains were
screened using a lethal dose of 500 spores/mouse of B. anthra-
cis Ames strain. The dose was empirically selected, guided in
part by available published information (35), and verified in an
initial trial challenge experiment designed to test its potential
to cause 100% lethality in selected inbred strains (data not
Survival differences among inbred mouse strains. Following
exposure to the same lethal dose of B. anthracis spores (500
spores/mouse), significant differences in susceptibility (mea-
sured as TTD) were observed among the 14 inbred strains.
Using the average TTD of both sexes, three susceptibility
groups were identified (Fig. 1): the most susceptible group,
with a TTD of ?60 h (DBA/2J ? NOD/ShiLtJ ? DBA/1J ? A/J
? FVB/NJ); the intermediate susceptible group, with a TTD of
60 to 90 h (NON/ShiLtJ ? 129S1/SvImJ ? BPL/1J ? SPRET/
EiJ ? C3H/HeJ ? CAST/EiJ ? C57BL/10J); the least suscep-
tible (or most resistant) group, with a TTD of ?90 h
(C57BL/6J ? BALB/cJ). The susceptibility grouping based on
the empirical cutoff limits was verified using pairwise statistical
comparisons of the means. TTD means and standard errors of
the strains among susceptibility groups obtained from analysis
of variance are shown in Table S1A of the supplemental ma-
terial. The statistical evaluation of paired differences between
TTD means of the 14 strains supported the linearly increasing
levels of the means and susceptibility group clustering (see
Table S1B in the supplemental material). Each susceptibility
group differed significantly (P ? 0.01) from the other groups.
The intermediate group included three subgroups, which are
separated by the ? signs in the above susceptibility order.
Mean TTD values of the 14 strains were significantly different
(P ? 0.001; degrees of freedom, 13). The ratio of between-
strain to within-strain variability for sex-specific and combined
sexes TTD was 26.9, implying a greater interstrain than intras-
Sex bias in the most resistant group of inbred strains. The
resistant (least susceptible) group of strains (BALB/cJ and
C57BL/6J) showed a significant difference (P ? 0.01) in sur-
vival times between males and females (Fig. 1). A significant
sex difference (P ? 0.01) was also observed in CAST/EiJ mice,
a member of the intermediate group. The BALB/cJ strain
showed a greater sex difference (?50 h higher mean TTD for
female than male mice) compared to the C57BL/6J strain (?30
h higher mean TTD for females). Furthermore, female
BALB/cJ mice groups showed partial mortality (40%) com-
pared to 100% mortality observed for the male counterparts,
following the same dose of 500 spores/mouse. Hence, to de-
termine the minimum effective lethal dose causing 100% mor-
tality in female BALB/cJ mice, various doses (500, 750, 1,000,
2,000, and 2,500 spores/mouse) were compared using 10 ani-
mals per dose. Dose-response curves showed a decreasing sur-
vival percentage with an increasing spore dose. A minimum
dose of 2,000 spores per mouse was required (Fig. 2) to achieve
100% mortality (mean TTD of ?80 h) in female BALB/cJ
mice. In contrast, the partial mortality (4 of 10 mice) observed
for the 500-spores/mouse dose was characterized by a mean
TTD of ?130 h for the animals who succumbed. Probit regres-
sion analysis revealed the following values for the lethal doses:
LD50, 584 spores; LD100, 1,978 spores. Based on these obser-
vations, a test dose of 2,000 spores was used for further studies
on BALB/cJ (female) mice and F1 or F2 progeny derived from
using BALB/cJ as a progenitor strain.
Organ colonization and infection kinetics differences among
inbred strains. At the time of death, a marked variation in
CFU values was noted in the lungs among the 14 inbred strains
challenged with the same lethal dose (500 spores). Mean val-
ues of the 14 strains were significantly different (P ? 0.001;
degrees of freedom, 13). The ratio of between-strain to within-
strain variability for sex-specific and total log CFU was 31.5,
implying a greater interstrain than intrastrain variability. Based
on the overall means, the following ascending order of suscep-
tibility to pathogen accumulation in the lung was obtained:
BPL/1J ? 129S1/SvImJ ? C57BL/6J ? NOD/ShiLtJ ?
FVB/NJ ? DBA/1J ? NON/ShiLtJ ? BALB/cJ ? A/J ?
DBA/2J ? SPRET/EiJ ? CAST/EiJ ? C3H/HeJ ? C57BL/10J
(data not shown). However, no clear correlation was observed
between the susceptibility orders for lung CFU and survival
time among these 14 inbred strains.
To better understand the differences in organ colonization,
FIG. 1. Comparative susceptibility of inbred mouse strains of both
sexes to pulmonary anthrax infection with spores of B. anthracis Ames.
