This study is the first to track a comprehensive set of parameters indicative of toxemia and bacteremia over the course of inhalation anthrax in rhesus macaques. It used the most sensitive assays for toxin detection (LF) and bacterial load (PGA) available. The results yield important insights that will support anthrax diagnostics, therapeutics development, and clinical treatment. The results also lead to an improved understanding of anthrax pathogenesis and innate cell changes which may reflect the host's early immune responses to B. anthracis nascent infection.
The results indicate that the MS detection of functional LF provides an improved diagnostic tool for inhalation anthrax in nonhuman primates. The detection of LF in rhesus macaques as early as 24 h postexposure and over the course of a 96-h to 120-h (4- to 5-day) infection period demonstrates the importance of this approach for early diagnosis of inhalation anthrax (Table ). Kobiler et al. (22
) previously reported using a PA capture immunoassay to monitor progression of anthrax toxemia in serum from infected rabbits and guinea pigs. However, measurements with this assay were achieved only over the last 24 h of infection and ranged from 1 ng/ml up to approximately 105
). Mabry et al. used capture immunoassays for PA and LF in infected guinea pigs and rabbits (25
). However, PA was measured only in late infection or at death (<9 h before death) in guinea pigs (0.1 to 1.7 μg/ml), and PA and LF were measured only in rabbits at terminal time points at 80 to 100 μg/ml and 11 to 15 μg/ml, respectively (25
). A recent report on a europium nanoparticle-based ELISA for PA has described detection limits of 0.01 ng/ml (40
), but in agreement with previous studies (22
), PA was detectable only in the later stages of infection in mice (40
). With the improved detection limits of the functional LF MS assay at 0.005 ng/ml and higher levels of LF demonstrated in early infection, we have demonstrated a clear advantage of using LF for earlier detection of systemic anthrax. Functional LF levels as low as 0.006 ng/ml were detected at 24 h using MS, levels that would not be detectable using previously published technologies (25
). These data emphasize that LF is the better target for early detection of anthrax.
PA in this study was not detectable until euthanasia and in only three animals, which indicates that PA levels at all time points preceding death were below the 4.84 ng/ml detection limit for the PA ELISA reported here. Nonetheless, PA detection provided an additional confirmation of anthrax-specific toxemia. The late-infection PA levels reported here of 147 to 19,434 ng/ml correspond with those reported previously for guinea pigs at 100 to 1,700 ng/ml but are much lower than reported for rabbits at 80 and 100 μg/ml (25
Although there are differences in detection limits between the LF and PA assays described here, the measured LF levels at the early time points 48 to 72 h were higher than the 4.84 ng/ml detection limit for PA, and therefore, LF levels were higher than PA at these early time points. These data suggest that either more LF is produced or this antigen is less rapidly sequestered by host tissues than PA at earlier time points, and thus the LF/PA ratios in serum are high. Conversely, we observed that LF/PA ratios were lower in late infection. These lower ratios during late infection in rhesus macaques may be due to saturation of host receptors, decreased cellular uptake, and thus accumulation of PA in serum (30
). Accumulation of serum PA in late infection is in agreement with previous studies in rabbits (22
), guinea pigs (25
), and mice (40
). Consequently, lower serum LF/PA ratios will reflect the uptake of LF via the PA-primed host cells.
These findings raise interesting questions about appropriate interventions after exposure to or infection with B. anthracis
spores. One interpretation of the low serum PA levels at 24 to 72 h is that PA is rapidly adsorbed to host tissues (30
), and thus circulating PA is the rate-limiting toxin component for potentiating early infection and the associated bacteremia of anthrax. In this context, a potential concern might be raised that an rPA vaccine given postexposure during this phase might facilitate additional uptake of LF and thus exacerbate the infection.
