B. anthracis Challenge Isolates
The selection of B. anthracis
isolates used in animal models of anthrax vaccine efficacy is dependent on several factors including the virulence of the isolate, the animal species, and the type of anthrax vaccine (cell-free versus modified live spore vaccine). Nonhuman primates, guinea pigs, and rabbits are susceptible to B. anthracis
infection when exposed to aerosolized spores by inhalation. The rhesus macaque has a long history as a model of human inhalation anthrax, dating back to the 1950s (7
). Guinea pigs and rabbits have also been used to test the efficacy and potency of various cell-free anthrax vaccine preparations (3
). The initial studies performed in the United States, designed to determine the efficacy of the first human cell-free anthrax vaccines in rhesus macaques, utilized the Vollum isolate of B. anthracis
to challenge the animals (86
). Early studies of inhalation anthrax in macaques performed in the United Kingdom utilized the M.36 B. anthracis
isolate, which was derived from the Vollum isolate by repeated passage in monkeys (7
). Auerbach and Wright were the first to report that vaccination with cell-free anthrax vaccine produced variable protection in guinea pigs challenged with various B. anthracis
isolates except for the Vollum isolate (3
). Further analysis of the comparative efficacy of both modified live spore and cell-free anthrax vaccines in guinea pigs revealed that a modified live spore vaccine could protect guinea pigs against multiple isolates of B. anthracis
while the cell-free anthrax vaccine provided good protection only against the B. anthracis
Vollum isolate and Vollum derivatives (56
). Table lists survival data for guinea pigs vaccinated with the cell-free anthrax vaccine or the modified live spore vaccine and challenged with six virulent B. anthracis
isolates. Fellows et al. also examined the efficacy of the cell-free anthrax vaccine in guinea pigs challenged with 100 50% lethal doses (LD50
) (Ames isolate equivalents) of 8 human B. anthracis
isolates, 21 animal B. anthracis
isolates, and 4 environmental B. anthracis
isolates inoculated intramuscularly (22
). As expected, survival rates varied by B. anthracis
isolate from 6 to 100%, indicating that protection induced by the cell-free anthrax vaccine was limited in guinea pigs to only a few B. anthracis
isolates. Because of these studies, the B. anthracis
Ames isolate was referred to as a vaccine-resistant isolate and was selected as the challenge isolate for most of the vaccine efficacy studies performed in the United States and the United Kingdom after 1986. Furthermore, the guinea pig is not suitable as an animal model for testing the efficacy of cell-free anthrax vaccines due to the limited protection afforded by these vaccines in this species. However, guinea pigs continue to play a role in determining the virulence of B. anthracis
isolate in vivo and in testing the potency of newly manufactured lots of human cell-free anthrax vaccines prior to efficacy studies with larger and higher-order species such as rabbits or nonhuman primates. We, along with other researchers, have observed that the virulence of particular isolates can vary over time and between laboratories (unpublished data). Coker et al. have hypothesized that virulence among pX01- and pX02-containing isolates of B. anthracis
may be due to the clonality of the bacteria and the plasmid copy number (15
). Complete characterization and standardization of the challenge isolates used for vaccine efficacy studies may be required to interpret the results.
Survival of guinea pigs after immunization with the human cell-free anthrax vaccine or Sterne live-spore vaccinea
The 50% Lethal Dose
The LD50 of a toxin or biological agent is defined as an estimate of the dose at which death occurs in 50% of the population to which the agent was administered. The LD50 of B. anthracis varies both by animal species and by the route of administration within a species. In addition, it often varies between strains or isolates of B. anthracis and may change over time because of passage in the laboratory.
