Influence of particle size on the pathology and efficacy of vaccination in a murine model of inhalational anthrax

Particle size affects deposition site

Particle size-related deposition of B. anthracis endospores has previously been shown to increase MLD and mean time-to-death (MTD) in the guinea pig model (Druett et al., 1953). A murine model has not been reported for the investigation of particle size-dependent effects of inhalational anthrax and the subsequent assessment of therapeutics. Since the A/J murine model has been reported for the evaluation of therapeutics against inhalational anthrax using B. anthracis STI delivered by small-particle aerosol (Flick-Smith et al., 2005), we utilized A/J mice to investigate inhalational anthrax caused by deposition of B. anthracis within the upper respiratory tract (URT) by using larger particles.

The Collison nebulizer and FFAG were used to expose A/J mice to 1 and 12 μm particle aerosols containing B. anthracis STI endospores from an initial starting concentration of 1.5±0.16×10⁹ c.f.u. ml⁻¹. The site of deposition was determined within 30 min of exposure (Fig. 1⇓). Differential deposition between the lower respiratory tract (LRT) and URT was observed for the 1 and 12 μm particle aerosols. The 1 μm particle aerosol deposited primarily in the lung tissue (19.7 %) compared to the nasal passages (0.2 %; P=0.0002). In contrast, the 12 μm particle aerosol deposited primarily in the nasal passages (40.9 %) compared to the lungs (1.7 %; P=0.0001). Comparably low numbers of endospores were present in the trachea and oesophagus while similarly higher levels were enumerated in the stomach for mice exposed to small- and large-particle aerosols. Large numbers of endospores were cleared to the stomach, equating to 77.3 % and 56.67 % of the total number deposited in the mice for small- and large-particle aerosols, respectively.

The deposition profiles obtained for the aerosols generated by the Collison nebulizer and FFAG are comparable to previously obtained profiles for Escherichia coli and Yersinia pestis (Thomas et al., 2008, 2009). The profile obtained in this study had much greater levels of B. anthracis in the gastrointestinal tract compared to Y. pestis. This is probably due to the higher resistance of B. anthracis endospores to the acidity of the stomach. Endospores, including those of B. anthracis, are known to be highly resistant to a range of stressful conditions including low pH (Nicholson et al., 2002; Setlow, 2006). In contrast, Y. pestis is much less tolerant and requires a large dose to initiate infection via the oral route in the murine model (Butler et al., 1982).

Inhalation of greater numbers of endospores within 12 μm particles is required to induce infection

The number of B. anthracis STI endospores necessary to induce lethal infection in the A/J murine model was determined as a function of particle size. A suspension of endospores at a concentration of 1.35±0.22×10⁹ c.f.u. ml⁻¹ was aerosolized using either the Collison nebulizer or FFAG to generate 1 and 12 μm particle aerosols, respectively. A calculated retained lung dose of 2.5×10⁴ c.f.u. for the 1 μm particle aerosol was obtained using impinger samples. This equated well to the actual retained dose of 1.13±0.14×10⁴ c.f.u. g⁻¹ from the lungs and 1.13±0.47×10² c.f.u. ml⁻¹ in the nasal passages after necroscopy. In contrast, when a 12 μm particle aerosol was generated, quantities of 3.7±0.52×10² c.f.u. g⁻¹ were recovered from the lungs and 4.2±1.31×10⁴ c.f.u. ml⁻¹ from the nasal passages upon necroscopy. Tenfold serial dilutions of the initial suspension used for challenge resulted in approximate 10-fold reductions in the numbers of endospores deposited in the lungs or nasal passages (data not shown).

Mice that inhaled a retained dose of 10⁴ c.f.u. within a 1 μm particle aerosol displayed 78 % mortality with a MTD of 101.6±10.4 h. Inhalational challenge with 10- and 100-fold lower doses resulted in reduced mortality of 33.3 % and 11.1 %, respectively. Less concentrated challenge doses resulted in no mortality over the course of experimentation. Deposition of 10⁴ c.f.u. within the nasal passages as a consequence of inhalation of 12 μm particle aerosols resulted in 56 % mortality with a MTD of 161±16.1 h. Challenge with 10- and 100-fold lower doses resulted in reduced mortality of 44.4 % and 33.3 %, respectively, whilst all animals survived challenge with lower doses. Irrespective of whether the mice were challenged with 1 or 12 μm survivors cleared the infection as evidenced from bacteriological analysis of lung and spleen tissues.