Animals for each inbred strain (n ? 5 to 7 mice of each sex; 6 to 8
weeks of age) were intratracheally challenged with 500 spores (per
mouse) of B. anthracis Ames and monitored for survival time, as
described in Materials and Methods. Survival time was measured as
TTD. The TTD values (in hours) were plotted as means and standard
errors of the means. All strains showed 100% lethality except BALB/cJ
(females); arithmetic means and standard errors of the plotted TTD
were therefore calculated based on the nonsurviving mice. The arith-
metic mean (146.9 h) and harmonic mean (145.3 h) for BALB/c (fe-
males), calculated based on the available TTD values for the mice who
succumbed, were comparable. Three susceptibility groups (most sen-
sitive, intermediate, and most resistant) were identified based on sur-
vival time, as shown using inverted brackets. Mean values of 14 strains
were significantly different for TTD (P ? 0.001; degrees of freedom,
13). Significant differences (P ? 0.01) in the TTD values between sexes
are indicated by an asterisk.
VOL. 79, 2011MULTIGENIC CONTROL OF MURINE SUSCEPTIBILITY TO ANTHRAX 3207
we compared predeath kinetics of pathogen accumulation and
dissemination in the most resistant strain (BALB/cJ) and most
sensitive strain (DBA/2J) (Fig. 3) at the site of spore deposi-
tion (lung) and in systemic locations (spleen and liver). Female
mice were used for both strains. While the lung bacterial load,
measured up to 24 h postinfection, in both strains was compa-
rable (P ? 0.05), the final bacterial load at death was ?20-fold
higher (P ? 0.01) in resistant BALB/cJ mice. In spleen, live
bacilli were first detectable at the same time point (24 h)
postinfection for both strains, with the sensitive DBA/2J strain
showing significantly higher CFU (P ? 0.05). However, the
final bacterial load in the spleen at death was significantly (P ?
0.01) higher (?10-fold) in BALB/cJ mice. No CFU were de-
tected in the liver until at the time of death in DBA/2J mice
(TTD, ?30 h), unlike BALB/cJ mice, which did not show
bacilli until shortly before death (72 h postinfection) and at the
time of death (TTD, ?80 h). Significantly different (P ? 0.01)
bacterial loads at 72 h and TTDs implied rapid dissemination
and/or multiplication and/or poorer clearance of bacilli at the
time of death as possible mechanisms for the BALB/cJ strain.
The rate of pathogen dissemination to spleen and liver was
greater in the DBA/2J mice, leading to an early and higher
systemic bacterial load in this strain at 24 to 30 h postinfection,
coinciding with its near-death/death time point. Nevertheless,
the absolute systemic load of the pathogen at the time of death
was significantly lower (P ? 0.01) in DBA/2J mice (?30 h)
than in BALB/cJ mice (?80 h), implying the former’s greater
susceptibility to bacterial accumulation during infection as a
FIG. 3. Kinetic changes in pathogen dissemination and accumula-
tion during pulmonary infection of the selected resistant (BALB/cJ)
and susceptible (DBA/2J) strains of mice with B. anthracis Ames
spores. Female mice (6 to 8 weeks of age) for the two inbred strains
were intratracheally challenged with 2,000 spores/mouse of B. anthra-
cis Ames and periodically monitored for organ pathogen burden until
death, as described in Materials and Methods. Homogenized lung,
spleen, and liver from each animal harvested at each time point (n ?
3 mice) were subjected to microbiological analyses by agar plating in
triplicate. Pathogen load in the infected tissue was expressed as CFU.
Values are plotted on a log scale based on the geometric mean and
standard error of the mean (the error bars have been plotted but are
too small to be visible on the log scale). Abbreviations: TTD(B), time
to death for BALB/cJ (?80 h); TTD(D), time to death for DBA/2J
(?30 h). Time zero corresponds to data measured within the first hour
(lag) period, when multiple inoculations and surgeries were per-
formed. Some of the significant differences in organ CFU values be-
tween the two strains are indicated by a single (P ? 0.01) or double
(P ? 0.05) asterisk. Other within-strain and between-strain organ CFU
differences across different time points were significant at the 1% or
5% level; however, the following were not significant (P ? 0.05): lung
CFU, DBA/2J (24 h versus TTD), BALB/cJ (72 h versus TTD), and
BALB/cJ versus DBA/2J for 0-h, 12-h, and 24-h time points; spleen
CFU, DBA/2J (24 h versus TTD), BALB/cJ (72 h versus TTD),
BALB/cJ (48 h) versus DBA/2J (24 h), and BALB/cJ (48 h) versus
FIG. 2. Dose-response analysis for female BALB/cJ mice for selec-
tion of the lethal dose (LD100) of B. anthracis Ames spores. Groups of
10 female mice were intratracheally inoculated with various doses of B.
anthracis spores (500 to 2,500 spores/mouse) and monitored for TTD,
as described in Materials and Methods. TTD values (in hours) were
plotted against the survival percentage for each dose. The mortality
ratio (number of dead animals divided by the total number of animals
infected) achieved with each dose is represented as n on the respective
survival curves. ?, censored data (i.e., not all mice died).
