Murine Model of Pulmonary Anthrax: Kinetics of Dissemination, Histopathology, and Mouse Strain Susceptibility

Bioweapons are most often designed for delivery to the lung,although this route is not the usual portal of entry for manyof the pathogens in the natural environment. Vaccines and therapeuticsthat are efficacious for natural routes of infection may notbe effective against the pulmonary route. Pulmonary models areneeded to investigate the importance of specific bacterial genesin virulence, to identify components of the host immune systemthat are important in providing innate and acquired protection,and for testing diagnostic and therapeutic strategies. Thisreport describes the characteristics of host and Bacillus anthracisinteractions in a murine pulmonary-infection model. The infectivedose varied depending on the route and method of inoculation.The germination process in the lung began within 1 h of inoculationinto the lung, although growth within the lung was limited.B. anthracis was found in the lung-associated lymph nodes $~$ 5h after infection. Minimal pneumonitis was associated with thelung infection, but significant systemic pathology was notedafter dissemination. Infected mice typically succumbed to infection $~$ 3 to 4 days after inoculation. The 50% lethal doses differedamong inbred strains of mice, but within a given mouse strain,neither the age nor the sex of the mice influenced susceptibilityto B. anthracis.

INTRODUCTION

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Introduction

Bacillus anthracis is a gram-positive, spore-forming, rod-shapedbacterium that causes the disease anthrax (21). Anthrax is predominantlya disease of herbivores, and humans usually become infectedthrough incidental exposure during animal handling. B. anthracisresides in the soil as a spore, and the spores typically enterthe herbivore through ingestion and germinate into a vegetativestate in tissues. The signals for germination include aminoacids, nucleosides, and glucose (2, 30, 31). Once in the vegetativestate, the organism multiplies rapidly and produces two importanttoxins that contribute to the pathology of the infection. Pathogenesisis thought to be due to the effects of toxins and to the highnumber of organisms that reach critical target organs (3, 19,35, 41).

Anthrax in humans may take three forms, i.e., cutaneous, gastrointestinal,and pneumonic. The most common form of human anthrax is cutaneousanthrax, typically caused by the inoculation of spores intoopen wounds on the extremities of animal handlers, e.g., woolsorter's disease (28, 37). Cutaneous anthrax is a disease witha mortality approaching 20% in the untreated. Naturally acquiredpulmonary anthrax is very unusual, occurring about 1/20 as oftenas cutaneous anthrax. However, when respiratory anthrax occurs,it has a mortality approaching 100% if not treated early (21).

The United States has been aware of the potential use of B.anthracis spores as inhaled weapons of bioterrorism for manyyears and was exploring its use as a bioweapon until offensivebioweapons work was terminated in 1969 and 1970 (8). Awarenessof this threat was heightened following the use of the U.S.mail system to disseminate a weaponized form of B. anthracisspores and to induce pulmonary disease in those receiving andhandling contaminated mail in the fall of 2001 (11).

The pathogenesis of anthrax, particularly inhalational anthrax,is complex, and most studies have examined in vitro cell culturemodels in an attempt to understand the early stage of infection.B. anthracis spores that are dispersed into 1- to 5-µm-diameterparticles are likely inhaled into the alveolar spaces of thelung (6). The spores are presumed to be phagocytosed by alveolarmacrophages (20). The phagosomes fuse with lysosomes to producephagolysosomes. Many spores may be killed in the phagolysosomes,but surviving spores may remain in the lung, or those escapingphagocytosis may drain via lymphatics into the mediastinum,where they may exist as spores for an undetermined length oftime. Once spores are inside macrophages, the surviving sporescan undergo germination to a vegetative state (13), althoughwhether macrophages are absolutely required for germinationin vivo is unclear. After germination, the symptoms of infectionfollow as a result of the rapid replication of the organismsand the release of toxins. The toxins of B. anthracis consistof combinations of two different proteins: either protectiveantigen (PA) plus lethal factor or PA plus edema factor (5,25, 29). An additional known virulence factor is the capsuleof the vegetative form. The genes for the three protein componentsof the toxins are carried on the plasmid pXO1, and the capsuleis encoded by the plasmid pXO2 (24). PA is a receptor-bindingprotein that facilitates the transport of edema factor and lethalfactor into target cells. The toxins act to inhibit an effectiveimmune response against B. anthracis and directly damage cells(7, 25, 29). The role of the capsule has not been extensivelystudied, but experiments using an acapsular Sterne B. anthracisstrain that lacks the plasmid pXO2 showed that the capsule hasantiphagocytic properties that facilitate the infectious process(40). Host-related signals that affect the transcription ofthe toxin and capsule genes include temperature and the bicarbonate-CO₂balance. Additional genes that may play roles in B. anthracisvirulence include a germination operon, gerX, and a stress transcriptionfactor gene (24).