The MLD calculated for the unencapsulated B. anthracis STI strain was 2432 and 7656 endospores for the 1 and 12 μm particle aerosols, respectively, equating to a 3.15-fold increase in the numbers of endospores required to produce infection with the larger particles. The MLD obtained for 1 μm particles is similar to that obtained for the fully virulent B. anthracis Ames strain in the A/J model of 1908 endospores with a 95 % CI range of 948–3687 endospores (Heine et al., 2007) but lower than that obtained for the unencapsulated B. anthracis Sterne 7702 of 2×10⁵ endospores (Loving et al., 2007). In contrast with the results of this study, Drysdale et al. (2005) described the attenuation of B. anthracis mutants lacking the capsule biosynthetic and regulatory genes. The MLDs in the BALB/c mice were greater than 5×10⁷ c.f.u. for B. anthracis strains lacking the capsule synthetic genes (capBACD) or the capsule regulatory genes (acpAB). One possibility for the observed differences may be the influence of immunodeficiency in the two strains of inbred mice used in these studies. BALB/c mice possess a fully functional immune system, whilst A/J mice have a mutation in the Hc gene causing a deficiency in C5 complement (Welkos et al., 1986). A/J mice also have a defect in the Naip5 protein, a NOD-like receptor that activates caspase-1 to cleave proIL-1β to IL-1β. Such activity is essential for effective macrophage killing of intracellular pathogens, including B. anthracis (Kang et al., 2008, 2010). A further difference is that the mice in the study of Drysdale et al. (2005) were inoculated intratracheally. This delivers the challenge dose to the lungs, bypassing any alternative routes of infection that may be utilized by pathogens deposited into the respiratory tract by inhalation (e.g. URT lymphoid tissues or gastrointestinal tract). These alternative portals of entry for infection may not be as dependent upon capsule expression as infection via the lungs.

Utilizing all the animal data from challenge doses that deposited 10⁴ c.f.u. within the lungs or nasal passages and proved fatal (n=89), the MTD was determined as 98.7±7.5 h and 136.2±10.6 h for the 1 and 12 μm particle aerosols, respectively, significantly greater for the 12 μm particle aerosol (P=0.005). The range was 3–7 days and 3–14 days for the 1 and 12 μm particle inhalational infections, respectively. Taken together, these data indicate that compared to the infection generated by the inhalation of B. anthracis within 1 μm particles, inhalation of greater numbers of endospores is required to generate the more protracted infection via URT deposition. Similar conclusions were drawn from the study of particle size-dependent inhalational studies in B. anthracis using guinea pigs as the animal model where the respiratory lethal dose was 0.23–3.4×10⁵ and 0.7–5.7×10⁶ c.f.u. for 1 and 12 μm particle aerosols, respectively (Druett et al., 1953).

Pathological progression of inhalational anthrax differs with respect to particle size

The progression of inhalational anthrax was investigated as a function of particle size by bacteriological and histopathological analysis. The differences and similarities between the two inhalational infections are summarized in Table 1⇓, indicating greater involvement of the LRT in the 1 μm particle infection, whilst greater involvement of the gastrointestinal tract was observed in the 12 μm particle infection. Involvement of the URT was observed irrespective of the size of the particles inhaled. Immediately after aerosol deposition (0 h), endospores were primarily localized to either the lungs or nasal passages depending on the size of the inhaled particles (Fig. 2⇓). All samples at the 0 h time point were resistant to heat treatment, indicating the presence of endospores rather than vegetative bacilli. Germination of the endospores predominantly occurred within 24 h, indicated by the predominance of heat-sensitive vegetative cells at this time point. Thereafter, from the 48 h time point all samples were heat-sensitive, indicating germination of the endospores into vegetative bacilli (data not shown). Lung involvement occurred in both infections but was more pronounced in mice that inhaled endospores within 1 μm particle aerosols with numbers of vegetative bacteria approaching 10⁷ c.f.u. g⁻¹ at 96 h post-infection. In contrast, approximately 10⁵ c.f.u. g⁻¹ bacteria were present at 96 h post-infection in the lungs of mice that inhaled the 12 μm particle aerosol. Histologically, no evidence of germination was observed in the lung parenchyma, and these vegetative cells are likely to be the result of bacteraemia. Colonization of the mediastinal lymph node was significantly greater in the 1 μm particle infection (Fig. 2⇓). Mediastinitis has been observed in inhalational anthrax caused by the lung deposition of small particle aerosols in other animal models, including rabbits and non-human primates (Fritz et al., 1995; Lever et al., 2008; Twenhafel et al., 2007; Vasconcelos et al., 2003; Zaucha et al., 1998), and widening of the mediastinum is one of the clinical signs of inhalational anthrax in humans.