While many statistical methods may be used to estimate the LD50
, two of the more common statistical methods are dose-response models, such as probit analysis and the Spearman-Karber Method (23
). Published LD50
s are estimates of the “true” value for a given animal model, particular B. anthracis
isolate, and route of administration. Briefly, in the probit dose-response model, an underlying assumption is that each member of the population has a threshold tolerance for the agent being tested and that, if exposed to that level or greater, the individual responds or dies. A normal distribution is used to model the population tolerances to a specified dose. Dose-response models may be used to estimate other percentiles of the dose-response curve, as well as the LD50
. The Spearman-Karber method is a nonparametric statistical method that provides a point estimate of the LD50
with confidence intervals, which makes no assumptions about the distribution of the population survival probabilities. Confidence intervals are typically reported side by side with an established LD50
, with the 95% confidence interval being the most common. The 95% confidence interval is interpreted as the probability (0.95) that the interval straddles the population LD50
(i.e., an interval in which we are reasonably confident that the “true” value will fall somewhere within those limits) (24
). A more detailed explanation of these two statistical methods for determining LD50
values can be found in references 23
The reported LD50
of B. anthracis
spores in rabbits and nonhuman primates are presented in Table . The inhalation LD50
s reported for the Ames and Vollum isolates of B. anthracis
in rhesus macaques and cynomolgus monkeys are comparable (5.0 × 104
to 6.2 × 104
), with the exception of the data reported by Glassman (31
). The specific B. anthracis
isolate was not reported and may be partially responsible for the low LD50
(4.1 × 103
) reported by Glassman for the cynomolgus monkey. The LD50
for the chimpanzee was determined from the information reported by Albrink and Goodlow, using the Spearman-Karber method (2
). The 95% confidence interval could not be determined due to the small number of animals used on the study. LD50
s for other routes of administration or different isolates of B. anthracis
in nonhuman primates are not available. The single reported inhalation LD50
for the Ames isolate of B. anthracis
in rabbits is approximately twofold higher than the reported inhalation LD50
in nonhuman primates; this may not be significant, given that the upper limit of the 95% confidence interval for the B. anthracis
Ames isolate in cynomolgus monkeys overlaps with the rabbit LD50
). Limitations on these published LD50
s include the following the data (i) were generated several decades ago in some cases, (ii) the age of the animals varied between studies, (iii) various spore preparations and production methods were used, (iv) the health status of the animals was unknown or poorly documented, (v) some of the nonhuman primates were used in previous non-anthrax-related studies, and (vi) there was no standardized method for the generation, quantification, and particle sizing of the aerosol.
Published LD50s for rabbits and nonhuman primates
The characteristics of infection with B. anthracis
in humans, as seen by gross and microscopic observations, have been reproduced in multiple animal models. The most thorough documentation of the pathology in human anthrax cases revolves around the Sverdlovsk epidemic of 1979, in which a large quantity of B. anthracis
spores was released from a military installation in the former Soviet Union (62
). In addition, several sporadic cases of inhalation anthrax in humans have been documented over the years (6
). The comparison of the documented pathologic changes in human infection with those due to experimentally induced anthrax in laboratory animals can provide useful information concerning the suitability of a species for a model.
The gross and microscopic observations of human inhalation anthrax focus primarily on mediastinal, hemic-lymphatic, and pulmonary changes. Changes in the brain associated with hemorrhagic meningitis have also been a focus of pathologic study. Gross mediastinal lesions in human beings consist primarily of edema and hemorrhage, with similar changes within the parenchyma of mediastinal lymph nodes. This finding is considered typical; however, its presence can be variable (1
). Histologically, these findings appear as a fibrin-rich fluid exudation within the connective tissue of the mediastinum as well as “low-pressure” hemorrhage (34
). Mediastinal lymph nodes often exhibit hemorrhage and necrosis characterized by lymphocytolysis. The presence of gram-positive bacilli has been demonstrated in these lesions by conventional staining and immunohistologic techniques. Vasculitis within the mediastinal lymph nodes is characterized by fibrinoid necrosis and infiltration by neutrophils and histiocytes.
Gross splenic pathology in cases of human infection is variable and can be insignificant. Histologically, the spleens have exhibited lymphocytolysis within the periarteriolar lymphoid sheaths and the lymphoid follicles. Variable severity between the lymphoid follicles (B-cell rich) and the periarteriolar lymphoid sheaths has been documented (1
). Moderate neutrophil infiltration has been observed, as well as the presence of extracellular and intracellular bacilli. Vasculitis appears to be a minimal characteristic in the spleen.
The literature describes a great deal of variation in the pulmonary lesions associated with inhalation anthrax. Some of this variability appears to be associated with preexisting pathology in the pulmonary parenchyma. Clinically, anthrax patients with preexisting illnesses, such as chronic obstructive pulmonary disease and pulmonary fibrosis, have been described (6
). Acute bronchopneumonia was described histologically in a significant percentage of the Sverdlovsk cases (62
). However, the association of pneumonia with infection by B. anthracis
in antibiotic-treated and untreated humans is poorly characterized, since primary infection of the lung parenchyma is usually absent. The gross findings in the lungs are commonly unspectacular, being limited to hemorrhage, edema, and atelectasis, with no apparent change in consistency and weight. Histologically, mild fibrinous exudate, hemorrhage, and alveolar histiocytosis are predominant, along with some characteristics of interstitial pneumonia, characterized by interstitial fibrin deposition. Bacilli have been identified in alveolar air spaces, with a majority being found within the alveolar exudate. It has been suggested that this accumulation of bacilli is hematogenous and is due to rupture from the interstitium rather than aerogenous deposition (28
). Vasculitis is typically fibrinoid but minimal.