The perceived risk of potentiating the infection due to postexposure vaccination with the current U.S. licensed anthrax vaccine and for rPA vaccines in development is low. First, in the course of the overall infection the rate limitation is most likely PA processing and turnover at the host cell receptor rather than the levels in serum. Second, PA is not readily released from an aluminum hydroxide vaccine formulation (15
). Third, the quantity of PA contributed by a vaccine given intramuscularly to a patient would be insignificant in comparison to the quantity produced by the bacterium at that stage of infection. Fourth, the anthrax vaccine is formalin treated which is anticipated to inactivate its protein components (18
), thereby reducing the risk of additional toxin uptake. Furthermore, the likelihood of vaccination during infection is low; the anthrax vaccine is contraindicated in persons who have recovered from anthrax (9
), and where vaccination is recommended for use in a postexposure setting prior to onset of infection, it is coadministered with antibiotics (8
The PGA capture immunoassay also represents a valuable diagnostic tool and facilitates a surrogate measurement of bacterial load (23
). PGA was consistently detected from 48 h onwards postexposure. The rapid increases in PGA at 48 h and declines at 72 h are reflected in the positive 48-h and negative 72-h bacteremia. Compared to LF, PGA was much higher than LF at 48, 96, and 120 h. This probably reflects the accumulation of PGA in the blood when bacteremia is positive, whereas some LF secreted by the organism is sequestered intracellularly. PGA declined much more rapidly at 72 h than LF, which may be due to the rapid clearance of bacilli; the cellular uptake of LF may be slower, allowing a portion of LF to linger in the blood in the absence of bacteremia (44
). Future studies planned may define how quickly PGA clears from the blood with antibiotic treatment.
LF and PGA were highly correlated with a coefficient of linearity (r2
) of 0.869, which is similar to that reported previously for PA and bacteremia in rabbits (r2
= 0.864) (22
). Since PGA correlates with bacterial levels (23
) and LF correlates with PGA, it is likely that LF may also correlate with bacterial levels and therefore may represent an alternative measurement for the stage or severity of infection. Studies in progress may yield additional measurements to support this association.
While LF and PGA were consistently detectable in RMS, reversion in bacteremia and pagA PCR reactivity over time provides a key insight to the onset and progression of systemic anthrax. Although most animals were bacteremic and had detectable pagA at 48 h, some reverted to a negative result at 72 h. These changing response patterns for pagA detection and bacteremia define a potential window of diagnostic uncertainty for these methods. Culture-dependent methods may fail if a patient is presented during this critical window of infection. This illustrates the importance of applying multiple analyses for diagnosis of anthrax and emphasizes the central role that the high-sensitivity and high-throughput capability of MS provides as a diagnostic technology.
A triphasic kinetics profile for B. anthracis infection in rhesus macaques was defined with a 48-h increase in LF, PGA, and percent neutrophils, followed by 72-h declines, before the final surge in parameters of infection and subsequent death. Importantly, the reversal of bacteremia from positive detection at 48 h to negative detection at 72 h provides compelling evidence supporting these quantitative declines. All elements define a 72-h decline in infection and represent tangible measures of antigen (LF) and microbial (PGA and culture) clearance in early anthrax infection. A similar but shorter triphasic profile for LF was also observed in rabbits and suggests that they may also be mounting innate cell changes that lead to antigen clearance (unpublished data).
Our data from rhesus macaques suggest an important contribution by neutrophils in the innate cell changes leading to temporary reduction in bacterial burden and antigenemia. The 48- and 72-h kinetics of neutrophils mirrored those of the parameters of infection with a 48-h increase and 72-h decline. Since the percent neutrophil kinetic profiles were consistent between all five infected animals, it is the most likely immune source responsible for the temporary clearance of microbial products observed at 72 h. We conclude from our data that there are two critical time periods during the infection process in rhesus macaques. The earliest period is at the spore-tissue interface. This first interaction of microbes with tissues initiates signaling events that sequentially recruit neutrophils (1 to 2 h) and then macrophages and other phagocytic cells to the site of tissue inflammation (10
). The second critical period coincides with the appearance of bacilli in the blood. Mature neutrophils are released from the marrow pool into the blood in response to increased demands for neutrophils during tissue inflammation (12
). Thus, blood neutrophil levels may be used as an indicator of increases in neutrophils at the inflammatory sites, similar to that reported to occur for monocytes (13
). In this context, the increase in neutrophils at 2 h in four of the five animals suggests a role for neutrophil involvement in the earliest stages of infection at the spore-tissue interface. However, the second increase in the neutrophils at 48 h in all five animals was consistent with the onset of bacteremia and increase in LF and PGA. This suggests that the neutrophils responded appropriately to the initial infection threat at 2 h and to the escalating sepsis at 48 h, which was followed by comprehensive declines in parameters of infection. It is interesting to note that animal D, with the highest neutrophil response at 2 h and throughout infection, had the lowest parameters of infection and survived an additional 96 to 120 h (4 to 5 days) longer than the other animals, indicating that the neutrophils may have contributed to the delay of fulminant bacteremia in this animal.