Deposition in the nasal passages was significantly higher with the 12 μm particle aerosol and bacterial counts remained higher than those associated with the infection caused by the inhalation of 1 μm particle aerosols over the 96 h time-course. Gross facial oedema was observed in 83 % (15/18) of mice that inhaled the 12 μm particle aerosol. This was previously observed in guinea pigs and monkeys exposed to B. anthracis in 12 μm particle aerosols indicating a localized infection (Druett et al., 1953). Interestingly, histological involvement of the NALT and degradation of the nasal mucosa was observed only on rare occurrences in both the 1 and 12 μm particle inhalational infections (Fig. 3⇓). In 8 % (1/12) and 17 % (3/18) of mice that inhaled 1 and 12 μm particle aerosols, respectively, ulceration and colonization of the nasal mucosa were evident with masses of vegetative B. anthracis cells present. Concomitantly, massive structural degradation of NALT was observed with lymphocyte necrosis by large numbers of proliferating bacteria. This represented a primary route of entry into the body for the endospores within these animals. However, this is in contrast to previous reports where NALT involvement was much more commonly observed (Glomski et al., 2007b; Loving et al., 2009). These differences may be linked to the phenotype of the bacterium: Glomski et al. (2007b) used the non-toxigenic, capsulated strain, 9602P, whilst this study used the unencapsulated strain, STI. Using the unencapsulated Sterne strain, Loving et al. (2009) demonstrated infection of NALT and the draining SLNs with dissemination to the visceral organs. Compared to our study using a 1 μm particle aerosol that deposited 10⁵ endospores in the lungs, Loving et al. (2009) deposited up to 40 times more endospores. Based on the presented deposition data (Fig. 1⇑), this would translate to increased deposition within the nasopharyngeal region. Specialized cells termed M cells are located in the epithelial layer of mucosal-associated lymphoid tissues such as NALT and Peyer's patches capable of transporting antigens across the epithelial layer. Greater interaction with these cell types is more likely with increased deposition of endospores. Indeed, M cells located in NALT have been shown to uptake group A streptococci and influenza virions (Fujimura et al., 2004; Park et al., 2003). Furthermore, it has been postulated that endospores are within the correct size range to be taken up by the M cells of Peyer's patches (Duc et al., 2003a).

Pathological changes in the SLNs were observed in 75 % (9/12) and 83 % (15/18) of those animals infected for the 1 and 12 μm particle inhalational infections, respectively. Supporting these results, colonization of the SLNs was observed in 82 % of rabbits exposed to small-particle aerosols of B. anthracis endospores (Zaucha et al., 1998). At 48 h post-infection, distension of the subcapsular sinuses was observed with the presence of abundant bacteria and focal areas of necrosis and increased numbers of tingible body macrophages (Fig. 4a⇓). By 72–96 h post-infection, structural degradation of the lymph node architecture was observed presenting as a necrotizing lymphadenitis within the subcapsular sinus and cortex. Large numbers of bacteria, severe haemorrhages and hyperaemia were present throughout the SLNs including the medullary sinuses permitting lymphatic dissemination (Fig. 4b⇓). The results presented here with the toxigenic, unencapsulated B. anthracis STI strain support the study of Loving et al. (2009) where dissemination from the draining lymph nodes into visceral organs in the A/J mouse model was linked to expression of the lethal toxin subunit (lef). Histopathological analysis indicated that the SLNs are a primary site of infection for both forms of inhalational infection. This raises the question, how do the endospores reach the SLNs? This area requires further research; however, one answer may be via the NALT, and certainly this would appear to be the case in those animals with histopathological involvement of this tissue. Supporting this theory, the URT mucosa of rodents and humans contains antigen-presenting cells (APCs) such as macrophages and dendritic cells that are known to phagocytose bacteria such as Bordetella spp. and transport them to the regional lymph nodes (Gueirard et al., 2003; Jahnsen et al., 2004; Dunne et al., 2009). Furthermore, orally delivered endospores have been recovered from the SLNs (Duc et al., 2003a). In those animals where NALT involvement was not observed histologically, it is possible that APCs within the mucosa could phagocytose the deposited endospores and transport them via the NALT to the SLNs where germination occurs. In addition, spores deposited in the oral cavity, pharynx and URT would also be phagocytosed and drained to those regional lymph nodes.