The development of well-characterized models of pneumonic anthraxis essential in order to understand its pathogenesis, as wellas to discover new, more effective vaccines and microbicidalagents. The majority of animal models for anthrax were usedto test the efficacy of vaccines against anthrax, and littleimmunopathology was described. Mice, in particular, were usedextensively for testing vaccines (4, 7, 10, 23, 33). Most murinestudies used a subcutaneous (s.c.) infection route, but onestudy described the use of an intratracheal (i.t.) infectionmodel to examine the role of irradiation in pulmonary anthraxinfection (14). For virulent B. anthracis, the s.c. 50% lethaldose (LD₅₀) was reported to be 6 to 25 spores (40). The presentreport compares the intranasal (i.n.) and i.t. routes for modelinginhalation anthrax in mice with the Ames strain of B. anthracisspores and describes the rate of germination, kinetics of dissemination,and histopathology. Finally, the model was used to test thehypothesis that genetics and age are determinants of susceptibilityto B. anthracis.

MATERIALS AND METHODS

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Materials and Methods

B. anthracis spore preparation. The original B. anthracis (Ames strain) spore stock (BA-O) wasobtained from the U.S. Army Medical Research Institute of InfectiousDiseases, Frederick, Md. To provide a new working stock (BA-NS-1),a sample of the original strain was streaked onto a blood agarplate (Remel, Lenexa, Kans.) and incubated overnight at 37°C.Prewarmed 2x SG medium (24a) was then inoculated with one colonyfrom the overnight plate culture and incubated at 37°C ona shaker. A sample from this preculture (1.25 ml) was addedto 50 ml of 2x SG medium (prewarmed) in a 500-ml filter-topplastic flask and shaken for 24 h. At the end of the 24-h period,200 ml of sterile double-distilled H₂O was added to the culture,and incubation was continued with shaking for 40 more hours.After the 40-h incubation, the sporulation of the culture wasevaluated, and no vegetative cells were observed by phase-contrastmicroscopy. The culture was then transferred to a sterile Oakridgetube and spun in a Sorvall Superspeed centrifuge using an SS-34rotor at 4,000 rpm and 4 to 10°C for 20 min, washed onetime with sterile phosphate-buffered saline (PBS), and resuspendedin 50 ml of sterile PBS. The culture was heated to 68°Cfor 40 min prior to further preparation to remove potentiallycontaminating vegetative cells. Aliquots of the final solutionwere transferred into vials and frozen at –80°C. Thetiter of the new stock was determined by plating serial dilutionsonto blood agar plates using a spiral plater (Spiral Biotech,Bethesda, Md.).

Inoculum. An aliquot of the BA-NS-1 working stock was removed from thefreezer and thawed to room temperature in a biohazard hood.The stock was then diluted in serial dilutions with sterilePBS to a desired concentration.

Animals. Mice were purchased and maintained as specific pathogen free.For most studies, female BALB/c mice were obtained from Harlan(Madison, Wis.) and were between 6 and 8 weeks of age. For analysisof strain sensitivity, mice were obtained from Harlan or JacksonLaboratories (Bar Harbor, Maine), depending on availability,and were between 6 and 10 weeks of age. Both male and femalemice were tested. For the experiments evaluating differencesamong inbred strains of mice, all mice in the experiment wereage and sex matched. The mice were housed five per cage in thevivarium located at the University of New Mexico in autoclaved,individually ventilated (HEPA-filtered) cages (Tecniplast, Phoenixville,Pa.) with autoclaved Tek-Fresh (Harlan) bedding. The mice hadaccess to irradiated food (Harlan) and autoclaved water ad libitum.The animals were allowed to acclimate for at least 7 days priorto being used in experimental studies. All protocols were approvedby the University of New Mexico Institutional Animal Care andUse Committee.