3208 YADAV ET AL.INFECT. IMMUN.
Susceptibility inheritance in the F1 progeny. Matings using
two strain-pair combinations of the most resistant and most
susceptible strains, BALB/cJ ? DBA/2J and C57BL/6J ?
DBA/2J, were performed to investigate the heritability of sus-
ceptibility in the F1 generation. F1 hybrid mice were compared
with their respective parents for TTD using the same lethal
spore dose for the parents and progeny (500 spores/mouse for
C57BL/6J crosses and 2,000 spores/mouse for BALB/cJ
crosses). The B6D2F1 progeny showed an intermediate TTD
(Fig. 4) compared to the C57BL/6J and DBA/2J parental
strains. The TTD differences between the B6D2F1 progeny
and the parents were statistically significant (P ? 0.01), as was
the difference between the two parental strains (P ? 0.01). The
CD2F1 and D2CF1 reciprocal crosses also showed TTD values
that were intermediate (Fig. 5A) to the values for their parents
(BALB/cJ and DBA/2J), and the differences were significant
(P ? 0.01). Specifically, the TTD value for the CD2F1 progeny
fell between the two parental values and was significantly (P ?
0.01) lower (?53 h) compared to the TTD for the reciprocal
cross D2CF1 (?67 h). The difference between reciprocal F1
mice suggested that one or more genes related to the overall
trait may be imprinted. More interesting, the survival times of
offspring from these reciprocal F1 crosses correlated more
closely with the survival time for the strain of the male breeder,
suggesting a possible paternal inheritance pattern. In fact, sur-
vival times for D2CF1 males and females (from BALB/cJ sires)
did not differ from the mean survival time for BALB/cJ fe-
males (P ? 0.22 for both). Besides a significant difference (P ?
0.01) from its reciprocal F1 group, survival times for CD2F1
males and females differed significantly (P ? 0.01) from both
parental strains (Fig. 5B) but were more similar to the CD2F1
sire strain (DBA/2J). Survival times of female versus male
CD2F1 progeny showed a trend toward a sex bias (P ? 0.15),
whereas no such trend was found in the reciprocal D2CF1
cross (Fig. 5B).
Susceptibility assessments in the BXD ARI strains. To iden-
tify genetic loci responsible for host susceptibility to B. anthra-
cis, we investigated BXD ARI strains, originally generated
from female C57BL/6J and male DB/2J mice (48), a parental
pair that represents a resistant-susceptible strain pair identified
in the current study. Comparison of the individual BXD ARI
strains showed an interstrain variability in survival time (Fig.
6). The range of the TTD values for the BXD strains approx-
imated the survival time range for the two progenitor strains
(?40 to ?80 h) and showed the following descending order of
susceptibility: BXD 90 ? BXD 97 ? BXD 87 ? BXD 98 ?
BXD 44 ? BXD 89 ? BXD 86 ? BXD 96 ? BXD 66 ? BXD
68 ? BXD 100 ? BXD 48 ? BXD 50 ? BXD 62 ? BXD 55 ?
BXD 69. The ratio of between-strain to within-strain variabil-
ity, calculated from the ANOVA by dividing the model mean
square by the residual mean square (406.01/62.03 [6.55]), in-
dicated a statistically significant between-strain variability (P ?
The BXD ARI strains also showed variability for the patho-
gen burden (CFU) at the site of infection (lung), as well as at
two systemic locations (spleen and liver). However, the order
FIG. 5. Comparative susceptibilities of the reciprocal F1 progeny and
the resistant BALB/cJ (C) and susceptible DBA/2J (D2) parental strains.
(A) Comparison of the reciprocal F1 crosses D2CF1 and CD2F1 (based
on mean values of both sexes) and the parental strains. (B) Comparison
strains. Results are consistent with a possible paternal mode of inheri-
tance, with reciprocal F1 offspring having survival times most similar to
those for the inbred strain of the sire. A lethal dose of B. anthracis spores
(2,000 spores/mouse) optimized for BALB/cJ was used for all inocula-
tions. Survival time, expressed as TTD (in hours) is plotted as the mean
and standard error of the mean for the number of animals in a given
group. Abbreviations: M, male; F, female; M?F, both sexes averaged; n,
total number of animals tested for each mouse strain.
