Animal models of human disease play an important role in providing insight to the mechanisms of pathogenic disease. Mice provide a very useful model for studying pulmonary B. anthracis
pathogenesis because they are relatively inexpensive and easy to maintain and handle. In addition, mice are valuable for studying mechanisms of host defense during infection because of the availability of immunological reagents and knockout strains. For studying B. anthracis
pathogenesis in mice, it is critical to use a nonencapsulated strain of anthrax because previous research indicates that capsule expression is lethal in mice (21
). To circumvent capsule-associated lethality in mice, pX02−
strains of B. anthracis
have been used, but these nonencapsulated strains are attenuated in many strains of mice (49
). Complement-deficient mice, such as A/J mice or cobra venom factor-treated C57BL/6 mice, are sensitive to challenge with B. anthracis
, regardless of capsule expression (20
). The current study outlines the course of inhalational B. anthracis
disease in A/J mice after exposure to aerosolized Sterne spores, and data indicate that the dissemination and outcome of disease as well as the pathological changes in various organs are similar to other animal models (non-human primates and rabbits) challenged with fully virulent spores. Thus, using complement-deficient mice with a nonencapsulated strain of B. anthracis
allows for investigating disease pathogenesis without using fully virulent bacterial strains (pXO1+
). This makes the murine model used in the current study valuable because disease progression is similar to the progression that has been described in other animal models, and the need for high-containment facilities and use of a select agent are eliminated.
Inhalational anthrax is the most deadly form of the disease, resulting in a high mortality rate after exposure. Although other methods of spore inoculation have been used for studying inhalational B. anthracis
pathogenesis, including i.n. and i.t. instillation, the current study used exposure to aerosols for anthrax infection. The aerosol exposure method is preferable to other methods of pulmonary introduction because exposure to aerosols results in a uniform distribution of inhaled particles throughout the lung, whereas i.t. or i.n. inoculation results in a nonuniform distribution (1
). The dispersal of B. anthracis
spores in the lungs after challenge by the various routes of pulmonary exposure may impact disease progression because it can affect the ability of the macrophage to control bacilli outgrowth. Alveolar macrophages are the primary cell to phagocytose B. anthracis
spores during pulmonary infection, and once the spore germinates inside the cell, the macrophage can kill vegetative cells (23
) and control disease progression. As macrophage uptake is affected by the route of administration (7
) and the number of spores engulfed by a single alveolar macrophage has a significant effect on bacilli replication and macrophage survival (39
), selecting the appropriate mechanism of spore administration used for the study of inhalational B. anthracis
pathogenesis is important. Following aerosol delivery, spores are dispersed evenly throughout the lung, and a large number of macrophages are involved in controlling infection by engulfing spores and killing vegetative bacilli. However, an identical inoculum delivered by a method resulting in the nonuniform dispersal of spores (e.g., i.t. or i.n.) throughout the lung may alter disease progression because the number of spores engulfed by individual macrophages would be greater, and the probability of bacteria overcoming macrophage defenses would be higher (39
). Different pulmonary administration routes (i.t and i.n. versus aerosol exposure) can vary the course or kinetics of the host response, subsequently altering disease pathogenesis (31
). As pulmonary anthrax after exposure to spore aerosols is the most likely route of infection to be used in any future biowarfare or bioterrorist attack, the aerosol delivery system is preferred for modeling inhalational anthrax and studying host pathogen interactions.