Animal inoculations. All mice were inoculated and kept in an animal biosafety level3 containment area. For i.n. inoculations, mice were lightlyanesthetized with isoflurane, and a 50-µl inoculum wasplaced on the nares for inhalation into the lungs. For i.t.inoculations, mice were anesthetized with avertin and restrainedon a small board, a small incision was made through the skinover the trachea, and the underlying tissue was separated. Abent 30-gauge needle attached by sterile polypropylene tubingto a tuberculin syringe was used; the needle was inserted intoand parallel with the trachea, and a 50-µl inoculum wasdelivered into the lung. Thirty minutes after i.n. or i.t. inoculations,lungs from three random mice were harvested and homogenized,and dilutions were plated on sheep blood agar plates to determinethe number of organisms deposited into the lung. For s.c. inoculation,doses of spores were delivered in a total volume of 200 µlof PBS using a 29-gauge needle attached to an insulin syringe.

LD₅₀ and mean time to death (MTD). For determination of LD₅₀s, 10 mice per group per dose of B.anthracis were used. The LD₅₀ was calculated according to themethod of Reed and Muench (34). The MTD was calculated by averagingthe time of death for all individuals that died within a group.

Determination of organ CFU. Lungs were removed after euthanasia by opening the chest cavityand cutting the trachea at the bifurcation. Lung CFU were determinedby homogenizing the entire lung in 1 ml of sterile PBS in tubesusing a bead beater. The homogenates were serially diluted andplated on sheep blood agar plates using a spiral plating system.Colony counts were determined using an automated counter scannersystem (Spiral Biotek). For some studies, half of the homogenatewas heated (40 min at 68°C) prior to plating. Viabilityafter heat treatment was taken as a measure of the populationof dormant spores. To control for the potential of cross-contaminationduring the harvesting and plating procedures in the lung-associatedlymph node (LALN) harvest, naïve uninfected mice were randomlyharvested along with infected mice, and their LALNs were platedas well. All uninfected mice had zero colonies in their LALNs(data not shown).

Histopathology. Each mouse received 10,000 spores i.t., and groups of threemice were euthanized at selected intervals with an overdoseof avertin. The lungs were inflated with $~$ 1 ml of 10% bufferedformalin prior to being removed from the chest cavity. The lungsand other organs were then placed in 10% formalin for >24h prior to being processed. Sectioned tissues were stained withhematoxylin and eosin prior to evaluation. A minimum of threemice were evaluated at each time point by a board-certifiedveterinary pathologist (J.H.).

Statistical analysis. Comparisons among multiple groups of nonparametric data werestatistically evaluated by Kaplan-Meier and Logrank (Mantel-Cox)analyses using the StatView statistical program (version 5.0.1;SAS Institute, Cary, N.C.). Differences were considered significantat P values of <0.05.

RESULTS

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Results

Determination of LD₅₀s for i.n. versus i.t. delivery methods. Previous studies had reported that the LD₅₀ for spores delivereds.c. was 6 to 25 spores (40). Our results support this finding(Table 1). We next compared two different methods for lung deliveryand determined the LD₅₀ for each. BALB/c mice were challengedeither i.n. or i.t. with a range of doses of B. anthracis spores,and survival was monitored (Fig. 1). The efficiency of establishingan infection varied significantly between the two methods. TheLD₅₀ for BALB/c mice was found to be $~$ 37,000 spores using thei.n. method and $~$ 1,000 spores using the i.t. method (Table 1);100,000 spores were commonly required for 80 to 90% mortalityvia an i.n. inoculation, while only 5,000 spores were neededfor 100% mortality when given i.t. In addition, the MTD wasslightly increased for the i.n. route compared to the i.t. route.

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TABLE 1. LD₅₀s for B. anthracis delivered i.n., i.t., or s.c.^a

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FIG. 1. Survival curves for mice infected i.n. (A) versus i.t. (B) with different doses of B. anthracis Ames strain spores. Groups of female mice (10/group) were infected i.n. or i.t. (as described in Materials and Methods) with the doses shown. Lung depositions were determined in three random mice from each group to assess the actual dose that was delivered to the lung.