FIG. 4. Comparative susceptibilities of the B6D2F1 progeny and
the resistant female C57BL/6J (B6) and susceptible male DBA/2J (D2)
parental strains. A dose of 500 spores/mouse of B. anthracis Ames was
used for all inoculations of age-matched (6 to 8 weeks old) F1 progeny
and parental strains. Survival time, expressed as TTD (in hours), is
plotted as the mean and standard error of the mean. Abbreviations: M,
male; F, female; n, number of animals tested for each strain.
VOL. 79, 2011MULTIGENIC CONTROL OF MURINE SUSCEPTIBILITY TO ANTHRAX3209
of pathogen accumulation (data not shown) did not correlate
with the survival susceptibility order (Fig. 6) across the BXD
ARI strains. Also, the order of bacterial accumulation in the
BXD strains did not correlate among the three organs. As an
example, comparison of bacterial buildup showed a significant
difference (P ? 0.01) between the most resistant (BXD 69) and
most susceptible (BXD 90) ARI strains, with a 2-log-higher
CFU in the lung but a 1-log-lower CFU in the spleen of the
sensitive strain. The pathogen dissemination to the liver was,
however, comparable. This indicated a variable rate and extent
of both the bacterial multiplication in the lung and systemic
dissemination to spleen in these two polar responding BXD
Genome-wide mapping of anthrax susceptibility in BXD
ARI mice. To gain insight into the QTLs underlying the dif-
ferential susceptibility of C57BL/6 and DBA/2J inbred strains
to a pulmonary instillation of B. anthracis Ames spores, median
survival time was mapped in 16 BXD ARI strains (n ? 76 total
mice; 4 to 5 mice per ARI strain) by using WebQTL (65) and
the integrated 3,795 polymorphic SNP and microsatellite
markers. Significant and suggestive threshold limits of linkage
were established at alpha values of 0.05 and 0.1, respectively,
and were derived from 2,000 permutations of the data set. A
genome-wide QTL map for median survival time in BXD mice
is presented in Fig. 7. Five QTLs were identified on chromo-
somes 5, 6, 9, 11, and 14. The chromosome 6 QTL had the
highest peak LOD frequency in a set of 2,000 random genome-
wide bootstrap runs. In all cases, the B allele increased the
To further assess these QTLs we performed CIM, in which
the variance for each of the best 2 QTLs (chromosomes 6 and
14) was individually removed and the genome rescanned to
identify further loci explaining the phenotypic variance (Fig.
7B and C). Interestingly, removing the variance of the chro-
mosome 6 QTL markedly increased the signals of the peaks on
chromosomes 5 and 9 and maintained comparable signals on
chromosomes 11 and 14 (Fig. 7B). Similarly, chromosome 6
and 11 signals remained when the variance for chromosome 14
was removed from the analysis (Fig. 7C). Unlike survival time,
no appreciable QTLs were associated with organ CFU differ-
ences across the BXD ARI strains.
QTL analysis of the CD2 F2 population. We further as-
sessed the genetic linkage of anthrax susceptibility by using
traditional QTL analysis on a recombinant F2 population de-
rived from the polar responding BALB/cJ and DBA/2J strain
pair. All 258 F2 mice were inoculated with 2,000 spores of
Ames strain and phenotyped for survival time and for bacterial
burden in the spleen, liver, and lung at the time of death. All
F2 mice were also genotyped for 71 microsatellite markers
across the genome, and QTL analysis was performed for each
of these 4 traits. QTL plots are presented in Fig. 8. Significant
linkage was identified on chromosome 17 for survival time,
near D17Mit89 (?64 Mbp), with a peak LOD score of 3.7 (Fig.
8A and C).
QTL analyses of organ CFU accumulation did not identify
linkage for spleen or liver. However, a chromosome 11 QTL
was revealed for lung CFU, with a LOD score of 2.8 (Fig. 8B
and D). Because this QTL coincided with the previously iden-
tified region on chromosome 11 in BXD SNP analysis (Fig.
7A), there is added confidence for its importance. A separate
genome-wide scan of all two-locus pairs did not reveal notable
additive or epistatic interactions.
Anthrax infection is a complex process that may vary with
the specific pathogen strain, as well as host factors. Murine
models of anthrax infection have demonstrated an important
role for the route of exposure (7, 33, 35, 71), in addition to
immune cell function (11, 15). However, little is known about
the role of host genetic background in anthrax infection; avail-
able studies in this regard are primarily based on the use of
avirulent strains of B. anthracis (1, 24, 35, 55, 68, 69). These
previous studies of inbred mice did not assess possible sex or
parental effects, nor were any genetic loci associated with sus-
ceptibility to virulent B. anthracis. Results of this study can
begin to fill this information gap.