In both inhalational infections, minimal lung pathology with minimal or no alveolar inflammation was observed, indicating that infection initiated elsewhere despite obvious endospore deposition in the lungs in the case of the 1 μm particle aerosols. Furthermore, histological evidence in both 1 and 12 μm particle inhalational infections at later stages of infection indicated the presence of bacteria within the alveolar capillaries rather than primary involvement of the alveoli (Fig. 4d⇑). This supports observations from previous studies investigating murine pulmonary anthrax where involvement of the local lung-associated lymph nodes occurred rather than the lung parenchyma (Lyons et al., 2004; Glomski et al., 2007b, 2008) and is consistent with trafficking from the lungs to the local thoracic lymph nodes within alveolar macrophages and interstitial lung dendritic cells (Cleret et al., 2007). Germination has been demonstrated to occur rapidly within the alveolar tissue prior to uptake and transport of the bacteria to the local lymph nodes within professional phagocytes (Sanz et al., 2008).

Gastrointestinal involvement was more pronounced in mice that inhaled B. anthracis endospores within 12 μm particles (Table 1⇑). Whether this was the result of primary involvement due to clearance from endospores deposited in the respiratory tract into the gastrointestinal tract or secondary due to bacteraemic or lymphatic dissemination is difficult to ascertain. Primary gastritis with mucosal ulceration was observed in 17 % (3/18) of mice that inhaled B. anthracis STI within 12 μm particles. This presentation was not observed in mice that inhaled 1 μm particles. At 96 h post-infection, large numbers of vegetative bacteria were observed within the distended gastric submucosa with focal necrosis of the mucosal epithelium (Fig. 5a⇓). Intravascular bacteria were observed in the capillaries of the stomach mucosa and there was colonization of the lamina propria (Fig. 5b⇓). Supporting these findings, in cynomolgus monkeys that developed inhalational anthrax, 7 % presented with stomach ulceration and 29 % with mucosal and submucosal haemorrhages (Vasconcelos et al., 2003). Interestingly, despite differences in the size of the particles inhaled, there was no significant difference in the numbers of endospores that reached the oesophagus and stomach (Fig. 1⇑). It is known for Bacillus atrophaeus that 12 μm particles contain aggregates of endospores as opposed to the singletons that occur in 1 μm particles (Thomas et al., 2008). This may prompt the observed differences in infection kinetics through two potential mechanisms. Firstly, aggregates of endospores may survive better within the transit through the stomach and intestine. This is unlikely given that based on tolerance to the gastrointestinal tract Bacillus subtilis endospores have been evaluated as vaccine delivery systems (Duc et al., 2003a, b). A more likely mechanism could centre on the comparative ability of endospores within singlets or aggregates to germinate. Singlet endospores may germinate within the stomach and small intestine leading to killing of the resultant vegetative cells by the low pH, proteases and bile salts present in these environments. Those present in aggregates may be inhibited from germination due to the higher local concentration of enzymes that regulate germination. Enzymes such as alanine racemase and nucleoside hydrolase are known to occur in the exosporium of B. anthracis and proposed to hydrolyse small molecule germinants (Todd et al., 2003; Redmond et al., 2004). Stomach colonization was also observed in A/J mice that were exposed to aerosolized endospores of an unencapsulated strain (Glomski et al., 2008). These mice were exposed to 1 μm particle aerosols that reached concentrations of 10⁵ c.f.u. in the lungs.