Efficiency of spore germination induction between the two routes of inoculation. We hypothesized that one of the reasons for the difference betweenthe LD₅₀s for the i.n. and i.t. inoculation methods was thatthe i.t. method delivered a higher number of spores deeper inthe lung, where the environment may be optimal for rapid germination.To test this hypothesis, we examined the efficiencies of germinationfor the two inoculation methods. We took advantage of the factthat spores are resistant to heating at 68°C for 40 min,while spores that have started the germination process are sensitiveto this heating process. Lungs were harvested in two differentways to assess whether significant numbers of spores remainedin the trachea and large bronchi after delivery by the two differentmethods: (i) individual lobes were removed from the bronchialtree as close to the lung parenchyma as possible, and (ii) whole-lunghomogenates were collected by cutting across the bronchi atthe bifurcation of the trachea. No significant difference wasobserved between the numbers of CFU recovered by the two methods,indicating that both methods delivered similar numbers of sporesinto the lung airways. Lung tissues were subsequently homogenizedat different time points after i.n. or i.t. inoculation, andhalf of the sample was heated, while the remaining sample wasnot. Both samples were plated, and the numbers of CFU were determined.The germination efficiency was defined as follows: number ofCFU recovered from the unheated sample – number of CFUrecovered from the heated sample/number of CFU recovered fromthe unheated samples x 100. Within 1 h postinoculation, thegermination efficiencies were $~$ 90% for the i.n. route and 97%for the i.t. route. These kinetic data were comparable to theheat sensitivity of spores in in vitro experiments where sporeswere exposed to germinants (22).

Pathology associated with i.t. and i.n. spore administration. The pulmonary and extrapulmonary lesions that developed followingeither an i.t. or an i.n. inoculation of B. anthracis were similar.Figures 2 and 3 demonstrate the typical histologic lesions ofanthrax in the lung and spleen, respectively, from BALB/c miceexposed to B. anthracis by the i.t. route. At 3 to 5 h postexposure,there were no significant lesions detected in the lungs (datanot shown). After 24 h, there was acute, mild, widespread swellingand vacuolar degeneration with occasional sloughing and necrosisof conducting airway epithelial cells (compare Fig. 2A and B).Multifocal, mild attenuation of conducting airway epithelium,interpreted as epithelial sliding to cover defects left by sloughedand dead epithelial cells, was also evident. Infrequently, smallfoci of minimal fibrinosuppurative to histiocytic alveolitiswere evident in random alveoli. In the spleen at 24 h, a mildincrease in intravascular neutrophils was evident, along withinfrequent to occasional foci, consistent with lymphocyte apoptosis,i.e., nuclear fragmentation without necrosis (data not shown).

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FIG. 2. Anthrax-induced lung pathology in BALB/c mice. The top image in each panel is a low-magnification view showing the area of focus for the high magnifications below. (A) Uninfected control mice. (B) Twenty-four hours postexposure. (C) Forty-eight hours postexposure. Note the vacuolar degeneration and sloughing of bronchiolar epithelium at 24 h (arrowheads). At 48 h, the epithelium is attenuated (large arrows). Myriads of vegetative B. anthracis bacilli are present within alveolar capillaries (small arrows). Magnifications, x40 (top), x220 (middle), x400 (bottom).

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FIG. 3. Anthrax-induced spleen pathology in BALB/c mice 48 h postexposure. There are necrosis of the red pulp with hemorrhage, neutrophilic inflammation, fibrin exudation, and myriads of bacilli (large arrow). Magnifications, x50 (top), x280 (middle), x500 (bottom).