The concept of same-dose–variable response is the basis of
investigating the genetic differences in host susceptibility.
However, the delivery of a consistent dose for anthrax infec-
tion can be challenging, depending on the route of adminis-
tration. In anthrax murine models, different routes of pulmo-
nary and extrapulmonary infection have been reported (7, 35,
69, 71). The following rationale was used to select the route for
infection in these studies. The pulmonary route of B. anthracis
infection is closest to a real world scenario of exposure in
bioterrorism situations; thus, a pulmonary route is most rea-
sonable. In addition, a pulmonary challenge requires a more
manageable lethal dose of spores (?1,000 to ?50,000, depend-
ing on the intratracheal versus intranasal versus aerosol deliv-
ery procedure), compared to the subcutaneous route of infec-
tion, which has a very low LD50and therefore requires only 5
to 50 spores (35, 69). Importantly, slight fluctuations of such a
low lethal dose may confound the variability differences due to
host genetic background. Given these facts for pulmonary an-
thrax spore dosing regimens, we chose to use the intratracheal
route of infection to deliver a consistent and reliable dose. A
dose of 500 spores/mouse was selected to induce a discernible
FIG. 6. Relative survival times of different BXD ARI strains intra-
tracheally challenged with B. anthracis Ames spores. A dose of 500
spores/mouse of B. anthracis Ames was used for all strains (5 mice per
strain). Survival time was measured as TTD in hours. Values are
expressed as means and standard errors of the means. Statistical anal-
ysis showed that interstrain variability was significant (P ? 0.001) and
the intrastrain variability was lower, as indicated by the ratio of the
interstrain variability to intrastrain variability (6.55; P ? 0.001).
3210YADAV ET AL.INFECT. IMMUN.
range of survival time and infection responses expected over
the entire spectrum of the inbred strain panel tested.
Interstrain variability. The mean and median survival time
differences among the 14 inbred strains supported a possible
genetic basis for susceptibility. The most resistant group (i.e.,
C57BL/6J and BALB/cJ strains) showed an ?3-fold-higher
overall mean survival period (based on TTD of the animals
that succumbed to infection) than the most susceptible group,
comprised of five equally sensitive strains, DBA/2J, A/J, NOD/
ShiLtJ, DBA/1J, and FVB/NJ (Fig. 1). Identification of multi-
ple sensitive strains in this study provides several additional
possible combinations to design susceptible-resistant models
for future genetic linkage analyses and therapeutics evaluation
research. Comparison of the results with the limited previous
studies available indicated that the relative extent and/or order
of murine interstrain susceptibility varies with the route of
FIG. 7. Genome-wide QTL linkage maps for median survival time of BXD ARI strains after Bacillus anthracis pulmonary infection. Blue plot, LOD
ratio; yellow histogram, frequency of peak LOD from 2,000 random bootstrap iterations; pink horizontal line, significant LOD threshold value at
genome-wide P level of ?0.05 (using 2,000 permutations); gray horizontal line, suggestive threshold value at genome-wide P level of ?0.63 (using 2,000
permutations). (A) A genome-wide LOD plot for median survival time suggested QTLs on chromosomes 5, 6, 9, 11, and 14. CI was then performed after
fixing each of the two strongest peaks (chromosomes 6 and 14) to remove their variance and rescanning the genome for linkage. (B) CIM results after
removing the chromosome 6 peak marker (gnf06.037.785) variance. Suggestive linkages on chromosomes 5, 9, and 11 were identified. (C) CIM results
after removing the chromosome 14 peak marker (rs3691815). Suggestive linkages on chromosomes 6 and 11 were identified.
VOL. 79, 2011 MULTIGENIC CONTROL OF MURINE SUSCEPTIBILITY TO ANTHRAX3211
infection and virulence status of the B. anthracis strain (viru-
lent versus avirulent) (35, 69). However, this generalization
does not seem to be universally true for all inbred strains. For
instance, DBA/2J and A/J mice showed higher susceptibility
regardless of the bacterial strain virulence or the route of
Previous murine infection studies using the Sterne strain
(24, 68) correlated anthrax susceptibility to natural comple-
ment C5 (encoded by the Hc gene; also called hemolytic com-
plement) deficiency genotype, as in other microbial infections
(19, 27). The studies using the Sterne strain were based either
on C5 replenishment (68) in a C5-deficient strain (A/J) or C5
depletion with cobra venom factor (24) in a C5-sufficient strain
(C57BL/6 strain). Our study using the virulent Ames strain,
however, showed that correlation between hereditary C5 defi-
ciency and anthrax susceptibility is not universal among differ-
ent inbred mouse strains. For instance, the C5-sufficient
DBA/1J strain (72) demonstrated equally high susceptibility
(i.e., decreased survival time) to pulmonary anthrax as the
C5-deficient strains DBA/2J, A/J, and NOD/ShiLtJ (Fig. 1).