In order to compare the pathology observed in other animal models of inhalational B. anthracis
to that of mice in the current study, five moribund mice challenged with Sterne spores were sacrificed, and hematoxylin- and eosin-stained sections of the lungs, liver, spleen, and heart were examined microscopically. The observations made from moribund mice were compared to previous reports detailing pathology in rabbits (51
), rhesus monkeys (12
), and cynomolgus macaques (44
). In mice, bacteria were generally present in one or more organs, with the heart, liver, and lungs being more frequently colonized than the spleen. In most instances bacteria were observed within blood vessels with little to no inflammatory response, suggesting fulminate bacteremia. Although inhalation anthrax symptoms in humans frequently include pneumonia with pleural effusions and hemorrhagic mediastinitis, pneumonia is less common in several animal models, specifically, in rabbits and macaques, which have previously been established as viable inhalation anthrax models (44
). The lower incidence of pneumonia in these animal models may be due to the relative absence of preexisting lung conditions in laboratory animals (versus humans), as was suggested previously by Zaucha et al., and may extend to laboratory mice in particular, due to the use of healthy mice with typically pristine lungs (51
In our study, two out of five mice exposed to aerosolized B. anthracis
exhibited clusters of bacteria within pulmonary blood vessels in the absence of an inflammatory response. Pulmonary hemorrhages were observed in two mice, with occasional attenuation of airway epithelium. One mouse presented with extensive pulmonary lesions which were primarily regions of hemorrhage with necrosis and consolidation. Similar findings were noted in an inhalation anthrax model using cynomolgus macaques, as 100% of non-human primate lung sections showed hemorrhages (44
). Vegetative B. anthracis
bacilli were apparent in 79% of lungs, while congestion (64%), edema (50%), and inflammation (29%) were also noted in many of the affected macaques. In a rhesus monkey model of inhalation anthrax, 15% of infected animals showed anthrax-related pneumonia with mild neutrophilic infiltrates and bacilli in the alveoli. Alveolar hemorrhages were observed in 31% of monkeys (12
). Bacterial loads in the lungs of moribund mice varied, with most animals exhibiting changes in lung CFU values with heat treatment, indicating the presence of vegetative bacilli in the lungs of most of these animals. Interestingly, although the total number of CFU in the lungs of moribund mice was high, the number of heat-resistant CFU (spores) was not reduced relative to day 1 (Fig. and ). This observation suggests that the increase in the lung CFU counts in moribund mice is due to the presence of vegetative bacilli in the pulmonary vasculature. This finding is consistent with histological changes in the lungs, as bacilli were present in the vessels of most moribund mice. As it is difficult to determine the time at which bacteremia occurs, some mice may exhibit signs of death early after bacterial dissemination, whereas others may not become moribund until the systemic bacterial load is extremely high (108
CFU) and the pathology more severe. Mice typically exhibit moribund symptoms for up to 12 h, and the length of time animals displayed these symptoms before being identified and euthanized for tissue collection were likely different. This may help to explain pathological differences observed between moribund mice.
The liver histopathology observed in cynomolgus macaques challenged with fully virulent anthrax were similar to those seen in mice in this study, with 71% of macaques and 60% (3/5) of mice exhibiting bacteria in hepatic sinusoids and with 30% of the macaques and 20% (1/5) of the mice displaying hepatic necrosis. However, acute inflammation (leukocytosis) was apparent in nearly all cynomolgus monkey livers, which was not observed in the mice. Interestingly, the most common hepatic finding in the rhesus model was Ito cell hypertrophy (6 of 13 monkeys), which has not been reported in any other animal model, nor was it noted in our mice.
Other hepatic findings in rhesus macaques were foci of hepatocellular degeneration and necrosis in 23% of monkeys (3/13), which was similar to findings reported in other animal models, and acute hepatitis, which was present in 23% of rhesus monkeys. Focal hepatic necrosis was present in one out of five moribund mice in the current study. Specific hepatic findings were not reported in the rabbit model (51
). The differences between the hepatic findings observed in mice in the current study and other animal models may be due to the sensitivity of A/J mice to anthrax infection, as these mice do not survive long enough to mount a strong, inflammatory immune response. Though TNF-α levels were elevated in the liver early after infection, the levels were only twofold higher than in uninfected animals. In addition, the bacterial loads in the livers of moribund mice were substantially higher than loads in mice on days 1 and 3 after challenge, in which TNF-α levels were measured. Taken together, early bacterial loads in the liver of mice may not induce a robust proinflammatory cytokine response that results in the infiltration of inflammatory cells, which likely explains the differences in acute inflammation and hepatic pathology observed in other animal models.
The splenic lesions in our mice most commonly exhibited lymphocytic depletion that was often accompanied by necrosis and occasional hemorrhage. In the rabbit model of B. anthracis
infection, all of the rabbits exhibited an acute fibrinous splenitis (51
). Rhesus monkeys showed lymphoid depletion, histiocytosis, hemorrhage, or acute splenitis with or without necrosis (12
) while the spleens of cynomolgus macaques had suppurative inflammation and fibrin (44
). Through the first 5 days of infection in mice, on average, less than 40% of challenged mice had CFU in the spleen, and those that were culture positive had relatively low CFU counts (5.1 × 104
). However, nearly all moribund mice had approximately 106
CFU in the spleen, indicating bacterial dissemination at the end stages of disease. The large bacterial burden in the spleen of moribund mice is likely accompanied by large toxin production by the bacteria, which may result in lymphoid depletion.