Bacteriological analysis indicated that the 12 μm particle infection was characterized by greater colonization of the Peyer's patches and gastrointestinal lymph nodes (mesenteric, jejunal and colonic) at 72 and 96 h post-infection (Fig. 2⇑). Histologically, Peyer's patches showed greater activation in the 12 μm particle infection with increased numbers of tingible body macrophages evident in 72 % (13/18) of infected mice (Fig. 5c⇑). In 67 % (12/18) of mice, the mesenteric lymph nodes showed considerable pathology. Numerous bacteria were visible within the distended subcapsular sinuses with necrosis of the underlying cortex and a large increase in tingible body macrophages within the cortex (Fig. 5d⇑). These data support the findings of Glomski et al. (2007a), where Peyer's patches were found to be the primary site of infection after intragastric inoculation of mice with a non-toxigenic strain of B. anthracis. The efferent lymphatics within the Peyer's patches drain into the mesenteric lymph nodes. Both lymphoid tissues are involved in oral tolerance and immune responses to gastrointestinal infection (Kwa et al., 2006; Macpherson & Smith, 2006; Tam et al., 2008) and have been demonstrated to be targeted by orally delivered endospores (Duc et al., 2003a). Gastrointestinal involvement was not noted in the study of Druett et al. (1953), where guinea pigs and monkeys were exposed to B. anthracis within large-particle aerosols. Observations on the difficulties in initiating gastrointestinal anthrax by the oral route and the low numbers of endospores deposited (10⁶) in the animals prompted the authors to ‘conclude that anthrax developed by way of the respiratory tract and not to any significant extent through the digestive tract’. However, supporting bacteriological and histological evidence was not supplied making direct comparisons with this study difficult. Without doubt, the URT represents the major portal of entry into the body; however, in susceptible individuals the gastrointestinal tract may offer an additional route if a sufficiently large dose is inhaled. Pathology of the Peyer's patches and mesenteric lymph nodes in rabbits after aerosol exposure was observed in 15 and 59 % of cases, respectively (Zaucha et al., 1998). Colonization of the mesenteric lymph nodes has been observed in a number of non-human primate models after exposure to small-particle aerosols (Fritz et al., 1995; Vasconcelos et al., 2003).

Temporal involvement of the visceral organs (spleen, liver, kidneys, thymus and thyroid) progressed similarly irrespective of the size of the inhaled particles, coinciding with bacteraemia at 48–72 h post-infection (Table 1⇑). Irrespective of the size of the inhaled particles, in 86 % (12/14) of terminal cases, focal areas of necrotic debris and vegetative bacteria were observed in the red and white pulp of the spleen. Diffuse hyperaemia was observed in the red pulp of the spleen with increased numbers of tingible body macrophages within the white pulp (Fig. 4c⇑). In contrast, Drysdale et al. (2005) and Glomski et al. (2007a, 2008) observed that B. anthracis strains lacking the ability to express capsule did not disseminate to the spleen. Conversely, the study of Loving et al. (2007) demonstrated dissemination to the spleen using the unencapsulated Sterne 7702 strain. Bacterial concentrations reached 10⁶ c.f.u. per organ at the terminal stage of infection, comparable to the concentrations obtained in this study (Fig. 2⇑). At 96 h post-infection, significantly higher titres of bacteria were observed in the blood of animals that inhaled endospores within 1 μm particle aerosols (Fig. 2⇑). During the late stages of the inhalational infections, plugs of bacteria were observed in the kidney glomeruli with interstitial haemorrhages exemplifying the bacteraemic nature of the terminal disease. Generally, liver pathology was localized to the intravascular presence of bacteria during the bacteraemia. However, in 64 % (9/14) of terminal cases, focal necrosis of the hepatic parenchyma was observed with bacterial colonization of the sinusoids. At 72–96 h post-infection, increased numbers of tingible body macrophages were observed in the thymus with intravascular bacteria and in some cases pyknotic and karyorrhectic debris.

Meningoencephalitis was a complication in 50 % of human inhalational anthrax cases occurring in the 1972 outbreak at Sverdlosk, Russia (Meyer, 2003). Indeed, 38–77 % of rhesus macaques, African green and cynomolgus monkeys that inhaled small-particle aerosols of B. anthracis presented with suppurative or haemorrhagic meningitis (Fritz et al., 1995; Twenhafel et al., 2007; Vasconcelos et al., 2003). However, no evidence of meningoencephalitis was observed in any animals that inhaled either 1 or 12 μm particle inhalational infections. In the terminal stages of infection, bacteria were observed within the meningeal capillaries due to bacteraemic dissemination. This observation is similar to that of Zaucha et al. (1998), where much lower numbers of rabbits (18 %) presented with infection of the meninges, attributed to the rapidly fatal progression of the systemic infection limiting the opportunity for meningeal anthrax to develop. Furthermore, there was no involvement of the olfactory neurone, present in the nasal passages providing direct access to the brain. The olfactory neurone has recently been observed to be infected after the intranasal deposition of Burkholderia pseudomallei into the upper airways producing encephalitis (Owen et al., 2009).