Data supporting the importance of neutrophils and their products in anthrax infection (3
) and other important infections (26
) are substantial. In vitro, human neutrophils engulf B. anthracis
spores, induce germination, and kill vegetative B. anthracis
). Neutrophils also produce abundant antimicrobial peptides called defensins (20
), which can kill vegetative bacilli (29
), neutralize anthrax lethal toxin, and protect against lethal toxin killing (20
). Related θ defensins, found in primates, kill both spores and vegetative bacilli and neutralize lethal toxin (45
). In cutaneous anthrax, neutrophils are the first cell type recruited to infected tissues (46
). Resistant mice with enhanced neutrophil and reduced macrophage function had an accumulation of neutrophils at the infection site (3
). Neutrophils were also prominent in the cutaneous anthrax cases from 2001 (38
). For inhalation anthrax, neutrophil recruitment in the lungs of dogs and pigs was associated with survival after high exposure doses (28
). Furthermore, the inhalation anthrax patients from the 2001 event had elevated circulating neutrophils or band forms even though overall WBC counts were normal (17
), consistent with other microbial infections (2
). Most rhesus macaques in this study also fit this description, with elevated neutrophils and normal WBC counts as late as 72 h. Therefore, current evidence suggests that early recruitment of neutrophils is an important feature of cutaneous and inhalation anthrax. It may be presumed that this is also the case for gastrointestinal anthrax (28
Our study provides additional information on neutrophil involvement in the early stages of inhalation anthrax. We can deduce that the substantial neutrophil recruitment observed at 2 h in animal D may have contributed to its extended survival. Importantly, since it is well established that neutrophils are the first cell type recruited (within 1 to 2 h) to the lung tissues in other infections (10
), it is reasonable to assume that the same is true for anthrax.
Whereas neutrophils increased during inhalation anthrax in this study, we observed that the percentage of lymphocytes decreased over time. Many microbial infections, including those caused by B. anthracis
spp., Staphylococcus aureus
, and Listeria monocytogenes
, are characterized by decreased lymphocytes along with increased neutrophils (7
). The lymphocyte declines occur because microbial products trigger lymphocyte cell death by apoptosis (6
). In our study, lymphocyte profiles declined early and at 48 h, which was consistent with the expected response (7
). The increase in lymphocytes observed at 72 h may reflect their recovery, concurrent with declines in bacterial load.
In this study, we have observed a classical response to microbial infections with elevated neutrophils and reduced lymphocytes during B. anthracis
infection. Their combination, expressed as the N/L ratio (49
), yielded a clear measure of the changes in inflammation over time. While excess inflammation is considered detrimental, capable of causing widespread tissue damage, it is often associated with resistance to serious infections (26
). This study included one animal with enhanced early inflammation and extended survival. The enhanced inflammation at 48 h in all five macaques led to a temporary clearance of microbial products. The temporary clearance of microbial products was longer in the 9-day survivor and might be viewed clinically as a “false” recovery, which has also been reported for human inhalation anthrax cases (5
). Ongoing animal studies are suggesting that the levels of circulating LF are associated with the length of survival and may be of prognostic value. These studies may also further explain the triphasic kinetics of toxemia described here.
This study is the first to track both LF and PGA levels in inhalation anthrax in rhesus macaques and to compare these with bacteremia and innate immune cell recruitment. A triphasic kinetic profile of LF and PGA, but not PA antigenemia, was observed in rhesus macaques. This study also identified a potential source of early innate defense, an association between neutrophil recruitment and temporary clearance of infection. Our data support the previously defined role for neutrophils in protection from cutaneous infections (3
). These data also suggest that neutrophils may attempt to clear bacilli early in systemic B. anthracis
inhalation infection, before they are overwhelmed or “disarmed” by the effects of the anthrax toxins, which leads to the characteristic exponential increase in bacteremia, antigenemia, and death. Brachman described human inhalation anthrax as a “biphasic” disease with two distinct periods of illness (5
). The first period is manifest as mild and flu-like and is followed by an improvement in clinical status (the second phase described in our study). The final clinical stage is acute, sudden, and usually fatal (5
). We have defined the central stage of clinical improvement observed by Brachman as a brief remission with reduced PGA and LF and absence of bacteremia. This period also represents a window of potential false negative diagnoses for culture-based tests and illustrates the importance of using a combination of tests for diagnosis. The sensitivity of the MS LF assay circumvents this problem with early and consistent detection of infection. The MS LF assay has provided an improved understanding of anthrax infection dynamics with this first description of the initial acute phase, through the remission phase, and beyond. The ability to detect infection in the initial phase prior to the onset of high-density bacteremia is important for successful intervention. Use of the LF MS assay in emergencies may give the earliest and most reliable measure of anthrax, leading to earlier and more successful intervention.