After 48 h, mild, widespread attenuation of the conducting airwayepithelium was still evident in the lungs, although the vacuolardegeneration and death of epithelial cells were substantiallyreduced (compare Fig. 2B and C). Most mice had many bacterialrods diffusely distributed within the alveolar capillaries and,to a lesser extent, within the larger pulmonary vessels (Fig.2C). Alveolar inflammation was typically infrequent to nonexistent(Fig. 2C). Occasionally, there were minimal to mild fibrinosuppurativeand histiocytic infiltrates in the adventitia of large pulmonaryveins late in the infection. Finding bacilli in alveolar capillariescorrelated with massive numbers of bacilli in the spleen aswell. These mice had massive necrotizing splenitis (red pulp)with variable congestion, hemorrhage, and fibrin depositionand aggregates of cells with nuclear fragmentation consistentwith apoptotic lymphocytes (Fig. 3). Occasionally, the onlysplenic lesion after 48 h was a slight increase in intravascularneutrophils and minimal to mild lymphocyte apoptosis, with onlysmall numbers of intrasinusoidal bacterial rods. The lungs ofthese same mice typically had only rare bacilli or no evidenceof bacilli within alveolar septae, suggesting that the disseminationphase in these mice was in its early phase.

On rare occasions, the lungs showed evidence of vasculitis andperivasculitis with bacilli in the absence of systemic dissemination(Fig. 4). In these mice, the growth of bacilli in the adventitiaof large vessels suggested the possibility of local spread ofbacilli to or from the mediastinum.

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FIG. 4. Pulmonary blood vessel pathology in anthrax-infected mice. Focal vasculitis with abundant perivascular bacilli (arrows) was observed, although rarely, in the lungs after 24 to 48 h, without evidence of dissemination. Magnifications, x50 (top), x280 (middle), x500 (bottom).

Tissues other than spleen and lung were examined histologicallyin a subset of mice. In mice with disseminated B. anthracis,nuclear fragmentation was consistently observed in several lymphnode groups, whether or not bacteria were evident. Abundantbacteria were also evident in the kidneys of mice with disseminatedB. anthracis, especially in the glomeruli, but inflammatorychanges were consistently absent. Other lesions occasionallyobserved in mice with disseminated anthrax included nuclearfragmentation within endothelial cells lining the hepatic andadrenal sinusoids, with a few bacterial rods occasionally observedin the sinusoids. In addition, there were several small intracerebralvessels in one mouse that had intravascular fibrin thrombi withfibrinoid necrosis of the vessel wall. Rare intravascular bacteriawere evident within vessels in this mouse when gram-stainedsections were examined. The brains of other mice were withoutdetectable bacteria or lesions.

Kinetics of dissemination following pulmonary spore administration. B. anthracis extrapulmonary spread is likely a multistep processrequiring germination, establishment of local infection, andthen dissemination. Previous studies by Ross in a guinea pigmodel demonstrated that B. anthracis organisms reached the draininglymph nodes of the lung between 4 and 5 h after i.t. inoculationof 50,000 spores (35). We determined whether this timing wassimilar in the murine i.t. model. We inoculated mice with twodifferent dosages of B. anthracis (5,000 and 50,000 spores),and the LALNs were harvested 5 h later. Two out of five micereceiving 5,000 spores had detectable bacteria (1 to 2 CFU)in their LALNs, while five out of five mice that received 50,000spores had detectable colonies (range, 5 to 387 CFU) in theirLALNs. No LALNs were detected on the second day of infection,suggesting that they had undergone complete cytolysis. Thisis consistent with reports of severe lymphocytolysis that occursduring human inhalational anthrax (18).

We also examined the kinetics of extrapulmonary disseminationin the murine model. BALB/c mice were challenged i.t., and lungsand spleens were harvested at different time intervals afterinoculation. Figure 5 shows that dissemination occurred betweendays 1 and 2, which was consistent with the onset of severeclinical symptoms usually observed on day 2 or 3, followed bydeath 4 to 8 h later. The number of CFU recovered from the lungsremained constant and did not increase prior to detection ofCFU in the spleen. We speculate that the significant increasein lung-associated CFU noted on days 2 and 3 was likely dueto organisms trapped in the pulmonary capillaries followingsystemic dissemination, as observed in the histological specimens.

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FIG. 5. Increases in numbers of lung CFU following i.t. inoculation of B. anthracis occurs coincident with colonization of the spleen. Mice were inoculated i.t. with $~$ 5,000 spores of B. anthracis. The data represent two separate experiments. Mice (five per group) were euthanized at 5, 20, and 40 h postinoculation, and the numbers of CFU in the lung and the spleen were determined. In experiment 2, only two mice were alive at the 40-h time point. The upper graphs show numbers of lung CFU at the three time points, and the lower graphs show numbers of splenic CFU. Each lane in the upper graphs represents a single mouse, and the lower bars show the corresponding splenic CFU.