Grossly, multifocal to coalescing areas of hemorrhage often form the extent of the neural lesions. Histologically, minimal to mild fibrin exudation, acute low-pressure hemorrhage, and infiltration by neutrophils, histiocytes, and small numbers of lymphocytes and plasma cells is documented. Mild diffuse neuronal necrosis has been observed (34
). Bacillary infiltration appears to be limited to the intravascular and Virchow-Robins space.
Lesions in other organs besides the lymphoid tissues, lungs, and brain have been described, but many appear to be secondary to shock and agonal changes. The presence of bacilli in the sinusoids and glomerular capillaries appears to the most significant finding, further demonstrating the presence of septicemia.
The significance of the vasculitis found in multiple organ systems is uncertain. The histopathologic characteristics appear to be uniform among organs, indicating a generalized condition. Fibrinoid vascular necrosis is often associated with severe endothelial damage but is not a specific finding in generalized bacterial septicemia. In the Sverdlovsk outbreak, vasculitis was a variable finding depending on the organ system; however, in certain tissues, such as the lungs, vasculitis was a significant finding. The etiology of this vasculitis is not apparent. It is possible that it is induced directly by anthrax toxins or is secondary to cytokine release.
Inhalation anthrax has been studied in a variety of animal models including guinea pigs, rabbits, rhesus macaques, cynomolgus macaques, and chimpanzees. It is important to note that many of the gross and microscopic lesions found in humans with inhalation anthrax are similar to those found in experimental animal models of inhalation anthrax, suggesting a shared pathogenesis. However, variations do exist among animal models.
The experimental pathology of anthrax in primates is the most thoroughly documented. The gross findings of primate inhalation anthrax reflect the documented human lesions. Mediastinal enlargement due to different degrees of edema and hemorrhage is consistent in chimpanzees, rhesus macaques, and cynomolgus monkeys; however, in the last of these, this lesion was found in less than 40% of the experimental group (83
). The lesions in hemic-lymphatic organs consist primarily of grossly observable hemorrhagic lymphadenitis. Histologically, these lesions appear as lymphocytolysis and hemorrhage with intralesional bacilli, similar to the lymphadenopathy in humans. However, splenic pathology was different between human beings and nonhuman primates, since all of the nonhuman primates studied demonstrated splenomegaly. In addition, the severity of the neutrophilic inflammation and the fibrin exudation appears to be more severe in the spleens of nonhuman primates. These lesions are in addition to the lymphoid follicular necrosis, which takes place in both nonhuman primates and humans. The pulmonary findings in primates are remarkably similar to those in humans. It is interesting that preexisting disease in these experimental animals appears to compound the anthrax-associated lesions. The presence of Pneumonyssus simicola
in the lungs of rhesus macaques, as reported by Gleiser et al., may enhance the phagocytosis of B. anthracis
spores by alveolar macrophages or allow the bacilli easier access to the systemic circulation (32
). This mirrors our earlier statement regarding preexisting injury in lungs of humans and susceptibility to pulmonary B. anthracis
infection. However, other studies of rhesus macaques have shown a lack of tropism to preexisting lesions, suggesting that specific lung lesions may not increase the susceptibility to infection. The meningeal and cerebral lesions in rhesus and cynomolgus macaques, when present, appear similar to those described in humans. The clinical signs associated with inhalation anthrax in rhesus macaques, cynomolgus monkeys, and chimpanzees have been described as nonspecific and include lethargy, anorexia, and depression (2
). The proportion of nonhuman primates with inhalation anthrax that develop overt clinical signs associated with meningitis is considerably smaller than would be expected, considering that meningitis is often a frequent histologic finding in these animals (28
). In some reports, mild perivascular suppurative encephalitis was a microscopic feature in addition to the characteristic fibrin exudation and hemorrhage. In all organs with lesions, bacilli were seen in touch impressions, with routine hematoxylin and eosin staining, or by immunohistochemistry. Other lesions of interest seen in nonhuman primates but not in humans have been noted. Multifocal myocardial necrosis has been seen infrequently in cynomolgus monkeys (83
). Gastrointestinal lesions have ranged from mild to moderate hemorrhage and occasional coagulation necrosis in cynomolgus monkeys to transmural acute colitis with necrotizing vasculitis. Models of inhalation anthrax for pure pathologic studies with nonprimate laboratory animals are infrequently documented in the literature. A model utilizing rabbits has been described recently (22
). Studies of guinea pigs and dogs were described approximately 50 years ago. Although some of the lesions observed in rabbits were similar to those found in primates, the lesions in rabbits appear to be less severe (88
). These lesions include mediastinal hemorrhage, splenomegaly, and typical pulmonary changes. Encephalitis is minimal to nonexistent. This discrepancy may be attributed to the rapid progression of disease in this species. Presumably, the inflammation and necrosis do not have time to develop in rabbits, in contrast to primates.