Two rhesus monkeys exhibited extensive foci of hemorrhage in the myocardium (15%), and acute myocarditis was found in one of the monkeys (12
). Cynomolgus monkeys showed myocardial hemorrhages (29%) and inflammation (29%) (44
). In our mouse model, four out of five mice (80%) had bacteria present in the vessels or myocardium with little to no inflammatory response. Histopathological findings were not specifically reported for the heart in the rabbit model although the authors noted that bacilli were observed within the vasculature of nearly all tissues examined (51
Similarities in disease progression and lesions in mice challenged with aerosolized Sterne spores compared to rabbit and cynomolgus or rhesus macaques challenged with fully virulent spores suggest that the mouse is a valuable model of inhalation anthrax. Using an aerosol challenge will allow for characterization of immune responses in the lungs and lymph nodes of infected animals that is most relevant to inhalational anthrax. Dissemination of B. anthracis
after inhalation of spores is suggested to occur after spores are phagocytosed by APCs in the lung and transported to the draining lymph nodes (15
). During this time, germination and vegetative outgrowth occur in the cell. Data from the current study concur with previous findings and show that spores do not germinate in the lung, but spores and vegetative bacilli can be found in the draining lymph nodes within a day of infection. In addition, even 5 days after challenge, minimal pathology in the lung was observed. Only when bacteria have disseminated throughout the body and vegetative bacilli return to the lungs, as seen in moribund mice, are histological changes observed in the lungs. Together, these data indicate that the lungs serve as a mucosal port of entry, but the germination and outgrowth of spores in the pulmonary draining lymph nodes are likely the primary sites of activation of the host immune response after pathogen recognition.
The utility and application of the described murine aerosol challenge model was used to investigate in vivo cytokine responses after inhalation of spores. TNF-α is a potent proinflammatory cytokine produced after host recognition of invading microbes by innate Toll-like receptors. Although TNF-α is primarily produced by macrophages, it can also be secreted by lymphoid and endothelial cells. TNF-α stimulates endothelial cells to produce proteins that alter vascular permeability in an attempt to control microbial spread into the bloodstream. In addition, TNF-α can stimulate the maturation and subsequent migration of dendritic cells to the draining lymph nodes. TNF-α has a role in controlling intracellular and extracellular microbial pathogens, as TNF-knockout mice exhibit heightened sensitivity after challenge with Mycobacterium tuberculosis
), Listeria monocytogenes
), or Leishmania
). Following inhalational B. anthracis
challenge, the draining lymph nodes showed a marked increase in TNF-α 1 day after challenge; however, these levels decreased by day 3. TNF-α may play a role in inducing the migration of infected APCs to the draining lymph nodes early after challenge. It has previously been shown that murine macrophages infected with B. anthracis
spores produce TNF-α (34
). However, as the bacteria replicate and begin producing toxins, the toxins may block TNF-α production by blocking mitogen-activated protein kinase kinase signaling (13
). These data, in conjunction with data that show the presence of both spores and vegetative bacilli in the lymph nodes of challenged animals, suggest that lymph node TNF-α levels decrease at nearly the same time bacterial burdens in distal organs (liver and spleen) increase. Normally, TNF-α is involved in controlling disease progression, and B. anthracis
toxins may block proinflammatory cytokine production in the lymph nodes which allows for bacterial dissemination (13
). Further studies are warranted to investigate TNF-α levels in moribund mice, as well as levels of other proinflammatory cytokines, such as interleukin-1 and interleukin-6, which have shown to be increased in primary cells infected with spores (33
This report summarizes the course of inhalational anthrax disease in mice after aerosol exposure and demonstrates that the murine aerosol challenge model is both relevant and useful for the study of B. anthracis pathogenesis. The course of anthrax disease in complement-deficient mice (A/J) challenged with aerosolized Sterne spores (pX01+ pX02−) is similar to that observed in other animal species (rabbits, guinea pigs, and non-human primates) challenged with fully virulent B. anthracis, as bacterial dissemination and pathological changes observed in mice were similar in comparison. We were able to use the aerosol challenge model to show changes in TNF-α levels in lungs, liver, and lymph nodes during the early stages of disease. This challenge model will be valuable in understanding inhalational B. anthracis pathogenesis and identifying components of the host immune system required for bacterial clearance and survival after aerosol infection. In addition, it can serve as a model for screening a new generation of vaccines and postexposure therapeutics.