LD₅₀ differences among different mouse strains. Previous work on the genetic differences in anthrax susceptibilityamong inbred mouse strains concentrated on the s.c. route ofinfection (39, 40). Very little information is available aboutwhether the infective dose varies among different strains ofmice when delivered into the lung. We challenged mice of differentgenetic backgrounds with B. anthracis spores by the s.c., i.n.,or i.t. route and compared the LD₅₀s (Table 2). The s.c. routeshowed the lowest LD₅₀, but no significant difference was observedamong the tested mouse strains. In contrast, significant LD₅₀differences were noted among different strains of mice afterpulmonary challenge. BALB/c mice were the most resistant, whereasDBA/2, C3H, and A/J mice were the most sensitive. The patternof sensitivity was identical for the i.n. and i.t. routes, althoughas previously shown (Table 1), more organisms were consistentlyrequired for a productive infection via the i.n. route. Separatestudies were conducted in male and female mice. No differencein susceptibility was observed between males and females inany mouse strain (data not shown).

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TABLE 2. LD₅₀s for different mouse strains challenged by different routes of infection^a

Effect of age on pulmonary infection. A potential influence of age on susceptibility to aerosol anthraxwas investigated, due to the high incidence of lethal anthraxcases that developed in middle-aged humans exposed to anthraxduring the 1979 accident at Sverdlosk or in the 2001 post officeexposures in the United States (16, 17, 26). Previous studiesanalyzing a variety of immune parameters have suggested thatboth mice and humans develop immune defects with increasingage (9, 32). Therefore, 3- and 22-month-old C57BL/6 mice purchasedfrom the National Institute on Aging were challenged with fourdifferent doses of B. anthracis spores, as shown in Fig. 1,and the LD₅₀s were determined. The LD₅₀ of 22-month-old micewas 12,500 spores, and that for the 3-month-old mice was 15,800spores.

DISCUSSION

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Discussion

We compared two pulmonary routes of infection for B. anthracis,and described the rates of germination, extrapulmonary spread,and histopathology with the goal of establishing a well-characterizedmodel for understanding the pathobiology of pulmonary anthrax.We also showed that there is genetic variability in susceptibilityto anthrax. However, no difference was observed between thesexes, and the data from one mouse strain suggest that age isnot a factor.

Pulmonary models of infectious agents have typically utilizedone of three strategies to deliver organisms into the lung,i.e., aerosolization, i.n. inoculation, or i.t. inoculation.Each of these methods has advantages and disadvantages. Nose-onlyaerosolization has the advantage of delivering the bacteriain a manner most similar to a bioweapon and, if performed correctly,will deliver organisms to the deep lung. The disadvantages includethe technical challenge of delivering identical doses reproducibly,the safety hazards associated with aerosolization of biothreats,and the fact that the delivery of particles to the alveolarregion is very inefficient in rodents (36). Mice often swallowthe majority of inhaled particles rather than depositing themin the lung. Thus, the exposure to the gut during an aerosolexposure is great and may influence the infectious process.The i.n. method requires manipulations and the ability to getthe mouse to breathe at an appropriate rate to inhale the majorityof inoculum. Depending on the characteristics of the bacteriaand vehicle, some bacteria are delivered to the upper airwaysas well as the lower airways. There is a small amount of liquidthat accompanies the inoculum that may also be swallowed bythe mouse. The primary advantage is that the technique is easilytransferred from laboratory to laboratory, and the doses deliveredare reproducible once the technique is mastered. The i.t. routeis also technically challenging and invasive but delivers reproduciblenumbers of CFU to the lung while minimizing delivery to theupper airways or gut. Still, we recognize that some of the i.t.bolus may be exhaled and then swallowed. For this reason weuse the absolute deposition dose in the lungs from random mice,rather than the inoculum, to describe i.t. or i.n. doses. Forthis report, we chose to compare the i.n. and i.t. routes ofinfection primarily because they deliver a more accurate andreproducible dose to the lungs than aerosol delivery and areless hazardous.