The rPA vaccine is protective against both small- and large-particle inhalational infections

A subunit vaccine based on the rPA constituent of anthrax toxin has been developed. The rPA vaccine is efficacious against anthrax caused by the inhalation of endospores within a small-particle aerosol in murine and non-human primate models (Flick-Smith et al., 2005; Williamson et al., 2005). This study determined the efficacy of the rPA vaccine against the infection caused by the inhalation of B. anthracis endospores within large particles that deposit in the URT (Table 2⇓). The mean total anti-rPA IgG concentration was determined pre-challenge as 3.04±1.52 μg ml⁻¹ (n=30). Those mice that were challenged with B. anthracis endospores within 1 or 12 μm particle aerosols displayed anti-rPA IgG concentrations of 4.80±2.99 and 1.28±0.32 μg ml⁻¹, respectively (n=15). There was no significant difference between particle size groups (P=0.26).

Mice were exposed to approximately 10 MLDs of B. anthracis endospores within either 1 or 12 μm particle aerosols. Significant differences between the control and immunized mice were observed for the infections caused by the inhalation of B. anthracis within both 1 and 12 μm particle aerosols (P<0.05). All of the control mice that were immunized with diluent alone and challenged with a 1 μm particle aerosol containing B. anthracis died, with a MTD of 100±8.89 h. In contrast, only 87 % of mice that were challenged with a 12 μm particle aerosol containing B. anthracis died, with a MTD of 124.6±8.65 h. However, particle size did not significantly alter survival of vaccinated mice (P>0.05). Immunization resulted in 73 % (11/15) and 80 % (12/15) survival when mice were challenged with 1 and 12 μm particle aerosols, respectively. The fact that some immunized mice succumbed to infection was attributable to the immunization schedule employed with these animals exhibiting non-protective IgG titres ranging from undetectable levels to less than 0.1 μg ml⁻¹ (data not shown). Clearance of B. anthracis within survivors was confirmed by bacteriological and histological analysis of spleen and lung tissues. No evidence of immunization-associated pathology was observed in immunized mice.

Optimization of rPA formulations and immunization schedules by intranasal, intramuscular and oral delivery routes in A/J mice has previously been undertaken, demonstrating efficacy against small-particle inhalational anthrax (Flick-Smith et al., 2002, 2005; Stokes et al., 2007). This study employed a suboptimal single-dose immunization schedule to enable differentiation between groups challenged with either small- or large-particle aerosols. Based on the results of Flick-Smith et al. (2002, 2005), implementation of a two-dose schedule or increased rPA dose would have increased the level of protection provided against inhalational challenge from 73–80 % to closer to 100 % by boosting antibody titres.

This study represents the first investigation of the effect of therapeutic intervention on respiratory anthrax infection caused by deposition of endospores within the URT. The data indicate that depending on the site of deposition the infection kinetics resulting from inhalational exposure to B. anthracis endospores may differ. However, the terminal point of an overwhelming bacteraemia is the same irrespective of whether the infection initiates in the LRT or URT; merely the site of initial dissemination into the bloodstream may differ. The small-particle inhalational infection presents as a necrotizing lymphadenitis of the SLNs with primary involvement of the lung lymphoid tissue. In contrast, the large-particle inhalational infection presents as a necrotizing lymphadenitis of the SLNs and mesenteric lymph nodes with primary gastritis in a number of cases. Indeed, human atypical presentations of inhalational anthrax have been observed with primary involvement of the nasopharyngeal or laropharyngeal regions of the respiratory tract. Symptoms such as rhinorrhoea, nasal congestion and epistaxis were more prevalent than in patients suffering from typical inhalational anthrax whilst the fatality rates were not statistically different (Holty et al., 2006a, b). A recent study simulating the bioterrorist attacks in the USA using Bacillus atrophaeus endospores demonstrated that particle sizes in the range of at least 1–9 μm could be disseminated by opening a letter (Duncan & Ho, 2008). Deposition after inhalation was generally within the lungs of swine; however, in some cases, considerable nasal deposition was observed (Duncan et al., 2009). Furthermore, clearance to the gastrointestinal tract or to local URT lymph nodes was not considered. The presence of different presentations of anthrax resulting from inhalational exposure requires consideration from the perspective of an intentional release and therapeutic intervention.