Similarly, an earlier study (35) reported comparable suscepti-
bility of a complement C5-deficient inbred strain (DBA/2) and
a C5-sufficient inbred strain (C3HeB/FeJ) to the virulent strain
of B. anthracis. These observations collectively highlight the
genetic complexities of the overall host response to anthrax
Sex bias and parent-of-origin effect. The significant (P ?
0.01) sex difference we observed for survival time (Fig. 1) of
BALB/cJ and C57BL/6J strains contrasts with earlier findings
from murine studies of anthrax susceptibility (35, 69). For
BALB/cJ, a sex bias was observed for the lethal dose (Fig. 2)
required to cause 100% mortality, an observation that may or
may not extrapolate as a general phenomenon for the other
resistant strains. Although the resistant group strains did not
show a common sex bias for lung bacterial burden (data not
shown), C57BL/6J resistant female mice (individual animals
that succumbed to infection) had lower bacterial burdens than
their male counterparts, implying divergent genetic determi-
nants for the two phenotypes (survival and pathogen multipli-
cation in the host lung). The sex difference could be the result
of hormonal differences or other sex-related expression
changes. Although anthrax spore infection and anthrax LT
showed opposing susceptibilities (69), a role for a perturbed
balance of endocrine function in a purified toxin-induced mu-
rine anthrax model has been indicated (41). However, no such
hormonal effect information has yet been identified for anthrax
infection. Importantly, the physiological basis for, and thera-
peutic implications of, this sex disparity could be further in-
vestigated using the sex bias models optimized in this study.
Comparing total progeny from the reciprocal CD2F1 and
D2CF1 crosses revealed a significant difference in TTD (Fig.
5A); however, males and females within each F1 cross did not
differ (Fig. 5B). This differential TTD in reciprocal F1 popu-
lations supports a possible parent-of-origin effect, such as im-
printing in response to anthrax infection. A parent-of-origin
effect was also demonstrated for susceptibility differences in
parasitic infections, including toxoplasmosis (28), Plasmodium
falciparum malaria (18), and trypanosomiasis (10). Of interest,
the parent-of-origin effect described for a mouse model of
trypanosomiasis was also associated with overall survival time
(10), with resistance attributed to the paternal allele.
Interestingly, the mean survival times of offspring from the
two reciprocal F1 crosses were consistent with the susceptibil-
ity of the male strain in each cross. Specifically, offspring de-
rived from the F1 cross using the sensitive DBA/2J sire
(CD2F1) were sensitive, and offspring from the F1 cross with
the resistant BALB/cJ sire (D2CF1) were resistant. Although
relatively uncommon in the literature until recently, such epi-
genetic inheritance through the paternal lineage was elegantly
demonstrated over multiple generations in mice evaluated for
obesity resistance of Obrq2a and for reduced food intake (73).
Data for survival time to hyperoxic acute lung injury was also
found to be consistent with imprinting and transgenerational
inheritance (64). But, epigenetic inheritance can also result
FIG. 8. LOD plots for the CD2F2 population (generated from
BALB/cJ and DBA/2J progenitors) infected with Bacillus anthracis
Ames spores. A total of 258 F2 mice were exposed to 2,000 Ames
spores and monitored for survival time and total lung burden (CFU).
(A and C) Results of survival time analysis for genome-wide (A) and
chromosome 17 only (C). (B and D) Lung CFU results following
infection for genome-wide (B) and chromosome 11 only (D) analysis.
Genome-wide threshold values that were significant (solid lines; LOD,
3.37) or suggestive (dashed lines; LOD, 3.00) are shown, as established
by 10,000 permutations of the data set. Symbols with numbers repre-
sent positions of the listed Mit microsatellite markers genotyped in the
full F2 population. The 95% confidence interval for the QTLs (chro-
mosomes 11 and 17) were established by the area marked with a
1.5-LOD drop from the peak.
3212 YADAV ET AL.INFECT. IMMUN.
from a heritable epigenetic effect that is independent of inher-
itance through the paternal lineage (43, 73), so additional
testing of cross populations with paternal strain variation and
strict environmental controls is needed to better understand
the exact mode of inheritance for anthrax susceptibility.