The pathogeneses of pulmonary anthrax were similar followingeither the i.n. or i.t. route of infection. The primary differencesbetween the two models were the lower LD₅₀ associated with thei.t. route of delivery and, in our hands, the improved efficiencyof establishing an infection with the i.t. method. The differencein LD₅₀ may be due to the mechanics of delivery. The i.t. methodlikely delivers a more reproducible dose of organisms to thedeeper lung than the i.n. route, although increasing the i.n.dose appears to overcome this problem. While fewer mice werereproducibly infected by the i.n. route than by the i.t. route,the kinetics of morbidity and mortality were similar for themice that did become infected. Mice inoculated by either routedied $~$ 48 to 96 h postinoculation, although in general, mice thatreceived an i.t. dose died closer to the 48-h time point andmice inoculated i.n. died closer to the 72-h time point.

For purposes of studying host-pathogen interactions, the optimalmodel would be one in which the paces of infection in each organand tissue compartment are nearly synchronized in all groupsof infected mice following inoculation. Unfortunately, the pulmonary-anthraxmodel demonstrates variability even among members of a singleinbred mouse strain inoculated at the same time. This variabilityshould probably be expected because of the complicated pathwayB. anthracis requires for dissemination (i.e., germination,virulence factor production, and lung penetration). The bestrepresentation of the lack of synchrony in the infection wasthe variability in the number of organisms that had disseminatedto the LALN 5 h after an i.t. delivery of 50,000 spores to thelung. Because the bacteria grow exponentially postdissemination,differences are likely amplified over the course of infection,as demonstrated by the splenic CFU data and the differencesin the times to death within a single inoculum dose.

The pulmonary and splenic lesions observed in BALB/c mice afteri.t. inoculation with B. anthracis spores were qualitativelysimilar to the lesions observed after i.n. inoculation. Mostmice showed evidence of disseminated anthrax infection by 48h. Differences between mice in the timing of lesion developmentand the overall severity of lesions were probably the resultof variations in the rate of dissemination. The most consistentlyidentified pathological lesions of disseminated anthrax appearedin the spleen. After 48 h, these lesions ranged in severityfrom mild lymphoid necrosis with few or no detectable bacillito the more common lesion of severe necrotizing and hemorrhagicsplenitis with many bacilli. Pulmonary changes at 48 h consistedprimarily of diffuse distention of septal capillaries with bacilli,with minimal to no parenchymal inflammation. In a few cases,there was mild, acute, perivascular fibrinosuppurative and histiocyticinflammation involving the hilar aspects of large pulmonaryvessels. Those mice with mild splenic lesions and few splenicbacilli often were without bacilli in the pulmonary septal capillaries,and those mice with severe splenic lesions and many splenicbacilli always had many bacilli in the pulmonary septal capillaries.None of the mice had diffuse septal bacilli without detectablebacilli in the spleen. The tight correlation between the presenceof splenic bacilli and lung bacilli and the diffuse nature ofthe intraseptal distribution of lung bacilli are consistentwith hematogenous spread of the bacilli back to the lungs aftersystemic dissemination.

Focal pulmonary vasculitis and perivasculitis with abundantperivascular bacilli occurred, but without the presence of diffusepulmonary septal bacilli or splenic bacilli. We interpretedthese cases as representing local proliferation of bacilli withinthe lungs without systemic distribution. Alternatively, thisfocal finding might indicate local spread from the mediastinumto the lungs via the connective tissue associated with the majorpulmonary vasculature.

The splenic and pulmonary lesions described in humans or nonhumanprimates that died following inhalation exposure to B. anthracisspores are very similar to those observed in mice after i.t.inoculation (1, 12, 15, 38). Splenic lesions are characterizedby splenomegaly, with congestion, hemorrhage, necrosis, andmany intrasinusoidal bacilli. Lung changes are characterizedby pulmonary septal bacilli with minimal to no inflammationin most humans and primates with disseminated anthrax. In additionto the splenic and pulmonary changes, humans and primates withdisseminated anthrax also demonstrate hemorrhagic mediastinallymphadenitis, mediastinal edema and hemorrhage, hemorrhagicmeningitis, and gastrointestinal submucosal hemorrhages. Scatteredfoci of necrotizing vasculitis have been identified in multipleorgans in more severe cases.

Recently, the pathological effects of B. anthracis lethal toxinexposure were explored in BALB/c mice (27). Major lesions wereidentified in the spleen, bone marrow, and liver. Prominenthepatocellular centrilobular necrosis was attributed to lethaltoxin-mediated liver hypoxia, with subsequent hypoalbuminemialeading to pleural effusion. The prominent hepatocellular necrosisobserved with lethal toxin exposure in mice is in contrast withour results using spores inoculated i.t. We noted minimal pathologicalchanges other than infrequent sinusoidal endothelial necrosis.The differences are likely due to the presence of other B. anthracisvirulence factors in the spore inoculation model that contributeto host pathology and death before lethal toxin production reacheshigh enough levels to cause significant liver pathology. Futurestudies are planned to quantitate the toxin levels followinginfection with live spores.

The consequences of B. anthracis delivery via the pulmonaryroute have been examined histologically in different animalmodels and following two different human population exposureevents. Information from these studies suggests that sporesintroduced into the lung are phagocytosed by alveolar macrophagesand taken to lymph nodes, where germination and replicationoccur. Our studies are compatible with these earlier histologicalfindings and provide a model for addressing host-pathogen interactionsusing molecular and immunological tools.

As an example of how the pulmonary-infection model might beused to explore host susceptibility factors, we examined whetherthe mouse strain and age might affect the ability of the hostto resist pulmonary-anthrax infection. No difference in LD₅₀was detected between male and female mice or among the differentage groups of C57BL/6 mice. However, significant differenceswere observed among different inbred mouse strains in susceptibilityto pulmonary infection with the Ames strain of B. anthracis.DBA/2 and C3HeB/FeJ or C3H/HeN mice were consistently more susceptibleto i.t. and i.n. inoculation of B. anthracis. A/J and C57BL/6mice were moderately susceptible, while BALB/c and 129X1/SVJmice were the most resistant. DBA/2 and A/J mice were also previouslyshown to be more susceptible to the avirulent Sterne strain(40). Both of these mouse strains are C5 deficient, and replenishingC5 in A/J mice produced A/J mice with a Sterne-resistant phenotype(39). In our model, C3HeB/FeJ mice, which are not C5 deficient,are as sensitive as DBA/2 mice. Therefore, genetic factors,in addition to C5 deficiency, must play a role in establishinga resistant phenotype. The strain differences in susceptibilityto the virulent Ames strain may provide a model for determiningwhat genetic factors are important for producing a more resistantlung environment against B. anthracis.

The generalized public fear that resulted from the identificationof anthrax infections caused by contamination of the U.S. mailresulted in mass disruption of normal daily life. Whether B.anthracis spores are capable of being an agent for mass destructionof human life is unclear. However, one important lesson fromthe human immunodeficiency virus epidemic is that the early,nearly paralyzing fear of this fatal disease in the 1980s lessenedonce the public understood the cause of infection and how itsspread might be prevented. Furthermore, understanding the molecularmechanisms of productive infection helped in the identificationof therapeutic targets. Crucial to developing control measuresfor B. anthracis is the recognition that the organism will beintroduced through the lungs as a bioweapon. Therefore, murinelung models of anthrax that can be used to explore pathogenicand resistance mechanisms are essential. Our studies show thatwhile the dosage that induces disease when spores are deliveredto the lung may differ depending on whether an i.t. or i.n.route is used, the outcomes and general host responses appearto be very comparable.

ACKNOWLEDGMENTS

This work was funded by the Department of Research AdvancedProjects Agency of the Department of Defense (N00178-01-C-3069to C.R.L.) and the National Institutes of Health (NIAID 1U54AI057156-01 and NHLBI RO1 HL64548 to C.R.L. and NHLBI P50 HL56384 to M.F.L.).

FOOTNOTES

* Corresponding author. Mailing address: Infectious Disease and Inflammation Program, Department of Internal Medicine, University of New Mexico Health Science Center 1, University of New Mexico, Ms.c.10 5550, Albuquerque, NM 87131. Phone: (505) 272-4450. Fax: (505) 272-9912. E-mail: clyons{at}salud.unm.edu.

Editor: J. D. Clements

REFERENCES

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