Pathogen fate in a resistant versus susceptible genetic back-
ground. Lung pathogen burden, when measured at death,
showed interstrain variability but did not correlate with the
survival pattern for inbred strains or BXD ARI strains (data
not shown). Our comparative infection kinetics data for the
most resistant (BALB/cJ) versus most susceptible (DBA/2J)
strains showed that the initial steps in bacterial establishment
in the lung (germination of spores, initial multiplication) are
independent of the genetic background, but the rate of sys-
temic dissemination was greater in the sensitive host back-
ground. However, once the pathogen-host interaction under-
lying the disease pathology comes into play, the resistant host
genetic background seems to confer greater tolerance to bac-
terial buildup, possibly leading to the longer survival. These
observations for inbred mice contrast with those of previous
studies (31) in other animal species, which reported that nat-
urally resistant (e.g., rats) or actively immunized animals had a
lower systemic load of anthrax bacilli at death than naturally
susceptible (e.g., guinea pigs) animals. Our observed infection
kinetics pattern may be a net outcome of innate immune de-
fense (pathogen clearance) and pathogen growth or dissemi-
nation rate (31), one or both of which may vary in the two
genetic backgrounds. The identified resistant and susceptible
strains of inbred mice thus offer potential infection models for
evaluations of vaccines and therapeutics.
Multigenic nature of host susceptibility. Genetic matings
(F1 crosses) using two progenitor pairs of resistant and sensi-
tive parents, e.g., C57BL/6J ? DBA/2J and BALB/cJ ? DBA/
2J, suggested multigenic control of susceptibility to Ames in-
fection. F1 progeny from either pair showed an intermediate
survival phenotype, consistent with the involvement of multiple
genes in host susceptibility to anthrax infection process. This
observation based on the fully virulent Ames strain of B. an-
thracis is in contrast to previous observations with the avirulent
B. anthracis strains Sterne or 34F2 (55, 68, 69). The previous
Sterne studies did not observe an intermediate phenotype in
the F1 progeny, which implied that host resistance is geneti-
cally linked to a dominant autosomal locus or gene complex.
Although it is also possible that the intermediate F1 response
is due to partial dominance, decreased penetrance, or variable
expressivity of a single gene, our subsequent genetic analyses
lend strong support to a role for multiple genes.
This study showed a reverse survival time phenotype for
pulmonary anthrax spores in inbred strains compared to that
reported for anthrax LT (32, 69). In addition, comparison of
our data with those from subcutaneous challenge studies (35)
demonstrated that a different route of administration of the
same anthrax strain spores can lead to opposing responses in
the same inbred mouse strain. For example, data here showed
that C57BL/6J mice are resistant to a pulmonary instillation of
B. anthracis spores, but this same strain died quickly when
these spores were given via a subcutaneous route (35). There-
fore, data suggest that a response to pulmonary infection with
B. anthracis spores is likely controlled by a different set of
genes than that controlling the response to anthrax LT or to a
subcutaneous challenge. It is reasonable to expect, however,
that one or more of the genes involved in each of these re-
sponses may overlap.
Genome-wide QTLs underlying host susceptibility to pul-
monary anthrax. In an initial effort to identify a genetic link to
susceptibility after pulmonary instillation of anthrax spores, we
performed genome-wide SNP analysis for the survival time of
16 BXD ARI strains. Several QTLs linked to median survival
time were identified, with loci mapping to chromosomes 6, 11,
and 14. Additional loci were identified on chromosomes 5 and
9 when CIM was used to remove the variance of the QTL on
chromosome 6. As a second strategy to identify genetic regions
linked to anthrax susceptibility, QTL analysis was performed
on an F2 population derived from the BALB/cJ-DBA/2J resis-
tant-sensitive mouse pair. A significant QTL was identified on
distal chromosome 17, with support for a second locus on
chromosome 11, based on its concurrence with the results of
the BXD analysis.
An earlier report (55) proposed that resistance of inbred
mice to a subcutaneous injection of spores of the avirulent
strain 34F2 (Sterne) of B. anthracis was controlled by a single
dominant gene in the C57BL/6 (sensitive) and C3H/He (resis-
tant) mouse model. If a single gene was relevant in our infec-
tion model (with the fully virulent strain), a small ARI panel
generated from C57BL/6 (resistant) and DBA/2 (sensitive)
mice would have been sufficient to detect a significant linkage.
In fact, if under the control of a single gene, the BXD strains
would be expected to show a biphasic (i.e., sensitive or resis-
tant) survival time. The continuous distribution of survival
times for the panel of 16 BXD strains strongly supports a
multigenic trait. A set of 16 RI strains has the power to detect
linkage with 95% probability if the QTL accounts for about
60% of the between-strain variance (44). Given that ARI lines
were used in this study, the power to detect a major locus was
further improved (44). But, because several loci of apparent
similar strength were detected in this set of 16 ARI strains, it
is not surprising that a significant linkage was not identified
using this strategy. Thus, unlike the subcutaneous route, a
single major effect locus controlling survival to a pulmonary
instillation of B. anthracis is not supported by our results for
BXD strains and for a separate CD2 F2 population.
Several preliminary observations can be made for the iden-
tified QTLs. A similar chromosome 11 locus was identified for
survival time in the BXD ARI strains and for lung burden at
death (CFU) in the CD2 F2 QTL analysis. This chromosome
11 linkage maps near Ltxs1, a major QTL identified for mac-
rophage susceptibility to anthrax LT in an in vitro model using
C57BL/6 (sensitive) and C3H/He (resistant) inbred strains
(52). The gene for Ltxs1 was recently identified as Nalp1b (an
NLR family pyrin domain containing 1B) (4). The effects of
allelic variation of this gene on the outcome of LT challenge
and infection have been recently investigated (39, 61). How-
ever, the four SNPs that define the chromosome 11 QTL peak
in our study map about 7 Mbp proximal to Nalp1b, suggesting
one or more possible new candidate genes. The QTL on chro-
mosome 6 coincides with the confidence interval of the re-
cently reported Rsvs1 locus (59). This QTL controls the differ-
ential susceptibility to respiratory syncytial virus infection, as
identified in C57BL/6J and AKR/J mice by measuring lung
viral titers (CFU) at 4 days postinoculation.
VOL. 79, 2011MULTIGENIC CONTROL OF MURINE SUSCEPTIBILITY TO ANTHRAX 3213
The significant QTL on distal chromosome 17 has been
tentatively designated Rpai1, for resistance to pulmonary an-
thrax infection, locus 1. This QTL is distinct from the plague
resistance locus, Prl1, which maps to the major histocompati-
bility complex (MHC) region on proximal chromosome 17
(62). This distinction was expected, given that C57BL/6J mice
are resistant to B. anthracis but sensitive to Yersinia pestis.
Many other QTLs for differential host defense map to the
MHC region of chromosome 17, including susceptibility/resis-
tance loci for retroviruses (6), malaria (20), tuberculosis sever-
ity (54), chlamydial pneumonia (38), leishmaniasis (53), and
mouse cytomegalovirus (14). Based on mapping location, these
loci must differ from Rpai1, identified in this study. Survival
time of mice to Trypanosoma congolense infection maps just
distal to the MHC region at ?30 Mbp (21) but is still consid-
erably proximal to Rpai1. Therefore, the mapped location for
Rpai1 identifies a heretofore-unrecognized region of chromo-
some 17 for host susceptibility and thus represents a distinct
gene or set of closely linked genes affecting the differential
response to pulmonary anthrax.
In conclusion, in a survey of 14 inbred mouse stains, this
study identified several potential mouse models of spore-in-
duced pulmonary anthrax susceptibility and also revealed a
possible role of sex and the parent of origin in anthrax suscep-
tibility. These mouse models will allow us to further investigate
sex-specific factors underlying anthrax disease susceptibility
and progression, as well as the responses to treatment thera-
peutics, vaccines, and other countermeasures. Results of ge-
netic analysis of the BXD ARI strains and a separate CD2 F2
population supported multiple genes in the host susceptibility
to pulmonary anthrax, with at least four genetic loci identified
on chromosomes 6, 11, 14, and 17. The chromosome 11 locus
was identified for both survival time and lung burden post-
anthrax spore infection and was also in the vicinity (7 Mbp) of
the previously identified Ltxs1 locus for anthrax LT suscepti-
bility. The significant novel QTL on chromosome 17 appears to
be distinct from all other mapped QTLs for host defense. The
striking differences between the genome-wide linkage profiles
from the two susceptibility mouse models support our hypoth-
esis that the anthrax phenotypes are multigenic. The data fur-
ther highlight that the genetic basis for this differential suscep-
tibility likely differs for the different inbred strains and suggest
that there is not a single common genetic contribution for
resistance to pulmonary anthrax across strains. Future research
will focus on efforts to characterize these genetic loci and to
identify positional candidate genes affecting host susceptibility
to pulmonary anthrax.
This study was supported by NIH grant AI070865 (J.S.Y.) from the
National Institute of Allergy and Infectious Diseases.
We thank Manish Gupta for occasional backup technical assistance
and Shu Zheng for assistance in statistical analysis. We also thank
William Gibbons, Jr., for training and technical support with the geno-
typing of microsatellite markers. We gratefully acknowledge Babetta
Marrone of the Los Alamos National Laboratory for providing the
Bacillus anthracis Ames strain.
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Editor: S. R. Blanke
VOL. 79, 2011MULTIGENIC CONTROL OF MURINE SUSCEPTIBILITY TO ANTHRAX 3215